The Signal Transduction Pathway Mechanisms Of Rats With Liver Fibrosis Regulated By Leptin And Interfering Effects Of Baoganning

Source of the research work:This research work was funded by National Natural Sciences Fund and Guangdong Medicine Bureau of Guangdong province.Research background:Liver fibrosis results from chronic damage to the liver in conjunction with the accumulation of ECM proteins,which is a characteristic of most types of chronic liver diseases and also is the important middle key to chronic hepatitis and deteriorating hepatocirrhosis.According to statistics there are over 30000000 patients suffering from chronic B hepatitis in China.Each year about 400000 patients die of posthepatitic cirrhosis.And the only way to cure hepatocirrhosis is to prevent liver fibrosis.Until now there is no authorized anti-fibrosis drug in the world including China.The clinical and experimental studies on effectiveness of TCM against fibrosis have demonstrated its special advantages. The multi-target mechanisms on anti-fibrosis with compound prescriptions are especially extraordinary.Abstract:Aiming at the pathogenesis characteristics of liver stasis and spleen deficiency,blood stasis and damp wet of hepatic fibrosis among chronic liver diseases,we have invented a new formula,Baoganning,which has the functions to relieve stagnancy of the liver Qi,reinforce Qi and invigorate the spleen,promote blood circulation to eliminate stasis,soften and resolve hard masses,concurrently clear up toxic heat and dampness.Clinic and experimental research have confirmed the efficacy of Baoganning in treating liver fibrosis.The main mechanisms are to decrease the inflammation,suppress the proliferation and promote the apoptosis of hepatic stellate cells by inhibiting the expression of nuclear factorκB.Leptin is a newly discovered cytokine which promotes liver frbrosis.Currently research on the molecular mechanism of leptin in promoting liver fibrosis has just started overseas and in China there are few publications in this field.Research on the signal transduction mechanism is insufficient and the existing studies are limited to the exsomatized cells.To the author's knowledge there is no open literature about vivo experiments.Therefore we believe research on signal transduction mechanism of leptin to stimulate the HSCs to proliferate in vivo and in vitro has great significance in understanding the mechanisms of liver fibrosis.Investigations on effects of Chinese medicine Baoganning on leptin,leptin-receptor and its signal transduction are helpful in finding the anti-fibrosis working principles.It will also provide the theoretical basis for Baoganning to prevent and treat the liver fibrosis.Objectives and purposes:to investigate the molecular mechanism of anti-liver-fibrosis treatment with Baoganning,thus providing a theoretical foundation for applying Baoganning to the clinic,and establishing a solid theoretical platform for developing the traditional Chinese medicine on anti-liver fibrosis.Methods:The fibrosis in the rats model was induced by multiple factors.And Baoganning consisting of Chinese herbs Huangqi,Taoren,Danshen,Huangqin,Biejia, etc was given to the rats.The effects of Baoganning on fibrosis were studied.Fifty Wistar rats were randomly divided into normal group(A),model group(B), Colchicine group(C),high dose of Baoganning group(D)and medium dose of Baoganning group(E).All rats except rats in group A were subcutaneously injected with 40%carbon tetrachloride in peanut oil with a dose of 0.5ml/100g according to the body weight once on the first day,then with a dose of 0.3ml/100g once every three days for six weeks.Meanwhile,they were fed with normal fodder.5%alcohol was used as drinking water.Rats in group A were subcutaneously injected with equal peanut oil and fed with normal fodder and water.Rats in group C,D and E were respectively given Colchicine(0.011mg/100g),Baoganning(3.54g/100g)and Baoganning(1.77g/100g)per day for six weeks via gastrogarage.Rats in group A and B were served with distilled water at the same time.At the end of the sixth week, blood and tissue specimens were taken from all the rats.Some serum were used for analysis of aspartate transaminase(AST)and alanine transaminase(ALT)activities according to the direction of the kit and others for the analysis of total cholesterol (TC),Triglyceride(TG)with an automatic biochemical machine and leptin(LP)by radioimmunoassay(RIA).Some liver specimens were mixed in a 10%solution of formaldehyde in 0.1 mol/L phosphatobuffered saline,and embedded in paraffin. Five-micron thick sections were prepared to evaluate the degree of fibrosis under an optical microscope and analyze the expression of leptin(LP),leptin receptor(OB-R) and signal transducer and activator transcription 3(STAT3)with immunohistochemistry(IH)methods and western blotting and analysis.As to statistical analysis,data were analyzed with the software of SPSS14.0.Quantitative data were presented as mean±SD and compared using one way ANOVA procedure.Results and Discussions:1.To observe the influence of Baoganning on symptoms,physical signs and indices(liver wet weight/100g body weight)of liver and spleen(spleen wet weight/100g body weight)in liver fibrosis rat model.Rats in model group displayed lassitude,thinness,anorexia and their stool was loose,the urine was yellow and less.The surface of livers in model group was not smooth,the colour was lightly red,the edge was blunt and the quality was hard.There were even some little tubercles on the liver surface.The spleens were obviously swollen.Compared with the rats in the model group,rats in the treatment groups were in better conditions.High and medium dosages of Baoganning and colchicines were effectual in improving weight(P＜0.01,P＜0.01,P＜0.05),There was no major difference between high,medium dosages of Baoganning and colchicines(P＞0.05),and all significantly decreased the indices of liver and spleen(P＜0.01 in both groups).High dosages of Baoganning were better than colchicines(P＜0.05).2.To observe the influence of Baoganning on liver morphology in liver fibrosis rat model.Under a microscope damaged lobules of liver degeneration,necrosis of liver cells, lots of inflammatory cells,collage fibers and even pseudolobules were observed for the model group,while for the treatment groups there were less damaged lobules of liver degeneration,necrosis of liver cells,inflammatory cells and collage fibers.But there were still incomplete pseudolobules in very few rats.3.The effects of Baoganning on serum hepatic function and lipid in liver fibrosis rat model.Compared with the normal group,levels of ALT,AST and AST/ALT in the model group went up sharply(P＜0.01);Compared with the model group,levels of ALT and AST in treatment groups went down obviously(all P＜0.01).The effects of a high dosage of Baoganning on decreasing the levels of AST were also more significant than colchicines(P＜0.05);AST/ALT significantly was decreased with medium dosages of Baoganning(P＜0.05).Compared with the normal group,levels of TC and TG were significantly decreased(P＜0.01).Compared with the model group,levels of TC and TG with high and medium dosages of Baoganning went up significantly(P＜0.01);The effects of high dosage Baoganning on increasing the levels of TC was greater than colchicines (P＜0.05);Baoganning with high and middle dosages was also more effective than colchicines in increasing the levels of TG(P＜0.05). 4.The influence of Baoganning on serum leptin in liver fibrosis rat model.Compared with the normal group,levels of leptin in the model group went up noticeably(P＜0.01);Compared with the model group,levels of leptin went down obviously(P＜0.01,P＜0.01,P＜0.05)for both Baoganning and colchicines groups with high and middle dosages.5.The effects of Baoganning on the expression of Leptin and its signal pathway in liver tissue in fibrosis rat model.Only a mild positive staining of leptin was found at central vein and Disse's areas but none at hepatocytes on sections of normal controls,whereas on sections of the model group,the positive staining was seen at interstitial cells,inflammatory cells, impaired hepatocytes as well as normal hepatocytes.Fibrous c septa were only slightly stained.In the treatment groups the positive staining cells decreased and the staining was evidently light.Normal liver tissue hardly expressed OB-Rb or only a mild positive staining was found at portal vein,central vein and portal tract.Whereas in the model group OB-Rb mostly was located in the portal tract,around sinusoid and fibrotic septa.Expression of OB-Rb exhibited as brown particles predominantly in cytoplasm or cell membrane.A few positive expressions were found in nucleus.Contrast to the model group,the positive staining cells and the staining degree in the treatment groups were not obviously changed.Normal liver tissue hardly expressed Stat3,only a mild positive staining was found at matrix in portal tract and at cytoplasm in interstitial cells,whereas in the model group Smad4 dominated.It was detected in interstitial cells at fibrous septa,portal tract, portal vein,central vein and around sinusoid,especially at fibrous septa.In the treatment groups the number of positive staining cells decreased,mainly expressed at portal tract,portal vein and central vein. Statistical data analysis indicated that the expression level of leptin,OB-Rb and Stat3 were increased significantly in the model group(P＜0.01).Compared with the model group,the Immunostaining score of leptin in high and middle dosage Baoganning,colchicines groups was remarkably decreased(P＜0.01 in both groups), Stat3 was also remarkably decreased(P＜0.01,p＜0.051 P＜0.05).OB-Rb was not noticeably decreased(P＞0.05 in all treatment groups).6.The effects of Baoganning on the expression of OB-Rb and JAK2-STAT3 signal pathway in liver tissue in fibrosis rat model.The expression of OB-Rb,JAK₂,STAT₃ and the activity of phosphor-JAK₂, phosphor-STAT₃ were detected by Western blotting analysis.All of these in normal liver tissue were barely detected,whereas in the model group expressed strongly. Compared with the model group,the expression of them in all treatment groupsConclusions:Baoganning can inhibit the process of liver fibrosis in rats model, the mechanisms are as follows.1.Baoganning can improve the life quality through improving the spirits, increasing the body weight,food and water intake and decreasing the indices of liver and spleen2.Morphological observation shows Baoganning can improve the damaged structure of heptocyte,decrease the fibrosis tissue.3.Baoganning can significantly decrease the levels of serum ALT,AST and increase the levels of TC and TG.4.Baoganning can reduce the expression of Leptin,OB-Rb and its signal transduction molecule JAK2 and STAT3 in the liver tissue,so the signal transduction pathway of leptin is inhibited by the EHAWE therapy.In conclusion,Baoganning has the complicated pharmacological action on preventing the liver fibrosis.It has multi-way and multi-target anti-fibrosis mechanisms.One of the molecular mechanisms is to inhibit the expression of leptin and its signal transduction which is concerned to inhibit the inflammation,protect the liver cell,decrease the waterfall action of the cytokine and inhibit the start of the fibrosis.