Study The Effect And Mechanism Of Danggui-shaoyao-san Containing Serum On Endothelin-1-mediated Inhibition Of Hepatic Stellate Cell Contraction

Objective: This experiment by activation of rat hepatic stellate cells(HSC-T6 cell line)as the research object,observation after the treatment Danggui shaoyao san(DSS)containing serum on the influence of ET-1 gene in HSC-T6; Further explore DSS containing serum on HSC-T6 induced by ET-1 cell contraction effect and possible mechanism of action, to provide a new theoretical basis of DSS containing serum reduce cirrhotic portal hypertension.Methods: 1.Using MTT method was developed for the determination of the cell vitality,select suitable rat serum concentration range.2. Detection of DSS containing serum on activation of HSC-T6 cell ET-1 molecular biology: by ELISA test groups in the cell culture supernatant on protein expression of ET-1; q RT-PCR to detect each group ET-1 m RNA expression in HSC-T6. The experimental group: blank serum control group, DSS drug-containing serum group, 3.The detection of DSS containing serum on the HSC-T6 contraction induced by ET-1:the FITC-ghost pen cyclic peptide cytoskeleton staining to observe HSC-T6 cell morphological changes; with the collagen lattice method is used to observe the contraction of HSC-T6. The experiment is divided into six groups:blank control group,model group, the DSS containing serum low dose group, DSS containing serum dosage group, DSS containing serum levels of high dose group, Y-27632 inhibitor group.4. Using Western blot method to detect different concentrations of DSS containing serum on Rho A, ROCK?, p-MLC?, MLC? protein expression in HSC-T6 cells induced by ET-1; Respectively the q RT-PCR and Western blot method to detect different concentrations of DSS containing serum on the HSC-T6 cells induced by ET-1in the expression of e NOS m RNA and protein level, study the effects of DSS containing serum on HSC-T6 induced by ET-1 cell signal transduction mechanism of contraction.The experiment is divided into six groups: blank control group, model group, the DSS containing serum low dose group, DSS containing serum dosage group, DSS containing serum levels of high dose group, Y-27632 inhibitor group.Results: 1. Determined by MTT method to detect the results show that, within the scope of 5%-15%, cell proliferation and vitality is stronger and the serum concentra- tion was positively proportional growth. Therefore, in the later experiment, selects the serum concentration of 5%, 10% and 15% as a medicated serum concentration.2. ELISA method to detect the ET-1 in HSC-T6 cells cultures of the protein expression of the results showed that compared with the blank serum group, DSS containing serum group(final concentration of 5%, 10%, 15%) ET-1 protein expression in cells decreased obviously(P < 0.05 or P < 0.01); q RT-PCR method of quantitative detection of HSC-T6 cells ET-1 m RNA expression level results show that compared with the blank serum group, DSS containing serum group(final concentration of 5%, 10%, 15%) in the cell of ET-1 m RNA expression level with the increase of DSS containing serum concentration gradually decreases, the final concentration of 10% and 15%,significantly decreased(P < 0.01).3. Through the FITC-ghost pen cyclic peptide cytoskeleton staining to observe HSC- T6 cells morphological changes, inverted fluorescence microscope is visible by the dye FITC-ghost pen cyclic peptide model group in a large number of microfilament cytoskeleton F-actin, DSS containing serum(final concentration of 5%, 10%, 15%) and the Y-27632(including 100 ?mol/L) to different degree of inhibition of ET-1(10nmol/L) inducing HSC-T6 cytoskeleton in F-actin populations; Through the collagen lattice experiment results showed that ET-1(10 nmol/L) can lead to HSC-T6 cells contract obviously, and the blank serum compared to the control group(final concentration of 10%),(P<0.01); DSS containing serum group(final concentration of5%, 10%, 15%) can be induced by different degree of inhibition of ET-1 HSC-T6 contraction, and presents a certain concentration dependence, compared with model group, the final concentration of 10%, 15%, could inhibit the ET-1(10 nmol/L)inducing HSC-T6 cells contract,(P<0.05); Y-27632(100 ?mol/L) has a significant inhibitory effect on HSC-T6 cells contraction caused by ET-1(10 nmol/L) as well, the difference was statistically significant compared with model group(P<0.05).4. By Western blot method to detect Rho A, ROCK?, p-MLC?, MLC? protein expression levels, The results showed that compared with the blank serum group, model group Rho A, ROCK?, p-MLC?, MLC? protein expression were signifycantly higher(p < 0.01); Compared with model group, DSS containing serum each dose group decreased Rho A, ROCK?, p-MLC?, MLC?(p<0.05) protein expression, and present a dose dependent, Y-27632 inhibitor protein expression above group compared with model group were also significantly lower(p<0.05). At the same time, Western blot method according to the results, the amount of e NOS protein expression was obviously decreased in the model group cells(p<0.01); Compared with model group, DSS containing serum e NOS protein expression in cells each dose group were increased, and as the angelica peony drug-containing serum concentration increased each dose group of e NOS protein expression in the cell has a rising trend.Conclusion: 1. DSS containing serum can inhibit ET-1 m RNA expression in the activation of HSC-T6 cells.2. DSS containing serum can inhibit HSC-T6 induced by ET-1 cell contraction.3. DSS containing serum inhibition of ET-1 inducing HSC-T6 cell contraction may be by activating Rh OA/ROCK, And through activation of e NOS, improve the NO level in the cell.