Study About ROCK1 Expression In Different Stages Of Hepatic Fibrosis Induced By Carbon Tetrachloride

Objective:Liver fibrosis is the main serious disease which is harmful to health .Liver fibrosis is the excessive accumulation of extracellular matrix (ECM) proteins that occurs in most types of chronic liver diseases. HSC is the core of hepatic fibrosis ,it can synthesize and secrete much more extracellular matrix(ECM) by autocrine or paracrine, at the same time it also synthesize and secrete much more TIMPs which can suppress the activity of MMPs (such as collagenase, and gelatinase )and reduce the degradation of ECM. Finally, it leads to accumulation of ECM in the liver, cause liver fibrosis. This complex pathological process is controlled by multiple signaling pathways and a series of cellular molecular networks. A lot of cytokines such as Transforming growth factor-β,ERK interact each other through TGFβ-Smad pathway, MAPKpathway, PI-3K pathway, and many other cell signaling pathways,which induced the ophysiological changes of liver fibrosis. Rho / Rho kinase signaling pathway is a common signal transduction pathway in the body which controll the biological behaviour, Such as cell morphological features, cell adhesion and migration, cell Proliferation and apoptosis, gene transcription by regulating the polymerization status of actin cytoskeleton . This cause cell contraction, adhesion and migration, growth and division, stress fiber produced, collagen synthesis and other biological effects. It has close relation with asthma, hypertension, atherosclerosis, tumor invasion and metastasis as well as multi-organ fibrosis and other diseases. Currently, changes of Rho-ROCK and their relationship with Hepatic fibrosis are not clear. To detect the expressions of ROCK1 in different phases of liver fibrosis, we can indicate the effects of Rho-ROCK on the formation of hepatic fibrosis.Methods:①experimental animals: healthy Wistar male rats were selected and randomly divided into control group and model group. The control group : fed normally, the model group : 2mL/kg of carbon tetrachloride(CCl4) is injected into the peritoneum of mice twice a week. The animals(n=6) are killed on3, 6 days and at 2, 4, 6, 8 weeks after injecting carbon tetrachloride. A portion of liver tissue was fixed in formalin and embedded with paraffin for HE staining and immunohistochemistry; the other liver tissues was stored in liquid nitrogen for PCR.②Observing parameter :HE staining was used to observe the pathological changes of live tissues.,Quantitative detection of ROCK1 gene transcripts in different periods were preformed by PCR. Immunohistochemistry were used to detect the location and expression of ROCK1, collagenⅠ, collagenⅢin the tissues.③Data Statistics and Analysis: The results were analyzed by variance analysis with SPSS10.0, P <0.05 have statistically significant.Results:1 HE staining :In the early stage , liver sinusoid was expansive and congestive, inflammatory cell infiltration with focal necrosis of liver cells.In medium-term , the hepatic cell was swelling, vacuole-like changes, fibrous tissue appeared in periportal.In later period ,a large number of hepatic cells were degenerative and necrotic and a lot of fibrous tissue was proliferate in interstitial.In this period pseudolobule was formed, deposition of collagen fibers increased significantly and the structure of liver was distorted.2 Fluorescence quantitative PCR: With the development of liver fibrosis, the mRNA transcription level of ROCK1 increased gradually, the△△CT value became bigger gradually ,and the eighth weeks ,it tends to be stable.3 Immunohistochemistry : With the formation of liver fibrosis ,the positive area of Rock1, collagenⅠ, collagenⅢincreased gradually, Rock1 had significantly positive correlation with collagenⅠand collagenⅢrespectively.Conclusion: The expression level of Rock1 protein increased gradually in the formation and development of liver fibrosis.This indicate that ROCK1 may play an important role in the occurrence of liver fibrosis , and the Rho / Rho kinase signaling pathway may be another important signaling pathway of liver fibrosis.