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[目的]研究二硫化碳(carbon disulfide,CS2)对大鼠睾丸生殖细胞凋亡线粒体通路的影响及其可能的分子机制,同时观察环孢素A(cyclosporin A,Cs A)对细胞凋亡的干预作用。[方法]选取清洁级雄性SD大鼠48只并随机分为6组:对照组、3个CS2染毒组(50、250、1 250 mg/m3)、干预组[CS2(1 250 mg/m3)+Cs A(12.5 mg/kg)]和Cs A(12.5 mg/kg)组。静式吸入染毒,每天2 h,每周5 d,共10周,对照组仅吸入新鲜空气,Cs A则依据大鼠体重并按相应比例先溶解在牛奶中然后灌胃。染毒结束后取睾丸组织做HE染色,观察其形态学改变;TUNEL荧光法检测大鼠睾丸组织细胞凋亡情况;Western Blot法检测各组细胞色素C(Cyt C)、半胱氨酸天冬氨酸酶原9(Procaspase-9)、Procaspase-3、Bcl-x L抗凋亡和促凋亡蛋白Bad的表达含量。[结果]随着CS2染毒浓度的增加,HE染色大鼠睾丸组织形态损伤情况不断加剧,中、高浓度组的凋亡指数高于对照组(P<0.05),Cs A干预对大鼠睾丸组织形态损伤有一定缓解;与高浓度CS2染毒组相比,干预组的凋亡指数明显下降(P<0.05),但仍高于Cs A组(P<0.05)。CS2染毒组Cyt C、Bad蛋白含量与对照组比较有所增加,而Procaspase-9、Procaspase-3和Bcl-x L蛋白含量则有下降的趋势(P<0.05)。Cs A干预后与高浓度CS2染毒组比较,干预组Cyt C蛋白含量下降(P<0.05),Bcl-x L蛋白含量则提高(P<0.05);Procaspase-9、Procaspase-3和Bad蛋白含量均有不同程度的变化,但与CS2高浓度组相比差异无统计学意义。[结论]CS2染毒可影响细胞凋亡线粒体通路继而诱导大鼠睾丸生殖细胞凋亡,而细胞凋亡线粒体通路相关蛋白的表达可能与Cs A的干预有关。
[Objective] To investigate the effect of carbon disulfide (CS2) on the mitochondrial pathway of germ cell apoptosis in testis and its possible molecular mechanism, and to observe the effect of cyclosporin A (Cs A) on apoptosis . [Methods] Forty-eight male Sprague-Dawley rats were randomly divided into 6 groups: control group, CS2 (50,250,1 250 mg / m3) and CS2 ) + Cs A (12.5 mg / kg)] and Cs A (12.5 mg / kg). The rats were inhaled with static inhalation twice a day for 5 days a week for 10 weeks. In the control group, only fresh air was inhaled. CsA was dissolved in milk according to the body weight of the rats and then orally in the corresponding proportion. Tissue sections were harvested at the end of the treatment for morphological changes. TUNEL staining was used to detect the apoptosis of testicular cells. Western Blot was used to detect the cytochrome C (Cyt C), cysteine aspartate Procaspase-9, Procaspase-3, Bcl-x L and anti-apoptotic protein Bad. [Results] With the increase of CS2 concentration, the morphological damage of testis in HE staining rats was aggravated. The apoptosis index of medium and high concentration groups was higher than that of control group (P <0.05) Compared with CS2 group, the apoptotic index of the intervention group was significantly decreased (P <0.05), but still higher than that of CsA group (P <0.05). The protein levels of Cyt C and Bad in CS2 group increased compared with those in the control group, while the protein levels of Procaspase-9, Procaspase-3 and Bcl-x L decreased (P <0.05). The levels of Cyt C, Procaspase-3 and Bad in the intervention group were significantly lower than those in the CS2-treated group (P <0.05) Content had varying degrees of change, but with CS2 high concentration group compared with no significant difference. [Conclusion] CS2 exposure can affect mitochondrial apoptosis pathway and induce the apoptosis of testicular germ cells in rat testes. The expression of mitochondrial pathway-related proteins may be related to the intervention of CsA.