The Protective Effect Of Notoginsenoside R1 On High Glucose Induced Epithelial-mesenchymal Transition In HK-2 Cells

Objective: To investigate the protective effect of Notoginsenoside R1 on high glucose induced epithelial-mesenchymal transition in HK-2 cells.Methods:The human renal tubular epithelial cells line-2(HK-2)were cultured in low glucose DMEM containing 10% fetal bovine serum.The HK-2 cells were randomly divided into 6 groups:(1)control(NG)group: 5.5 mmol/L D-glucose;(2)mannitol(MA)group: 24.5 mmol/L mannitol,the concentration of D-glucose was still 5.5mmol/L,and the osmotic pressure was the same as that of HG group;(3)high sugar(HG)group: 30 mmol/L D-glucose;(4)high glucose+Notoginsenoside R1 low concentration(HG+C20)group: 20 ?mol/L Notoginsenoside R1,the concentration of D-glucose was the same as HG group;(5)high glucose+Notoginsenoside R1 low concentration(HG+C40)group: 40 ?mol/L Notoginsenoside R1,the concentration of D-glucose was the same as HG group;(6)high glucose+Notoginsenoside R1 low concentration(HG+C60)group: 60 ?mol/L Notoginsenoside R1,the concentration of D-glucose was the same as HG group.Each group of cells was cultured for 48 h and then collected protein and RNA for reserve.Morphological changes of each group was observed by inverted microscope.The drug safety range was screened by MTT assay,the optimum drug concentration was determined and the survival rate of each group was determined;Western blot and quantitative real-time PCR(RT-qPCR)were used to detect the expression levels of inflammatory cytokines NF-?B,TNF-? protein and mRNA.ELISA method was used to detect the expression of the protein ?-SMA and FN of fibrin related proteins.Results:(1)By the inverted microscope,it was found that NG group cells adhered to the wall,and the cells were round or elliptic,and the cells were connected to form a typical "pavement stone" pattern.Compared with NG group,the cell morphology of MA group was the same.The cells in the HG group became larger and longer,and showed a long fusiform change,which was expressed as fibroblast pattern,and the appearance of the original "pavement stone" was lost.HG+C20 group,HG+C40 group,HG+C60 group cells were gradually approaching normal cells.(2)The MTT results showed that the safe range of the Notoginsenoside R1 was below 60 ?mol/L,and the results were determined by the reference literature and the MTT experimental results,respectively,to interfere with the cells in the low,medium and high concentrations(20,40,60 ?mol/L).(3)Compared with NG group,the HG group of cell populations,suggest abnormal high sugar can promote cell proliferation,the difference was statistically significant(P<0.05),MA group was no statistically significant difference compared with NG group(P>0.05),so the follow-up experiments excluded high permeability groups,groups compared with NG group difference was statistically significant(P<0.05)from MTT results;(4)Compared with NG group,there was a significant up-regulation(P <0.05)between HG group,HG+C20 group,HG+C40 group,HG+C60 group of the NF-?B,TNF-? mRNA and protein expression(P<0.05),while the expression of ?-SMA and FN protein was also up-regulated(P<0.05).Compared with HG group,HG+C20 group,HG+C40 group,HG+C60 group of the NF-?B,TNF-? mRNA and protein expression significantly down-regulated(P<0.05),and ?-SMA,FN protein expression was also significantly down-regulated(P<0.05),and drug intervention effect was dose dependent(P < 0.05);(5)Correlation analysis showed that the inflammatory factor NF-?B was positively correlated with the fibrosis related factor ?-SMA(r=0.875,P<0.05)and the FN(r=0.857,P<0.05);TNF-? was positively correlated with the fibrosis related factor ?-SMA(r=0.975,P<0.05)and FN(r=0.971,P<0.05).Conclusion:(1)High glucose induced EMT and extracellular matrixc(ECM)accumulation in HK-2 cells,accompanied by inflammatory factors NF-?B and TNF-? production.(2)Notoginsenoside R1 inhibited the production of EMT and ECM-related proteins induced on high glucose induced HK-2 cells.The protective mechanism may be partly through down-regulatingNF-?B,TNF-?.