Inhibitory effect of CLC-2 chloride channel targeted blocking on fibrosis of human conjunctival fibroblasts

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.

Glaucoma; Filtering surgery; Chloride channels; Filtering bleb; Cicatrix; Apoptosis, cells; Cell migration; Collagen contraction