In vitro study on the role and mechanism of interferon α-1b in regulating the inhibition of protein kinase Cε and Cα on fibrosis of hepatic stellate cells

Abstract

Objective To investigate the regulation of interferon α-1b (IFNα-1b) on protein kinase Ce (PKCe) and protein kinase Cα (PKCα) which inhibit the fibrosis of hepatic stellate cells (HSC), and to explore its mechanism. Methods HSC-T6 cells were treated with different levels of IFNα-1b (100, 200, 400, 800 and 1 000 U/mL) and the proliferation of HSC-T6 cells was analyzed by methyl thiazol tetrazolium (MTT) assay. Changes of hydroxyproline level were analyzed. The expressions of PKCe and PKCα were detected by immunofluorescence staining. PKCe, PKCα, β-catenin and Survivin mRNA levels were detected by RT-PCR. PKCe, PKCα, β-catenin and Survivin protein levels were detected by Western blot. Variance analysis was conducted by using one-way ANOVA approach. Results The inhibition rates of 100, 200, 400, 800 and 1 000 U/mL IFNα-1b treatment after 24 hours of administration were (15.85±1.05)%, (36.59±1.03)%, (45.12±1.05)%, (50.00±1.01)% and (62.20±1.02)%, respectively, with statistically significant differences among groups (F=27.478, P<0.01). The 48h inhibition rates were (20.87±1.09)%, (43.96±1.08)%, (53.85±1.08)%, (64.84±1.06)% and (74.72±1.07)%, respectively, with statistically significant differences among groups (F=25.321, P<0.01). half maximal inhibitory concentration at 48 h was 343.47 U/mL. The levels of hydroxyproline in 100, 200 and 400 U/mL IFNα-1b groups were (7.48±0.28), (6.26±0.17) and (3.86±0.20) μg/mL, respectively, which were lower than that in control group (8.47±0.32) μg/mL. The differences were all statistically significant (t=4.033, 10.564 and 21.160, respective, all P<0.05). The fluorescence intensities of PKCe in 100, 200 and 400 U/mL IFNα-1b groups were all lower than that of control group. The differences were statistically significant (t=1.984, 2.457 and 7.771, respectively, all P<0.05). The fluorescence intensities of PKCα were also significantly lower than that of control group (t=9.232, 15.921 and 22.222, respectively, all P<0.01). With the increase of IFNα-1b level, the levels of HSC-T6 PKCe, PKCα, β-catenin and survivin were significantly lower than those of control group (t=7.020, 24.562, 45.701 and 14.241, respectively, all P<0.01). With the increase of IFNα-1b, the levels of HSC-T6 PKCe, PKCα, β-catenin and survivin were significantly lower than those of control group (t=9.564, 4.409, 10.036 and 6.794, respectively, all P<0.01). Conclusions IFNα-1b can down-regulate the expression of collagen in hepatic stellate cells in a dose-dependent manner, reduce the expressions of PKCe, PKCα, β-catenin and Survivin, and inhibit the proliferation of HSC-T6 hepatic stellate cells. Key words: IFNα-1b; hepatic stellate cells; proliferation; molecular mechanism