The Mechanism Of Ferroptosis Involved In Testicular Damage Caused By Varicocele And Its Treatment Strategy

Objective:Ferroptosis is a newly discovered from of programmed cell death,which is widely involved in the occurrence and development of many diseases.However,whether it is involved in the testicular spermatogenesis dysfunction caused by varicocele(VC)is still unclear,and there is no report on drugs targeting ferroptosis to inhibit testicular damage induced by VC.Methods:1)16 7-week-old male SD rats were randomly divided into a sham-operated group and a VC group,with 8 rats in each group.Rats’ testis tissue was isolated after 8 weeks.The structural changes of seminiferous tubules were detected by HE staining,and the alternations of ferroptosis-related markers were detected by transmission electron microscopy,ELISA,Western-Blot and RT-qPCR.2)The TM4 cell injury model was constructed using cobalt chloride(CoCl2),and RSL3,Erastin,Ferrostatin-1,Z-VAD-FMK and Necrosulfonamide were added to the plates.Cell proliferation ability was measured by CCK-8 assay,and the changes of ferroptosis-related markers were detected by immunofluorescence,transmission election microscopy and ELISA.3)Semen samples from 20 VC patients and 20 healthy people were collected,and sperm and seminal plasma were separated.The levels of iron,MD A and GSH in sperm were quantified by ELISA,and their diagnostic ability for VC was calculated.4)The changes of protein expression levels in testis tissue of rats in sham-operated group and VC model group were analyzed by TMT-labeled quantitative protein profiling technology,and the small molecule drugs targeting these differential proteins were analyzed by CMap database.5)The GeneCards database was queried to retrieve the potential therapeutic target genes of VC,and the SwissTargetPrediction database was used to collect the potential target genes of allicin.The intersection genes were taken and then analyzed through the KOBAS database.6)30 7-week-old male SD rats were randomly divided into sham-operated group,VC group and VC treatment group,10 rats in each group.After 60 days of modeling,the VC treatment group was given allicin(20 mg/kg)by gavage for 30 days,and then the rat testis tissue was taken.The changes of seminiferous tubules in rat testis were detected by HE staining,and the levels of ferroptosis markers were detected by ELISA,Western-Blot,transmission electron microscopy and immunofluorescence.7)The ferroptosis TM4 cells were treated with allicin(40μM).Subsequently,the changes of ferroptosis markers were detected by ELISA,and the levels of related pathway proteins were detected by Western-Blot.Results:1)The structure of seminiferous tubules in VC rats is disordered,the number of seminiferous epithelial cells is decreased,the density of mitochondrial bilayer membrane is increased and the mitochondrial cristae is decreased.The apoptosis of VC rats’ testis cells is increased,and the expression of GPX4 and SLC7A11 is decreased.The proliferation ability of TM4 cells treated with CoCl2 was significantly decreased,the level of ferroptosis was increased,and mitochondrial damage was obvious,and these changes could be inhibited by Ferrostatin-1.In the sperm of VC patients,the levels of iron ion,MDA and GSH were significantly altered,and they all showed high diagnostic ability for VC.2)181 proteins were differentially expressed in the testis tissue of rats in the sham operation group and the VC model group,and allicin had the highest targeting ability.Allicin have 8 underlying target genes in VC,which are significantly enriched in the ferroptosis pathway.After allicin treatment,the disorder of seminiferous tubules in testicular tissue of VC rats was significantly improved,the level of testicular ferroptosis was decreased,and mitochondrial damage was alleviated to a certain extent,and the expression level of EGFR among the 8 target genes was significantly decreased.Cell experiments displayed that allicin can inhibit TM4 cell ferroptosis by regulating the EGFR-mTOR signaling pathway.Conclusion:VC can cause ferroptosis in testicular cells of rats,and the ferroptosis markers MDA,GSH and iron ions in sperm have high diagnostic efficacy for VC in human.Allicin inhibits ferroptosis by regulating the EGFR-mTOR signaling pathway,and alleviates the testicular damage caused by VC.