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目的以棕榈酸(palmitic acid, PA)诱导小鼠睾丸支持细胞损伤为体外模型,探究睾酮对支持细胞的保护作用。方法体外培养小鼠睾丸支持细胞(TM4)并随机分为3组进行相应处理,分别为对照组(仅溶剂)、PA模型组(PA 1 mmol/L)、PA模型睾酮干预组(PA 1 mmol/L+睾酮2μg/ml)。培养的TM4细胞进行相应处理24 h后,CCK8检测细胞存活率,并使用实时定量PCR和Western blot检测TM4细胞中组成血睾屏障的主要连接分子Occludin和ZO-1的表达;采用试剂盒检测抗氧化酶活性和谷胱甘肽的含量;RT-PCR评价抗氧化因子的mRNA表达。结果与对照组相比,PA模型组TM4细胞存活率显著降低,Occludin mRNA和蛋白水平以及ZO-1 mRNA水平显著下降(P<0.05);与PA模型组相比,睾酮干预后,TM4细胞存活率显著升高,且Occludin的mRNA和蛋白水平以及ZO-1 mRNA表达显著升高(P<0.05)。与对照组相比,PA模型组超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性显著降低,谷胱甘肽(GSH)含量显著下降(P<0.05);与PA模型组相比,睾酮干预后,SOD和CAT活性显著提高,GSH含量显著升高(P<0.05)。与对照组相比,PA模型组抗氧化因子Nrf2、HO-1、CAT和NQO-1的mRNA表达水平显著下降(P<0.05);与PA模型组相比,睾酮干预后Nrf2、HO-1、CAT和NQO-1的mRNA表达均显著升高(P<0.05)。结论 PA可诱导体外培养的睾丸支持细胞(TM4细胞)损伤,睾酮可提高TM4细胞的抗氧化酶活性而行使保护作用。
Abstract：Objective:To explore the protective effects of testosterone on Sertoli cells by using an in vitro injury model of mouse testicular Sertoli cells induced by palmitic acid(PA).Methods:The mice Sertoli cells(TM4 cells)were cultured and randomly divided into 3 groups: the control group(0.1% DMSO),the PA model group(PA 1 mmol/L),and the testosterone+PA group(PA 1 mmol/L+testosterone 2 μg/ml). After TM4 cells were cultured for 24 hours according to the above experimental conditions, CCK8 was used to detect the cell viability and the expression of occludin and ZO-1 were determined by RT-PCR and Western blot. Antioxidant enzyme activity and glutathione content were detected by the kits. The mRNA expression of antioxidant factors was evaluated by RT-PCR.Results:TM4 cell viability was decreased in PA group compared with the control group(P<0.05),and the levels of occludin mRNA and protein and ZO-1 mRNA decreased significantly(P<0.05). Compared with PA model group, the survival rate of TM4 cells increased significantly and the mRNA and protein levels of occludin and the expression of ZO-1 mRNA increased significantly after testosterone intervention(P<0.05). Compared with the control group, the activities of superoxide dismutase(SOD)and catalase(CAT),and the content of glutathione(GSH)in PA model group decreased significantly(P<0.05). Compared with PA model group, the activities of SOD and CAT increased significantly, and the content of GSH increased significantly after testosterone intervention(P<0.05). Compared with the control group, the mRNA expression levels of antioxidant factors Nrf2,HO-1,CAT and NQO-1 in PA model group decreased significantly(P<0.05). Compared with PA model group, the mRNA expressions of Nrf2,HO-1,CAT and NQO-1 increased significantly after testosterone intervention(P<0.05).Conclusions:PA can induce the injury of testicular Sertoli cells(TM4 cells)in vitro, and testosterone exerts protective effects on PA-induced TM4 cell damage by increasing the antioxidant enzyme activity.
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基本信息:
中图分类号:R697.22
引用信息:
[1]穆杨,罗凌波,杨菁.睾酮对棕榈酸诱导的睾丸支持细胞受损的保护作用[J].生殖医学杂志,2021,30(10):1359-1364.
基金信息:
国家自然科学基金青年项目(82001609); 湖北省卫生健康委员会面上项目(WJ2021M146); 中央高校基本科研业务费专项资金青年教师资助项目(2042020kf0089)
2021-10-15
2021-10-15