Protective Role And Potential Mechanisms Of Rapamycin In Testicular Ischemia Reperfusion Injury Treatment

Background:testicular torsion is one of the most common urological emergencies.Ischemia reperfusion（I/R）injury is the main pathological mechanism of testicular torsion.Surgical intervention is the first choice.The success rate of the treatment is the highest within 6 hours of torsion.However,after 12 hours of torsion,the success rate of testicular rescue is reduced to 50%,and then it decreases to<10%after 24 hours.Male infertility and hypogonadism are the sequelae of testicular torsion.Drug therapy may play an important role in promoting the rapid recovery of spermatogenesis and reducing complications after testicular torsion,but there is no strong clinical evidence so far.In recent years,many drugs have been proved to play a protective role in the ischemia-reperfusion injury model in vivo and in vitro,for example,rapamycin.However,the protective effect of rapamycin on testis ischemia-reperfusion injury is still controversial,especially its protective effect on Sertoli cells is not clear.Objective:To investigate whether rapamycin has protective effect on Sertoli cells（TM4）in vitro after ischemia-reperfusion injury,and to further explore whether rapamycin exert protective effect through mTORC1 and mTORC2 complex.Then,the protective role of rapamycin in vivo model would be validated.Method:（1）Make the model.In vitro:in vitro,the hypoxia-reoxygenation（H/R）model induced by cobalt chloride was used to simulate the environment in vivo.The expression of HIF-1α,an hypoxia inducible factor,was detected under different concentrations of cobalt chloride to evaluate the success of hypoxia induction.Then,the function of rapamycin and other drugs was evaluated by changing into the complete medium and reoxygenated for 24 hours after administration of rapamycin and other drugs.In vivo model:the experimental group c57/bl6 mice were anesthetized and then separated the spermatic cord slightly after anesthesia and then clamped with microvascular.Rapamycin（1mg/kg）was given to the abdominal cavity half an hour after ischemia,and the clips were released after 2 hours of ischemia.The mice were killed 24 hours later.The control group only opened the abdomen without medication,the injury group was not administered rapamycin,the drug group was only administered rapamycin without ischemia-reperfusion treatment.（2）Biological function evaluation:apoptosis and autophagy were characterized by immunohistochemistry,WB,RT-q PCR,etc.;cell morphology was observed by electron microscope and light microscope;ROS,apoptosis and mitochondrial membrane potential were detected by flow cytometry with DCFH probe,apoptosis kit and JC-1.Cell viability was tested by CCK-8 kit。（3）To explore the relationship between mtorc1/autophagy pathway and the protective effect,3-m A autophagy inhibitor was used and then evaluate the function.（4）To explore the correlation between mTORC2/AKT/miR-21 pathway and the protection effect,RICTOR si RNA,AKT inhibitor MK-2206 2HCl,miR-21 mimics and miR-21 inhibitors were used.Meanwhile,the plasmid was constructed and the target gene of miR-21 was verified by the double luciferase reporter assay.Result:（1）CoCl₂induced the hypoxia of TM4 cells.With the increase of CoCl₂concentration,the expression level of HIF-1αwas also increased with respect to ACTIN,and there was significant difference between the groups（P<0.05）.Rapamycin promoted the viability of TM4 cells in H/R model.With the increase of rapamycin concentration（25nm,50nm,125nm,250nm,500nm）,the cell viability gradually increased,and the protective effect of 250nm rapamycin reached the maximum.However,rapamycin alone can reduce cell viability,indicating that rapamycin can inhibit the proliferation of TM4.（2）Rapamycin inhibited the apoptosis of TM4 in H/R model,which was related to the decrease of ROS and the recovery of mitochondrial membrane potential.The results of flow cytometry showed that rapamycin could inhibit the apoptosis induced by cobalt chloride.Compared with the control group,rapamycin alone did not increase the apoptosis rate（P>0.05）.Western blot showed that the anti-apoptotic protein BCL2 and BCL-XL in the treatment group increased significantly（P<0.05）,and the pro-apoptotic protein BAX decreased significantly（P<0.05）,and the downstream apoptosis signaling pathway proteins like cleaved caspase 3 and cleaved parp also decreased（P<0.05）,compared with the injury group.After the CoCl₂treatment,the mitochondrial membrane potential decreased significantly（P<0.05）,and the mitochondrial membrane potential increased after rapamycin intervention in reoxygenation stage（P<0.05）,but the single administration of rapamycin did not change significantly compared with the control group（P>0.05）.Similarly,the ROS level of the cells increased significantly after the CoCl₂administration compared with the control group（P<0.05）,and the ROS level of the cells decreased after rapamycin intervention in the reoxygenation stage（P<0.05）（3）The protective effect of rapamycin is related to mTORC1 and mTORC2.Rapamycin can activate autophagy and akt/mir-21 pathway through mTORC1 and mTORC2 respectively,which can relieve the ischemia-reperfusion injury of testis.When we treated with rapamycin,mTORC1 was significantly inhibited,while autophagy level was significantly increased（P<0.05）,and apoptosis decreased.The protective effect could be blocked by autophagy inhibitor 3-m A（P<0.05）;at the same time,mTORC2 was activated,and AKT/miR-21 was activated with the decreased ROS,recovered mitochodria membrane potential and reduced apoptosis.This protective effect could be partially blocked by Akt inhibitor and miR-21 inhibitor,but partially recovered by miR-21 mimic.（4）In vivo experiments showed that rapamycin could protect testis after ischemia-reperfusion injury,promote Sertoli cell survival and spermatogenesis recovery.The results showed that the Johnson score of testis in the treatment group was significantly higher than that in the injury group（P<0.05）,and the Sertoli cells in the tubules were more than those in the injured group（P<0.05）.Western blot and PCR showed that rapamycin reduced apoptosis by inhibiting mTORC1 with increased autophagy and activating mTORC2/AKT/miR-21 pathway.Conclusion:Rapamycin can inhibit apoptosis through mTORC1 and mTORC2 complexes,the former activating autophagy,the latter promoting AKT/miR-21 pathway,which stabilizes mitochondrial membrane potential,reduces ROS production,thus promoting the survival of Sertoli cells and the recovery of testicular spermatogenesis.