亚慢性摄入酒精诱导大鼠生精细胞凋亡及其分子机制研究

Apoptosis of germ cells in rat testis induced by sub-chronic alcohol intake

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。

Objective To explore chronic alcohol consumption-induced germ cells apoptosis in rat testis and its mechanism.Methods 40 healthy Sprague-Dawley adult male rats were randomly divided into four groups. Different doses of alcohol （0,3. 375 ml/kg, 5. 625 ml/kg,9. 375 ml/kg） were administrated to the rats for 13 weeks by oral garage. The histological changes of testicle tissue of rats were examined by light microscopy. The malonaldehyde （MD）k） content in testicular mitochondria was simultaneously determined. The incidence of germ cell apoptosis and the expression of Bcl-2 and Bax were evaluated by TUNEL and Flow Cytometry （ FCM）, respectively. The expression of iNOS in testes was detected by immunohistochemistry. Results Light microscopic evaluation of testes demonstrated the typical morphological changes of germ cell apoptosis including nuclear degeneration, condensation, and increased sloughed cellular element in tubular lumina especially in 9. 375 ml/kg alcohol-treated rats. And Furthermore, the degree of testicular injury was associated with the dose of alcohol. The MDA content of testicular mitochondrial in alcohol 5. 625 ml/kg and 9. 375 ml/kg group was markedly higher than that of control （P 〈 0. O1 ）. Compared with control group , the apoptosis index of alcohol 9. 375 ml/kg group was higher, the expression of iNOS and Bax in the 9. 375 ml/kg alcohol groups increased, the expression of Bcl-2 decreased （P 〈 0.05）. Conclusion Chronic alcohol intake leads to germ cells apoptosis. The mechanism might involve the elevated expression of iNOS and down-regulation of Bcl-2, up-regulation of Bax.