Effect Of Compound G-TOA On The Proliferation And Apoptosis Of Hepatic Stellate Cells

Prevention and treatment of liver fibrosis become the emphasis and difficulty spot in international study. The formation and development of prevent liver fibrosis of chronic liver disease to liver cirrhosis, to delay the transition has important significance. Chinese herbal compound in the treatment of chronic diseases have significant advantages, but its disadvantage limit its clinical application, such as the unclear ingredients and lack of awareness of the mechanism. The lead compound G-TOA is a new compounds, which is based on traditional Chinese medicine compound Biejiaruangan tablets compatibility guidance and used combination principle.Hepatic fibrosis is a cell in the liver tissue extracellular matrix components and structure of liver hyperplasia and abnormal deposition caused by abnormal function, and hepatic stellate cells is a major source of liver fibrosis is the central link of ECM, hepatic fibrosis.Inhibition of HSC activation, apoptosis is the main method for the treatment of liver fibrosis.Our previous studies found that the lead compound G-TOA had obvious inhibition effects onhepatic stellate cells. This study further to observe the effect of the compounds on the proliferation and apoptosis of HSC, to investigate the mechanism of anti liver fibrosis.Objective:The activation of hepatic stellate cells in vitro (HSC-T6) as the object of study, effect of lead compound G-TOA on cell proliferation and apoptosis, the effect of G-TOA on the expression of NF-κB/p65, COX-2, Bax, Bcl-2 protein, to explore the mechanism of G-TOA induced apoptosis of hepatic stellate cells may from cellular and molecular level. Methods: 1.MTT method for detection of G-TOA in hepatic stellate cells (HSC-T6) and normal liver cells(L02) cell proliferation:G-TOA of different concentrations (0.625μM、1.25μM、2.5μM、5μM,、10μM、20μM) effect on HSC-T6 and L02 cells, to observe the effect of G-TOA on HSC-T6 and the proliferation of L02 cells by MMT method in drug effects of 24t、48h、72h respectively after. Select the appropriate action time, concentration, in order to further explore the mechanism to prepare.2.Effect of flow cytometry detection of G-TOA on HSC-T6 cells apoptosis:the use of the effect of flow cytometry was used to detect G-TOA on HSC-T6 cells cycle and apoptosis; and to further explore the effect of G-TOA on mitochondrial membrane potential of HSC-T6 cells and free calcium level.3.Morphological method to detect the G-TOA effect on the morphology of HSC-T6 cells and its mechanism:using HE staining, to observe the morpholo gic change after the light microscope.The expression of NF-KB/p65, COX-2 protein was detected by HSC-T6 immunohistochemical method.4.To study the effect of G-TOA Western blot on the apoptosis of HSC-T6 cells: implications fordetection of G-TOA Western Blot on the expression of Bax and Bcl-2 protein in HSC-T6 cells.Results:1.G-TOA can significantly inhibit HSC-T6 cells proliferation, intensity showed a dose time dependent; G-TOA on the growth of L02 cells with bidirectional regulation effect, promote the proliferation of low concentration, high concentration(≥5μM) inhibited L02 cells proliferation, so select after administration of 48h to 2μM、3μM、4μM as the experimental concentration for subsequent experiments.2.Different doses (2μM、3μM、4μM) G-TOA in HSC-T6 cells: ①the proportion of cells in G1 phase and G2 phase did not change significantly, the difference was not statistically significant(P>0.05); ②the apoptosis of G-TOA induced by different concentrations of HSC-T6 were 7.70±0.290%、14.30±1.153%、 22.40±0.536%, and the control group(4.70±0.218%),the difference was statistically significant (P<0.05);③the concentration of G-TOA decreased the mitochondrial membrane potential of HSC-T6 fluorescence intensity were 18.83±0.546%、14.15±0.804%、8.97±0.940%,and the control group (21.30±0.681%),the difference was statistically significant(P<0.05);④the concentration of G-TOA in the HSC-T6 of intracellular free calcium fluorescent intensity increased, respectively,77.97±1.128%,91.13±3.763%,95. 30 ±1.840%, and the control group (70.40±.937%), the difference was statistically significant (P< 0.05).3.Morphological observation results were as follows: ①HE staining showed that the control group, HSC-T6 cells, ranging from the size of compact, connected, cytoplasm s pink. After administration of the smaller cell size, cell shrinkage, nuclear chromatin margination, nuclear pyknosis,or the formation of eosinophilic bodies; ②mmunocytochemistry showed:48 hours of G-TOA HSC-T6 cells,the expression of NF-κB/p65, COX-2 protein were lower than the control group.4.The results show that Western Blot,compared with the control group, the HSC-T6 expression of Bcl-2 protein was significantly decreased after drug intervention(P<0.05), the expression of Bax protein did not change obviously, reduce the proportion of Bcl-2/Bax(P<0.05).Conclusion:The lead compound G-TOA has four effects:①in vitro could obviously inhibit the proliferation of HSC-T6 cells, L02 cells and promote the proliferation of low concentration, high concentration(≥5uM) inhibited cell proliferation; ②it had no significant effect on the cell cycle of HSC-T6 cells, induce the apoptosis of HSC-T6 cells, and its mechanism may be related todecreased mitochondrial membrane potential, increased free calcium level; ③ the HSC-T6 cells by down regulating the expression of NF-icB/p65, COX-2 protein and down regulate the expression of HSC-T6 cells; ④Bcl-2 protein, decrease the ratio of Bcl-2/ Bax. G-TOA through the inhibition of HSC-T6 cell proliferation, induce HSC-T6 cell apoptosis, prevent the excessive activation of hepatic stellate cells, plays the role of anti hepatic fibrosis.