参杞强精颗粒对H2O2诱导小鼠睾丸间质细胞氧化应激损伤的作用

Effects of Shenqi Qiangjing granules against oxidative stress injury by H2O2 in TM3 cells

摘要:

目的 探讨参杞强精颗粒对过氧化氢H₂O₂诱导的小鼠睾丸间质细胞TM3氧化应激损伤的作用。方法 以小鼠睾丸间质细胞为细胞模型，用MTT法检测参杞强精颗粒对正常TM3的细胞毒性作用；以500 μmol/L H₂O₂诱导损伤TM3细胞4 h后复制氧化应激损伤模型，采用CCK-8法检测不同浓度参杞强精颗粒对H₂O₂损伤TM3细胞增殖活力的影响；分别给予参杞强精颗粒（0.4、0.8和1.6 mg/mL）干预处理24 h后，用酶联免疫吸附试验检测细胞上清液中睾酮分泌水平、一氧化氮（NO）、乳酸脱氢酶（LDH）、8-羟基脱氧鸟苷（8-OHdG）含量及谷胱甘肽-S转移酶（GST）的活性；同时检测细胞内活性氧（ROS）、丙二醛（MDA）、过氧化氢酶（CAT）、超氧化物歧化酶（SOD）及总抗氧化能力（TAOC）的含量；实时荧光定量聚合酶链反应（qRT-PCR）检测睾酮合成酶类固醇合成快速调节蛋白（StAR）、胆固醇侧链裂解酶（P450scc）、17β-羟基固醇脱氢酶（17β-HSD） mRNA表达。结果 参杞强精颗粒在浓度为0.05～2.50 mg/mL时对正常TM3细胞无毒性作用。与H₂O₂损伤模型组比较，参杞强精颗粒各剂量组能提高TM3细胞增殖活率（P <0.05）。与H₂O₂模型组比较，参杞强精颗粒各剂量组提高细胞上清液中睾酮水平和NO含量，增加GST活性，降低LDH和8-OHdG含量（P <0.05）。与H₂O₂损伤模型组比较，参杞强精颗粒各剂量组降低细胞内ROS、MDA含量，同时增强CAT、SOD和TAOC的活性（P <0.05）。与H₂O₂模型组比较，参杞强精颗粒各剂量组能提高睾酮合成酶StAR、P450scc、17β-HSD mRNA相对表达量（P <0.05）。结论 参杞强精颗粒对H₂O₂诱导睾丸间质细胞氧化应激损伤具有保护作用，其机制可能通过清除氧自由基、抗氧化应激损伤及促进睾酮合成有关。

Abstract:

Objective To investigate the protective effects and possible mechanism of Shenqi Qiangjing Granules (SQQJG) on oxidative stress injury of mouse leydig cells (TM3) induced by hydrogen peroxide (H₂O₂).Methods Using mouse leydig cells as cell model, determination of the cytotoxicity of SQQJG on normal TM3 cells by MTT method. Establishment of oxidative stress injury model by H₂O₂ (500 μmol/L) induced injury of TM3 cells for 4 h, CCK-8 method was used to detect the proliferation activity of TM3 cells injured by H₂O₂ with different concentrations of SQQJG. And then they were treated with different concentrations of SQQJG (0.4, 0.8 and 1.6 mg/mL) for 24 h, the content of nitric oxide (NO), lactate dehydrogenase (LDH), 8-hydroxydeoxyguanosine (8-OHdG), glutathione-S transferase (GST) and testosterone secretion in the cell supernatant were detected by ELISA, similarly, the levels of reactive oxygen species (ROS), malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (TAOC) in cells were measured by ELISA. The mRNA expression of StAR, P450scc and 17β-HSD were detected by real time fluorescent quantitative PCR.Results Within the concentration range of 0.05 to 2.5 mg/mL, non toxic effect of SQQJG on normal TM3 cells. Compared with the H₂O₂ model group, the survival rate of TM3 cells can be significantly improved in each dose group of SQQJG, SQQJG at 0.4, 0.8 and 1.6 mg/mL concentration could obviously increase the levels of NO, GST and testosterone, and reduce the content of LDH and 8-OHdG. Meanwhile, SQQJG could also enhance levels of CAT, SOD, TAOC, and reduce the expression of ROS and MDA in the cell, and the mRNA expression levels of testosterone synthetase StAR, P450scc, 17β-HSD were increased significantly. The results showed significant difference (P < 0.05 or < 0.01).Conclusion SQQJG has significant protective effect on H₂O₂ induced oxidative stress injury of TM3 cells, the mechanism may be related to scavenging oxygen free radicals, anti oxidative stress injury and promoting testosterone synthesis.