| Objective:This study examined the effect of the Haizhu Yigan Fang to hepatic fibrosis in mice induced by Con A(concanavalin A) and its possible mechanisms. It demonstrated the relationship between hepatic fibrosis with leptin, OB-Rb(leptin receptor), JAK2(Janus kinase 2), and STAT3(signal transducer and activator 3) and cleard the role of JAK2/STAT3 signaling pathway in the development of hepatic fibrosis after treatment with Haizhu Yigan Fang. It provided new ideas and new strategies for the prevention and treatment of hepatic fibrosis through the analysis of key signaling pathways involved in the process of this experiment.Methods: 48 SPF C57BL/6 mice were randomly divided into 4 groups: blank control group, model group, colchicine group, Haizhu Yigan Fang group.There were 12 mice in each group. Experimental model of hepatic fibrosis was induced by intravenous injection Con A with 6mg.kg-1 W-1 dose for 12 weeks.The distilled wate, colchicine, Haizhu Yigan Fang decoction were orally administered for 4 weeks in different groups respectively. Mice were given0.25 mg.kg-1d-1 dose of colchicine by intragastric administration in colchicine group. Mice were given 9.86 g.kg-1d-1 dose of Haizhu Yigan Fang decoction by intragastric administration in Haizhu Yigan Fang group. Mice were given same dose distilled wate by intragastric administration in blank control groupand model group. They were treated by intragastric administration at 9 am and 3 pm respectively two times daily for 4 weeks. Mice fasted on the night after intragastric administration. The blood, liver, spleen tissues specimen of mices were collected in the next morning.The pathological slices were made from the liver tissue and checked by HE staining under optical microscop. Integrals of mice liver tissue lesions were evaluated in all groups. Masson staining was used to observe the liver fibrosis of mice in each group under the optical microscope. Electron microscopy was used to observe the ultrastructural changes of liver tissues of mice in each group. Indicators of each group were detected by automatic biochemical analyzer, such as ALT(alanine aminotransferase), AST(aspartate aminotransferase), TG(triglyceride),TC(total cholesterol) of the serum and TG,TC, MDA(malondialdehyde), SOD(superoxide dismutase) of the liver homogenate supernatant. Double antibody sandwich ELISA method was applied to detect the HA(hyaluronie acid), LN(laminin), PC III(procollagen III), CIV(collagen type IV) of liver homogenate supernatant and LP(leptin)of the serum in each group of mice. RT-PCR method was applied to detect leptin m RNA 、 OB-Rb m RNA 、 JAK2 m RNA 、 STAT3 m RNA of liver homogenate supernatant in each group of mice.Western blot analysis method was applied to detect the leptin, OB-Rb, JAK2, STAT3 of liver homogenate supernatant in each group of mice.Results:1 Comparison of the general situation of mice in each group Mice in blank control group were in good mental states and lively. They had a good appetite and the food intakes were normal. Their furs were smooth and body weights increased gradually. Mice in the model group were listlessness and didn’t like activities. Compared with the blank control group,they ate less and their furs were clutter, dim and dark. Their urine was yellowand their body weight all increased slowly. Their mental state, activity and food intake were acceptable in colchicine group and Haizhu Yigan Fang group.Their furs were shiny and their weight increased. Their general situations were obviously improved.2 Observation the hepatic tissue of mice in each group The liver textures of mice in the blank control group were soft, slightly dark red, shiny, no spots, projections. Their surfaces were smooth and their edges were sharp. The liver textures of mice in the model group were hard,uneven, color dark red, margin obtusely. Their surfaces were less smooth and part of the specimens had rough surfaces. The liver textures of mice in colchicine group and Haizhu Yigan Fang group were fragile, normal color,almost no spots, protruding etc. Their surfaces were smooth.3 Comparison of liver and spleen indexs of mice in each group Compared with the blank control group, the liver indexs of mice significantly increased in model group, colchicine group and Haizhu Yigan Fang group. The difference was significant(P<0.05). Compared with blank control group, the spleen indexs of mice significantly increased in model group, colchicine group and Haizhu Yigan Fang group. The difference was significant(P<0.05). The liver and spleen indexs of mice in colchicine group and Haizhu Yigan Fang group decreased compared with model group. The difference was significant(P<0.05). The decline of the liver and spleen indexs in Haizhu Yigan Fang group was superior to that of colchicine group(P<0.05).4 Effect of Haizhu Yigan Fang on hepatic tissue pathological changes in mice with liver fibrosis Hepatic lobules were clear and complete in blank control group. The liver cells were around the central veins and arranged radially. There were no degeneration and necrosis, no fibrous tissue hyperplasia and inflammatory cell infiltration. The structure of hepatic lobules were damaged in model group,liver cells cord arranged in disorder, liver cells degeneration, partial liver cell necrosis and associated with neutrophil and lymphocyte infiltration, fibrous tissue hyperplasia, the pseudo lobule formation, more fatty drops in liver cells.The degree of liver injury in colchicine group and Hai Zhu Yi Gan Fang group was mild compared that of model group. The damage of hepatic lobule structure and the collagen fiber hyperplasia of the liver reduced. Fatty drops in liver cells and the infiltration of inflammatory cells lessened.5 Effect of Haizhu Yigan Fang on the serum ALT, AST and the liver homogenate SOD, MDA in mice with liver fibrosis Compared with blank control group, the serum ALT, AST and the liver homogenate MDA levels in mice of model group, colchicine group, Haizhu Yigan Fang group were elevated(P<0.05) and the level of the liver homogenate SOD was decreased(P<0.05). Compared with model group, the serum ALT, AST levels in mice of colchicine group were lower(P<0.05) but the level of the liver homogenate SOD, MDA had no obvious change(P>0.05). Compared with the model group, the serum ALT, AST and the liver homogenate MDA levels in mice of Haizhu Yigan Fang group were lower(P<0.05) and the level of the liver homogenate SOD increased(P<0.05).Compared with the colchicine group, the serum ALT, AST and the liver homogenate MDA levels in mice of Haizhu Yigan Fang group were lower(P<0.05) and the level of the liver homogenate SOD increased(P<0.05).The difference was significant(P<0.05).6 Effect of Haizhu Yigan Fang on the serum TG, TC and the liver homogenate TG,TC in mice with liver fibrosis Compared with blank control group, the serum TG, TC levels in mice of model group, colchicine group, Haizhu Yigan Fang group were decreased(P<0.05). Compared with the model group, the serum TG, TC levels in mice of colchicine group increased(P<0.05). Compared with the model group, theserum TG, TC levels in mices of Haizhu Yigan Fang group increased(P<0.05).Compared with colchicine group, the serum TG, TC levels in mice of Haizhu Yigan Fang group increased(P<0.05).Compared with blank control group, the liver homogenate TG,TC levels in mice of model group, colchicine group, Haizhu Yigan Fang group increased(P<0.05). Compared with model group, the liver homogenate TG,TClevels in mice of colchicine group had no obvious change(P>0.05).Compared with model group, the liver homogenate TG,TC levels in mice of Haizhu Yigan Fang group were lower(P<0.05). Compared with colchicine group, the liver homogenate TG,TC levels in mice of Haizhu Yigan Fang group were lower(P<0.05).7 Effect of Haizhu Yigan Fang on the liver homogenate HA, LN, PC III, CIV in mice with hepatic fibrosis Compared with blank control group, the liver homogenate HA, LN, PC III, CIV levels in mice of model group, colchicine group, Haizhu Yigan Fang group increased(P<0.05).Compared with model group, the liver homogenate HA, LN, PC III, CIV levels in mice of colchicine group, Haizhu Yigan Fang group were lower(P<0.05). Compared with the colchicine group, the liver homogenate HA, LN, PC III, CIV levels in mice of Haizhu Yigan Fang group were lower and the difference was statistically significant(P<0.05).8 Effect of Haizhu Yigan Fang on the serum leptin level in mice with hepatic fibrosis Compared with blank control group, the serum leptin level in mice of model group, colchicine group, Haizhu Yigan Fang group increased(P<0.05).Compared with model group, the serum leptin levels in mice of colchicine group, Haizhu Yigan Fang group were lower and the difference was statistically significant(P<0.05).Compared with the colchicine group, the serum leptin levels in mice of Haizhu Yigan Fang group were lower and thedifference was statistically significant(P<0.05).9 Effect of Haizhu Yigan Fang on the protein and gene expression levels in liver tissue of leptin and OB-Rb in mice with hepatic fibrosis Compared with blank control group, the protein and gene expression levels in liver tissue of leptin in mice of model group, colchicine group,Haizhu Yigan Fang group all increased(P<0.05).Compared with model group,the protein and gene expression levels in liver tissue of leptin in mice of colchicine group, Haizhu Yigan Fang group were lower and the difference was statistically significant(P<0.05).Compared with colchicine group, the protein and gene expression levels in liver tissue of leptin in mice of Haizhu Yigan Fang group were lower and the difference was statistically significant(P<0.05).Compared with blank control group, the protein and gene expression levels in liver tissue of OB-Rb in mice of model group, colchicine group,Haizhu Yigan Fang group all increased(P<0.05).Compared with the model group, the protein and gene expression levels in liver tissue of OB-Rb in mice of colchicine group, Haizhu Yigan Fang group were lower and the difference was statistically significant(P<0.05).Compared with colchicine group, the protein and gene expression levels in liver tissue of OB-Rb in mice of Haizhu Yigan Fang group were lower and the difference was statistically significant(P<0.05).10 Effect of Haizhu Yigan Fang on the protein and gene expression levels in liver tissue of JAK2 and STAT3 in mice with hepatic fibrosis Compared with blank control group, the protein and gene expression levels in liver tissue of JAK2 in mice of model group, colchicine group,Haizhu Yigan Fang group all increased(P<0.05).Compared with model group,the protein and gene expression levels in liver tissue of JAK2 in mice of colchicine group, Haizhu Yigan Fang group decreased(P<0.05).Comparedwith colchicine group, the protein and gene expression levels in liver tissue of JAK2 in mice of Haizhu Yigan Fang group decreased(P<0.05).Compared with blank control group, the protein and gene expression levels in liver tissue of STAT3 in mice of model group, colchicine group,Haizhu Yigan Fang group all increased(P<0.05).Compared with model group,the protein and gene expression levels in liver tissue of STAT3 in mice of colchicine group, Haizhu Yigan Fang group decreased(P<0.05).Compared with colchicine group, the protein and gene expression levels in liver tissue of STAT3 in mice of Haizhu Yigan Fang group decreased(P<0.05).Conclusions:Haizhu Yigan Fang could improve the general situation of the model mice with liver fibrosis such as: improving mental health, increasing body weight, improving appetite and drinking water and other symptoms,significantly reducing the liver and spleen indexs in mice with hepatic fibrosis.Haizhu Yigan Fang could obviously improve the damage of the structure of hepatic lobules and inhibit proliferation of fibrous tissue. It could lessen the necrosis of liver cells and the infiltration of inflammatory cells significantly.After the treatment with Haizhu Yigan Fang, the levels of the serum ALT and AST, the liver homogenate MDA could be reduced and the level of the liver homogenate SOD could be increased. It could also reduce the content of the liver homogenate HA, LN, PC III, IV in mice with hepatic fibrosis. Haizhu Yigan Fang had an action to protect mice’s hepatic cell from injury caused by Con A. Haizhu Yigan Fang exertd functions of anti-hepatic fibrosis effectively by alleviating the inflammatory reaction, enhancing ECM’s degradation,inhibiting fibrous tissue hyperplasia, improving the pathological organization structure of mice with hepatic fibrosis. Haizhu Yigan Fang could early inhibit the initiation of liver fibrosis by regulating blood lipids(TG, TC) levels,reducing the accumulation of fat in the liver cells and protect the liver cells.The mechanism of Haizhu Yigan Fang’s effect of anti hepatic fibrosis may be:1 Haizhu Yigan Fang could significantly reduce the serum leptin level of mice with hepatic fibrosis and effectively prevent the combination of leptin and OB-Rb. It played an important role in HSC activation and proliferation.2 Haizhu Yigan Fang could significantly inhibit the protein and gene expression levels in liver tissue of leptin and OB-Rb in mice with hepatic fibrosis. It suggests that leptin is one of the targets of Haizhu Yigan Fang’s effect of anti hepatic fibrosis.3 It can illustrate the effect of Haizhu Yigan Fang’s anti liver fibrosis through the down regulation of expression of JAK2, STAT3 induced by Haizhu Yigan Fang, the inhibition of HSC activation and proliferation and the reduction of the collagen expression and secretion. Haizhu Yigan Fang could inhibit the expression of OB-Rb. This may be the molecular mechanism of Haizhu Yigan Fang’s anti liver fibrosis. The transduction pathway of regulating JAK2 /STAT3 signal by leptin may be the pathways and targets which Haizhu Yigan Fang works on anti liver fibrosis.Hepatic fibrosis(fibrosis Hepatic, HF) is the synthesis of matrix(ECM),which is caused by many pathogenic factors, which is greater than the degradation of extracellular, which leads to excessive deposition of ECM in the liver. It is the common pathological feature of most chronic liver disease. It is also an important link to the development of chronic hepatitis, hepatic cirrhosis and liver cancer. The formation of hepatic fibrosis is a complex changing dynamic process of multi factors and multi stage. A variety of pathways and cytokines are involved in the complex regulatory networks which effect continuously the formation and prognosis of liver fibrosis.Therefore it is important to study the formation mechanisms of liver fibrosis from the cytokine and the signal transduction pathway. It will continue to deepen the drug treatment study of liver fibrosis. Leptin is found as a pro fibrotic cytokine in recent years. Leptin and its receptor(OB-Rb) play animportant role in the formation and development of hepatic fibrosis. Leptin is one of the initial factors of liver fibrosis. leptin can activate hepatic stellate cells(hepatic stellate cell, HSC) to promote liver fibrosis. The normal liver tissue dosen’t expresse leptin but can expresse the m RNA of its receptor.Leptin exists only in the activaed HSC. The increased expression of leptin/OB-Rb m RNA and protein were found in the activaed HSC.The leptin level in serum and tissue was significantly increased mainly by expression of the activated HSC. After leptin combines with the OB-Rb of the activaed HSC, by regulating the expression of HSC cytokines, the transcription level of collagen m RNA was significantly increased and it promotes the progress of liver fibrosis disease. The expression of leptin is closely related to the staging and progression of hepatic fibrosis. The serum leptin level is positively related to the degree of hepatic fibrosis. At present, the study about leptin is less on the molecular mechanism of promoting liver fibrosis. The mechanism analysis is’ t clear. Therefore, we think the study of the Haizhu Yigan Fang’s effects on leptin, ob-Rb and the signal transduction JAK2/STAT3 pathway can give us an in-depth undersanding on the molecular mechanism of Haizhu Yigan Fang’s antifibrosis properties. It can provide more sufficient theoretical support for the prevention and reversal of hepatic fibrosis. At the same time it has very important theoretical significance to widen and deepen the study on the mechanisms of liver fibrosis.It still lacks of the antifibrosis drugs and methods in the clinic. But the study of anti liver fibrosis and clinical medicine experiments have demonstrated that traditional Chinese medicine has unique advantages in anti hepatic fibrosis. Traditional Chinese medicine has characteristics of multi-level and multi-channel, multi targets, in treatment of liver fibrosis and it has obtained fruitful results.The intrinsic phlegm, liver collateral stasis is the main pathogenesis ofhepatic fibrosis in TCM. We base on the full understanding of liver fibrosis in treating chronic hepatitis B from the phlegm. The previous clinical and experimental researches suggest that Haizhu Yigan Fang has obvious the antiviral effect, reducing liver damge, protecting liver and reducing enzyme.Through the observation of Haizhu Yigan Fang on hepatic tissue pathological morphology of mice, HA, LN, PCIII, CIV, TC, TG, leptin, its receptor(OB-Rb), Janus kinase 2(JAK2) and signal transducer and activator of transcription factor 3(STAT3) expression, this paper investigates its effect on leptin regulation of hepatic fibrosis in mice JAK2/STAT3 signal transduction pathway. Through the observation of the above aspects, we want to deeply analyze the relationship between serum leptin and liver fibrosis process, to explore the mechanism of treating liver fibrosis from the liver and spleen, to comprehensively assess Haizhu Yigan Fang’s value on anti liver fibrosis,further to reveal the scientific connotation of the theory of treating hepatic fibrosis from sputum, to update experimental basis for treating hepatic fibrosis,to explore new treatment direction. |
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