The effect and mechanisms of chloride channel blocker NPPB on TGF-β1 induced HConF fibrosis / 中华实验眼科杂志

ABSTRACT

Objective To study the effect and mechanisms of chloride channel blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) on thansforming growth factor β1 (TGF-β1) induced human conjunctival fibroblasts (HConF) fibrosis.Methods Cell counting kit (CCK-8) was used to screen out the optimal TGF-β1 treatment time and the optimal NPPB concentration.The cells were divided into control group,TGF-β1 treatment group and TGF-β1+NPPB group.Cell proliferation and cell cycle were detected by CCK-8 and flow cytometer,respectively.Cell migration ability were observed by scratch and transwell migration assays.Western blot and Real time-PCR were used to detect the expression of collagen Ⅰ (COL-Ⅰ),fibronectin (FN) and α-smooth muscle actin (α-SMA).The phosphorylation level of PI3K and Akt were measured by Western blot.Results TGF-β1 promotes cell proliferation in a time-dependent manner.There was no statistically significant difference in A values between 48 hours and 72 hours after TGF-β1 treatment (P =0.064).Forty-eight hours was selected as the most appropriate time for TGF-β1 treatment.NPPB inhibited HConF cell proliferation in a concentration-dependent manner.Compared with the control group,the proliferation A values of cells in the 50 mol/L and 100 mol/L NPPB groups were significantly reduced (P =0.020,0.000),and 100 mol/L was selected as the optimal concentration of NPPB.The cell proliferation A value,migration area and migration cell number of TGF-β1 +NPPB group were significantly lower than those of TGF-β1 treatment group (all at P＜0.05).Compared with the control group and TGF-β1 +NPPB group,the proportion of G1 phase cells in the TGF-β1 treatment group was reduced,and the proportion of cells in the S phase and G2/M phase were increased,with statistically significant differences between them (all at P ＜ 0.05).The protein and mRNA expression of α-SMA,COL-Ⅰ and FN in the TGF-β1 treatment group were higher than those in the control group and TGF-β1+NPPB group,with statistically significant differences between them(all at P＜0.05);the ratios of p-PI3K/PI3K and p-Akt/Akt in the TGF-β1 treatment group were significantly higher than those in the control group and TGF-β1 +NPPB group,with statistically significant differences between them (all at P＜0.05).Conclusions NPPB may inhibit TGF-β1 induced HConF fibrosis process by inhibiting phosphorylation of PI3K and Akt.