Mechanism Of Yishen Granules Inducing Autophagy Of Podocyte Cells To Improve Diabetic Nephropathy By LncRNA MALAT1/mTOR

Diabetic kidney disease(DKD)or diabetic nephropathy(DN)is a chronic kidney disease caused by diabetes.In the developed countries,DN becomes the main cause of end-stage renal disease(ESRD).DN pathogenesis is complex,the role of long chain noncoding RNA and autophagy in the pathogenesis realized by more and more researchers.Due to the uncertainty of the mechanism,there is still a lack of effective treatment plan.Yishen Granule(YSG)is professor zhang ning’s empirical scheme for the treatment of DN.It has been clinically proven to be effective and has now become a hospital preparation,but the pharmacological components and mechanism of YSG are still unclear.Therefore,the subjects of this study are the DN rat model constructed by high glucose and high fat feed combined with STZ intramuscular injection and the model of glycolipid-induced podocyte injury.To observe the role and mechanism of YSG in activating autophagy,protecting podocyte,alleviating pathological damage and delaying DN progression through LncRNAMALAT1/mTOR signaling pathway.Moreover,the compounds of YSG were identified by HPLC.Experiment 1:YSG component identificationObjective:To identify the chemical constituents of YSG by high performance liquid chromatography(HPLC).Methods:YSG was pretreated by water extraction,and after decompression and concentration,constant volume was carried out.The obtained filtrate was passed through a 0.45μm porous membrane,and 10ul was inj ected into the high-performance liquid chromatograph.At the same time,the standard solution was prepared,followed by 0.45 μm microporous membrane,10ul was taken and injected into the high-performance liquid chromatograph.Optimize the detection conditions of high performance liquid chromatography analyzer:ODS chromatographic column,mobile phase:acetonitrile(A)-water(B)gradient elution:0-10min 2-10%A,10-20min 10-20%A,20-30min 20-30%A,30-40min 30-60%A,40-50min 60-80%A,50-60min 80-95%A,60-65min 95%A;The flow rate was lml/min,the column temperature was 300℃ the injection amount was 10ul(YSG/standard),the detection wavelength was 254nm/203nm,and the compounds were isolated and purified.The wave peak of YSG was compared with that of standard products,and make sure the comparison.Results:At different wavelengths of 254nm and 203nm,clear wave peaks can be separated and the chromatogram of YSG can be obtained.After comparison with standard products,the four components of tanshinone IIA tanshinone I were determined at the wavelength of 254nm.At the wavelength of 203nm,4 components of astragaloside IV ligligolide were identified.Conclusions:Eight components of yishen granules were identified by HPLC,which were astragaloside IV,triflavone,glucoside tanshinone,IIA,tanshinone I,tanshinone,ligligolide,catalpa galactate,respectively.Experiment 2:The effect of YSG on prevention and treatment of glomerulosclerosis in DN model rats induced by HFSD+STZ.Objective:to observe the effect of YSG on fibronectin(FN)smooth muscle actin(FN)and to investigate the effect of YSG on prevention and treatment of DN glomerulosclerosis.Methods:Diabetic nephropathy model was constructed by feeding high fat/high sucrose diet(HFSD)with STZ intraperitoneal injection.HFSD was fed for 6 weeks,followed by intraperitoneal injection of 30mg/kg 1%STZ solution.After 72 hours,non-fasting glucose in tail vein for 3 times was≥16.7mmol/L,which was the model standard of diabetes mellitus.They were divided into model group,losartan potassium group,YSG low dose group,YSG medium dose group,YSG high dose group,and normal group.Gavage intervention was performed for 10 weeks.After the intervention,anaesthesia was performed.The 24-hour urinary protein quantification(24hU-Pro)was detected by biuret method,and the fasting blood glucose(FBG),plasma albumin(ALB),serum creatinine(Scr),blood urea nitrogen(Bun),cholesterol(CHO),triglyceride(TG)and so on were detected by enzyme method.HE,PAS staining and FN,α-SMA immunohistochemistry,,desmin immunofluorescence and caspase-3 western blotting were performed.Results:Compared with the normal group,24hU-Pro Bun FBG CHO TG in the model group increased(P<0.01 or P<0.05),and ALB decreased(P<0.01).After the intervention of yishen granules,24hU-Pro Bun FBG CHO TG decreased(P<0.01 or P<0.05),while ALB increased(P<0.05).The effect of YSG in high dose was the most obvious,and the effect was better than that of losartan potassium group.HE PAS staining showed that in the model group,increased glomerular volume and thickened mesangial matrix in the glomerular basement membrane were observed.No late lesions such as hyaline degeneration of small artery with k-w nodular glomerular collapse were found.The YSG group of losartan potassium group showed different degree of renal tissue improvement after the administration of 10W.The immunohistochemistry of a-SMA and FN showed that the glomerular tubules and stroma of the normal group had less positive expression of a-SMA and FN.In the model group,the glomerular basement area,mesangial area,renal tubular epithelial interstitial area,and other positive signals of different degrees were observed(brown-yellow staining).After the intervention of losartan potassium and YSG,the positive signal was weakened to different degrees,with the most significant effect in the high-dose YSG group(P<0.01).Immunofluorescence of desmin protein and caspase-3 in podocytosis showed that the expression of desmin and caspase-3 increased in the model group,and the expression of desmin and caspase-3 was down-regulated after YSG intervention(P<0.05)Conclusions:YSG can reduce the urinary protein of DN model rats,reduce nitrogenemia,improve glucose and lipid metabolism,and can reduce the expression of FN and a-SMA,reduce the pathological damage of renal tissues,and finally delay the progression of glomerulosclerosis.Moreover,YSG may play these roles by reducing the cytoskeleton damage of podocytes,protecting the podocyte structure and reducing the generation of apoptosisExperiment 3:The mechanism of YSG regulated autophagy through LncRNA MALATl/mTOR signaling pathway to improve of DN and podocyte injury.Objective:To observe the effect of YSG on podocytes LncRNA MALAT1 and mTOR-mediated autophagy,as well as the relationship between MALAT1 and mTOR,and to explore the mechanism of YSG.Methods:In vivo,Atg5 immunohistochemistry,MALAT1 expression detected by qRT-PCR,LC3,Atg5,SRSF1,p-mtor,c-myc expression detected by WB,through the kidney tissue of experiment two.In vitro,the injury model of podiocyte was induced by glycolipid toxin,and YSG freeze-dried powder was used after 48h intervention with 25mM sugar+0.08mm PA,and losartan potassium was used as the positive control.①The proliferation activity of podocyte cells was detected by cck-8 method,the migration ability of podocytes was detected by scratch test,cytoskeleton was stained with FITC ghost pen ring peptide,Lyso-tracker Red performed autophagosomal staining,Annexin v-fitc/PI flow cytometry was used to detect apoptosis of podiocytes,the expression of MALAT1 was detected by qRT-PCR,and the expression of desmin,Synaptopodin,ATG5,SRSF1,p-mtor and c-myc detected by WB.②ATG5,P62 expression was detected by WB after autophagy activator rapamycin(Rap)or inhibitor(CQ)was added,and changes of YSG expression on autophagy were observed.Under Rap conditions,the expression of MALAT1 was detected by qrt-pcr,and the effect of mTOR on MALAT1 and YSG was observed.③The expression of MALAT1 was detected by qrt-pcr after siRNA interference on MALAT1,and the effect of YSG on MALAT1 was observed.The expression of p-mTOR,SRSF1 and c-myc detected by WB,and observed the effect of MALAT1 on mTOR and the effect of YSG in it.Results:In vivo,compared with the normal group,the Atg5 immunohistochemical expression decreased in the model group.After YSG intervention,desmin expression decreased and Atg5 expression increased.WB results showed that LC3 and ATG5 expression in the model group were down-regulated(P<0.05),p-mTOR expression was up-regulated(P<0.05),and SRSF1 c-myc did not change significantly.After YSG intervention,LC3 and ATG5 expression increased(P<0.05),and p-mTOR expression decreased(P<0.05),suggesting that YSG activated autophagy.In vitro,①YSG can increase the activity of podiocyte proliferation and promote cell migration.Compared with the model group,YSG increased the expression of Synaptopodin,decreased the expression of desmin,made the actin bundles arranged neatly,and reduced cytoskeletal rearrangement.②Compared with the model group,YSG could increase the expression of Atg5 and reduce the accumulation of P62.Compared with the Rap group,the expression of Atg5 was higher in the YSG+Rap group(P<0.01),but there was no decrease in P62,and the same situation occurred in the YSG+CQ group and the CQ group.Annexin v-fitc/PI apoptosis showed that after the intervention of YSG,the apoptosis rate was significantly lower than that of the model group,with no statistical difference from the normal group(P>0.05).③After YSG intervention,the expression of MALAT1 increased compared with that of the model group(P<0.05),and the expression of p-mTOR was down-regulated.After silencing MALAT1,p-mTOR increases,after glycolipid toxicity injury,p-mTOR was further increased(compared with negative control,p<0.05)and after YSG intervention,p-mTOR was significantly down-regulated,with no statistical difference compared with the negative control(p>0.05).Under the condition of glycolipid toxicity,the expression of SRSF1 was decreased compared with that of the normal group.After the intervention of YSG and losartan potassium,the expression increased,but there was no statistical difference(P>0.05).SRSF1 expression increased after siRNA interference,after YSG intervention,the trend of expression increase was higher than that of the model group,but there was no statistical difference(P>0.05).Under different conditions,c-myc does not change much and is irregular.Conclusions:MALAT1 and mTOR can interact with each other and show negative correlation.MALAT1 down-regulation can activate mTOR,thereby inhibiting autophagy and increasing apoptosis.YSG can increase autophagy by activating the MALAT1/mTOR signaling pathway,mainly to promote autophagosome formation and autophagosomal binding,so as to reduce cell apoptosis and reduce podocytocytoskeletal rearrangement.The regulatory effect of MALAT1 on mTOR may not be related to the regulatory effect of SRSF1 splicing factor.