线粒体凋亡通路参与香烟烟雾暴露所致的大鼠睾丸凋亡

The Mitochondrial Apoptosis Pathway Is Involved in Testicular Apoptosis Induced by Cigarette Smoke Exposure in Rats

近年来,伴随全球吸烟人数剧增,对吸烟所致雄性生殖毒性损伤关注也日益增多。研究表明,长期大量吸烟可致男性睾丸损伤,表现为精液质量下降等,然而其具体机制不详。为探讨线粒体凋亡通路在香烟烟雾暴露所致大鼠睾丸凋亡损伤中的调控作用,选用SPF级SD雄鼠并随机分为高、中、低香烟烟雾暴露组(30支/d、20支/d和10支/d)和对照组,采用静式染毒法处理各组,分别于2、4、6、8和12周后麻醉处死动物,计算睾丸脏器系数,检测睾丸组织病理学改变及细胞凋亡情况。同时检测细胞凋亡激活因子1(cell apoptosis activation factor 1,Apaf-1)、胱天蛋白酶9(caspase-9)及Bcl-2家族中Bim、Bcl-w和Bak在mRNA和蛋白质水平的表达。结果显示,随香烟烟雾暴露剂量增加,暴露时间延长,暴露组大鼠的体重逐渐下降,睾丸组织呈现出明显的病理学改变,睾丸脏器系数逐渐减少。染毒第8周,中、高剂量组及第12周各剂量组睾丸脏器系数较对照组显著减少(P<0.05);睾丸阳性凋亡细胞数也随染毒时间延长而逐渐增多,从第6周起呈现统计学差异(P<0.05);香烟烟雾暴露12周后,睾丸Apaf-1和胱天蛋白酶9表达均较空白组显著上调(P<0.05);促凋亡基因Bak的mRNA转录水平在染毒初期逐渐下调,从第8周开始,其mRNA转录水平逐渐上调;Bak的蛋白质水平上调,在染毒12周时,Bak的mRNA和蛋白质水平均出现显著性上调(P<0.05);抗凋亡基因Bcl-w的mRNA转录表达在染毒第6周开始出现显著性下调(P<0.05),Bcl-w在染毒12周时出现显著性下调(P<0.05),BH3 only基因Bim表达虽有上调趋势,但未出现统计学差异(P>0.05)。以上结果提示,长期大量吸烟可激活线粒体凋亡通路,促使睾丸组织的不可逆性凋亡损伤。这为进一步探讨吸烟所致男性生殖损伤的分子机制提供了科学依据。

In recent years,with the rapid increase of smoking in the world,male reproductive toxicity induced by cigarette smoking(CS)has attracted increasing attention.Studies have shown that long-term heavy smoking can lead to testicular damage in men,resulting in decreased semen quality,but the specific mechanism is still unknown.This study aimed to investigate the regulation mechanism of the Bcl-2 signaling pathway in rat testicular apoptosis induced by CS exposure.SPF male SD rats were randomly divided into high,medium and low CS exposure groups(30,20 and 10 non-filtered cigarettes/day,respectively)and control groups.The rats in each group were treated with static exposure methods and were anesthetized after 2,4,6,8 and 12 weeks-CS exposure,respectively.The testicular organ coefficient was calculated,and the testicular histopathological changes and apoptosis in rats were detected.The mRNA and protein expressions of Apaf-1,Caspase-9,Bim,Bcl-w and Bak in the mitochondrial apoptosis pathway were detected.Results indicated that with the increase of exposure dose and duration,the weight of rats in exposure groups gradually decreased,the testis in exposure groups showed obvious pathological changes,and the testicular organ coefficient gradually decreased.Compared with the control groups,the testicular organ coefficient in the middle,high-dose groups at the 8 th week and all groups at the 12 th week significantly decreased(P<0.05).The number of positive apoptotic cells in the testis increased with the prolongation of exposure time,and it showed statistical difference since six weeks(P<0.05).After 12 weeks of CS exposure,the mRNA transcription and protein expression of Apaf-1 and Caspase-9 in the rat testis were significantly up-regulated compared with control groups(P<0.05).The mRNA expression level of the pro-apoptotic gene Bak gradually decreased in the early stage of exposure,and increased since the eighth week of exposure;the protein expression of Bak was up-regulated,and mRNA transcription and protein expression of Bak were significantly up-regulated at 12-week exposure groups compared with control groups(P<0.05).The mRNA expression of the anti-apoptotic gene Bcl-w was significantly down-regulated since the 6 th week of exposure(P<0.05),the protein of Bcl-w was significantly down-regulated since the 12 th week of exposure(P<0.05).The mRNA and protein expression of BH3-only gene Bim was up-regulated,but there was no statistical difference compared with control groups(P>0.05).In conclusion,long-term heavy smoking activated the mitochondrial apoptosis pathway and induced testicular irreversible apoptotic damage.These results provide a scientific basis for further study of the molecular mechanism of male reproductive injury induced by cigarette smoking.