Resveratrol Attenuates Acute Oxidative Injury in Mouse Leydig Cell TM3 via Silent Information Regulator 1/Uncoupling Protein 2 Signaling Pathway

ZHANG Xiaochun, CHEN Zhilong, FANG Juan, LI Chen, WU Xiaosong, YANG Qing

doi:10.3969/j.issn.1006-267x.2018.07.023

2018 , Vol. 30 >Issue 7: 2632 - 2640

DOI: https://doi.org/10.3969/j.issn.1006-267x.2018.07.023

Molecular Nutrition

Resveratrol Attenuates Acute Oxidative Injury in Mouse Leydig Cell TM3 via Silent Information Regulator 1/Uncoupling Protein 2 Signaling Pathway

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1. College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128, China;
2. College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China

Received date: 2018-01-03

Online published: 2018-07-20

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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.

Key words： resveratrol; oxidative injury; SIRT1; UCP2; mouse Leydig cell TM3

Cite this article

ZHANG Xiaochun, CHEN Zhilong, FANG Juan, LI Chen, WU Xiaosong, YANG Qing . Resveratrol Attenuates Acute Oxidative Injury in Mouse Leydig Cell TM3 via Silent Information Regulator 1/Uncoupling Protein 2 Signaling Pathway[J]. Chinese Journal of Animal Nutrition, 2018 , 30(7) : 2632 -2640 . DOI: 10.3969/j.issn.1006-267x.2018.07.023

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