*Article* **Neuroprotective Effects of Fingolimod in a Cellular Model of Optic Neuritis**

**Amritha A. Candadai 1,2,3, Fang Liu 1,2,3 , Arti Verma 1,2, Mir S. Adil 1,2 , Moaddey Alfarhan 1,2,3 , Susan C. Fagan 1,2, Payaningal R. Somanath 1,2 and S. Priya Narayanan 1,2,3,\***


**Abstract:** Visual dysfunction resulting from optic neuritis (ON) is one of the most common clinical manifestations of multiple sclerosis (MS), characterized by loss of retinal ganglion cells, thinning of the nerve fiber layer, and inflammation to the optic nerve. Current treatments available for ON or MS are only partially effective, specifically target the inflammatory phase, and have limited effects on long-term disability. Fingolimod (FTY) is an FDA-approved immunomodulatory agent for MS therapy. The objective of the current study was to evaluate the neuroprotective properties of FTY in the cellular model of ON-associated neuronal damage. R28 retinal neuronal cell damage was induced through treatment with tumor necrosis factor-α (TNFα). In our cell viability analysis, FTY treatment showed significantly reduced TNFα-induced neuronal death. Treatment with FTY attenuated the TNFα-induced changes in cell survival and cell stress signaling molecules. Furthermore, immunofluorescence studies performed using various markers indicated that FTY treatment protects the R28 cells against the TNFα-induced neurodegenerative changes by suppressing reactive oxygen species generation and promoting the expression of neuronal markers. In conclusion, our study suggests neuroprotective effects of FTY in an in vitro model of optic neuritis.

**Keywords:** optic neuritis; multiple sclerosis; oxidative stress; neuroprotection; fingolimod

#### **1. Introduction**

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) prevalent in about 400,000 people in the US and 2.1 million people worldwide [1–4]. Approximately 20% of MS patients present with vision deficits associated with optic neuritis (ON) [5,6], and neurodegeneration characterized by loss of retinal ganglion cells, thinning of the nerve fiber layer, and axonal damage [7,8]. Parameters of visual function are utilized as necessary outcome measures in MS studies [9]. Although the current MS therapies target the inflammatory pathology, effects on the long-term neurodegenerative phases of the disease have not been shown. A treatment that effectively targets both aspects of MS would likely achieve preferred status as a disease-modifying agent.

Fingolimod (FTY720 or FTY), a sphingosine analog that functions as a potent immunosuppressive agent in the CNS, is approved for MS therapy, especially for highly active disease [10,11]. Once phosphorylated into its active form by the sphingosine-1-kinase, it acts as an agonist on sphingosine-1-phosphate (S1P) receptors [12–15] and induces internalization of S1P receptors after binding [16]. Pharmacologically FTY is known as an immunomodulatory drug. FTY exerts its immunosuppressive effect by lymphocyte sequestration and thus reduces the numbers of T and B cells in circulation [17,18]. FTY has been shown to exhibit neuroprotective properties in experimental models of Alzheimer's, stroke,

**Citation:** Candadai, A.A.; Liu, F.; Verma, A.; Adil, M.S.; Alfarhan, M.; Fagan, S.C.; Somanath, P.R.; Narayanan, S.P. Neuroprotective Effects of Fingolimod in a Cellular Model of Optic Neuritis. *Cells* **2021**, *10*, 2938. https://doi.org/10.3390/ cells10112938

Academic Editors: Maurice Ptito and Joseph Bouskila

Received: 10 September 2021 Accepted: 21 October 2021 Published: 28 October 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and Parkinson's disease [19–24]. A recent study showed that FTY exerts neuroprotective and anti-inflammatory effects on the retina and optic nerve in a mouse model of MS, perhaps explaining the potent protective effects in patients [25]. However, the molecular mechanisms regulating the neuroprotective properties have not been studied in retinal neurons. The current study was performed to assess the neuroprotective potential of FTY in an in vitro model of optic neuritis.

The R28 rat neuro-retinal cell line treated with TNFα was standardized to mimic MS-mediated neuronal injury in vitro. The cellular model was chosen based on the studies by Seigel et al. demonstrating the activity and/or expression of neuronal markers at the mRNA, protein, and functional levels in response to various stimuli [26,27]. The expression of neuron-specific markers such as microtubule associated protein 2 (MAP2), Syntaxin, neuron-specific enolase [NSE], Nestin, and receptors for neurotransmitters such as dopamine, serotonin, acetylcholine, and glycine justify the use of these cells to study CNS function [28]. Utilizing the in vitro experimental model of optic neuritis standardized in our laboratory using the tumor necrosis factor-α (TNFα) as an insult, the current study investigated the neuroprotective properties of FTY in reducing the TNFα-induced injury in R28 cells.

#### **2. Materials and Methods**

#### *2.1. Cell Culture*

Immortalized R28 postnatal day 6 rat neuro-retinal cells (heterogeneous population of cells derived from the parent cell line) (Cat # E1A-NR.3, Kerafast, Inc., Boston, MA, USA) were maintained in low-glucose DMEM medium (Cat # SH30021.01, Hyclone, Logan, UT, USA) supplemented with 10% fetal calf serum (Cat # SH30073.02, Hyclone, Logan, UT, USA), 0.225% Sodium bicarbonate (Cat # S8761, Sigma, St. Louis, MO, USA), 1X MEM nonessential amino acids (Cat # 11140-050, GIBCO, Waltham, MA, USA), 1X MEM vitamins (Cat # 11120-052, GIBCO, Waltham, MA, USA), 0.5 mM l-glutamine (Cat # 25030-081, GIBCO, Waltham, MA, USA), and 50 µg gentamicin (Cat # 15750-060, GIBCO, Waltham, MA, USA). The cells were differentiated to a neuronal phenotype with the help of 25 µg/mL laminin (Cat # 11243217001, Sigma, St. Louis, MO, USA) and 250 mM modified cyclic adenosine monophosphate (pCPT-cAMP) (Cat # C3912, Sigma, St. Louis, MO, USA) treatments, according to the published methods [29,30]. All other chemicals were purchased from Fisher Scientific, Waltham, MA, USA, unless otherwise mentioned.

#### *2.2. The In Vitro Model of Optic Neuritis*

Dose-response experiments were conducted to standardize the in vitro treatment with TNFα (recombinant rat tumor necrosis factor-α) (Cat # 510-RT, R&D Systems, Minneapolis, MN, USA) to induce neuronal injury in R28 cells. On day 0, cells were differentiated on 6-well culture plates (24 h), as described above. Treatments with TNFα at doses of 5, 10, 25, 50 ng/mL were initiated on day 1, followed by a 24 h incubation period. Cell viability with various doses of TNFα was compared against the control group with no treatment that depicted average growth and differentiation.

#### *2.3. Treatments with Fingolimod*

Once the effective dose of TNFα was established, experiments were conducted to identify an appropriate treatment concentration of FTY (Cat # 11975, Cayman Chemicals, Ann Arbor, MI, USA). Cells were pre-treated with FTY at concentrations of 2.5, 5, 10, 25, 50, and 100 nM for a 1 h incubation period before the TNFα treatment. Cell viability differences were compared among control (no treatment), TNFα-treated group, and TNFα co-incubated with varying FTY concentrations. FTY treatment alone at higher doses of 100, 200, and 500 nM were performed to test its cytotoxicity.

#### *2.4. Cell Viability*

The degree of viability of R28 cells post-treatment with TNFα and/or FTY was determined using the Trypan blue method [31]. Cells were seeded on six well plates at a density of 0.5 × 10<sup>6</sup> per well. Following treatments, they were trypsinized and collected in labeled tubes respective to their grouping. Equal volumes of a sample of cell suspension and trypan blue dye were thoroughly mixed using a micropipette, from which 10 µL was injected into a cell counting chamber (Cat # 02-671-55A, Fischer Scientific, Waltham, MA, USA) for manual counting. Trypan blue dye stains dead cells blue, and the number of viable cells in all four 16-squared tiles of the chamber were counted. This was repeated in triplicates for each cell suspension sample, and cell viability was plotted as the percentage with respect to 100% control. All graphs are represented as Mean ± SEM.

#### *2.5. Western Blot Analysis*

R28 cells seeded at a density of differentiated on six well plates and treated with TNFα and/or FTY as described earlier, resulting in four groups: Control (no treatment), TNFα, TNFα + FTY, and Control + FTY groups. Cells were homogenized, and the lysate was collected in RIPA buffer (Cat # 20-188, EMD Millipore, Burlington, MA, USA) containing protease (Cat # 78430, Fischer Scientific, Waltham, MA, USA) and phosphatase inhibitors (Cat # 78428, Fischer Scientific, Waltham, MA, USA). Protein estimation was performed using the Bradford's protein assay kit (Cat # 5000201, Bio-Rad Laboratories, Hercules, CA, USA). Samples with an equal amount of protein were prepared by using 4X Laemmli buffer (Cat # 161-0747, Bio-Rad Laboratories, Hercules, CA, USA) containing β-mercaptoethanol (Cat # O3446I-100, Fischer Scientific, Waltham, MA, USA). Samples were separated on SDS-PAGE and transferred to nitrocellulose membranes (Cat # 1620112, Bio-Rad Laboratories, Hercules, CA, USA). Membranes were blocked in 5% milk (Cat # 1706404, Bio-Rad Laboratories, Hercules, CA, USA) in tris-buffered saline with tween-20 (TBS-T) and incubated with respective primary antibodies (Table A1) overnight at 4 ◦C. Membranes were washed with 1× TBS-T and incubated in appropriate secondary antibodies. Signals were detected using enhanced chemiluminescence (ECL) (Cat # 32106, Fischer Scientific, Waltham, MA, USA). NIH Image J software was utilized to conduct densitometric analysis, and the intensity measurements were normalized to loading control. Experiments were repeated for a minimum of three times.

#### *2.6. Immunofluorescence Staining*

Cells seeded at a density of 15,000 to 20,000 cells per well on 8 well glass chamber slides (Cat # 154941, Fischer Scientific, Waltham, MA, USA) were treated according to the study design described previously. Following the 24 h incubation period, the culture media was removed, cells were washed with 1× PBS and fixed with 2% paraformaldehyde for 10 min. This was followed by a wash with PBS, and the chamber slides were stored in humidified containers at 4 ºC. Slides were brought to room temperature and washed with PBS before initiating the staining protocol. Permeabilization was achieved using 0.1% Triton X-100 in PBS for 5 min, followed by a PBS wash, and blocking with 10% donkey serum at room temperature for 1 h. Cells were washed and incubated with respective primary antibodies (Table A1) overnight. The next day, the cells were incubated with appropriate secondary antibodies for 2 h. The Slides were washed, and the chambers were separated from the glass slide. Cells were covered with a coverslip using a mounting medium containing DAPI and stored in 4 ◦C. Images were taken using a confocal microscope (LSM 780; Carl Zeiss, Thornwood, NY, USA) available at the Augusta University imaging core facility. For the Tuj1 and NSE quantitative analysis, similar thresholds were set for all the images. Three to five regions of interest (ROI) were randomly selected per chamber slide images, and the fluorescent intensity of immunoreactivity was measured (Integrated Density) using NIH Image J software. Along with the average fluorescence intensities of a given ROI, the average fluorescence intensity of areas without staining (background) was measured as well. The Corrected Fluorescence was calculated as Integrated Density—(Area of selected

cells X Mean fluorescence of background readings). The values were then normalized relative to percentage of control group. Experiments were repeated a minimum of three times and details are provided under figure legends.

#### *2.7. Cellular ROS Formation Using DCF Assay*

CM-H2DCFDA (General Oxidative Stress Indicator, Thermofisher Scientific, cat# C6827) was used to measure reactive oxygen species (ROS) formation in TNFα treated cells and any changes in response to FTY treatment, as per the manufacturer's instructions. Briefly, cells were washed with ice-cold PBS (three times), and then incubated with 10 µm CM-H2DCFDA (working solution) at 37 ◦C for 30 min under dark. Washed with ice-cold PBS (three times), cover-slipped using the mounting medium with DAPI stain (Vector laboratories), and the images were immediately taken by confocal microscope (LSM 780; Carl Zeiss, Thornwood, NY, USA). NIH Image J software was used for the analysis of fluorescent intensity. For single-cell quantification, single-cell was delineated and sampled at 40× from a random start point. Only cells with precise neuronal shape and specific nuclear staining with DAPI were analyzed. Three to five chamber slides per treatment were examined and the experiments were repeated three times.

#### *2.8. Mitochondrial ROS Measurement*

The production of superoxide by mitochondria in response to TNFα treatment and the impact of FTY on the mitochondrial ROS generation was measured using the MitoSOX™ Red reagent (Thermo Fisher Scientific, cat# M36008), following the manufacturer's instructions. Briefly, cells on chamber slides were washed with ice-cold PBS (three times), incubated with 5 µM MitoSOX™ reagent working solution, and incubated for 10 min at 37 ◦C, protected from light. Chamber slides were then carefully washed with ice-cold PBS (three times), cover slipped, and images were immediately taken by confocal microscope (LSM 780; Carl Zeiss, Thornwood, NY, USA). NIH Image J software was used for the analysis of fluorescent intensity. Three to five chamber slides per treatment were examined and the experiments were repeated three times.

### *2.9. H2O<sup>2</sup> Treatment and LDH Assay*

Oxidative stress was induced on differentiated R28 cells using H2O<sup>2</sup> (SigMA, USA) following the method of Song et al. [32] with some minor modifications. Briefly, R28 cells were treated with multiple concentrations of H2O<sup>2</sup> (0.0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.5 mM) for 24 h and cellular cytotoxicity was measured. In order to determine the effect of FTY on H2O2-induced oxidative stress, cells were pre-treated with 25 nM FTY (as described previously) and changes in cytotoxicity was measured.

Lactate dehydrogenase (LDH) assay was used to determine cellular cytotoxicity. LDH released into the culture media from damaged cells was measured following the manufacturer's instructions (CytoTox 96 non-radioactive cytotoxicity assay kit; Promega Corporation, Madison, WI, USA). The level of LDH release was normalized to the total LDH content following cell lysis in a medium. The absorbance was determined at 490 nm using a Multimode Microplate Reader (Berthold Technologies, Bad Wildbad, Germany). LDH release was expressed as a percentage of the maximum LDH released after cell lysis.

#### *2.10. Statistical Analysis*

All statistical analyses were performed with GraphPad Prism 7 (GraphPad Software Inc., La Jolla, CA, USA). Student t-test or Two-way ANOVA followed by Tukey's multiple comparisons test was employed to analyze the groups. A *p* value less than 0.05 was considered as statistically significant. Results are presented as Mean ± SEM.

#### **3. Results**

#### *3.1. Fingolimod Treatment Reduces TNFα-Induced Neuronal Injury*

We found that TNFα treatment resulted in a significant reduction (*p* < 0.05) in cell viability at doses of 10 ng/mL (44.6 ± 24.8%), 25 ng/mL (39.8 ± 8.7%), and 50 ng/mL (60.2 ± 13.0%) compared to the untreated control (Figure 1A). Our findings suggested that TNFα at 10 ng/mL desirably reduced the percentage of viable cells by nearly half that of the control group. In the next step, cells were pre-treated with fingolimod at concentrations of 0, 2.5, 5, 10, 25, 50, and 100 nM for 1 h prior to TNFα induction (10 ng/mL). Our results showed that FTY concentrations at 25 nM (79.7 ± 17.7%) and 50 nM (71.0 ± 32.3%) significantly prevented the TNFα-induced injury (*p* < 0.001) (Figure 2B). Experiments with high-dose FTY treatment alone resulted in cytotoxicity at doses above 100 nM. Doses of 100, 200, and 500 nM showed a decreasing viable cell count of 14.5 × 10<sup>4</sup> , 3.5 × 10<sup>4</sup> , and 2 × 10<sup>4</sup> , respectively, compared to an average viable cell count of 47.5 × 10<sup>4</sup> in control cells without FTY treatment (not shown). Based on our findings, 25 nM was chosen as the dose of FTY to be used in further studies (Figure 1C). *α* α α α

α

α α α α α α α α **Figure 1.** Dose-response effect of TNFα treatment on R28 cell survival and its reversal by co-treatment with fingolimod. (**A**) Neuronal damage was induced by treating R28 cells with various doses of TNFα for 24 h assessed by Trypan blue method. TNFα at 10ng/mL showed a marked reduction in cell survival and was chosen as the effective dose for further analysis. [*n* = 3; \* *p* < 0.05 vs. dose 0]. (**B**) Cells were pre-treated with different doses of FTY, followed by TNFα (10 ng/mL), and cell survival was assessed at 24 h. Bar graph showing the effect of 25, 50, and 100 nM FTY on improving the rate of R28 cell survival [# *p* < 0.005 vs. Con; \* *p* < 0.05 vs. TNFα]. (**C**) Bar graph indicating 25 nM as the optimal dose of FTY in protecting R28 cells from TNFα-induced injury in vitro [*n* = 3; \* *p* < 0.001 vs. Con; # *p* < 0.01 vs. TNFα].

α α **Figure 2.** Fingolimod co-treatment mitigates TNFα-induced activation of cellular stress signaling. (**A**) Western blot images showing upregulation of phospho-p38 MAPK levels in response to TNFα (10 ng/mL) treatment in R28 cells, which was reduced in the presence of FTY. (**B**) Bar graph showing band densitometry quantification of Western blots indicating the effects of FTY in suppressing P38

α

α

α α

α α

α

α

α

α

α

α

MAPK activation induced by TNFα treatment [*n* = 6; \* *p* < 0.05 vs. Con; # *p* < 0.05 vs. TNFα]. (**C**) Western blot images showing no changes in phospho-Akt (Ser473) levels in response to either TNFα (10 ng/mL) and/or FTY in R28 cells. (**D**) Bar graph showing band densitometry quantification of Western blots indicating the no changes in phospho-Akt (Ser473) levels in response to either TNFα (10 ng/mL) and/or FTY in R28 cells [*n* = 6; NS vs. TNFα].

#### *3.2. Fingolimod Attenuates Cellular Stress and Survival Signaling*

Changes in phosphorylated p38 MAP kinase expression were assessed to characterize cellular stress by Western blotting. Figure 2A shows that TNFα augmented the level of p-P38 MAPK, and this increase was markedly prevented in the presence of FTY. Moreover, we found that FTY treatment alone did not affect levels of p-P38 MAPK. Our quantification data demonstrated that in the presence of FTY, levels of p-P38/total-P38 were significantly reduced (*p* < 0.05) versus the TNFα group (Figure 2B). Changes in phosphorylated Akt levels were tested to assess cell survival using Western analysis. Figure 2C shows no changes in the expression of p-AKT with TNFα induction. Consistently, our quantification data also showed no significant decrease in levels of p-Akt/t-Akt in the presence of TNFα versus control (Figure 2D). α α α

α

α α

#### *3.3. Effect of Fingolimod on Neuronal Cell Death*

To evaluate apoptotic changes, Western blot analyses using apoptotic marker cleaved caspase-3, and anti-apoptotic marker Bcl-xL were performed. An upregulated expression on cleaved caspase-3 along with a decreased level of Bcl-xL in the presence of TNFα was observed. In the co-treatment group with TNFα and FTY, we observed a reversal in these changes (Figure 3). Quantification data showed a significant increase in the levels of cleaved caspase-3 (*p* < 0.01) versus control, while FTY treatment significantly reversed this effect (*p* < 0.05) versus the TNFα group (Figure 3B). TNFα caused a significant decrease in levels of Bcl-xL (*p* < 0.05) compared to the control group. However, the difference observed in response to FTY co-treatment was not statistically significant compared to the TNFα group (Figure 3D). α α α α α

α

α α **Figure 3.** Fingolimod co-treatment blunted TNFα-induced activation of cleaved caspase-3 and expression of Bcl-xL. (**A**) Representative Western blot data showing increased expression of cleaved caspase-3 with TNFα treatment, which was reduced in the presence of FTY. (**B**) Bar graph showing band densitometry quantification indicating increased cleaved caspase-3 expression with TNFα treatment [\* *p* < 0.05 vs. Con; *n* = 3] and its reversal by co-treatment with FTY [\* *p* < 0.005 vs. control; # *p* < 0.05 vs. TNFα; *n* = 4]. (**C**) Western blot images showing reduced expression of Bcl-xL with TNFα treatment, which was increased in the presence of FTY. (**D**) Bar graph showing band densitometry quantification of Western blots indicating reduced Bcl-xL expression with TNFα treatment [\* *p* < 0.05, *n* = 3]. The changes observed in response to FTY, however, were not statistically significant.

α

#### *3.4. Effect of Fingolimod on TNFα-Induced Neuronal Damage α*

α

α α

Immunofluorescence studies were conducted to study the neurodegenerative changes observed in R28 cells in response to the different treatments. β-tubulin III, also called Tuj1 contributes to microtubule formation in neuronal cell bodies and axons and plays important roles in axonal transport and cell differentiation. It is a useful marker for the detection of injury-related alterations [33]. Neuron specific enolase (NSE) is widely used and accepted as a neuronal marker, and is expressed by mature neurons and cells of neuronal origin [34]. In the present study, immunostaining with Tuj1 and NSE revealed the degenerative changes induced by TNFα, which was attenuated with FTY treatment (Figure 4A,C). Tuj1 expression was downregulated in TNFα-treated cells; however, we observed that FTY was able to protect the cells against neurofilament damage (Figure 4A lower panels). Consistently, the NSE marker was found to be reduced in the presence of TNFα, and the reduction was prevented by FTY treatment (Figure 4B,D). β α α α

α β α α α α α **Figure 4.** Fingolimod depicts protection against neuronal damage evidenced by immunofluorescence staining of marker proteins. (**A**) Representative confocal images showing the impact of TNFα treatment on neurofilament, Tuj1 (β-tubulin class III) indicating neurodegeneration, which was reduced by co-treatment with FTY. High magnification images of the boxed areas, indicating reduced Tuj1 expression, are presented in the lower panel. Scale bar 50 µm. (**B**) Bar graph showing the quantification of Tuj1 level in response to TNFα treatment, and the protective effect by co-treatment with FTY (**C**) Representative confocal images showing the changes in neuronal enolase (NSE) in response to TNFα and FTY treatments. Lower panel show high magnification images of the boxed areas, indicating reduced levels in TNFα treated group and the improved NSE expression in response to FTY co-treatment. Scale bar 50 µm. (**D**) Bar graph showing the quantification of Tuj1 and NSE levels in response to TNFα treatment, and the effect by co-treatment with FTY [\* *p* < 0.01 vs. control; # *p* < 0.01 vs. TNFα, *n* = 3 per group].

#### *3.5. Effect of Fingolimod on ROS Formation*

Changes in ROS formation were studied using DCF assay. As shown in Figure 5A, ROS levels were observed to be markedly elevated in the neuronal cells in response to TNFα treatment. However, treatment with FTY downregulated the TNFα induced ROS generation. Our quantification studies demonstrate that the ROS levels are significantly higher in the TNFα group compared to control and are significantly reduced in response to FTY co-treatment (Figure 5B).

α α

α α **Figure 5.** Fingolimod reduced TNFα-induced ROS formation. (**A**) Representative images showing the impact of FTY on reactive oxygen species (ROS) formation. H2DCFDA (DCF) assay was used to assess the generation of ROS. Scale bar 50 µm. (**B**) Quantification of single cell fluorescence intensity showing of the increased ROS formation in response to TNFα treatment, which was reduced in the presence of FTY [\* *p* < 0.01 vs. control; # *p* < 0.01 vs. TNFα, n = 3 per group].

α

#### *3.6. Effect of Fingolimod on Mitochondrial Dynamics and ROS Formation*

α

α α α α α α In the present study, we investigated the effect of FTY treatment on the changes in proteins related to mitochondrial dynamics, including DRP-1 (dynamin related protein-1), Mitofusin 2 and OPA-1 (optic atrophy 1). As illustrated in Figure 6A,D, expression of Mitofusin 2 was significantly reduced in TNFα treated R28 cells, while FTY treatment normalized the level of Mitofusin 2 in TNFα treated cells, similar to control levels. An increase in the level of p-DRP1 was observed in TNFα treated group, while treatment with FTY reversed this effect. However, these changes were not statistically significant. No marked differences were seen in the other mitochondrial proteins studied. Further, we investigated the impact of FTY treatment on mitochondrial ROS formation using MitoSox assay (Figure 6G,H). TNFα treatment resulted in the generation of mitochondrial ROS as evidenced by elevated fluorescence indicator. FTY treatment markedly reduced the level of mitochondrial ROS formed in response to TNFα treatment. Quantification results demonstrate that the mitochondrial ROS level is significantly upregulated in TNFα treated group and the treatment with FTY significantly reduced the effect (Figure 6H).

### *3.7. Fingolimod Attenuates H2O2-Induced Cellular Damage and Stress Signaling in R28 Cells*

α To further assess the neuroprotective effect of FTY, experiments were performed using H2O2, another cellular stressor. Our results show that H2O<sup>2</sup> induces cytotoxicity in differentiated R28 cells in a dose-dependent manner (Figure 7A). Treatment with FTY significantly reduced the cytotoxicity induced by TNFα at the various concentrations studied (Figure 7B). Our results indicate that H2O<sup>2</sup> treatments significantly elevated p-P38 levels at all the concentrations studied, while FTY treatment significantly reduced the effect at two different contentions of H2O2. FTY treatment did not offer protection at a higher concentration, 1mM of H2O<sup>2</sup> (Figure 7C).

α

α α α α α α **Figure 6.** Changes in mitochondrial protein dynamics and ROS formation in response to Fingolimod treatment. (**A**–**C**) Representative Western blot data showing changes in expression of proteins associated with mitochondrial dynamics with TNFα treatment, and the effect of FTY cotreatment. (**D**–**F**) Respective bar graphs showing quantification of these proteins expression changes with TNFα treatment and any changes by co-treatment with FTY [\* *p* < 0.05 vs. control; # *p* < 0.05 vs. TNFα; N = 3]. (**G**) Representative images of Mitosox Red staining showing the impact of TNFα on mitochondrial reactive oxygen species (ROS) formation and the effect of FTY on the treatment. Scale bar 20 µm. (**H**) Quantification of fluorescence intensity showing of the increased mitochondrial ROS formation in response to TNFα treatment, which was reduced in the presence of FTY [\* *p* < 0.01 vs. control; # *p* < 0.01 vs. TNFα, *n* = 3 per group]. α α α α α

**Figure 7.** Fingolimod attenuates H2O<sup>2</sup> -induced cellular damage and stress signaling in R28 cells. (**A**) LDH assay results showing the dose-dependent effect of H2O<sup>2</sup> treatment on R28 cells. \* *p* < 0.05 vs control; # *p* < 0.01 vs. control and *n* = 3. (**B**) LDH assay showing the cytotoxic effect of H2O<sup>2</sup> at

selected concentrations on R28 cells and the protective effect of FTY on the treatments. \* *p* < 0.05; \*\* *p* < 0.01. (**C**) Representative Western blot data showing changes in p-P38 MAPK with H2O2 treatment, and the protective effect of FTY co-treatment on differentiated R28 cells. (**D**) Bar graph showing quantification of p-P38MAPK proteins expression changes with H2O<sup>2</sup> and FTY treatments. \* *p* < 0.01 and the experiment was repeated three times.

#### **4. Discussion**

The present study was conducted to assess the potential neuroprotective action of FTY in an in vitro model of optic neuritis. Lack of effective treatment strategies to reduce neurodegeneration continues to be a major problem in the field of MS research. It is vital to understand the underlying mechanisms of MS-induced neuronal damage and dysfunction. Even though MS research on the pathophysiology and associated molecular mechanisms have evolved over decades of research, including our laboratory [35,36], the field lacks reliable in vitro models to study neurodegeneration. Synthetic molecules such as trimethyltin [37], oxaliplatin [38], and cuprizone [39], although successful in creating a neurodegenerative environment, do not accurately represent the neuroinflammatory changes observed in an MS brain. In MS, inflammatory leukocytes are believed to infiltrate the CNS to mediate demyelination and neuronal degeneration via cytokines upon activation of T lymphocytes and antigen-presenting cells (APCs) [40]. tumor necrosis factor-α (TNFα) is one of the primary cytokines that are present in elevated levels in active MS lesions, serum, and cerebrospinal fluid of MS patients [41]. Studies conducted on BV-2 microglial cell lines [42] and primary mixed neuronal and glial cultures [43] show the effect of TNFα-induced damage and apoptosis. Hence, utilizing the R28 neuro-retinal cells, we standardized an in vitro experimental model of neurodegeneration to assess the impact of fingolimod on TNFα-induced neuronal damage. Using a set of functional and biochemical analyses, our study demonstrates the neuroprotective properties of fingolimod in MS-associated optic neuritis in vitro.

We first demonstrated the dose-dependent effects of TNFα on neuronal cell viability in R28 neuro-retinal cells in vitro and identified the optimal dose of TNFα for further molecular characterization. The R28 cells are immortalized, rat retinal origin, heterogenous, precursor cells with differentiation potential [26]. According to the published methods, R28 cells were differentiated to neuronal phenotype with the addition of a modified form of cAMP and laminin and grown using DMEM [44–46]. Studies by Seigel et al. utilizing R28 cells demonstrate the expression of neuron-specific markers such as MAP2, Syntaxin, NSE, and Nestin, along with neurotransmission receptors [26,28]. Our study characterized the expression of neurofilament marker Tuj1, along with NSE in these cells, thus demonstrating the reliability of this model in evaluating the impact of FTY on the neuronal injury. In MS, one of the major clinical presentations observed in patients is optic neuritis [47]. Studies have shown that approximately 20% of patients present with inflammation of the optic nerve as their first symptom of MS [5,6]. Another study conducted by the North American Research Committee on multiple sclerosis (NARCOMS) showed that of the 9107 patients participating in the study, 60% reported signs of vision impairment, and 14% of these depicted moderate/severe/very severe impairment of vision [9]. Based on the available research, visual dysfunction is a common component of MS disease progression and an important determinant of quality of life.

Current MS therapies function by suppressing the inflammatory pathways and have unknown impact on the long-term neuronal damage, causing a major knowledge gap and emphasizes the need to identify a neuroprotective therapeutic agent. Therefore, our study focused on assessing the neuroprotective effect of FTY in an in vitro model of MS-induced optic neuritis. Fingolimod, a sphingosine-1-phosphate (S1P) receptor modulator, has previously been shown to prevent neurodegenerative mechanisms targeting an inflammatory CNS state in vitro, in vivo, and clinical settings, as detailed below. Studies on Parkinson's disease models have shown a positive impact with FTY [19–21]. Mechanistically, it was found that the protective effects of FTY in Parkinson's disease were correlated with the activation of survival pathway mediated by Akt/ERK1/2 and increased expression of a

neuron-specific brain-derived neurotrophic factor (BDNF). In a model of Alzheimer's, FTY was able to reverse the effect of damage by modulating the levels of different markers such as the Glial fibrillary acidic protein (astrogliosis marker), taurine (anti-inflammatory marker), and neuronal markers such as the N-acetyl aspartate and glutamate [23]. A meta-analysis was conducted by Liu et al. which included nine studies that focused on quantification of infarct volume and neurological deficit scoring in a transient middle cerebral artery occlusion model of ischemic stroke challenged with FTY [24]. The study concluded that FTY could be a possible candidate for stroke due to its protective effects on neurological deficit and infarct volume in eight of the nine included studies. Promising outcomes with FTY represent the need for further investigation to confirm the theories on its action as a neuroprotective agent. Utilizing the EAE (Experimental Autoimmune Encephalitis) model of MS, a recent study by Yang et al. investigated whether FTY is beneficial to the visual system [25]. Their results showed that FTY treatment offered neuroprotective and anti-inflammatory effects on the retina and optic nerve. FTY treatment alleviated EAE-induced gliosis, inflammation and reduced the apoptosis of RGCs and oligodendrocytes.

In response to FTY treatment, our studies found a reduction in the phosphorylation of p38 MAP kinase (a cellular stress signaling pathway) in TNFα-treated R28 cells. However, fingolimod treatment did not induce any changes in the levels of phosphorylated Akt, indicating its effect on cell survival is independent of Akt, with the conditions studied. TNFα-induced cell death was confirmed by the upregulation of cleaved caspase-3 (a cell death marker) expression along with reduced levels of Bcl-xL (an anti-apoptotic protein). These changes were reversed in response to FTY treatment, supporting its neuroprotective and anti-apoptotic function. FTY treatment also protected the retinal neurons against the TNFα-induced neuronal damage determined by the expression of neuronal markers.

Oxidative stress plays a critical part in the pathogenesis of various neurodegenerative diseases and neuroinflammation. There exists an increasing amount of data indicating that oxidative stress plays a major role in the pathogenesis of MS and optic neuritis [48]. Results from preclinical studies show that suppression of oxidative stress is a promising strategy for optic neuritis [49–53]. In the present study, we show that FTY mediated neuroprotection of R28 cells involves the regulation of ROS formation. A recent study performed on serum samples from patients with relapsing-remitting MS and healthy controls demonstrated that TFY treatment increased total antioxidant capacity [54]. Furthermore, FTY treatment reduced oxidative stress in experimental models of cardiomyopathy [55], multiple system atrophy [56], autism [57], and vitamin K-induced neurotoxicity [20].

Oxidative damages along with mitochondrial dysfunction are common characteristics of neurodegenerative diseases. Mitochondrial dysfunction is increasingly recognized as a major mechanism of MS associated pathologies [58–60]. The present study investigated the impact of FTY treatment on the changes in protein levels related to mitochondrial dynamics. Alterations in mitochondrial dynamics affect mitochondrial size and shape and impact mitochondrial metabolism and cell death. These events are controlled by mitochondrial dynamin-related GTPases, including mitofusin-1, mitofusin-2, OPA1, and DRP1. Our results show that one possible mechanism by which FTY offers neuroprotection of R28 neuronal cells via regulation of mitochondrial fusion. Altered levels of mitochondrial proteins were reported in retinal neurons in models of diabetic retinopathy [61] and glaucoma [62]. Results from our study are consistent with studies on neuroprotective properties of FTY in other models where FTY improved mitochondrial stability and restored mitochondrial dynamics under oxidative stress conditions [20,63–65]. Our present study did not investigate the changes in mitochondrial function.

Our study showed that FTY treatment reduces TNFα induced mitochondrial ROS formation in R28 cells. Other studies recently reported that FTY reduced mitochondrial dysfunction in a rat model of chronic cerebral hypoperfusion, reduced neuroinflammation and restored mitochondrial function in a model of Multiple System Atrophy [65], prevented mitochondrial dysfunction and protected neurons in prion protein-disease model [66]. Furthermore, we investigated the neuroprotective effect of FTY on R28 cells treated with H2O2, a cellular stressor known to induce oxidative stress. Our results reveal that FTY offers neuroprotection in response to oxidative stress-induced cellular damage. While FTY treatment rescued R28 cells at all concentrations of H2O<sup>2</sup> studied, FTY did not significantly reduce phospho-P38 levels at higher concentration of H2O2, suggesting the possibility of other signaling pathways involved. Another possibility is that FTY at a different dose/time could reduce the phospho-P38 levels at higher concentrations of H2O2. Overall, these results indicate that FTY mediated neuroprotection could be offered through multiple mechanisms, including P38MAPK signaling. However, further studies are needed to confirm this observation.

Evidence as a regulator of oxidative stress, along with its immunomodulatory function, offers significant therapeutic potential to FTY in neuroinflammatory diseases such as optic neuritis. Figure 8 depicts the possible mechanisms of FTY mediated neuroprotection in response to TNFα-induced damage. Further studies are needed to define whether the effect of FTY on ROS level is direct or indirect and to delineate the molecular mechanisms of FTY mediated neuroprotection. α

α **Figure 8.** Proposed mechanism of FTY mediated neuroprotection. It is postulated that TNFα treatment induces cellular stress and cell death in retinal neurons by elevating oxidative stress. Treatment with FTY reduces TNFα-induced oxidative stress and improved neuronal survival.

α

α One limitation of our study is that it did not elucidate the role of cell survival pathways that are directly associated with TNFα and FTY action. The concentration of FTY used in our study (25 nM) corresponds to 7.6875 ng/mL. However, as per the manufacturer's (Gilenya®) package insert, the concentration of active fingolimod phosphate in adult MS patients is 1.35 ng/mL. Lower concentrations used in our study, did not offer any neuroprotective effect. It is likely that the neurovascular unit could respond differently in response to other cytokines or injury mediators. This difference in human systemic concentration versus in vitro concentration in our models is an interesting aspect of the study and suggests that repurposing may be needed for the neuroprotective action of FTY in MS patients. Another limitation of the current study is that it is purely performed in a cellular model in vitro, which is not a true reflection of what happens in a complex in vivo set up. Nevertheless, the study provides reasonable optimism on the potential therapeutic benefits of FTY to treat ON. Studies performed on EAE model by Yang el tal demonstrated neuroprotective and anti-inflammatory effects on the retina and optic nerve, with no direct negative effects at the two different doses of FTY (0.3 and 1.0 mg/kg) utilized [25]. Results from our study complements the findings of Yang et al., where stronger neuroprotective effects on the visual system.

#### **5. Conclusions**

Overall, our study investigated the potential neuroprotective effects of FTY in an in vitro model of neurodegeneration. The R28 neuro-retinal cells are characterized as a successful platform for evaluating neuronal damage in the presence of TNFα, and its suppression with FTY. Furthermore, our studies demonstrated the antioxidant properties of FTY, a possible mechanism of neuroprotection. However, further studies are required to confirm the results. Based on the cellular and molecular analysis, FTY demonstrated the potential to be investigated as a novel neuroprotective strategy in conditions like MS and associated pathologies.

**Author Contributions:** Conceptualization, S.P.N. and S.C.F.; methodology, A.A.C., F.L., A.V., M.A. and M.S.A.; formal analysis, A.A.C., F.L., A.V. and M.S.A.; investigation, A.A.C., F.L., A.V. and M.A.; resources, S.P.N. and P.R.S.; writing—original draft preparation, A.A.C.; writing—review and editing, F.L., M.A., M.S.A., S.C.F., P.R.S. and S.P.N.; supervision, S.P.N.; project administration, S.P.N.; funding acquisition, S.P.N. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported in part by University of Georgia startup funds and the National Eye Institute to SPN (R01EY028569). This work has been accomplished using the resources and facilities at the Charlie Norwood VA Medical Center, Augusta, GA, and a core grant from the NIH/NEI to the Augusta University Vision Discovery Institute (P30EY031631). The funders had no role in study design, data collection, analysis, and decision to publish the data. The contents of the manuscript do not represent the views of the Department of Veteran Affairs or the United States.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors are thankful to Chithra Palani for her assistance with the R28 cell culture studies.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A**

**Table A1.** List of antibodies used in the study.


#### **References**


### *Article* **Annexin A1 Mimetic Peptide and Piperlongumine: Anti-Inflammatory Profiles in Endotoxin-Induced Uveitis**

**Ana Paula Girol 1,2,3 , Caroline de Freitas Zanon <sup>2</sup> , Ícaro Putinhon Caruso <sup>4</sup> , Sara de Souza Costa 1,2 , Helena Ribeiro Souza 1,2, Marinônio Lopes Cornélio <sup>4</sup> and Sonia Maria Oliani 2,3,5,\***


**Abstract:** Uveitis is one of the main causes of blindness worldwide, and therapeutic alternatives are worthy of study. We investigated the effects of piperlongumine (PL) and/or annexin A1 (AnxA1) mimetic peptide Ac2-26 on endotoxin-induced uveitis (EIU). Rats were inoculated with lipopolysaccharide (LPS) and intraperitoneally treated with Ac2-26 (200 µg), PL (200 and 400 µg), or Ac2-26 + PL after 15 min. Then, 24 h after LPS inoculation, leukocytes in aqueous humor, mononuclear cells, AnxA1, formyl peptide receptor (fpr)1, fpr2, and cyclooxygenase (COX)-2 were evaluated in the ocular tissues, along with inflammatory mediators in the blood and macerated supernatant. Decreased leukocyte influx, levels of inflammatory mediators, and COX-2 expression confirmed the anti-inflammatory actions of the peptide and pointed to the protective effects of PL at higher dosage. However, when PL and Ac2-26 were administered in combination, the inflammatory potential was lost. AnxA1 expression was elevated among groups treated with PL or Ac2-26 + PL but reduced after treatment with Ac2-26. Fpr2 expression was increased only in untreated EIU and Ac2-26 groups. The interaction between Ac2-26 and PL negatively affected the anti-inflammatory action of Ac2-26 or PL. We emphasize that the anti-inflammatory effects of PL can be used as a therapeutic strategy to protect against uveitis.

**Keywords:** eye inflammation; lipopolysaccharide; natural bioactive extracts; Ac2-26; FPR receptor; inflammatory mediators

### **1. Introduction**

Uveitis is an intraocular inflammation of different etiologies [1–5] characterized by leukocyte accumulation in ocular tissues and cytokine release. It is a painful condition and is associated with redness, photophobia, impaired vision, and blindness [6–10]

Pharmacological treatment for uveitis includes corticosteroids, chemotherapeutic agents, and tumor necrosis factor (TNF)-α inhibitors [3,7,8,11], but the use of these drugs is limited by their serious side effects, such as increased ocular pressure or cytotoxicity [9,12]. However, recent advances in the mechanisms of inflammation and the discovery of several endogenous anti-inflammatory mediators have provided new therapeutic possibilities for uveitis treatment [5,7,10,13–15].

**Citation:** Girol, A.P.; de Freitas Zanon, C.; Caruso, Í.P.; de Souza Costa, S.; Souza, H.R.; Cornélio, M.L.; Oliani, S.M. Annexin A1 Mimetic Peptide and Piperlongumine: Anti-Inflammatory Profiles in Endotoxin-Induced Uveitis. *Cells* **2021**, *10*, 3170. https://doi.org/ 10.3390/cells10113170

Academic Editors: Maurice Ptito and Joseph Bouskila

Received: 12 September 2021 Accepted: 28 October 2021 Published: 15 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

In particular, the endogenous protein annexin A1 (AnxA1) may represent an alternative therapy for uveitis [16–20]. AnxA1 is an anti-inflammatory 37 kDa protein, which exhibits calcium and membrane phospholipid binding sites and is involved in the inhibition of glucocorticoid-induced eicosanoids and phospholipase A2 synthesis [21–24]. Structurally, AnxA1 comprises a specific small N-terminal region and a central domain formed by four to eight replicates of a highly conserved 70 to 80 amino acids sequence [25–27]. The N-terminal domain contains sites for post-translational processes, such as phosphorylation, glycosylation, and proteolysis [17,24,28,29].

Over the years, our research group has investigated the effect of AnxA1 on different ocular inflammatory conditions [17,19,30–35]. Positive modulation of endogenous AnxA1 in inflammatory cells in the eyes of mice and retinal pigment epithelial cells (ARPE-19) infected with *Toxoplasma gondii* suggests the protein can be used as a therapeutic target in ocular toxoplasmosis [30]. AnxA1 is involved in the signaling cascades of inflammatory processes, leading to decreased cell proliferation and increased migration by modulation of connective tissue growth factor (CTGF) and lecithin retinol acyltransferase (LRAT) gene expression in ARPE-19 cells [19]. In an experimental allergic conjunctivitis model using wild and AnxA1-null Balb/c mice, administration of the AnxA1-N-terminal region mimetic peptide (Ac2-26) was effective in reducing interleukin (IL)-2, IL-4, IL-10, IL-13, eotaxin, and regulated upon activation normal T cell expressed and presumably secreted (RANTES) [32]. In addition, the potential involvement of the formyl peptide receptor (fpr) family in the protective effect of Ac2-26 was investigated in the same allergic conjunctivitis model [35]. In a *Pseudomonas aeruginosa* keratitis model, the overexpression of AnxA1 and fpr2 occurred in the corneas of Balb/c mice and especially C57BL/6 mice, which is more susceptible to pathogens and infectious antigens [34]. Concerning the uveitis, the expression of AnxA1 in leukocytes and aqueous humor (AqH) was observed in endotoxin-induced uveitis (EIU) in rats [16], with this protein noted as one of the essential mediators in the inflammatory homeostasis process. Moreover, the mechanism of action and potential use of AnxA1 and Ac2-26 were demonstrated in EIU in rodents and lipopolysaccharide (LPS)-activated ARPE-19 cells [17]. The results of this investigation showed that following specific serine phosphorylation, AnxA1 can be translocated to the cell surface, where it interacts with fpr2 and inhibits the release of inflammatory mediators independent of the nuclear factor (NF)-kB signaling pathway and in a post-translational manner.

In recent years, another potent anti-inflammatory mediator, piperlongumine (PL) (5,6-dihydro-1-[(2E)-1-oxo-3-(3,4,5-trimethoxyphenyl)-2-propenyl]-2(1H)pyridinone), a biologically active component of *Piper* species (Piperaceae), has attracted the attention of our research group for its possible interaction with AnxA1 [36]. In particular, PL is the main alkaloid of long pepper (*Piper longum* L.), and its pharmacological actions include cytotoxic, antitumor, antiangiogenic, antiplatelet, antibacterial, antidiabetic, antianxiolytic, antiatherosclerotic, and antifungal effects [36–43]. PL induces apoptosis by interfering with redox and reactive oxygen species (ROS) homeostatic regulators, such as glutathione S-transferase pi 1 (GSTP1) and carbonyl reductase (CBR1) [44]. Moreover, on nonsmall cell lung cancer (NSCLC) in vivo and in vitro, *PL* suppressed lung cancer cell growth in a dose-dependent manner via inhibition of the NF-κB signaling pathway [45].

Regarding inflammation, LPS insult on PL protected the vascular barrier integrity by inhibiting hyperpermeability, expression of cellular adhesion molecules (CAMs), and adhesion and migration of leukocytes, thus endorsing its usefulness as a therapy for vascular inflammatory diseases [46]. Moreover, PL and derivatives reduced the amount of nitric oxide (NO) in LPS-stimulated RAW264.7 macrophages [47]. The protective effect of *P. longum* alkaloid extract containing piperine and PL on dopaminergic neurons against inflammatory reaction was observed in LPS-induced damage. The active extract attenuated the depletion of dopamine in the striatum, facilitated the survival of damaged neurons by inhibiting microglial activation, suppressed the release of neurotoxic factors, and improved LPS-induced behavioral dysfunctions [48]. For neuroinflammation caused by LPS in a model of amyloidogenesis, PL exhibited anti-inflammatory and antiamyloidogenic effects by inhibiting NF-κB [49].

Our investigations indicated that PL attenuated systemic and pulmonary inflammatory changes, partially by modulating the expression of the endogenous AnxA1, in lung inflammation induced by cigarette smoke [42]. The potential of PL as a therapeutic immunomodulator for cancer prevention and progression was reinforced by analyzing PL administration in human cancer cells from an epidermoid carcinoma of the larynx (Hep-2) and umbilical vein endothelial cells (HUVEC), in which PL modulated the expression of genes involved in inflammatory processes [36]. Although there are several publications related to PL, few studies have focused on its anti-inflammatory role, and the actions of PL in ocular inflammation are not known. Building upon these observations, we decided to investigate the role of PL as an alternative therapy for EIU.

Ac2-26 and PL interaction has been explored by our group through different molecular or computational screening techniques, such as phage display and molecular docking [36]. In silico analyses showed that, among other PL molecules, there was a terpene that appeared to interact with lysine 9 from AnxA1 in the region corresponding to the N-terminal peptide Ac2-26 [36]. However, the physiological reason for this interaction, whether positive or negative in vivo, with regard to the anti-inflammatory effects of AnxA1 is not yet understood, which opens up a new and stimulating field for research.

Given the above, we tested PL in EIU either alone or in coadministration with the peptide Ac2-26, followed by analyses of the leukocyte influx and inflammatory mediators, to verify the following hypotheses: (1) there is anti-inflammatory potential of PL in EIU; (2) Ac2-26 + PL coadministration may interfere with the anti-inflammatory response profile of Ac2-26, favoring or attenuating the effects of its administration on experimental uveitis.

#### **2. Materials and Methods**

#### *2.1. Experimental Model of Uveitis and Treatment Protocols*

Male Wistar rats, 6 to 8 weeks of age (200 g), were distributed in 7 groups (*n* = 10/group). The animals were kept in cages in a temperature-controlled environment (22 to 25 ◦C) and received water and food ad libitum. The experimental procedures were conducted according to the guidelines for biomedical research stated by the Brazilian Societies of Experimental Biology and also approved by the Ethic Committee on Animal Use of University Center Padre Albino (Certificate (No. 11/14). The experiments were designed to minimize the number of animals used and their suffering during the execution of the protocols. All animals were evaluated daily by the institution's veterinarian.

For the development of EIU, rats were anesthetized with isoflurane (1%) and inoculated subcutaneously in the right footpad with 200 µg (1 mg/kg) of lipopolysaccharide (LPS type *Escherichia coli* serotype 0127: B8, Sigma Chemical Co. Poole, Dorset, UK) diluted in 100 µL of phosphate-buffered saline (PBS) [16,17].

The anti-inflammatory effects of Ac2-26 peptide (Ac-AMVSEFLKQAWFIENEEQE-YVQTVK, Thermo Fisher Scientific, Grand Island, NY, USA) and PL (C17H19NO5, CAS number: 20069-09-4, Sigma-Aldrich/Merck, Darmstadt, Hesse, Germany) administered singly or in combination were tested by intraperitoneal (ip) injection 15 min after LPS induction in five EIU groups (*n* = 10/group) (Figure 1) [17]. The dosage of Ac2-26 at 200 µg (1 mg/kg or 539 µM) diluted in 100 µL of PBS was based on previous studies [17]. For the selection of the PL dosage and for the purpose of comparison with the peptide, we chose two dosages. The lowest dosage of 200 µg (1 mg/kg or 1.57 mM) was equal to the one used for the peptide, while the highest dosage was based on another investigation by our group, which tested the therapeutic efficacy of PL by ip administration at a dosage of 400 µg (2 mg/kg or 3.15 mM) diluted in 100 µL of 10% dimethyl sulfoxide (DMSO, Gold Lab; Ribeirão Preto, São Paulo, Brazil) [42].

Rats that received an intraperitoneal injection of 10% DMSO were used as the control group. The animals were anesthetized with isoflurane (1%) before each experimental treatment and euthanized 24 h after LPS inoculation by excessive dose of the anesthetic.

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**Figure 1.** Schematic representation of experimental groups. Induced by (**1**) LPS, (**2**) LPS and treated with Ac2-26 200 µg diluted in PBS, (**3**) LPS and treated with PL 200 µg diluted in 10% DMSO, (**4**) LPS and treated with PL 400 µg diluted in 10% DMSO, (**5**) LPS and treated with Ac2-26 200 µg diluted in PBS and PL 200 µg diluted in 10% DMSO, (**6**) LPS and treated with Ac2-26 200 µg diluted in PBS and PL 400 µg diluted in 10% DMSO, and (**7**) uninduced, administered with 10% DMSO (*n*  = 5/group). **Figure 1.** Schematic representation of experimental groups. Induced by (**1**) LPS, (**2**) LPS and treated with Ac2-26 200 µg diluted in PBS, (**3**) LPS and treated with PL 200 µg diluted in 10% DMSO, (**4**) LPS and treated with PL 400 µg diluted in 10% DMSO, (**5**) LPS and treated with Ac2-26 200 µg diluted in PBS and PL 200 µg diluted in 10% DMSO, (**6**) LPS and treated with Ac2-26 200 µg diluted in PBS and PL 400 µg diluted in 10% DMSO, and (**7**) uninduced, administered with 10% DMSO (*n* = 5/group).

*2.2. Histopathological and Quantitative Analyses*  AqH was collected by puncturing the anterior chamber of the left eyes, and 10 µL Rats that received an intraperitoneal injection of 10% DMSO were used as the control group. The animals were anesthetized with isoflurane (1%) before each experimental treatment and euthanized 24 h after LPS inoculation by excessive dose of the anesthetic.

#### samples were used and stained in Turk's solution (90 µL). Blood was collected by cardiac puncture, and 10 µL samples were diluted in 190 µL of Turk's solution. Neutrophils and *2.2. Histopathological and Quantitative Analyses*

monocytes were quantified in the Neubauer chamber. Values for quantification of AqH and blood leukocytes were expressed as mean ± standard error (SEM) of the average number of cells × 10<sup>5</sup> /mL in the AqH samples and the number of cells × 10<sup>3</sup> /mL in the blood samples [31]. After AqH collection, the left eyes were fixed in 4% formaldehyde, dehydrated in increasing order of alcohol content, and placed in paraffin for histopathological analyses and immunohistochemistry. These analyses were performed in the Leica DM500 micro-AqH was collected by puncturing the anterior chamber of the left eyes, and 10 µL samples were used and stained in Turk's solution (90 µL). Blood was collected by cardiac puncture, and 10 µL samples were diluted in 190 µL of Turk's solution. Neutrophils and monocytes were quantified in the Neubauer chamber. Values for quantification of AqH and blood leukocytes were expressed as mean ± standard error (SEM) of the average number of cells × 105/mL in the AqH samples and the number of cells × 103/mL in the blood samples [31].

scope (Leica, Wetzlar, Hessen, Germany). *2.3. Immunohistochemical and Densitometric Studies*  Detection of the AnxA1 protein, fpr1 and fpr2 receptors, cyclooxygenase (COX)-2 en-After AqH collection, the left eyes were fixed in 4% formaldehyde, dehydrated in increasing order of alcohol content, and placed in paraffin for histopathological analyses and immunohistochemistry. These analyses were performed in the Leica DM500 microscope (Leica, Wetzlar, Hessen, Germany).

#### zyme, and phagocytic mononuclear cells (macrophages and monocytes) were performed in 5 µm sections of the paraffin-embedded material. After an antigen retrieval step using *2.3. Immunohistochemical and Densitometric Studies*

citrate buffer (pH 6), the endogenous peroxide activity was blocked, and the sections were incubated overnight at 4 °C with the primary rabbit polyclonal antibodies anti-AnxA1 (1:2000) (Invitrogen, Camarillo, CA, USA), anti-fpr1 and anti-fpr2 (1:1000) (Bioss Inc, Woburn, MA, USA), anti-COX-2 (1:1000) (Bioss Inc, Wo-burn, MA, USA) and with monoclonal anti-ED-1 (monocytes and macrophages) (1:1000) (Millipore, Temecula, CA, USA) diluted in 1% BSA. Subsequently, the slides were incubated with biotinylated secondary antibody (Histostain kit, Invitrogen, Carlsbad, CA, USA). Positive staining was detected using a peroxidase-conjugated streptavidin complex, and color was developed using diaminobenzidine substrate (DAB Kit, Invitrogen, Carlsbad, CA, USA). The sections were counterstained with hematoxylin. Detection of the AnxA1 protein, fpr1 and fpr2 receptors, cyclooxygenase (COX)-2 enzyme, and phagocytic mononuclear cells (macrophages and monocytes) were performed in 5 µm sections of the paraffin-embedded material. After an antigen retrieval step using citrate buffer (pH 6), the endogenous peroxide activity was blocked, and the sections were incubated overnight at 4 ◦C with the primary rabbit polyclonal antibodies anti-AnxA1 (1:2000) (Invitrogen, Camarillo, CA, USA), anti-fpr1 and anti-fpr2 (1:1000) (Bioss Inc., Woburn, MA, USA), anti-COX-2 (1:1000) (Bioss Inc, Wo-burn, MA, USA) and with monoclonal anti-ED-1 (monocytes and macrophages) (1:1000) (Millipore, Temecula, CA, USA) diluted in 1% BSA. Subsequently, the slides were incubated with biotinylated secondary antibody (Histostain kit, Invitrogen, Carlsbad, CA, USA). Positive staining was detected using a peroxidase-conjugated streptavidin complex, and color was developed using diaminobenzidine substrate (DAB Kit, Invitrogen, Carlsbad, CA, USA). The sections were counterstained with hematoxylin.

ED-1 positive cells were quantified in the anterior segment of the eyes of different groups with the aid of the Leica Image Analysis software, and the values obtained were

expressed as number of cells per mm<sup>2</sup> . For the protein densitometric analyses, 3 different slides from each animal were used, and 15 points were analyzed in 3 regions of the cornea, iris, and ciliary processes for an average related to the intensity of immunoreactivity. The values were obtained as arbitrary units [17].

#### *2.4. Inflammatory Mediator Levels*

The intact right eyes of all the studied groups were macerated in liquid nitrogen and placed in eppendorfs, which were added with 500 µL of protease (Protease Inhibitor Cocktail Set I, Cat. No. 53391, Millipore Corporation, CA, USA) and phosphatase (PhosphoSafe, Cat. No. 7,126-3-3, Novagen, Millipore Corporation, Billerica, CA, USA) inhibitor solution prepared according to the manufacturer's instructions. The material was incubated for 20 min at 4 ◦C under constant stirring and centrifuged at 14,000 RPM for 10 min at 4 ◦C. The supernatants were then collected and immediately frozen at −80 ◦C. The protein concentration in the supernatant was measured using a Bradford assay (Bio-Rad, Hemel Hempstead, UK).

IL-1β, IL-6, IL-10, monocyte chemoattractant protein (MCP)-1, and TNF-α inflammatory mediators were quantified in the eye macerate supernatant and in blood plasma using the rat cytokine MILLIPLEX MAP Kit (RECYTMAG-65K; Millipore Corporation, Billerica, CA, USA) according to the manufacturer's instructions and analyzed on the LUMINEX xMAP MAGPIX (Millipore Corporation, Billerica, CA, USA) equipment. The concentration of analytes was determined by MAGPIX xPONENT software (Millipore Corporation, Billerica, CA, USA). Results were expressed as mean ± SEM of cytokine concentrations (pg/mL).

#### *2.5. Statistical Analyses*

The results were first submitted to descriptive analysis and normality determination. As the samples presented normal distribution, the analysis of variance (ANOVA) was used, followed by the Bonferroni test. All values were expressed as mean ± SEM and *p* values less than 0.05 were considered statistically significant.

#### **3. Results**

*3.1. Singly Administered, the Treatments Inhibited the Influx of Leukocytes, Indicating Protective Effects of PL, Especially at 400 µg Dosage, and Confirming the Anti-Inflammatory Action of Ac2-26, but These Effects Were Lost with Coadministration*

Transmigrated leukocytes were absent in the control eyes (Figure 2A), but a high influx of these cells, mainly neutrophils, occurred 24 h after LPS inoculation without treatment (Figure 2B). The anterior eye segment was the most affected, and the inflammatory cells were observed in AqH, anterior and posterior chambers, and also in iris, ciliary body, and ciliary process stroma (Figure 2B). Except for Ac2-26 + PL 400 µg group (Figure 2G), fewer transmigrated leukocytes were presented after treatments (Figure 2C–F), especially in Ac2-26 (Figure 2C) and PL 400 µg (Figure 2E) groups.

Decreased neutrophil transmigration into AqH was verified after Ac2-26 (*p* < 0.001) and PL 400 µg (*p* < 0.01) administrations compared to the untreated LPS group (Figure 2H). The influx of monocytes into AqH was also reduced by treatments (*p* < 0.001), except in the Ac2-26 + PL 400 µg group (Figure 2I). Similarly, in blood quantifications, higher numbers of neutrophils and monocytes were observed, especially in the LPS and Ac2-26 + PL 400 µg groups, with a marked reduction after Ac2-26 and PL 400 µg treatments (Figure 3A,B).

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showed a large number of ED-1 positive cells in the LPS and Ac2-26 + PL 400 µg (Figure 3C) groups (*p* < 0.001) compared to the control but significant decrease in the other groups. Analyses related to influx of neutrophils and monocytes in AqH and blood and phagocytic mononuclear cells in the anterior eye segment showed anti-inflammatory action of Ac2-26 and dose-dependent effects of PL, which was more efficient at 400 µg dosage. In contrast, Ac2-26 + PL coadministration abrogated the peptide inhibitory action on neutro-

phil and monocyte extravasation, especially in the Ac2-26 + PL 400 µg group.

**Figure 2.** Histopathological analyses of ocular tissues in EIU**.** Absence of leukocytes in control tissues (**A**). Influx of neutrophils after 24 h LPS (**B**) and treated with Ac2-26 + PL 400 µg (**G**). Significant decrease in cellular extravasation after systemic treatments with Ac2-26 (**C**) and PL 400 µg (**E**) and moderate inflammatory influx reduction after treatments with PL 200 µg (**D**) and Ac2-26 + PL 200 µg (**F**). The details show enlargements of dashed areas. Sections: 5 µm, stain: HE, bars: 100 µm. Quantitative analyses of neutrophils (**H**) and monocytes (**I**) in the aqueous humor. The data show mean ± SEM of neutrophils and monocytes × 10<sup>5</sup> mL in the eyes of control, untreated (LPS), and treated (Ac2-26 200 µg, PL 200 µg, PL 400 µg, Ac2-26 + PL 200 µg, and Ac2-26+ PL 400 µg) rats (*n* = 10 animals/group). \*\*\* *p* < 0.001, \*\* *p* < 0.01, and \* *p <* 0.05 versus control; ### *p* < 0.001 and ## *p* < 0.01 versus LPS; and χχχ *p* < 0.001 versus Ac2-26 200 µg, PL 200 µg, PL 400 µg, and Ac2- 26 200 µg + PL 200 µg. *3.2. Ac2-26 and PL Singly Administered Reduced the Release of Proinflammatory Mediators in EIU, but These Effects Were Abrogated with Coadministration, Especially Ac2-26 + PL 400 μg*  The supernatants of macerated ocular tissues of LPS animals and those treated with **Figure 2.** Histopathological analyses of ocular tissues in EIU. Absence of leukocytes in control tissues (**A**). Influx of neutrophils after 24 h LPS (**B**) and treated with Ac2-26 + PL 400 µg (**G**). Significant decrease in cellular extravasation after systemic treatments with Ac2-26 (**C**) and PL 400 µg (**E**) and moderate inflammatory influx reduction after treatments with PL 200 µg (**D**) and Ac2-26 + PL 200 µg (**F**). The details show enlargements of dashed areas. Sections: 5 µm, stain: HE, bars: 100 µm. Quantitative analyses of neutrophils (**H**) and monocytes (**I**) in the aqueous humor. The data show mean ± SEM of neutrophils and monocytes × 10<sup>5</sup> mL in the eyes of control, untreated (LPS), and treated (Ac2-26 200 µg, PL 200 µg, PL 400 µg, Ac2-26 + PL 200 µg, and Ac2-26+ PL 400 µg) rats (*n* = 10 animals/group). \*\*\* *p* < 0.001, \*\* *p* < 0.01, and \* *p <* 0.05 versus control; ### *p* < 0.001 and ## *p* < 0.01 versus LPS; and χχχ *p* < 0.001 versus Ac2-26 200 µg, PL 200 µg, PL 400 µg, and Ac2-26 200 µg + PL 200 µg.

Ac2-26 + PL 400 µg showed significant increase in total protein levels (*p <* 0.001) compared to the control. In the other treated groups, the protein concentration was reduced in relation to the LPS group (Figure 4A). Phagocytic mononuclear cells were studied following immunohistochemical reaction with anti-ED-1 antibody in the ciliary body and iris (Figure 3D). Quantification showed a large number of ED-1 positive cells in the LPS and Ac2-26 + PL 400 µg (Figure 3C) groups (*p* < 0.001) compared to the control but significant decrease in the other groups.

Analyses related to influx of neutrophils and monocytes in AqH and blood and phagocytic mononuclear cells in the anterior eye segment showed anti-inflammatory action of Ac2-26 and dose-dependent effects of PL, which was more efficient at 400 µg dosage. In contrast, Ac2-26 + PL coadministration abrogated the peptide inhibitory action on neutrophil and monocyte extravasation, especially in the Ac2-26 + PL 400 µg group.

**Figure 3.** Quantitative analyses of neutrophils (**A**) and monocytes (**B**) in blood**.** Data show mean ± SEM of neutrophils and monocytes × 10<sup>3</sup> mL in the blood of control, untreated (LPS), and treated (Ac2-26 200 µg, PL 200 µg, PL 400 µg, Ac2-26 + PL 200 µg, and Ac2-26 + PL 400 µg) rats (*n* = 10 animals/group). \*\*\* *p <* 0.001, \*\* *p* < 0.01, and \* *p <* 0.05 versus control; ## *p <* 0.01 versus LPS; αα *p* < 0.01 and α *p* < 0.05 versus Ac2-26 200 µg; and & *p* < 0.05 versus PL 400 µg. Quantification of phagocytic mononuclear cells in the anterior segment of the eye (**C**). Data show mean ± SEM of ED-1 positive cells per mm<sup>2</sup> in the eyes of control, untreated (LPS) and treated (Ac2-26 200 µg, PL 200 µg, PL 400 µg, Ac2-26 + PL 200 µg and Ac2- 26 + PL 400 µg) rats. (*n* = 10 animals/group). \*\*\* *p <* 0.001 versus control; # *p <* 0.05, ## *p* < 0.01, and ### *p* < 0.001 versus LPS; and χχ *p <* 0.01 versus Ac2-26 200 µg, PL 400 µg, and Ac2-26 200 µg + PL 200 µg. ED-1 positive cells (arrows) on iris induced to uveitis and untreated (**D**). Sections: 5 µm, counterstain: hematoxylin, bars: 10 µm. **Figure 3.** Quantitative analyses of neutrophils (**A**) and monocytes (**B**) in blood. Data show mean ± SEM of neutrophils and monocytes × 10<sup>3</sup> mL in the blood of control, untreated (LPS), and treated (Ac2-26 200 µg, PL 200 µg, PL 400 µg, Ac2-26 + PL 200 µg, and Ac2-26 + PL 400 µg) rats (*n* = 10 animals/group). \*\*\* *p <* 0.001, \*\* *p* < 0.01, and \* *p <* 0.05 versus control; ## *p <* 0.01 versus LPS; αα *p* < 0.01 and α *p* < 0.05 versus Ac2-26 200 µg; and & *p* < 0.05 versus PL 400 µg. Quantification of phagocytic mononuclear cells in the anterior segment of the eye (**C**). Data show mean ± SEM of ED-1 positive cells per mm<sup>2</sup> in the eyes of control, untreated (LPS) and treated (Ac2-26 200 µg, PL 200 µg, PL 400 µg, Ac2-26 + PL 200 µg and Ac2-26 + PL 400 µg) rats. (*n* = 10 animals/group). \*\*\* *p <* 0.001 versus control; # *p <* 0.05, ## *p* < 0.01, and ### *p* < 0.001 versus LPS; and χχ *p <* 0.01 versus Ac2-26 200 µg, PL 400 µg, and Ac2-26 200 µg + PL 200 µg. ED-1 positive cells (arrows) on iris induced to uveitis and untreated (**D**). Sections: 5 µm, counterstain: hematoxylin, bars: 10 µm.

#### matory cytokine IL-10, which are all multifunctional molecules that play important roles in host defense in acute phase inflammatory reactions, were analyzed in supernatants of *3.2. Ac2-26 and PL Singly Administered Reduced the Release of Proinflammatory Mediators in EIU, but These Effects Were Abrogated with Coadministration, Especially Ac2-26 + PL 400 µg*

The proinflammatory mediators IL-1β, IL-6, TNF-α, and MCP-1 and the anti-inflam-

the ocular tissues after maceration and in the blood plasma. The results indicated low levels of the proinflammatory cytokines in the control eyes and, as expected, significant increased levels in the untreated LPS group, both in the macerated supernatant (Figure 4B,D,F,H) and blood plasma (Figure 4C,E,G,I). The supernatants of macerated ocular tissues of LPS animals and those treated with Ac2-26 + PL 400 µg showed significant increase in total protein levels (*p <* 0.001) compared to the control. In the other treated groups, the protein concentration was reduced in relation to the LPS group (Figure 4A).

Treatments with PL at 200 µg dosage singly administered or in combination with Ac2-26 peptide were able to reduce the levels of IL-1β only in the blood plasma (Figure 4E), while the combined administration of the AnxA1 mimetic peptide and PL at 400 µg dosage did not reduce the proinflammatory cytokine levels (Figure 4B–K). In contrast, when the peptide Ac2-26 or PL at 400 µg dosage were singly administered, IL-1β, IL-6, and TNF-α levels were reduced compared to the LPS group (Figure 4B–K), indicating resolution of the inflammatory process. Regarding IL-10, increased levels were verified in the LPS and Ac2-26 + PL 400 µg The proinflammatory mediators IL-1β, IL-6, TNF-α, and MCP-1 and the anti-inflammatory cytokine IL-10, which are all multifunctional molecules that play important roles in host defense in acute phase inflammatory reactions, were analyzed in supernatants of the ocular tissues after maceration and in the blood plasma. The results indicated low levels of the proinflammatory cytokines in the control eyes and, as expected, significant increased levels in the untreated LPS group, both in the macerated supernatant (Figure 4B,D,F,H) and blood plasma (Figure 4C,E,G,I).

groups in blood plasma and macerated supernatant (Figure 4J,K). Treatments with Ac2- 26 peptide and PL (200 and 400 µg) reduced the cytokine concentration in blood plasma

*Cells* **2021**, *10*, x FOR PEER REVIEW 9 of 19

**Figure 4.** Effects of peptide Ac2-26 and PL, administered singly or in combination, on EIU. Levels of total proteins in supernatants after maceration of ocular tissues (**A**) and dosages of anti-inflammatory mediators TNF-α (**B**,**C**), IL-1β (**D**,**E**), IL-6 (**F**,**G**), and MCP-1 (**H**,**I**) and proinflammatory cytokine IL-10 (**J**,**K**) in the macerated eye supernatant and blood plasma. Data expressed as mean ± SEM of mg of proteins/mL and pg of cytokines/mL of control, untreated (LPS), and treated (Ac2-26 200 µg, PL 200 µg, PL 400 µg, Ac2-26 + PL 200 µg, and Ac2-26 + PL 400 µg) rats (*n* = 10 animals/group). \*\*\* *p* < 0.001, \*\* *p* < 0.01, and \* *p* < 0.05 versus control; # *p* < 0.05, ## *p* < 0.01, and ### *p* < 0.001 versus LPS; αα *p* < 0.01 and α *p* < 0.05 versus Ac2-26 200 µg; ββ *p* < 0.01 versus PL 200 µg; && *p <* 0.01 versus PL 400 µg; and χχχ *p* < 0.001 versus all other groups.

Treatments with PL at 200 µg dosage singly administered or in combination with Ac2-26 peptide were able to reduce the levels of IL-1β only in the blood plasma (Figure 4E), while the combined administration of the AnxA1 mimetic peptide and PL at 400 µg dosage did not reduce the proinflammatory cytokine levels (Figure 4B–K). In contrast, when the peptide Ac2-26 or PL at 400 µg dosage were singly administered, IL-1β, IL-6, and TNF-α levels were reduced compared to the LPS group (Figure 4B–K), indicating resolution of the inflammatory process.

Regarding IL-10, increased levels were verified in the LPS and Ac2-26 + PL 400 µg groups in blood plasma and macerated supernatant (Figure 4J,K). Treatments with Ac2-26 peptide and PL (200 and 400 µg) reduced the cytokine concentration in blood plasma and eye supernatant, while Ac2-26 + PL 200 µg administration decreased IL-10 in blood plasma.

#### *3.3. COX-2 Expression Is Not Inhibited after Treatments with PL 200 µg and Ac2-26 + PL 400 µg*

In the anterior ocular segment of the control rats, especially in iris, ciliary body, and ciliary processes (Figure 5A), the expression COX-2 enzyme was not detected. However, in the same regions, in the untreated EIU animals (Figure 5B) and after systemic treatments with PL 200 µg and Ac2-26 + PL 400 µg (Figure 5D,G), strong COX-2 immunolabeling was observed. The Ac2-26, PL 400 µg, and Ac2-26 + PL 200 µg groups showed reduced enzyme expression (Figure 5C,E,F), mainly in the singly peptide treated group. There was no immunoreactivity for COX-2 in the reaction control (Figure 5H), confirming antibody specificity. *Cells* **2021**, *10*, x FOR PEER REVIEW 11 of 19

**Figure 5.** Expression of the COX-2 enzyme in ciliary processes in EIU. Absence of immunostaining in the control eyes (**A**). Strong immunostaining in the untreated, induced EIU animals (LPS) (**B**) and those treated with PL 200 µg (**D**) and Ac2-26 + PL 400 µg (**G**). Decreased expression after systemic treatments with Ac2-26 (**C**), PL 400 µg (**E**), and Ac2-26 + PL 200 µg (**F**). Absence of immunostaining in the control eyes (**H**). Counterstaining: hematoxylin, bars: 10 µm. Densitometric analysis of COX-2 (**I**). Results were obtained as mean ± SEM of the densitometric index of the eyes of control rats, untreated uveitis (LPS), and treated groups (Ac2-26 200 µg, PL 200 µg, PL 400 µg, Ac2-26 + PL 200 µg, and Ac2-26 + PL 400 µg) (*n* = 10 animals/group). \*\*\* *p* < 0.001 and \*\* *p* < 0.01 versus control; # *p* < 0.05, ## *p* < 0.01, and ### *p* < 0.001 versus LPS; αα *p* < 0.01 and α *p* < 0.05 versus Ac2-26 200 µg*. 3.5. Expression of the fpr2 Receptor Is Modulated by Treatment with AnxA1 Peptide but Not with PL Singly Administered or Ac2-26 in Combination with PL 400 μg* **Figure 5.** Expression of the COX-2 enzyme in ciliary processes in EIU. Absence of immunostaining in the control eyes (**A**). Strong immunostaining in the untreated, induced EIU animals (LPS) (**B**) and those treated with PL 200 µg (**D**) and Ac2-26 + PL 400 µg (**G**). Decreased expression after systemic treatments with Ac2-26 (**C**), PL 400 µg (**E**), and Ac2-26 + PL 200 µg (**F**). Absence of immunostaining in the control eyes (**H**). Counterstaining: hematoxylin, bars: 10 µm. Densitometric analysis of COX-2 (**I**). Results were obtained as mean ± SEM of the densitometric index of the eyes of control rats, untreated uveitis (LPS), and treated groups (Ac2-26 200 µg, PL 200 µg, PL 400 µg, Ac2-26 + PL 200 µg, and Ac2-26 + PL 400 µg) (*n* = 10 animals/group). \*\*\* *p* < 0.001 and \*\* *p* < 0.01 versus control; # *p* < 0.05, ## *p* < 0.01, and ### *p* < 0.001 versus LPS; αα *p* < 0.01 and α *p* < 0.05 versus Ac2-26 200 µg.

The expression of fpr2 receptor in ocular tissues overlapped the location of the

ure 7A,J). However, after other treatments, there was no significant change in receptor expression (Figure 7D–G,J). In contrast, we observed no immunoreactivity for fpr1 (Figure 7H) in all the studied groups. The control of the reaction indicated antibody specificity

(Figure 7I).

Densitometric analyses corroborated the immunohistochemical observations (Figure 5I), reinforced the anti-inflammatory activities of PL in a dose-dependent manner, and showed that the combination of Ac2-26 and PL, especially at the higher dosage, inhibited the protective action of the peptide and PL singly administered.

#### *3.4. Endogenous AnxA1 Increased during Inflammation in Ocular Tissues, but Ac2-26 Administered Singly or in Combination with PL at Lower Dosage Reduced AnxA1 Immunoreactivity*

The immunohistochemical and densitometric analyses of AnxA1 expression in the anterior eye segment showed significant increase in the endogenous protein, especially in the ciliary processes, 24 h after uveitis induction in the untreated group (*p* < 0.001) compared to the control (Figure 6A,B,I). *Cells* **2021**, *10*, x FOR PEER REVIEW 12 of 19

**Figure 6.** Expression of AnxA1 in ciliary processes in EIU. Increased expression after 24 h of induction of inflammation in the untreated (LPS) (**B**) and treated groups with PL (**D**,**E**) and Ac2-26 + PL 400 µg (**G**) relative to the control (**A**). Reduction of immunoreactivity after treatments with Ac2-26 (**C**) and Ac2-26 + PL 200 µg (**F**) compared to LPS. Absence of immunoreactivity in the control of the reaction (**H**). Counterstaining: hematoxylin, bars: 10 µm. Densitometric analysis of AnxA1 (**I**). Results were obtained as mean ± SEM of the densitometric index of the eyes of control rats, untreated uveitis (LPS), and treated groups (Ac2-26 200 µg, PL 200 µg, PL 400 µg, Ac2-26 + PL 200 µg, and Ac2-26+ PL 400 µg) (*n* = 10 animals/group). \*\* *p* < 0.01 and \* *p* < 0.05 versus control; ## *p* < 0.01 and ### *p* < 0.001 versus LPS; ααα *p* < 0.001 and αα *p* < 0.01 versus Ac2-262 00 µg. **Figure 6.** Expression of AnxA1 in ciliary processes in EIU. Increased expression after 24 h of induction of inflammation in the untreated (LPS) (**B**) and treated groups with PL (**D**,**E**) and Ac2-26 + PL 400 µg (**G**) relative to the control (**A**). Reduction of immunoreactivity after treatments with Ac2-26 (**C**) and Ac2-26 + PL 200 µg (**F**) compared to LPS. Absence of immunoreactivity in the control of the reaction (**H**). Counterstaining: hematoxylin, bars: 10 µm. Densitometric analysis of AnxA1 (**I**). Results were obtained as mean ± SEM of the densitometric index of the eyes of control rats, untreated uveitis (LPS), and treated groups (Ac2-26 200 µg, PL 200 µg, PL 400 µg, Ac2-26 + PL 200 µg, and Ac2-26+ PL 400 µg) (*n* = 10 animals/group). \*\* *p* < 0.01 and \* *p* < 0.05 versus control; ## *p* < 0.01 and ### *p* < 0.001 versus LPS; ααα *p* < 0.001 and αα *p* < 0.01 versus Ac2-262 00 µg.

AnxA1 immunolabeling remained increased after treatments with PL singly administered (Figure 6D,E,I) or in combination with Ac2-26 at 400 µg dosage (Figure 6G,I). In contrast, in the Ac2-26 (Figure 6C) and Ac2-26 + PL 200 µg groups (Figure 6F) decreased AnxA1 expression was observed relative to the LPS group (Figure 6I). In the group treated only with the peptide, the expression of the protein was also reduced compared to control group (*p* < 0.05). The specificity of the immunolabeling was confirmed by the reaction control (Figure 6H).

#### *3.5. Expression of the fpr2 Receptor Is Modulated by Treatment with AnxA1 Peptide but Not with PL Singly Administered or Ac2-26 in Combination with PL 400 µg*

The expression of fpr2 receptor in ocular tissues overlapped the location of the AnxA1 with increased labeling in the LPS group (*p* < 0.05) (Figure 7B,J) and mainly in animals treated with AnxA1 peptide (*p* < 0.001) (Figure 7C,J) compared to the control (Figure 7A,J). However, after other treatments, there was no significant change in receptor expression (Figure 7D–G,J). In contrast, we observed no immunoreactivity for fpr1 (Figure 7H) in all the studied groups. The control of the reaction indicated antibody specificity (Figure 7I). *Cells* **2021**, *10*, x FOR PEER REVIEW 13 of 19

**Figure 7.** Specific expression of fpr2 in ocular tissues. Strong immunoreactivity for fpr2 in the LPS (**B**) and Ac2-26 (**C**) groups. Similar expressions were found among the control (**A**), PL (**D**,**E**), and Ac2-26 + PL (**F**,**G**) groups. Absence of labeling for fpr1 receptor (**H**) and in control of the reaction (**I**). Counterstaining: hematoxylin, bars: 10 µm. Densitometric analysis of fpr2 (**J**). Results were obtained as mean ± SEM of the densitometric index of the eyes of the control rats, untreated uveitis (LPS), and treated groups (Ac2-26 200 µg, PL 200 µg, PL 400 µg, Ac2-26 + PL 200 µg, and Ac2-26 + PL 400 µg) (*n* = 10 animals/group). \*\*\* *p* < 0.001 and \* *p <* 0.05 versus control. **4. Discussion**  Several investigations have explored the anti-inflammatory and protective activities **Figure 7.** Specific expression of fpr2 in ocular tissues. Strong immunoreactivity for fpr2 in the LPS (**B**) and Ac2-26 (**C**) groups. Similar expressions were found among the control (**A**), PL (**D**,**E**), and Ac2-26 + PL (**F**,**G**) groups. Absence of labeling for fpr1 receptor (**H**) and in control of the reaction (**I**). Counterstaining: hematoxylin, bars: 10 µm. Densitometric analysis of fpr2 (**J**). Results were obtained as mean ± SEM of the densitometric index of the eyes of the control rats, untreated uveitis (LPS), and treated groups (Ac2-26 200 µg, PL 200 µg, PL 400 µg, Ac2-26 + PL 200 µg, and Ac2-26 + PL 400 µg) (*n* = 10 animals/group). \*\*\* *p* < 0.001 and \* *p <* 0.05 versus control.

#### of AnxA1 protein and its mimetic peptides, especially Ac2-26 [25,29]. In recent years, our **4. Discussion**

group has researched the role of AnxA1 in the eye by means of in vivo and in vitro studies and highlighted the potential of the protein in controlling the ocular inflammatory process [16,17,19,34,35]. Recently, the understanding that AnxA1 could interact with PL [36] has opened up a new and exciting field of research. Thus, we proposed an investigation into the effects of PL administered alone in the EIU, and our findings indicate an important anti-inflammatory profile of PL in LPS-induced uveitis. Moreover, we evaluated the coadministration of Ac2-26 + PL in the experimental uveitis. We speculated whether their coadministration would have synergistic or antagonistic effects. It would be expected that the combination would enhance the action against uveitis, but the result proved to be the opposite. It follows from this result that both might also act in close proximity and that a possible peptide structure conformational change occurred. Our initial analysis, as expected, showed that inflammatory stimuli induced by LPS released inflammatory mediators IL-1β, IL-6, TNF-α, and MCP-1 and increased expression of COX-2 by promoting disruption of the blood–ocular barrier and intense influx of leukocytes, reinforcing previous studies [17,31]. Neutrophils were the predominantly ex-Several investigations have explored the anti-inflammatory and protective activities of AnxA1 protein and its mimetic peptides, especially Ac2-26 [25,29]. In recent years, our group has researched the role of AnxA1 in the eye by means of in vivo and in vitro studies and highlighted the potential of the protein in controlling the ocular inflammatory process [16,17,19,34,35]. Recently, the understanding that AnxA1 could interact with PL [36] has opened up a new and exciting field of research. Thus, we proposed an investigation into the effects of PL administered alone in the EIU, and our findings indicate an important anti-inflammatory profile of PL in LPS-induced uveitis. Moreover, we evaluated the coadministration of Ac2-26 + PL in the experimental uveitis. We speculated whether their coadministration would have synergistic or antagonistic effects. It would be expected that the combination would enhance the action against uveitis, but the result proved to be the opposite. It follows from this result that both might also act in close proximity and that a possible peptide structure conformational change occurred.

> travasated inflammatory cells, especially near ciliary processes. In EIU, neutrophil transmigration occurs at the base of the ciliary body, whereas the infiltrate of phagocytic mon-

> *like receptor 4* (TLR4), preferably by cells in the anterior region of the eye, may explain the

Our initial analysis, as expected, showed that inflammatory stimuli induced by LPS released inflammatory mediators IL-1β, IL-6, TNF-α, and MCP-1 and increased expression of COX-2 by promoting disruption of the blood–ocular barrier and intense influx of leukocytes, reinforcing previous studies [17,31]. Neutrophils were the predominantly extravasated inflammatory cells, especially near ciliary processes. In EIU, neutrophil transmigration occurs at the base of the ciliary body, whereas the infiltrate of phagocytic mononuclear cells and lymphocytes occurs in the iris vessels [16,17]. The expression of *Toll-like receptor 4* (TLR4), preferably by cells in the anterior region of the eye, may explain the apparent susceptibility of anterior uvea to disruption of the blood–ocular barrier and development of uveitis [50,51].

Data obtained after systemic treatments with Ac2-26 and PL in EIU confirmed the anti-inflammatory action of AnxA1 mimetic peptide in experimental ocular inflammation [18,32], including uveitis, as previously reported by our research group [17]. However, as a novelty, our current results also indicated the protective effects of PL at 400 µg dosage and therefore a dose-dependent manner. Both Ac2-26 and PL at 400 µg dosage promoted decrease in the influx of neutrophils and monocytes into AqH and blood as well the reduction of the phagocytic mononuclear cells in the iris and ciliary body. Furthermore, the anti-inflammatory effects of the mimetic peptide and PL at higher dosage stimulated the reduction of IL-1β, IL-6, TNF-α, and MCP-1 levels, which are produced especially by neutrophils and phagocytic mononuclear cells [52,53].

Our findings in relation to PL in EIU are in agreement with other investigations that showed the anti-inflammatory potential of PL, such as in the reduction of ear edema induced by croton oil [54], analgesia and suppression of the stress response caused by pain in a dose-dependent manner [55], and decreased release of TNF-α and IL-1β in collagen-induced arthritis [56]. In particular, in LPS-induced-inflammation, PL suppressed leukocytes migration, TNF-α and IL-6 production, NF-κB and regulated extracellular kinases (ERK) 1 and 2 activation [46], and reduced mortality in sepsis [44]. Moreover, in LPSinduced neuroinflammation, PL protected dopaminergic neurons against inflammation by inhibiting microglial activation and decreasing levels of TNF-α, IL-1β, IL-6, and the production of ROS and NO [48] as well as inhibiting NF-κB and amyloidogenesis [49].

However, when Ac2-26 was administered in combination with PL, anti-inflammatory effects were abrogated, especially in combination with PL at 400 µg dosage. Interestingly, the groups that showed increased levels of the anti-inflammatory cytokine IL-10 were LPS and Ac2-26 + PL 400 µg, whereas treatment with peptide singly administered led to reduction of this cytokine level. In the model of allergic conjunctivitis, low levels of IL-10 were also observed after treatment with Ac2-26, and a significant increase in this cytokine occurred in AnxA1-null animals [32], indicating the importance of Th1/Th2 balance in the development of allergic inflammatory responses and suggesting that the protective role of AnxA1 in ocular allergy occurs through downregulation of both cytokine profiles. The same seems to happen in EIU.

The efficacy of Ac2-26 and PL at 400 µg dosage was also verified by the reduction in COX-2 proinflammatory enzyme expression. Again, the administration of PL at 200 µg dosage and the combination of Ac2-26 + PL 400 µg did not revert the inflammatory process. In a previous research, we had shown the exacerbated inflammatory response, characterized by COX-2 overexpression in the eyes of AnxA1-null mice, reinforcing the actions of AnxA1 in the resolution of ocular inflammation [17]. Concerning PL, the importance of its analogs in COX-2 inhibition after LPS induction was demonstrated in the RAW264.7 lineage of macrophages [38]. More recently, our research group showed that PL administration decreased expressions of COX-2, NF-κB, and neutrophil elastase and recovered lung tissues in a model of lung inflammation [42].

We studied the endogenous expression of AnxA1 in the different experimental groups, especially in the anterior eye segment (iris, ciliary body, and ciliary processes). The immunoreactivity for AnxA1 in the ocular tissues overlapped with the sites of TLR4 expression and production of inflammatory mediators [57,58]. Indeed, TLR4 in the eye

is particularly expressed by epithelial cells (cornea and pigmented epithelia of the ciliary body), iris endothelial cells [50,51], and resident antigen presenting cells of the uvea [52,59]. Moreover, in uveitis, cytokines are produced mainly by inflammatory and endothelial cells as well as by corneal epithelial cells and retinal pigmented cells [57,58].

The immunohistochemical analysis of untreated EIU eyes showed an increase in the intensity of immunolabeling for AnxA1, corroborating our previous findings in ocular tissues [17,34] and ocular inflammatory cells [16]. The higher expression of AnxA1 was also observed in neutrophils in ocular toxoplasmosis in mice and culture of retinal pigmented cells infected with *Toxoplasma gondii* [30]. In contrast, in animals treated with Ac2-26 singly or in combination with PL at lower dosage, there was a decrease in immunoreactivity, probably due to a negative feedback mechanism, as hitherto observed in the uveitis [17] and allergic conjunctivitis [32] models.

The fact that endogenous AnxA1 is strongly induced by LPS has already been reported in other investigations and reinforces the action of AnxA1 as a proresolving mediator in inflammation [23,26,27]. In the systemic inflammatory reaction induced by LPS, higher AnxA1 expression was observed and associated with the combined actions of endogenous glucocorticoids IL-6 and TNF-α [60]. Intense increase in *AnxA1* gene activity and protein synthesis in hepatocytes, endothelial cells, and leukocytes at the beginning of the inflammatory process, followed by reduction in AnxA1 expression in the late phase of inflammation, was verified by means of the *LacZ* reporter gene in AnxA1-null mice after LPS endotoxemia [61]. Similar to our findings, AnxA1 expression increased during lung inflammation induced by LPS but decreased after peptide Ac2-26 treatment [62]. Moreover, in the model of LPS-induced pleurisy, glucocorticoid-induced leucine zipper (GILZ) deficiency was associated with an early increase in AnxA1, so the lack of endogenous GILZ during the resolution of inflammation was compensated by AnxA1 overexpression [63].

Although our results indicated that the combination of the peptide with PL promoted a decrease in the effects of Ac2-26, the dosage of 200 µg of PL still allowed the peptide actions in a moderate manner, which may explain the lower expression of the endogenous AnxA1 in this group. Modulation in the expression of AnxA1 after LPS inoculation found in this investigation reinforces the involvement of the protein in ocular tissue physiology during inflammatory [16,17], infectious [34], allergic [32,35], and autoimmune [18,20] processes in experimental models. Interestingly, the expression of AnxA1 remains increased after treatments with PL singly administered or at 400 µg dosage in combination with the peptide. This finding could reflect the results found by other researchers who used herbal medicines, as in rats induced with sepsis and treated with Xuebijing (XBJ) [64] and culture of lung tumor cells administered with *Camellia simensis* [65]. Similar to our results, in a model of lung inflammation induced by cigarette smoke, PL administration promoted increased expression of AnxA1, concomitant with the reduction in COX-2 [42].

Following the study, we investigated the expression of fpr1 and fpr2 receptors in all groups. In previous studies on inflamed ocular tissues, it was shown that the expression of fpr2 perfectly overlapped the distribution of AnxA1 and that it was increased after treatment with Ac2-26 [17,34]. Again, our results strengthen the possible specificity of the AnxA1/fpr2 interaction as the expression of the fpr1 receptor did not occur in any of the experimental groups. In contrast, intense expression of both fprs were detected in conjunctival epithelial cells in an allergic conjunctivitis model [35], but the lack of AnxA1 protein in the ovalbumin-sensitized mice produced a marked increase only in fpr2 expression.

In addition, we found that the fpr2 expression was not altered by PL singly administered or in combination with Ac2-26, suggesting that the attenuation of the protective effects of mimetic peptide by interaction with PL probably is not related to receptor expression changes. In the light of these data and our previous findings about the interaction between Ac2-26 and PL [36], we speculate that conformational alteration may have occurred in the Ac2-26 + PL complex and may have interfered with the fpr2 binding receptor, which impaired the anti-inflammatory actions of the peptide. This reasoning is supported by

recently reported results as we demonstrated that the interaction of PL with the AnxA1 derived peptide Ac2-26 occurred spontaneously, was enthalpically driven, and that the forces governing the interaction were hydrophobic. Moreover, Ac2-26 peptide binds to PL via two hydrogen-bonding interactions at lysine 9 but not at tryptophan 12 [36]. Previous data from our group have allowed us to report that the anti-inflammatory activities of AnxA1 occur after a specific serine phosphorylation event in the N-terminal region [17]. Therefore, as the interaction between Ac2-26 and PL occurs on tyrosine, the serine site, which is an important post-translational modification related to the translocation of AnxA1 from cytoplasm to cell surface [28], remains free for phosphorylation. region [17]. Therefore, as the interaction between Ac2-26 and PL occurs on tyrosine, the serine site, which is an important post-translational modification related to the translocation of AnxA1 from cytoplasm to cell surface [28], remains free for phosphorylation. At first, this could indicate that PL does not affect the action of endogenous AnxA1. This thought was supported by our current findings, which showed increased expression of AnxA1 when PL was administered alone. However, with coadministration in vivo,

*Cells* **2021**, *10*, x FOR PEER REVIEW 16 of 19

At first, this could indicate that PL does not affect the action of endogenous AnxA1. This thought was supported by our current findings, which showed increased expression of AnxA1 when PL was administered alone. However, with coadministration in vivo, Ac2-26 and PL promoted the reversal of the anti-inflammatory effects when administrated alone (Figure 8). Moreover, the higher the concentration of PL, the greater the possibility of interaction of this molecule with Ac2-26 and, consequently, the lower the anti-inflammatory response. These findings indicate that the impairment of the anti-inflammatory action of Ac2-26 may be related to a conformational change, which prevents the peptide from binding to the fpr2 receptor. Relevantly, the PL sequestered by the complex is also prevented from entering the cells and performing its anti-inflammatory role. Thus, there is a competition between Ac2-26 and PL in the anti-inflammatory action against uveitis. This important result implies there are other factors to be considered, such as molecular weight, size of the molecules, charge distribution on the peptide, possible conformational structure adopted by the peptide during the action, and localization of the interaction site. Thus, a reasonable next step will be studies requiring new approaches, both from the point of view of action against uveitis as well as experimental evidence. Ac2-26 and PL promoted the reversal of the anti-inflammatory effects when administrated alone (Figure 8). Moreover, the higher the concentration of PL, the greater the possibility of interaction of this molecule with Ac2-26 and, consequently, the lower the anti-inflammatory response. These findings indicate that the impairment of the anti-inflammatory action of Ac2-26 may be related to a conformational change, which prevents the peptide from binding to the fpr2 receptor. Relevantly, the PL sequestered by the complex is also prevented from entering the cells and performing its anti-inflammatory role. Thus, there is a competition between Ac2-26 and PL in the anti-inflammatory action against uveitis. This important result implies there are other factors to be considered, such as molecular weight, size of the molecules, charge distribution on the peptide, possible conformational structure adopted by the peptide during the action, and localization of the interaction site. Thus, a reasonable next step will be studies requiring new approaches, both from the point of view of action against uveitis as well as experimental evidence.

**Figure 8.** Schematic model of Ac2-26 and PL actions in EIU. (**1**) LPS triggers the release of cytokines (IL1-β, IL-6, IL-10, TNF-α, MCP-1), leukocyte influx (neutrophils and monocytes), and the production of endogenous COX-2, AnxA1, and fpr2. (**2**) These inflammatory responses are mitigated by Ac2-26, mediated by fpr2 receptor, and (**3**) PL, especially at a higher dosage. (**4**) The coadministration of Ac2-26 and PL abrogates the anti-inflammatory effects of the singly administered compounds. (**5**) Conformational changes on the Ac2-26 peptide due to its interaction with PL may impair its binding to the fpr2 receptor. Larger arrows directed upwards or downwards indicate an increase or decrease, respectively, of cytokines and endogenous proteins produced by the cell. Smaller arrows pointing upwards indicate increased expression of fpr2. Outside the cell, pink shape **Figure 8.** Schematic model of Ac2-26 and PL actions in EIU. (**1**) LPS triggers the release of cytokines (IL1-β, IL-6, IL-10, TNF-α, MCP-1), leukocyte influx (neutrophils and monocytes), and the production of endogenous COX-2, AnxA1, and fpr2. (**2**) These inflammatory responses are mitigated by Ac2-26, mediated by fpr2 receptor, and (**3**) PL, especially at a higher dosage. (**4**) The coadministration of Ac2-26 and PL abrogates the anti-inflammatory effects of the singly administered compounds. (**5**) Conformational changes on the Ac2-26 peptide due to its interaction with PL may impair its binding to the fpr2 receptor. Larger arrows directed upwards or downwards indicate an increase or decrease, respectively, of cytokines and endogenous proteins produced by the cell. Smaller arrows pointing upwards indicate increased expression of fpr2. Outside the cell, pink shape represents neutrophils, purple shape represents monocytes, light green circle depicts PL at 200 µg dosage, dark green circle depicts PL at 400 µg dosage, and blue oval represents Ac2-26.

Our study sheds light on the protective effects of PL, revealing it as a potential therapeutic target in ocular inflammation. Furthermore, the results show that Ac2-26 + PL combination abrogates the anti-inflammatory actions of Ac2-26 and PL singly adminis-

**5. Conclusions** 

tered.

#### **5. Conclusions**

Our study sheds light on the protective effects of PL, revealing it as a potential therapeutic target in ocular inflammation. Furthermore, the results show that Ac2-26 + PL combination abrogates the anti-inflammatory actions of Ac2-26 and PL singly administered.

**Author Contributions:** Conceptualization, A.P.G., M.L.C. and S.M.O.; methodology, A.P.G., C.d.F.Z., Í.P.C., S.d.S.C. and H.R.S.; formal analysis, A.P.G., C.d.F.Z., Í.P.C., S.d.S.C. and H.R.S.; investigation, A.P.G., C.d.F.Z., Í.P.C., S.d.S.C., H.R.S., M.L.C. and S.M.O.; resources, A.P.G., M.L.C. and S.M.O.; data curation, A.P.G.; writing—original draft preparation, A.P.G. and H.R.S.; writing—review and editing, M.L.C. and S.M.O.; supervision, A.P.G.; project administration, S.M.O.; funding acquisition, A.P.G. and S.M.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), grant numbers 2015/03359-5 (APG) and 2019/19949-7 (SMO); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grant number 404190/2016-2 (APG); and Advanced Research Center in Medicine, CEPAM, Unilago, Brazil (56.569.197/0001-39) (SMO).

**Institutional Review Board Statement:** The rat experimental model was conducted according to the Brazilian Law 11.794 of 8 October 2008, Decree 6899 of 15 July 2009, and the rules issued by the National Council for Control of Animal Experimentation (CONCEA) and approved (Certificate No. 11/14) by the Ethic Committee on Animal Use at University Center Padre Albino (CEUA/UNIFIPA) in the meeting of 20 November 2014 (protocol code 14.11.03-13). The study did not involve humans.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data available on request due to restrictions.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

