摘要
目的:明确疏肝补肾毓麟汤抑制电压依赖性阴离子通道2(VDAC2)基因甲基化,通过环磷酸腺苷/蛋白激酶A(cAMP/PKA)通路,调节线粒体功能,提高精子活力的作用机制。方法:雄性SD大鼠40只随机分成空白组、模型组、疏肝补肾毓麟汤高、低剂量组及左卡尼汀组,每组8只。采用腺嘌呤(0.05 g·kg;)灌胃14 d,复制少弱精症大鼠模型;疏肝补肾毓麟汤组给予疏肝补肾毓麟汤高、低剂量(32.4,8.1 g·kg;)灌胃;左卡尼汀组予以左卡尼汀(0.27 g·kg;)灌胃。全自动精子分析仪检测精子活力;苏木素-伊红(HE)染色观察睾丸组织病理形态学变化;流式细胞法检测精子线粒体膜电位差;免疫荧光法检测睾丸VDAC2的表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测睾丸组织VDAC2 mRNA表达;亚硫酸氢盐测序法检测睾丸组织VDAC2基因甲基化;酶联免疫吸附测定法(ELISA)检测睾丸组织cAMP表达;蛋白免疫印迹法(Western blot)检测睾丸组织PKA蛋白表达。结果:与空白组比较,模型组精子密度及活动率均显著降低(P<0.01),线粒体膜电位显著增高(P<0.01),睾丸组织VDAC2的mRNA及蛋白,PKA蛋白,cAMP含量均显著降低(P<0.01),VDAC2甲基化程度显著增高(P<0.01);与模型组比较,左卡尼汀组、疏肝补肾毓麟汤高、低剂量组精子密度及活动率、线粒体膜电位均显著升高(P<0.01),睾丸组织VDAC2的mRNA及蛋白,PKA蛋白,cAMP含量均显著升高(P<0.01),VDAC2甲基化程度显著降低(P<0.01);与左卡尼汀组比较,疏肝补肾毓麟汤低剂量组精子密度及活动率、线粒体膜电位均显著降低(P<0.01),睾丸组织VDAC2的mRNA及蛋白,PKA蛋白,cAMP含量表达均显著降低(P<0.01),VDAC2甲基化程度显著升高(P<0.01)。结论:疏肝补肾毓麟汤可能通过抑制VDAC2基因甲基化,增加VDAC2表达,调节cAMP/PKA通路,改变线粒体膜电位增强精子活力。
Objective:To investigate the mechanism of Shugan Bushen Yulin decoction in inhibiting voltage-dependent anion-selective channel protein 2(VDAC2)gene methylation,affecting sperm mitochondria function,and improving sperm motility through the cyclic adenosine monophosphate/protein kinase A(cAMP PKA)pathway.Method:Forty male SD rats were randomly divided into the blank group,model group,highand low-dose Shugan Bushen Yulin decoction groups,and L-carnitine group,with eight rats in each group Adenine(0.05 g·kg;)was administered by gavage for 14 d for inducing oligospermia and asthenospermia.Rats in the Shugan Bushen Yulin decoction groups were treated with intragastric administration of 32.4,8.1 g·kg;Shugan Bushen Yulin decoction,respectively,while those in the L-carnitine group received0.27 g·kg;L-carnitine by gavage.Following the measurement of sperm motility using an automatic sperm analyzer,the pathological changes in testicular tissue were observed by hematoxylin-eosin(HE)staining.Sperm mitochondrial membrane potential was detected by flow cytometry.The expression of VDAC2 in the testicular tissue was determined by immunofluorescence assay.Real-time polymerase chain reaction(Real-time PCR)was conducted for detecting VDAC2 mRNA expression in testicular tissue.The methylation of VDAC2 gene was examined using bisulfite sequencing.The cAMP expression in testicular tissue was detected by enzyme-linked immunosorbent assay(ELISA),and the PKA protein expression in testicular tissue by Western blot.Result:Compared with the blank group,the model group exhibited significantly decreased sperm density and motility(P<0.01),increased mitochondrial membrane potential(P<0.01),down-regulated VDAC2 mRNA and protein expression,PKA protein expression,and cAMP content in testicular tissue(P<0.01),and elevated VDAC2gene methylation(P<0.01).Compared with the model group,L-carnitine and Shugan Bushen Yulin decoction at the high and low doses all remarkably increased the sperm density and motility and mitochondrial membrane potential(P<
作者
萧闵
王威
魏巍
周艳艳
陈曙辉
姜兴宇
赵敏
曹继刚
XIAO Min;WANG Wei;WEI Wei;ZHOU Yan-yan;CHEN Shu-hui;JIANG Xing-yu;ZHAO Min;CAO Ji-gang(Hubei University of Chinese Medicine,Wuhan 430065,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2021年第23期72-79,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
2021湖北中医药大学科研团队建设项目
第四批全国中医优秀人才研修项目(国中医药人教发[2017]24号)
武汉市中青年中医名医项目(武卫[2019]17号)
湖北省卫计委资助项目(81ZY2019F047)。
关键词
弱精症
疏肝补肾毓麟汤
电压依赖性阴离子通道2
甲基化
线粒体膜电位
asthenospermia
Shugan Bushen Yulin decoction
voltage-dependent anion-selective channel protein 2(VDAC2)
methylation
mitochondrial membrane potential
