Effect of Nrf2 overexpression damaged bone marrow mesenchymal stem cells induced by Paraquat

Abstract

Objective To observe the effects of nuclear factor erythroid 2-related factor 2 (Nrf2) overexpression on damaged bone marrow mesenchymal stem cells (BMSCs) induced by paraquat (PQ). Methods BMSCs were randomly (random number) divided into six groups: normal BMSCs group, BMSCs-mCherry (Red flurescent protein) group, BMSCs-Nrf2 group, BMSCs+ PQ group, BMSCs-mCherry+ PQ group and BMSCs-Nrf2+ PQ group. The BMSCs group, BMSCs-mCherry group and BMSCs-Nrf2 group were cultured by medium. The BMSCs+ PQ group, BMSCs-mCherry+ PQ group and BMSCs-Nrf2+ PQ group were cultured by 1.0 mmol/L final concentration PQ. All cells and supernatant were collected at 24 h after culture in medium or in PQ. The nucleus protein level of Nrf2 was measured by Western blot; the cell viability was measured by CCK-8 assay; apoptosis of cells was detected by flow cytometry; the levels of malondialdehyde (MDA) , catalase (CAT) in supernatant were determined by chemical colorimetry; the levels of interleukin (IL) -6, IL-10 and tumor necrosis factor (TNF) -α in supernatants were detected by Enzyme-linked immunosorbent assays (ELISA). One-way analysis of variance (AVOVA) was employed for statistical analysis by using SPSS version 20.0 to compare values among all groups. Results There were no significant differences in all biomarker variables between BMSCs + PQ group and BMSCs-mCherry+ PQ group (P>0.05). Compared with BMSCs group, the nucleus protein level of Nrf2 in BMSCs+ PQ group was higher markedly (P=0.008) ; compared with BMSCs+ PQ group, the nucleus protein level of Nrf2 in BMSCs-Nrf2+ PQ group was also higher (P=0.031). The cell viability was much lower in BMSCs+ PQ group than that in BMSCs group, while the cell apoptosis was much higher in BMSCs+ PQ group (P=0.000) ; compared with BMSCs+ PQ group, the cell viability in BMSCs-Nrf2+ PQ group increased remarkably [(75.20±2.92) % vs. (53.27±2.40) % (P=0.000)], and the cell apoptosis decreased significantly [(16.30±1.73) % vs. (26.43±2.21) % (P=0.000)]. The levels of MDA, IL-6, TNF-α in supernatant were much higher in BMSCs+ PQ group than those in BMSCs group, while the levels of CAT and IL-10 were much lower in BMSCs+ PQ group (P=0.000) ; compared with BMSCs+ PQ group, the MDA, IL-6, TNF-α levels decreased remarkably in BMSCs-Nrf2+ PQ group [MDA: (37.29±1.88) nmol/L vs. (43.88±0.89) nmol/L, P=0.000; IL-6: (52.00±5.87) pg/mL vs. (75.57±11.63) pg/mL, P=0.001; TNF-α: (124.62±2.95) pg/mLvs. (164.51±9.29) pg/mL, P=0. 000], whereas the CAT and IL-10 levels in BMSCs-Nrf2+ PQ group were markedly increased [CAT: (38.45±1.24) U/mL vs. (22. 82±0.63) U/mL, P=0.000; IL-10: (15.59±1.73) pg/mL vs. (6.96±0.93) pg/mL, P=0.000]. Conclusion The Nrf2 gene can increase the nucleus protein level of Nrf2 in BMSCs and up-regulate Nrf2-ARE pathway, which have the obvious effects on protecting BMSCs against oxidative damages. Key words: Nrf2; Paraquat; Bone marrow mesenchymal stem cells; Oxidative stress