Experimental Study On The Molecular Mechanism Of Curcumin Against Renal Interstitial Fibrosis Based On MiRNAs

Chronic kidney disease(CKD),which is caused by a variety of pathogenic factors,will develop through renal fibrosis to end-stage chronic renal failure(chronic renal failure,CRF).A large number of studies have confirmed that the overexpression of transforming growth factor-?1(TGF-?1)plays a key role in the mechanism of fibrosis,so it is also considered as the main target for the treatment of fibrosis,and Smad protein mediates the intracellular signal transduction of TGF-?.Curcumin mainly comes from traditional Chinese medicinal herbs,such as turmeric,zedoary turmeric and tulip.It not only has strong pharmacological activity in anti-tumor,anti-inflammatory,antioxidation,antiviral,neuroprotection and so on,but also has a good application prospect in anti fibrosis.Our previous study found that curcumin inhibited the expression of alpha smooth muscle actin(?-SMA)and collagen type ?(COL ?)protein in renal interstitial fibroblasts.MicroRNAs is an endogenous noncoding small RNA,which regulates cell proliferation,differentiation and apoptosis,and affects the development of disease.Recent studies show that miR-21?miR-29s plays an important role in renal fibrosis.In this study,the molecular mechanism of curcumin on the anti fibrosis effect of curcumin was further investigated by observing the effect of curcumin on the expression of miR-21?miR-29s and fibrosis related genes.Objective:This subject uses recombinant human recombinant TGF-?1 to induce rat renal fibroblast(NRK49F)transdifferentiation in vitro to construct a renal interstitial fibrosis cell model1.To explore the role and pathway of miR-21?miR-29s in the process of renal interstitial fibrosis.2.To explore the molecular mechanism of curcumin against renal interstitial fibrosis.Method:1.The effect of TGF-?1 on activation and function of NRK49F cells.TGF was used to stimulate NRK49F cells to establish a renal fibrosis cell model,to observe the cell morphology,and to detect the expression of ?-SMA,Smad3,COL I,COL III and miR-21 and miR-29s in cells after TGF-?1 was detected by real-time fluorescence quantitative PCR(real-time PCR).2.The effect of miR-21 on the activation and function of rat renal fibroblasts after it was induced by TGF-?1.Cell transfected with miR-21 inhibitor,then addition of TGF-?1,the expression of a-SMA,Smad3,COL ?,COL ?,miR-21,miR-29s expression in cells was detected by real-time PCR and Western blot(Western Blot).After transfection of miR-21 inhibitor,the cells were then stimulated by TGF-?1,and the expression of ?-SMA,Smad3,COL ? and COL ? were detected by real-time PCR and Western Blot.3.The effect of curcumin on miR-21,miR-29s and fibrosis related factors induced by TGF-?1 in NRK49F cells.TGF-p 1 was used to stimulate NRK49F cells to establish a renal fibrosis cell model,adding different concentrations of curcumin,and using cell immunofluorescence,real-time PCR,Western Blot to detect the changes in the expression of miR-21 and COL ?,COL ?,?-SMA,Smad3 protein.Result:1.TGF-?1(10ng/ml)can induce cell activation in NRK49F cells.The activated cells express a high expression of a-SMA,and the expression of COL ? and COL ? of the extracellular matrix is increased,and the expression of Smad3 in the downstream of TGF-?1 pathway is enhanced.In particular,the expression of miR-21 in cells was significantly enhanced,and the expression of miR-29s decreased significantly.2.After transfection of miR-21 inhibitor(50nM)in cells,TGF-?1(lOng/ml)was given,and the expression of COL ?,COL ?,?-SMA,Smad3 was significantly lower than that of the only group of TGF-?1.3.When cells were induced with TGF-?1(10 ng/ml),different concentrations of curcumin(20,10,5,M)were added.The expressions of COL ?,COL ?,?-SMA and Smad3 were obviously down regulated in each group,and they were drug concentration dependent.The expression of miR-21 in each group decreased significantly,and showed a drug concentration dependent manner.There was no significant difference in miR-29s expression.conclusion1.miR-21 and miR-29s is closely related to the fibrosis process induced by TGF-?1;miR-21 plays a role in promoting fibrosis by regulating TGF-?1/Smads signaling pathway.2.Blocking the expression of miR-21 and its related fibrosis related genes may be one of the ways that curcumin exerts anti fibrosis effect.