1. Introduction
More than 4.9 million people globally suffer from inflammatory bowel diseases (IBDs), which include Crohn’s disease and ulcerative colitis [
1]. These are chronic idiopathic diseases that arise from a complex interplay of factors such as individual susceptibility, interactions with gut microflora, and the immune system’s role [
2,
3]. The dysfunction between effector and regulatory cells leads to an uncontrolled release of inflammatory molecules [
4]. The enteric nervous system (ENS), composed of the myenteric plexus and the submucosal plexus, is a division of the nervous system, forming a neural network located in the wall of the organs of the gastrointestinal tract [
5,
6]. The neural network of the myenteric plexus is involved with the reflex regulation of the contractile activities of the external musculature, while the motor neurons of the submucosal plexus are related to the control of the secretomotor and vasomotor activities of the tunica mucosa [
7].
Inflammation and oxidative stress are strongly interconnected. In IBD, oxidative stress is exacerbated due to the increased production of ROS/RNS by activated immune cells and impaired antioxidant defenses. Inflammation can induce oxidative stress through various mechanisms, including the activation of NADPH oxidases, nitric oxide synthases, and inflammatory pathways such as NF-kB. Conversely, oxidative stress can exacerbate inflammation by promoting the release of pro-inflammatory cytokines, activation of immune cells, and disruption of barrier function in the intestine [
8]. Neutrophils and monocytes accumulate in the gastrointestinal wall and participate in IBD pathogenesis by producing inflammatory cytokines and soluble mediators such as ROS (e.g., neutrophil-myeloperoxidase (MPO) catalyzes the production of potent ROS) [
9].
Current treatments involve pharmacotherapy, including immunosuppressants, with groundbreaking results seen with tumor necrosis factor (TNF) inhibitors [
10]. However, challenges like non-response and loss of effectiveness have led to the exploration of emerging therapies like small molecules, apheresis, improved gut ecology, cell therapy, and exosomes. The ultimate aim is to achieve mucosal healing, linked to improved long-term outcomes [
11]. Strategies that target the ENS are promising by potentially modulating gastrointestinal motility, maintaining mucosal barrier function, regulating immune responses, and restoring neuroimmune communication.
Nanotechnology has the potential to yield the most effective ways to deliver compounds or treat diseases directly, in a more controlled and safe manner, giving the possibility of the targeted delivery of molecules and accumulation of nanoparticles and bioactive compounds on the desired tissue [
12]. Furthermore, it also facilitates overcoming certain barriers such as membranes and extreme pH [
13], of particular relevance to IBD. Nanoparticles show promise in IBD therapy due to their small size, large surface area, and stability in the gastrointestinal tract. Various types, including polymeric, lipid-based, metallic nanoparticles, exosomes, and plant-derived nanoparticles, are being investigated for their potential in targeted drug delivery [
14].
Gold nanoparticles (AuNPs) hold significant promise for treating IBD. Gold has a long history in therapy, thanks to its high biocompatibility and well-known antioxidant properties. With the advancements in nanotechnology, AuNPs have found applications not only in imaging but also in therapy [
15]. They have been widely explored for treating various inflammatory conditions [
16] such as neuroinflammation [
17], autoimmune inflammation [
18], and skin inflammation [
19]. Numerous studies have highlighted their efficacy in managing inflammatory bowel diseases [
19,
20,
21,
22]. A notable advantage of AuNPs is their exceptional stability, especially at very low pH levels. This stability is crucial for their oral administration, enabling them to reach the intestine where they can exert their therapeutic effects on IBD pathology. This characteristic makes them particularly suitable for targeted interventions in IBD treatment [
23].
Extracts from algae have already been used for alleviating IBD due to their potent anti-inflammatory properties. These are conferred by a high content in polysaccharides [
24] which can counteract damage to the physical, chemical, immune, and biological barriers of the intestine [
25]. Brown algae are especially promising for these applications since they have a polysaccharide content higher than 50% of their dry weight, with some species reaching values over 70% [
6,
26].
Ericaria selaginoides (ES), previously known as
Cystoseira tamariscifolia, has a 50% content of polysaccharides, lower than some other closely related species [
27], but its anti-inflammatory potential has been described to be higher than most algae species [
28]. This high polysaccharide content also allows for the efficient production of gold nanoparticles by green synthesis, since the accumulation and reduction of gold ions is crucial for the obtention of these nanoparticles [
29]. The green synthesis of gold nanoparticles in ES extract (Au@ES) was previously optimized and their cytocompatibility was validated [
30]. The ES aqueous extract, rich in fucoidan and total phenolic content, revealed very high reducing and good scavenging activities [
30].
In this study, we address the anti-inflammatory and antioxidant properties of ES and Au@ES in acetic acid-induced experimental colitis, a highly reproducible animal model. Macroscopic and histopathological data revealed that colitis induced by acetic acid causes the rupture of the superficial epithelium, multiple erosions, and mucosal ulcers, accompanied by a severe inflammatory reaction and abscess, thus resembling human colitis [
31].
To our knowledge, this is the first study that addresses these properties in vivo in a murine model of IBD, which allows for a detailed evaluation of the positive effects observed upon oral administration of both the extract and derived nanoparticles.
2. Materials and Methods
Preparation of the algae extract and gold nanoparticles:
Euricaria selaginoides was collected at the lower intertidal rocky shore in the NW coast of Portugal (N 41 47.858′ W 008 52.423′). ES extract and Au@ES were prepared and characterized as previously reported [
30]. As reported, Au@CT exhibited a mean diameter of 7.6 ± 2.2 nm and were spherical, polycrystalline, and stable.
Animals: The experiments used male Swiss mice (Mus musculus) 25 to 35 days old, with a body mass of 25 to 30 g, obtained from the Department of Physiology and Pharmacology-Universidade Federal do Ceará. The animals were kept in the vivarium of the Center for Studies in Microscopy and Image Processing, 6–8 animals per cage, at a temperature of 22–24 °C, in a 12 h light/dark circadian cycle, receiving standard feed and water and libitum. The standard food used was Nuvilab CR-1 rat and mouse food with the following nutritional composition: moisture (max) 125 g/kg, crude protein (min) 220 g/kg, ether extract (min) 50 g/kg, mineral material (max) 90 g/kg, raw fiber (max) 70 g/kg, calcium 10–14 g/kg, and phosphor 6000 mg/kg. The animals were fasted for 16 h before the experiments.
Experimental groups: A total of 75 animals were distributed in groups of 9 animals, for a total of 9 experimental groups, as in
Table 1.
An experimental Crohn’s disease (CD) model was induced by the administration of acetic acid in mice.
The experimental protocol was based on previously published articles available in the literature, with modifications [
31]. The animals were fasted for 12 h and given water ad libitum. To induce CD, 8% of acetic acid (AA) was diluted in distilled water (100 mL) and this solution was administered in a volume of 0.2 mL per 10 g of animal weight. The animals were previously anesthetized intraperitoneally (IP) with a mixture of ketamine (100 mg/kg) and xylazine (20 mg/kg) and placed in the left lateral decubitus position. A polyethylene catheter number 6 measuring 4 cm in length was inserted rectally to administer the 8% AA or saline solutions. Each animal remained suspended by the tail for 30 s to prevent the solutions from returning.
Microscopic and macroscopic evaluation: 12 h after inducing colitis, the animals were euthanized, followed by exsanguination. Using standard operating techniques, laparotomy was performed through a median incision, dissection and removal of the colonic segment for macroscopic evaluation. The specimens were opened longitudinally, washed with saline solution and distended on a flat surface. They were evaluated in a blinded manner according to the microscopic and macroscopic inflammation scores using a stereoscopic magnifying glass (
Table 2) [
32].
Determination of IL-1β and TNF-α cytokine levels: The levels of pro-inflammatory cytokines IL-1β and TNF-α present in the intestinal mucosa were quantified by ELISA. The best dose of EACP (3 mg/kg) was used to measure both. The collected tissues were homogenized in PBS 1X. The cytokines were detected using the DuoSet Kit (R&D Systems, Minneapolis, MN, USA), the primary antibodies for IL-1β and TNF-α were both acquired from Invitrogen. The 96-well ELISA plates were incubated with the capture antibody for 18 h at room temperature (100 μL of antibody per well). Subsequently, the plates were washed three times with 200 μL of wash buffer and blocked with 200 μL of 1% BSA for 1 h. After blocking, 100 μL of the samples or the standard curve preparations were added to each well and incubated for 2 h at room temperature. The plates were then washed three times with 200 μL of wash buffer and then incubated with cytokine detection antibody at room temperature for 2 h. After three washes, they were incubated with 100 μL of streptavidin at room temperature for 20 min. After another three washes, 100 μL of developer substrate solution was added to each well and incubated for 20 min at room temperature protected from light. The enzymatic reaction was stopped by adding 50 μL of stop solution (H2SO4). The absorbance was measured at 450 nm and the results were expressed in pg/mL.
Immunohistochemistry for S100 β and GFAP: To assess the localization of the inflammatory markers S100 β and GFAP in the intestinal mucosa, immunohistochemistry was carried out. Collected colon segments were isolated, separated and fixed in 10% buffered formalin for 20 h. The tissues were embedded in paraffin and cut into 4 μm thick cuts using a microtome, inserted into salinized histological slides. These slides were deparaffinized by oven at 60 °C overnight followed by three baths with xylene of five min each. The colons were then hydrated with two baths of absolute ethanol, one bath in 90% ethanol, one bath in 80% ethanol, and one bath in 70% ethanol, for three min each. The sections were submerged in a distilled water bath for 10 min and antigen recovery was carried out with citrate buffer (DAKO, Singapore; pH 7.0) for 20 min in a water bath (95 °C). The tissues were washed with phosphate-buffered saline (PBS 1X) for 5 min after which the peroxidase was blocked with 3% hydrogen peroxide (Abcam, Oxford, UK) for 30 min. The slides were washed with PBS 1X and incubated with the primary antibodies for S100 beta and GFAP (both from DAKO) for 1 h at room temperature. In the negative controls, the primary antibodies were omitted. The slides were washed three times with PBS 1X and incubated with the polymer (DAKO) for 30 min. The slides were then washed three times with PBS 1X for three minutes each, dried and DAB was applied (DAKO, 3,3-diaminobenzidine, one drop of DAB to 1 mL of diluent). DAB is a chromogen that reacts with the peroxidase of the target antigen, resulting in a brown color. In this way, the slides were observed until a brown coloration appeared, after which the reaction was stopped immediately by immersing them in distilled H2O. Finally, the slides were counterstained with Mayer’s hematoxylin and processed to insert the coverslip. The immunohistochemical images were captured (eight fields per slide for each animal) using a light microscope coupled to a camera with a LAZ 3.5 acquisition system (LEICA DM1000, Wetzlar, Germany). To quantify the number of cells positive for S100 beta and GFAP, the Image J program (version 1.53k) was used. The results were expressed as the mean ± SEM of the number of cells positive for GFAP or S100 beta per field.
Evaluation of Myeloperoxidase Activity (MPO): For this assay, 50 to 100 mg of colon from each animal was placed in buffer 0.1 M NaCl + 0.015 M Disodium EDTA and 0.02 M Na3PO4 at pH 4.7. After homogenization in a Polytron at 18,516 g, they were centrifuged for an additional 15 min at 986 g. The supernatant was then removed, the precipitate was resuspended in 0.1 M NaCl + 0.015 M Disodium EDTA and 0.02 M Na3PO4 at pH 4.7, and it was again centrifuged for 15 min at 986 g. The supernatant was removed, and the precipitate was homogenized in a Politron at 18,516 g in HTAB 0.05% diluted in 200 mL of Na3PO4 0.05 M. This homogenate was then frozen and thawed in liquid nitrogen twice. The homogenate was centrifuged at 10,956–21,475 g for 15 min and the supernatant was pipetted onto a plate (5–10 μL) to which 45 μL of 0.08 M Na3PO4 was added. Then, we added 25 μL of TMB and 100 μL of H2O2 for 5 min. The reaction was completed with the addition of 50 μL of a 4 M of H2SO4 and the absorbance were read on a plate reader at 450 nm (FLUOstar OPTIMA-BMG LABTECH). The neutrophil infiltrate was obtained from a neutrophil standard curve and the results are expressed as MPO units per mg tissue.
Determination of Malondialdehyde (MDA) Levels: MDA levels were determined using the method of Uchiyama and Mihara [
33]. An amount of 50 mg of intestinal mucosa tissue samples corresponding to the colon was homogenized in 500 µL of 1.15% KCl solution at 4 °C, to obtain a 10% homogenate. From this, 250 µL aliquots of the homogenate were added to tubes containing 1.5 mL of 1% H
3PO
4 solution and 500 µL of TBARS solution (0.6%). The tubes were heated in a water bath at 100 °C for 45 min, then cooled in an ice-cold water bath, followed by the addition of 2 mL of n-butanol. The samples were then vortexed (PHOENIX Instrument, Garbsen, Germany) for 1 min and then centrifuged at 158 g for 15 min. The supernatant absorbance was measured at 520 and 535 nm in a plate reader (FLUOstar OPTIMA - BMG LABTECH, Offenburg, Germany), and the results correspond to the difference between absorbances which were converted to nmol/g of intestinal tissue.
Determination of Glutathione (GSH) levels: The colon samples were homogenized in 0.02 M EDTA (1 mL/100 mg tissue). Subsequently, 400 μL of homogenate was mixed with 300 μL of distilled water and 80 μL of trichloroacetic acid (50%, w/v) and centrifuged at 986 g for 15 min. Then, 400 μL of supernatant was mixed with 800 μL of Tris buffer (0.4 M, pH 8.9), 20 μL of 0.01 M DTNB was added, and the samples were subsequently stirred for a period of 3 min. A reading spectrophotometer with absorbance adjustment measured at 412 nm was used. The results are expressed as μg of GSH/g tissue.
Statistical analysis: All quantitative results were expressed as the mean ± standard deviation (SD), except for the histopathology scores, which were expressed as the median. The data were statistically analyzed using GraphPad Prism® software, version 5.0. The following tests were used to carry out the statistical analysis between the groups: analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test, and for the histological scores, the Kruskal–Wallis test followed by Dunn’s test. The significance level adopted was 5% (p < 0.05) and descriptive levels (p) below this value were considered significant.
4. Discussion
Inducing colitis in rodents using a chemical agent, such as AA, offers the advantage of instigating inflammation in animals with a normal immune system [
34]. Rectally administered AA can replicate colitis in a diffuse and dose-dependent manner, observed in the distal portion of the rodent colon [
35,
36]. This induces inflammation akin to ulcerative colitis and Crohn’s disease in humans, considering molecular changes, histological features, and clinical characteristics [
37]. Our findings align with the existing literature, revealing an increase in macroscopic lesions in the colons of rats, 12 h after the administration of 8% AA [
38], which could be treated with dexamethasone as a control treatment.
The administration of ES demonstrated a reduction in overall inflammatory aspects of AA-induced colitis, in terms of macro- and microscopic scoring, most significantly at 50 mg/kg. Interestingly, with 25 and 50 mg/kg Au@ES, the animals also significantly ameliorated in terms of inflammation scores and histology markers. However, Au@ET 100 mg/kg did not exhibit a similar effect. It is noteworthy that ES and Au@ES yielded comparable results to the standard drug, dexamethasone.
ES is a brown macroalgae rich in polysaccharides and phenolic compounds [
39]. Therefore, it has been associated with anti-inflammatory [
28] and antioxidant activities [
27]. As previously reported by our group, ES aqueous extract has a high content in phenolic compounds, revealing extremely high reducing activity and good free radical scavenging capacity [
30]. This observation was in line with characterization data of extracts with organic solvents [
27,
40]. Together, they support the concept that ES can directly counteract oxidative stress and mitigate oxidative-mediated damage to biomacromolecules. This antioxidant activity facilitates the green synthesis of metallic nanoparticles in aqueous extracts of this algae [
30,
41]. Macroalgae polysaccharides are considered a potential therapeutic option for patients with IBD [
4]. Sulfated polysaccharides from the macroalgae
Gracilaria caudata have been shown to improve macro- and microscopic scores of histopathology of colitis, induced with ethanol, in mice [
42]. A similar positive effect on colon damage was observed with polysaccharide extracted from red algae
Gracilaria birdiae and
Hypnea musciformin on trinitrobenzenesulfonic acid (TNBS)-induced colitis [
43,
44]. Interestingly, the application of naked AuNPs on rodent models of colitis has also reportedly led to the improved status of the colon [
45].
A key feature of AA-induced colitis is immune cell infiltration, producing high levels of proinflammatory cytokines such as TNF-α and IL-1β [
46]. These chemoattractant mediators are essential for cytokine-dependent neutrophil activation, associated with elevated MPO levels. In this study, animals with colitis induced by AA displayed a notable increase in the concentration of both IL-1β and TNF-α, assessed through ELISA and immunohistochemistry, as reported by others [
38]. Administration of dexamethasone, ES at a dose of 50 mg/kg, and Au@ES at doses of 25 mg/kg and 50 mg/kg leads to a significant reduction in the expression of these proinflammatory cytokines in the colon of animals with induced IBD. This observation suggests that the protective effect of ES and Au@ES may be mediated by hampering the inflammatory process. This is in agreement with reports linking the administration of algal extracts or components (mostly polysaccharides, e.g., fucoidan), particularly brown algae, to the diminished expression of these inflammatory mediators [
24,
46,
47]. A previous study proved that the ethanolic extract of ES had some anti-inflammatory properties, with a better activity in extracts collected in the summer [
28]. Sulfated polysaccharides of related species of the
Cystoseira genus, formerly the genus of ES, are recognized for their anti-inflammatory, gastroprotective, and antiradical activities [
47]. Mhadhbei et al. showed that aqueous extracts of
Cystoseira amentacea, Cystoseira crinita, Cystoseira sedoides, and
Cystoseira compressa reduced inflammation in rat paw oedema, with doses of 25 mg/kg and 50 mg/kg, a few hours after incubation [
48]. The concentrations used, although in the context of other clinical applications, are comparable to the ones used by our group, indicating that this family of brown algae has potential for multiple applications in inflammatory diseases. With
Sargassum hemiphyllum, the capacity to inhibit the expression of IL-1β and TNF-α, while promoting IL-10 and IFN-γ release, was directly associated with better intestinal epithelial barrier and immune function [
49]. Polysaccharides from
G. caudata also improved the disease status in AA-induced colitis by regulating the production of these inflammation mediators [
38]. Interestingly, in clinical practice, the inhibition of TNF-α serves as a widely adopted standard treatment for IBD [
50], emphasizing the importance of finding natural products with a similar activity.
AuNPs have also been associated with reduced inflammation in chronic diseases, with a particular emphasis on colitis. AuNPs have been shown to attenuate colonic inflammation, reduce oxidative stress, inhibit pro-inflammatory cytokines, and promote tissue repair in experimental models of colitis [
9]. Other studies also proved that the expression of interleukin-17, a key mediator in the pathogenesis of intestinal inflammation, was sensitive to AuNPs [
20]. Notably, AuNPs produced by green synthesis in algae have already been used to suppress cytokine production in vitro [
51]. AuNPs produced in extracts of three seaweeds,
C. myrica,
C. trinodis, and
C. prolifera, were also able to diminish inflammation in vitro by inhibiting egg albumin denaturation [
52]. Curiously, Zhu et al. proved that the incubation of monocytes with AuNPs for 5 h resulted in a significant reduction in ROS-mediated activation of the inflammation signaling pathway [
22]. As the current study did not aim to draw a comprehensive profile of the modified cytokine expression patterns, it would be important in future studies to clarify if ES and Au@ES can interfere with IL17-driven inflammatory processes, and even upstream cytokines IL12 and IL23, emergent therapeutic targets in gut inflammation [
53].
In IBD, oxidative stress is exacerbated due to the increased production of ROS/RNS by activated immune cells and impaired antioxidant defenses. This can cause oxidative damage to cellular components such as lipids, proteins, and DNA. Conversely, oxidative stress can exacerbate inflammation by promoting the release of pro-inflammatory cytokines, activation of immune cells, and disruption of barrier function in the intestine. Neutrophil activation and infiltration in AA-induced disease [
54] reflect the importance of this feature in IBD. These cells release granular MPO in the inflamed bowel, which leads to the production of powerful ROS and perpetuates inflammation [
55]. MPO is thus considered a biomarker for IBD. Colons from mice treated with ES 50 mg/kg and Au@ES 25 or 50 mg/kg displayed a striking impairment of neutrophil activation, with significantly lowered MPO activity, and consequently very reduced lipid peroxidation and augmented glutathione levels. This observation is in agreement with the already discussed antiradical activity of ES [
27,
30,
40] but also hints at the concomitant induction of antioxidant enzymatic defenses. This strong antioxidant activity, unquestionably beneficial to interrupt the pathological cascade in IBD, is comparable to that of purified sulfated polysaccharides from
G. caudata in AA-induced and from
G. birdiae in TNBS-induced models, previously cited [
38]. The first was effective at 1.0 to 10.0 mg/kg administered intraperitoneally, which is very invasive, while the latter showed positive results at 30, 60, and 90 mg/kg by oral administration, more approximate to the current study.
The lower dose of Au@ES (25 mg/kg) leading to a maximal effect on antioxidant biomarkers is possibly related to the antiradical activity of AuNPs. Orally administered gold nanoclusters showed ROS scavenging, with a consequent reduction in the expression of proinflammatory cytokines, beneficial for inflammatory bowel disease treatment [
23]. Oral administration of naked AuNPs (2.5 mg Au/kg) was also shown to effectively target colonic tissue in dextran sodium sulphate (DSS)-induced ulcerative colitis, reducing lipid peroxidation and displaying anti-inflammatory potential [
20]. Interestingly, the current work used similar dosages of gold (25 mg/kg of Au@ES containing 3.4 mg Au/kg). Another study reported that orally given AuNPs (25 μg Au/kg) can prevent colitis by attenuating the inflammatory response mediated by Toll-like receptor 4 and oxidative stress but may lead to an imbalance of the intestinal flora of mice [
22]. The green synthesis of AuNPs as in the present study may mitigate the risk of dysbiosis, as macroalgae are considered a source of prebiotics [
56], thereby further supporting our approach as a more viable option for IBD management. Although AA-induced colitis mimics the hallmarks of human IBD, it would be useful to corroborate the positive effects of ES and Au@ES in future investigations using, for example, ex vivo systems, as it is known that interspecies differences may make it difficult to predict treatment outcome of strategies validated in in vivo models [
57]. This approach could also be useful in identifying the putative modulation of intestinal epithelial barrier function, which is central to IBD pathology.
The myenteric plexus neural network plays a vital role in the reflex regulation of contractile activities in the intestinal external musculature [
6]. Intestinal ischemia/reperfusion in the digestive tract leads to a substantial loss of myenteric and submucosal plexus neurons, resulting in changes in neuron density and size, influencing intestinal motility [
58]. In Crohn’s disease, transmural inflammation is associated with enteric nervous system lesions, such as hypertrophy and hyperplasia of neurons, an irregular increase in the number of nerve fibers, ganglia, and an augmented number of glial cells. The increase in the number of cells may reflect the severity of inflammation. Although the myenteric plexus is a crucial player in IBD pathology, few studies include its monitoring. Treatment with ES 50 mg/kg but more strikingly Au@ES 50 mg/kg strongly reduced the numbers of enteric glial cells, signifying that the benefits verified in the colon of mice also imply an impact on the myenteric plexus. To our knowledge, this is the first study indicating that ES and biosynthetic AuNPs may have this effect. Beyond its anti-inflammatory role, ES is known to be neuroprotective in other instances [
10], which agrees with our results in terms of glial cell preservation.