Study On The Mechanism Of Hepatoprotective Action Of Extract From Isodon Iophanthoides Var. Gerardianus(Benth.) Har

As a common disease, epidemic chronic hepatitis has strongly influenced health of humanbeing in China. The epidemic investigation shows that chronic hepatitis B is a mainlyreason causing hepatic cirrhosis and primary hepatic carcinoma. Therefore, to develop anew and advanced means to prevent and treat chronic hepatitis is always a hot button in theworldwide medical science.Nowadays there are 3 kinds of commonly used medicine in the clinic:①antiviral drug,IFN and several nucleoside analogues as main treatment. The each kind of medicine haverespective merits and demerits. Firstly, the former has definitive effects, high conversionrate in virus of blood serum, certain healing ending, however, the fairly abundant sideeffects and inconvenient use influence its wide use; the latter has convenient administration,definitive effects, meanwhile, it also has a few of demerits which need long term adm-inistration, without certain healing ending and easily induce drug resistance.②im-munomodulator, thymic peptide and remedial vaccine as main represantive medicines ofthe kind. The former may inhibit even eliminate the virus in the blood by regulating theimmunological function, strengthening clearance ability of immune system. It is often usedwith IFN or nucleoside analogues. However, effects of the kind of drugs are still con-troversial. As for remedial vaccine, it is still in the period of experimental research.③thedrugs with the function of protecting liver, such as bifendate, preparation from fructusschizandrae and some kind of Chinese herbal medicine. The kind of drugs has some effecton lowering the level of transaminase in blood and eliminates jaundice, specially bifendatedeveloped by china. The drugs easily caused bounce-back after drug withdrawal. It can beused as an adjunctive treatment with antiviral drugs.To search a kind of drug with certain curative effect is a difficult problem all over the worldfor the reason of HBV replication in the cell of host. The immunologic response of patient to hepatitis B plays a very important role on pathogenesis of virus hepatitis and it is alsomain factor that will give rise to impairment of Hepatic tissue. Apart from antiviral agent,discovering a new drug with repairing Hepatic tissue and regulating immune function isalso an important aspect. Nowadays, to discover a drug with protecting liver function iswell prospective, for example, the glycyrrhizin made by Japanese and silymarin developedby Germany are important products in the international herb product market. Scientists andenterprises attach much importance to develop antihepatitis drug from Chinese traditionaland herbal drugs recent years. However, product with high efficacy and low toxicity cannotbe discovered, meanwhile, development of this kind drug still is in low level and status ofimitating each other between enterprise.The animal model similar to clinic pathomechanism is the key to screen antihepatitis drugand research the mechanism of drug. As has been said before, the hepatic cell damagecaused by the immunologic response of patient to hepatitis B is chief pathomechanism ofchronic hepatitis B, establishing a animal model of immune liver injury is relatively idealmodel on developing antihepatitis drug. Recent years, Scientists built up a model ofimmune liver injury by administered Con A intravenously, that has many resemblances tohuman virus hepatitis in causative characteristic. The model has been proved to be immuneliver injury mediated by T cell. To sum up, our research choose a model of immune liverinjury induced by Con A, and discuss mechanism of action of Isodon lophanthoidesvar. gerardianus(Benth) Har.As a species of habitual medicinal herb in Guang dong Province, I. serra have effect withclearing heat, eliminating dampness, reducing jaundice, cooling blood and removing bloodstasis. Its main current species is Isodon lophanthoides var. gerardianus through resourcesinvestigation. Previous research suggested that the water soluble extract and alcohol—soluble extract have effect on protecting liver, inhibiting bacterium and relievinginflammation. Then the protective effect on immune liver injury of water-soluble extractwas studied, the results showed that it had marked effect to protect mice from immune liverinjury induced by Con A. Scientists in other research unit of our university set up a series ofstudy on I. serra (Maxim.) Kudo., the result showed that its water-soluble extract andcompounds derived from ethyl acetate may inhibit content of DHBV-DNA in duck andinhibit the level of HBs-Ag secreted from HepG2.2.15 cell in vitro.Based on what have been mentioned above, the main aims of this research are to establish the immune liver injury model, and study the mechanism of action of water-soluble extractfrom Isodon lophanthoides var. gerardianus(Benth.) Har.on the following aspects:①effect on the level of ALT and AST in mice blood serum and histopathologic examinationof liver tissue;②influence on gene expression of important medium such as TNF-αandICAM-1;③influence on NF-κB protein expression.Ⅰeffect of extract from Isodon lophanthoides var. gerardianus on the level of ALT andAST in mice blood serum and histopathologic examination of liver tissue.Objective: To establish immune liver injury model induced by Con A to study influence ofwater-soluble extract on content of ALT and AST in mice blood serum and histopathologicexamination of liver tissue. Methods: after breeded on SPF grade animal room for 3 days,male Balb/c mouse were randomly divided into 5 groups, which included normal group,model group, positive control group (PDTC group) and two treatment group by traditionalChinese medicine (high-dosage group and low -dosage group). The normal control group,model group and positive control group were fed with sterile normal saline, while the twoTCM treatment group (water-soluble extract from lsodon lophanthoides var. Gerardian-us(Benth.) Har.) were administrated at dosage 9.2g/kg/d, 4.1g/kg/d respectively. These hadbeen fed for 7 days. Except for normal control group and positive control group, everygroup were challenged by a single tail intravenous injection of Con A (25mg/kg) at half anhour after administration, the positive control group were treated with PDTC (120mg/kg)half an hour before Con A injection. Collected blood serum at 4h and 8h point afterchallenged, determined the level of ALT and AST in the blood and histological examinationof liver tissue, while preserve liver tissue for detection of RT-PCR and immunohistoc-hemistry detection. Results: (1) treatment of mice with 25mg/kg ConA resulted in immuneliver injury model, compared with normal control group, ConA can markedly increase thelevel of ALT, AST in blood, while histological examination suggested, model groupappeared that structure of liver cell cord were destroyed, stagnated blood in sinus hepaticus,lamellar necrosis area; at 8h point, much more acidophilia than 4h point, mulberry stainingin cytoplasm. (2) Compared with model group, the extract group could significantly lowerthe high level of ALT and AST in blood, improve the histological change of liver tissue ofthe immune liver injury model. Conclusion: the water soluble extract from Isodonlophanthoides var. gerardianus can prevent mice from immune liver injury by ConA, ithave definitive effect of lowering level of ALT and AST and protecting liver.Ⅱeffect of extract from Isodon lophanthoides var. gerardianus on level of TNF-αmRNA and protein expressionObjective: TNF-αis a important inflammatory mediator, we investigated its roles onimmune liver injury model induced by ConA, and determined influence of extract of Isodonlophanthoides var. gerardianus on its mRNA and protein expression.Methods: the levels of the protein expression of TNF-αin liver tissue were detected byimmunohistochemistory examination. The mRNA expression of TNF-αwere detected byreverse transcription polymerase chain reaction (RT-PCR). Results: ConA administrationcaused increased hepatic TNF-αeither mRNA or protein expression. Compared withmodel group, the extract could significantly lower the protein expression of TNF-α,furthermore, the mRNA expression of in liver tissue were lower than those of model group.Conclusion: TNF-αplay an important role in the immune liver injury model, thewater-soluble extract from Isodon lophanthoides var. gerardianus could inhibit TNF-αexpression at the level of mRNA transcription.Ⅲeffect of extract from Isodon lophanthoides var. gerardianus on level of ICAM-1 mRNA and protein expressionobjective: TNF-αcould promote production of ICAM-1, ICAM-1 also mediate theliver injury induced by Con A. we investigated its roles on immune liver injury model, anddetermined influence of extract of Isodon Iophanthoides var. gerardianus on ICAM-1mRNA and protein expression. Methods: immunohistochemistry examination was used todetermine the protein expression of ICAM-1 in liver tissue, and reverse transcriptionpolymerase chain reaction (RT-PCR) was performed to analyze the mRNA expression ofICAM-1. Results: high protein and mRNA expression were detected in model group,correlated with increases in serum ALT and AST levels and histological examinationappearance, that demonstrated ICAM-1 acted as a significant mediated role on pathomec-hanism induced by Con A. compared with model group, the protein and mRNA expressionof ICAM-1 were markedly inhibited in extract group. Conclusion: extract from Isodonlophanthoides var. gerardianus may suppress ICAM-1 gene expression by inhibitingICAM-1 transcription.Ⅳeffect of extract from Isodon lophanthoides var. gerardianus on nuclear factor-κB inimmune liver injuryObjective: The transcription factor, nuclear factor kappa B (NF—κB) were found to beinvolved in many pathophysiology processes such as inflammation and cancer. We studyits relevance with liver damage, and evaluate extract of Isodon lophanthoides var.Gerard- ianus's effect on NF—κB expression and expression translocation. Methods: proteinexpression and expression translocation of NF—κB were detected by immunohisto-chemistry examination. Results: up-regulation of protein expression of NF—κB weredetected in model group, and NF—κB expression were translocated from cytoplasm intonucleus can be observed. PDTC can inhibit the translocation of NF—κB to the nuclear byinhibiting the IκB degradation. Correspondingly, the levels of ICAM-1 and TNF-αmRNA expression reduced, the results indicated that NF—κB regulate some geneexpression such as ICAM-1 and TNF-αmRNA, thereby contributing to immune liverinjury. Extract group also observed that down-regulation of NF—κB protein expression,and extract can keep NF—κB in cytoplasm, reduce its translocation from cytoplasm intonucleus. Conclusion: it is suggested that extract from Isodon lophanthoides var. gera-rdianus can act against immune liver damage induced by ConA, that is associated withinhibition of the activation of NF—κB, thereby down-regulate the transcription andexpression of important inflammatory factor such as ICAM-1 and TNF-αin immune liverinjury model.