Study On The Mechanism And Sensitizing Effect Of Yougui Pills In Rats With Steroid Resistant Nephrotic Syndrome Based On MicoRNA

Background and objective:Steroid resistant nephrotic syndrome(SRNS)is a types of refractory nephrotic syndrome which is lack of response to a standardized corticosteroid therapy.The pathogenesis of SRNS is not completely known.As a high-risk factor for nephrotic syndrome events,Steroid resistance is an important deter-minant of future risk for end-stage renal disease and endanger the life of the patient.Podocyte injury is closely related with chronic kidney disease(CKD).podocyte genes mutations and abnormal expression are the key factor of in the occurrence of Abnormal SRNS miRNA expression has been shown to cause glomerular disease.Some miRNAs regulate the expression of slit diaphragm molecules through the mediation of Dicer enzyme.Yougui Pills can be proved to improves the expression of SD molecules and reduces proteinuria,which have strengthened the sensitivity of glucocorticiod in treatment of SRNS.but its molecules mechanism is not clear.It was hypothesized that Yougui Pills could enhance the expression of SD molecules to reduce urinary protein by Dicer-microRNA.The clinical basis of this study is that Yougui Pills have strengthened the sensitivity of glucocorticiod effect on SRNS.With the expression of SD molecule as target,and miRNA as the regulatory factors,the mechanism that microRNA regulate slit diaphragm molecules expression and Yougui pills induce SD molecules expression,protecting renal function,maintaining SD function and structure of podocytes via miRNA is explored to provide experiment basis for the development of SRNS sensitizing drugs,by observing morphology change of model rats,expression of miRNA,the apoptosis factors,Dicer,Slit diaphragm molecules mRNA and proteins with intervention of Yougui Pills.Methods:1.Gene chip and mechanism 64 male SPF Wistar rats were reared adaptively for one week,and were randomly divided into blank group(Control,12)and model group(52)according to body weight.Successfully establish SRNS rats model,and the SRNS rats were randomly divided into model group,PAT group,high,medium,and low dose groups of Yougui Pill.SRNS model groups could be injected of Adriamycin(6mg/kg/day)by tail intravenous,and normal group rats could be injected of physiological saline with equal volumeby tail intravenous.and,SRNS model was made by muscular injection of Prednisone Acetate Tablets(PAT,25mg/kg/once),Meanwhile control group rats was made by muscular injection of physiological saline once a day for two weeks.2.By irrigation stomch every day for 6 weeks after successful modeling.on days14,28,42,and56,24-h urine sample were collected,on day 55,measured the number of activities and heads up in 5 minutes by autonomy device.On the 55th day of the experiment,the rats'autonomous activity was measured at 8 o'clock in the evening.The autonomic activity was turned on to measure the number of activities and heads up for 5 minutes.After 42 days of intervention,the blood biochemical parameter and kidney tissues histopathological were observed respectively.3.All rats were killed,the kidney was isolated by local anesthesia,and the left kidney of the rat was excised and the upper and lower stages were respectively added with 4%glutaraldehyde and 4%paraformaldehyde fixative to fix tissues for electron microscopy.The right kidneys were isolated and stored in-70~oC refrigerator.To extract total RNA,and validate 3 miRNAs differentially expressed in normal and model groups by Real-time PCR.4.The changes of kidney tissues were observed by HE,PAS,Masson and PASM staining.On the other hand,detect the expression of dicer,CD2AP,synaptopodin,Bax,Bcl-2,caspase-3protein and mRNA in renal tissueby RT-PCR and Western Blotting.Results:1.SRNS rats were made successfully,it could be injected of Adriamycin(6mg/kg/day)by tail intravenous.Senven days after modeling,the model rats could significiency lowered spirits,decrease urine volume,increase ascites and lose weight.In two weeks,steroid resistant nephrotic syndrome were worst in model rats.2.The nephrotic syndrome of SRNS rats treated withYougui Pill was significantly improved compared with the model group.The effect of reducing urinary protein and blood lipids levels in the medium dose of Yougui Pill group was past beyond other treatment groups.Serum creatinine and blood urea nitrogen in PAT+YGh and PAT+YGl groups were not as low as those in PAT+YGm group(p<0.05).Levels of high cholesterol/triglyceride,and serum albumin was significantly changed after 6-week treatment in the PAT+YGm group.3.Compared with the control group,the glomerulus became larger,the basement membrane thickened and irregular,the mesangial membrane slightly proliferated,and the renal tubules showed clear tubular shapes in SRNS rat.moreoever,the fusion of the foot processes of podocytes were found in the model group by electronic microscop.And,Yougui Pills group reduced the podocyte fusion,and the effect of podocyte recovery was obvious.4.Compared with control group,there were 15 miRNAs up-regulation and 8 miRNAs down-regulation in SRNS rat.In PAT+YGm group,the expression of rno-mir-29b-3P and rno-mir-26b-5P was up-regulated and the expression of rno-mir-205 was down-regulated.After treatment with Yougui Pill,the rno-mir-29b-3P and rno-mir-26b-5P were inhibited,while the rno-mir-205 was enhanced,which have been validated by real-time PCR analysis.5.Compared with the control group,the immunofluorescence of Dicer enzyme up-regulated,and the expression of protein and miRNA increased in the model group.Compared with the model group,the nephrin,podocin,CD2AP,synaptopodin protein and mRNA significantly decreased(p(27)0.05).hint that Dier-miRNA negatively regulates podocyte injury and podocyte apoptosis.6.The immunofluorescence intensity of Dicer and miRNA and protein the expression of Dicer were all significantly decreased compared with SRNS model group before treatment by Yougui pill.Moreover,Yougui pill could improve the immunofluorescence intensity of SD molecules,and raise the expression of mRNA and protein.7.Yougui pill can improve the expression of synaptopodin,Bax,Bcl-2 and caspase-3 of immune fluorescence intensity,miRNA and protein.Compared with the control group,the Bax/Bcl-2,caspase-3 of immune fluorescence intensity were enhanced in model group,protein and mRNA expressions were increased(p<0.05),and synaptopodin,Bcl-2 expression was decreased(p<0.05).Compared with SRNS rats,the Bax,caspase-3 the high expression of immune fluorescence intensity,protein and mRNA were significantly inhibit by PAT+YGm(p<0.05).And increased the expression of synaptopodin,Bcl-2(p<0.05).Conclusion:1.In the experiment combining the disease and syndrome,nephrosis rats can be made successfully,the method of injection of 6mg/kg via the tail vein,with muscular injection of Prednisone Acetate Tablets(25mg/kg/once)was used to successfully establish SRNS model rat.2.15 miRNAs up-regulation and 8 miRNAs down-regulation were detected by miRNA array in SRNS rats.In these specific miRNAs,The up-regulated miRNAs of rno-mir-29b-3P,rno-mir-26b-5p may cause podocytes injury and proteinuria,while the down-regulated miRNAs of rno-mir-205 may be protective factors against apoptosis and development of SRNS sensitizing drugs.3.Dicer negatively regulates the expression of nephrin,CD2AP,podocin and cytoskeletal proteins in model sample via miRNA.Dicer-miRNA may also be involved in the regulation of podocyte injury and podocyte apoptosis.It is suggested that Dicer-miRNA may be a key target for the regulation of SD molecules and proteinuria.4.Yougui Pills can down-regulate the expression of Dicer in rats with steroid resistance nephrotic syndrome,which may be raised the mRNA and protein expression profile of nephrin,podocin and synaptopodin by triggering Dicer-miRNA.It is suggested that Dicer,rno-mir-29b-3P,and rno-mir-26b-5P may be therapeutic targets for Yougui Pills to treat SRNS,protect podocytes,and reduce proteinuria.5.Yougui Pills may improve injury of podocytes in rats with SRNS through up-regulating the expression of Bax/Bcl-2 mRNA and protein,and down-regulating the expression of caspase-3mRNA and protein.which may be played the promoting podocyte autophagy role by inhibit podocyte apotosis.6.Yougui Pills can reduce toxity and strengthened the sensitivity of glucorticoids to treat steroid resistant nephrotic syndrome by reducing proteinuria,improving hypoalbuminemia,remissioning high cholesterol/triglyceride levels,slowing down the space of podocyte injury,reducing epithelial cells degeneration renal of renal tubular and the degree of glomerular sclerosis.