Experimental Study Of Hujin Granules On Anti-immunity Hepatic Fibrosis

Chonic liver disease caused by various pathogenesis, and liver damage by some toxic materials, will bring the liver cell inflammation or putrescence, and subsequently cause the pathology characteristics of fibrosis. Hepatic fibrosis is the pathology process of abnormal hyperplasia of the fibre integrated tissue, when the liver cell occur to be putrescence and activated from inflammation. It is called fibrosis when the symptom is small. When the structure of the small part of the liver is Preudolobular and knur formed, we called it hepatocirrhosis.Recently, with the wide application of the medical molecule biotechnology, deep research has been made on the happening mechanism of Hepatic fibrosis, and many important achievements has been procured, as change the previous point that the liver fibrosis can' t be reversed, and bring out the conclusion that it' s completely possible for the liver fibrosis to be reversed.Now, the research on the Hepatic fibrosis has becomed a hot topic in the field of the liver disease in the world.Our country is a highly epidemic area by Virosis hepatitis. According to the recent statistical data, there are 20 million sufferers infected with the liver disease. There is a high incidence of Hepatic fibrosis in our country, as has become a healthy problem for us to face seriously. Therefore, to find the valid way and the valid medicine for curing Hepatic fibrosis, is in urgent need by our people, and have deep significance and great value to our society.Now the research on the pathogenesis mechanism of Hepatic fibrosis mainly focus on HSC, Extracellulaur matrix, and Cytokin closely concerned with Hepatic fibrosis. Much work have been done.Researches show, that the reason that the amount of HSC increase markedly when the liver becomes fibrosis, is concerned with the HSC s excessive activation, proliferation and insufficent apoptosis.Hepatic fibrosis mainly presents that the ECM compound and excrete a great deal, but decompose insufficiently, which make the ECM deposit pervadingly in the liver. The elements of ECM contain collagen, non colloidal sugar albumen and albumen amylase, and etc. Collagen is the most primary element of ECM, which includes five types: type I, type III, type IV, type V and type VI. When the liver becomes fibrosis, the amount of collagen will increase several times, thereinto type I and type III take up larger portion than the others, and they are the primary element of collagen .Non colloidal sugar albumen has many varieties, for example, Fibronectin, Laminin, Secreted protein, acidic and rich in cystekne, and etc. but primarily contains the FN and LN. Albumen amylase is one type of large molecule material with kernel albumen and a great deal of amylase molecule. Albumen amylase, together with the colloid, distributes on the stroma and the floor theca outside the cell, and play a wide part in the liver cell, among the liver cell and through the reciprocity between the liver cell and stroma. Albumen amylase mainly includes Hyaluronic acid, Heparan sulfate and other elements. Thereinto, HA has been widely applied in the clinical diagnosis, and has positive relations with Hepatic fibrosisin, which reflects the increase of the compound of the myofibroblast and decrease of the dissolution of the endothlial cell. Heparan sulfate, as the main part of the floor theca .exists outside the cell, and has the function of proliferation and tropism to stromal cell. MMPs and TIMPs play an important part in adjusting the metabolization of collagen. MMPs can dissolve Extracellulaur matrix, but that TIMPs combines with MMPs equally will preventMMPs from dissolving Extracellulaur matrix. The prevention to the dissolvability of the TIMPs to the MMPs is considered one of the main reason for Hepatic fibrosis. The happening, development of Hepatic fibrosis have close relations with the exudation of the Cytokin. The Cytokin comes into being from the activated cell in the blood supply system, immunity system and inflammation reaction, and can adjust the differentiation and proliferation of the cell.Some Cytokin stimulate the inflammation and fibrosis, while some other ones restrain these reaction. The Cytokin stimulating fibrosis includes TGF-β., PDGF, TNF-a), IL-1, IL-6, IL-8) , ET-1, and etc. The Cytokin restraining hepatic fibrosis includes IFN, IL-10, and etc. When the exudation of the stimulative Cytokin is too much, and that from the restraining one is comparatively little, Hepatic fibrosisin will take place and develop.The diagnosis of hepatic fibrosis contain pathologic diagnosis, serum diagnosis and imaging diagnosis, and etc. Up to present, the most reliable method is liver biopsy, but this method is not widly accepted by the patients because it has some trauma. So research on untraumaic diagnosis gets more recognition.Up to present, The drugs used to cure the hepatic fibrosis are primarily Colchicine, γ-interferon, and etc. Although these drugs have some effect on the treatment of hepatic fibrosis, they are not widely used in the clinical activities because their side-effect and their unclear mechanism.Up to present, the common experimental animal models of liver fibrosis that have been established as follows:①carbon tetrachloride induced animal model of liver fibrosis;②serum of heterogeneity animal induced animal model of liver fibrosis;③ethanol induced animal model of liver fibrosis;④dimethylnitrosamine induced animal model of liver fibrosis;⑤multiples factor induced animal model of liver fibrosis;⑥rat bile duct occlusive fibrosis model. We should choose appropriate animal modelsaccording to experimints .Because different model has different trait.Scientists on traditonal chineses medicine in China have carried out a lot of clinical and experimental studies in the area of anti-liver fibrosis in recent years, and have achieved many effective resluts. Many Chinese herbs have been found to have good effect on anti-liver fibrosis, for example radix salviae miltiorrhixae, Sophora flavescens, Radix Notoginserg, Astragulus, Coryceps sinensis, and etc. Some effective compounds have also been found, for example Fufang Biejia Ruangan Tablet, compound 861,Ruan Gan Hua Zheng Wan, Fuzheng Huayu Decotion, Ruangansuopiwan, Handanganle Capsules , and etc. The Chinese medicine carry it effect on anti-liver fibrosis through the following effects: 1,anti-lipid peroxidative action; 2, rebulate Cytokines; 3, protcect hepatic cell; 4, promote exertion of bile; 5, reduce the acivation and Proliferation of HSC; 6, reduce sedimentation of ECM, et al.Hujin Granules is a experienced compound of Chinese drugs of professor Huang Zhaosheng. This compound is cosisted with six herbs of Rhizoma Polygonicuspidati, Radix Curcunae, Fructus Crataegi, Radix Notoginserg, Rhizoma Alismatis, Ganoderma.. In this compound, Rhizoma Polygonicuspidati and Radix Curcunae could promot blood to resolve stagnation and promot qi flow to stop pain as principal drugs. Fructus Crataegi and Radix Notoginserg could promt blood to resolve stagnation; Rhizoma Alismatis could promting urination to remove dampness. Both of them were minister drug. Ganoderma could replenish qi and reinforce deficiency as adjuvant drug. We used HJG to treat Chronic hepatitis Hepatic fibrosis and received good effect in recernt years . Experimental results revealed that HJG had protective effect against CCL4-induced Hepatic fibrosis in rat. This thesis was base on the optimizing the arts and crafts and improving on the from of prepared drugs, later the Hujin Granules (HJG) were gotten. The aim of study was to investigate the possible effects and mechanism of HJG on experimintal hepatic fibrosis.Methods:Male SD rats wre randomly divided into six groups, ie, normalcontrol group, model control group, Colchicine group and the high dose (2.3g/kg),median dose(1.2g/kg), low dose(0.6g/kg) group of HIG.. The immunohepatic fibrosis model was induced by intraperitoneal injection of porine serum. Except the control group, the other 5 groups rats were given an intraperitoneal injection of porcin serum(0.5ml,twice a week for 12 weeks, drugs were given from the next day, once for 12 week, The experimintal rats were killed at the end of the 12nd week. The changes of HE of the rats' s liver were observed with optic microscope. The ultrastructure was observed with electronic microscope. Hyp and MDA in the liver, ALT, AST, ALB, TP, A/G HA, LN, IV-C,PCⅢ,IL-1,TNF levels in the serum were measured respectively. TGFH31> TGF-β1 RⅡ TIMP-1 mRNA were detected in liver section by RT-PCR. The exepression of type Ⅰ, Ⅲ collagen in liver were detected by immunohistochemical examinations. Proliferation of HSC-T6 cells were evaluated by MTT assay.The results of the study indicated that:Firstly, Effects of HUJIN granules (HJG) on function of the liver and the indexes on the hepatic fibrosis of the immunity hepatic fibrosis rats.1. The serum level of ALT (Alanine transaninase) and AST (Aspartic acid transaminase) rise obviously in the immunity hepatic fibrosis rat which is induced with pig serum, compared with the normal group, the difference seems significant(P<0.01), but the content of ALT and AST in serum in the HUJIN granules group decreased obviously, and has the dose-dependent effect. In the aspect of decreasing ALT and AST, the HUJIN granule is better than the control medicine Colchicine.2. The levels of serum albumin in the model group decreased obviously, the ratio of albumin and globulin(A/G) descend, but the serum albumin in the HUJIN granules treated group resumed in different degree , especially the high dose HUJIN granules treated group, albumin content and the ratio of A/ G come near to the normal group.3. The serum levels of HA, LN, IV- C and the PCⅢin hepatic fibrosis rat which was induced with the pig serum increased more significant than that in the normal group , the difference shows the significance ( P<0.01).Compared with the model sets, each group with different doses of HUJIN granules can reduce the PCⅢ, HA, LN, IV- C content of the serum, and shows some effect, of dose-dependent.secondly,The study of effects on morphology in immunity hepatic fibrosis rat.The optical microscope observation results indicated that it can be obviously seen that fibre tissue proliferated obviously and extended to the hepatic lobules in the liver tissue in model group, but in the HUJIN granules treated sets it can not be seen clearly, which reveal that the HUJIN granules may have a inhibiting effect on the fibroplastic proliferation in the liver tissue.The elecron microscope observation result indicated that in model group, liver cells have obvious pathologic changed, intercelular space broaden, mitochondrion swelling, irregularity, fat droplets can be seen easily in hepatocytes, the lessen rough endoplasmic reticulum, karyopyknosis, nucleolus disappeared; in the HUJIN granules treated group , the hepatic cell denaturation, necrosis rarely , there are abundance mitochondrion in it, and a bit swelling , lessen rough endoplasmic reticulum, fat-storing cells a bit enlarge, fat droplets can not see easily, the resuls indicated that the HUJIN granules have the effect of protecting the hepatic cell.The third, Effect on the content of Hyp, MD in the immunity hepatic fibrosis rat' s liver tissueTo compare with the normal group, the content of Hyp, MDA increases obviously in the model group ;Compared with the model set, the control group have lower content of the Hyp, MDA. The results indicated that HUJIN granules can decrease the content of collagen in liver tissue of the immunity hepaticfibrosis rat , and to some extent have the effect of anti- oxygenation, indicated that the effect of anti-hepatic fibrosis of the HUJIN granules concerned withthe inhibiting effect on the reaction of lipid peroxidation.The fourth, Effect on immunity hepatic fibrosis rat' s serum cytokine IL-1.. TNF.The serum levls of IL-1, TNF all increased significantly than the normal set in hepatic fibrosis rat which was induced by the pig serum:Compared with the model set, through the treatment of Colchicine and HUJIN granules, the level of IL-1, TNF decreased obviously in the rat serum. The results indicated that HUJIN granules has the effect of inhibiting serum IL-1, TNF in immunity hepatic fibrosis rats .The fifth, Immunohistochemical study on anti-immunity hepatic fibrosisIn the immunity hepatic fibrosis rat induced by pig serum the injured hepatic cells, (mainly show cloudy, steatosis, apoptosis, focal necrosis)can be seen and stroma fibroplastic proliferation, Ⅰ-collagen increased significantly, the Ⅲ-collagen increased slightly, some hepatic cells damaged can be seen in the group treated with Chinese medicine , interstitial fibers is weaker than the model group. The results indicated that HJG has the effect of protecting the hepatic cell and certain anti-hepatic fibrosisThe sixth,The effect on proliferation of HSC-T6When the HSC- T6 was cultivated to 12h, the HUJIN granules- containing serum have no obvious influence to the HSC-T6 proliferation. But when it was cultivated to 24h and 48h, the proliferation of HSC- T6 is obvious. HUJIN granules -containing serum can inhibit the proliferation of HSC more significantly .with the enhance of medicine-containing serum concentration, the inhibitory effect enhanced, indicating that HUJIN granules -containing serum has the dose-dependent inhibitory effect on HSC-T6 cells. The results indicated that HUJIN granules' s effect on anti-hepatic fibrosis contributes to the inhibition on the proliferation of HSC.