Mechanism Of Xu Duan Zhong Zi Formula In The Treatment Of Oligoasthenozoospermia Via P38MAPK/ERK1/2 Pathway

Objective:1.The objective of this part is to generalize the research progress of oligoasthenospermia in traditional Chinese medicine and explore the relationship between kidney essence deficiency and oligoasthenospermia;2.Through network pharmacology research,the key active components,main targets and pathways of Xuduan Zhongzi Formula in the treatment of oligoasthenospermia were explored,and analyse its potential pharmacological mechanisms;3.To explore the effect of Xu Duan Zhong Zi Formula on the proliferation and differentiation of testicular spermatogenic cells in oligoasthenospermia model rats and its possible mechanism of action.Methods:1.Literature research: By searching Chinese databases such as CNKI,VIP and Wanfang,the literature on the treatment of oligoasthenospermia by traditional Chinese medicine in recent years were summarized.In addition,the records of kidney essence deficiency and oligoasthenospermia in previous works were sorted out.2.Network pharmacology study: Gene Cards,OMIM,Pharm Gkb,TTD and Drug Bank databases were utilized to search for disease targets associated with oligoasthenospermia.The intersection targets between drug active ingredients and oligoasthenospermia were identified,followed by GO and KEGG enrichment analysis.This part study aimed to analyze the potential pharmacological mechanism of Xu Duan Zhong Zi Formula in treating oligoasthenospermia from the perspectives of active ingredients,key targets,and key pathways.3.Experimental study: According to the random number table method,44 male SD rats were divided into normal group,model group,levocarnitine group and Xu Duan Zhong Zi Formula group,with 11 rats in each group.The rats in the normal group were fed daily,while those in the other groups received intraperitoneal injections of cyclophosphamide to induce oligoasthenospermia.After successful establishment of the oligoasthenozoospermia model,both the normal and model groups were administered 2ml of 0.9% sodium chloride solution via gavage.The rats in the levocarnitine group received levocarnitine oral liquid at a dose of 100mg/kg/d dissolved in 2ml of 0.9%sodium chloride solution via gavage.The Xu Duan Zhong Zi Formula group was treated with free granules of Xu Duan Zhong Zi Formula(10g/kg/d)dissolved in 2ml of 0.9% sodium chloride solution via gavage,once a day for four weeks.In terms of observation indicators,the sperm concentration and viability of rats were detected respectively,and the testicular tissue structure was observed by HE staining and transmission electron microscopy.Biochemical methods were used to detect the activities of MDA,SOD,and GSH in testicular tissue samples.Additionally,ATP content in testicular tissue was measured,while Western blot analysis was employed to detect Cyt-C and Caspase3 protein expressions within testicular tissue samples.Furthermore,RT-PCR and Western blot were used to detect the expression of p38 MAPK,ERK1 and ERK2.Results:1.Literature research: The occurrence of oligoasthenospermia is closely related to kidney essence deficiency,liver depression and spleen deficiency.The pathogenesis of oligoasthenospermia is mixed with deficiency and excess,and the method of tonifying kidney and producing sperm is often used in the treatment of oligoasthenospermia.2.Network pharmacology study: we obtained 181 active components and286 targets of Xu Duan Zhong Zi Formula,as well as 1317 disease targets,and we also identified 113 drug-disease common targets.KEGG pathway analysis showed that the treatment of oligoasthenospermia with Xu Duan Zhong Zi Formula was mainly related to antioxidant and apoptotic pathways such as PI3K-AKT signaling pathway and MAPK signaling pathway.The main active components of Xu Duan Zhong Zi Formula in the treatment of this disease were kaempferol,wogonin,quercetin and luteolin.The target proteins were MAPK1,AKT1,MAPK14,EGFR,TP53 and NOS3.3.Experimental study:After 4 weeks of treatment,compared with the normal group,the body weight,testicular wet weight,sperm concentration and viability of the model group were significantly decreased(P<0.01).Compared with the model group,the body weight,testicular wet weight,sperm concentration and viability of the rats in the Xu Duan Zhong Zi Formula group increased(P<0.05).Pathological sections showed that compared with the normal group,the number of spermatogenic cells in the seminiferous tubules of the model group decreased,the cell arrangement was disordered,and the number of sperm in the tubules decreased significantly.Compared with the model group,the structure of the seminiferous tubules of the rats in the Xu Duan Zhong Zi Formula group was basically normal,the layers of spermatogenic cells at all levels of the tube wall were basically neat and dense,and a large number of mature sperms were seen in the lumen.Ultra-micro electron microscopy showed that compared with the normal group,the mitochondria of the Leydig cells in the model group were swollen,the nucleus center was lysed,the structure between the supporting cells was blurred,the smooth endoplasmic reticulum was partially collapsed,the Golgi complex was not seen,the cytoplasm of the lumen surface was obviously detached,and the abnormal sperm was scattered in the seminiferous tubules.Compared with the model group,the sub-structure of testicular cells in the Xu Duan Zhong Zi Formula group was basically complete,such as the Golgi complex,endoplasmic reticulum,and mitochondrial morphology were all regular.There were a large number of mature sperm,primary sperm in the lumen of the testis,and the basic structure of seminiferous tubules was complete.In terms of oxidase: compared with the normal group,the activities of SOD and GSH in the model group were significantly decreased,and MDA was significantly increased(P<0.01).Compared with the model group,the activity of SOD and GSH in the Xu Duan Zhong Zi Formula group increased,and the activity of MDA decreased(P<0.05).ATP content in testis: Compared with the normal group,the ATP content in the testis of the model group was significantly decreased(P<0.01);Compared with the model group,the ATP content in the testicular tissue of the Xu Duan Zhong Zi Formula group was significantly increased(P<0.01).Protein and m RNA expression: Compared with the normal group,the expression of Caspase3,Cyt-C,p38 MAPK and ERK1/2 protein in the testis of the model group were significantly increased,and the expression of p38 MAPK m RNA and ERK1 m RNA、ERK2 m RNA were significantly increased(P<0.01).Compared with the model group,the expression of Caspase3 and Cyt-C protein were significantly decreased(P<0.01),the expression of p38 MAPK and ERK1/2 protein were decreased,and the expression of p38 MAPK m RNA,ERK1 m RNA and ERK2 m RNA were decreased(P<0.05).Conclusions:1.The pathogenesis of oligoasthenospermia is mostly mixed with deficiency and excess,mainly manifested as deficiency of kidney essence.Tonifying kidney and producing essence is an important treatment for oligoasthenospermia.2.The key compounds in Xu Duan Zhong Zi Formula are kaempferol,wogonin,quercetin,luteolin and so on,which may be involved in the treatment of oligoasthenospermia by regulating PI3K-AKT,MAPK and other signaling pathways,interfering with the expression of core targets,such as MAPK1,AKT1,MAPK14,EGFR,TP53,NOS3,etc.3.Xu Duan Zhong Zi Formula may be involved in maintaining the homeostasis of the testicular environment and regulating the apoptosis of spermatogenic cells by down-regulating the expression of p38MAPK/ERK1/2pathway and apoptosis-related proteins Caspase3 and Cyt-C,thereby improving sperm quality and achieving the purpose of treating oligoasthenospermia.