| Renal Fibrosis?RF?which is developed by a variety of chronic kidney disease,is a kind of progressive kidney disease leading to end-stage renal damage and giving rise to the high mortality.One crucial feature of kidney fibrosis is that the renal tubular epithelial cells transition into mesenchymal cells,which is called epithelial-mesenchymal transition?EMT?.The clinical treatment of RF is extremely expensive.Thus,it is very vital to find out the effective treatment drug for RF.Salvianolic Acid B?SalB?which is extracted from traditional Chinese medicine Dan Shen is a high active constituent with water-soluble property.There are lots of researches of SalB and studies mainly focus on pharmacological effects of anti-inflammatory,antioxidant and anti-fibrosis.The resistance to organ fibrosis has been widely concerned.SalB has an activation effect of Sirt1 which paly a variety of functions roles in body.Sirt1 protein has deacetylation of different proteins.In the process of autophagy,activation proteins related autophagy depending on the acetylation of Sirt1.Autophagy has a protective mechanism of organism which remove harmful proteins,cytokines and organelles in cells.In the study of RF,autophagy can effectively remove the collagen deposition in the cells and improve RF.Taken together,this study is going to finding the pharmacological effects of SalB and the function mechanisms of Sirt1 and autophagy in RF so as to provide new ideas for clinical medicine and basic experiment.Objective1.To determine pharmacologic actions and the mechanism of SalB on RF which progressed by chronic nephrosis,RF model of rats subjected to ADR accompanied with unilateral nephrectomy was established and SalB was used to treat RF rats.2.To find out the effect and the mechanism of SalB on TGF-?1 induced EMT model in HK-2 cells,a TGF-?1 induced EMT model in HK-2 cells was established and SalB was used to treat HK-2 cells subjected to EMT.3.In order to illuminate the key role of Sirt1 and autophagy in treatment of SalB on TGF-?1 induced EMT model in HK-2,Sirt1 and autophagy inhibition or activation in TGF-?1 induced EMT model in HK-2 cells was used.Methods1.SD rats were taken unilateral nephrectomies and 3mg·kg-1 and 5mg·kg-1?in saline?Adriamycin was given in one week and one month respectively later.After the model completed,rats were given SalB with high?200 mg·kg-1?,medium?100 mg·kg-1?,low?50mg·kg-1?doses and benazepril as a drug control daily for six weeks.Serum contents of albumin?ALB?,total protein?TP?,creatinine?CRE?,urea nitrogen?UREA?,triglyceride?TC?and cholesterol?CHOL?were detected to evaluate the protective effect of SalB on renal function in rats.HE and Masson staining were uesd to analyse the protection of the pathological changes of SalB in rats.Protein expressions of FN,?-SMA,TGF-?and E-Cad were detected to evaluate the SalB impact on EMT by western blot and immunohistochemistry.LC3,p62,mTOR,Beclin1 and Sirt1 were used to analyse the autophagy on RF rats after treated with SalB.2.The EMT model induced by TGF-?1 in HK-2 cells was established.Cell morphology,MTT,and Anexin V/FITC were usd to analyse the protection of SalB in TGF-?1 induced HK-2 cells.Protein expressions of FN,?-SMA,TGF-?and E-Cadherin were detected to evaluate the SalB impact on EMT induced by TGF-?1 of HK-2 cells with Western blot and immunohistochemistry.LC3,p62,mTOR,Beclin1 and Sirt1 were used to analyse the autophagy on TGF-?1 induced HK-2 cells treated with SalB.3.Rapamycin,chloroquine,resveratrol and nicotinamide were used to co-treated the TGF-?1 induced HK-2 cells treated with SalB.EMT,autophagy and Sirt1 level were detected.The key role of autophagy and Sirt1 in the improvement of TGF-?1 induced EMT in HK-2cells was discussed.Results1.Excretion of 24h urinary protein began to increase after modeling.While this increased 24h urinary protein could to some extent be mitigated by both benazepril and SalB in different dose.Histopathological analyses visualized by HE and Masson staining revealed obvert changes in RF rats.Specifically,there are obvious atrophic necrosis in glomerular and renal tubular epithelial cells and hyperplasia of interstitial collagen.Whereas benazepril and SalB could ensure against these damages in rats suffered from ADR with unilateral nephrectomy.Changes of renal function revealed increased serum Urea,Crea,Chol and TG and reduced ALB and TP which indicated decreased glomerular filtration function and disordered metabolism of the protein and lipid in the RF models.Treatment with benazepril and SalB reversed these changes.The level of EMT in rats with RF was significantly reduced after treated with SalB,which showed significant decrease in the content of FN,?-SMA,TGF-?protein expressions,and significantly increased E-cadherin protein expression compared with the model group.SalB can promote autophagy and protein expression of Sirt1,mainly manifested in SalB compared with model group can increase the ratio of LC3II/I,the expression of Beclin1,Sirt1 protein expression,and reduce the expression of mTOR and p62in kidney tissues.2.The SalB can protect the cellular morphology of TGF-?1 induced HK-2 cells,increase cell activity and inhibit cell apoptosis.The level of the EMT in TGF-?1 was reduced by SalB.Compared with the model group,protein expression of FN,?-SMA,TGF-?were decreased significantly,and protein expression of E-cadherin was significantly increased.SalB can promote autophagy and Sirt1 expression in TGF-?1 induced HK-2 cells.SalB can increase the ratio of kidney tissue LC3II/I,the expression of Beclin1,Sirt1 protein expression,and reduce the expression of mTOR and p62 in the kidney tissues.3.The inhibition effect of SalB on EMT in TGF-?1 induced HK-2 cells depend mainly on the activation of Sirt1 and autophagy,and SalB can be induced Sirt1 activate autophagy and improve EMT in TGF-?1 induced HK-2 cells.Conclusion1.SalB could improve kidney dysfunction and renal pathological damage in RF rats.The protective effect of SalB on RF rats was mainly by inhibiting renal EMT and promoting renal autophagy and Sirt1.2.The SalB can protect the cellular morphology of TGF-1-induced HK-2 cells,increase cell activity and inhibit cell apoptosis.The protective effect of SalB on TGF-?1 induced HK-2 cells mainly by inhibiting renal EMT and promoting renal autophagy and Sirt1.3.SalB Attenuates EMT in HK-2 cells through Activating Sirt1-mediated Autophagy. |
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