Substance-P Restores Cellular Activity of ADSC Impaired by Oxidative Stress

Oxidative stress induces cellular damage, which accelerates aging and promotes the development of serious illnesses. Adipose-derived stem cells (ADSCs) are novel cellular therapeutic tools and have been applied for tissue regeneration. However, ADSCs from aged and diseased individuals may be affected in vivo by the accumulation of free radicals, which can impair their therapeutic efficacy. Substance-P (SP) is a neuropeptide that is known to rescue stem cells from senescence and inflammatory attack, and this study explored the restorative effect of SP on ADSCs under oxidative stress. ADSCs were transiently exposed to H2O2, and then treated with SP. H2O2 treatment decreased ADSC cell viability, proliferation, and cytokine production and this activity was not recovered even after the removal of H2O2. However, the addition of SP increased cell viability and restored paracrine potential, leading to the accelerated repopulation of ADSCs injured by H2O2. Furthermore, SP was capable of activating Akt/GSK-3β signaling, which was found to be downregulated following H2O2 treatment. This might contribute to the restorative effect of SP on injured ADSCs. Collectively, SP can protect ADSCs from oxidant-induced cell damage, possibly by activating Akt/GSK-3β signaling in ADSCs. This study supports the possibility that SP can recover cell activity from oxidative stress-induced dysfunction.


Introduction
Oxidative stress is an imbalance of free radicals and antioxidants in the body, which can lead to cell and tissue damage. There are several factors that cause oxidative stress and excess free radical production, including obesity, smoking, alcohol consumption, pollution, and chemicals. These risk factors can activate immune cells, which subsequently produce free radicals and impair normal cells. Consistent accumulation of free radicals and cellular damage causes inflammation, contributing to the development of serious diseases including cancer, diabetes, Alzheimer's, and cardiovascular diseases.
Aging is the progressive loss of tissue and organ function over time. Oxidative stress is believed to be closely related to the aging process because age-associated functional losses are caused by the accumulation of oxidative damage to macromolecules. Although the exact mechanism of oxidative stress-induced aging is unclear, increased free radical production obviously leads to cellular senescence, decreased cell proliferation, and increased inflammatory cytokine production [1]. In addition, several studies have found that with aging, endogenous antioxidant levels, antioxidant enzyme activity, gene expression, and protein levels decrease. This alteration in the antioxidant defense system worsens ROS imbalances and contributes to oxidative-stress-induced aging [1][2][3][4].

Hydrogen Peroxide Exposure Procedure and SP Treatment
Cells were seeded in a 96-well plate at a density of 1 × 10 4 cells/well or in a 6-well plate with a density of 3 × 10 4 cells/well. These cells were allowed to adhere to the bottom of the well. Twenty-four hours later, different concentrations of H 2 O 2 (50, 100, 200, 300, and 400 µM) were added to the wells for 2 h and then removed by changing the culture media. After 24 h, SP was added to each well at a final concentration of 100 nM, and this was repeated 24 h later.

Wst-1 Assay
Ten microliters of water-soluble tetrazolium salt (WST-1; Roche) solution was added to each well at 10% the total volume of the medium, and the 96-well-plate was incubated for 1 h at 37 • C in 5% CO 2 . After incubation, the optical density values were measured at a wavelength of 450 nm using an Enzyme Linked Immunosorbent Assay (ELISA) microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Enzyme Linked Immunosorbent Assay (ELISA)
The total TGF-β1 and VEGF levels in the supernatants were quantified using ELISA kits, according to the manufacturer's instructions. In brief, standards and samples were added to the wells of anti-TGF-β1 or anti-VEGF antibody-coated 96-well plates and incubated for 2 h at room temperature. After discarding the supernatant, a horseradish peroxidase-conjugated secondary antibody was added to each well and incubated again for 2 h at room temperature. After rinsing with washing solution three times, 100 µL of substrate solution was added, followed by the addition of 100 µL of stop solution. The optical density was measured at 450 nm using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Preparation of Cell Extracts and Western Blot Analysis
Cells were rapidly washed with chilled 1× PBS and lysed with 1× lysis buffer/1 mM phenylmethylsulfonyl fluoride (PMSF) solution. Cells were then scraped and supernatants were collected by centrifugation (Rotor radius: 70 mm) at 12,000 rpm for 10 min at 4 • C. Protein concentrations of lysates were determined using the bicinchoninic acid (BCA) assay (Thermo Fisher, Rockford, IL, USA). Ten micrograms of lysates were denatured and electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After blocking with 5% skim milk, membranes were incubated with primary anti-Akt, anti phospho-Akt, anti-GSK-3β, anti-phospho-GSK-3β, or anti-GAPDH antibodies, followed by an anti-IgG horseradish peroxidase-conjugated secondary antibody. The blots were processed using enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK).

Statistical Analysis
All data are presented as the mean ± standard deviation (SD) of more than three independent experiments. p values of less than 0.05 were considered statistically significant. Statistical analysis of the data was carried out using an unpaired, two-tailed Student's t-test. Antioxidants 2020, 9, 978 4 of 10

H 2 O 2 Impairs the Viability and Morphology of ADSCs
Previous reports have induced oxidative stress in vitro by treating cells with H 2 O 2 for 24 h [25,26]. However, ADSCs appear to be particularly susceptible to H 2 O 2 -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 H 2 O 2 for 2 h ( Figure 1A). Antioxidants 2020, 9, 978 4 of 10

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]. 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). 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 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 H 2 O 2 primarily affected cell proliferation and morphology ( Figure 1B). At 24 h after the removal of H 2 O 2 , cell density was significantly different between nontreated and H 2 O 2 -treated conditions. While nontreated cells started to proliferate and became confluent at 72 h, H 2 O 2 -treated ADSCs displayed suppressed cell proliferation. Notably, a distinct difference in cellular density and shape was observed at H 2 O 2 concentrations above 100 µM. High concentrations of H 2 O 2 (i.e., 200, 300, and 400 µM) severely impaired the cellular activity of ADSCs, significantly reducing density at 72 h. H 2 O 2 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). H 2 O 2 treatment decreased cell viability in a dose-dependent Antioxidants 2020, 9, 978 5 of 10 manner ( Figure 1D,E). Consistent with our results on cell shape and density, ADSCs treated with 50 µM H 2 O 2 spontaneously restored their activity within 48 h ( Figure 1E). However, concentrations above 100 µM H 2 O 2 reduced cell viability and this did not return to normal levels. Based on these data, we determined that 100 µM H 2 O 2 induced substantial oxidative damage in ADSCs in vitro. This indicates that transient H 2 O 2 treatment impairs the cellular viability of ADSCs, which might affect cell survival and the function of ADSCs.

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 H 2 O 2 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][16][17].
Antioxidants 2020, 9,978 5 of 10 determined cell viability at 24 and 48 h after oxidative stress ( Figure 1C). H2O2 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 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.

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 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][16][17]. 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  Figure 2C; H 2 O 2 treated: 73.16 ± 1.83%, H 2 O 2 + 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, H 2 O 2 treated: 58.79 ± 1.96%, H 2 O 2 + 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).
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).

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.
Antioxidants 2020, 9, 978 6 of 10 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). 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).

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. 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, H 2 O 2 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, H 2 O 2 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, H 2 O 2 + SP-treated: 312.6 ± 8.1 pg/mL; Figure 3D; 72 h after oxidative stress, H 2 O 2 + 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).
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.

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 H 2 O 2 and then with SP for 20 min ( Figure 4A). ADSCs treated with H 2 O 2 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.
Antioxidants 2020, 9, 978 7 of 10 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 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.

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 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.

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 H 2 O 2 for 2 h. We found that this transient exposure to H 2 O 2 was sufficient to affect ADSC activity and function, but was not completely detrimental to the ADSC population. H 2 O 2 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][35][36]. SP could restore VEGF and TGF-beta production from ADSC with H 2 O 2 . 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.