Kangxian Ruangan Granule On Rat Hepatic Stellate Nucleolar Non-histone Protein

ObjectiveFrom the regulation of protein level studies in the liver of KXRG (anti-fiber soft granule) on hepatic stellate cells (HSC) proliferation control mechanism, to further explore the role of anti-hepatic fibrosis KXRG mechanism so as to further clarify the anti-Chinese medicine pharmacological mechanism of liver fibrosis and the role of the target for the development of new anti-hepatic fibrosis drugs to provide theoretical basis.MethodsUse of activated hepatic stellate cells (HSC-T6) culture passage, and then were added to traditional Chinese medicine KXRG (1.25mg/ml,2.5mg/ml,5mg/ml) and the rats were drug-containing serum (5%,10%,20%) were cultured, and established a control group. 72 hours later, each group a portion of HSC cells observed by flow cytometry DNA content in each cycle, and used electron microscopy to observe the changes of ultrastructure of the study KXRG of HSC cycle; the same time, another part of the cells for nuclear protein extraction, using Western blot method (Western blot) observed after treatment of non-histone proteins HSC nucleolus (B23, C23) activity level changes, explore KXRG level of protein expression in HSC activation in vitro. Results1. KXRG and its drug-containing serum on activated HSC proliferation in MTT results showed that KXRG containing various concentrations of groups and their serum concentrations can inhibit the proliferation of activated HSC, and the drug-containing serum group was significantly better than the drug group (P< 0.01); the drug group and drug-containing serum group was superior to the control group (P<0.01, P<0.05), and 2.5mg/ml treatment group separately with 1.25mg/ml,5mg/ml drug group is a difference (P <0.01, P<0.05); 10% respectively containing serum and 5%,20% drug-containing serum group significant difference (P<0.01).2. KXRG and its drug-containing serum on the activation of HSC cell cycle measured by flow cytometry in each group of cells in different cell cycle DNA content showed that serum containing different concentrations of group and drug group acting on the cells, after 72h with the increase in the concentration of content of G0/G1 phase cells increased gradually with the control group is a difference (P<0.05, P<0.01), while the S phase and G2/M phase cell content was gradually reduced. That KXRG can prevent cells from G0/G1 phase to S phase, but G2/M phase had little impact which can show KXRG a major role in the G1/S checkpoint, that is, the cell cycle restriction point, while the M phase restriction point, no effect3. KXRG and its drug-containing serum on the activation of HSC electron microscope, the ultrastructure of the normal HSC cell membrane integrity and organelle structure, clear cytoplasm, nuclear membrane integrity, a clear nucleolus, chromatin evenly distributed in nucleus; The KXRG pretreatment, the cell volume reduced, the membrane surface, uneven, cytoplasmic electron density deepening contraction in the nucleus, nucleolus loose, and gradually dispersed around the nuclear membrane, and can see the formation of intracellular vacuoles.4. KXRG and its drug-containing serum on the non-histone proteins nucleolar B23, C23 activity levels Western blot results showed that KXRG can reduce the HSC nucleolar B23, C23 expression of protein activity, and with the KXRG and its drug-containing serum concentration increases.Conclusion:1. KXRG can inhibit the proliferation of activated HSC; block the cell cycle of normal to stay in the G0/G1 phase; and had a significant effect on their ultrastructure.2. KXRG can reduce the HSC nucleolar non-histone proteins B23, C23 activity level expression.3. The study results suggest that:KXRG possible by reducing the non-histone proteins HSC nucleolar B23, C23 activity level of the expression of cell cycle regulation, inhibit the proliferation of activated HSC to achieve the anti-hepatic fibrosis, which may be KXRG anti-liver fibrosis mechanisms.