Effect Of JAK3/STAT1 Pathway On CTGF-induced EMT In HK-2 Cells

Background: Renal interstitial fibrosis plays an important role in the development of diabetic nephropathy.In the interstitial fibrosis process,myofibroblasts which are mainly derived from human renal tubular epithelial cells exert predominant effects.Objective: To investigate the effect of tyrosine kinases Tofacitinib,a kinase inhibitor,on the process of mesenchymal transdifferentiation of human renal tubular epithelial cells,which is incuded by CTGF.And to explore the role of signaling pathway JAK3/STAT1 in renal interstitial fibrosis.Methods: Mesenchymal transdifferentiation cell model of human renal tubular epithelial(HK-2 cells)was constructed with connective tissue growth factor.HK-2 cells were divided into 4 groups according to the CTGF dosage,listed as follows: CTGF(5?g/L),CTGF(10?g/L),CTGF(20?g/L),CTGF(40?g/L).In order to observe the dose-effect relationship of CTGF induction,the expression of cell transdifferentiation markers were taken as indicators.Moreover,HK-2 cells were divided into the following groups based on the duration of action: CTGF(12h),CTGF(24h),CTGF(36h),and CTGF(48h).In order to investigate the time-effect relationship of CTGF induction,the expression of cell transdifferentiation markers were taken as indicators.Subsequently,optimal concentration of CTGF was selected for further experiments(10?g/L).Pretreated with 10?g/L CTGF for 24 hours,HK-2 cells were then harvested and the expression of E-cadherin and ?-smooth muscle actin(?-SMA)were determined by Western blot.To explore the effect of JAK3/STAT1 pathway inhibitor CP690550,HK-2 cells were divided into the following groups: control group(Con),CTGF(10?g/L)group,CTGF+CP690550(20?mol/L)intervention group(CTGF+CP),and CP690550(20?mol/L)group(CP).After 24 hours of culture,the expression of E-cadherin,?-SMA,phosphorylated JAK3(P-JAK3)and phosphorylated STAT1(P-STAT1)were evaluated by Western blot and the release of lactic acid was measured by enzymelinked immunosorbent assay(ELISA).Results:(1)At a certain concentration(5 ?g/L,10 ?g/L),CTGF could induce the expression of E-Cad and ?-SMA in a dose-dependent manner.The expression of ECad protein was down-regulated as the CTGF concentration increased,while the expression of ?-SMA increased with the CTGF concentration.In a certain period of time(12h,24h),CTGF could induce the expression of E-Cad and ?-SMA in a time-dependent manner.The expression of E-Cad protein decreased with time while the expression of ?-SMA protein increases with time.(2)Compared with the control group,the expression of E-Cad protein was significantly decreased while the expression of ?-SMA protein was significantly increased in CTGF group.(3)Compared with control group,the expression level of P-JAK3 and PSTAT1 were up-regulated significantly in the CTGF group while the phosphorylation of JAK3/STAT1 were remarkably inhibited in the CTGF + CP group;no significant change was observed regarding the expression of P-JAK3 and P-STAT1 in the CP group.(4)Compared with control group,the protein expression of E-Cad decreased significantly while the protein level of ?-SMA increased markedly in both CTGF and CTGF+CP group;no significant change was observed regarding the expression of E-Cad and ?-SMA in CP group.Compared with CTGF group,the protein expression of E-Cad increased significantly while the protein level of ?-SMA decreased significantly in CTGF+CP group.(5)Compared with control group,the expression of lactic acid was obviously increased in the CTGF group.Compared with the CTGF group,the expression of lactic acid was obviously decreased in the CTGF+CP group.Conclusions:(1)CTGF can induce the transdifferentiation of renal tubular epithelial cells and activate the JAK3/STAT1 pathway.(2)JAK3/STAT1 pathway inhibitor CP690550 can inhibit the phosphorylation of JAK3/STAT1 involved in the transdifferentiation of renal tubular epithelial cells.(3)The JAK3/STAT1 pathway inhibitor CP690550 can inhibit the transdifferentiation of renal tubular epithelial cells,indicating an anti-fibrosis function.