Effects Of Rapamycin On Autophagy,ERS And Apoptosis Of CCSMCs Induced By Glucose In SD Rats

Background: With the increasing incidence of diabetes year by year,WTO predicts that the number of patients with diabetes mellitus(DM)will reach about300 million by 2025,and erectile dysfunction(ED)is one of the complications of diabetes.Compared with ordinary people,the overall incidence of diabetic mellitus induced erectile dysfunction is 3 to 5 times higher,and the onset time is about 10 years earlier than ordinary people,the course of the disease is more complex,treatment is also more difficult.The pathogenesis of diabetic mellitus induced erectile dysfunction is not clear.In erectile tissue,the smooth muscle of corpus cavernosum is the main structure.In recent years,researchers have gradually focused on the corpus cavernosum smooth muscle cells(CCSMCs)of the penis to study the pathogenesis of diabetic mellitus induced erectile function.The functional,morphological and quantitative changes of CCSMCs in diabetic mellitus erectile dysfunction have been confirmed,but the mechanism is not clear.Some studies have confirmed that autophagy,endoplasmic reticulum stress(ERS)and apoptosis are the main pathways for the occurrence and development of diabetes mellitus.While rapamycin can promote autophagy and inhibit ERS and apoptosis,do autophagy,ERS and apoptosis also exist in CCSMCs of diabetic mellitus induced erectile dysfunction? what are the effects of rapamycin on autophagy,ERS and apoptosis in CCSMCs of diabetic mellitus induced erectile dysfunction? At present,there are few studies,which are worthy of further study,in order to provide ideas for the study of the pathogenesis and treatment of diabetic mellitus induced erectile dysfunction Objective: To investigate the effects of rapamycin on autophagy,endoplasmic reticulum stress and apoptosis of CCSMCs induced by glucose in SD rats.Methods:(1)To detect the changes of related indexes of autophagy,ERS and apoptosis after the intervention of different concentrations of glucose on CCSMCs: CCSMCs was isolated and purified from corpus cavernosum of SD rats and identified by immunofluorescence of α-smooth muscle actin(α-SMA)and alkaline calcitonin,and then cultured in complete medium containing 5.6mmol/L,10mmol/L,20mmol/L,30mmol/L and 40mmol/L glucose for CCSMCs 48 hours.Cell viability was detected by MTT,autophagy flow was detected by mCherry-GFP-LC3 B adenovirus transfection,and the protein expression of autophagy related genes 5(ATG5),Beclin1,p62,microtubule associated protein 1 light chain 3(LC3)and ERS related indexes glucose regulated protein 78(GRP78)and C/EBP homologous protein(CHOP)were detected by Western blot.Autophagy related indexes ATG5,Beclin1,P62,LC3 mRNA expression were detected by PCR,and apoptosis of cells in each group was detected by flow cytometry.(2)To detect the effects of rapamycin on autophagy,ERS and apoptosis of CCSMCs induced by high concentration of glucose in SD rats: cultured in complete medium containing 5.6mmol/L and40mmol/L glucose for 48 hours and pretreated with rapamycin in 600nmol/L for2 hours and then cultured in complete medium with 40mmol/L glucose for 48 hours.Western blot and PCR were used to detect the expression of autophagy related index p62,LC3,Beclin1 and endoplasmic reticulum stress related index GRP78,CHOP protein and mRNA,and flow cytometry was used to detect the apoptosis of cells in each group.Results:(1)After subculture and purification,the cells were identified by immunofluorescence.Both calponin and α-SMA were strongly positive.CCSMCs primary culture was successful.MTT detection showed that compared with 5.6mmol/L glucose group,CCSMCs activity in 10mmol/L glucose group did not decrease significantly,but CCSMCs activity decreased significantly with the increase of glucose concentration(P < 0.01).The results of mCherry-GFP-LC3 B adenovirus transfection,Western blot and PCR showed that autophagy occurred after treatment with different concentrations of glucose.With the increase of glucose concentration,autophagy was the strongest in 10mmol/L glucose group(P<0.05),and then decreased with the increase of glucose concentration(P < 0.05).The results of Western blot showed that endoplasmic reticulum stress increased gradually with the increase of glucose concentration(P<0.05).Flow cytometry showed that compared with 5.6mmol/L glucose group,the apoptosis rate of 10mmol/L glucose group did not increase significantly(P=0.302),but the apoptosis rate increased gradually with the increase of glucose concentration(P<0.01).(2)After pretreatment with rapamycin,Western blot assay showed that the protein expression of LC3 and Beclin1 increased(P<0.05),while the protein expression of p62 decreased(P<0.05),indicating autophagy,while the protein and mRNA expression of GRP78,CHOP and mRNA decreased after pretreatment with rapamycin(P<0.05).Flow cytometry showed that the apoptosis rate decreased after pretreatment with rapamycin(P<0.01).Conclusion:(1)When CCSMCs were cultured in vitro with different concentrations of glucose,grape concentration in a certain range may promote autophagy,thus protect the CCSMCs and maintain cell viability and function.however,when the concentration of glucose exceeds a certain concentration,it may exceed the range of CCSMCs,reduce autophagy,activate endoplasmic reticulum stress and promote apoptosis.Thus impairing the vitality and function of cells.(2)High concentration of glucose acts on CCSMCs.Rapamycin may protect CCSMCs by enhancing autophagy and inhibiting endoplasmic reticulum stress and apoptosis.