| ObjectiveMale infertility is a refractory disease,especially in the contemporary society with the gradual decline of fertility.It is one of the most popular male reproductive research programs.The majority of them are idiopathic male infertility(male infertility caused by unknown factors),which makes physicians and patients uncomfortable.The current therapy of this ailment by Western medicine has several limits,while traditional Chinese medicine offers some advantages.Based on the present state of the disease and the tutor’s effective clinical experience,this study investigated the mechanism of Yishen Huoxue Decoction in the treatment of idiopathic male infertility from network pharmacology,clinical,animal,cell,and other perspectives.(1)Network pharmacology research:using system biology research methods,analyze the effective active components and potential targets of Yishen Huoxue Decoction,select intersection targets of drugs and diseases for correlation enrichment analysis,and then investigate the possible mechanism of Yishen Huoxue Decoction in the treatment of male infertility.(2)Clinical investigation:To investigate the probable mechanism of action of Yishen Huoxue Decoction in the treatment of idiopathic male infertility.(3)Animal experiment:To investigate the impact of Yishen Huoxue Decoction on enhancing spermatogenic function in rats suffering from bisphenol A-induced reproductive harm,as well as its potential molecular biological mechanism.(4)Cell experiment:To investigate the mechanism of damage caused by bisphenol A on TM3 cells as well as the improvement impact of Yishen Huoxue Decoction.(5)This research examines the efficacy and scientific validity of instructor Professor Zhou Shaohu’s use of Tonifying the kidney and stimulating blood circulation in the treatment of idiopathic male infertility using the "prescription and syndrome approach"system.Method(1)Network pharmacological analysis:The TCMSP database was searched for the effective active components and probable target genes of Yishen Huoxue Decoction.The databases OMIM,CTD,DisGeNET,and Genecards were utilized to find male infertility target genes.The intersection of illness and drug target genes was obtained,string tool was used to analyze protein interactions,clusterprofiler tool was used to analyze GO enrichment,and David tool was used to analyze KEGG enrichment.(2)Clinical trial:For 12 weeks,20 patients with idiopathic male infertility were treated with Yishen Huoxue Decoction.Before and after treatment,the associated sperm parameters,redox potential,sperm autophagy,and PI3K/Akt/mTOR pathway related protein levels were compared.(3)Animal experiment:60 male SD rats were randomly assigned to one of six groups:normal control(NC),model control(BPA),low dosage Yishen Huoxue Decoction(YSHXD-L),medium dose Yishen Huoxue Decoction(YSHXD-M),high dose Yishen Huoxue Decoction(YSHXD-H),and positive control(CoQ10).The intervention lasted 8 weeks.Body weight,gonadal index,sperm parameters,histopathological alterations,oxidative stress level,autophagy route,and molecular protein expression associated to the PI3K/Akt/mTOR signaling pathway were all measured.(4)Cell experiment:①The mouse Leydig cell line(TM3 cell line)was treated to various doses of bisphenol A to investigate the effects of bisphenol A on cell viability,reactive oxygen species levels,autophagy,and the expression of PI3K/Akt/mTOR signal pathway associated molecules and proteins.②To investigate the mechanism by which bisphenol A damages TM3 cells,the cells were pretreated with a ROS inhibitor and a PI3K agonist.③The effects of Yishen Huoxue Decoction on TM3 cell viability,reactive oxygen species,autophagy,and the PI3K/Akt/mTOR signal pathway were studied.ResultNetwork pharmacology:(1)Yishen Huoxue Decoction yielded 253 active components and 318 putative target genes.(2)A total of 2136 target genes for male infertility were identified.(3)A total of 206 intersection target genes were identified.PPI analysis yielded ten key target genes:JUN,MAPK3,TP53,AKT1,STAT3,MAPK1,RELA,TNF,FOS,and ESR1.(4)According to GO CC analysis,the top ten are:cyclin dependent protein kinase holoenzyme complex,serine/threonine protein kinase complex,protein kinase complex,transcription regulator complex,transfer complex,transferring phosphorus containing groups,membrane raft,membrane microdomain,membrane region,RNA polymerase II transcription regulator complex,plasma membrane raft.In GO MF analysis,the top ten are DNA binding transcription factor binding,RNA polymerase II specific DNA-binding transcription factor binding,cytokine receptor binding and phosphatase binding,Protein serine/threonine kinase activity,cytokine activity,ubiquitin like protein ligase binding,protein phosphatase binding,signaling receptor activator activity,Growth factor receptor binding.In GO BP analysis,the top ten are:cellular response to chemical stress,response to reactive oxygen species,response to oxidative stress,peptidyl-serine phosphorylation,cellular response to oxidative stress,Peptidyl-serine modification,cellular response to reactive oxygen species,muscle cell proliferation,response to metal ion,regulation of DNA-binding transcription factor activity.According to the analysis of KEGG signaling pathway,the top ten are:pathways in cancer,lipid and atherosclerosis,PI3K-Akt signaling pathway,chemical carcinogenesis receptor activation,human cytomegalovirus infection AGE-RAGE signaling pathway in diabetic complications,Hepatitis B,Kaposi sarcoma-associated herpesvirus infection,Fluid shear stress and atherosclerosis,Proteoglycans in cancer.Clinical study:(1)The clinical efficacy rate after therapy was 85%;(2)Semen volume:before therapy,the semen volume was(3.61 ± 1.08)ml,and after treatment,it was(3.70±1.19)ml.There was no statistically significant difference between them(P>0.05).(3)Sperm concentration:before treatment,sperm concentration was(8.47±2.76)×106ml,after treatment,sperm concentration was(13.42±3.86)×106ml,the change was statistically significant(P<0.01);(4)The forward motile sperm rate was(18.35±9.18)%before treatment and(24.62±11.35)%after treatment(P<0.01);(5)The percentage of normal sperm:the percentage of normal sperm before treatment was(2.13±0.83)%,and the percentage of normal sperm after treatment was(2.78±1.11)%(P<0.01);(6)Redox potential level:the redox potential level was(2.08±1.16)mV/106 sperm/ml before treatment and(1.31±0.40)mV/106 sperm/ml after treatment(P<0.01);(7)Autophagy:following treatment,the relative expression levels of LC3-II and Beclin-1 protein were reduced.(8)PI3K/Akt/mTOR signal pathway expression:following treatment,the expression of PI3K,p-Akt,and p-mTOR protein was increased.(9)Safety analysis:two individuals experienced temporary diarrhea following therapy,but there were no significant adverse effects.Animal experiments:(1)Rat body weight:there was no significant difference in the beginning body weight of the rats in any group(P>0.05);When compared to the NC group,rats in the BPA group gained weight slower.There was a significant difference between the YSHXD-H group and the BPA group after the fourth week(P<0.05),and a significant difference between the CoQ10 group and the BPA group after the sixth week(P<0.05).At the eighth week,there was no significant difference between the low,medium,and high dosage groups as compared to the CoQ10 group(P>0.05).(2)Rat gonadal organ index:Testicular index:as compared to the NC group,the BPA group’s testicular index reduced dramatically(P<0.001).When compared to the BPA group,each dose of traditional Chinese medicine and CoQ10 was considerably greater(P<0.001).There was no significant difference in either dosage group of traditional Chinese medicine compared to the CoQ10 group(P>0.05);Epididymal index:as compared to the NC group,the epididymal index in the BPA group was considerably lower(P<0.01);Each dosage of traditional Chinese medicine and CoQ10 group rose when compared to the BPA group,however the difference was not statistically significant(P>0.05).There was no significant difference in either dosage group of traditional Chinese medicine compared to the CoQ10 group(P>0.05);(3)Rat semen parameters:Rat sperm concentration:as compared to the NC group,the sperm concentration in the BPA,YSHXD-L,and YSHXD-M groups was considerably lower(P<0.01);The sperm concentrations of the YSHXD-H and CoQ10 groups rose considerably(P<0.01);the YSHXD-H group exhibited no significant change(P>0.05)when compared to the BPA group.Rat sperm forward movement rate:compared to the NC group,PR in the BPA group,YSHXD-L group,and YSHXD-M group dropped considerably(P<0.01);When compared to the BPA group,PR in the YSHXD-H and CoQ10 groups rose considerably(P<0.01);There was no significant difference in each dosage group of traditional Chinese medicine compared to the CoQ10 group(P>0.05).Rat total sperm motility:as compared to the NC group,the total sperm motility of the BPA,YSHXD-L,and YSHXD-M groups reduced considerably(P<0.01);The total sperm motility of the YSHXD-H group and the CoQ10 group rose considerably(P<0.01);There was no significant difference in either dosage group of traditional Chinese medicine compared to the CoQ10 group(P>0.05);The rate of normal sperm morphology in rats:as compared to the NC group,the rate of normal sperm morphology in the BPA,YSHXD-L,and YSHXD-M groups was considerably lower(P<0.01);YSHXD-H and CoQ10 groups had considerably greater rates of normal sperm than the BPA group(P<0.01);There was no significant difference in either dosage group of traditional Chinese medicine compared to the CoQ10 group(P>0.05);(4)Serum sex hormones in rats:serum T,FSH,LH,and INH-B concentrations in the BPA group were considerably lower(P<0.01);The amounts of serum T,FSH,LH,and INH-B in the YSHXD-H and CoQ10 groups were considerably greater(P<0.01);YSHXDH demonstrated no significant change(P>0.05)when compared to the CoQ10 group.(5)Rat testis histopathology:in the NC group,the seminiferous tubules were clear,the stromal cell morphology was normal,and the arrangement of spermatogenic cells and Sertoli cells at all levels in the cavity was normal and rich in sperm.The creation of cavities,the integrity of seminiferous tubules,spermatocytes and spermatogonia,and the amount of sperm in the cavity were all considerably decreased in the BPA group.Each dosage of traditional Chinese medicine and CoQ10 group was better as compared to the BPA group.(6)Rat oxidative stress level:ROS immunofluorescence in testicular tissue:ROS level in BPA group was higher than in NC group;The amount of ROS in each dosing group of traditional Chinese medicine and CoQ10 was lower than in the BPA group.Serum SOD,MDA,and cat concentrations:as compared to the NC group,the BPA group’s serum SOD and cat concentrations declined considerably,while the MDA concentration rose dramatically(P<0.01);Serum SOD,MDA,and cat concentrations in the YSHXD-H and CoQ10 groups were considerably higher than in the BPA group(P<0.01);There was no significant difference(P>0.05)between the YSHXD-H and YSHXD-M groups when compared to the CoQ10 group.(7)Autophagy level of testicular tissue:Transmission electron microscopy revealed that the BPA group had a higher number of autophagy bodies and autophagy lysosomes than the NC group.When compared to the BPA group,the number of autophagosomes and lysosomes in the YSHXD-H and CoQ10 groups reduced,and an early isolation membrane with a crescent shape was seen.Western blot:LC3-Ⅱ and Beclin-1 protein levels in the BPA group were substantially higher than in the NC group(P<0.05);In comparison to the BPA group,the levels of LC3II and Beclin-1 protein in the YSHXD-H and CoQ10 groups dropped(P<0.05);in comparison to the CoQ10 group,there was no significant change(P>0.05).(8)PI3K/Akt/mTOR pathway levels in rat testis:when compared to the NC group,the levels of PI3K,p-Akt,and p-mTOR protein in the BPA group were significantly decreased(P<0.05);when compared to the BPA group,the levels of PI3K,p-Akt,and p-mTOR protein in the YSHXD-H group and CoQ10 group were up-regulated(P<0.05);Cell experiment:(1)Cell vitality:bisphenol A suppressed TM3 cell viability,with the degree of inhibition linked with time and concentration;Yishen Huoxue Decoction increased the viability of bisphenol A-induced TM3 cells.(2)Intracellular reactive oxygen species levels:bisphenol A caused intracellular ROS generation,which was positively linked with concentration;Yishen Huoxue Decoction reduced bisphenol A-induced reactive oxygen species formation in TM3 cells.(3)Cell autophagy level:when the concentration of bisphenol A rose,the relative expression level of cell LC3-Ⅱ and Beclin-1 protein increased and was positively connected with the concentration;Yishen Huoxue Decoction inhibited the autophagy level produced by bisphenol A in TM3 cells.(4)PI3K/Akt/mTOR signaling pathway levels:as BPA concentration increased,protein expression levels of cellular PI3K,p-Akt,and p-mTOR were down regulated and positively correlated with concentration;Yishen Huoxue Decoction up-regulated the PI3K/Akt/mTOR pathway level induced by bisphenol A in TM3 cells.(5)Following NAC pretreatment,the protein expression levels of PI3K,p-Akt,and pmTOR in Tm3 cells were up-regulated,while the protein levels of LC3-Ⅱ and Beclin-1 were down-regulated compared to the bisphenol a group,Following 740 Y-P pretreatment,the relative expression levels of LC3-Ⅱ and Beclin-1 protein in cells were down-regulated.Conclusion(1)BPA may harm TM3 cells by causing oxidative stress in the male reproductive system,blocking the PI3K/Akt/mTOR signaling pathway,and triggering autophagy.(2)Yishen Huoxue Decoction has been shown to increase spermatogenic activity in the male reproductive system via the ROS-PI3K/Akt/mTOR-autophagy pathway. |
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