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目的探讨亚慢性摄入酒精诱导大鼠生精细胞凋亡及其分子调控机制。方法 40只健康雄性成年SD大鼠随机分为4组,每组10只,各组每日分别灌胃给予酒精0、3.375、5.625和9.375 ml/kg,连续13周。光学显微镜观察睾丸组织形态学,测定睾丸线粒体中丙二醛(MDA)的含量。原位末端标记(TUNEL)检测睾丸生精细胞凋亡,流式细胞仪检测Bcl-2和Bax蛋白的表达。免疫组化法检测睾丸生精细胞中诱导型一氧化氮合酶(iNOS)的表达。结果光学显微镜观察显示酒精组尤其是9.375 ml/kg组动物睾丸出现生精细胞核固缩、变性等细胞凋亡形态学改变,曲细精管腔中脱落细胞增多;且随剂量增大损伤加重。酒精5.625和9.375 ml/kg组动物睾丸线粒体丙二醛含量明显高于对照组(P<0.05);生精细胞凋亡指数明显升高,Bcl-2表达降低,Bax表达升高,Bcl-2/Bax比值减小,iNOS表达明显增强(与对照组相比,均P<0.05)。结论亚慢性摄入酒精可以诱发生精细胞凋亡,其机制可能与iNOS表达增强,过量NO产生,同时凋亡调控基因Bcl-2表达上调,Bax表达下调有关。
Aim To investigate the apoptosis of spermatogenic cells induced by subchronic alcohol ingestion and its molecular mechanism. Methods Forty healthy adult male Sprague-Dawley rats were randomly divided into 4 groups with 10 rats in each group. The rats in each group were orally administered with 0,3.375, 5.625 and 9.375 ml / kg orally by gavage for 13 consecutive weeks respectively. The morphological changes of testis were observed with light microscope, and the content of malondialdehyde (MDA) in testicular mitochondria was measured. Apoptosis of spermatogenic cells in testis was detected by TUNEL, and the expression of Bcl-2 and Bax protein was detected by flow cytometry. Immunohistochemistry was used to detect the expression of inducible nitric oxide synthase (iNOS) in testicular spermatogenic cells. Results Optical microscopy showed that morphological changes of apoptotic cells such as nucleus pyknosis and degeneration of spermatogenic cells were observed in testis of alcohol group, especially in testis of 9.375 ml / kg group. Exfoliated cells in seminiferous tubules increased in alcohol group, and increased with dose increasing. The content of malondialdehyde in testis mitochondria of alcoholic 5.625 and 9.375 ml / kg groups was significantly higher than that of control group (P <0.05); apoptosis index of germ cell was significantly increased, Bcl-2 expression was decreased, Bax expression was increased, Bcl-2 / Bax ratio decreased, iNOS expression was significantly enhanced (compared with the control group, P <0.05). Conclusion Subchronic alcohol intake can induce the apoptosis of spermatogenic cells. The mechanism may be related to the increase of iNOS expression and excessive NO production, and the up-regulation of Bcl-2 and Bax expression.