五子衍宗丸调控睾丸线粒体融合蛋白2减轻内质网应激改善肥胖大鼠生精功能

Wuzi Yanzong Pill improves spermatogenic function in obese rats by regulating mitochondrial fusion protein 2 and reducing endoplasmic reticulum stress in testis

目的观察五子衍宗丸对高脂饮食诱导肥胖大鼠生精功能的影响,并从睾丸内质网应激角度探讨可能机制。方法30只雄性SD大鼠随机分为对照组、模型组和五子衍宗丸低、中、高剂量(0.5、1.0、2.0g·kg^(-1))组。对照组给予普通饮食,其余组给予高脂饲料喂养,16周后各组ig给予相应剂量药物,连续4周;取附睾进行精子功能分析;血清ELISA分析性激素水平变化;苏木素-伊红染色观察睾丸组织病理改变;透射电镜观察睾丸支持细胞线粒体和内质网结构改变;免疫荧光染色检测睾丸线粒体融合蛋白2(Mfn2)及内质网应激标识蛋白葡萄糖调节蛋白78(GRP78)的定位及表达;Westernblotting检测睾丸组织Mfn2和蛋白激酶R样内质网激酶(PERK)通路相关蛋白相对表达。结果与对照组比较,模型组大鼠体质量明显增加(P<0.01),血清雌二醇水平明显升高(P<0.01),睾酮水平明显降低(P<0.01);睾丸生精细胞排列稀疏,细胞变性;精子活力明显降低(P<0.05),精子畸形率明显升高(P<0.01);睾丸支持细胞内质网和线粒体肿胀,呈空泡状,部分网膜断裂,Mfn2表达明显降低,GRP78荧光表达明显增加;PERK、活化转录因子-4(ATF4)和CHOP蛋白量明显升高(P<0.01)。与模型组比较,五子衍宗丸高剂量组体质量减轻明显(P<0.05);中、高剂量组血清雌二醇水平明显降低(P<0.05、0.01),睾酮水平明显升高(P<0.05、0.01);高剂量组精子活力明显升高(P<0.05),精子畸形率明显降低(P<0.05);睾丸病变减轻,排列较致密,支持细胞内质网和线粒体肿胀改善;Mfn2荧光表达逐渐增强,蛋白相对表达明显增加(P<0.05、0.01),GRP78荧光表达逐渐减弱;低剂量组ATF4蛋白量明显降低(P<0.01),中剂量组PERK、ATF4蛋白量明显降低(P<0.05、0.01),高剂量组PERK、ATF4和CHOP蛋白量均明显降低(P<0.01)。结论五子衍宗丸可改善高脂饮食诱导肥胖引起的大鼠生精障碍,其机制可能与调控睾丸支持细胞Mfn2表达,减轻内质网应激有关。

Objective To observe the effect of Wuzi Yanzong Pill(WYP)on spermatogenic function in obese rats induced by high fat diet,and to explore the possible mechanism from ERS in testis.Methods Totally 30 male SD rats were divided into control group,model group,low,middle and high dose(0.5,1.0,and 2.0g·kg^(-1))groups of WYP randomly.The control group was fed with normal diet,the rest were fed with high-fat diet.After 16 weeks,the corresponding doses of drugs in each group were gavaged for four weeks continuously.the rats were dissected,and sperms in comepididymis were collected for the function analysis.Serum ELISA was used to analyze the changes of sex hormone levels.The pathological changes of testis were observed by hematoxylin-eosin staining.Transmission electron microscopy(TEM)was used to observe the structural changes of the mitochondria and endoplasmic reticulum in sertoli cells.The localization and expression of Mfn2 and GRP78 in testis were detected by immunofluorescencestaining.The expression of Mfn2 and PERK pathway related proteins in testis were detected by Western blotting.Results Compared with control group,the model group rats showed a significant increase in body weight(P<0.01),a marked increase in serum estradiol level(P<0.01),and a marked decrease in serum testosterone level(P<0.01);the arrangement of spermatogenic cells in the testes was sparse,and the cells were degenerated;the sperm motility was significantly lower(P<0.05),and the sperm malformation rate was markedly increased(P<0.01);the rough endoplasmic reticulum and mitochondria in the Leydig cells were swollen and hollow,some of the network was broken,the expression of Mfn2 was markedly reduced,and the GRP78 fluorescence expression was markedly increased;the protein levels of PERK,activated transcription factor-4(ATF4),and CHOP were markedly increased(P<0.01).Compared with the model group,the high-dose group of Wuzi Yanzong Pill showed a significant reduction in body weight(P<0.05);the serum estradiol level was markedly lower in the middle and high-dose groups(P<0.05,0.01),and the serum testosterone level was markedly higher(P<0.05,0.01);the sperm motility was significantly improved in the high-dose group(P<0.05),and the sperm malformation rate was markedly reduced(P<0.05);the pathological changes in the testes were alleviated,the arrangement was denser,and the rough endoplasmic reticulum and mitochondria in the Leydig cells were improved;the fluorescence expression of Mfn2 gradually increased,and the relative protein expression was markedly increased(P<0.05,0.01),while the GRP78 fluorescence expression gradually decreased;the ATF4 protein level was markedly reduced in the low-dose group(P<0.01),the PERK and ATF4 protein levels were markedly reduced in the middle-dose group(P<0.05,0.01),and the PERK,ATF4,and CHOP protein levels were all markedly.Conclusion Wuzi Yanzong Pill can ameliorate spermatogenesis disorder induced by high fat diet in rats,which may be related to the regulation the expression of Mfn2 and alleviate the endoplasmic reticulum stress in sertoli cells.