雄蚕益肾方对小鼠睾丸间质细胞氧化应激损伤后铁死亡的影响

Add to Favorite Effect of Xiongcan Yishen Formula on ferroptosis in mouse TM3 Leydig cells after oxidative stress injury

目的:探讨雄蚕益肾方含药血清对H2O2诱导小鼠睾丸间质细胞氧化应激损伤后铁死亡的影响。方法:应用H2O2诱导小鼠TM3睾丸间质细胞建立氧化应激损伤模型,将诱导成模的TM3细胞随机分为模型组、雄蚕益肾方组、铁死亡抑制剂Ferrostatin-1组、Ferrostatin-1联合雄蚕益肾方组,分别用空白血清、20%含药血清、2μmol/L Ferrostatin-1、2μmol/L Ferrostatin-1+20%含药血清干预,另设置对照组(正常TM3细胞+空白血清)。观察各组细胞形态,检测各组细胞分泌的睾酮、超氧化物歧化酶(SOD)、活性氧(ROS)、丙二醛(MDA)、铁蛋白重链1(FTH1)、溶质载体家族7成员11(SLC7A11)、谷胱甘肽(GSH)、谷胱甘肽过氧化物酶4(GPX4)、脂肪酸辅酶A连接酶4(FACL4)、总铁离子及亚铁离子含量。结果:与模型组比较,对照组ROS、MDA、FACL4、总铁和亚铁离子表达均显著降低(P<0.05),睾酮、SOD、谷胱甘肽、FTH1、SLC7A11及GPX4表达均显著升高(P<0.05);雄蚕益肾方组能显著促进TM3细胞分泌睾酮并上调TM3细胞FTH1、SLC7A11、GPX4、GSH和SOD表达(P<0.05),显著下调ROS、MDA、FACL4、总铁离子和亚铁离子表达(P<0.05)。结论:H2O2暴露后可通过氧化应激诱导小鼠TM3睾丸间质细胞发生铁死亡,雄蚕益肾方可能通过激活SLC7A11/GSH/GPX4轴拮抗TM3细胞氧化应激损伤及铁死亡,这可能是雄蚕益肾方治疗男性迟发性性腺功能减退症的潜在作用机制。

Objective:To investigate the effects of Xiongcan Yishen Formula(XYF)on ferroptosis in mouse TM3 Leydig cells after oxidative stress injury(OSI)induced by H_2O_2.Methods:An oxidative stress injury model was established in mouse TM3 Leydig cells using H_2O_2 induction.The modeled TM3 cells were randomly divided into OSI group,XYF group,the ferroptosis inhibitor Ferrostatin-1(F-1)group,and F-1+XYF group,which were respectively intervened with blank serum,20%drug-containing serum,2μmol/L F-1,and 2μmol/L F-1+20%drug-containing serum.A control group(normal TM3 cells+blank serum)was also set up.The morphology of cells in each group was observed,and the levels of testosterone,superoxide dismutase(SOD),reactive oxygen species(ROS),malondialdehyde(MDA),ferritin heavy chain 1(FTH1),solute carrier family 7 member 11(SLC7A11),glutathione(GSH),glutathione peroxidase 4(GPX4),fatty acid CoA ligase 4(FACL4),total iron ions,and ferrous ions were detected.Results:Compared with the model group,the control group showed significantly decreased expression of ROS,MDA,FACL4,total iron,and ferrous ions(P<0.05),and significantly increased levels of testosterone,SOD,GSH,FTH1,SLC7A11,and GPX4(P<0.05).The male silkworm kidney-tonifying formula group significantly promoted testosterone secretion by TM3 cells and upregulated the expression of FTH1,SLC7A11,GPX4,GSH,and SOD in TM3 cells(P<0.05),while significantly downregulating ROS,MDA,FACL4,total iron ions,and ferrous ions(P<0.05).Conclusion:Following H_2O_2 exposure,oxidative stress can induce ferroptosis in mouse TM3 Leydig cells.XYF can antagonize OSI and ferroptosis in TM3 cells by activating the SLC7A11/GSH/GPX4 axis,which may underlie the mechanism of XYF in the treatment of male late-onset hypogonadism.