摘要
目的 明确疏肝补肾毓麟汤畅通自噬流,通过PTEN诱导假定激酶1(PTEN-induced putative kinase 1,PINK1)/帕金蛋白(Parkin)通路,调节线粒体功能,提高精子活力的作用机制。方法 通过随机数字表法将SD大鼠分为正常组、模型组、疏肝补肾毓麟汤高剂量组、疏肝补肾毓麟汤低剂量组、五子衍宗丸对照组。采用腺嘌呤(0.05 g·kg^(-1))灌胃14 d,复制少弱精症大鼠模型。正常组与模型组给予等容0.9%氯化钠溶液,疏肝补肾毓麟汤高、低剂量组给药32.4 g/(kg·d)、8.1 g/(kg·d),五子衍宗丸对照组给药2.6 g/(kg·d)。全自动精子分析仪检测精子活力,透射电镜观察睾丸组织中线粒体自噬的超微结构变化、JC-1线粒体膜电位检测试剂盒与流式细胞仪检测线粒体膜电位水平变化、免疫荧光标记线粒体外膜易位酶20(Translocase of the outer membrane ofmitochondrion 20,TOMM20)与微管相关蛋白1轻链3II(Microtubule-associated protein 1 light chain 3II,LC3II)的共定位、PCR与Western Blot检测睾丸组织PINK1、Parkin、选择性自噬接头蛋白62(Selective autophagy adaptor protein 62,P62)、微管相关蛋白1A/1B-轻链3(LC3II/I)的表达。结果 与正常组相比,模型组的精子活动力下降(P<0.05),线粒体膜电位比例下降(P<0.05),线粒体超微结构出现核周聚集,嵴受损或丢失,除了碎裂外,还有暗基质,以及外膜不规则,并且受损的线粒体与包括线粒体吞噬体在内的线粒体空泡相关,免疫双染中发现LC3II的存在量明显增多,LC3II与TOMM20的双重标记也增加,自噬相关指标PINK1、Parkin、LC3II/I、P62的基因与蛋白表达增加(P<0.05)。与模型组相比,疏肝补肾毓麟汤高剂量组、疏肝补肾毓麟汤低剂量组、对照组的精子活动力升高(P<0.05),线粒体膜电位比例增加(P<0.05),超微结构下正常形态的线粒体比例明显增多,且线粒体溶酶体的数量也减少,免疫双染中发现LC3II的存在量明显减少,LC3II与TOMM20的双重�
Objective To clarify the mechanism of Bushen Shugan Yulin Decoction(疏肝补肾毓麟汤)regulating autophagy flow and improving sperm motility through PTEN-induced putative kinase 1(PINK1)/Parkin pathway.Methods SD rats were divided into normal group,model group,Bushen Shugan Yulin Decoction high dose group,Bushen Shugan Yulin Decoction low dose group and Wuzi Yanzong Pill(五子衍宗丸)control group by random number table method.The rats with oligospermia were administrated with adenine(0.05 g·kg^(-1))for 14 days.Normal group and model group were given isovolume normal saline,Chinese medicine high and low dose groups were given the corresponding Chinese medicine[32.4 g/(kg·d),8.1 g/(kg·d)].The Wuzi Yanzong Pill control group was given Wuzi Yanzong Pill[2.6 g/(kg·d)].Automatic sperm analyzer was used to detect sperm motility.Ultrastructural changes of mitochondrial autophagy in testicular tissue were observed by transmission electron microscopy,and the mitochondrial membrane potential level was detected by JC-1 mitochondrial membrane potential detection kit and flow cytometry,co-location of immunofluorescence labeled mitochondrial outer membrane translocation enzyme 20(TOMM20)and microtubule-associated protein 1A/1B-light chain 3II(LC3II).PCR and Western Blot were used to detect the expressions of PINK1,Parkin,selective autophagy junction protein 62(P62)and microtubule-associated protein 1A/1B-light chain 3(LC3II/I)in testicular tissue.Results Compared with that of the normal group,the sperm motility of the model group decreased(P<0.05)and the proportion of the mitochondrial membrane potential decreased(P<0.05).The mitochondrial ultrastructure gathered a nuclear week,the crest was damaged or lost.In addition to shatter,there was dark matrix as well as the irregular outer membrane.And the damaged mitochondria were associated with mitochondrial vacuoles,including mitochondrial phagosomes.In immune double staining,it was found that the presence of LC3II increased significantly,and the double labeling of LC3I
作者
陈曙辉
萧闵
姜兴宇
蔚文垚
曹继刚
CHEN Shuhui;XIAO Min;JIANG Xingyu;YU Wenyao;CAO Jigang(Hubei Provincial Hospital of Integrated Chinese and Western Medicine,Wuhan 430015,Hubei,China;Hubei University of Chinese Medicine,Wuhan 430065,Hubei,China)
出处
《中华中医药学刊》
CAS
北大核心
2024年第3期178-183,I0022-I0024,共9页
Chinese Archives of Traditional Chinese Medicine
基金
国家自然科学基金项目(82104704)
湖北名师工作室项目(鄂教师函[2022]1号)
武汉中青年名中医项目(武卫[2019]17号)
湖北中医药大学科研团队建设项目(中医校[2021]183号)
湖北中医药大学校级科研平台项目(中医校[2022]80号)
湖北省中西医结合医院院级科研项目(H2023Q008)。
关键词
精子活力低下
线粒体自噬
自噬流
疏肝补肾毓麟汤
low sperm motility
mitochondrial autophagy
autophagy flow
Bushen Shugan Yulin Decoction(疏肝补肾毓麟汤)
