Metformin Enhanced Autophagy In High Glucose Cultured Rat Mesangial Cells Via MiR-124a-3p/AMPK Alpha

Background:Diabetic kidney disease(diabetic kidney diseases,DKD)is one of the most common chronic microvascular complications of diabetes(diabetes mellitus,DM),and also is the leading cause of end-stage renal disease,which contributes to diabetic patients’ fatality rate.From high glucose state to DKD and progress to end-stage renal disease is a series of pathophysiological processes of continuous changes.Usually,early state showed high reversibility glomerular filtration,mesangial expansion and thickening of basement membrane,and developed gradually with the progress of the illness to irreversible glomerular mesangial cells and extracellular matrix proliferation,and eventually leading to glomerular sclerosis,fibrosis,and shrink,which could cause renal failure.Therefore,exploring the pathogenesis of DKD and to take effective measures early to prevent DKD,reversing early kidney damage,blocking the progress of the DKD has become one of the important tasks of clinical therapeutics.Autophagy is one of the important ways of cell protein decomposition and organelles degradation.Autophagy played an important role in regulating metabolism,maintaining cellular homeostasis and removing damaged organelles,etc.Studies showed that LC3-Ⅱ was known as an common in mammalian cells autophagosome one marker protein was involved in the formation of autophagy.Metformin is a common kind of oral glucose-lowering agent.Studies showed that besides treating hypoglycemia,metformin had renal protection effect,could reduce urinary albumin excretion rate in the DKD patients.Metformin could protect kidney from oxidative stress,inflammation,and fibrosis.But whether metformin could regulatie miR-124 or autophagy in rat glomerular mesangial cells cultured with high glucose and its mechanism still needs further research.MicroRNA(miRNA)is a kind of small non-coding RNA,the length of which is about 19 to 25 nucleotides.miRNA could adjust silencing of post-transcription of target genes.One single miRNA could target hundreds of mRNA and effect usually participate in the way of function and interaction between the expression of many genes.Studies showed that miR regulated autophagy by different pathways,played a vital role in the treatment of various diseases.miRNA acted in the basic cell biological processes such as proliferation,important regulator of autophagy and apoptosis.MiR-124 is an important member of miRNAs,highly expressed in mammalian central nervous system,also in the islet beta cells.MiR-124 negative regulated of insulin mRNA level,and also acted important in the secretion of insulin.Metformin treatment was closely related to the AMPK activation,but there was no direct connect between AMPK and lysosomes.Connect between miR-124a-3p and AMPKα needs exploring.Objective:This study aims to explore modulating effect of metformin on the function of rat glomerular mesangial cells,and to explore the regulation affect of metformin on autophagy via miR-124a-3p and AMPKα in high glucose cultured rat glomerular mesangial cells and to provide new targets of DKD therapy.Methods:Part 1.The regulation of metformin on type 2 diabetes mellitus SD rat renal glomerulus.After two weeks of adaptive feeding,SPF male five-week-old SD rats weighted(162.5+12.5)g,were randomly divided into control group amount of 10 with the normal diet.The remaining 20 rat were feed by high fat and glucose diet.After 4 weeks,intraperitoneal injection with streptozocin(STZ)30 mg/kg.BW.Detection of fasting blood glucose levels,takeing fasting plasma glucose more than 11.1mmol/L or random blood glucose more than 16.7mmol/L as diagnosis standards.16 rats were successfully established T2DM model and randomly devided into diabetes group for 8 rats(DM),8 for metformin group(Met).Rats in met group were administered metformin(250 mg/kg BW)intragastrically.DM group and NC group rats received an equal volume of vehicle(pure water)intragastrically for 8 weeks.General symptoms were observed and body weight fasting blood glucose(FBG)were measured.Kidney index was calculated,the rat kidney and liver tissue fixed paraffin section,HE,D-PAS,Masson staining,the pathological changes was observed.The expressions of Lamp2 and Tomm20 in renal biopsy were observed by laser confocal microscope.Part 2.Metformin enhanced autophagy in high glucose cultured rat mesangial cells via miR-124a-3p/AMPK alpha.5-9 passages of the rat messengial cells(HBZY-1)were used in this study.1 X 106 cells inoculated for cell concentration in 25 cm2 culture plate.After serum-free medium MEM synchronization for 24h,cells were divided in the following groups:(1)normal glucose control group(NG,5.5mmol/L D-glucose);(2)osmotic control group(HM,5.5mmol/L D-glucose and 24.5mmol/L mannitol);(3)high glucose group(HG,30mmol/L D-glucose),(4)HG+miR-124a-3p inhibitor(HG+miR-i),(5)HG+miR-124a-3 p NC group(HG+miR-NC),(6)HG+miR-i+Metformin group(Metformin,Met)(7)HG+miR-NC+Met group.Exogenous addition of metformin processed high glucose-cultured RMC;Gene silencing by RNA interference technology was used to restrain the expression of miR-124a-3p;the CCK-8 kit was used to detect cell proliferation 24,48 and 72 hours cultured.Real-Time qPCR was used to measured the expression of miR-124 a-3p;Western Blot was used to measured autophagy related proteins’ expression levels,including P62,LC3B Lamp2,BECN-1,AMPKα and p-AMPKα.Results:Part 1.Metformin’s effects on pathological changes of T2DM SD rats renal glomerulus.1.General symptoms:metformin relieved the symptoms of T2DM rat body weight increase.2.Indicators of glucose metabolism:metformin reduced fasting blood glucose(FBG)level of T2DM rats,(p<0.05).3.Kidney damage index:T2DM rats decreased kidney weight index after metformin treatment(p<0.05).4.Morphological changes:(1)HE staining results:NC groups:showed a normal kidney tissue structure,mesangial cells,mechanism of mesangial proliferation,basement membrane thickening,glomerular size,normal renal tubule showed no obvious change.DM groups:glomerular swelled,mesangial matrix proliferation,with a small amount of mesangial cell proliferation,swelled in the renal tubular epithelium.Met groups:proliferation of glomerular mesangial cells was not obvious,a small amount of mesangial matrix proliferation,renal tubular epithelial vacuoles degeneration.(2)D-PAS staining results:metformin to improve type 2 diabetes mellitus in SD rat glomerular glycogen deposition.(3)Masson staining results:metformin to improve fiber accumulation in SD rat glomerular type 2 diabetes mellitus.(4)Immunohistochemical observed that metformin auttenated the reduction of BECN-1,LC3Bexpression(p<0.05)and reduces the increased expression of P62(p<0.05)in DM rats renal cortex.5.Laser confocal microscope observed the NC group,DM group and the group Met in rat glomerular Lamp2,Tomm20 deposition.DM group showed fluorescence intensity increased of the two factors compared with the NC group,the Met group showed fluorescence intensity decrease of the above factors compared with the DM group(p<0.01).6.Protein immunoblot observed that metformin auttenated the reduction of BECN-1,LC3Bexpression(p<0.05)and reduces the increased expression of P62(p<0.05)in DM rats renal cortex,as while increased the reduced phosphorylation level of AMPKαprotein in T2DM rats renal cortex(p<0.05).Part 2.Metformin enhanced autophagy in high glucose cultured rat mesangial cells via miR-124a-3p/AMPK alpha.1.Proliferation of RMCs increased in HG group(p<0.01).2.Lamp2 and P62 protein expression significantly increased(p<0.01),BECN 1(p<0.01),Atg12(p<0.01)and the LC3B(p<0.01)expression significantly decreased in HG group.3.RMCs miR-124a-3p mRNA expression levels increased in HG group(p<0.05).4.Phosphorylation level of AMPKα significantly decreased(p<0.01)in high glucose cultured RMCs.5.Phosphorylation levels of AMPKα(p<0.01)and BECN-1(p<0.01)expression significantly increased by using gene silencing technology inhibition of miR-124a-3p in high glucose cultured RMCs.6.MiR-124a-3p represses AMPKα gene expression by interaction with 3’-UTR.7.Gene silencing technology inhibition of miR-124a-3p in combination with metformin treatment activated autophagy in high glucose cultured RMCs.(1)Metformin reduced proliferation rate high glucose cultured RMCs(p<0.01).(2)Metformin reduced miR-124a-3p expression in high glucose cultured RMCs(p<0.01).(3)Inhibition of miR-124a-3p and metformin treatment increased phosphorylation level of AMPKα in high-glucose cultured RMCs(p<0.01),and reduced BECN-1 expression(p<0.05).Conclusions:Part1.1.Metformin released glomerular hypertrophy,mesangial area broadening,mesangial matrix proliferation and expansion of renal tubule pathologic changes and reduced average body weight of DM group rats.Metformin might play a role of kidney protection by activating autophagy in glomeruli.2.Metformin acted a protective effect by activating autophagy in DM group rat renal cortical tissue.Part 2.1.High glucose restrained autophagy in RMCs via upregulateing miR-124a-3p expression.2.Dual luciferase report gene affirmed the miR-124a-3p negative regulation on AMPKαby combining with the AMPKα mRNA 3’-UTR.3.Metformin increased miR-124a-3p expression and phosphorylation levels of AMPKαin high glucose cultured RMCs via activating autophagy and acted a role of prevention and treatment in DKD.