Dynamic Changes Of IGF-1 In The Development Of Hepatic Fibrosis In Rats And Effects Of Fuzhenghuayu On Hepatic Fibrosis

Objective:Hepatic fibrosis is a complicated pathological course, in which all kinds of etiological factors cause hepatic cells to damage, and then lead to hepatic inflammation and tissue reparation. The imbalance between synthesis and degradation of ECM can result in excessive ECM deposition in liver, which leads to formation of hepatic fibrosis and even progression to liver cirrhosis. Hepatic fibrosis may be reversed and is a common pathological process in which all kinds of chronic liver diseases may develop into cirrhosis. It is proposed that cytokines may play critical roles in the pathogenesis of hepatic fibrosis. Hepatic stellate cell (HSC) secretes various cytokines and is the source of ECM production. The HSC activation and its proliferation play a pivotal role in the formation of hepatic fibrosis. Several cytokines, such as TGF-β1 and IGF-1, can regulate the activation and proliferation of HSC as well as the production of ECM. Under the physiological conditions, the structure and function of liver may be dependent on the balance between synthesis and degradation of ECM. However, the HSC can be activated by various cytokines and can secrete cytokines, as a consequence of liver inflammation due to all kinds of etiologies. These cytokines promote the HSC activation and its proliferation, leading to the production of ECM by HSC. The ECM deposits in the liver, and then hepatic fibrosis or even cirrhosis forms. IGF-1 regulates cell growth, differentiation, proliferation, and anabolic metabolism as a polypeptide growth factor. In cultured HSC, IGF-1 increases proliferation and collagen synthesis, providing evidence that IGF-1 could play a role in proliferation of activated HSC and in liver fibrosis. However, the change of IGF-1 is not clear in the process of liver fibrosis. Liver fibrosis progresses to cirrhosis which is a final phase of a wide number of chronic liver diseases and a high risk of liver failure and hepatocellular carcinoma. Many complications may occur and cause patients suffering at the stage of cirrhosis. Cirrhosis is the inevitable result of liver fibrosis progression. Therefore, the inhibition and prevention of the development of fibrosis might be an effective strategy to improve the prognosis of cirrhosis patients. Recent clinical trials have revealed that treatment with interferon and nucleoside analogue prevents or delays the development of liver fibrosis by inhibiting inflammation reaction. The therapeutic effect of drug is limited and the category of drug is few now. Traditional Chinese drug play a unique advantage of treatment of fibrosis by bi-directional, multi-channel, multi-target regulation.Fuzhenghuayu is composed of Danshen Root, Peach Seed, Pine Pollen, Gynostemma pentaphylla and so on. In Chinese medical science, it can promote blood flow, remove blood stasis, enhance immunity and nourish the liver. In modern research, it can reverse hepatic fibrosis effectively and safely. We study the change of IGF-1 during the course of the model of hepatic fibrosis and observe the Fuzhenghuayu efficacy on hepatic fibrosis and the effect on IGF-1, which is a strong theoretical basis for the seeking of the effective anti-hepatic-fibrosis drug.Method:The experiment was divided into two parts.Part 1:Dynamic changes of IGF-1 in the development of hepatic fibrosis66 male Wistar rats were randomly divided into control group and model group. The rats from the model group were injected intraperitoneally with 30%CCL4 plus olive oil and the control group was injected intraperitoneally with normal saline alone. This process was twice every week. Since the 4th week of model establishment, six rats from the model group and three rats from the control group were sacrificed every two week and their specimens were kept. All rats from the control group and the model group were sacrificed at the 14th week.Part 2:Effects of Fuzhenghuayu on hepatic fibrosis and IGF-157 male Wistar rats were randomly divided into control group, model group and prevention group. The rats from the model group were injected intraperitoneally with 30%CCL4 plus olive oil. The prevention group was injected intraperitoneally with 30%CCL4 plus olive oil, and the control group was injected intraperitoneally with normal saline alone. This process was twice every week and should last eight weeks. The prevention group was poured the feed water incorporated with Fuzhenghuayu into stomach, which was once every day. Since the 4th week of model establishment, six rats from the model and prevention group and three rats from the control group were sacrificed every two week and their specimens were kept. All groups should be observed until the 8th week.Serum ALT and AST levels were detected. Pathological changes in hepatic tissue were observed by H&E and Masson trichrome stainings. The protein expressions ofα-SMA and IGF-1 and their distributionbs in hepatic tissue were examined by immunohisochemistry. The expression of IGF-1 mRNA was analyzed by RT-PCR. The content of hydroxyproline was examined by alkaline hydrolysis.Result:Part 1:Dynamic changes of IGF-1 in the development of hepatic fibrosis1 The biochemical parameters of rats in all study groupsThe serum ALT and AST in the model group were gradually increased at the 4th week,6th week and 8th week and then started to decline from the 10th week, but still remained at higher levels compared with the control group at 14th week. The model group animals showed a significant increase of serum ALT and AST levels as compared with the control group.2 The histopathologic changes of the liverH&E and Masson trichrome staining demonstrated that the structure of the hepatic lobules was normal, the line up of the hepatic plate regular, inflammatory cell infiltrate none, little the fibrous connective tissue in vessel wall and portal area in the control group. The model group showed that the structure of the hepatic lobules was damaged, and hepatic cells were degeneration and necrosis, and many inflammatory cells were seen at the 4th week. The model group demonstrated the liver steatosis and a great quantity of fibroplasia at the 6th week on the basis of liver tissue of model group at the 4th week. Liver steatosis disappeared and fibroplasia was deteriorated at the 8th week. Inflammatory cells were gradually reduced in the late period, so was the collagen fibers.3 The immunohistochemical staining ofα-SMA and IGF-13.1 The protein expression ofα-SMA in hepatic tissueThe a-SMA of model group was significantly increased compared with control group. Theα-SMA was only expressed in the vessel walls in the control group. Theα-SMA expression in the model group was gradually increased. Theα-SMA protein existed among the vessel walls, portal area and the spacing of the fiber and decreased ten weeks later in the model group.3.2 The protein expression of IGF-1 in hepatic tissueThe IGF-1 expression was little in hepatocellular endochylema in the control group. The expression of IGF-1 in the model group presented a gradual increase between the 4th week and the 8th week, exsiting among hepatocellular endochylema and cytomembrane, and was gradually lessen with the improvement of inflammation and fibrosis.4 The expression of the IGF-1 mRNA in hepatic tissue detected by RT-PCRIt was increased that the IGF-1 mRNA of the rats were in the model group compared with the control group. The level of IGF-1 mRNA from the model group was the highest at the 8th week and gradually reduced with stop of CCL4 and reversal of fibrosis.5 The content of hydroxyproline measured by alkaline hydrolysisThe hydroxyproline content from the model group was gradually increased with application of CCL4 and to the highest at the 8th week. The level of hydroxyproline was decreased with stop of CCL4 and reversal of fibrosis. The model group presented a significant increment of hydroxyproline compared with the control group at the corrseponging period.6 The correlation between hydroxyproline content and IGF-1 orα-SMA expressionIt was linear correlation between content of hydroxyproline and expression of IGF-1 orα-SMA. The correlation coefficient was 0.953 and 0.944 respectively.Part 2:Effects of Fuzhenghuayu on hepatic fibrosis and IGF-11 The biochemical parameters in all study groupsThe model and prevention group animals showed a significant increment of serum ALT and AST levels compared with the control group at the 4th week,6th week and 8th week. The ALT and AST in the prevention group were significantly lower than that in the model group at the 4th week,6th week and 8th week.2 The histopathologic changes of the liverH&E and Masson trichrome staining demonstrated that the structure of the hepatic lobules was normal, the line up of the hepatic plate regular, inflammatory cell infiltrate none, little the fibrous connective tissue in vessel wall and portal area in the control group. The model group showed that the structure of the hepatic lobules was damaged, and hepatic cells were degeneration and necrosis, and many inflammatory cells were seen at the 4th week. The model group demonstrated the liver steatosis and a great quantity of fibroplasia at the 6th week on the basis of liver tissue of model group at the 4th week. Liver steatosis disappeared and fibroplasia was deteriorated at the 8th week. Compared with the hepatic tissue in the model group at the same stage, the degree of inflammation and fibrosis in the prevention group was lessened.3 The immunohistochemical staining ofα-SMA and IGF-13.1 The protein expression ofα-SMA in hepatic tissueTheα-SMA was only expressed in the vessel walls in the control group. Theα-SMA expression in the model group was gradually increased. Theα-SMA protein exsited among the vessel walls, portal area and the spacing of the fiber in the model group. The expression of theα-SMA in the prevention group was significantly decreased compared with the model group at the same period.3.2 The protein expression of IGF-1 in hepatic tissueThe IGF-1 expression was little in hepatocellular endochylema in the control group. The expression of IGF-1 presented a marked increment in the model group compared with that of the control group. The expression of the IGF-1 in the prevention group was a significant reduction compared with the model group at the same period.4 The expression of the IGF-1 mRNA in hepatic tissue detected by RT-PCRIt was increased that the IGF-1 mRNA of the rats were in the model group and prevention group compared with the control group. The IGF-1 mRNA of the rats in the model group was consisted with the expression of the IGF-1 at the corresponding period. The expression of the IGF-1 mRNA of the prevention group was apparently lower than that of the model group.5 The content of hydroxyproline measured by alkaline hydrolysisThe model and prevention groups presented a significant increment of hydroxyproline compared with the control group. The prevention group was a significant reduction of hydroxyproline at the same stage compared with model group.Conclusion:1 Intraperitoneal injection with the CCL4 on the rats for eight weeks can lead to the spontaneous reversal hepatic fibrosis model. The pathologic change is consistent with the hepatic fibrosis features in human.2 The level of IGF-1 is a considerable correlation with the degree of the hepatic fibrosis, inflammation and necrosis. The IGF-1 may promote the formation of hepatic fibrosis.3 The IGF-1 expression is parallel with the contents of a-SMA and hydroxyproline. The IGF-1 may facilitate the HSC activation and its proliferation, which is helpful to fibrosis.4 Fuzhenghuayu can obviously restrain the liver inflammation and its fibrous degeneration and improve the liver function.5 Fuzhenghuayu therapy is associated with a decrease of the expression of IGF-1,α-SMA and hydroxyproline, a factor involved in mechanisms of hepatoprotection. Fuzhenghuayu may lessen the HSC activation and its proliferation and then restrain hepatic fibrosis.