The Renoprotective Effects Of Berberine On Diabetic Nephropathy Rats And Its Regulatory Effect On Podocyte Autophagy By The MTOR/P70S6K/4EBP1 Signaling Pathway

Diabetic nephropathy(DN),the manifestation of diabetic systemic microvascular lesions in the kidney,is one of the main causes of end-stage renal disease(ESRD).The pathogenesis of DN is complex,and more and more studies have found that the abnormal autophagy behavior of glomerular podocytes are associated with diabetic nephropathy.On the basis of previous studies of DN,this study aimed to investigate the effects of berberine(BBR)on podocyte autophagy,apoptosis and m TOR/P70S6K/4EBP1 signaling pathway.In addition,this study verified the intervention of BBR on podocytes of DN in vivo and in vitro.Objective:To investigate the renoprotective effects of BBR on DN rats,we observed the effects of BBR on renal function such as,blood glucose,hematuria,and histopathological in DN rats.In order to investigate whether BBR exerts renoprotective effects on DN rats by affecting autophagy and its potential mechanisms,we also detected the level of autophagy in kidney tissue and the level of m TOR/P70S6K/4EBP1 signaling pathway in the treatment of BBR.To investigate the effects of BBR on podocyte autophagy,apoptosis under high glucose conditions,we detected the levels of podocyte autophagy,apoptosis in the treatment of BBR.In order to investigate the potential mechanism of berberine on podocyte autophagy and apoptosis,we also detected the level of m TOR/P70S6K/4EBP1 in the treatment of BBR,providing a theoretical basis for further exploring the pathogenesis and treatment of DN.Methods:In vivo,the diabetic models were induced by streptozocin(STZ,30mg/kg)combined with high-sugar and high-fat diet.After 3d,7d,fasting blood glucose(FBG)was detected in rats.Once the FBG level of the rats was ≥11.1mmol/L,then the rats were included in the experiment group.The experiment was divided into 6 groups:Diabetic nephropathy group(DN),Metformin(200mg/kg)group,Captopril(15mg/kg)group,BBR(100mg/kg)group,BBR(200mg/kg)group.In addition,the normal group(NC)was set and fed with ordinary feed.After 12 weeks of administration,FBG,urine creatinine(Ucr),urine albumin(UAlb),and serum creatinine(Scr)were detected in rats,and albumin creatinine ratio(ACR)and creatinine clearance ratio(Ccr)were calculated.The histopathological changes of kidney tissue were observed by HE and PAS staining.The expression levels of LC3,p62,cleaved caspase-3 were detected by immunohistochemistry.The expression level of p-m TOR in podocytes of kidney tissue was observed by co-localization fluorescence staining of p-m TOR-Synapotodin.The expression levels of nephrin,desmin,cleaved caspase-3,LC3,p62 in kidney tissue were detected by western blot.The expression levels of p-m TOR/m TOR,p-P70S6k/P70S6 K and p-4EBP1/4EBP1 in kidney tissue were detected by western blot.In vitro,the levels of LC3II/I in podocytes stimulated with HG were detected at 0,2,4,8,12,24,36 and 48 h by western blotting.CCK-8 was used to detect the viability of podocytes.The level of autophagy was detected by western blotting,transmission electron microscopy and immunofluorescence.Podocyte apoptosis was analysed by using Hoechst staining,western blotting,flow cytometry,and confocal microscopy.Then,podocytes were transfected with si RNA to silence m TOR,and the expression levels of proteins and m RNA involved in the m TOR/P70S6K/4EBP1 pathway were further investigated by western blotting and q RT-PCR.Results:In vivo:1.BBR can significantly alleviate hyperglycemia,increase creatinine clearance rate(Ccr),reduce urinary albumin creatinine ratio(ACR).2.BBR can significantly alleviate glomerular hypertrophy,reduce the increase of mesangial matrix and reduce the thickening of basement membrane and other pathological features.In addition,BBR can increase the level of autophagy and reduce apoptosis in kidney tissue.3.BBR can significantly increase the expression of LC3,reduce the expression of p62,cleaved caspase-3 in kidney tissue,and reduce the expression of p-m TOR in podocytes of kidney tissue,thereby increasing autophagy and reducing apoptosis.4.BBR can significantly inhibit the phosphorylation level of m TOR/P70S6K/4EBP1 signaling pathway.In vitro:1.The level of podocyte autophagy reached a minimum at 24 h under the stimulation of high glucose,and the apoptosis rate of podocyte increased significantly.After the intervention of BBR,BBR can significantly increase autophagy and reduce apoptosis.2.The effects of autophagy inhibitors 3-MA and HCQ further indicated that BBR can reduce podocyte apoptosis by increasing autophagy.3.Podocytes were transfected with si RNA to silence m TOR,and further study indicated that BBR can increase podocyte autophagy and reduce apoptosis through the m TOR/P70S6K/4EBP1 signaling pathway.Conclusion:1.BBR can increase level of autophagy in renal tissue of diabetic nephropathy rats.2.BBR can increase podocyte autophagy and reduce apoptosis through the m TOR/P70S6K/4EBP1 signaling pathway.