The Effect Of Salvia Acid B On Chronic Glomerulonephritis Via MiR-145/Rab14/Autophagy Pathway

Chronic Glomerulonephritis(CGN)is a clinically multiple immune inflammatory disease that seriously threatens human health.Infiltration of inflammatory factors and abnormal proliferation of mesangial cells are the most common pathological changes of CGN.Reducing inflammatory immune responses in the kidney is the key of the treatment of CGN.Autophagy is a lysosome-dependent intracellular degradation system commonly found in eukaryotic cells.It can maintain the homeostasis in cells by eliminating the damaged or senescent organelles and biological macromolecules.Autophagy is closely related to glomerular mesangial cell,podocyte and tubular cell injury.It is widely involved in the occurrence and development of various types of renal diseases and may be used as a new approach to treat kidney diseases.In addition,mi RNA family members are involved in almost every aspect of autophagy regulation,and they can regulate the autophagy process through targeted regulation of autophagy-related gene expression.In the previous study of our group,we found that Salvianolic Acid B significantly upregulated the expression of mi R-145 in mesangial cells,while mi R-145 was closely related to the abnormal proliferation and inflammatory infiltration of mesangial cells.Further,bioinformatics software predicts that Rab14 protein may be the target protein of mi R-145.Rab14 protein can accumulate in the phagosome membrane,leading to increased phagosome binding to early endosomes,interfering the combination of phagosomes and lysosomes,inhibiting autophagosome maturation,and ultimately inhibiting autophagy activity.Therefore,this study explores the mechanism of Salvianolic Acid B in the treatment of CGN by establishing a rat model of cationized bovine serum albumin(C-BSA)chronic nephritis to observe the effects of Salvianolic Acid B on CGN,and to observe the regulation of Salvianolic Acid B on inflammation,also to observe the effect of related signaling molecules in the pathway of mi R-145/Rab14/autophagy.ObjectiveObjective 1.Established the C-BSA-induced CGN rat animal model.24 h urine protein content,renal function,oxidative stress injury,the pathological changes of the kidney and the expression levels of inflammatory factors in the kidney were observed to evaluate the protective effect of Salvianolic Acid B on the kidneys of CGN rats.2.Detected the expression level of mi R-145,Rab14 and autophagy-related protein in rat kidney by q PCR and Western Blot,explored the effect of Salvianolic Acid B on C-BSAinduced nephritis mechanism of kidney protection in model rats.3.Human mesangial cells(HMC)were cultured and stimulated with lipopolysaccharide(LPS)to establish an HMC inflammatory proliferation model.Salvianolic acid B was used for intervention to observe and evaluate the proliferation of Salvianolic Acid B on HMC and the effects of apoptosis and growth cycle.4.The mimics and inhibitors of mi R-145-5p were transfected into HMC.The expression of mi R-145-5p in HMC was detected to observe the intervention effect of Salvianolic Acid B,and the regulation effect of Salvianolic Acid B on mi R-145 was clarified.At the same time,the changes of mi R-145/Rab14/autophagic signaling pathway were explored to reveal the molecular mechanism of Salvianolic Acid B in protecting HMC induced by LPS stimulation.Methods 1.Observing the effect of Salvianolic Acid B on CGN rat model.Male Sprague-Dawley(SD)rats were injected with C-BSA emulsion subcutaneously at multiple sites for pre-immunization,and CGN models were established by continuous tail vein injection of C-BSA.Since the third week of model establishment,low and high doses of salvianolic acid B(50 mg/kg,100 mg/kg)were administered continuously for 3 weeks.The urine protein,serum creatinine,blood urea nitrogen,SOD,MDA,and GSH were measured to evaluate the protective effect of Salvianolic Acid B on renal function in CGN rats.Rats' kidneys were stained with HE and PAS.Pathological changes of rat kidneys and proliferation of mesangial cells were observed.The expression of Ki 67 in kidney tissues was measured by immunohistochemistry.Ig G and C3 deposition in glomeruli were observed by immunofluorescence.The expression of IL-6 and IL-1? in renal tissues was used to evaluate the effect of Salvianolic Acid B on immune inflammation in CGN rats,and a comprehensive evaluation of the intervention effect of Salvianolic Acid B on CGN model rats was performed.2.Investigating the effect of Salvianolic Acid B on renal mi R-145/Rab14/autophagy signaling pathway in CGN rats.After 3 weeks of drug intervention,total RNA was extracted from rat kidney.the expression of mi R-145 was detected by q RT-PCR.Total rat kidney protein was extracted,Rab14 and autophagy related proteins p62,Beclin1,and LC3 were detected by Western Blot.The expression of referred protein were studied to investigate the effect of salvianolic acid B on the mi R-145/Rab14/autophagy signaling pathway in kidneys of CGN model rats.3.Observing the effect of Salvianolic Acid B on proliferation and apoptosis of HMC stimulated by lipopolysaccharide(LPS).HMC were cultured and stimulated by LPS to establish an inflammatory proliferation model.Salvianolic Acid B was administered to intervene.MTT and CCK8 methods were used to detect the effect of Salvianolic Acid B on the proliferation of HMC;The growth cycle of HMC was detected by PI flow cytometry.The effect of Salvianolic Acid B on HMC apoptosis was detected by Anexin V/FITC.4.Investigating the effect of Salvianolic Acid B on mi R-145/Rab14/autophagy signaling pathway in LPS-stimulated HMC model.To study the effect of Salvianolic Acid B on mi R-145/Rab14/autophagy signaling pathway,Mimics(30 n M)and inhibitor(100 n M)of mi R-145-5p were transfected into HMC.The expression of mi R-145-5p was detected by q RT-PCR.The expression of Rab14,p62,beclin1 and LC3 were detected by Western Blot.Results1.Effect of Salvianolic Acid B on CGN rat model.The results showed that compared with the rats in the model group,low and high doses of Salvianolic Acid B significantly improved the activity,feeding,and hair luster of the CGN model rats,reduced the urinary protein excretion of the rats for 24 hours.The serum creatinine,urea nitrogen,MDA content were reduced.The serum SOD,GSH expression levels were increased.And kidney disease due to swelling was inhibited.Kidney histopathology results showed that low and high doses of Salvianolic Acid B could inhibit the proliferation of mesangial cells in the kidneys of CGN rats,repair renal tubular injury,reduce renal interstitial infiltration,and reduce the expression of Ki67 in renal tissues.At the same time,the immunofluorescence results showed that Salvianolic Acid B could improve the deposition of glomerular Ig G and C3 complement in CGN model rats.The results of ELISA showed that the contents of IL-6 and IL-1? in renal tissue were significantly reduced in the low-and high-dose Salvianolic Acid B groups.2.Effect of Salvianolic Acid B on renal mi R-145/Rab14/autophagy signaling pathway in CGN rats.According to the results of q RT-PCR,the expression of mi R-145 in the kidney tissue of rats with low and high Salvianolic Acid B increased significantly compared with the model control group;Western Blot test results showed that the expression of Rab14 in renal tissue of rats was decreased with Salvianolic Acid B in low-and high-dose.The expression of autophagy-related protein p62 was decreased,while the expression of Beclin1 and LC3 protein was increased.3.Effect of Salvianolic Acid B on proliferation and apoptosis of HMC stimulated by LPS.Compared with the LPS model group,it was found that Salvianolic Acid B can significantly inhibit the abnormal proliferation of HSMC stimulated by LPS,but it has no significant effect on cell apoptosis.4.Effect of Salvianolic Acid B on mi R-145/Rab14/autophagy signaling pathway in LPSstimulated HMC model.The PCR results suggested that Salvianolic Acid B could promote the expression of mi R-145-5p in HMC;Western Blot results suggested that Salvianolic Acid B could synergize with mi R-145 to inhibit the expression of Rab14 and enhance the autophagic activity of cells.Conclusions 1.Salvianolic Acid B have a good therapeutic effect on CGN model rats in the 24 h urinary protein content reducing,kidney function protecting,oxidative stress injury reducing,renal inflammatory response inhibiting,and renal pathological changes improving.2.Salvianolic Acid B can inhibit the abnormal proliferation of HMC stimulated by LPS,but it has no obvious effect on cell apoptosis.3.Salvianolic Acid B can up-regulate the expression of mi R-145 in CGN rat model and LPS-stimulated HMC model,inhibit Rab14 expression and enhance autophagy.The results suggest that Salvianolic Acid B may inhibits the proliferation of mesangial cells and regulates the immune and inflammatory response in the kidney through the mi R-145/ Rab14/autophagy signaling pathway.