| Actually, hepatic fibrosis(HF) is a repairing process of the body towards the injury. Now it is widely accepted that HF is the inevitable and preexisting stage for lots of chronic hepatic disease developing into liver cirrhosis(LC)eventually. Investigation of mechanism of liver fibrosis and reversing the disease are very important for clinical treatment. Hepatic stellate cell(HSC)plays a key role in the pathogenesis of HF. Following chronic liver injury, HSC undergos transdifferentiation to an activated myofibroblastic phenotype with expression ofα-smooth muscle actin (α-SMA) and synthesizes various extracellular matrix protein components, which has the ability of contraction, adhesion and migration. All sorts of cytokines and signal transduction pathway exert very important roles in the activation of HSC.RhoA family proteins are important regulatory molecules of actin cytoskeleton, which take part in all kinds of cell signal transduction associated with cytoskeleton. RhoA mainly increases cell contractility by promoting the assembly of focal adhesion and actin stress fibers. Rho-kinase(ROCK)is now the most distinct downstream signal molecule of RhoA, which can increase the level of myosin light chain phosphorylation though inactivation of MLC phosphatase, cause cross linking of actin and myosin, which promotes actin microfilament polymerization and results in cell contraction, migration and adhesion.RhoA proteins generally cycle between an active GTP-bound, conformation and an inactive GDP-bound conformation. Lysophosphatidic acid (LPA) is first identifyed to activate Rho; C3 transferase can inactivate RhoA by making ribosylation of ADP; and fasudil is an inhibitor of ROCK. Recent years some studies have confirmed that Rho family proteins play potent roles in cell migration. Cell migration related with RhoA/ROCK signaling pathways has correlation with cardiovascular diseases, tumours and fibrotic diseases. In our past study, integrin-FAK signal pathway was found to take part in the HSC migration process, Focal adhesion related non-kinase (FRNK) inhibited the HSC migration. Studies on integrin signalling suggested that the substratum continuously feed signals to Rho proteins in migrating cells to influence migration rate. But RhoA/ROCK signaling pathways how to affect HSC migration process has not been proved.This research will focus on the basis of activation of HSC, through experiments in vivo and in vitro, discuss the roles of RhoA/ROCK signaling pathways in the hepatic fibrogenesis and the effects on the contraction, adhesion and migration of HSC. We hope that it would provide theoretical basis for therapy of liver fibrosis. The experiments contain three parts as below:Part 1: The Dynamic Expressions of RhoA,p-MLC andα-SMA during Hepatic Fibrogenesis in RatsObjective: To observe the expression changes of the key signaling molecules RhoA and phosphorylated myosin light chain belonged to RhoA/ROCK signaling pathways in rat liver tissue during hepatic fibrogenesis, meanwhile, to observe the expression ofα-SMA which represents cytoskeleton element and to discuss the role of Rho/ROCK signaling pathways in the hepatic fibrogenesis.Methods:A rat model of common bile duct ligation (BDL)-induced hepatic fibrosis was used to assess the formation of liver fibrosis. Liver tissues were obtained at 1st,2nd,3rd and 4th week, respectively, in the operation group, and at 4th week in the sham operation group. The liver histopathological changes were evaluated by hematoxylin and eosin staining, and by Masson's trichrome method. Western blotting and Immunohistochemistry were used to determine the expressions of RhoA,p-MLC andα-SMA. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expression of RhoA mRNA.Results:①Building of liver fibrosis model: The results of Hematoxylin and eosin staining and Masson's trichrome methods showed that in sham-operated group, the structure of liver lobule was integrin and the array of the liver flank was in order. But in model groups, the connective tissue hyperplasiaed broadly and the collagen fiber deposited around the central vein. The structure of the lobule was disturbed even the pseudo-lobules were formed.②The outcome of immunohistochemistry showed that the expression of RhoA protein was negative. After 1st week, there becomed some spot-like staining mainly in the interstitial cells in portal tract, the positive cells of RhoA increased a lot after BDL 2nd and 3rd week, they were mainly situated in portal ducts, fiber septa, perisinusoidal cells and around the bile ducts, the staining area enlarged at 4th week. The positive areas of RhoA in the rat livers in model groups at week 1 to 4 (5.32%±0.41%,13.43%±2.13%,22.14%±3.22%,31.48%±2.31%) were all larger than that in control group(0.23%±0.14%), P<0.05; The expression ofα-SMA was weak located in the smooth muscle cells of vessel wall, with the development of liver fibrosis, the expression ofα-SMA increased, there was brown staining in portal ducts, disse space, fiber septa, and around the bile ducts. There was weak staining for p-MLC in vessel walls in the sham operation group, with the development of hepatic fibrosis, the expression of p-MLC was increased especially in the walls of venules, and there was positive staining in the perisinusoidal cells. The positive areas of p-MLC in the rat livers in model groups at week 1 to 4 (13.47%±1.02%,20.05%±2.22%,28.31%±2.05%,35.41%±3.08%) were all larger than that in control group(2.01%±0.51%),and the difference was significant, P<0.05.③By RT-PCR analysis, RhoA mRNA appeared in the liver of sham-operated group (43%±5%), and was upregulated after bile duct ligation, during the liver fibrosis, the expression of RhoA mRNA was 71.22%±1.06%,78.49%±2.24%,81.31%±3.21%,96.38%±2.12%, respectively, after BDL1-4 week, compared with the sham-operated group, and the difference was significant, P<0.05. The expression of RhoA mRNA reached the peak value at the 4th week, which was 2.23 times of that of the sham-operated group.④RhoA, the molecular weight is 24 kD, Western blot analysis showed that in the sham-operated group, the RhoA was expressed a little, but with the development of liver fibrosis, the expression of it increased in model group, the expression increased 4.34, 9.95, 19.85, 22.12 times than that of the sham-operated group. p-MLC , the molecular weight is 18 kD, Western blot analysis showed that the p-MLC was expressed weak in the sham-operated group, The expression level of it was highest in the fourth week and 5 times of that of 1 wk and 43.16 times of that of the sham-operated group. With the development of liver fibrosis, the expression ofα-SMA in model group at week 1 to 4 increased 2.37, 7.08, 13.14, 17.62 times than that of the sham-operated group, the difference was significant, P<0.05. RhoA and p-MLC correlated withα-SMA positively, respectively (r=0.981, P<0.01, r=0.976, P<0.01).Conclusions: In liver fibrogenesis, the expression of RhoA,p-MLC andα-SMA increased obviously. It suggested that RhoA/ROCK signaling pathways and cytoskeleton changes play a pivotal role in the formation and development of liver fibrosis.Part 2: Effects of RhoA/ROCK signaling pathway inhibitors-C3 transferase and fasudil on the rat HSC adhesion and migration Objective: To observe the effects of RhoA/ROCK signaling pathway agonist-LPA, RhoA inhibitor-C3 transferase and ROCK inhibitor-fasudil on the HSC adhesion and migration.Methods: The adhesive effects of LPA, C3 transferase and fasudil on HSC were examined by toluidine blue colorimetric assay,and the effects of migration were evaluated by improved Boyden chamber. Morphological alteration of HSC treated with C3 transferase and fasudil was observed with laser scanning confocal microscopy (LSCM).Results:①Normal activited HSC was well spread. By contrast, HSC treated with C3 transferase changed to round and retractile shape, HSC treated with fasudil (50μM) changed to an elongated morphology with prominent dendritic processes, and appeared powerless shape.②Compared with the control group, LPA increased the HSC proliferation, the increation rate was 12.24%, while C3 transferase inhibited the HSC proliferation, the inhibition rate was 73.26%. C3 transferase inhibited LPA-induced HSC proliferation, compared with the control group, the inhibition rate was 46.51%. Fasudil inhibited HSC proliferation, the inhibition rates were 11.6%, 26.7%, 37.2%, 51.2%, at 12.5, 25, 50 and 100μmol/L, respectively. HSC proliferation correlated with fasudil concention negatively (r=-0.949, P<0.01).③compared with the control group, the adhesion rate increased 25.58% in LPA group, and decreased 50% in C3 group. While C3 transferase inhibited LPA-induced HSC adhesion, the adhesion rate decreased 40.63%. Fasudil inhibited LPA-induced HSC adhesion, and it was in concention-dependent manner; compared with the control group. The adhesive inhibition rates of fasudil 12.5, 25, 50, 100μmol/L were 13.31%, 36.72%, 41.71%, 62.43%, respectively. There was significant difference among the different groups, P<0.05.④LPA induced HSC migration, the cell number of migrating to the bottom chamber increased 6 times in LPA group compared with that of the control group. C3 transferase inhibited HSC migration, simultaneously, inhibited LPA-induced HSC migration, compared with the control group, and there was statistic difference, P<0.01. Fasudil inhibited LPA induced HSC migration, and it was in concention-dependent manner; the cell number of migrating of fasudil 100μmol/L decreased 80% compared with that of the LPA group.Conclusions: RhoA inhibitor-C3 transferase and ROCK inhibitor-fasudil inhibited the LPA-induced HSC proliferation, adhesion and migration. Part 3: The molecular mechanism of the inhibiting migration of RhoA/ROCK signaling pathway inhibitors on rat HSCObjective: To explore the molecular mechanism of RhoA/ROCK signaling pathway inhibitors inhibiting HSC migration induced by LPA.Methods: HSC was cultured in vitro, and the expressions of RhoA, p-MLC andα-SMA were assessed using Western blot and RT-PCR assay. The intracellular free calcium was observed with laser scanning confocal microscopy (LSCM). Results:①Western blot showed: RhoA expression (4326.48±213.32) increased obviously after cultivated with LPA, compared with the control group (2335.54±211.81), up to 46.02%,P<0.01.②When HSC was cultured with C3 transferase for 24 h, The protein expression of RhoA decreased obviously, compared with the control group, down to 73.40 %, meanwhile C3 transferase inhibited LPA-induced HSC RhoA expression, compared with the control group, down to 66.21%.③HSC was induced by LPA for 24 h, the expression of RhoA mRNA was examined by RT-PCR. Compared with the control group, the expression of RhoA mRNA increased 47.92%.④When HSC was cultured with C3 for 24 h, the expression of RhoA mRNA decreased obviously, Compared with the control group, it reduced 68 %. C3 transferase inhibited LPA-induced expression of HSC RhoA mRNA, compared with the control group, it reduced 64 %.⑤LPA increased the protein expressions of p-MLC andα-SMA, compared with the sham-operated group, it increased 33.85%, 36.81%, respectively.⑥Western blot analysis showed: when HSC was cultured with C3 transferase for 24h, the protein expressions of p-MLC andα-SMA decreased 70.45%, 68.34%, respectively, than that in the control group. While C3 transferase inhibited LPA-induced protein expressions of p-MLC andα-SMA, compared with the control group, they decreased 63.24%, 61.83%, respectively.⑦Western blot showed: the expression number ofα-SMA was 2336.53±329.51 in the control group. The protein expressions ofα-SMA in the fasudil 12.5,25,50,100μmol/L group were 2015.78±231.56, 1865.37±203.12, 1549.64±187.13 and 1364.72±140.88, respectively, compared with the control group, it reduced 13.74%, 20.16%, 33.69%, 41.61%, respectively. Fasudil inhibited the expression of p-MLC, the protein expression number of p-MLC in the fasudil 12.5,25,50,100μmol/L group were 2105.49±305.73, 1783.57±216.83, 1544.31±158.67, 1042.83±146.09, respectively, so it reduced 21.66%, 33.64%, 42.54%, 61.22% than that in the control group. The expressions ofα-SMA and p-MLC were in fasudil concention-dependent manners.⑧Compared with HSC in the control group, calcium fluorescence intensity of HSC treated by C3 for 24 h mildly decreased 2.6%, there was no statistic difference between them, P>0.05; C3 transferase had no effect on LPA-induced HSC calcium fluorescence intensity, compared with the control group, there was no significant difference, P>0.05.⑨HSC was treated with fasudil for 24 h, calcium fluorescence intensities of fasudil 12.5,25,50,100μmol/L group were the same on the whole. The results showed that there was no difference among 4 different groups, P>0.05, and compared with the control group, there was no statistic difference, P>0.05.Conclusions: Activated HSC expressed the key signaling molecules RhoA and p-MLC belonged to RhoA/ROCK signaling pathways. RhoA inhibitor-C3 transferase inhibited the expression and transcription of RhoA, and the expression of downstream factor p-MLC was inhibited in the meantime, the expression ofα-SMA was also inhibited. ROCK inhibitor-fasudil inhibited the expressions ofα-SMA and p-MLC, and they were in concention manners. RhoA/ROCK signaling pathway inhibitors had no effect on intracellular free calcium. It belonged to Ca2+-independent signaling pathway.
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