Study On The Mechanism Of Lowering Transaminase Effect Of Ronggan Mixture On Immunological Liver Injury Model Of Mice Induced By Concanavalin A

Objective:1. Theoretical study part:To explore the relationship between the principle of QingLiHuoXueJianPi (Which is principally clearing away heat evil and promoting diuresis, and activating blood circulation, invigorating the spleen, strengthening healthy qi as additional treatment) and chronic hepatitis B(CHB), which can provides a thinking for the therapy of CHB and provides the basis for recipe construction of RongGang mixture.2. Part of discussion on the research ideas:To clarify some concepts of the therapy of "protecting liver and lowering transaminase" and confirm the connotation, which can make the Traditional Chinese Medicine(TCM) therapy for CHB more comprehensive and objectivity.3. Experiment research part:To investigate the lowering transaminase effect and the mechanism of RongGang mixture on immunological liver injury model (both acute and chronic) in mice induced by concanavalin A (Con A).4. Clinical research part:To investigate the effect of the RongGang mixture on the quality of life(QOL) on patients with CHB.Methods:1. Theoretical research:The etiopathogenisis and therapeutical principle of CHB were discussed by reviewing the literatures of the ancient Chinese and western medicine documents and works to analyse the relationship between the principle of QingLiHuoXueJianPi and CHB. Meanwhile, analyse the principles of formulating prescription of RongGang mixture. 2. Summarise experiences of the therapeutic methods of TCM on "protecting liver and lowering transaminase" therapy in recent years, analyze the problems and discuss the ways of deal with these problems.3.(1) Acute Liver Injury:HBV transgenic mice were randomly divided into control group, model group, RongGang mixture group, Yinchen group, Yinchenhao decotion group and Bifendate group,10 mice in each group. The acute immunological liver injury model in mice were established by tail vein injection of Con A. Fourteen days before the model established, the herbal intervention were given as follows:normal saline (control group and model group), RongGang mixture, Yinchen extract, Yinchenhao decotion and Bifendate. One hour after the last herbal intervention, the control group were given PBS by tail vein injection, while the others were given ConA on dose of 3μg/g by the tail vein injection. Eight hours after the modeling, the mice were sacrificed, the serum and tissue samples were collectd to detect various kinds of lab indications:ALT, AST, TBiL, histopathological changes of the liver, HBV DNA, HBsAg, lipid over-oxdizing reaction of hepatocyte (SOD,MDA), T-lymphocyte subsets (CD4+, CD8+, CD4+/CD8+), cytological apoptosis genes (Fas, FasL, Bax, bcl-2),cytokines in liver tissue(TNF-α, INF-γ, IL-4, IL-10).(2) Chronic Liver Injury:HBV transgenic mice were randomly divided into normal group(10) and Con A model group(59). The normal group were given PBS by the tail vein injection(0.3ml), while the others were given Con A by the tail vein injection on the dose 6μg/g. Eight weeks after the model established, the Con A model group were randomly divided into model group, RongGang mixture group, Yinchen group, Yinchenhao decotion group and Bifendate group. Then the mice were filled the stomaches with corresponding therapeutic interventions for 4 weeks, while the normal group and model group were given normal saline. Twenty-four hours after the last gastric perfusion, the mice were sacrificed; the serum and tissue samples were collectd to detect various kinds of lab indications as the acute liver injury.4. Randomized controlled clinical trial was adopted. Sixty CHB patients were divided into two groups(treatment group and control group) by the block randomization method. Treatment group were treated by RongGang mixture, while the control group were treated by the nucleoside/nucleotide analogues (NUCs). The period of treatment is 6 months, and 3 months as a visiting point of time. The indications of ALT, AST, HBV DNA in the serum were detected in each visiting point of time. And the QOL of the patients were obseved by the 36-items short form health survey (SF-36) and Chronic Liver Disease Questionnaire (CLDQ).Result:1. The CHB is a syndrome of excessive Biao and deficiency Ben. The excessive Biao is dampness-heat syndrome and blood stasis syndrome, while the deficiency Ben is the spleen deficiency syndrome. The Liver-Qi stagnation syndrome is the concomitant syndrome sporadically and prolonged disease always involve the kidney.2.The therapy of "protecting liver and lowering transaminase" now is not the combination of the effect of lowering transaminase and improved histopathological changes, and do not combine with antiviral therapy, treatment of enhance immune function, the preventive effects of liver fibrosis. The "protecting liver and lowering transaminase" therapy pay no attention on the QOL of CHB in recent years.3.(1) Acute Liver Injury:①Serum enzyme changes:Compared with model group or the other groups, RongGang mixture and Bifendate can decrease the level of ALT and AST(P≤0.01), RongGang mixture can decrease the level of TBiL(P<0.01).②Histopathological changes:Compared with model group, liver cell degeneration, cellular necrosis and inflammatory cell infiltration in RongGang mixture group were lighter(P<0.05),which showed RongGang mixture can improve liver histopathological changes. While the histopathological changes degree of liver injury in Bifendate group and Yinchen group were similar with the model group on these aspects with no significant difference (P>0.05).③HBsAg, HBV DNA:Compared with Yinchen group, RongGang mixture can decrease the level of HBV DNA with significant difference (P< 0.05).④Lipid over-oxdizing reaction of hepatocyte (SOD,MDA):The index of SOD in the model group is lower than the normal group(P<0.01), while MDA is higher than the normal group(P<0.05). Compared with the other group, RongGang mixture can increase the level of SOD (P<0.01). Compared with model group and Yinchen group, RongGang mixture can decrease the level of MDA (P<0.05).⑤T-lymphocyte subsets(%):Compared with model group, all of the drug intervention group can increase the percentage of CD4+CD8-T cells, but RongGang mixture is the best(P<0.05). Compared with model group and Bifendate group, the percentage of CD4-CD8+T cells in RongGang mixture group is lower(P<0.05). Compared with model group, the ratio of CD4+T cells to CD8+T cells increase evidently in therapeutics group, while RongGang mixture is superior to Yinchenhao decotion group(P<0.05).⑥cytological apoptosis genes (Fas, FasL, Bax, bcl-2):In the normal group, there is no express of FasL and Bax and the expression of Fas and bcl-2 is just a little, while with significant difference compared with the normal group(P<0.05). Compared with the model group, all of the expression of Fas, FasL, Bax, bcl-2 gene in the RongGang mixture changed with significant difference, and the effect of RongGang mixture is superior to the other groups partly (P<0.05).⑦cytokines in liver tissue(TNF-a, INF-y, IL-4, IL-10):Compared with the normal group, TNF-a and INF-y in the model group is higher (P<0.05), while IL-4 and IL-10 is lower(P<0.05). Compared with the model group, all of the expression of TNF-a, INF-y, IL-4, IL-10 in the RongGang mixture group changed with significant difference, and the effect of RongGang mixture is superior to the other groups partly (P<0.05).(2) Chronic Liver Injury:①Compared with the model group, RongGang mixture and Bifendate can decrease the level of ALT, AST, TBiL (P<0.01).②Histopathological changes:Compared with model group, liver fibrosis, liver cell degeneration, cellular necrosis and inflammatory cell infiltration in RongGang mixture group and Yinchenhao decotion group were lighter(P<0.05), which showed they can improve liver histopathological changes, and RongGang mixture is superior to Yinchenhao(P<0.05).③HBsAg, HBV DNA:Compared with model group, RongGang mixture can decrease the level of HBV DNA with significant difference (P≤0.05). Compared with other groups, RongGang mixture can decrese the level of HBsAg with significant difference(P<0.05).④Lipid over-oxdizing reaction of hepatocyte (SOD,MDA):The index of SOD in the model group is lower than the normal group, while MDA is higher than the normal group(P<0.01). Compared with the model group, the RongGang mixture,Yinchenhao decotion and Bifendate can increase the level of SOD (P<0.01), while the RongGang mixture and Yinchen extracting solution can decrease the level of MDA (P<0.05).⑤T-lymphocyte subsets(%):Compared with model group, all of the drug intervention group can increase the percentage of CD4+CD8-T cells, but RongGang mixture is the best(P<0.05). Compared with model group, the percentage of CD4-CD8+T cells in RongGang mixture group and Bifendate group are lower(P<0.05). Compared with model group, the ratio of CD4+T cells to CD8+T cells increase evidently in therapeutics group, while the RongGang mixture is superior to Yinchenhao decotion group and Bifendate group (P≤0.05).⑥cytological apoptosis genes (Fas, FasL, Bax, bcl-2):In the normal group, there is no express of FasL and Bax.the expression of Fas and bcl-2 is just a little,while with significant difference compared with the normal group(P<0.05). Compared with the model group, all of the expression of Fas, FasL, Bax, bcl-2 gene in the RongGang mixture changed with significant difference, and the effect of RongGang mixture is superior to the other groups. RongGang mixture group is superior to model group on the index of ratio of bcl-2 to Bax (P<0.05).⑦cytokines in liver tissue(TNF-a, INF-y, IL-4, IL-10):The index of TNF-a, IFN-y in the model group is higher than the normal group, while IL-4, IL-10 is lower than the normal group(P<0.05). The RongGang mixture can decrease the level of TNF-a and IFN-y, while increase the level of IL-4 and IL-10 in the liver tissue(P<0.01-0.05), but the other therapeutic interventions are just partially effective on those indexes.4.After the drug intervention, in each visiting point of time, the level of ALT, AST in each group decrease dramaticly, the recover rate of ALT and AST of patient with CHB were above 80% in 3months and 100% in 6months, while there have no difference between the two groups. As time went on, the load of HBV DNA in each group is declined, but the NUCs have more remarkable anti-virus effect than RongGang mixture. On the QOL aspects, RongGang mixture can significantly improve the QOL of CHB patients in all of the domins of SF-36 and CLDQ persistently, while the NUCs just improve the QOL of CHB patients in part of the domins, NUCs do not works in the domin such as RP(Role-Physical), RE(Role-Emotional), BP(Bodily Pain), etc. Comparison among the two groups in each visiting point of time is as follow:①3months:the improvement of RongGang mixture is superior to the NUCs in those domins(Physical Functioning, RP, Vitality, Physical Component Summary, Mental Component Summary, overall score of SF-36 and all domins of CLDQ)with significant difference (P≤0.05).②6months:Except the domins of Abdominal symptoms, the improvement of RongGang mixture is superior to the NUCs with significant difference (P<0.05).Conclusion:1.The therapeutic methods of CHB is principally clearing away heat evil and promoting diuresis, and activating blood circulation, invigorating the spleen, strengthening healthy qi as additional treatment, and the formala principle of RongGang mixture is consistent with these therapeutic methods.2. The therapy of "protecting liver and lowering transaminase" must be the combination of the effect of lowering transaminase and improved histopathological changes, must combine with antiviral therapy, treatment of enhance immunefunction and preventive effects of liver fibrosis. The "protecting liver and lowering transaminase" therapy must pay great attention on the QOL of CHB.3.The conclusion from the experiment research:①The effect of protecting liver and lowering transaminase of RongGang mixture is not only decreasing the transaminase in the peripheral blood, but also can change the histopathology of the liver:reverse the liver fibrosis, reduce cellular necrosis and inflammatory cell infiltration,etc.②The principle of QingLiHuoXueJianPi is effective for the treatment of CHB, and the compound Chinese medicine do great works than the single herb (Yinchen) and Bifendate.③The mechanism of protecting liver and lowering transaminase of RongGang mixture can be various:resisting the lipid over-oxdizing reaction of hepatocyte, regulate the balance of T lymphocyte subsets and Thl/Th2 factor level, inhibited apoptosis of hepatocyte,etc.4.The RongGang mixture can enhance the recover rate of ALT and AST of patient with CHB, and can decrease the load of HBV DNA, while the effect for HBV DNA is inferior to NUCs. RongGang mixture can improve the QOL of CHB, and the effect is superior to the NUCs.