基于“肝肾同源”理论探讨“地五养肝方”治疗HBeAg阴性慢性乙型肝炎的机理
发布时间：2018-09-15 19:03
【摘要】：目的从“肝肾同源”理论探讨“地五养肝方”治疗HBe Ag阴性慢性乙型肝炎的作用机制,为临床应用提供依据。方法本研究从理论研究及实验研究两个方面进行。理论研究:从肝肾同源理论的源流及内涵来梳理肝肾同源发展的脉络;从肝肾同源理论的临床应用及实验研究来论述现代对肝肾同源理论实质的探讨和发挥;最后对慢性乙型肝炎(CHB)从肝肾论治的病因病机及可能的作用机制进行了归纳和探讨。实验研究:120例入组的HBeAg阴性CHB患者,随机分为对照组、治疗组,每组均为60例。治疗组口服地五养肝方及恩替卡韦;对照组予中药安慰剂口服,同时口服恩替卡韦,疗程为48周。并另选同期健康人30例作为正常组。比较对照组和治疗组治疗前后肝功能、凝血功能、血清Ⅳ型胶原(Ⅳ-C)、Ⅲ型前胶原(PCⅢ)、层粘蛋白(LN)、透明质酸(HA)、乙型肝炎三系定量、HBV DNA定量及肝脏瞬时弹性。同时采用酶联免疫吸附法(ELISA)检测治疗组、对照组治疗前后及正常组血清TGF-β1、EGF、HGF、IFN-γ及IL-9、IL-10的水平,并计算IFN-γ/IL-10值。检测治疗组、对照组治疗前后及正常组血清SOD、GSH、CAT、8-OHdG的水平。用流式细胞技术检测外周血CD3+、CD4+及CD8+T淋巴细胞的表达情况。用real time PCR技术、免疫组化法测定治疗组和对照组治疗前后肝组织EGF、EGFR、HGF、c-met mRNA量及蛋白表达水平。结果理论研究:肝肾同源理论萌芽于《黄帝内经》,深化于汉唐金元,形成于明清。肝肾同源理论包含有肝肾精血相关,肝肾母子相生,肝肾共司相火,肝肾同居下焦、阴阳互补,肝肾藏泄相协及肝肾经脉相通,而现代医家将肝肾同源与现代医学的下丘脑-垂体-靶腺、神经-内分泌-免疫网络联系起来,并延伸至下丘脑-垂体-肝轴及睾丸-睾酮-hgf,更进一步丰富了肝肾同源理论的内涵。chb的发生、发展、变化、转归等一系列过程均与肝肾功能状态密不可分,其根本病机为肝肾失调,脏腑正气不足,因此从肝肾论治chb能取得卓越疗效。实验研究:1.hbvdna阴转情况:治疗组hbvdna阴转率明显高于对照组,差异有统计学意义(p0.05)。2.对照组和治疗组肝功能及凝血功能的比较:两组治疗后alt、ast水平较治疗前均明显下降(p0.01),且治疗组治疗后alt、ast水平较对照组治疗后明显降低(p0.01)。两组治疗后pt较治疗前有所降低,但差异无统计学意义(p0.05)。3.对照组和治疗组肝纤维化指标的比较:两组治疗后Ⅳ-c、pcⅢ、ln、ha水平较治疗前均明显下降(p0.01),且治疗组治疗后Ⅳ-c、pcⅢ、ln、ha水平低于同期对照组(p0.01)。4.对照组和治疗组肝脏瞬时弹性值(lsm)的比较:两组治疗后lsm较治疗前明显下降(p0.05或p0.01),且治疗组治疗后lsm明显低于同期对照组(p0.05)。5.对照组和治疗组tgf-β1、ifn-γ及il-9、il-10水平的比较:两组治疗后tgf-β1、ifn-γ及il-9、il-10的水平均较治疗前显著减低(p0.01);治疗组治疗后tgf-β1、ifn-γ及il-9、il-10的水平明显低于对照组治疗后(p0.05)。两组治疗后ifn-γ/il-10值较治疗前显著升高(p0.01);治疗组治疗后ifn-γ/il-10值明显高于同期对照组(p0.05)。6.对照组和治疗组血清egf、hgf水平的比较:两组治疗后血清egf及hgf的水平较治疗前均明显升高(p0.01);治疗组治疗后egf及hgf的水平高于同期对照组(p0.05)。7.对照组和治疗组血清gsh、cat、sod及8-ohdg水平的比较:两组治疗后血清gsh、cat、sod水平较治疗前明显升高,8-ohdg水平显著下降(p0.01)。治疗组治疗后血清8-ohdg水平明显低于同期对照组,gsh、cat、sod水平明显高于同期对照组(p0.01)。8.对照组和治疗组外周血t淋巴细胞亚群的比较:对照组治疗后cd4+及cd4+/cd8+较治疗前均显著增加(p0.05),cd3+及cd8+差异无统计学意义(p0.05)。治疗组治疗后cd4+及cd4+/cd8+较治疗前均显著增加(p0.01或p0.05),cd8+显著下降(p0.01),cd3+差异无统计学意义(p0.05)。与对照组治疗后比较,治疗组治疗后cd4+及cd4+/cd8+升高更明显(p0.05),cd8+下降更明显(p0.05)。9.对照组和治疗组肝组织egf、egfr表达的比较:hbeag阴性chb患者肝组织内可见egf、egfr的表达,肝细胞胞浆及细胞膜区域呈浅棕色。治疗组治疗后egf、egfr在肝细胞胞浆、细胞膜及血窦区域表达呈强阳性,为棕黄色,egf、egfr的表达量较治疗前明显增加(p0.01)。且治疗组治疗后egf、egfr的表达量较同期对照组显著增加(p0.01)。10.对照组和治疗组肝组织hgf、c-met表达的比较:hbeag阴性chb患者肝组织内可见hgf、c-met的表达,肝窦及窦周细胞胞浆、肝细胞膜等区域呈浅棕色。经过地五养肝方治疗,hgf、c-met在肝窦及窦周细胞胞浆、肝细胞膜等区域的表达显著增强,呈强阳性,表现为棕黄色,hgf、c-met的表达量较治疗前明显增加(p0.01)。且治疗组治疗后hgf、c-met的表达量较同期对照组显著增加(p0.01)。11.对照组和治疗组肝组织egf、egfr、hgf、c-metmrna表达的比较:治疗组治疗后肝组织egf、egfr、hgf、c-metmrna的表达量较治疗前明显增加,差异有统计学意义(p0.01);对照组治疗后肝组织egf、egfr、hgf、c-metmrna的表达量较治疗前有所增加,但差异无统计学意义(p0.05);治疗组治疗后egf、egfr、hgf、c-metmrna的表达量较同期对照组明显增加(p0.01)。结论理论研究:肝肾同源理论历史悠久,内涵丰富,立足肝肾同源来探讨慢性乙型肝炎的病因病机、治疗及药物作用机制等具有非常重要的临床意义。实验研究:1.地五养肝方能有效治疗hbeag阴性chb。2.hbeag阴性chb患者存在细胞因子网络、神经-内分泌-免疫网络及氧化应激等的紊乱,相当于祖国医学之“精髓失调”、“骨髓失调”和“脑髓失调”。hbeag阴性chb患者存在的上述各种紊乱失调导致肝组织炎症损害,影响肝细胞正常再生,与祖国医学之“髓失生肝”意义相通。3.地五养肝方通过补肾能有效调控hbeag阴性chb患者机体细胞因子网络,改善外周血t淋巴细胞亚群状态,调整神经-内分泌-免疫网络,并提高抗氧化能力,减轻氧化应激损伤,提高肝组织egf、egfr、hgf、c-met的表达,改善肝内微环境,从而有利于肝细胞正常再生,促进肝功能恢复,初步揭示了肝肾同源“补肾生髓成肝”的物质学基础。
[Abstract]:Objective To explore the mechanism of "Diwu Yanggan Decoction" in treating HBe Ag-negative chronic hepatitis B from the theory of "liver-kidney homology" and to provide evidence for clinical application. The clinical application and experimental study of the theory of renal homology discuss the essence of the modern theory of liver and kidney homology. Finally, the etiology, pathogenesis and possible mechanism of the treatment of chronic hepatitis B (CHB) from the liver and kidney were summarized and discussed. The treatment group was given Diwuyanggan Recipe and Entecavir orally, while the control group was given Chinese herbal placebo and Entecavir orally for 48 weeks. Another 30 healthy people were selected as the normal group at the same time. The liver function, blood coagulation function, serum collagen IV-C (IV-C), procollagen III (PCIII) and lamina were compared between the control group and the treatment group before and after treatment. The serum levels of TGF-beta 1, EGF, HGF, IFN-gamma, IL-9 and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA) before and after treatment in the treatment group and the control group as well as in the normal group, and the IFN-gamma/IL-10 values were calculated. The levels of SOD, GSH, CAT and 8-OHdG in normal serum were measured by flow cytometry. The expression of CD3 +, CD4 + and CD8 + T lymphocytes in peripheral blood was detected by flow cytometry. The expression of EGF, EGFR, HGF and c-Met mRNA and protein in liver tissue were measured by real-time PCR and immunohistochemistry before and after treatment in treatment group and control group. The theory of homology of liver and kidney includes the correlation of liver and kidney essence and blood, the intergrowth of liver and kidney mother and son, the co-operation of liver and kidney for fire, the co-habitation of liver and kidney in lower jiao, complementary Yin and yang, the coordination of liver and kidney storage and drainage, and the communication of liver and kidney meridians and channels. Secretory-immune network is connected and extended to hypothalamus-pituitary-liver axis and testis-testosterone-hgf, which further enriches the connotation of the theory of liver-kidney homology. The occurrence, development, changes, and prognosis of CHB are closely related to the state of liver and kidney function. The fundamental pathogenesis of CHB is liver-kidney disorder and insufficiency of vital energy of the viscera. Experimental study: 1. HBV DNA negative conversion: the treatment group HBV DNA negative conversion rate was significantly higher than the control group, the difference was statistically significant (p0.05). 2. control group and treatment group liver function and coagulation function comparison: two groups after treatment alt, AST levels were significantly lower than before treatment (p0.01), and the treatment group after treatment alt, AST levels were significantly lower than the control group (p0.01). After treatment, the levels of IV-c, PC III, LN and HA in the control group and the treatment group were significantly lower than those before treatment (p0.01). After treatment, the levels of IV-c, PC III, LN and HA in the treatment group were lower than those in the control group (p0.05). Comparison of liver transient elasticity (lsm) between the control group and the treatment group: after treatment, the LSM of the two groups decreased significantly (p0.05 or p0.01), and the LSM of the treatment group was significantly lower than that of the control group (p0.05). 5. comparison of TGF-beta 1, ifn-gamma, il-9, IL-10 levels between the control group and the treatment group: after treatment, the levels of TGF-beta 1, IFN-gamma and il-9, IL-10 After treatment, the levels of tgf-1, ifn-gamma, IL-9 and IL-10 in the treatment group were significantly lower than those in the control group (p0.05). The levels of serum EGF and HGF in the treatment group were higher than those in the control group (p0.01). the levels of EGF and HGF in the treatment group were higher than those in the control group (p0.05). 7. the levels of serum gsh, cat, SOD and 8-OHdG in the control group and the treatment group were compared. the levels of serum gsh, cat, SOD in the two groups were significantly higher than those before treatment, and the levels of 8-OHdG were significantly lower than those before treatment. Serum 8-OHdG levels in the treatment group were significantly lower than those in the control group, gsh, cat, SOD levels were significantly higher than those in the control group (p0.01). 8. CD4 + and CD4 + / CD8 + in the treatment group after treatment were significantly increased (p0.01 or p0.05), CD8 + significantly decreased (p0.01), CD3 + no significant difference (p0.05). compared with the control group after treatment, the treatment group after treatment CD4 + and CD4 + / CD8 + increased more significantly (p0.05), CD8 + decreased more significantly (p0.05). 9. Comparison: the expression of EGF and EGFR in liver tissue of HBeAg negative CHB patients, and the expression of EGF and EGFR in the cytoplasm, membrane and sinusoidal region of hepatocytes were strongly positive after treatment in the treatment group. the expression of EGF and EGFR was brown yellow, EGF and EGFR were significantly increased after treatment (p0.01). The expression of HGF and c-Met in the liver tissues of patients with HBeAg-negative CHB showed that the expression of HGF and c-Met in the liver tissues of patients with HBeAg-negative chb, the cytoplasm of sinusoidal and perisinusoidal cells, and the membranes of hepatocytes were pale brown. The expression of egf, egfr, hgf, c-Met in the liver tissue of the control group and the treatment group were significantly increased after treatment (p0.01). 11. the expression of egf, egfr, hgf, c-met mRNA in the liver tissue of the control group and the treatment group were compared after treatment: the expression of egf, egfr, hgf, c-met mRNA in the liver tissue of the treatment group was significantly increased after treatment (p0.01). The expression of egf, egfr, hgf, c-met mRNA in the liver tissue of the control group increased after treatment, but the difference was not statistically significant (p0.05); after treatment, the expression of egf, egfr, hgf, c-met mRNA in the treatment group increased significantly compared with the control group (p0.01). Conclusion: The theory of liver-kidney homology has a long history and rich connotation. It is of great clinical significance to explore the pathogenesis, treatment and drug mechanism of chronic hepatitis B based on the theory of liver-kidney homology. The disorders of the trans-endocrine-immune network and oxidative stress are equivalent to the "essence disorder", "bone marrow disorder" and "brain-marrow disorder" in Chinese medicine. Diwuyanggan recipe can effectively regulate the cytokine network of HBeAg negative CHB patients, improve the state of peripheral blood T lymphocyte subsets, regulate the neuroendocrine immune network, improve the antioxidant capacity, reduce oxidative stress injury, increase the expression of egf, egfr, hgf, c-Met in liver tissue, and improve the liver microenvironment by Tonifying the kidney. Normal cell regeneration promotes the recovery of liver function, which preliminarily reveals the material basis of "tonifying kidney and regenerating marrow to form liver".
【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259
[Abstract]:Objective To explore the mechanism of "Diwu Yanggan Decoction" in treating HBe Ag-negative chronic hepatitis B from the theory of "liver-kidney homology" and to provide evidence for clinical application. The clinical application and experimental study of the theory of renal homology discuss the essence of the modern theory of liver and kidney homology. Finally, the etiology, pathogenesis and possible mechanism of the treatment of chronic hepatitis B (CHB) from the liver and kidney were summarized and discussed. The treatment group was given Diwuyanggan Recipe and Entecavir orally, while the control group was given Chinese herbal placebo and Entecavir orally for 48 weeks. Another 30 healthy people were selected as the normal group at the same time. The liver function, blood coagulation function, serum collagen IV-C (IV-C), procollagen III (PCIII) and lamina were compared between the control group and the treatment group before and after treatment. The serum levels of TGF-beta 1, EGF, HGF, IFN-gamma, IL-9 and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA) before and after treatment in the treatment group and the control group as well as in the normal group, and the IFN-gamma/IL-10 values were calculated. The levels of SOD, GSH, CAT and 8-OHdG in normal serum were measured by flow cytometry. The expression of CD3 +, CD4 + and CD8 + T lymphocytes in peripheral blood was detected by flow cytometry. The expression of EGF, EGFR, HGF and c-Met mRNA and protein in liver tissue were measured by real-time PCR and immunohistochemistry before and after treatment in treatment group and control group. The theory of homology of liver and kidney includes the correlation of liver and kidney essence and blood, the intergrowth of liver and kidney mother and son, the co-operation of liver and kidney for fire, the co-habitation of liver and kidney in lower jiao, complementary Yin and yang, the coordination of liver and kidney storage and drainage, and the communication of liver and kidney meridians and channels. Secretory-immune network is connected and extended to hypothalamus-pituitary-liver axis and testis-testosterone-hgf, which further enriches the connotation of the theory of liver-kidney homology. The occurrence, development, changes, and prognosis of CHB are closely related to the state of liver and kidney function. The fundamental pathogenesis of CHB is liver-kidney disorder and insufficiency of vital energy of the viscera. Experimental study: 1. HBV DNA negative conversion: the treatment group HBV DNA negative conversion rate was significantly higher than the control group, the difference was statistically significant (p0.05). 2. control group and treatment group liver function and coagulation function comparison: two groups after treatment alt, AST levels were significantly lower than before treatment (p0.01), and the treatment group after treatment alt, AST levels were significantly lower than the control group (p0.01). After treatment, the levels of IV-c, PC III, LN and HA in the control group and the treatment group were significantly lower than those before treatment (p0.01). After treatment, the levels of IV-c, PC III, LN and HA in the treatment group were lower than those in the control group (p0.05). Comparison of liver transient elasticity (lsm) between the control group and the treatment group: after treatment, the LSM of the two groups decreased significantly (p0.05 or p0.01), and the LSM of the treatment group was significantly lower than that of the control group (p0.05). 5. comparison of TGF-beta 1, ifn-gamma, il-9, IL-10 levels between the control group and the treatment group: after treatment, the levels of TGF-beta 1, IFN-gamma and il-9, IL-10 After treatment, the levels of tgf-1, ifn-gamma, IL-9 and IL-10 in the treatment group were significantly lower than those in the control group (p0.05). The levels of serum EGF and HGF in the treatment group were higher than those in the control group (p0.01). the levels of EGF and HGF in the treatment group were higher than those in the control group (p0.05). 7. the levels of serum gsh, cat, SOD and 8-OHdG in the control group and the treatment group were compared. the levels of serum gsh, cat, SOD in the two groups were significantly higher than those before treatment, and the levels of 8-OHdG were significantly lower than those before treatment. Serum 8-OHdG levels in the treatment group were significantly lower than those in the control group, gsh, cat, SOD levels were significantly higher than those in the control group (p0.01). 8. CD4 + and CD4 + / CD8 + in the treatment group after treatment were significantly increased (p0.01 or p0.05), CD8 + significantly decreased (p0.01), CD3 + no significant difference (p0.05). compared with the control group after treatment, the treatment group after treatment CD4 + and CD4 + / CD8 + increased more significantly (p0.05), CD8 + decreased more significantly (p0.05). 9. Comparison: the expression of EGF and EGFR in liver tissue of HBeAg negative CHB patients, and the expression of EGF and EGFR in the cytoplasm, membrane and sinusoidal region of hepatocytes were strongly positive after treatment in the treatment group. the expression of EGF and EGFR was brown yellow, EGF and EGFR were significantly increased after treatment (p0.01). The expression of HGF and c-Met in the liver tissues of patients with HBeAg-negative CHB showed that the expression of HGF and c-Met in the liver tissues of patients with HBeAg-negative chb, the cytoplasm of sinusoidal and perisinusoidal cells, and the membranes of hepatocytes were pale brown. The expression of egf, egfr, hgf, c-Met in the liver tissue of the control group and the treatment group were significantly increased after treatment (p0.01). 11. the expression of egf, egfr, hgf, c-met mRNA in the liver tissue of the control group and the treatment group were compared after treatment: the expression of egf, egfr, hgf, c-met mRNA in the liver tissue of the treatment group was significantly increased after treatment (p0.01). The expression of egf, egfr, hgf, c-met mRNA in the liver tissue of the control group increased after treatment, but the difference was not statistically significant (p0.05); after treatment, the expression of egf, egfr, hgf, c-met mRNA in the treatment group increased significantly compared with the control group (p0.01). Conclusion: The theory of liver-kidney homology has a long history and rich connotation. It is of great clinical significance to explore the pathogenesis, treatment and drug mechanism of chronic hepatitis B based on the theory of liver-kidney homology. The disorders of the trans-endocrine-immune network and oxidative stress are equivalent to the "essence disorder", "bone marrow disorder" and "brain-marrow disorder" in Chinese medicine. Diwuyanggan recipe can effectively regulate the cytokine network of HBeAg negative CHB patients, improve the state of peripheral blood T lymphocyte subsets, regulate the neuroendocrine immune network, improve the antioxidant capacity, reduce oxidative stress injury, increase the expression of egf, egfr, hgf, c-Met in liver tissue, and improve the liver microenvironment by Tonifying the kidney. Normal cell regeneration promotes the recovery of liver function, which preliminarily reveals the material basis of "tonifying kidney and regenerating marrow to form liver".
【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R259
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