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 of mitochondrion 20，TOMM20)与微管相关蛋白 1 轻链3Ⅱ(Microtubule-associat-ed 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(Se-lective 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的双重标记减少，自噬相关指标PINK1、Parkin、LC3Ⅱ/Ⅰ、P62的基因与蛋白表达减少(P＜0。05)。与对照组相比，高剂量组的线粒体膜电位比例增加(P＜0。05);疏肝补肾毓麟汤高剂量组、疏肝补肾毓麟汤低剂量组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的双重标记减少，自噬相关指标PINK1、Parkin、LC3Ⅱ/Ⅰ、P62的基因与蛋白表达减少(P＜0。05)。结论 疏肝补肾毓麟汤能改善精子活力低下症模型大鼠的精子活动力，可能是通过提高模型大鼠的线粒体膜电位水平，保护线粒体功能，下调PINK1、Parkin、LC3 Ⅱ/Ⅰ、P62的表达，并畅通自噬流，减轻线粒体的过度自噬来发挥作用。
Bushen Shugan Yulin Decoction(疏肝补肾毓麟汤)Regulating Autophagy Flow and Improving Sperm Vitality through PINK1/Parkin Pathway
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(PINK 1)/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.Ul-trastructural changes of mitochondrial autophagy in testicular tissue were observed by transmission electron microscopy,and the mi-tochondrial 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 propor-tion 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 LC3Ⅱ and TOMM20 also increased.The expres-sions of genes and proteins related to autophagy,such as PINK1,Parkin,LC3Ⅱ/Ⅰ and P62 increased(P＜0.05).Compared with those of the model group,the sperm motility of high dose group,low dose group and the control group increased(P＜0.05),the mi-tochondrial membrane potential increased(P＜0.05),the ultrastructure of mitochondria in the form of normal ratio increased obvi-ously and the number of mitochondria and lysosomes reduced.The immune double staining found LC3Ⅱ significantly reduced,the presence of the double markers of LC3Ⅱ and TOMM20 were decreased,and the gene and protein expressions of autophagy related in-dicators PINK1,Parkin,LC3Ⅱ/Ⅰ and P62 were decreased(P＜0.05).Compared with those of the control group,the proportion of mitochondrial membrane potential in the high dose group was increased(P＜0.05).In the high dose and low dose groups,the pro-portion of mitochondria with normal morphology increased significantly in ultrastructure,and the number of mitochondrial lysosomes also decreased.The presence of LC3Ⅱ was significantly reduced in immunodouble staining,and the double labeling of LC3Ⅱ and TOMM20 decreased.The gene and protein expressions of autophagy related indicators PINK1,Parkin,LC3Ⅱ/Ⅰ and P62 decreased(P＜0.05).Conclusion Bushen Shugan Yulin Decoction can improve the sperm motility in model rats with hyposperm motility,possibly by increasing the level of mitochondrial membrane potential,protecting mitochondrial function,down-regulating the expressions of PINK1,Parkin,LC3 Ⅱ/Ⅰ and P62,and obstructing the autophagy flow and alleviating excessive autophagy of mitochondria.
万方数据
维普
