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

Abstract

Human mesenchymal stem cells (MSCs) may be used in cell-based therapy to promote neovascularization for the treatment of ischemic diseases. However, high levels of reactive oxygen species (ROS) derived from the pathophysiological ischemic environment induce senescence and apoptosis of MSCs, resulting in reduced functionality and defective neovascularization. Therefore, the present study aimed to determine the protective effects of Cirsium setidens, a natural product, on oxidative stress‑induced apoptosis in MSCs. The present study investigated for the change of ROS levels in MSCs using ROS assays. In addition, cell viability determined by MTT and TUNEL assays. Western blot analysis was performed to investigate the change of apoptosis‑associated proteins in MSCs. Treatment of MSCs with hydrogen peroxide (H2O2; 200 µM) significantly increased intracellular ROS levels and cell death; however, pretreatment with C. setidens (100 µg/ml) suppressed H2O2‑induced ROS generation and increased the survival of MSCs. H2O2‑induced ROS production increased the levels of phosphorylated‑p38 mitogen activated protein kinase, c‑Jun N‑terminal kinase, ataxia telangiectasia mutated and p53; these increases were inhibited by pretreatment with C. setidens. In addition, C. setidens inhibited ROS‑induced apoptosis of MSCs by increasing the expression levels of the anti‑apoptotic protein B‑cell lymphoma 2 (BCL‑2), and decreasing the expression levels of the proapoptotic protein BCL‑2‑associated X protein. These findings indicated that pretreatment of MSCs with C. setidens may prevent ROS‑induced oxidative injury by regulating the oxidative stress‑associated signaling pathway, and suppressing the apoptosis‑associated signal pathway. Therefore, C. setidens may be developed as a beneficial broad‑spectrum agent for enhancing the effectiveness of MSC transplantation in the treatment of ischemic diseases.

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).

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.

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.

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.