白藜芦醇对Gc-1 spg细胞氧化应激损伤的作用研究

Effect of resveratrol on oxidative stress injury of Gc-1 spg cells

摘要:

目的 观察白藜芦醇（RES）对过氧化氢H₂O₂诱导的小鼠精原细胞（Gc-1 spg）氧化应激损伤的作用。方法 H₂O₂复制Gc-1 spg细胞氧化应激损伤模型，设置空白组、H₂O₂组（800 μmol/L）、H₂O₂ +5 μmol/L RES组、H₂O₂ +10 μmol/L RES组和H₂O₂+15 μmol/L RES组。检测超氧化物歧化酶（SOD）、丙二醛（MDA）、乳酸脱氢酶（LDH）和谷胱甘肽过氧化物酶（GSH-Px）水平，CCK-8法检测细胞活力，JC-1试剂盒检测线粒体膜电位，Mito Tracker^?? Green FM试剂盒检测活细胞线粒体水平，Hoechst 33342染色检测细胞核凋亡率，Western blotting检测凋亡蛋白Bax、Bcl-2和Caspase-3表达。结果 浓度为800 μmol/L H₂O₂处理Gc-1 spg细胞6 h时达到实验要求（P <0.05）。与空白组比较，各H₂O₂组细胞活力降低（P <0.05），与H₂O₂组比较，H₂O₂ +5 μmol/L RES组、H₂O₂+10 μmol/L RES组、H₂O₂ +15 μmol/L RES组细胞活力升高（P <0.05）。与空白组比较，各H₂O₂组的GSH-Px和SOD水平降低（P <0.05），LDH和MDA水平升高（P <0.05）；与H₂O₂组比较，H₂O₂ +5 μmol/L RES组、H₂O₂+10 μmol/L RES组、H₂O₂ +15 μmol/L RES组GSH-PX和SOD水平降低（P <0.05），LDH和MDA水平升高（P <0.05）。与空白组比较，H₂O₂组细胞的红/绿荧光比值降低，提示线粒体膜电位降低（P <0.05）；与H₂O₂组比较，H₂O₂+5 μmol/L RES组、H₂O₂ +10 μmol/L RES组、H₂O₂ +15 μmol/L RES组JC-1红/绿荧光比值升高（P <0.05）。与空白组比较，H₂O₂组细胞线粒体数明显减少（P <0.05）；与H₂O₂组比较，H₂O₂ +5 μmol/L RES组、H₂O₂+10 μmol/L RES组、H₂O₂ +15 μmol/L RES组细胞荧光强度升高（P <0.05）。与空白组比较，H₂O₂组大量细胞核皱缩、碎裂，染色程度明显增强，细胞凋亡严重；与H₂O₂组比较，H₂O₂ +5 μmol/L RES组、H₂O₂+10 μmol/L RES组、H₂O₂ +15 μmol/L RES组细胞凋亡率降低（P <0.05）。与空白组比较，H₂O₂组凋亡蛋白Bax和Caspase-3相对表达量增加（P <0.05），Bcl-2相对表达量减少（P <0.05）；与H₂O₂组比较，H₂O₂ +5 μmol/L RES组、H₂O₂ +10 μmol/L RES组、H₂O₂ +15 μmol/L RES组Bax和Caspase-3相对表达量降低（P <0.05），Bcl-2相对表达量增加（P <0.05）。结论 RES对H₂O₂诱导的Gc-1 spg细胞氧化应激损伤具有保护作用，这种保护作用与RES浓度有关。

关键词:白藜芦醇;过氧化氢;Gc-1 spg细胞;氧化应激;线粒体损伤;凋亡

Abstract:

Objective To observe the effect of resveratrol (RES) on oxidative stress injury of mouse spermatogonia (Gc-1 spg) induced by hydrogen peroxide (H₂O₂).Methods Set the blank control group, H₂O₂ group and H₂O₂ + low concentration RES group (5 μmol/L), H₂O₂ + medium concentration RES group (10 μmol/L) and H₂O₂ + high concentration RES group (15 μmol/L), the RES group was treated with RES of different concentrations for 24 h, and the rest groups were treated with H₂O₂ for 6 h except the control group. The levels of oxidative stress indicators SOD, MDA, GSH-Px and LDH were detected, and the cell viability was detected by CCK-8, detection of mitochondrial membrane potential by JC-1, Mito Tracker? Green FM was used to detect the level of mitochondria in living cells, Hoechst 33342 staining for detection of nuclear apoptosis rate and Western blotting was used to detect the expression level of apoptotic proteins Bax, Bcl-2 and Caspase-3.Results Concentration is 800 μmol/L when Gc-1 spg cells were treated with H₂O₂ for 6 h, the experimental requirements were met (P < 0.05). Compared with the control group, H₂O₂ treatment significantly decreased cell viability (P < 0.05), inhibited SOD and GSH-Px activities, increased MDA and LDH levels (P < 0.05), significantly decreased mitochondrial membrane potential (P < 0.05), damaged mitochondria (P < 0.05) and destroy nucleus (P < 0.05), increased the expression levels of Bax and Caspase-3, and decreased the expression level of Bcl-2 (P < 0.05). Below 20 μmol/L RES had no significant effect on cell viability (P > 0.05), partially restored cell viability (P < 0.05), increased SOD and GSH-Px activities, decreased MDA and LDH levels (P < 0.05), increased mitochondrial membrane potential (P < 0.05), reduced mitochondrial damage (P < 0.05) and apoptosis rate, the expression of Bax and Caspase-3 was decreased and the expression of Bcl-2 was increased in a concentration dependent manner (P < 0.05).Conclusion RES can protect Gc-1 spg cells from oxidative stress injury induced by H₂O₂, which is related to the concentration of RES.

Key words:resveratrol;hydrogen peroxide;Gc-1 spg cells;oxidative stress;mitochondrial damage;apoptosis