**3. Results**

**3. Results** 

#### *3.1. H2O<sup>2</sup> Impairs the Viability and Morphology of ADSCs 3.1. H2O2 Impairs the Viability and Morphology of ADSCs*  Previous reports have induced oxidative stress in vitro by treating cells with H2O2 for 24 h [25,26].

Previous reports have induced oxidative stress in vitro by treating cells with H2O<sup>2</sup> for 24 h [25,26]. However, ADSCs appear to be particularly susceptible to H2O2-dependent oxidative stress, with 1 or 2 h of exposure being sufficient to impair ADSC activity [27,28]. Thus, in this study, we induced oxidative stress in ADSCs by exposing these cells to different concentrations of H2O<sup>2</sup> for 2 h (Figure 1A). However, ADSCs appear to be particularly susceptible to H2O2-dependent oxidative stress, with 1 or 2 h of exposure being sufficient to impair ADSC activity [27,28]. Thus, in this study, we induced oxidative stress in ADSCs by exposing these cells to different concentrations of H2O2 for 2 h (Figure 1A).

**Figure 1.** H2O2 treatment impairs cellular activity of adipose derived stem cells (ADSCs). (**A**,**C**) Experimental scheme for oxidative stress induction in ADSCs. (**B**) Alterations in ADSC cellular morphology depending on the concentration of H2O2. (**D**–**E**) The viability of ADSCs treated with H2O2 at 24 h (**D**) and 48 h (**E**) after the removal of H2O2. The untreated control was set as 100%, and cell viability was expressed as the percentage relative to the activity of the control group. Values of *p* < 0.05 were interpreted as statistically significant (\* *p* < 0.05, \*\*\* *p* < 0.001). The data are expressed as the mean ± SD of three independent experiments. **Figure 1.** H2O<sup>2</sup> treatment impairs cellular activity of adipose derived stem cells (ADSCs). (**A**,**C**) Experimental scheme for oxidative stress induction in ADSCs. (**B**) Alterations in ADSC cellular morphology depending on the concentration of H2O<sup>2</sup> . (**D**–**E**) The viability of ADSCs treated with H2O<sup>2</sup> at 24 h (**D**) and 48 h (**E**) after the removal of H2O<sup>2</sup> . The untreated control was set as 100%, and cell viability was expressed as the percentage relative to the activity of the control group. Values of *p* < 0.05 were interpreted as statistically significant (\* *p* < 0.05, \*\*\* *p* < 0.001). The data are expressed as the mean ± SD of three independent experiments.

The transient treatment of ADSCs with H2O2 primarily affected cell proliferation and morphology (Figure 1B). At 24 h after the removal of H2O2, cell density was significantly different between nontreated and H2O2-treated conditions. While nontreated cells started to proliferate and became confluent at 72 h, H2O2-treated ADSCs displayed suppressed cell proliferation. Notably, a distinct difference in cellular density and shape was observed at H2O2 concentrations above 100 μM. High concentrations of H2O2 (i.e., 200, 300, and 400 μM) severely impaired the cellular activity of ADSCs, significantly reducing density at 72 h. H2O2 at 50 μM seemed to damage ADSCs, but these cells appeared to recover as time passed. To compare cellular impairment quantitatively, we The transient treatment of ADSCs with H2O<sup>2</sup> primarily affected cell proliferation and morphology (Figure 1B). At 24 h after the removal of H2O2, cell density was significantly different between nontreated and H2O2-treated conditions. While nontreated cells started to proliferate and became confluent at 72 h, H2O2-treated ADSCs displayed suppressed cell proliferation. Notably, a distinct difference in cellular density and shape was observed at H2O<sup>2</sup> concentrations above 100 µM. High concentrations of H2O<sup>2</sup> (i.e., 200, 300, and 400 µM) severely impaired the cellular activity of ADSCs, significantly reducing density at 72 h. H2O<sup>2</sup> at 50 µM seemed to damage ADSCs, but these cells appeared to recover as time passed. To compare cellular impairment quantitatively, we determined cell viability at 24 and 48 h after oxidative stress (Figure 1C). H2O<sup>2</sup> treatment decreased cell viability in a dose-dependent

manner (Figure 1D,E). Consistent with our results on cell shape and density, ADSCs treated with 50 µM H2O<sup>2</sup> spontaneously restored their activity within 48 h (Figure 1E). However, concentrations above 100 µM H2O<sup>2</sup> reduced cell viability and this did not return to normal levels. Based on these data, we determined that 100 µM H2O<sup>2</sup> induced substantial oxidative damage in ADSCs in vitro. This indicates that transient H2O<sup>2</sup> treatment impairs the cellular viability of ADSCs, which might affect cell survival and the function of ADSCs. and density, ADSCs treated with 50 μM H2O2 spontaneously restored their activity within 48 h (Figure 1E). However, concentrations above 100 μM H2O2 reduced cell viability and this did not return to normal levels. Based on these data, we determined that 100 μM H2O2 induced substantial oxidative damage in ADSCs in vitro. This indicates that transient H2O2 treatment impairs the cellular viability of ADSCs, which might affect cell survival and the function of ADSCs. *3.2. Substance-P Restores Cell Viability of ADSCs Injured by Oxidative Stress* 

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cell viability in a dose-dependent manner (Figure 1D,E). Consistent with our results on cell shape

#### *3.2. Substance-P Restores Cell Viability of ADSCs Injured by Oxidative Stress* In order to determine the effect of SP on damaged ADSCs, cells were treated with 100 μM H2O2

In order to determine the effect of SP on damaged ADSCs, cells were treated with 100 µM H2O<sup>2</sup> for 2 h and then provided with fresh media. After 24 h, SP was added to the damaged ADSCs at a concentration of 100 nM (Figure 2A). This dose of SP was determined based on previous reports [15–17]. for 2 h and then provided with fresh media. After 24 h, SP was added to the damaged ADSCs at a concentration of 100 nM (Figure 2A). This dose of SP was determined based on previous reports [15– 17].

**Figure 2.** Substance-P improves the viability of ADSCs damaged by oxidative stress. (**A**) Experimental scheme for inducing oxidative stress in ADSCs and the subsequent SP treatment. (**B**,**C**) Cell viability was measured by WST assay at 24 (**B**) and 48 h (**C**) after SP treatment. The untreated control was set as 100%, and cell viability was expressed as the percentage relative to the activity of the control group. (**D**) The representative image of cellular morphology. (**E**) Final cell yield was measured by counting total cell number. Values of *p* < 0.05 were interpreted as statistically significant (\* *p* < 0.05, \*\*\* *p* < 0.001). The data are expressed as the mean ± SD of three independent experiments. **Figure 2.** Substance-P improves the viability of ADSCs damaged by oxidative stress. (**A**) Experimental scheme for inducing oxidative stress in ADSCs and the subsequent SP treatment. (**B**,**C**) Cell viability was measured by WST assay at 24 (**B**) and 48 h (**C**) after SP treatment. The untreated control was set as 100%, and cell viability was expressed as the percentage relative to the activity of the control group. (**D**) The representative image of cellular morphology. (**E**) Final cell yield was measured by counting total cell number. Values of *p* < 0.05 were interpreted as statistically significant (\* *p* < 0.05, \*\*\* *p* < 0.001). The data are expressed as the mean ± SD of three independent experiments.

Substance-P treatment started to improve ADSC viability within 24 h (Figure 2B; H2O2 treated: 67.47 ± 1.29%, H2O2 + SP-treated: 70.4 ± 1.4%, *p* < 0.05) and fully restored cell viability at 48 h (Figure 2C; H2O2 treated: 73.16 ± 1.83%, H2O2 + SP-treated: 84.7 ± 3.6%, *p* < 0.001). Cell viability is directly related to cell repopulation. Therefore, we examined the cell yield by counting the total number of Substance-P treatment started to improve ADSC viability within 24 h (Figure 2B; H2O<sup>2</sup> treated: 67.47 ± 1.29%, H2O<sup>2</sup> + SP-treated: 70.4 ± 1.4%, *p* < 0.05) and fully restored cell viability at 48 h (Figure 2C; H2O<sup>2</sup> treated: 73.16 ± 1.83%, H2O<sup>2</sup> + SP-treated: 84.7 ± 3.6%, *p* < 0.001). Cell viability is directly related to cell repopulation. Therefore, we examined the cell yield by counting the total number of cells and

comparing it with that of the control. The analysis of cell yield confirmed the restorative effect of SP on damaged ADSCs (Figure 2E, H2O<sup>2</sup> treated: 58.79 ± 1.96%, H2O<sup>2</sup> + SP-treated: 72.97 ± 4.9%, *p* < 0.001, relative to control). SP treatment did not influence cell morphology, but enhanced cell proliferation (Figure 2D). cells and comparing it with that of the control. The analysis of cell yield confirmed the restorative effect of SP on damaged ADSCs (Figure 2E, H2O2 treated: 58.79 ± 1.96%, H2O2 + SP-treated: 72.97 ± 4.9%, *p* < 0.001, relative to control). SP treatment did not influence cell morphology, but enhanced cell proliferation (Figure 2D).

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This revealed that SP treatment enhances the viability of ADSCs impaired by oxidative stress. This effect of SP finally led to the reduced cellular senescence of ADSCs (Figure S2). This revealed that SP treatment enhances the viability of ADSCs impaired by oxidative stress. This effect of SP finally led to the reduced cellular senescence of ADSCs (Figure S2).

#### *3.3. E*ff*ect of SP on Paracrine Potential of ADSCs Exposed to Oxidative Stress 3.3. Effect of SP on Paracrine Potential of ADSCs Exposed to Oxidative Stress*

Oxidative stress is well known to decrease cell viability and negatively affect cell function. Stem cells exert their function via paracrine factors, and thus, it is critical to evaluate ADSC cytokine production when investigating the effects of oxidative stress (Figure 3A). VEGF and TGF-β are constitutively produced from MSCs, and their levels are typically reduced by aging or cellular damage [29]. Therefore, VEGF and TGF-β were selected as surrogate markers to represent the paracrine action of ADSCs in this experiment. Oxidative stress is well known to decrease cell viability and negatively affect cell function. Stem cells exert their function via paracrine factors, and thus, it is critical to evaluate ADSC cytokine production when investigating the effects of oxidative stress (Figure 3A). VEGF and TGF-β are constitutively produced from MSCs, and their levels are typically reduced by aging or cellular damage [29]. Therefore, VEGF and TGF-β were selected as surrogate markers to represent the paracrine action of ADSCs in this experiment.

**Figure 3.** Substance-P recovers the paracrine potential of oxidatively damaged ADSCs. (**A**) Experimental scheme for ADSC with oxidative stress and SP treatment. (**B**–**E**) VEGF in the conditioned medium of ADSCs was quantified using ELISA at 24 (**B**,**C**) and 48 h (**D**,**E**) after the first SP treatment. The absolute concentration of VEGF (**B**,**D**) and secreted amount per 1 × 105 ADSCs (**C**,**E**) were evaluated. (**F**–**I**) TGF-β in the conditioned medium of ADSCs was quantified by ELISA at 24 h (**F**,**G**) and 48 h (**H**,**I**) after the first SP treatment. The absolute concentration of TGF-β (**F**,**H**) and the secreted amount per 1 × 105 ADSCs (**G**,**I**) were evaluated. Values of *p* < 0.05 were interpreted as statistically significant (\*\* *p* < 0.01, \*\*\* *p* < 0.001). The data are expressed as the mean ± SD of three **Figure 3.** Substance-P recovers the paracrine potential of oxidatively damaged ADSCs. (**A**) Experimental scheme for ADSC with oxidative stress and SP treatment. (**B**–**E**) VEGF in the conditioned medium of ADSCs was quantified using ELISA at 24 (**B**,**C**) and 48 h (**D**,**E**) after the first SP treatment. The absolute concentration of VEGF (**B**,**D**) and secreted amount per 1 <sup>×</sup> <sup>10</sup><sup>5</sup> ADSCs (**C**,**E**) were evaluated. (**F**–**I**) TGF-<sup>β</sup> in the conditioned medium of ADSCs was quantified by ELISA at 24 h (**F**,**G**) and 48 h (**H**,**I**) after the first SP treatment. The absolute concentration of TGF-<sup>β</sup> (**F**,**H**) and the secreted amount per <sup>1</sup> <sup>×</sup> <sup>10</sup><sup>5</sup> ADSCs (**G**,**I**) were evaluated. Values of *p* < 0.05 were interpreted as statistically significant (\*\* *p* < 0.01, \*\*\* *p* < 0.001). The data are expressed as the mean ± SD of three independent experiments.

independent experiments. VEGF levels from ADSCs significantly decreased after oxidative stress (Figure 3B, 48 h after oxidative stress, Control: 651.8 ± 8.7 pg/mL, H2O2 treated: 251.4 ± 4.7 pg/mL), and this reduction was sustained by 72 h after oxidative stress (Figure 3D; 72 h after oxidative stress, Control: 721.8 ± 12.9 VEGF levels from ADSCs significantly decreased after oxidative stress (Figure 3B, 48 h after oxidative stress, Control: 651.8 ± 8.7 pg/mL, H2O<sup>2</sup> treated: 251.4 ± 4.7 pg/mL), and this reduction was sustained by 72 h after oxidative stress (Figure 3D; 72 h after oxidative stress, Control: 721.8 ± 12.9 pg/mL, H2O<sup>2</sup> treated: 321.74 ± 9.77 pg/mL). This indicates that VEGF secretion

did not increase significantly between 48 and 72 h, and that impairment of cytokine secretion occurred early. However, SP treatment elevated VEGF production in ADSCs (Figure 3B; 48 h after oxidative stress, H2O<sup>2</sup> + SP-treated: 312.6 ± 8.1 pg/mL; Figure 3D; 72 h after oxidative stress, H2O<sup>2</sup> + SP-treated: 477 ± 5.4 pg/mL). pg/mL, H2O2 treated: 321.74 ± 9.77 pg/mL). This indicates that VEGF secretion did not increase significantly between 48 and 72 h, and that impairment of cytokine secretion occurred early. However, SP treatment elevated VEGF production in ADSCs (Figure 3B; 48 h after oxidative stress, H2O2 + SPtreated: 312.6 ± 8.1 pg/mL; Figure 3D; 72 h after oxidative stress, H2O2 + SP-treated: 477 ± 5.4 pg/mL).

In this experiment, oxidative stress decreased the total cell number (Figure 2), and thus, it could be inferred that the reduction in cytokine secretion was due to the low cell number. To clarify this, the amount of cytokines was assessed per cell. It was found that oxidative stress disabled the paracrine function of ADSCs (Figure 3C,E). Interestingly, SP treatment increased the concentration of VEGF in the conditioned medium of ADSCs, which might be attributed to their improved ability to produce VEGF as well as the increased cell number. This phenomenon was also observed for TGF-β secretion (Figure 3F,H), with TGF-β levels decreasing following oxidative stress and then recovering after SP treatment (Figure 3G,I). In this experiment, oxidative stress decreased the total cell number (Figure 2), and thus, it could be inferred that the reduction in cytokine secretion was due to the low cell number. To clarify this, the amount of cytokines was assessed per cell. It was found that oxidative stress disabled the paracrine function of ADSCs (Figure 3C,E). Interestingly, SP treatment increased the concentration of VEGF in the conditioned medium of ADSCs, which might be attributed to their improved ability to produce VEGF as well as the increased cell number. This phenomenon was also observed for TGFβ secretion (Figure 3F,H), with TGF-β levels decreasing following oxidative stress and then recovering after SP treatment (Figure 3G,I).

This suggests that SP is able to improve the paracrine action of ADSCs under oxidative stress, and that repeated SP treatment can intensify the restorative function of SP in damaged ADSCs. This suggests that SP is able to improve the paracrine action of ADSCs under oxidative stress, and that repeated SP treatment can intensify the restorative function of SP in damaged ADSCs.

#### *3.4. SP Activates Akt Signaling in ADSCs Injured by Oxidative Stress 3.4. SP Activates Akt Signaling in ADSCs Injured by Oxidative Stress*

Typically, when cells are under oxidative stress, signaling associated with cell survival is activated, allowing the cells to survive. The phosphoinositide 3-kinase (PI3K)-Akt pathway is a pro-survival pathway regulated by ROS. When oxidative stress is exerted on cells, Akt is phosphorylated in a PI3K-dependent manner, which induces the phosphorylation and subsequent inactivation of pro-apoptotic factors, including glycogen synthase kinase (GSK)-3 [30,31]. To examine whether the increase in ADSCs viability by SP was accompanied by the activation of Akt signaling, we determined the phosphorylation state of Akt and GSK-3β following ADSCs treatment with H2O<sup>2</sup> and then with SP for 20 min (Figure 4A). ADSCs treated with H2O<sup>2</sup> failed to maintain phosphorylated Akt levels, whereas SP treatment promoted Akt phosphorylation (Figure 4B). Additionally, GSK-3β, a downstream effector of Akt signaling and a pro-apoptotic molecule [14], was phosphorylated and inactivated following SP treatment. The expression levels of phospho-Akt and phospho-GSK-3β were quantified relative to the levels of total Akt and GSK-3β (Figure 4B). Taken together, these results demonstrate that SP can activate Akt/GSK-3β signaling, which contributes to the SP-induced recovery of oxidatively damaged ADSCs. Typically, when cells are under oxidative stress, signaling associated with cell survival is activated, allowing the cells to survive. The phosphoinositide 3-kinase (PI3K)-Akt pathway is a prosurvival pathway regulated by ROS. When oxidative stress is exerted on cells, Akt is phosphorylated in a PI3K-dependent manner, which induces the phosphorylation and subsequent inactivation of proapoptotic factors, including glycogen synthase kinase (GSK)-3 [30,31]. To examine whether the increase in ADSCs viability by SP was accompanied by the activation of Akt signaling, we determined the phosphorylation state of Akt and GSK-3β following ADSCs treatment with H2O2 and then with SP for 20 min (Figure 4A). ADSCs treated with H2O2 failed to maintain phosphorylated Akt levels, whereas SP treatment promoted Akt phosphorylation (Figure 4B). Additionally, GSK-3β, a downstream effector of Akt signaling and a pro-apoptotic molecule [14], was phosphorylated and inactivated following SP treatment. The expression levels of phospho-Akt and phospho-GSK-3β were quantified relative to the levels of total Akt and GSK-3β (Figure 4B). Taken together, these results demonstrate that SP can activate Akt/GSK-3β signaling, which contributes to the SP-induced recovery of oxidatively damaged ADSCs.

**Figure 4.** (**A**) Experimental scheme for ADSC oxidative stress and SP treatment. (**B**) Level of phospho-Akt (**B**) and phospho-GSK-3β as detected by Western blotting. Phospho-Akt and phospho-GSK-3β protein expression levels relative to total Akt and GSK-3β were quantified using the Image J program. Expression levels were represented relative to that of the untreated control. The data are expressed as the mean ± SD of three independent experiments. **Figure 4.** (**A**) Experimental scheme for ADSC oxidative stress and SP treatment. (**B**) Level of phospho-Akt (**B**) and phospho-GSK-3β as detected by Western blotting. Phospho-Akt and phospho-GSK-3β protein expression levels relative to total Akt and GSK-3β were quantified using the Image J program. Expression levels were represented relative to that of the untreated control. The data are expressed as the mean ± SD of three independent experiments.

#### **4. Discussion**

Aging and oxidative stress are highly associated with inflammation and the development of mortal diseases [1,21]. To combat oxidative stress-related diseases, antioxidants are typically applied from natural compounds or various medicines; however, their effects were equivocal, and are often accompanied by unwanted side effects. Therefore, additional treatment options with increased efficacy are urgently needed.

Most studies deal with oxidative stress to study retinal disease [6,16]. Stem cell therapies have emerged as an exciting option in the treatment of a variety of diseases including those that are related to oxidative stress. ADSCs are among the most popular stem cells to be used in novel therapies [12,32], as they have a high repopulation potential and their tissue of origin (adipose) is easily accessible. Ideally, therapies involving ADSCs would involve their autologous transplantation; however, ADSCs from diseased or aged patients may not be fully functional given the heightened levels of free radicals and inflammation in the individual. Indeed, we found that ADSCs from diseased animals have low repopulation rates and decreased cytokine secretion with a lack of differentiation potential even in early passages [33]. Moreover, a recent study corroborated the impairment of ADSCs by oxidative stress. Several studies have also indicated that, compared to other cells, stem cells are more susceptible to damage due to free radicals [27]. Thus, the effect of oxidative stress on stem cells should be taken into consideration for the application of stem cell therapy and endogenous regeneration.

In this study, oxidative stress was induced by treating ADSCs with H2O<sup>2</sup> for 2 h. We found that this transient exposure to H2O<sup>2</sup> was sufficient to affect ADSC activity and function, but was not completely detrimental to the ADSC population. H2O<sup>2</sup> treatment altered cell morphology, reduced cell viability, inhibited the proliferation of ADSCs, and decreased their paracrine potential. This impairment could not be restored by removing the oxidant, which might lead to cellular senescence and death.

In an attempt to rescue ADSCs from oxidative stress, SP was employed. SP treatment of injured ADSCs enhanced cell viability and restored the paracrine potential of ADSCs. Moreover, at an early time point, SP activated the Akt/Gsk-3β pathway, which was downregulated by oxidative stress, and might contribute to the improvement of cell survival. Differentiation potential was also improved by SP but its effect was so slight, comparing to that of cell viability and cytokine secretion. Therefore, it was inferred that SP can augment cell survival and secretome production (rather than stemness), which might contribute to enhanced differentiation potential, to some extent. Notably, paracrine factors including VEGF and TGF-beta are deeply involved in osteogenesis [34–36]. SP could restore VEGF and TGF-beta production from ADSC with H2O2. This might suggest the possibility for the correlation between paracrine potential and osteogenesis (Figure S3)

In conclusion, this study demonstrated that SP could stimulate the recovery of ADSCs under oxidative stress, possibly by promoting cell proliferation through the activation of Akt/GSK-3β signaling. SP is anticipated to enhance the activity of ADSCs from aged or diseased individuals. Constant treatment of SP is anticipated to further augment the restoration of impaired stem cells.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3921/9/10/978/s1, Figure S1: The analysis for marker expression of ADSC; Figure S2: Beta-galatoxidase staining of ADSC with H2O<sup>2</sup> and SP; Figure S3: The differentiation ability of ADSC with H2O<sup>2</sup> and SP.

**Author Contributions:** Conceptualization: H.S.H., J.S.P.; Methodology: H.S.H., J.S.P., J.P., G.P., Validation: H.S.H., J.S.P.; Formal analysis: H.S.H., J.S.P., J.P.; Investigation: H.S.H., J.S.P., G.P.; Data curation: H.S.H., J.S.P., J.P., G.P.; writing—original draft preparation: H.S.H., J.S.P., supervision: H.S.H.; project administration: H.S.H.; funding acquisition: H.S.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1D1A1B0704104813) and by a Korean Health Technology R&D Project grant [HI18C1492] from the Ministry of Health and Welfare (Sejong, Republic of Korea).

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