Wuzi-Yanzong-Wan(WZYZW)is a classic prescription for male infertility.Our previous investigation has demon-strated that it can inhibit sperm apoplosis via afecting mitochondria,but the underlying mechanisms are unclea...Wuzi-Yanzong-Wan(WZYZW)is a classic prescription for male infertility.Our previous investigation has demon-strated that it can inhibit sperm apoplosis via afecting mitochondria,but the underlying mechanisms are unclear.The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore(mPTP)in mouse spermatocyte cell line(GC-2 cells)opened by atractyloside(ATR).At first,WZYZW-mediated serum was prepared from rats following oral adminis-tration of WZYZW for 7 days.GC-2 cells were divided into control group,model group,positive group,as well as 5%,10%,15%WZYZW-medicated serum group.Cyclosporine A(CsA)was used as a positive control.50 μmol·L^(-1) ATR was added afer drugs in-cubation.Cell viability was asessed using CCK-8.Apoptosis was detected using flow cytometry and TUNEL method.The opening of mPTP and mitochondrial membrane potential(MMP)were dected by Calcein AM and JC-1 fuorescent probe respectively.The mRNA and protein levels of voltage-dependent anion channel I(VDACI),cyelophilin D(CypD),adenine nucleide translocator(ANT),cytochrome C(Cyt C),caspase 3,9 were dected by RT-PCR(real time quantity PCR)and Western blotting respectively.The results demonstrated that mPTP of GC-2 cells was opened alpter 24 hours of ATR treatment,resulting in decreased MMP and increased apoptosis.Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associ ated with increased MMP and decreased apoptosis.Morcover,the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDACI and CypD,Caspase-3,9 and CylC,as well as a increased ra-tio of BclBax.However,ANT was not significantly ffected.Therefore,these findings indicated that WZYZW inhibited mitochondri-al mediated apoptosis by atenuating the opening of mPTP in GC-2 cells.WZYZW-medicated serum inhibited the expressions of VDACI and CypD and increased the expression of Bcl-2,which afected the opening of mPTP and exerted protective and anti-apop-totic ffects on GC-2 cell induced by ATR.展开更多
目的:分析鉴定大鼠口服五子衍宗丸后的血中移行成分,并初步探讨其治疗少弱精子症的网络药理机制。方法:建立UPLC-ESI-LTQ-Orbitrap方法,分析五子衍宗丸的体内成分,鉴定其血中移行成分。随后利用Stitch、DrugBank、OMIM数据库分别获得药...目的:分析鉴定大鼠口服五子衍宗丸后的血中移行成分,并初步探讨其治疗少弱精子症的网络药理机制。方法:建立UPLC-ESI-LTQ-Orbitrap方法,分析五子衍宗丸的体内成分,鉴定其血中移行成分。随后利用Stitch、DrugBank、OMIM数据库分别获得药物入血成分靶标及与少弱精子症相关的靶标信息。采用String数据库和Cytoscape软件构建五子衍宗丸入血成分-入血成分靶标-少弱精子症靶标网络,再根据网络拓扑结构特征值筛选核心靶标并明确其对应的入血成分并通过Systems Dock Web Site对预测结果进行分子对接验证。最后借助DAVID数据库对核心靶标进行生物学功能和KEGG通路富集分析。结果:鉴定了五子衍宗丸大鼠血中移行成分42个,网络药理分析共筛选到五子衍宗丸入血成分20个,核心靶标78个。其治疗少弱精子症可能主要涉及信号转导、分子功能、催化活性、内环境稳态、生物合成及代谢等生物学过程和神经活性配体-受体相互作用、钙信号、甾体激素生物合成、甘氨酸、丝氨酸及苏氨酸代谢等信号通路。结论:本研究初步阐明了五子衍宗丸的潜在药效物质基础,探讨了五子衍宗丸治疗少弱精子症的作用机制,为更进一步深入研究其药效物质及其药理机制提供了有益参考。展开更多
基金This work was supported by the National Natural Science Foundation of China(No.81473674)and Natural Science Foundation of Anhui Provincial Department of Education(No.KJ2020A0386).
文摘Wuzi-Yanzong-Wan(WZYZW)is a classic prescription for male infertility.Our previous investigation has demon-strated that it can inhibit sperm apoplosis via afecting mitochondria,but the underlying mechanisms are unclear.The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore(mPTP)in mouse spermatocyte cell line(GC-2 cells)opened by atractyloside(ATR).At first,WZYZW-mediated serum was prepared from rats following oral adminis-tration of WZYZW for 7 days.GC-2 cells were divided into control group,model group,positive group,as well as 5%,10%,15%WZYZW-medicated serum group.Cyclosporine A(CsA)was used as a positive control.50 μmol·L^(-1) ATR was added afer drugs in-cubation.Cell viability was asessed using CCK-8.Apoptosis was detected using flow cytometry and TUNEL method.The opening of mPTP and mitochondrial membrane potential(MMP)were dected by Calcein AM and JC-1 fuorescent probe respectively.The mRNA and protein levels of voltage-dependent anion channel I(VDACI),cyelophilin D(CypD),adenine nucleide translocator(ANT),cytochrome C(Cyt C),caspase 3,9 were dected by RT-PCR(real time quantity PCR)and Western blotting respectively.The results demonstrated that mPTP of GC-2 cells was opened alpter 24 hours of ATR treatment,resulting in decreased MMP and increased apoptosis.Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associ ated with increased MMP and decreased apoptosis.Morcover,the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDACI and CypD,Caspase-3,9 and CylC,as well as a increased ra-tio of BclBax.However,ANT was not significantly ffected.Therefore,these findings indicated that WZYZW inhibited mitochondri-al mediated apoptosis by atenuating the opening of mPTP in GC-2 cells.WZYZW-medicated serum inhibited the expressions of VDACI and CypD and increased the expression of Bcl-2,which afected the opening of mPTP and exerted protective and anti-apop-totic ffects on GC-2 cell induced by ATR.
文摘目的:分析鉴定大鼠口服五子衍宗丸后的血中移行成分,并初步探讨其治疗少弱精子症的网络药理机制。方法:建立UPLC-ESI-LTQ-Orbitrap方法,分析五子衍宗丸的体内成分,鉴定其血中移行成分。随后利用Stitch、DrugBank、OMIM数据库分别获得药物入血成分靶标及与少弱精子症相关的靶标信息。采用String数据库和Cytoscape软件构建五子衍宗丸入血成分-入血成分靶标-少弱精子症靶标网络,再根据网络拓扑结构特征值筛选核心靶标并明确其对应的入血成分并通过Systems Dock Web Site对预测结果进行分子对接验证。最后借助DAVID数据库对核心靶标进行生物学功能和KEGG通路富集分析。结果:鉴定了五子衍宗丸大鼠血中移行成分42个,网络药理分析共筛选到五子衍宗丸入血成分20个,核心靶标78个。其治疗少弱精子症可能主要涉及信号转导、分子功能、催化活性、内环境稳态、生物合成及代谢等生物学过程和神经活性配体-受体相互作用、钙信号、甾体激素生物合成、甘氨酸、丝氨酸及苏氨酸代谢等信号通路。结论:本研究初步阐明了五子衍宗丸的潜在药效物质基础,探讨了五子衍宗丸治疗少弱精子症的作用机制,为更进一步深入研究其药效物质及其药理机制提供了有益参考。
