Effects Of Glucose Concentrations On The Autophagy And Phenotype Of SD Rats

Background: In recent years,the incidence of Diabetes melitus(DM)has increased,the number of patients of Diabetes melitus induced Erectile dysfunction(DED)also gradually improved,and this disease occupies a considerable proportion in Sexual dysfunction.However,as far as the complexity of DM mechanism,and the differences between DED pathogenesis and normal erectile dysfunction pathogenesis,the pathogenesis of DED has not been fully illustrated.In numerous studies about the appearance,development mechanism of Erectile dysfunction(ED),even researches of Corpus cavernosum smooth muscle cells(CCSMCs)started relatively late,it has hopeful prospects,and which may bring new ideas for the diagnosis and treatment of ED.CCSMCs is the most important component of corpus cavernosa,and it is also the last point where various neuro-vascular signaling molecules work during penile erection process,it is also vital for the maintenance of the microstructure in cavernous body.This has been proved by the changes in the function,morphology and quantitatity of the CCSMCs in patients who have been diagnosed with DED,but its mechanism is not fully understood.Autophagy,as an important mechanism for removing metabolic products and promoting cells to adapt to external stimuli,has been confirmed to be related to the structural remodeling of body organs in many fields.For cardiovascular in the body of the diabetes,a number of studies hold the opinion that change in microstructure of Vascular smooth muscle is associated with the participation of cell autophagy,and this change is derived from the regulation of autophagy for phenotype in Vascular smooth musle cells(VSMCs).It is confirmed that there is autophagy phenomenon in CCSMCs for diabetic patients,but its influences and mechanism for CCSMCs need further study.In terms of CCSMCs autophagy caused by diabetes,more studies need to be done for the question whether it has the same effects and same working mechanism as VSMCs autophagy in diabetes patients.This may provide new ideas for pathogenesis of erectile dysfunction in person with diabetes.Objective:To study effects of glucose concentrations on the autophagy andphenotype of SD rats,and find out the connections between them.Methods:To find out autophagy level changes of CCSMCs after dealing with different concentrations of glucose: the original materials were taken from the CCSMCs,and the cells were treated with 25mmol/L glucose,30mmol/L glucose,and 45mmol/L glucose for 24 h.Electron microscopy is employed to observe autophagy and autophagy lysosomes,mFRP-GFP-LC3 adenovirus transfection observes the autophagy flow in each group,immunofluorescence detection is to show LC3 level,Western blot detects protein LC3,P62,Beclin1 levels which are related to autophagy;(2)to investigate how different concentrations in glucose levels influence phenotypic indexes of CCSMCs: based on CCSMCs,the above three levels of glucose cells are processed 24 h,phenotypic protein ?-SMA,hlcalponin,OPN levels of CCSMCs are tested by means of immunofluorescence,Western blot,at the same time,kits of CCK-8 are used to test cell proliferation ability,scratch tests for its migration ability;(3)to investigate transformation relationships between autophagy and phenotype of CCSMCs brought by different concentrations of glucose: according to the processing above,changes of autophagy and phenotype index can be found.In different groups set by different glucose concentrations,the glucose group which has the most obvious transition phenomenon lies in 30mmol/L,they are respectively processed with autophagy inhibitor hydroxychloroquine and3-Methyladenine(3-MA).Western blot and mFRP-GFP-LC3 adenovirus transfection are used to detect autophagy index in each group.Western blot is used to detect levels of smooth muscle phenotype in each group.Results:(1)primitive separation of CCSMCs was successful,test results from electron microscope,mFRP-GFP-LC3 adenovirus transfection,immunofluorescence,Western blot indicated that there were different degrees of autophagy after processing glucose in different concentrations,the autophagy level was increased after processing CCSMCs with higher concentration of glucose than normal level(25mmol/L),and 30mmol/L group had the highest autophagy level(P < 0.05),and autophagy level in the group of 45mmol/L wasreduced(P < 0.05).(2)after being processed with higher concentration of glucose than normal level(25mmol/L),under the condition of 30mmol/L and45mmol/L in glucose concentration,immunofluorescence and Western blot showed a decrease of typical protein ?-SMA and hlcalponin levels in Contractile smooth muscle,which was compared with 25mmol/L(P < 0.05),typical protein of OPN levels in synthetic smooth muscle increased when compared with25mmol/L(P < 0.05),proliferation ability(P < 0.05),the migration ability of CCSMCs also increased;These changes were more significant in glucose group with 30mmol/L than in glucose group with 45mmol/L(P < 0.05).(3)after dealing with Hydroxychloroquine and 3-MA,mFRP-GFP-LC3 adenovirus transfection and Western blot indicated that autophagy flow was blocked;at the same time,when given two different autophagy inhibitors,the Western blot results indicated that typical protein ?-SMA,hlcalponin amount in Contractile smooth muscle increased slightly when compared with glucose of 30mmol/L(P <0.05),but protein level of OPN in synthetic smooth muscle lowered when compared with glucose of 30mmol/ L(P < 0.05),namely phenotypic transition caused by glucose in CCSMC was restrained.Conclusion:(1)under the condition of conventional culture,autophagy exists in CCSMCs,when given a higher concentration of glucose intervention,autophagy can be further enhanced,but when glucose level is higher than a critical value,level of autophagy starts to wane;(2)phenotypic transformation occurs in CCSMC when glucose concentration is higher than glucose concentration in conventional culture conditions,and the change tendency is in consistent with the tendency in which cell's autophagy level changes with the glucose's concentration;(3)there is an inhibition of autophagy,and phenotype autophagy transformation of CCSMCs caused by glucose was inhibited,suggesting that autophagy of CCSMC caused by glucose may be involved in phenotypic transformation process.