Polydatin Protects Bone Marrow Stem Cells against Oxidative Injury: Involvement of Nrf 2/ARE Pathways

Abstract

Polydatin, a glucoside of resveratrol, has been reported to possess potent antioxidative effects. In the present study, we aimed to investigate the effects of polydatin in bone marrow-derived mesenchymal stem cells (BMSCs) death caused by hydrogen peroxide (H2O2), imitating the microenvironment surrounding transplanted cells in the injured spinal cord in vitro. In our study, MTT results showed that polydatin effectively prevented the decrease of cell viability caused by H2O2. Hochest 33258, Annexin V-PI, and Western blot assay showed H2O2-induced apoptosis in BMSCs, which was attenuated by polydatin. Further studies indicated that polydatin significantly protects BMSCs against apoptosis due to its antioxidative effects and the regulation of Nrf 2/ARE pathway. Taken together, our results indicate that polydatin could be used in combination with BMSCs for the treatment of spinal cord injury by improving the cell survival and oxidative stress microenvironments.

Figure 1

Representative fields of BMSCs morphologies.

Figure 2

Effects of polydatin on the cell viability of BMSCs exposed to H₂O₂. Cells were pretreated with different concentrations of polydatin for 2 h followed with H₂O₂ (600 μM) for 24 h. (a) Structure of polydatin. (b) Cells were treated with different concentrations of H₂O₂ for 24 h. (c, d) Cell viability was measured by MTT assay and cells were photographed under phase-contrast optics. (A) CT, (B) H₂O₂, (C) polydatin 3 μM + H₂O₂, (D) polydatin 10 μM + H₂O₂, and (E) polydatin 30 μM + H₂O₂. (e) Cell death was measured by LDH assay. Bar graph represents independent experiments, each performed in triplicate. One-way ANOVA followed by Tukey's test. Data are presented as means ± S.D. ^∗ p < 0.05 and ^∗∗ p < 0.01 versus control group. ^# p < 0.05 versus H₂O₂-treated group.

Figure 3

Polydatin attenuated H₂O₂-induced apoptosis in BMSCs. (a) Hochest 33258 staining was applied to detect the nuclear condensation of BMSCs, pretreated with polydatin in presence of H₂O₂. Fluorescence images (A–D) were observed by fluorescence microscope. (A)–(D) represented CT, polydatin, H₂O₂, and H₂O₂ + polydatin group, respectively. (b) BMSCs were pretreated with 30 μM polydatin for 2 h and followed by exposing to H₂O₂ (600 μM) for 12 h. The induction of apoptosis was determined using Annexin V-FITC/PI staining. (c) Quantitative analysis of apoptotic cells in Figure 3(b). (d) The expression of Bcl-2, cleaved caspase-3, and Bax of BMSCs exposed to H₂O₂ with or without polydatin. Data are presented as means ± S.D. ^∗ p < 0.05 versus control group; ^# p < 0.05 versus H₂O₂-treated group.

Figure 4

Polydatin scavenges ROS produced by H₂O₂. (a) ROS production induced by H₂O₂ was detected by H₂DCF-DA staining. (b) Quantitative analysis of DCF fluorescent intensity. (c) The level of GSH was measured using GSH assay kit. The basal contents of GSH in untreated control cells were taken as 100%. Data are collected from 3 independent experiments and presented as means ± S.D. One-way ANOVA followed by Tukey's test. ^∗ p < 0.05 and ^∗∗ p < 0.001 versus control group; ^# p < 0.05 versus H₂O₂-treated group.

Figure 5

Polydatin did not inhibit the proliferation of BMSCs. (a) Proliferation rate of BMSCs was detected by EdU and Hochest 33258 staining. Fluorescence was visualized by a laser-scanning confocal microscope. Scale bar represents 20 μM. (b) Quantitative analysis of the EdU incorporation rate of BMSCs. ^∗ p < 0.05 versus control group.

Figure 6

Polydatin protected BMSCs against H₂O₂-induced cell death partly through Nrf 2/ARE pathway. BMSCs were pretreated with polydatin for 2 h and further exposed to H₂O₂ for 12 h. (a) Effects of polydatin on NQO-1 and the phosphorylation of Nrf 2. (b, c) Quantitative analysis of the blots was shown in panel after being normalized by α-tubulin. (d) Cells were treated with different concentration of brusatol for 24 h. Effects of brusatol on phosphorylation of Nrf 2 were detected by Western blot and (g) the bands were normalized by α-tubulin. (e) Cell viability was tested in the presence of different concentration of brusatol. (h) BMSCs were pretreated with brusatol (100 μM) for 1 h followed by incubating with/without polydatin and H₂O₂ for 24 h. (f) ROS production was detected by H2DCF-DA staining. (b) Quantitative analysis of DCF fluorescent intensity. One-way ANOVA followed by Tukey's test. ^∗ p < 0.05 and ^∗∗ p < 0.001 versus control group; ^# p < 0.05 and ^## p < 0.01 versus H₂O₂-treated group.