Wuziyanzong Pills reduce apoptosis of oligoasthenozoospermia via down-regulating IL-17 pathway
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 enzyme-linked 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 factor κB(p-NF-κB),nuclear factor κB(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 significantly reduced apoptosis in testicular tissues of OAS rats(P＜0.05);Immunohistochemistry results showed that 1.5 g·kg-1 WZYZP significantly reduced testicular IL-17 expression in OAS rats(P＜0.001);ELISA results showed that IL-1β(P＜0.05)in the testicular tissues of rats in the 1.5 g·kg-1 WZYZP group compared with the model group,IL-6(P＜0.01)and TNF-α(P＜0.01)were significantly lower in the testicular tissues of rats in the 1.5 g·kg-1 WZYZP group compared with the model group;the results of Western Blotting showed that the expression levels of Caspase-3(P＜0.001)and HSP90AA1(P＜0.01)were significantly lower in the 1.5 g·kg-1 WZYZP group compared with the model group,and the expression level of NF-κB(P＜0.05),ERK1/2(P＜0.05)and p38(P＜0.05)phosphorylation levels were significantly reduced.Molecular docking simulations revealed that the binding energies of the eight main active ingredients(quercetin,schisandrin A,chrysin,kaempferol,zingiber officinale,schisandrin,isoquercitrin,and chlorogenic acid)and the five relevant targets of the IL-17 pathway(HSP90AA1,ERK,p38,NF-κB,and Caspase3)were all less than-20.92 kJ·mol-1,with good binding.Conclusion WZYZP can reduce apoptosis by down-regulating IL-17 pathway to treat OAS.
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