褪黑素对氧化应激损伤小鼠睾丸支持细胞的作用及机制

Effect and Mechanism of Melatonin on Oxidative Damage of Mouse Testicular Sertoli Cells

目的探讨褪黑素对过氧化氢诱导的小鼠睾丸支持细胞TM4细胞氧化损伤的保护作用及相关调控机制。方法体外培养小鼠睾丸支持细胞TM4细胞,采用随机数字表法分为对照组、过氧化氢组、过氧化氢+褪黑素组以及褪黑素组,采用甲氮甲唑蓝法检测褪黑素和过氧化氢对TM4细胞生长的影响以及褪黑素对过氧化氢诱导的TM4细胞氧化损伤的保护作用;采用试剂盒检测细胞内丙二醛、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)的含量;酶联免疫吸附法检测上清液中睾酮和促卵泡生成素(FSH)含量;Western blot检测细胞磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/Akt)信号通路相关分子PI3K、p-PI3K、Akt、p-Akt的蛋白表达。结果对照组、过氧化氢组、过氧化氢+褪黑素组、褪黑素组细胞存活率分别为(1.00±0.17)%、(0.73±0.05)%、(0.92±0.04)%、(1.14±0.08)%,各组比较差异有统计学意义(P<0.05),与对照组相比,过氧化氢组细胞存活率降低(P<0.01);与过氧化氢组相比,过氧化氢+褪黑素组细胞存活率升高(P<0.05)。与对照组相比,过氧化氢组丙二醛表达升高,SOD、CAT、GSH-Px表达降低(P<0.01);与过氧化氢组相比,过氧化氢+褪黑素组丙二醛降低(P<0.05),SOD、CAT、GSH-Px表达升高(P<0.05或P<0.01);与过氧化氢+褪黑素组相比,褪黑素组丙二醛表达降低(P<0.05),SOD、CAT表达升高(P<0.05)。与对照组相比,过氧化氢组睾酮和FSH表达降低(P<0.01);与过氧化氢组相比,过氧化氢+褪黑素组睾酮和FSH表达升高(P<0.01)。与对照组相比,过氧化氢组PI3K、p-PI3K、p-Akt蛋白表达明显下降(P<0.01);与过氧化氢组相比,过氧化氢+褪黑素组PI3K、p-PI3K、p-Akt蛋白表达明显升高(P<0.01);各组Akt蛋白表达比较差异无统计学意义(P>0.05)。结论褪黑素能有效抑制过氧化氢诱导的TM4细胞氧化应激损伤,其机制可能与激活PI3K/Akt信号通路相关。

Objective To investigate the protective effect of melatonin on oxidative damage induced by hydrogen peroxide in mouse testicular Sertoli(TM4)cells.Methods The routinely cultured TM4 cells in vitro were divided into a control group,a hydrogen peroxide group,a hydrogen peroxide+melatonin group and a melatonin group by random number table methods,and MTT was used to detect the effect of melatonin and hydrogen peroxide on TM4 cell growth as well as the protection of melatonin on TM4 cells induced by hydrogen peroxide oxidation damage;test kits were used to detect intracellular malondialdehyde,superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)content;the levels of testosterone and follicle-stimulating hormone(FSH)in the supernatant were detected by enzyme linked immunosorbent assay;the protein expressions of phosphatidylinositol-3-kinase(PI3K),p-PI3K,protein kinase B(PKB/Akt)and p-Akt related molecules in the PI3K/AKt signaling pathway were detected by Western blot.Results The cell survival rates of the control group,the hydrogen peroxide group,the hydrogen peroxide+melatonin group and the melatonin group were(1.00±0.17)%,(0.73±0.05)%,(0.92±0.04)%and(1.14±0.08)%respectively,and the differences among different groups were statistically significant(P<0.05).The survival rate of hydrogen peroxide group was decreased than the control group(P<0.01).Compared with the hydrogen peroxide group,the survival rate of hydrogen peroxide+melatonin group was increased(P<0.05).Compared with the control group,the expression of malondialdehyde was increased in hydrogen peroxide group,and the expression of SOD,CAT and GSH-Px was decreased(P<0.01).Compared with hydrogen peroxide group,malondialdehyde in the hydrogen peroxide+melatonin group was decreased(P<0.05),while SOD,CAT and GSH-Px expression was increased(P<0.05 or P<0.01).Compared with the hydrogen peroxide+melatonin group,the malondialdehyde expression in the melatonin group was decreased(P<0.05),while SOD and CAT expression was increased(P<0.05).Compared with the control group,the expression of testosterone and FSH in the hydrogen peroxide group was decreased(P<0.01).Compared with the hydrogen peroxide group,the expression of testosterone and FSH in the hydrogen peroxide+melatonin group was increased(P<0.01).Compared with the control group,the protein expression of PI3K,p-PI3K and p-Akt in the hydrogen peroxide group was significantly decreased(P<0.01).Compared with the hydrogen peroxide group,the protein expression of PI3K,p-PI3K and p-Akt in the hydrogen peroxide+melatonin group was significantly increased(P<0.01).There was no significant difference in Akt protein expression among all groups(P>0.05).Conclusion Melatonin can effectively inhibit hydrogen peroxide-induced oxidative stress injury of TM4 cells,and its mechanism may be related to the activation of PI3K/Akt signaling pathway.