Ergothioneine Attenuates Varicocele-induced Testicular Damage By HSP90AA1 In Rats

Objective:Varicocele(VC)is one of the most important causes of infertility.This study investigates the therapeutic effect and the underlying mechanisms of ergothioneine(EGT)on the testicular damage caused by varicocele.Methods:1)A total of 18 Sprague Dawley(SD)male rats were randomly and averagely divided into three groups,including the sham,VC model,and VC model with EGT treatment(VC+EGT)groups.In the sham group,the left renal vein was only separated.The VC model and the VC+EGT groups were half-ligated left renal vein for 4 weeks.The VC+EGT group was intragastrically administrated with EGT(10 mg/kg)for 4 weeks.2)The cells were divided separately into 3 groups:the NC group,the H2O2 with EGT treatment(H2O2+EGT)group,and the H2O2 model group.The cells of the NC group were only treated with the medium.For the H2O2 model group,cells were treated with H2O2 for 12h,and the cells in the H2O2+EGT group were treated with H2O2 and EGT for 12h.3)The network pharmacology was used to explore the potential targets of EGT in the treatment of VC.HSP90AA1 was screened out as a key gene,and the interaction network of its common target proteins was established by the STRING database.4)Western blotting(WB)and immunohistochemistry(IHC)were used to test the HSP90AA1 level in vivo and in vitro.5)GeneMANIA(http://genemania.org/)was used to visualize gene networks of HSP90AA1 and its related top 100 genes.The R package clusterProfiler(https://guangchuangyu.github.io/clusterProfiler)was utilized in performing gene ontology(GO)and pathway enrichment analysis(Kyoto Encyclopedia of Genes and Genomes(KEGG)).And p-value or q-value≤0.05 will be considered as statistically significant.Results:1)By testing the apoptosis related protein Bax and Bcl-2,we found that EGT could reduce the VC-induced testicular cell apoptosis levels.The HE sections also indicated that EGT could repair the seminiferous tubule’ s structure,shown as the addition of Johnsen scores.And EGT improved the sperm count and progressive motility.In vitro,EGT could also reduce the apoptosis in GC1 and GC2 cells.2)Based on the results of network pharmacology and bioinformatic analysis,we found HSP90AA1 was one of the most important target genes by which EGT treat VC.By testing HSP90AA1 protein level through WB and IHC both in vivo and in vitro,it was found that EGT could protect HSP90AA1 protein in VC.Conclusion:EGT treatment ameliorated the testicular injury in the VC model both in vivo and in vitro by HSP90AA1.