| Hepatic fibrosis, of which cirrhosis is the most advanced stage, can result from liver disease due to any cause. It is the important link that affects the chronic liver disease. Therefore, prevention and treatment of hepatic fibrosis is the focus of chronic liver disease. HBV and HCV type hepatitis are the most mainly chronic liver diseases in our country. Currently, we are yet under the condition of not having good countermeasure for chronic liver disease and so prevention and therapy of hepatic fibrosis are the important process for those diseases.Hepatic stellate cells (HSCs) play a pivotal role in this process. It is the important production source of extracellular matrix (ECM). So the proliferation and activation of HSCs play a key role in the process of formation of hepatic fibrosis.Migration of cells is the important process of development, damnification and inflammation. During the recondition of liver tissue and formation of hepatic fibrosis, aggregation of HSCs in inflammation areas is correlated with HSCs migration. In cell biology, cell adhesion, spread and migration are a pathological process which is not unknown today.Great importance event has been attached to the contribution of Rho/ROCK signaling pathway to chronic inflammatory fibrosis of several organs, such as lungs and heart. Evidence accumulated that this signaling pathway had protective effects on liver fibrosis, however, the mechanisms underlying are to be elucidated. Now, the inhibitor of Rho/ROCK signaling pathway can be produced. It maybe a new choice of treating of liver fibrosis if we can use this inhibitor in clinical work.Hence, we set about our research from the migration of HSCs. Through experiments in vivo and in vitro, the role of Rho/ROCK signaling pathway in hepatic fibrosis and migration of HSCs were studied. The experiments contained three parts as below:Part 1: Increased Expression of ROCK-I, p-MBS Thr-697 andα-SMA During Hepatic FibrogenesisObjective:To explore the dynamic expression of ROCK-I, p-MBS Thr-697,α-SMA and their mRNA in the hepatic fibrogenesis and the changes of actin cytoskeleton.Methods:The Sprague Dawley rats were randomly divided into two groups, i.e., sham operation group and model group (bile duct ligation, BDL). Livers in model group were harvested at fixed timepoints: 1wk, 2wk, 3wk and 4wk after operation. Livers in sham operation group were harvested at 4wk after operation. Histopathological changes were evaluated by hematoxylin and eosin staining and by Masson's trichrome method. ROCK-I, p-MBS Thr-697, protein in the livers were determined by Western blot and their mRNA were examined by reverse transcription-polymerase chain reaction (RT-PCR), while the distribution of ROCK-I,α-SMA in the livers were assessed immunohistochemistically. The changes of actin cytoskeleton were studied by fluorescence.Results:①With the development of hepatic fibrosis, the positive cells of ROCK-I andα-SMA increased obviously. They mainly resided in the cells of portal ducts, fiber septa, perisinuses and around the proliferation bile ducts. The positive areas in model groups at week 1 to 4 of ROCK-I of the rat livers (14.71%±4.01%,20.66%±2.34%,26.21%±1.74%,32.50%±2.55%) andα-SMA of the rat livers (13.62%±1.48%,23.25%±2.02 %,27.92%±1.61%,33.69%±1.99%) were larger than that in control group (5.35%±1.30%,6.22%±1.56%) respectively, P<0.05.②ROCK-I protein of the rat livers in model groups at week 1 to 4 were increased by 83%, 114%, 196%, 233% than that in control group respectively. p-MBS Thr-697 protein at week 1 to 4 were increased by 80%, 137%, 229%, 334% than that in control group respectively andα-SMA protein were increased by 48%, 87%, 149%, 210% than that in control group respectively.③α-SMA mRNA expressed in normal rat livers and were up-regulated at day 2 and reached the peak at day 21 after operation. ROCK-I mRNA expressed as well in normal rat livers and reached the peak at day 14 after operation. ROCK-I mRNA expression correlated withα-SMA, r=0.718, P<0.05.④Image of fluorescence using FITC-phalloidin to stain F-actin showed, peripheral filament band is smooth and thin in normal liver tissue and with the development of liver fibrosis, the images of fluorescence were inhanced.Conclusions: With the development of liver fibrosis, both protein and mRNA of ROCK-I andα-SMA were increased. At the same time, p-MBS Thr-697 protein was increased as well. It suggested ROCK-I occurred up-regulated expression and activated function. ROCK-I played an important role in liver fibrogenesis which induced the formation of myofibroblast. During hepatic fibrogenesis, actin was proliferated and there was cytoskeleton recombination.Part 2: Y-27632, inhibitor of ROCK-I, Inhibits the HSCs Adhesion and MigrationObjective: To investigate the effects of ROCK-I inhibitor—Y-27632 on expression of ROCK-I, p-MBS Thr-697,α-SMA and adhesion and migration of HSCs and changes of cytoskeleton.Methods: The adhesive inhibition of Y-27632 on HSCs was examined by toluidine blue colorimetric assay,and the inhibition of migration of Y-27632 on HSCs was evaluated by improved Boyden chamber. And the levels of ROCK-I, p-MBS Thr-697,α-SMA in HSCs were assayed by Western blot on protein level and RT-PCR on mRNA level. Meanwhile, the changes of cytoskeleton of HSCs were determined by fluoresence.Results:①Compared with control group, Y-27632 at concentration of 10μmol/L, 20μmol/L and 30μmol/L inhibited the adhesion of HSCs. The inhibition rates were 2.56%, 31.21%, and 60.91%, respectively, P<0.05.②Compared with control group, Y-27632 at concentration of 10, 20, 30μmol/L inhibited the proliferation of HSCs. The inhibition rates were 5.98%, 25.36% and 46.93% respectively, P<0.05; and the inhibition rates of 20μmol/L at 12hr, 24hr and 48hr were 17.58%, 25.36%, 46.93% respectively. So the Y-27632 could dramatically inhibit the proliferation of HSCs in dose-dependent and time-dependent manners.③Compared with control group, the cell number of migration to the bottom chamber decreased in Y-27632 group. The numbers of crossing membrane of HSCs at 12hr, 24hr and 48hr were 19.30±4.19,13.00±3.32,7.9±2.42, P<0.05.④Compared with control group, ROCK-I protein level and mRNA level were reduced by 25% and 52% respectively.α-SMA protein level and mRNA level were reduced by 47% and 47% respectively. Under the influence of Y-27632, p-MBS Thr-697 protein level was reduced by 36%.⑤Image of fluorescence using FITC-phalloidin to stain F-actin showed, under the influence of LPA, peripheral filament band was disappeared and stress fiber was long and thick. Under the influence of Y-27632, peripheral filament band was smooth and thin and stress fiber was obscure.Conclusions: These results suggest that Y-27632 might inhibit proliferation, adhesion and migration of HSCs. At the same time, Y-27632 could inhibit expression of ROCK-I, p-MBS Thr-697,α-SMA and recombination of cytoskeleton.Part 3: Fasudil Inhibits the Formation of Liver Fibrosis and Migration of HSCsObjective: To explore the effects of ROCK-I inhibitor-fasudil on the expression of ROCK-I, p-MBS Thr-697 andα-SMA in liver tissue and the changes of cytoskeleton. To investigate the effects of fasudil on the expression of ROCK-I, p-MBS Thr-697 andα-SMA of HSCs and the changes of cytoskeleton.Methods: The distributions of ROCK-I andα-SMA in livers were assessed by immunohistochemistry. The expression of ROCK-I, p-MBS Thr-697 andα-SMA protein were determined by Western blot. ROCK-I andα-SMA mRNA were determined by RT-PCR. The changes of cytoskeleton were determined by flurorescence.Results:①The positive areas in BDL+fasudil group at week 1 to 4 of ROCK-I of the rat livers (6.49%±3.78%,9.32%±2.05%,10.37%±2.56%,12.41%±3.79%) andα-SMA of the rat livers (7.16%±1.89%,9.53%±2.18%,12.39%±3.46%,15.15%±4.68%) were lower than that in BDL group, P<0.05.②The expressions of ROCK-I, p-MBS Thr-697 andα-SMA protein at week 4 in BDL+fasudil group decreased by 38.68%, 20.50% and 30.69%, respectively. The expressions of ROCK-I andα-SMA mRNA at week 4 in BDL+fasudil group were reduced by 32.39% and 34.46% respectively.③Compared with control group, fasudil inhibited the adhesion of HSCs. The inhibition rates at 12hr, 24hr and 48hr were 19.25%, 34.55% and 68.22% respectively. The numbers of crossing membrane of HSCs at 12hr, 24hr and 48hr were 17.54±3.26, 11.25±2.55, 8.13±1.24 respectively, P<0.05.④Compared with control group, ROCK-I protein level and mRNA level were reduced by 43% and 55%,α-SMA protein level and mRNA level were reduced by 48% and 52%. And p-MBS Thr-697 protein level was reduced by 43%.⑤Image of fluorescence using FITC-phalloidin to stain F-actin showed, compared with BDL group, peripheral filament band of liver tissue became regular in BDL+fasudil group. Peripheral filament band of HSCs under the influence of fasudil became smooth and thin and stress fiber was obscure.Conclusions: These results suggst that fasudil might inhibit the expression of ROCK-I, p-MBS Thr-697 andα-SMA of liver tissue and HSCs. At the same time, fasudil could inhibit proliferation, adhesion and migration of HSCs and recombination of cytoskeleton.
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