五子衍宗丸通过下调IL-17通路减少细胞凋亡治疗少弱精子症  

Wuziyanzong Pills reduce apoptosis of oligoasthenozoospermia via downregulating IL-17 pathway

作　　者：毛静 张立新[1] 王文凯 陈霞[1] MAO Jing;ZHANG Lixin;WANG Wenkai;CHEN Xia(Medical College of Nantong University,Nantong 226019,China)

机构地区：[1]南通大学医学院,江苏南通226019

出　　处：《药物评价研究》2024年第7期1540-1555,共16页Drug Evaluation Research

基　　金：南通市科技计划项目(JC2021011)。

摘　　要：目的探究五子衍宗丸(WZYZP)治疗少弱精子症(OAS)的药效与作用机制。方法应用环磷酰胺(CTX)诱导的SD大鼠作为OAS的体内模型,通过ig 0.5、1.5 g·kg^(-1) WZYZP探究其治疗OAS大鼠的体内药效与作用机制。通过全自动精子分析系统检测大鼠精子参数;应用酶联免疫吸附法测定大鼠血清中睾酮的量,通过HE染色法观察给药后OAS大鼠睾丸组织形态学改变。采用网络药理学预测WZYZP治疗OAS的具体靶点。应用酶联免疫吸附法测定大鼠睾丸组织炎症因子白细胞介素1β(IL-1β)、白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)水平;通过TUNEL染色测定大鼠睾丸组织中细胞凋亡情况;免疫组织化学染色检测睾丸组织白细胞介素17(IL-17)表达情况;Western blotting检测Caspase-3、热休克蛋白90AA1(HSP90AA1)、磷酸化核因子κB(p-NF-κB)、核因子κB(NF-κB)、磷酸化细胞外调节蛋白激酶1/2(p-ERK1/2)、细胞外调节蛋白激酶1/2(ERK1/2)、p-p38和p38的表达水平。使用HPLC检测WZYZP中的主要活性成分并与网络药理学所得靶点进行分子对接模拟以探索其具体机制。结果体内实验结果表明,WZYZP可显著改善CTX诱导的OAS大鼠精子参数、血清睾酮水平(P<0.001)和睾丸组织形态学。网络药理学结果表明WZYZP与OAS的关键通路为IL-17,关键靶点为Caspase3、p38、NF-κB、HSP90和ERK1/2。TUNEL染色结果表明,1.5 g·kg^(-1) WZYZP可显著降低OAS大鼠睾丸组织细胞凋亡(P<0.05)。WesternBlotting结果显示,与模型组相比,1.5g·kg^(-1)WZYZP组Caspase-3(P<0.001)和HSP90AA1(P<0.01)的表达水平明显降低,NF-κB(P<0.05)、ERK1/2(P<0.05)和p38(P<0.05)磷酸化水平明显降低。分子对接模拟结果发现,8种主要活性成分(槲皮素、五味子醇甲、金丝桃苷、山柰酚、紫云英苷、五味子甲素、异槲皮素和绿原酸)和IL-17通路5种相关靶点(HSP90AA1、ERK、p38、NF-κB和Caspase3)的结合能均小于-20.92 kJ·mol^(-1),具有较好的结合亲�Objective To investigate the efficacy and mechanism of action of Wuziyanzong Pill(WZYZP)in treatment of oligoasthenozoospermia(OAS).Methods Cyclophosphamide(CTX)-induced SD rats were used as an in vivo model of OAS,and the in vivo efficacy and mechanism of action of WZYZP were investigated by ig 0.5 and 1.5 g·kg^(-1) WZYZP.The sperm parameters of rats were detected by automatic sperm analysis system;the amount of testosterone in serum of rats was measured by enzymelinked immunosorbent assay,and the histomorphometric changes of testes of OAS rats were observed by HE staining after drug administration.Network pharmacology was used to predict the specific targets of WZYZP in the treatment of OAS.The levels of inflammatory factors interleukin 1β(IL-1β),interleukin 6(IL-6)and tumor necrosis factor alpha(TNF-α)in rat testicular tissue were determined by enzyme-linked immunosorbent assay(ELISA);apoptosis in rat testicular tissue was determined by TUNEL staining;the expression of interleukin 17(IL-17)in testicular tissue was detected by immunohistochemical staining;and the expression of cysteine-containing cells in testicular tissue was detected by western blotting.Blotting was performed to detect the expression of Caspase-3,heat shock protein 90AA1(HSP90AA1),phosphorylated nuclear factorκB(p-NF-κB),nuclear factorκB(NF-κB),phosphorylated extracellular regulated protein kinase 1/2(p-ERK1/2),ERK1/2,p-p38 and p38 expression levels.The main active components in WZYZP were detected using HPLC and molecular docking simulations with targets obtained from network pharmacology were performed to explore the specific mechanisms.Results The results of in vivo experiments showed that WZYZP significantly improved sperm parameters,serum testosterone levels(P<0.001)and testicular histomorphometry in CTX-induced OAS rats.The results of network pharmacology showed that the key pathway of WZYZP with OAS was IL-17,and the key targets were Caspase-3,p38,NF-κB,HSP90 and ERK1/2.TUNEL staining results showed that 1.5 g·kg^(-1) WZYZP s

关 键 词：五子衍宗丸 少弱精子症 白细胞介素 细胞凋亡 分子对接 五味子醇甲 金丝桃苷 紫云英苷 五味子甲素 

分 类 号：R965[医药卫生—药理学]