Antioxidant effects of Cirsium setidens extract on oxidative stress in human mesenchymal stem cells

Introduction

Mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine due to their potential for self-renewal and multilineage differentiation into bone, cartilage, muscle, ligament, tendon, adipose, and endothelial cells (1). The isolation of MSCs from human donors or patients is relatively easy, and MSC cultures are able to expand rapidly for ≥30 population doublings. In addition, the differentiation of MSCs into various phenotypes is simple in lineage-specific culture conditions. These properties make MSCs attractive candidates for the treatment of various diseases, and >500 clinical trials are being conducted using MSCs to overcome a diverse range of diseases (2).

Despite their beneficial effects, the application of MSCs is limited due to pathophysiological environmental conditions, including oxidative stress, inflammation, low oxygen levels and restricted nutrient supply (3). Various stress conditions trigger reduced proliferation and loss of stemness, and are able to induce senescence, resulting in >99% cell death during the first few days following MSC transplantation (4–7). Therefore, protection against several stressors and optimization of MSC culture conditions are required to produce functional MSCs with high therapeutic efficiency. To address this issue, preconditioning, hypoxic culture, pretreatment and genetic manipulation have been suggested to increase the survival of MSCs (3).

Cirsium setidens is a wild perennial herb that possesses various bioactivities, including antitumor, antioxidant and hepatoprotective effects (8–10). C. setidens, which is a bioactive flavonoid, has previously been used to treat hemostasis, hematemesis, hematuria and hypertension (11). Although C. setidens exerts various biological activities, there is no evidence regarding the protective effects of C. setidens against oxidative stress in MSCs. The present study aimed to assess the effects of C. setidens on ROS-induced oxidative stress in MSCs, and to elucidate the mechanism underlying its anti-apoptotic effects against oxidative stress.

Materials and methods

MSCs culture conditions

Human adipose tissue-derived MSCs were obtained from the American Type Culture Collection (Manassas, VA, USA), and were confirmed to be pathogen- and mycoplasma-free. The supplier certified that the MSCs expressed specific cell surface markers [cluster of differentiation (CD)73 and CD105, but not CD31], and had adipogenic and osteogenic differentiation potential when cultured with specific differentiation media. MSCs were cultured in α-minimum essential medium (α-MEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin. MSC cultures were grown in a 5% CO2 humidified incubator at 37°C.

Preparation of C. setidens water extract

C. setidens was collected from the Jeongseon-Gondre Farming Association Corporation (Jeongseon-gun, South Korea). C. setidens was extracted by suspending 100 g into 5 L of hot distilled water for 3 h. The water extract was filtered to remove plant particles through filter paper and was then concentrated in a vacuum under reduced pressure and lyophilized using a freeze dryer for three days in order to fully dry the sample. In total, 19.2 g C. setidens extracted powder was recovered and was maintained at −20°C. The powder was then dissolved in phosphate buffer (pH 7.4, 20 mg/ml) and was stored at −80°C.

High-performance liquid chromatography (HPLC)

A Shimadzu LC-20A (Shimadzu Corporation, Kyoto, Japan), consisting of two LC-20 AD Pumps, a DGU-20A3 vacuum degasser, a SPD-M20A photodiode array detector (PDA) and a CTO 20A Autosampler, was employed. The chromatographic analysis for determination of pectolinarin was carried out using a Luna 5 u C18 100A column (250×4.6 mm; Phenomenex, Inc., Torrance, CA, USA). The mobile phase consisted of solvent A (0.1% formic acid in water) and 100% acetonitrile (solvent B). The solvent gradient elution conditions were 5% (B) for 0–5 min, 5–60% (B) for 5–22 min, 60–60% (B) for 22–24 min and 60-5% (B) for 24–30 min at a flow-rate of 1.0 ml/min, and the column oven was operated at 40°C throughout the study. Detection was conducted with different wavelengths of 273 nm. Injection volume of the sample solutions was 10 µl. Peak identity was confirmed by comparison of spectra obtained from the PDA detector. A spectrum of pectolinarin (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) and C. setidens water extract were compared and analyzed using PDA, at a wavelength of 200–400 nm. The data of chromatogram and spectrum were stored and displayed on a computer.

Chemical treatment of MSCs

MSCs were washed twice with phosphate-buffered saline, and the medium was replaced with fresh α-MEM supplemented with 10% FBS. To assess cell viability, MSCs were pretreated with C. setidens (100 µg/ml or 200 µg/ml) at 37°C for 30 min, and were then treated with hydrogen peroxide (H2O2; 200 µM) for the indicated durations (0, 4, 6 and 8 h). To investigate various cell signaling pathways, MSCs were treated with H2O2 (200 µM) for the indicated duration (0, 15, 30, 60 and 120 min). MSCs were treated with C. setidens (100 µg/ml) for 30 min and were then treated with H2O2 (200 µM) for 120 min.

Cell viability assay

Subconfluent, exponentially growing MSCs were incubated in a 96-well plate with C. setidens for various durations. Cell viability was determined using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, which is based on the conversion of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2-tetrazolium to formazan by mitochondrial NAD(P)H-dependent oxidoreductase enzymes. After 4 h formazan levels were quantified by measuring the absorbance at 575 nm using a microplate reader (Tecan Group AG, Männedorf, Switzerland).

Intracellular reactive oxygen species (ROS) assay

CM-H2DCFDA (DCF-DA), which acts as H2O2-sensitive fluorophore, was used to detect the H2O2-induced production of ROS. DCF-DA (10 µM) was added to the cells and incubated for 30 min at room temperature in the dark. The cells were subsequently observed under a laser confocal microscope (magnification, ×600; Fluoview 1000; Olympus Corporation, Tokyo, Japan) with excitation and emission wavelengths of 488 and 515–540 nm, respectively.

Western blot analysis

Total protein was extracted using RIPA Lysis Buffer (Thermo Fisher Scientific, Inc.). Bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.) was used to quantify proteins. The cell lysate (50 µg protein) was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were transferred to nitrocellulose membranes. The membranes were then blocked with 5% skim milk for 1 h at room temperature, and were incubated with the primary antibodies at the dilutions recommended by the supplier. Ataxia telangiectasia mutated (ATM; cat. no. sc-377293), phosphorylated (p)-ATM (cat. no. sc-47739), p38 (cat. no. sc-81621), p-p38 (cat. no. sc-101758), c-Jun N-terminal kinase (JNK; cat. no. sc-7345), p-JNK (cat. no. sc-6254), p53(cat. no. sc-126) and p-p53 (cat. no. sc-101762), B-cell lymphoma 2 (BCL-2;cat. no. sc-7382), BCL-2-associated X protein (BAX; cat. no. sc-6236), cleaved poly (ADP ribose) polymerase-1 (PARP-1; cat. no. sc-56196), cleaved caspase-3 (cat. no. sc-7272) and β-actin (cat. no. sc-47778) primary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All primary antibodies were diluted 1:1,000 in 5% skim milk and incubated with membranes overnight at 4°C, the membranes were washed, and the primary antibodies were detected following incubation with secondary antibodies horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG; cat. no. sc-2004; 1:10,000) or goat anti-mouse IgG (cat. no. sc-2005; 1:10,000; Santa Cruz Biotechnology, Inc.) for 1 h at 37°C. The bands were visualized using enhanced chemiluminescence reagents (Amersham; GE Healthcare Life Sciences, Little Chalfont, UK). The semi-quantification of western blotting bands were used Image J version 1.47 (National Institutes of Health, Bethesda, MD, USA) and compared to β-actin.

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay

A TUNEL assay was performed using the TdT Fluorescein In Situ Apoptosis Detection kit (Trevigen, Gaithersburg, MD, USA). The MSCs were pretreated with C. setidens for 30 min, and were then treated with H2O2 for 8 h. MSCs were labeled according to the manufacturer's instructions. Stained MSCs were visualized under a fluorescent microscope (Carl Zeiss, Oberkochen, Germany).

Statistical analysis

All data are presented as the mean ± standard error of the mean. All experiments were repeated 5 times and were analyzed by one-way analysis of variance, followed by a comparison of the treatment and control groups using the Bonferroni-Dunn test using SPSS version 19 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

C. setidens exerts protective effects against H2O2-induced cell death in MSCs

Examination of the HPLC chromatograms of C. setidens indicated that the extract contained pectolinarin (Fig. 1A and B). To explore the protective effects of C. setidens against H2O2-induced oxidative stress, MSCs were treated with H2O2 (200 µM) for the indicated time periods (0, 4, 6 and 8 h). The maximal effect of H2O2 on MSC cell death was observed following 8 h of treatment (Fig. 1C). MSCs were pretreated with C. setidens (0, 100 or 200 µg/ml) for 30 min and were then treated with H2O2 for 8 h. Pretreatment with C. setidens significantly increased the viability of MSCs, particularly when used at a concentration of 100 µg/ml (Fig. 1D).

Figure 1 Effects of Cirsium setidens on hydrogen peroxide (H2O2)-induced apoptotic cell death of mesenchymal stem cells (MSCs). (A and B) High-performance liquid chromatography (HPLC) chromatograms of (A) pectolinarin and (B) water extract of C. setidens. (C) MSCs were treated with H2O2 (200 µM) for 0–8 h, and cell viability was subsequently assessed. (D) Following pretreatment with C. setidens (100 and 200 µg/ml) for 30 min, cell viability was investigated following 8 h of exposure to H2O2. Data are presented as the mean ± standard error of the mean. *P<0.05 and **P<0.01 vs. control (untreated).

C. setidens mediates the inhibition of H2O2-induced ROS generation and phosphorylation of stress-associated mitogen-activated protein kinases (MAPKs)

To investigate the inhibitory effects of C. setidens on ROS generation in MSCs, DCF-DA was used as an indicator of ROS production, and alterations to intracellular peroxide levels were assessed. Following treatment of MSCs with H2O2 (200 µM), the intracellular ROS levels were markedly increased compared with in the untreated cells. Conversely, pretreatment of MSCs with C. setidens (100 µg/ml) markedly suppressed intracellular ROS levels (Fig. 2). To determine the regulation of stress-associated MAPKs in ROS-induced cell death, MSCs were treated with H2O2 for the indicated time periods (0, 15, 30, 60 and 120 min). H2O2 increased the phosphorylation of p38 and JNK MAPKs in a time-dependent manner (Fig. 3A and B), whereas the phosphorylation of p38 and JNK was significantly decreased following pretreatment with C. setidens (Fig. 3C and D).

Figure 2 Cirsium setidens inhibits intracellular reactive oxygen species (ROS) generation. Mesenchymal stem cells were treated with C. setidens (100 µg/ml) for 30 min prior to hydrogen peroxide (H2O2; 200 µM) treatment; cellular ROS levels were assessed using the ROS-sensitive fluorophore CM-H2DCFDA (DCF-DA). Images were captured under a confocal microscope and are representative of four independent experiments. Scale bar, 200 µm.

Figure 3 Cirsium setidens inhibits the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). (A and B) Mesenchymal stem cells (MSCs) were treated with hydrogen peroxide (H2O2; 200 µM) for 0–120 min, and the levels of phosphorylated (p)-p38 MAPK and p-JNK were assessed by western blot analysis. Bar graphs represent the relative expression levels of p-p38 and p-JNK normalized to total p38 and total JNK, respectively. (C and D) MSCs were treated with C. setidens (100 µg/ml), followed by H2O2, and the levels of p-p38 MAPK and p-JNK were assessed by western blot analysis. Bar graphs represent the relative expression levels of p-p38 and p-JNK normalized to total p38 and total JNK, respectively. Data are presented as the mean ± standard error of the mean. **P<0.01 vs. control (untreated); ##P<0.01 vs. treatment with H2O2 alone. ROD, relative optical density.

Effects of C. setidens on apoptosis regulation in MSCs

To elucidate the involvement of ATM and p53 activation in ROS-induced apoptosis, MSCs were treated with H2O2 for the indicated time periods (0, 15, 30, 60 and 120 min). H2O2 increased the phosphorylation of ATM and p53 in a time-dependent manner (Fig. 4A and B). However, the activation of ATM and p53 was significantly reduced following pretreatment with C. setidens (Fig. 4C and D).

Figure 4 Cirsium setidens inhibits the ataxia telangiectasia mutated (ATM) and p53 pathways. (A and B) Mesenchymal stem cells (MSCs) were treated with hydrogen peroxide (H2O2; 200 µM) for 0–120 min, and the levels of phosphorylated (p)-ATM and p-p53 were assessed by western blot analysis. Bar graphs represent the relative expression levels of p-ATM and p-p53 normalized to total ATM and total p53, respectively. (C and D) MSCs were treated with C. setidens (100 µg/ml), followed by H2O2, and the levels of p-ATM and p-p53 were assessed by western blot analysis. Bar graphs represent the relative expression levels of p-ATM and p-p53 normalized to total ATM and total p53, respectively. Data are presented as the mean ± standard error of the mean. **P<0.01 vs. control (untreated); ##P<0.01 vs. treatment with H2O2 alone. ROD, relative optical density.

Anti-apoptotic effects of C. setidens on the regulation of apoptosis-associated proteins in MSCs

To determine whether pretreatment with C. setidens may affect ROS-induced expression of apoptotic proteins in MSCs, MSCs were treated with H2O2 for the indicated time periods (0, 4, 6 and 8 h). Subsequently, the expression levels of BCL-2 (anti-apoptotic protein) and Bax (proapoptotic protein) were assessed using western blot analysis. H2O2-induced ROS decreased the expression levels of BCL-2 and increased the expression levels of Bax in a time-dependent manner (Fig. 5A); however, pretreatment with C. setidens significantly increased BCL-2 expression and decreased Bax expression (Fig. 5B). To further investigate the regulation of key signaling pathways against oxidative stress, following pretreatment with C. setidens the expression levels of proapoptotic regulators, cleaved PARP-1 and caspase-3, were detected by western blot analysis. H2O2-induced ROS activated cleaved PARP-1 and caspase-3, whereas pretreatment with C. setidens significantly inhibited their expression (Fig. 5C and D). In addition, the results of a TUNEL assay indicated that pretreatment with C. setidens significantly protected against oxidative stress-induced apoptosis of MSCs (Fig. 6A and B).

Figure 5 Effects of Cirsium setidens extract on hydrogen peroxide (H2O2)-induced regulation of apoptotic proteins in mesenchymal stem cells (MSCs). (A) MSCs were treated with 200 µM H2O2 for 0–8 h, and the expression levels of B-cell lymphoma 2 (BCL-2) and BCL-2-associated X protein (Bax) were detected by western blot analysis. Bar graphs represent the relative expression levels of BCL-2 and Bax normalized to β-actin. (B) MSCs were pretreated with C. setidens (100 µg/ml) for 30 min, followed by 200 µM H2O2 (8 h), and the expression levels of BCL-2 and Bax were detected by western blotting. Bar graphs represent the relative expression levels of BCL-2 and Bax normalized to β-actin. (C) MSCs were treated with 200 µM H2O2 for 0–8 h, and the expression levels of cleaved poly (ADP ribose) polymerase-1 (Cl-PARP-1) and cleaved caspase-3 (Cl-Caspase-3) were detected by western blot analysis. Bar graphs represent the relative expression levels of Cl-PARP-1 and Cl-caspase-3 normalized to β-actin. (D) MSCs were pretreated with C. setidens (100 µg/ml) for 30 min, followed by 200 µM H2O2 (8 h), and the expression levels of Cl-PARP-1 and Cl-caspase-3 were detected by western blotting. Bar graphs representsthe relative expression levels of Cl-PARP-1 and Cl-caspase-3 normalized to β-actin. Data are presented as the mean ± standard error of the mean. **P<0.01 vs. control (untreated); #P<0.05, ##P<0.01 vs. treatment with H2O2 alone. ROD, relative optical density.

Figure 6 Effects of Cirsium setidens on hydrogen peroxide (H2O2)-induced apoptosis in mesenchymal stem cells (MSCs). MSCs were pretreated with C. setidens for 30 min, followed by H2O2 (200 µM) for 8 h, and a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was conducted. (A) Representative images of TUNEL-positive cells (green). Scale bar, 100 µm. (B) Bar graph represents the number of TUNEL-positive cells per field. Data are presented as the mean ± standard error of the mean. **P<0.01 vs. control; ##P<0.01 vs. treatment with H2O2 alone.

Discussion

For applications such as preclinical and clinical trials, the cell source for stem cell-based therapy should be abundant, accessible, easy to deliver to the injured site, tested and optimized in suitable animal models, and non-tumorigenic and non-immunogenic (12). Among stem cell sources, MSCs exhibit an extensive differentiation potential and are easy to harvest from several tissues, including bone marrow, adipose tissue, and skeletal muscle (1). In particular, adipose-derived MSCs are an attractive cell source due to their abundance, accessibility, multipotency and loss of immunogenicity (13). However, the survival rate and differentiation of transplanted stem cells in injured sites is limited due to apoptosis caused by the pathophysiological environment (3). Therefore, identification of a strategy for the protection of transplanted MSCs is important for the development of MSCs-based therapy for regenerative medicine. C. setidens is a phenolic and flavonoid compound, which possesses anti-inflammatory, anticancer, anti-mutation, anti-fungal, hepatoprotective and neuroprotective activity, and is capable of immune enhancement (9,10,14–16). In the present study, the HPLC chromatogram detected pectolinarin in the C. setidens extract. Pectolinarin possess anti-inflammatory effects through the inhibition of eicosanoid formation (17). Despite these attractive properties, the protective effects of C. setidens on oxidative injury in MSCs have yet to be fully elucidated.

The present study demonstrated that pretreatment of MSCs with C. setidens inhibited H2O2-induced apoptosis, and reduced ROS generation. Ischemic conditions induce intracellular ROS production, resulting in the activation of inflammation and apoptosis. In addition, oxidative stress activates MAPKs, which contribute to modulation of the stress response (18). MAPK kinase kinases sense the degree of stress-induced cell damage and regulate the MAPK cascade to determine cell fate. Eventually, downstream MAPKs phosphorylate several effectors, including p38 MAPK and JNK, to induce apoptosis (19). H2O2-induced ROS increased the phosphorylation of p38 and JNK; however, pretreatment of MSCs with C. setidens suppressed the phosphorylation of p38 and JNK. These results suggested that pretreatment with C. setidens may inhibit ROS-induced apoptosis through the regulation of stress-associated MAPKs.

ATM is a pivotal regulatory molecule in ROS-induced apoptosis (20), which has a key role in the cellular response to DNA damage, particularly double strand breaks (21). The phosphorylation of ATM has previously been reported to differ between oxidative stress-induced phosphorylation and double strand break-dependent ATM-mediated phosphorylation (22). Treatment with H2O2 induces the phosphorylation of ATM (S1981) and its downstream target p53, but not histone 2AX, a double strand break marker, thus suggesting that ROS-induced ATM activation is independent of the double strand break process (23). The results of the present study revealed that treatment with C. setidens decreased the phosphorylation of ATM and p53 in response to oxidative stress, thus indicating that C. setidens may reduce oxidative stress-induced cell death via the modulation of ATM and p53 activity, as well as MAPK activation.

Apoptosis occurs under pathophysiological conditions via a cascade of cellular events, which involves various apoptosis-associated genes (24). Pathological apoptosis is induced by downregulation of anti-apoptotic proteins, such as BCL-2, or by the expression of proapoptotic proteins, such as Bax (25). In addition, the cleavage of caspase-3 results in DNA fragmentation, cytoskeletal and nuclear protein degradation, and the expression of ligands for phagocytic cell receptors (25). The cleavage of PARP-1 by caspases is one of the first biochemical markers of apoptosis (26). The present study indicated that H2O2 induced-ROS decreased the BCL-2/Bax ratio, and increased the cleavage of caspase-3 and PARP-1. Conversely, pretreatment of MSCs with C. setidens restored the BCL-2/Bax ratio and reduced activation of caspase-3 and PARP-1. These results suggested that C. setidens may inhibit ROS-induced apoptosis via regulation of the apoptotic cascade.

The present study identified the cytoprotective effects of C. setidens on MSCs exposed to oxidative stress. Under oxidative stress conditions, C. setidens protected MSCs from the harmful effects of intracellular ROS via regulation of stress-associated MAPKs, ATM and p53 phosphorylation, and apoptotic signal cascades. These findings suggested that C. setidens may be developed as a cytoprotective agent for use alongside MSCs-based therapy for the treatment of ischemic diseases.

Acknowledgments

The present study was supported by the National Research Foundation (NRF) grant funded by the Korean government (MEST) (grant no. 2011-0009610) and the Ministry of Education (grant. no. 2016R1D1A3B01007727). The funders had no role in study design, data collection or analysis, the decision to publish, or preparation of the manuscript.

References