The effect and mechanisms of chloride channel blocker NPPB on TGF-β1 induced HConF fibrosis

Authors: Sun Lixia,  Li Yingjun,  Cui Renzhe,  Lu Di,  Zheng Yajuan

DOI: 10.3760/cma.j.issn.2095-0160.2019.06.003
Published 2019-06-10
Cite as Chin J Exp Ophthalmol, 2019,37(6): 411-418.

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.

Key words:

Chloride channel; Filtering bleb scarring; Chloride channel blocker

Contributor Information

Sun Lixia
Department of Ophthalmology, Yanbian University Affiliated Hospital, Yanbian University, Yanji 133000, China
Li Yingjun
Department of Ophthalmology, Yanbian University Affiliated Hospital, Yanbian University, Yanji 133000, China
Cui Renzhe
Department of Ophthalmology, Yanbian University Affiliated Hospital, Yanbian University, Yanji 133000, China
Lu Di
Department of Ophthalmology, Yanbian University Affiliated Hospital, Yanbian University, Yanji 133000, China
Zheng Yajuan
Department of Ophthalmology, The Second Hospital of Jilin University, Jilin University, Changchun 130041, China
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Updated: September 25, 2019 — 2:27 am