Pharmacodynamic Study Of ATR Extracts Against HBV And Liver Injury

BackgroundHepatitis B is caused by the hepatitis B virus (HBV) and features in liver damage.It is such a kind of infectious diseases that mainly a serious hazard to human health,with characteristics of strong infection, a wide range of popularity and high incidence. HBV infection distributes worldwide, popular region including indigenous Asia, South Pacific, Australia, New Zealand, South America, the Middle East, sub Saharan Africa and Aboriginal of Arctic Circle.There are about2billion people suffer from hepatitis B virus infection in the world, the number of annual death due to acute and chronic HBV infection was as high as1million, to2004Global HBV chronic infection has reached400,000,000. China has120,000,000HBV carriers of hepatitis B infection, accounting for30%of the world. Liver injury is the pathological basis of all the liver diseases. The study found,60%-70%liver biopsy pathology reports of "asymptomatic hepatitis B virus carriers" present chronic persistent hepatitis or chronic active hepatitis;and long-term chronic hepatitis B virus infection may develop into liver cirrhosis, and even hepatocellular carcinoma. Therefore, anti-HBV treatment is the key to prevent the further development of the disease;and the research and development of new antiviral drugs of good effect and low side effect is to be imperative.The screening of anti HBV drugs needs to perform first in animal and cell model experiment. The most used animal models in vivo is duck hepatitis B virus (DHBV) model. For the reason of easy feeding, the DHBV model has been widely used. These models were divided into congenital infection model and acquired infection model, foreign countries use the former, while the latter is used in our country. The DHBV model is mainly used for three aspects:viral replication test, immunological tests and antiviral drug test. As for the research on anti-HBV pharmacodynamics, DHBV model has been a widely accepted animal model.The cell models are mainly HepG2.2.15cells. HepG2.2.15cell line, which was established by the The Mount Sinai Medical Center of America in1986, has been successfully applied to the screening of anti-HBV drugs. China Health Department has contained it as the anti-HBV effect index in the treatment of hepatitis in the pharmacodynamics guide of new traditional Chinese medicine. This model is suitable for studies of antiviral medicines.And it has been successful used in the experimental studies on dozens of ingredients of common leafflower herb, cress, rhubarb.Liver injury is the pathological basis of liver disease. The mechanism of liver injury is rather complicated, which can be divided into chemical and immunity.The chemical mechanism of injury is mainly through intermediate metabolites produced by cytochrome P450and conjugation reaction,such as the change of plasma membrane integrity, mitochondrial dysfunction in the intracellular concentration changes, the activity of enzymes and free radicals; while the immune mechanism by cytokines, nitric oxide, complement and immune allergic reaction and damage. Hepatitis B virus is the most common cause of liver injury.At present, most scholars think that immune response is mainly related with the liver injury induced the hepatitis B virus infection. At the same time, autoimmune stimulation, viral or parasitic infection are the main reasons for liver injury, especially hepatitis. Acute chemical liver injury induced by carbon tetrachloride, D-galactosamine or thioacetamide, and liver injury caused by chronic model have been widely used in screening medicine for liver protection. However, the damage is obviously external chemical factors rather than by the human host defense induced hepatitis, therefore they are not suitable for evaluation of liver immune modulators. Studies on the mechanism of liver injury pathogenesis and liver-protective drugs needs to establish the suitable animal models. Therefore, in the study of anti-hepatitis drug,multiple models of different damage mechanisms can be taken to observe the protective effect of drug, with the purpose of comprehensive judgment of therapeutic effect and mechanism of action.Con-A induced liver injury model, whose liver injury is mediated by T lymphocyte,has been developed in recent years. Concanavalin A (Con-A) is a plant lectin that has the effect of specific toxicity to liver cells in vivo.It induces the infiltration of inflammatory cell,predominantly CD4+T lymphocyte in liver cells after moving into the circulation, and than activates cytokines such as TNF-a and interleukin, and causes inflammation reaction,and damages live cell through a variety of ways such as liver cell apoptosis, resulting in immunological liver injury. This model requires only a single intravenous injection of Con-A, its main character is a rapid onset, obvious liver damage. The pathophysiology of liver cell damage is very similar with that mediated by T lymphocyte in human chronic hepatitis B, with the characteristics of liver-dependent and dose-dependent. The model well simulates the human viral hepatitis, autoimmune liver disease, and is an ideal animal model for the screening of drugs for the treatment of acute liver injury and fulminant hepatic failure. China is one of the countries with high incidence of liver disease. It has always been an important direction of research of traditional Chinese medicine to search for and study effective natural medicines which has both antiviral and liver-protective effect. Anti-viral, immunomodulatory drugs and protecting liver and lowering protein drugs are several drugs that usually used in the treatment of liver diseases by western medicine. Clinical results show that, these drugs are good at the control of virus replication, the restoration of normal liver function, and the protection of liver cells, and so on. In the mean time, their curative effects are rapid. However, the disadvantages of antiviral drugs are unstable long-term efficacy, easy relapse, and low HBsAg negative rate. Besides, some drugs are expensive and have certain side effects. On the other hand, modern medicine in the treatment of liver injury has no specific drug. It usually suggests to take a rest, strengthen nutrition, and supplement and symptomatic treatment. Severe cases were even forced to discontinue their medication. Whereas Traditional Chinese Medicine, with its unique characteristics of the overall concept, the harmony between man and nature,and treatment based on syndrome differentiation, gives full play to the advantages of Chinese traditional medicine which include multi-targets, multi-ways, multi-levels, low price, less side effect, good curative effect in the treatment of hepatitis and liver injury,thus more and more medical researchers pay great attention to the development of it.The root of Alchornea trewioides(Benth.)Muell.-Arg (ATR),which belongs to dicotyledonous plants botanicals Euphorbiaceae, is one of the commonly used Chinese herb in south of the Five Ridges area. In the civil application It has a long history and curative effect in the protection of liver. The previous results of our research group suggested for the first time that ATR has the functions of protecting liver, reducing enzyme, anti-liver fibrosis. And It has been applied for patent protection. In order to further investigate and screen the effective parts of ATR, and provide exact mechanism for its liver-protective, antiviral effect,we extracted and isolated4kinds of ATR extracts, do experimental research on them.ObjectiveTo study the mechanism of ATR against hepatitis B virus (HBV) and acute immunological liver injury and its mechanism of immune regulation, we used four extracts water extract (WE), petroleum ether extract (PE), ethyl acetate extract (EE), n-butanol extract (BE) of ATR and do experiments on anti-hepatitis B virus and anti-immune liver injury and immune regulation action by HepG2.2.15cell model,duckling models congenitally infected with DHBV and Con-A induced acute liver injury mice model, with the purpose of laying a foundation for the further development and utilization of ATR.Methods:Part one Anti-hepatitis B virus experimentsExperiment1:the experimental study of ATR extracts on the inhibition of HBsAg and HBeAg in vitro.Methods:the four methyl thiazolyl tetrazolium (MTT) method was used to observe the toxic effects of ATR extracts on HepG2.2.15cells; and the inhibitory effects of ATR extracts on the HBsAg and HBeAg secreted by HepG2.2.15cells was detected by ELISA analysis.Experiment2:The inhibitory effect of ATR extracts on the replication of the hepatitis B virusMethods:detecting the inhibitory effect of ATR extracts on HBV-DNA secretion of HepG2.2.15cells by fluorescence quantitative PCR-probe analysis. Experiment3:Experimental study of ATR extracts against duck hepatitis B virus in vivoMethods:Screening the duckling models congenitally infected with DHBV by the conventional PCR method.And randomly grouping the screened ducks. Drug groups were administered drugs once a day by intragastric gavage, totally14d. The control group did not do any processing. Ducklings in each group were drawn blood from tibial vein1d before administration,7d and14d after administration,5d after drug cessation, collecting the serum after centrifugation.Using dot blot hybridization to detect DHBV-DNA in duck serum.Part two Studies of the protective effects of4ATR extracts on Con-A induced acute liver injury in miceExperiment4:the influence of ATR extracts on the transaminases and hepatic histopathology of Con-A induced acute liver injury in miceMethods:after grouping randomly, the model group and normal control group were gavaged daily with the same volume of normal saline; each drug group was continually administrated for3d.lh after the last administration, the model group and the drug groups were injected with Con-A solution, which induces acute immune hepatic injury in mice,while the normal control group was injected equal saline, fasting, but not water deprivation for8h.Then drew blood from eyeballs of mice,and took the same part in the livers of mice after the mice were killed,10%formalin fixed. The collected blood is centrifuged to collect serum preparing for the measure of alanine aminotransferase, aspartate aminotransferase; the formalin-fixed liver tissue were used to do further pathological examination and observing the change of structure of liver tissue after HE staining.Experiment5:immunological regulation of ATR extracts on Con-A induced acute liver injury in mice Methods:the WST-1method was used to detect the activity of SOD in liver tissue; the TBA method was to measure the content of MDA in liver, and the activity of GSH-PX in liver tissue was detected by colorimetric assay; the ELISA method was used to analyze the contents of serum inflammatory cytokines IL-6, TNF-α, IFN-γ; the flow cytometry was used to detect TLR4, Tim-4expression on splenic macrophages.Statistical processingThe experimental results were presented by mean±standard deviation (mean±SD), multiple samples were analyzed by one-way ANOVA; repeated measurement data using repeated measures analysis of variance, multiple level/frequency table data with a non parametric test of K Independent Samples. Correlation between the two variables by using bivariate correlation analysis (Bivariate).Using the statistical software SPSS13.0to carry on the analysis, and P<0.05is confirmed as the difference has statistical significance.Results1. The inhibition rates of BE on the secretion of HBsAg, HBeAg on HepG2.2.15reached65.2%,94.4%respectively under non-toxic concentrations,and the therapeutic index were>9.8and>67.1respectively;the inhibition rate of EE on HBeAg was53.4%, the therapeutic index>2, while no inhibitory effect on HBsAg; whereas WE and PE had no effect on the inhibition of HBsAg, HBeAg.2. Results demonstrated that F=9.479, P=0.000. Compared with the control cells, WE, PE, EE and BE all could significantly inhibit the viral load of HBV-DNA secreted by HepG2.2.15, the difference was statistically significant (P<0.05).3. The results showed that, overall, no significant difference of DHBVDNA changes at different time points (F=2.296, P=0.110); there are significant differences among the groups DHBVDNA (F=12.416, P=0.000); a significant interaction existed between time and group (P=6.393, P=0.000).In the duckling models congenitally infected with DHBV, among the ATR extracts groups, only the high-dose of PE could inhibit DHBVDNA after two weeks’ treatment and5ds of drug cessation,the difference was statistically significant(T14and P5, P<0.05).4. The results showed that, ALT, F=34.616, P=0.000; AST, F=37.658, P=0.000. Degeneration of liver cells, X2=57.935, P=0.000; necrosis of liver cells, X2=41.751, P=0.000;the results above indicated that there was a significant difference in the curative effect of each group.4ATR extracts could significantly reduced the serum alanine transaminase, aspartate transaminase,the difference was statistically significant (P<0.01).In the liver tissue morphology, Compared with the normal group, model group liver degeneration and necrosis pathological changes were especially significant (P<0.05/14), which indicated that our animal model was established successfully. As for the degeneration of liver cells, there was no significant difference among drug groups in improving the liver degeneration. In the necrosis of liver cells, medium dose of PE group, EE high dose group, BE in low, middle dose group and the GLX group reduced necrosis of liver cells significantly (P<0.05/14); microscopy results are shown in Table4-2and figure4-(1-15)(HE staining,*200).5. The results showed that, SOD, F=34.961, P=0.000; MDA F=23.613P=0.000; GSH-PX, F=37.089, P=0.000. IL-6F=74.279, P=0.000; TNF-α F=28.023, P=0.000; IFN-γ F=374.800, P=0.000. TLR4F=4.552, P=0.015; Tim-4F=6.763, P=0.003.The4ATR extracts not only could significantly decreased the liver MDA content (P<0.01), but also could significantly improve the SOD activity of liver (P<0.05); as for the GSH-PX, except for EE, other three extracts could significantly improve the GSH-PX activity of liver (P<0.01). At the same time,4ATR extracts species not only could significantly inhibit the inflammatory cytokines IL-6, TNF-α, IFN-γ, the difference was statistically significant (P<0.05); but also could improve the percentage of expression of TLR4and Tim-4on spleen macrophages, the difference was statistically significant (P<0.05), suggesting that4ATR extracts could raised TLR4and Tim-4levels on splenic macrophages. TLR4and Tim-4were positively correlated (r=0.538, P=0.021)(P<0.05).ConclusionsAs for hepatitis B virus, study results in vitro showed that only EE and BE could inhibit the HBV antigen, and BE is better than EE;4ATR extracts could inhibit HBV-DNA;study results in vivo showed that only PE of high dose group in the treatment of7d,14d could reduce the level of serum DHBV-DNA, and the DHBV-DNA level remained decreased and showed no rebound after drug cessation for5days(P<0.05), other groups had no inhibitory effect of serum DHBV-DNA. Thus we can conclude that the HBV antigen and HBV-DNA are two targets of ATR against HBV.The results showed that, among the4ATR extracts, n-butanol extract not only could inhibit two HBV antigens, but also could inhibit HBVDNA level, so the n-butanol extract of ATR is the best in the effect of anti-HBV. As for acute liver injury, ATR extracts not only could reduce transaminase, improving liver tissue morphology, but also could reduce the content of liver MDA, liver SOD and GSH-PX activity increased. At the same time,4ATR extracts not only could significantly inhibit the inflammatory cytokines IL-6, TNF-α, IFN-gamma, but also increased the expression level of TLR4and Tim-4on splenic macrophages, there was a significant positive correlation between TLR4and Tim-4, thus we can conclude that reducing transaminase, improving liver histology, inhibiting lipid peroxidation, and inflammation cytokines, up-regulating the expression of TLR4and Tim-4are several liver-protective mechanisms for4ATR extracts.In addition, as it is known that TLR4 and Tim-4plays an important role in immune regulation, we found that there was a positive correlation between the two, so it could be inferred that the up-regulation of TLR4and Tim-4expression on splenic macrophages may be associated with the activity of anti-hepatitis B virus and anti-hepatic injury. And ATR has a rather good immune-regulating effect on acute liver injury. Among the4kinds of ATR extracts,BE was the best at the effect of anti-hepatitis B virus and anti-hepatic injury.We hypothesized that BE up-regulate the levels of TLR4and Tim-4through its immune regulation function. On the one hand, BE may act as a TLR4agonist, and inhibit the secretion of HBsAg, HBeAg and HBVDNA, thus playing the role of anti hepatitis B virus,;on the other hand, its up-regulation of Tim4lead to the inhibition of lipid peroxidation and inflammatory cytokines, thereby reducing transaminase, improving liver tissue degeneration and necrosis, thus playing the role of anti-hepatic injury.