基于“肝腎同源”理論探討“地五養(yǎng)肝方”治療HBeAg陰性慢性乙型肝炎的機理
發(fā)布時間：2018-09-15 19:03
【摘要】：目的從“肝腎同源”理論探討“地五養(yǎng)肝方”治療HBe Ag陰性慢性乙型肝炎的作用機制,為臨床應(yīng)用提供依據(jù)。方法本研究從理論研究及實驗研究兩個方面進行。理論研究:從肝腎同源理論的源流及內(nèi)涵來梳理肝腎同源發(fā)展的脈絡(luò);從肝腎同源理論的臨床應(yīng)用及實驗研究來論述現(xiàn)代對肝腎同源理論實質(zhì)的探討和發(fā)揮;最后對慢性乙型肝炎(CHB)從肝腎論治的病因病機及可能的作用機制進行了歸納和探討。實驗研究:120例入組的HBeAg陰性CHB患者,隨機分為對照組、治療組,每組均為60例。治療組口服地五養(yǎng)肝方及恩替卡韋;對照組予中藥安慰劑口服,同時口服恩替卡韋,療程為48周。并另選同期健康人30例作為正常組。比較對照組和治療組治療前后肝功能、凝血功能、血清Ⅳ型膠原(Ⅳ-C)、Ⅲ型前膠原(PCⅢ)、層粘蛋白(LN)、透明質(zhì)酸(HA)、乙型肝炎三系定量、HBV DNA定量及肝臟瞬時彈性。同時采用酶聯(lián)免疫吸附法(ELISA)檢測治療組、對照組治療前后及正常組血清TGF-β1、EGF、HGF、IFN-γ及IL-9、IL-10的水平,并計算IFN-γ/IL-10值。檢測治療組、對照組治療前后及正常組血清SOD、GSH、CAT、8-OHdG的水平。用流式細(xì)胞技術(shù)檢測外周血CD3+、CD4+及CD8+T淋巴細(xì)胞的表達情況。用real time PCR技術(shù)、免疫組化法測定治療組和對照組治療前后肝組織EGF、EGFR、HGF、c-met mRNA量及蛋白表達水平。結(jié)果理論研究:肝腎同源理論萌芽于《黃帝內(nèi)經(jīng)》,深化于漢唐金元,形成于明清。肝腎同源理論包含有肝腎精血相關(guān),肝腎母子相生,肝腎共司相火,肝腎同居下焦、陰陽互補,肝腎藏泄相協(xié)及肝腎經(jīng)脈相通,而現(xiàn)代醫(yī)家將肝腎同源與現(xiàn)代醫(yī)學(xué)的下丘腦-垂體-靶腺、神經(jīng)-內(nèi)分泌-免疫網(wǎng)絡(luò)聯(lián)系起來,并延伸至下丘腦-垂體-肝軸及睪丸-睪酮-hgf,更進一步豐富了肝腎同源理論的內(nèi)涵。chb的發(fā)生、發(fā)展、變化、轉(zhuǎn)歸等一系列過程均與肝腎功能狀態(tài)密不可分,其根本病機為肝腎失調(diào),臟腑正氣不足,因此從肝腎論治chb能取得卓越療效。實驗研究:1.hbvdna陰轉(zhuǎn)情況:治療組hbvdna陰轉(zhuǎn)率明顯高于對照組,差異有統(tǒng)計學(xué)意義(p0.05)。2.對照組和治療組肝功能及凝血功能的比較:兩組治療后alt、ast水平較治療前均明顯下降(p0.01),且治療組治療后alt、ast水平較對照組治療后明顯降低(p0.01)。兩組治療后pt較治療前有所降低,但差異無統(tǒng)計學(xué)意義(p0.05)。3.對照組和治療組肝纖維化指標(biāo)的比較:兩組治療后Ⅳ-c、pcⅢ、ln、ha水平較治療前均明顯下降(p0.01),且治療組治療后Ⅳ-c、pcⅢ、ln、ha水平低于同期對照組(p0.01)。4.對照組和治療組肝臟瞬時彈性值(lsm)的比較:兩組治療后lsm較治療前明顯下降(p0.05或p0.01),且治療組治療后lsm明顯低于同期對照組(p0.05)。5.對照組和治療組tgf-β1、ifn-γ及il-9、il-10水平的比較:兩組治療后tgf-β1、ifn-γ及il-9、il-10的水平均較治療前顯著減低(p0.01);治療組治療后tgf-β1、ifn-γ及il-9、il-10的水平明顯低于對照組治療后(p0.05)。兩組治療后ifn-γ/il-10值較治療前顯著升高(p0.01);治療組治療后ifn-γ/il-10值明顯高于同期對照組(p0.05)。6.對照組和治療組血清egf、hgf水平的比較:兩組治療后血清egf及hgf的水平較治療前均明顯升高(p0.01);治療組治療后egf及hgf的水平高于同期對照組(p0.05)。7.對照組和治療組血清gsh、cat、sod及8-ohdg水平的比較:兩組治療后血清gsh、cat、sod水平較治療前明顯升高,8-ohdg水平顯著下降(p0.01)。治療組治療后血清8-ohdg水平明顯低于同期對照組,gsh、cat、sod水平明顯高于同期對照組(p0.01)。8.對照組和治療組外周血t淋巴細(xì)胞亞群的比較:對照組治療后cd4+及cd4+/cd8+較治療前均顯著增加(p0.05),cd3+及cd8+差異無統(tǒng)計學(xué)意義(p0.05)。治療組治療后cd4+及cd4+/cd8+較治療前均顯著增加(p0.01或p0.05),cd8+顯著下降(p0.01),cd3+差異無統(tǒng)計學(xué)意義(p0.05)。與對照組治療后比較,治療組治療后cd4+及cd4+/cd8+升高更明顯(p0.05),cd8+下降更明顯(p0.05)。9.對照組和治療組肝組織egf、egfr表達的比較:hbeag陰性chb患者肝組織內(nèi)可見egf、egfr的表達,肝細(xì)胞胞漿及細(xì)胞膜區(qū)域呈淺棕色。治療組治療后egf、egfr在肝細(xì)胞胞漿、細(xì)胞膜及血竇區(qū)域表達呈強陽性,為棕黃色,egf、egfr的表達量較治療前明顯增加(p0.01)。且治療組治療后egf、egfr的表達量較同期對照組顯著增加(p0.01)。10.對照組和治療組肝組織hgf、c-met表達的比較:hbeag陰性chb患者肝組織內(nèi)可見hgf、c-met的表達,肝竇及竇周細(xì)胞胞漿、肝細(xì)胞膜等區(qū)域呈淺棕色。經(jīng)過地五養(yǎng)肝方治療,hgf、c-met在肝竇及竇周細(xì)胞胞漿、肝細(xì)胞膜等區(qū)域的表達顯著增強,呈強陽性,表現(xiàn)為棕黃色,hgf、c-met的表達量較治療前明顯增加(p0.01)。且治療組治療后hgf、c-met的表達量較同期對照組顯著增加(p0.01)。11.對照組和治療組肝組織egf、egfr、hgf、c-metmrna表達的比較:治療組治療后肝組織egf、egfr、hgf、c-metmrna的表達量較治療前明顯增加,差異有統(tǒng)計學(xué)意義(p0.01);對照組治療后肝組織egf、egfr、hgf、c-metmrna的表達量較治療前有所增加,但差異無統(tǒng)計學(xué)意義(p0.05);治療組治療后egf、egfr、hgf、c-metmrna的表達量較同期對照組明顯增加(p0.01)。結(jié)論理論研究:肝腎同源理論歷史悠久,內(nèi)涵豐富,立足肝腎同源來探討慢性乙型肝炎的病因病機、治療及藥物作用機制等具有非常重要的臨床意義。實驗研究:1.地五養(yǎng)肝方能有效治療hbeag陰性chb。2.hbeag陰性chb患者存在細(xì)胞因子網(wǎng)絡(luò)、神經(jīng)-內(nèi)分泌-免疫網(wǎng)絡(luò)及氧化應(yīng)激等的紊亂,相當(dāng)于祖國醫(yī)學(xué)之“精髓失調(diào)”、“骨髓失調(diào)”和“腦髓失調(diào)”。hbeag陰性chb患者存在的上述各種紊亂失調(diào)導(dǎo)致肝組織炎癥損害,影響肝細(xì)胞正常再生,與祖國醫(yī)學(xué)之“髓失生肝”意義相通。3.地五養(yǎng)肝方通過補腎能有效調(diào)控hbeag陰性chb患者機體細(xì)胞因子網(wǎng)絡(luò),改善外周血t淋巴細(xì)胞亞群狀態(tài),調(diào)整神經(jīng)-內(nèi)分泌-免疫網(wǎng)絡(luò),并提高抗氧化能力,減輕氧化應(yīng)激損傷,提高肝組織egf、egfr、hgf、c-met的表達,改善肝內(nèi)微環(huán)境,從而有利于肝細(xì)胞正常再生,促進肝功能恢復(fù),初步揭示了肝腎同源“補腎生髓成肝”的物質(zhì)學(xué)基礎(chǔ)。
[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".
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：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".
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R259
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