Resveratrol Attenuates Acute Oxidative Injury in Mouse Leydig Cell TM3 via Silent Information Regulator 1/Uncoupling Protein 2 Signaling Pathway
ZHANG Xiaochun1, CHEN Zhilong1, FANG Juan1, LI Chen1, WU Xiaosong2, YANG Qing1
1. College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China;
2. College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China
Abstract：This experiment was conducted to investigate the protective effect of resveratrol (RES) on oxidative-damaged mouse Leydig cell TM3, and to explore its possible mechanism. Firstly, TM3 cells were treated with different concentrations (0, 150, 200, 250 and 300 μmol/L) of hydrogen peroxide (H2O2), and the optimal concentration of H2O2 was selected to establish the oxidative-damaged cell model. Secondly, the normal cells were treated with different concentrations (0, 2.5, 5.0 and 10.0 μmol/L) of RES for 24 h to select the safe concentration of RES. Finally, the oxidative-damaged cells were treated with safe concentration of RES for 24 h. Cell proliferation was real-time monitored by iCELLigence cell function analyzer during the whole culture process. After the oxidative-damaged cells treated by safe concentration of RES, the reactive oxygen species (ROS) content in cell was detected by 2',7'-dichlorodi-hydrofluorescein diacetate (DCFH-DA) probe method, and the real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting method were used to detect the mRNA and protein relative expression levels of silent information regulator 1 (SIRT1) and uncoupling protein 2 (UCP2) which were the key factors in SIRT1/UCP2 signal pathway, respectively. The results showed as follows:1) the cell viability was extremely significantly reduced after treatment with a concentration of 150 μmol/L or more of H2O2 for 8 h (P<0.01). So, the concentration of 150 μmol/L was selected as the optimal concentration of H2O2 to establish the oxidative-damaged cell model. 2) The cell viability had no significant change when the normal cells treated by 10.0 μmol/L or less RES for 24 h (P>0.05); the cell viability was significantly increased when the oxidative-damaged cells treated by 2.5, 5.0 and 10.0 μmol/L RES for 24 h (P<0.05), and no significant difference among different concentration RES groups (P>0.05). So, the concentration of 5.0 μmol/L was selected as the safe concentration. 3) When the oxidative-damaged cells were treated by 5 μmol/L of RES for 24 h, the content of ROS in cells was extremely significantly increased (P<0.01); the relative expression levels of SIRT1 mRNA and protein were extremely significantly up-regulated (P<0.01), while the relative expression levels of UCP2 mRNA and protein were extremely significantly or significantly down-regulated (P<0.01 or P<0.05). It is concluded that RES can activate the expression of SIRT1 and inhibit the expression of UCP2; meanwhile, UCP2 can attenuate the production of ROS in cells through a negative feedback regulation, thereby inhibiting the oxidative injury of TM3 cells to some extent.
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