Clinical Meta-analysis And Mechanism Of Guishen Pill In The Treatment Of Diminished Ovarian Reserve

With the rapid changes in the social environment,people’s lifestyles and rhythms are also undergoing tremendous changes,and mental pressure is sharply increasing.The incidence rate of diminished ovarian reserve(DOR)remains high,and the trend of aging is evident.Guishen Pill has better advantages in treating DOR,polycystic ovary syndrome,and other diseases.The combination of traditional Chinese medicine and Western medicine has demonstrated a good effect on the treatment and recovery of DOR.This research aims to investigate the therapeutic effect and underlie the mechanism of Guishen Pill on DOR.The study potentially provides a theoretical basis and diagnostic criteria for the clinical application of Guishen Pill in DOR.Objective:In this study,Meta-analysis of relevant literature was investigated compared the efficacy and safety on the Guishen Pill in treating DOR with hormone therapy.Network pharmacology was utilized to predict the key targets and associated signal pathways of the classic prescription Guishen Pill in treating DOR.Subsequently,the results were further verified by in vivo experiment and underlied mechanism of Guishen Pill in treating DOR.Our study aims to provide a rational and scientific basis for the clinical use of Guishen Pill in treating DOR.Methods:1.Meta-analysis:Literature search of published clinical randomized controlled trials was conducted on the use of Guishen Pill,both alone and in combination with conventional western medicine,in the treatment of DOR from CNKI,Wanfang Data Knowledge Service Platform,VIP Information Chinese Journal Service Platform,PubMed,and Cochrane Library database up to January 12,2023.The literature was then strictly screened based on the inclusion and exclusion criteria,and extracted the relevant information.The Cochrane system evaluation method was employed to evaluate the risk bias,Meta-analysis was conducted using RevMan 5.4.1.2.Network pharmacology:Traditional Chinese Medicine Systems Pharmacology Database(TCMSP)was used to search for the effective chemical components and chemical names of the eight medicines in the Guishen Pill.The predicted targets of the Guishen Pill components were searched using GeneCards and Herb databases.Then,target genes related to DOR disease were searched using GeneCards,OMIM,and Disgent databases.Venn software was used to intersect the drug targets of the Guishen Pill with the disease targets of DOR,and imported the intersection targets into the STRING database.Cytoscape3.8.2 was used to to perform visual analysis and obtain a "drug-disease" protein-protein interaction(PPI)network.The MNC algorithm was used to calculate the top 10 genes,and the intersection targets were input into the interactive visualization platform Hiplot and David database,respectively,to perform GO enrichment analysis and KEGG pathway enrichment analysis.3.In vivo experiment:In this study,the therapeutic effect of Guishen Pill on reproductive damage of Caenorhabditis elegans induced by cyclophosphamide.The LC50 of cyclophosphamide was identified,the number of offspring and fertilized eggs in the Caenorhabditis elegans gonads and body length was detected.The key target genes and pathways of Guishen Pill in treating DOR were analyzed through transcriptomics.Results:1.Meta-analysis:The effective rate of Guishen Pill treatment was higher than that of the control group(Log RR=1.25,95%CI=0.78-1.73).The pregnancy rate within 12 months after treatment was significantly higher in the test group compared to the control group(Log RR=1.08,95%CI=0.67-1.48),and the FSH hormone level on the 2-3 days of the first menstrual cycle was lower in the test group(MD=-3.25,95%CI=-4.27-2.22).However,the Meta-analysis of the E2 hormone level on the 2-3 days of the first menstrual cycle after treatment showed that the 95%CI of the combined effects of the two groups overlapped with the invalid line(MD=-1.04,95%CI=-4.56-2.49),indicating no significant difference between the test and control groups.2.Network pharmacology:Guishen Pill contains 126 effective chemical components,and was associated with 246 targets related to DOR.Of these,181 targets overlapped with those of DOR,indicating that they are possible targets of Guishen Pill for interfering with DOR.10 targets were identified by PPI protein interaction network,including STAT3,PIK3R1,TP53,EP300,MAPK3,MAPK1,PIK3CA,SRC,JUN,and AKT1,which were predicted to be the most effective targets of Guishen Pill for treating DOR.GO and KEGG Enrichment analysis found that the fatty acid metabolism pathway was the potential activated site,and the membrane structure was a crucial component of cells that plays a vital role.3.In vivo experiment results:The determination of the median lethal dose of Caenorhabditis elegans in the CTX group showed that the optimal concentration of CTX was 2000 μg/mL;The number of offspring of C.elegans showed that the CTX group decreased the number of eggs,while the GSW group increased the number of eggs.The optimal concentration for the GSW treatment group was 62.5 mg/mL;The number of fertilized eggs in the uterus of C.elegans showed that CTX decreased the number of fertilized eggs in the uterus,while GSW treatment group strongly increased the number of fertilized eggs in the uterus;The body length measurement results of C.elegans showed that CTX decreased the body length,while GSW significantly increased the body length.Transcriptome sequencing of C.elegans showed that the key driving genes of TOP10 were R12E2.7,col-77,H10E21.4,col-125,col-12,ZK662.2,col-91,col-65,grd-14 and grd-16,respectively.Among them,only grd-14 homologous genes were found in humans,while the other 9 key driving genes were not found homologous genes in humans.Grd-14 is mainly homologous to the human Hh gene and has three subtypes:DHH,IHH,and SHH.In addition,GO enrichment analysis showed that the fatty acid metabolism pathway and the membrane structure are involved in the process.Conclusions:1.Based on the Meta-analysis,the experimental group receiving Guishen Pill plus hormone treatment showed a higher therapeutic effect than the control group receiving hormone treatment alone.The combined effect analysis revealed that compared to conventional Western medicine alone,the combination of Guishen Pill and Western medicine improved the total effective rate and pregnancy rate of DOR treatment,and reduced the serum E2 and FSH levels in patients.2.According to the network pharmacology analysis,the active compounds in Guishen Pill include sitosterol,stigmasterol,quercetin,and isorhamnetin et al.STAT3,PIK3R1,TP53,EP300,MAPK3,MAPK1,PIK3CA,SRC,JUN,and AKT1 are predicted to be the key targets of Guishen Pill in treating DOR,while PI3K-Akt,MAPK,FOXO,HIF-1,and P53 pathways are predicted to be the signaling pathways.GO and KEGG Enrichment analysis found that the membrane structure was a crucial component of cells that plays a vital role.3.The in vivo experiment revealed that the model of decreased reproductive function of C.elegans caused by CTX was successful,and GSW had an effective therapeutic effect on the reproductive damage model of C.elegans caused by CTX.Hh signaling pathway was identified as an important target for GSW treatment of CTX induced DOR models in C.elegans.Regulation of the fatty acid metabolism pathway and the membranous structure are involved in the action of Guishen Pill in treating DOR.