| It is well known that the critical event of liver fibrosis is that hepatic stellate cells(HSCs)become active, the increasing of production and secretion of extracellular matrix(ECM)and the decreasing of degradation of them result in the accumulation of over ECM in the liver. In recovery period, the apoptosis of HSCs increase significantly, on the one hand it eliminates the origin of ECM, on the other hand it also relieves the inhibitory action of tissue inhibitor of metalloproteinase to matrix metalloproteinase, thus promotes the degeneration of ECM. So the apoptosis of HSCs plays a key role in the process of the resolution of hepatic fibrosis. Bicyclol is the Chinese academy of medical sciences institute of drug resistance hepatitis drug development, the chemical name for a 1,5 a carbonyl group, which was a 2,3,2, 3pair of methylene 2 oxygen radicals 4,4 Dimeth yl oxygen radicals. Previous research shows, many experiments bicylol on acute and chronic liver injury were significantly protective effect. Clinical treatments for chronic hepatitis B, C viral hepatitis have achieved good effect. There are studies shown that bicyclol can reduce the level of procollage peptide (PIIIP) of the rats having liver fibrosis; also found that bicyclol have effective in treatment of chronic hepatitis B liver fibrosis clinically . We Speculated that bicyclol possible achieve the treatment of hepatic fibrosis by increasing the HSCs apoptosis.Objective: Through different concentration by design in vitro bicylol on the hepatic stellate cells of rat, the test investigates the effects on proliferation, apoptosis, mitochondrial membrane potential and caspase 3 activities of the activied Hscs. Methods: Rat Hscs was cultured in vitro and divided into 6 groups,①blank control group,②0.01mM bicylol group,③0.1mM bicylol group,④0.5mM bicylol group,⑤without DMSO group,⑥AC-DEVD-CHO group. Application of MTT method and 3H-TdR incorporation assay activated hepatic stellate cell proliferation; With Hoechst3325 staining by fluorescence microscope to detect cell apoptosis rate; With JC-1 dye laser confocal microscope and flow cytometry to detect HSCs mitochondrial membrane potential ; use microplate reader to detect Caspase3 of activity.Results:①The inhibition effect of different concentrations bicyclol on HSCs proliferation: The results of MTT assay showed that, no significant difference was found between the A490 value of the control group and the group without DMSO (P> 0.05). 0.02% DMSO has not significant effect on HSCs proliferation. Every A490 value overall mean of bicyclol groups was significantly higher than that of control , P<0.05.So, bicyclol have inhibition effect on aHSCs proliferation, 0.1mM bicyclol group and 0.5mM bicyclol group were the last. These inhibition effects were the strong on aHSCs proliferation. -TdR incorporation assay showed that, the overall mean of 0.1mM and 0.5mM bicyclol group were lower than the control group( P< 0.05),It show that bicyclol have inhibition effect on aHSCs proliferation .0.5mM bicyclol group was the lowest and the inhibition of aHSCs proliferation of the most Strong.②The inhibition effect of 0.5mM bicyclol on HSCs proliferation at different times: the difference of each group inhibition rate overall mean was statistically significant (p<0.05), it was show that different concentrations bicyclol ion times of bicyclol have effect on the inhibitory rate of HSCs proliferation. After 0.5mM bicyclol interval HSCs 12h, HSCs proliferation was inhibited ; At 24h and 36h , the inhibitory rate reached the peak; after 48h, the inhibitory rate begin decline.③Th promote effect of different concentrations bicyclol on HSCs apoptosis. After bicyclol intervene HSCs 24h, staining HSCs with Hoechest3325, imaging under fluorescence microscopy, We find that the nuclear enrichment and the formation of apoptotic bodies, find there were some HSCs apoptosis. The difference of each group apoptosis rate overall mean was statistically significant (p<0.05), it was show that bicyclol have effect on the rate of apoptosis. 0.5 mM bicyclol Group apoptosis rate over was significantly higher than that of control group, no DMSO group and 0.01mM bicyclol group ( P<0.05). 0.5 mM bicyclol can significantly increase HSCs apoptosis.④The effect of 0.5mM bicyclol on HSC apoptosis rate at different times: by analysis of variance, the difference of each group overall mean of apoptosis rate was statistically significant (P<0.05), indicating that intervention times of bicyclol have effect on the rate of HSCs apoptosis. The overall mean of apoptosis rates of 0h group and 8h group have no difference, after 0.5mM bicyclol interval HSCs 24h and 32h , the apoptosis rate reached the peak .⑤The effect of 0.5mM bicyclol on HSCs mitochondrial membrane potential: By JC-1 confocal laser scanning microscopy detecting mitochondrial membrane potential, the difference of each group of mitochondrial membrane potential was statistically significant (P<0.05);The mitochondrial membrane potentials of 8h Group, 16h group and the positive control group have no difference; all was lower than that of the negative control group. it was show that 0.5mM bicyclol can reduced aHSCs mitochondrial membrane potentials , reaching the peak at 8h; after 24h, the effect begin decline. By JC-1 flow cytometry assaying aHSCs mitochondrial membrane potential, the mitochondrial membrane potentials of 0.5mM Bicyclol group and the control group in after interval 8h and 16h, were statistically significant, were lower than the control group, showing that 0.5mM bicyclol can reduced aHSCs mitochondrial membrane potentials. The mitochondrial membrane potential of 8h, 16h, was statistically significant reduce.⑥Caspase3 activity detection Show that, the difference of each group Caspase3 overall mean was statistically significant (P<0.05), except the control group between 0.01mM group have no difference(P>0.05),, 0.1mM group and Ac-EDVE - CHO group are of non-discrimination, the other the overall mean difference between the two groups were statistically significant(P<0.05), 0.5mM group Caspase3 have highest activity, It was show that 0.5mM bicyclol can increase the activity of the aHSCs and Ac-EDVE-CHO can inhibite the effection .Conclusion:①cell culture in vitro studies have shown that, bicyclol can inhibit HSCs proliferation, the most effective concentration is of 0.5mmol / l, the inhibition on HSCs proliferation is of the strongest between 24 and 36h.②Caspase3 can promote apoptosis of HSCs.③Bicyclol can decrease mitochondrial membrane potential of aHSCs.④Bicyclol can make Caspase3 activity of aHSCs enhanced. |
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