The Signal Transduct Ion Pathway Mechanisms Of Rats With Liver Fibrosis Regulated By Leptin And Interfering Effects Of Modified Shengjiang Power

Liver fibrosis results from chronic damage to the liver in conjunction with the accumulation of extra cellular matrix (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. Its essence is excessive deposition of ECM components in liver, which occurs due to an imbalance between the production and degradation of matrix. It is the most common pathological features of chronic liver disease, further to the important link of the development of hepatocirrhosis. Liver fibrosis can be reversed. It can prevent serious consequences if hepatic fibrosis blocked and reversed. The imbalance between synthesis and degradation of ECM components is a leading cause of liver tissue remodeling, fibrosis, cytokine and polypeptide growth factor and regulation factor is an important factor in the development of liver fibrosis initiation regulation.Hepatic fibrosis is a complex process of multiple factors, multiple links,multiple stage. Its forming process is not static, but constantly changing. Complex regulatory network composed of various cytokines has influenced the formation and prognosis of Hepatic fibrosis. It is advantageous in HF treatment from the cell factor and its signal pathways to study mechanism of drug intervention. Leptin is considered a key factor to promote the formation of HF. Leptin levels often leads to fibrosis produced in liver injury. Leptin and leptin receptor play an important role in the formation of liver fibrosis as one of the initiating factor of liver fibrosis which be promoted by activation of hepatic stellate cells. Normal liver tissue did not express leptin but with its receptor mRNA. Leptin immune reactivity exists only in the activation of hepatic stellate cell. In the activation of hepatic stellate cells, leptin and its functional receptors express and synthesis of leptin mRNA and protein elevated. By binding with leptin receptor, it activates signal transduction pathway corresponding, promotes the occurrence and development of HF. JAK/STAT signal transduction pathway is the focus of the current study. Modified Shengjiang power based on theory of elevating clear qi to lower turbid qi has the effect of benefiting the qi and invigorating the circulation of blood, clearing heat and detoxicating,soothing liver and strengthening spleen, which obtains the good curative effect in previous clinical and experimental researches. In this experiment,we used hepatic fibrosis rats’model by intraperitoneal injection,we observed the effect of Modified Shengjiang power on the levels of serum AST、ALT、LN、Col-Ⅳand pathomorphology in hepatic fibrosis rats. We observed the expression ofleptin, OB-Rb, JAK2, STAT3and MMP-1in rats with HF, and then explored the effect of the JAK2/STAT3signal transduction pathway regulated by leptinin rats with HF. Investigated on possible Mechanism of the resistance to HF,so that provided theoretical and experimental basis for treating and preventing HF.ObjectivesThe aim was to observe the mechanism of liver fibrosis regulated by leptin sig nal transduction and the effect of Modified Shengjiang power, investigate the molec ular mechanism of anti-fibrosis.Methods1. The effect of Modified Shengjiang Power on the levels of serum the levels AS T、ALT LN、Col-Ⅳ.Total72clean male SD rats were randomly divided into five groups:the normal group, the model group,colchicine group, Modified Shengjiang power of low dose group(MSPL), Modified Shengjiang power of medium dose group(MSPM) and Modified Shengjiang power of high dose group(MSPH) with12rats for each group. The first dose was40%(volume fraction) CCL4peanut oil5ml/kg; every3dayafter injection of3ml/kg to established rats model. The drugs were given through stomach-perfusion to MSPL group(5.5g/kg.d),GFJM group(llg/kg.d),GFJH group(22g/kg.d) and colchicine treatment group(11mg/kg-d) after the model was successful. Gastric lavage liquid amount is lOml/kg,1times/d.The rats in the normal control and model group received intragastric administration of0.9%saline(10ml/kg).Observed rats’ordinary circumstances. At the8th weekend, We draw blood from abdominal aorta. The level of AST、ALT LN、Col-Ⅳ was measured with radioimmunoassay (RIA).2. The effect of Modified Shengjiang power on the pathomorphology in hepatic fibrosis rats.The were taken and the degree of hepatic fibrosis was judged by routine haematoxylin-eosin staining、VG staining and Gordon-Sweet staining.3. Effect of Modified Shengjiang power on leptin, OB-Rb in HF ratsTo detect the expression of leptin and leptin receptor in liver tissue of rats, and GF-β1in liver tissue in rats4. Effect of Modified Shengjiang power on TIMP-1mRNA expression The TIMP-1mRNA expression and the interventional effects of Modified Shengjiang power in6h,12h and24h on the expression of TIMP-1mRNA were determined Reverse transcription polymerase chain reaction (RT-PCR).5. Effect of Modified Shengjiang power on Reactive Oxygen Species in hepaticstellate cells By using Reactive Oxygen Species Assay Kit, the intracellular ROS level in HSC-T6cells was detected through the fluorescence intensity of fluorescence microscopy or flow cytometry DCF. The fluorescence intensity in HSC-T6cells of each treatment group was detected in1h,12h,24h.6. Effect of Modified Shengjiang power on JAK2/STAT3signal transduction pathway in HF ratsActivity of JAK2, STAT3protein was detected by immunocytochemistry method to explore Modified Shengjiang power’s effect on expression of JAK2, STAT3protein in n HSC-T6cells.Results1. The effect of Modified Shengjiang Power on the levels of serum the levels AS T、ALT LN. Col-Ⅳ.Compared with the normal control group, the levels of serum AST、ALT、LN、Col-IV all increased in model group, Modified Shengjiang power group and colchicines group(P<0.05). Compared with the model control group, the levels of serum AST、ALT LN、Col-Ⅳ could be decreased from all medicine intervention group significantly(P<0.05). The level of Modified Shengjiang power of high dose group decreased most significantly, better than the colchicine group, middle dose group and low dose group (P<0.05).The difference between the colchicines treatment group and MSPM group was not significant (P>0.05).The level of serum AST、ALT、LN、Col-IV in colchicine treatment group was significantly reduced compared with MSPL group (P<0.05).2. The effect of Modified Shengjiang power on the pathomorphology in hepatic fibrosis rats.Under the light microscope, the hepatic lobules of normal group were arranged in order and ranged radially around the central vein. There was not degeneration and necrosis in liver tissue. There was not collagen proliferation. The Structure of model control rats was destroyed. Most of hepatocytes were hydropic degeneration, ballooning degeneration and necrosis. There were neutrophilic granulocytes and lymphocytes around central vein and portal area. The fibrous tissue was in proliferation.Pseudolobule proliferation could be seen in individual animal. The degree of hepatocyte injury in all medicine intervention groups was less serious than model group. The destructions were lightened, proliferations of collagen fibers were also lightened, Swelling of liver cells and degeneration alleviated, infiltrating cells decreased. These improvements were most significantly in high dose group. 3. Effect of Modified Shengjiang power on leptin, OB-Rb in HF ratsCompared with the normal control group, the levels of serum Leptin all increased in model group, Modified Shengjiang power group and colchicines group(P<0.05).Compared with the model control group, the levels of serum Leptin could bedecreased from all medicine intervention group significantly(P<0.05). The serum leptin level of Modified Shengjiang power of high dose group decreased most significantly, better than the colchicine group, middle dose group and low dose group(P<0.05).The difference between the colchicine treatment group and MSPM group was not significant(P>0.05).The level of serum leptin in colchicine treatment group was significantly reduced compared with MSPL group (P<0.05).4. Effect of Modified Shengjiang power on TIMP-1mRNA expressionThe TIMP-1mRNA expression level is significantly higher than normal group after Leptin stimulation to the HSC, which is consistent with previous report. TIMP-1mRNA expression can be inhibited by JAK inhibitor called AG490,which showed that leptin regulates mRMA TIMP-1through JAK-STST signal transduction pathways. NAC and NOX inhibitor and antioxidant DPI can inhibit TIMP-1mRMA expression, combined with previous studies, leptin generates ROS mainly through inducing NOX, suggesting that ROS mediated by leptin in HSC-T6intracellular signal transduction, which affect the mrna expression of TIMP-1.5. Effect of Modified Shengjiang power on Reactive Oxygen Species in hepatic stellate cellsDCF fluorescence intensity increased in HSC-T6cells by leptin stimulates, com pared with blank control group increased significantly (P<0.001), indicating that le ptin can induce the HSC-T6intracellular ROS production; as Continuous stimulation of leptin, DCF fluorescence intensity of leptin in HSC T6cells within24h is high er than12h effect (P<0.05); Modified Shengjiang power group, the NAC group of DCF model in the cell fluorescence intensity are lower than the same time point1eptin stimulation group (P<0.01), at12,24h flavored elevator powder treatment group were lower than that of NAC treatment group (P<0.01); As the duration ext ension, flavored powder treatment group1,12,24hours of DCF model in the cell fluorescence intensity gradually reduce, the difference was statistically significant (P (0.01).6. Effect of Modified Shengjiang power on JAK2/STAT3signal transduction pathway in HF ratsThis experiment found that for the first time that the expression of JAK2, STAT3protein decreased in the in HSC-T6cells treating by Modified Shengjiang power serum before leptin, suggesting that the Modified Shengjiang power can inhibit the activation of JAK2,STAT3signal transduction pathway. Modified Shengjiang power can prevent the phosphorylation of STAT3forming into dimert to shift to the nucleus by inhibiting the phosphorylation of STAT3protein,which affects gene transcription,such as inhibiting of the expression of TIMP-1mRNA, up-regulating expression in MMP-1mRNA.ConclusionModified Shengjiang power could inhibit the expression of JAK2,STAT3.It can inhibit the activation, proliferation of HSC, reduce theexpression and secretion of col lagen, and ultimately resist hepatic fibrosis.JAK2/STAT3signal transduction pathway regulated by leptin is the new function way and target for Ganfujian formula anti HF.