ABSTRACT
Objective:To investigate the inhibitory effect of CLC-2 chloride channel targeted blocking on fibrosis of human conjunctival fibroblasts (HConF).Methods:HConF were divided into blank control group, lipofectamine 2000 (Lipo2000) group, nonsense small interfering RNA (siRNA) group, and CLC-2 siRNA transfected group.The HConF were cultured in medium containing the corresponding transfection reagents according to grouping.No intervention was given to blank control group.The expression level of CLC-2 mRNA of HConF was detected by real-time fluorescence quantitative PCR; absorbance ( A) value indicating the proliferative ability of HConF was determined by CCK-8 kit; the apoptosis ratio of HConF was tested by flow cytometry; the migration ability of HConF was identified by cell scratch test and Transwell migration assay; the contraction rate of HConF was assayed by collagen contraction test; the expression levels of collagenⅠ, collagen Ⅲ, PI3K, Akt, p-PI3K and p-Akt proteins were measured by Western blot. Results:Significant differences were found in relative expression levels of CLC-2 mRNA and A value among four groups ( F=90.110, 198.680; both at P<0.001). The relative expression level of CLC-2 mRNA and A value were significantly lower in CLC-2 siRNA transfected group than nonsense siRNA group, showing statistically significant differences (both at P<0.001). The proportion of apoptotic HConF in blank control group, Lipo2000 group, nonsense siRNA group, and CLC-2 siRNA transfected group was (4.78±1.10)%, (4.54±1.51)%, (4.82±0.88)% and (28.90±0.91)%, respectively, and a statistically significant difference was found ( F=363.260, P<0.001). The proportion of apoptotic HConF was significantly higher in CLC-2 siRNA transfected group than nonsense siRNA group, with a statistically significant difference ( P<0.001). Statistically significant differences were found in cell migration rate and the number of migrating cells among four groups ( F=74.493, 1 625.431; both at P<0.01). The cell migration rate of HConF in CLC-2 siRNA transfected group was significantly lower and the number of migrating cells was significantly smaller than those of nonsense siRNA group, with statistically significant differences (both at P<0.001). A statistically significant difference in contraction rate was found among four groups ( F=104.692, P<0.001). The contraction rate of HConF was significantly lower in CLC-2 siRNA transfected group than nonsense siRNA group, and the difference was statistically significant ( P<0.001). Statistically significant differences were found in relative expression levels of collagen Ⅰ and collagen Ⅲ proteins, p-PI3K/PI3K ratio, and p-Akt/Akt ratio among four groups ( F=112.073, 456.931, 340.889, 43.021; all at P<0.001). The relative expression levels of collagen Ⅰ and collagen Ⅲ proteins, p-PI3K/PI3K ratio and p-Akt/Akt ratio in CLC-2 siRNA transfected group were significantly lower than those of nonsense siRNA group, showing statistically significant differences (all at P<0.05). Conclusions:Targeted blocking of CLC-2 chloride channel gene expression can inhibit fibrosis of HConF by promoting apoptosis of HConF through PI3K/Akt signaling pathway and inhibit fibrotic processes such as cell migration, collagen synthesis and collagen contraction.