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

#### **Jeong Seop Park <sup>1</sup> , Jiyuan Piao <sup>2</sup> , Gabee Park <sup>2</sup> and Hyun Sook Hong 1,3,\***


Received: 15 September 2020; Accepted: 11 October 2020; Published: 12 October 2020

**Abstract:** 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. H2O<sup>2</sup> 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 H2O<sup>2</sup> 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.

**Keywords:** Substance-P; adipose-derived stem cells; oxidative stress; paracrine factors

### **1. 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 [1–4].

Oxidative stress and cellular senescence are involved in several acute and chronic pathological processes such as cardiovascular diseases (CVDs), acute and chronic kidney disease (CKD), neurodegenerative diseases (NDs), macular degeneration (MD), biliary diseases, and cancer. Cardiovascular (CV) risk factors are associated with the inflammatory pathway mediated by interleukin (IL)-1α, IL-6, IL-8, and with increased cellular senescence [5]. The associations between oxidative stress, inflammation, and aging produce a vicious cycle whereby chronic ROS production and inflammation feed each other and accelerate aging and age-related morbidity [6]. Thus, modulating oxidative stress-mediated cellular injury may be a fundamental solution to prevent the progression of lethal diseases in aged individuals.

Stem cells facilitate tissue repair in vivo, and many, such as bone marrow, adipose-derived, and umbilical cord blood stem cells are currently being used as novel treatments for various diseases. The application of mesenchymal stem/stromal cells (MSCs) in regenerative medicine has been intensively studied in many clinical trials, as these cells represent a promising source of multipotent adult stem cells for cell therapy and tissue engineering [7–12]. The therapeutic effects of MSCs are generally mediated by various secreted cytokines, growth factors, extracellular matrix proteins, and factors involved in matrix remodeling, as well as different types of extracellular vesicles.

Currently, as alternative of bone marrow stem cells, adipose-derived stem cells (ADSCs) are being applied extensively in the clinic because they can be easily isolated and cause lower donor-site morbidity [12–14]. Aging, oxidative stress, and/or inflammation can affect tissue-resident cells as well as circulating cells, which suggests that stem cells may also be influenced by oxidative stress in vivo. Considering that the aged population is the main population to be treated by stem cell therapy, the restoration of the cellular function of stem cells injured by oxidative stress is likely important.

Substance-P (SP) is an endogenous neuropeptide that interacts with neurokinin receptor 1 (NK-1R). SP has been reported to stimulate cell proliferation and prevent apoptosis under inflammatory or oxidative stress by activating the extracellular signal-regulated kinases 1/2 (ERK 1/2) or Akt and by translocating β-catenin to cell nuclei [15–18]. SP could suppress inflammation to promote tissue repair in severe diseases by modulating the immune cell profile in circulation-associated and lymphoid organs [19–22]. Moreover, SP can recover the cellular activity of senescent stem cells [23,24] and stimulate stem cell mobilization from the bone marrow to the peripheral blood [17].

Considering these functions of SP, we hypothesized that SP would be able to restore stem cells from oxidative stress-induced injury. To explore the potential recovery role of SP in injured stem cells due to oxidative stress, ADSCs were exposed to H2O<sup>2</sup> at various concentrations. Subsequently, SP was added to the damaged ADSCs and the effect of SP was assessed by evaluating cell viability, cell proliferation, paracrine factors, and early signaling molecules.

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

#### *2.1. Materials*

SP and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin/streptomycin, 0.25% trypsin-EDTA solution, and phosphate-buffered saline (PBS) were provided by Welgene (Daegu, Korea). Fetal bovine serum and alpha-MEM were purchased from Gibco (Grand Island, NY, USA). An anti-GAPDH antibody (Abcam, Cambridge, MA, USA), and anti-WST-1 antibody (Roche; Indianapolis, IN, USA) were used in this study. Cell lysis buffer, anti-Akt antibody, anti-phospho-Akt antibody, anti-GSK-3β antibody, and anti-phospho-GSK-3β antibody were purchased from Cell Signaling Technology (Danvers, MA, USA).

#### *2.2. Cell Culture*

Healthy adipose tissues were provided by the Kyung Hee University Hospital Institutional Review Board (eight donors, M6/F2; Seoul, Korea; (IRB# 2016-12-022)). All consents were informed. Fat tissue (two fat tissues (1 × 1 cm) from one donor; total 16 tissues) washed twice with PBS, and treated with

collagenase (GMP grade, Vivagen, Los Angeles, CA, USA) for 1 h at 37 ◦C. Red blood cells and debris were removed using a cell strainer (SPL Life, Pocheon, Korea). The vascular fraction was seeded in α-MEM supplemented with 10% FBS at 37 ◦C with 5% CO2. ADSCs were characterized by analyzing the expression of CD29, CD73, CD105, and CD90 using a fluorescence-activated cell sorting (FACS) Calibur flow cytometer and CELLQuest software (Becton Dickinson, San Jose, CA, USA) (Figure S1).

### *2.3. Hydrogen Peroxide Exposure Procedure and SP Treatment*

Cells were seeded in a 96-well plate at a density of 1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well or in a 6-well plate with a density of 3 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well. These cells were allowed to adhere to the bottom of the well. Twenty-four hours later, different concentrations of H2O<sup>2</sup> (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.

#### *2.4. 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% CO2. 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).

#### *2.5. 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).

#### *2.6. 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).

#### *2.7. 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.
