Effects And Mechanism Of Fuzhenghuayu On Hepatic Fibrosis

Objective: Hepatic fibrosis, the liver's wound healing response to a variety of acute or chronic liver injury, is an important pathologic character in many kinds of chronic liver disease. And it will induce hepatic cirrhosis. In some circumstance, hepatic fibrosis was reversible. The main pathological characteristic of hepatic fibrosis is increased irregular deposition of extracellular matrix (ECM) and tissue remodeling. Hepatic stellate cells (HSCs) is the main source of ECM. Currently, it is believed that the activation of HSCs, which play a pivotal role in fibrosis process, will lead to their proliferation and collagen synthesis. It is generally considered that the hepatic cirrhosis, the final stage of hepatic fibrosis, is irreversible. So try to find out the first incentive of fibrosis is an effective method of treating it.As an intracellular lipid second messenger, phosphatidylinositol 3-kinase (PI3K) is discovered resently. It is related with a variety of biological events, such as cytoskeletal reorganization, cell survival, apoptosis and tumor etc. PI3K can produce a variety of lipid products when activated. It not only led to protein kinase B (PKB, Akt) transfer from the cytosol to the plasma membrane, but also promote its conformational change while these products combination with Akt in its PH area. Then, Akt is activated. Activated Akt is considered a key downstream survival factor by regulation cell differentiation, proliferation and metabolism. Studies show that the PI3K/Akt signaling pathway involved in heart, lung, kidney, liver and other tissues and organs of the formation of fibrosis. Up to now, the mechanism of PI3K in hepatic fibrosis, especially the relationship between PI3K with the HSCs differentiation, proliferation and collagen deposition, remain unclear.In recent yeas, The research on the pathogenesis of hepatic fibrosis and its prevention has become one of the hottest problems in the biological domain and medical domain both at home and aborad. At present, there is no specific drugs for hepatic fibrosis. Scholars to look more and more toward the development and use of Chinese medicine. Its research has been furthered into molecular level. A large number of pharmacological studies confirmed that Fuzhenghuayu capsule, which compose of the Salvia miltiorrhiza, fermented Cordyceps powder, walnuts, pine pollen, Gynostemma, Schisandra and other drugs, has the functions of protect liver cells, against lipid peroxidation, inhibition of cytokines promoting liver fibrosis, inhibition of hepatic stellate cell proliferation and promote apoptosis of activated hepatic stellate cells, inhibition of collagen synthesis and secretion, and promote collagen degradation, and so on.This study used intraperitoneal injection with CCl₄ establish a rats model of hepatic fibrosis to investigate the mechanism of PI3K in hepatic fibrosis, especially the relationship between PI3K with the HSCs differentiation, proliferation and collagen deposition. And application Fuzhenghuayu capsule orally to investigate its effect and mechanism on prevention of hepatic fibrosis.Methods: Selected 90 male Wistar rats, body weight about 180～200g, randomly divided into normal control group, hepatic fibrosis model group and Fuzhenghuayu prevention group. Normal group were given 0.9% Sodium Chloride by intraperitoneal injection, two times a week for 8 weeks. Model group were given 30% CCL₄ olive oil (0.2ml/100g) by intraperitoneal injection,two times a week for 8 weeks. And then observed to 14weks. Modeling methods of prevention group in the same manner. Fuzhenghuayu capsule (0.092g/100g) was given by intragastric administration to prevention group, once a day for 8 weeks at beginning of 1st week. The rats, randomly taken out from model group for six, normal group for three at the 4^th, 6^th, 8^th, 10^th, 12^th and 14thweekend, and prevention group for six at the 4^th, 6^th and 8^th weekend, were killed after anesthesia by chloral hydrate. Serumal ALT were measured by automatic biochemical analyzer. Part of the fresh liver tissue fixed in 10% neutral formalin, then embedded in paraffin for routine HE, Masson and immunohistochemistry staining. And part of the fresh liver tissue store at -80℃for Western Blot, RT-PCR and other detection.Results:1 The common behavior of rats: The normal group rats in good mental status. And their hair was neat and shiny, weight increased gradually. With the injection of CCL₄, the model group rats gradual emergence of listlessness, hair loss of gloss, weight gain slowly, and irritably individual. And this state improved gradually after disabling CCL₄. The state of prevention group rats is generally good than that in model group's at the same time.2 Examination of biochemical markers: The serum ALT levels of rats in model group, 76.31±8.16 U/L,137.56±9.48 U/L,262.96±12.16 U/L,167.04±10.95 U/L,104.30±10.95 U/L,39.36±6.95 U/L (4, 6, 8, 10, 12 and 14 weeks, respectively), were all in defference, and were higher than the normal group's (21.35±4.73 U/L), P<0.05. The serum AST levels of rats in model group, 191.69±9.61 U/L,375.04±11.63 U/L,528.09±12.46 U/L,397.23±10.92 U/L,164.38±8.81 U/L,92.65±7.30 U/L (4, 6, 8, 10, 12 and 14 weeks, respectively), were all in defference, and were higher than the normal group's (26.62±7.16 U/L), P<0.05. The serum ALT and AST levels of rats in prevention group were all in defference, and were higher than the normal group's, P<0.05.3 HE and Masson staining showed that the model of hepatic fibrosis were established successfully. Fibrosis gradually increase with the extended of modeling. And degeneration, necrosis were appeared in liver cells. The histopathology improvement after stop using of CCL4. And the histopathology of prevention group rats generally good than that in model group's at the same time.4 Immunohistochemistry showed that there was only weakly expression ofα-SMA in the smooth muscle cells of vessel wall in hepatic tissues of normal group rats . But with the development of hepatic fibrosis, theα-SMA positive cells increased significantly in model group's. In contrast, theα-SMA positive cells decreased gradually after stop using of CCL₄. Theα-SMA positive cells of prevention group rats's is significantly less than that in model group's at the same time. It is similar to the prevention group's, 4 weeks after the injection of CCL₄, the optical density values ofα-SMA in model group rat hepatic tissues, 1.17±0.06,2.55±0.09,3.86±0.11 (4, 6, 8weeks, respectively), increased significantly with every two weeks, P<0.05. But it is reduced significantly with every two weeks after 10 weeks injection of CCL₄, 3.65±0.09,2.82±0.08,1.18±0.08 (10, 12, 14weeks, respectively) in the model group's, P<0.05. Furthermore, optical density values from model group and prevention group rats were all significantly higher than those from the normal group's, 0.52±0.08, P<0.05.5 Immunohistochemistry showed that there was only weakly positive expression of PI3K in hepatic tissues of normal rats. But with the development of hepatic fibrosis, the PI3K positive cells increased significantly in model group's. In contrast, it decreased gradually after stop using of CCL₄. The PI3K positive cells of prevention group rats's is significantly less than that in model group's at the same time. It is similar to the prevention group's, 4 weeks after the injection of CCL₄, the optical density values of PI3K in model group rat hepatic tissues, 0.84±0.06,1.92±0.14,3.01±0.09 (4, 6, 8weeks, respectively) increased significantly with every two weeks, P<0.05. But it is reduced significantly with every two weeks after 10 weeks injection of CCL₄, 2.70±0.09,2.38±0.13,1.19±0.10 (10, 12, 14weeks, respectively) in the model group's, P<0.05. Furthermore, the optical density values from model group and prevention group rats were all significantly higher than those from the normal group's (0.27±0.05), P<0.05.6 The PI3K mRNA increased significantly with the development of hepatic fibrosis in hepatic tissues of model group rats . In contrast, it decreased gradually after stop using of CCL₄. But it's significantly lower in prevention group's at the same time. It is similar to the prevention group's, 4 weeks after the injection of CCL₄, the PI3K mRNA expression levels in model group rat hepatic tissues, 0.88±0.07, 1.63±0.03, 2.26±0.06 (4, 6, 8weeks, respectively) increased significantly with every two weeks, P<0.05. But it is reduced significantly with every two weeks after 10 weeks injection of CCL₄, 1.95±0.07, 1.51±0.11, 1.16±0.11 (10, 12, 14weeks, respectively) in the model group's, P<0.05. Furthermore, the PI3K mRNA expression levels in the hepatic tissue of model and prevention group rats were all significantly higher than those from the normal group's, 0.63±0.08, P<0.05.7 The expression of PI3K protein is similar to that of PI3K mRNA. Western blot showed that the expression of PI3K protern, 0.66±0.08, 0.96±0.08, 1.54±0.08 (4, 6, 8weeks, respectively), increased significantly with every two weeks after 4 weeks injection of CCL₄ in the hepatic tissues of model group rats, P<0.05. And it's similar to the prevention group. But it is reduced significantly with every two weeks after 10 weeks injection of CCL4, 1.24±0.09, 0.99±0.11, 0.76±0.11 (10, 12, 14weeks, respectively), in the hepatic tissues of model group's, P<0.05. Furthermore, the PI3K protern expression levels in the hepatic tissue of model and prevention group rats were all significantly higher than those from the normal group's, 0.32±0.05, P<0.05.8 Alkaline hydrolysis measured HYP content in liver tissue displayed that HYP content increase gradually with the extended of modeling. And it decrease gradually after stop using of CCL₄. And the content of HYP in prevention group's is generally less than that in model group's at the same time.Conclusions:1 The rats hepatic fibrosis model could be established successfully by intraperitoneal injection with CCL4 for 8 weeks. And its pathological features is similar to that of human.2 The expression of PI3K mRNA and protern increased significantly with the development of hepatic fibrosis. And the level of increased is consistent with the degree of hepatic fibrosis. The expression of PI3K mRNA and protern decreased gradually with the reversion of hepatic fibrosis. And the level of decreased is consistent with the degree of reversal.3 Fuzhenghuyu capsules, which could reduce the liver injury by CCL₄, have an effect of prevent hepatic fibrosis. 4 Fuzhenghuayu capsule play a role of prevent hepatic fibrosis may be by blocking the PI3K/Akt signaling pathway.