| Objective:This study investigated the mechanism of Diwu Yangganfang in the treatment of HBe Ag negative chronic hepatitis B,and provide the basis for clinical application Method:This study included two parts, theoretical studies and experimental research.theoretical studies: Discussed the ancients understanding of hepatorenal homologous from the origin of hepatorenal homology theory, the connotation of hepatorenal homologous theory,explored the development of hepatorenal homology theory in modern times from the modern clinical application and experimental study,Finally investigated systematically etiology,pathogenesis of chronic hepatitis B and its possible mechanism from treating liver and kidney at the same time.experimental research: 120 cases of HBe Ag negative CHB patients were randomly divided into control group and treatment group, each group had 60 cases. The treatment group was given Diwu Yangganfang and entecavir,the control group were treated with traditionalChinese medicine placebo and entecavir.The treatment course was48 weeks. Over the same period, 30 cases of healthy people were selected as the normal group. The relative detections were performed in liver function, blood coagulation function, serum type IV collagen(IV-C), type III procollagen(PC III) and laminin(LN),hyaluronic acid(HA), hepatitis B, quantitative and quantitative HBV DNA.The LSM of all patients were recorded by fibroscan( FS)before and after treatment. At the same time, the levels of serum TGF-β1, EGF, HGF, IFN-γ, IL-9,IL-10 and the value of IFN-γ/IL-10 of all patients and healthy people were detected by enzyme-linked immunosorbent assay(ELISA).The levels of serum SOD, GSH, CAT and8-OHd G of all patients and healthy people were detected. The expression of CD3+, CD4+and CD8+T lymphocytes in peripheral blood were detected by flow cytometry. Determination of EGF, EGFR, HGF,c-met m RNA and protein expression in liver tissues of the control group and the treatment group before and after treatment by real-time PCR technique and immunohistochemical method.Result:theoretical studies: The theory of hepatorenal homologous originated from the emperor’s Internal Classic, which was deepened in Han and Tang Jin Yuan and formed in the Ming and Qing Dynasties.The theory of hepatorenal homologous including essence and blood,mother and child, controlling fire, common living in lower Jiao,Yin Yang complementarily, coordination of reservoir and vent spirit and meridians connected of liver and kidney,were further enriched in modern times by linking up hepatorenal homologous with the modern medicine of hypothalamus-pituitary-target gland,neuro- endocrineimmune network, and extending to the thalamus-pituitary-liver axisand testicular-testosterone-HGF. The occurrence, development,change and prognosis of a series of process of chronic hepatitis B are inseparable from the liver and kidney function.The basic pathogenesis of chronic hepatitis B were liver and kidney dysfunction and viscera deficiency. Excellent effects can be usually achieved on treatment of chronic hepatitis B by invigorating liver and kidney.experimental research:1. HBV DNA negative conversion: The rate of HBV DNA negative conversion of treatment group was significantly higher than that of the control group, the difference was statistically significant(P<0.05).2. Comparison of liver function and coagulation function: The levels of AST and ALT after treatment in the two groups were decreased significantly compared with those before treatment(P<0.01), and the levels of AST and ALT in treatment group were significantly lower than those in control group after treatment(P<0.01).the PT of the two groups after treatment was lower than those before treatment, but the difference was not statistically significant(P>0.05).3. Comparison of indexes of liver fibrosis: The levels of IV-C,PCIII, LN and HA in two groups after treatment were decreased significantly compared with those before treatment(P<0.01), and the levels of IV-C, PCIII, LN and HA in treatment group were significantly lower than those in control group after treatment(P<0.01).4. Comparison of the liver transient elastic value(LSM): The LSM of two groups after treatment were decreased significantly compared with those before treatment(P<0.05 or P<0.01), and The LSM of the treatment group were significantly lower than those of the control group after treatment(P<0.05).5. Comparison of the levels of TGF-β1, IFN-γ,IL-9 and IL-10 :The levels of TGF-β1, IFN-γ,IL-9 and IL-10 in two groups after treatment were decreased significantly compared with those before treatment(P<0.01), and the levels of TGF-β1, IFN-γ,IL-9 and IL-10 in treatment group were significantly lower than those in control group after treatment(P<0.05).The values of IFN- γ/IL-10 in two groups after treatment were significantly higher than those before treatment(P<0.01), and the values of IFN- γ/IL-10 in treatment group were significantly higher than those in control group after treatment(P<0.05).6. Comparison of the levels of serum EGF and HGF : The levels of serum EGF and HGF in two groups after treatment were increased significantly compared with those before treatment(P<0.01), and the levels of serum EGF and HGF in treatment group were significantly higher than those in control group after treatment(P<0.05).7. Comparison of the levels of serum GSH, CAT,SOD and 8-OHd G:Compared with those before treatment, the levels of serum GSH, CAT,SOD in two groups after treatment were elevated markedly and the level of serum 8-OHd G was decreased significantly, the difference was statistically significant(P<0.01). and the level of 8-OHd G in the treatment group was significantly lower than that of the controlgroup after treatment, the levels of GSH, CAT, SOD were significantly higher than those of the control group after treatment(P<0.01).8. Comparison of T lymphocyte subsets in peripheral blood:Compared with those before treatment, The percentages of CD4+and CD4+/CD8+T lymphocytes in two groups after treatment were increased significantly(P<0.05 or P<0.01).and there was no significant difference in CD3+ and CD8+ before and after treatment in the control group.but the percentage of CD8+T lymphocytes in the treatment group after treatment were decreased significantly compared with that before treatment(P<0.01).The percentages of CD4+and CD4+/CD8+T lymphocytes in the treatment group was significantly higher than that of the control group after treatment,the percentage of CD8+was significantly lower than that of the control group after treatment(P<0.01).9. Comparison of expression of EGF and EGFR in liver tissues:The expressions of EGF and EGFR were observed in liver tissues of HBe Ag negative CHB patients, and the cytoplasm of hepatocytes and the area of cell membrane were light brown. The expressions of EGFR,EGF in the cytoplasm, cell membrane and blood sinus were strongly positive after treatment. and the expressions of EGF, EGFR in liver tissues were increased significantly compared with those before treatment(P<0.01).10. Comparison of expression of HGF and c-met in liver tissues:The expressions of HGF and c-met were observed in liver tissues of HBe Ag negative CHB patients, and the areas of the hepatic sinusoidal,sinus cell cytoplasm and the hepatic cell membrane were light brown.The expressions of HGF and c-met in the hepatic sinusoidal and Disse’s cytoplasm, liver cell membrane area were strongly positive after treatment. and the expressions of HGF and c-met in liver tissues were increased significantly compared with those before treatment(P<0.01).11.Comparison of expression of EGF, EGFR, HGF and c-met m RNA in liver tissue: The expressions of EGF, EGFR, HGF and c-met m RNA in liver tissue were increased significantly in the treatment group after treatment compared with those before treatment(P<0.01).The expressions of EGF, EGFR, HGF and c-met m RNA in liver tissue were increased in the control group after treatment compared with those before treatment, but the difference was not statistically significant(P>0.05).The expressions of EGF, EGFR, HGF and c-met m RNA of the treatment group was significantly higher than those of the control group after treatment(P<0.01).Conclusion:theoretical studies: hepatorenal homologous theory has a long history and rich content. It was very important to explore the etiology, pathogenesis, treatment and drug action mechanism of HBV based on the hepatorenal homologous theory.experimental research:1. Diwu Yangganfang could be effective in the treatment of HBe Ag negative liver CHB.2. Patients with HBe Ag negative CHB exist the disorder of the cytokine network, nerve-endocrine-immune network and oxidative stress, which is equivalent to the motherland medicine " the disorders of essence, bone marrow and brain ". The variety ofdisorders in HBe Ag negative CHB patients lead to inflammation damage of the liver tissue, affect the normal regeneration of liver cells, which is the same meaning with the Chinese medicine "marrow failure to generate liver".3. Diwu Yangganfang by tonifying the kidney can regulate effectively cytokine network, improve the peripheral blood T lymphocyte subsets, adjust "nerve-endocrine-immune network" and increase the capacity of antioxidant, reduce oxidative stress and improve the expressions of EGF, EGFR, HGF and c-met in the liver tissue, improve liver micro environment of HBe Ag negative CHB patients, thus conducive to the normal regeneration of liver cells,promotion the recovery of liver function, which reflects the essence of tonifying kidney and marrow into liver of hepatorenal homologous. |
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