The Effects Of Interferon-α On The Early Apoptosis Of Activated Hepatic Stellate Cells

Live fibrosis is a common pathology process of chronic liver disease, considered as early stage of cirrhosis., Chronic hepatitis B is the major cause of hepatic fibrosis and cirrhosis in China. Recently, IFN-αhas been used to be antiviral drug for teatmeant of viral hepatitis clinically, meanwhile some scholars discovered that IFN-αhas an independent anti-hepatic fibrosis acton having nothing to do with antiviral action. But the mechanism of anti-hepatic fibrosis is still unclear. The purpose of this study is to figure out the change of apoptosis in activated rat HSC exposed to IFN-αin vitro by MTT and flow cytometry, recognize the mechanism of interferon-αanti-fibrosis in liver, and provide the theoretical basis for clinical treatment.Materials and methodsIn our study, the HSC of rat was cultured in vitro,and then selected respectively three generations of cells in growing well were selected respectively. The activated rat HSC was cultured in the DMEM nutrient fluid that contains the IFN-alpha density is 0, 1×10², 1×10³, 1×10⁴, 1×10⁵ the U/ml for 24 hours respectively, then HSC was continued to culture for 4 hours after 50ul MTT was added. The serum was removed, and 100ul DMSO was added and shaked for 10 minutes. The absorbance value of every holes was measured at the wave of 492nm with the enzyme-labelling equipment. The absorbance value represents the cell numbers in different holes. Again three generations of cells were selected in growing well, The activated rat HSC were cultured in DMEM of the same density IFN-αas previous described for 24 hours, 1ml cells were centrifuged for 10 minutes at 4℃with the speed of 1000rpm and then the supernatant was removed. They were shaked gently in order to keep the cells suspension after 1ml cold PBS was added, and then They were centrifaged for 10 minutes at 4℃with the speed of 1000rpm and then the supernatant was removed. The cells was suspended in 200ul Binding Buffer and mixed with 10ul Annexin V-FITC and 5ul PI, and then gently uniform mixing. They were put away from light at room temperature for 15 minutes. At last, 300ul Binding Buffer was added, they should be measured within one hour by flow cytometry. Datas were analysized by t-test using SPSS11．0 software.ResultsThe study of the effect of IFN-αon the apoptosis of activated rat HSC.1．MTT method result: After the activated rat HSC was cultured in DMEM culture solution with the concentration of IFN-α0, 1×10², 1×10³, 1×10⁴, 1×10⁵ the U/ml for 24 hours, the OD value was measured (0．493±0．0518,0．4497±0．0338,0．4273±0．0566,0．3657±0．0581and 0．2707±0．0387respectively). According to the results above, we can find after the stimulation of IFN-α, the activated rat HSC number decreased with increased IFN-αconcentration. Datas were analyzed using SPSS software. The differences between IFN-αgroups(1×10⁴andl×10⁵U/ml)and control groups were statistically significant. P value less than 0．05 has statistical significance. (t=2．82 and t=5．44>t_{0．05/2(4)} ＝2．132, P<0．05) It demonstrated that the IFN-αhad the effect of inducing rat HSC apoptosis at the concentration of 1×10⁴and1×10⁵U/ml.2．Flow gytometry method: The activated rat HSC was cultured in DMEM culture solution with the concentration of IFN-α0, 1×10², 1×10³, 1×10⁴, 1×10⁵ the U/ml for 24 hours respectively. After Annexin V-PI-staining, according to the result which was measured by flow cytometry, the red-marking-points in right inferior quadrant were more in IFN-αgroups and less in contral groups. The early apoptosis ratio was 0．0327±0．001652,0．0553±0．008921,0．0915±0．0200．The early stage apoptosis ratio was significantly different between the groups of 1×10⁴, 1×10⁵ U/ml and their corresponding control group by SPSS software. P value less than 0．05 has statistical significance.(t=4．31and t=5．07>t_{0．05/2(4)} ,P<0．05) It showed that IFN-αinduced the early apoptosis of rat HSC.Conclusions1．IFN-αhas the effect of inducing apoptosis of actived rat HSC cultured in vitro.2．IFN-αinducing the apoptosis of rat HSC is mainly in the early stage, that is promoting the early apoptosis of rat HSC.