Dynamic Changes Of Smad Protein In The Development Of Experimental Liver Fibrosis In Rat And Effects Of GanxianrongGranule On Liver Fibrosis

Hepatic fibrosis is a middle stage in the process of various hepatic diseases developing into hepatocirrhosis, which is characterized by deposition of intra and extra-cellularmatrix resulted from their increased synthesis and decreased degradation. The early phase is called hepatic fibrosis, and the sever phase called hepatocirrhosis involving re-configuration of the liver and formation of pseudo lobules. Some immune media or cell factors secreted by active hepatic stellate cell (HSC), damaged and regenerative hepatic cells, Kupffer cells, sinusoids endothelial cells, or natural killing cells, etc, when being stimulated by inflammatory and toxic factors, make biological effects through autocrine and paracrine on receptors of target cells. That constitutes the cytological basis of hepatic fibrosis. The developing of hepatic fibrosis is a gradual process and recent studies concluded that hepatic fibrosis is to some extent reversible, studies on the reverse factors will therefore contribute to better understanding of pathogenetic mechanism of hepatic fibrosis and hepatocirrhosis.Development of hepatic fibrosis is a complex process involving multiple cell factors and cell signaling pathways, one of which is TGFP as a major hepatic fibrosis-precipitating factor. Smads are proteins newly found in cells of humans and animals, and are regarded as the lower reaction element of TGF-pi. Existent studies indicated that activation of TGFp-Smad signaling pathways is closely linked to occurrence and development of hepatic fibrosis and abnormal expression of Smads plays an important role in occurrence and development of hepatic fibrosis, which is becoming hotspots in research concerning hepatic fibrosis. Chinese herbs have long been thought to be one of the effective therapies for treating hepatic fibrosis. while most studies were focused on their effects on inhibiting synthesis of collagen, enhancing degradation of collagen, reducing inflammation, killing virus, and improving blood circulation, no study is available concerning whether Chinese herbs are effective in activating the TGF $ /Smad signaling pathway. Ganxianrong keli is derived from professor Yang's years of experience, and has proved to be effective in prevention and treatment of hepatic fibrosis in clinical trials.This study, based on hepatic fibrosis (CC14) model in mice, continuously observed expression levels of Smad3 and Smad7 through RT-PCR and immunohistochemistry, in order to identify roles of Smad3 and Smad7 in the development of hepatic fibrosis. The other part of the study is the use of Ganxianrong keli for preventing and treating hepatic fibrosis in mice model which was newly established or had been established for 4 weeks, observing serum biochemistry, liver function, hydroxyproline level in liver cells, degree of pathology fibrosis, levels of Smad3, Smad7, TGF-J3 1 of the TGF P /Smad signaling pathway, to investigate effects of Ganxianrong keli on expression levels of Smad3, Smad7, TGF-0 1, hence highlighting a plausible acting mechanism of Ganxianrong keli. The following is the study outcomes:1) The expression levels of Smad3-protein and TGF-P 1-gene in liver tissue of fibrosis mice were higher than normal mice with significant difference (P < 0.05 ) . The expression level was in direct proportion to the severity of hepatic fibrosis in model mice.2) The expression level of Smad7-protein in liver tissue of hepatic fibrosis mice was lower than normal mice with significant difference (P < 0.05) . The expression level was in inverse proportion to the severity of hepatic fibrosis in model mice.3) The expression levels of Smad3mRNA and TGF- 0 lmRNA were in direct proportion of the severity of hepatic fibrosis in model mice. The expression level of Smad7mRNA in inverse proportion to the severity of hepatic fibrosis in model mice. The r value was 0.957> 0.894> 0.838 respectively, of which relativity of Smad3 with hepatic fibrosis was the highest (P < 0.01), and TGF- 0 1 and Smad7 with less relativity (P < 0.05 ) o4) Hepatic function: Compared with the model group, hepatic function of mice in treatment groups was significantly improved with respect to ALT,ALB and TBIL ( P < 0.01) with the more significant difference between Ganxianrong keli prevention group and treatment group (P < 0.05) .5) Serum hepatic fibrosis index: Compared with the model group, serum levels of HA, LN and PCIII of every treatment group were all significantly reduced (P < 0.05) .with Ganxianrongkeli prevention group more ruduced than treatment group (P <0.05) .6) Detection of level of hydroxyproline in liver: Compared with the normal group, the level of hydroxyproline of the model group was significantly increased, with the level of which in direct proportion to the severity of hepatic fibrosis. The level of hydroxyproline was significantly lowered in treatment groups(P < 0.05) Compared with the model group, Compared with treatment group, prevention group of Ganxianrong keli has significant difference (P<0.05) .7) Histo-pathology observation: With the trial going on, hepatic fibrosis gradually resulted from injections of CC14 after 4 weeks compared with 0 week (PO.05). severity of hepatic fibrosis in Ganxianrong keli prevention and treatment groups were less than model group (P < 0.05) with no significant difference between the prevention and treatment groups.8) There were decreased levels of Smad3 and TGF- 3 1 (P < 0.01) and increased level of Smad7 (P < 0.01) in Ganxianrong keli prevention group and treatment group compared with model group. There was significant difference between Ganxianrong keli prevention group and treatment group (P < 0.05) .Outcomes outlined above indicate that:1) Increased expression level of Smad3 and decreased expression level of Smad7 play key role in the development of hepatic fibrosis.2) The expression levels of Smad3mRNA> Smad7mRNA and TGF- J3 lmRNA were defferent degree relativity with the severity of hepatic fibrosis and level of hydroxyproline o3) Ganxianrong keli can protect liver from injury caused by CC14.4) Ganxianrong keli can greatly reduce the expression levels of TGF- ï¿¡ 1, Smad 3, and increase the expression level of Smad7, effectively deterring signal transformation of TGF- ï¿¡ /Smad, which is the molecular basis of effects of Ganxianrong keli on reducing hepatic fibrosis.5) Ganxianrong keli has anti-fibrosis effects. The earlier it is used, the better the effects. It works through inhibiting TGF- ï¿¡ and Smad3, prompting synthesis and release of Smad7, suppressing cell factors that precipitate hepatic fibrosis, preventing the1 activation of HSC, and reducing the deposit and synthesis of ECM.Significance and innovation of the study:1) The study is the first of its kind using RT-PCR and immunohistochemistry to observe the changes of expression levels of Smad3 and Smad7 continuously. In the development of hepatic fibrosis, the expression level of Smad3 is enhanced and the expression level of Smad7 is suppressed, which is consistent with the severity of hepatic fibrosis. Its mechanism is still unclear.2) The study tries to explain the anti-fibrosis mechanism of Ganxianrong keli from a new perspective of cell signaling pathway. Ganxianrong keli most likely works through regulating the expression of Smads, directly checking cell signaling pathways that induce hepatic fibrosis. That is a new explanation of the acting mechanism of Ganxianrong keli. The study provides new evidence for its clinical application and sound basis for finding new treatment options for hepatic fibrosis.