【作者】 张引强；

【导师】 唐旭东；

【作者基本信息】 中国中医科学院 ， 中医内科学， 2010， 博士

【副题名】荣肝合剂对慢性乙型肝炎患者生活质量影响的初步研究

【摘要】 目的：1.理论研究,探讨清利活血健脾法与慢性乙型肝炎(CHB)的关系,为CHB的治疗提供立法依据,为荣肝合剂提供组方基础。2.研究思路探讨,澄清“保肝降酶”治疗过程中的一些概念,明确其内涵,使中医药对CHB的治疗更为全面客观。3.实验研究,探讨荣肝合剂对刀豆蛋白A(ConA)介导的急、慢性免疫性肝损伤小鼠的降酶效应及其机制。4.临床病例观察,研究中药荣肝合剂对CHB患者生存质量的影响。方法：1.理论分析,收集古今、中西医相关文献,在回顾和总结的基础上,分析清利活血健脾法与CHB的关系,并与荣肝合剂相对应,分析其组方依据。2.解析“保肝降酶”的内涵,分析中医药保肝降酶治疗CHB中存在的问题,探讨其应对措施。3.(1)急性肝损伤实验：HBV转基因小鼠60只,随机分为6组(模型组、正常组、荣肝合剂组、茵陈蒿汤组、茵陈组、联苯双酯组),每组10只,采用ConA尾静脉注射制作急性免疫性肝损伤模型。造模前14天,模型组、正常组给予生理盐水灌胃,其余组分别给予：荣肝合剂、茵陈蒿汤、单味茵陈煎液、联苯双酯溶液,每日一次灌胃给药。末次给药后1h,正常组给予磷酸盐缓冲液(PBS)尾静脉注射,其余各组按照3μg/g剂量尾静脉注射造模。造模后8h,处死动物取血或组织标本检测ALT、AST、TBiL;观察病理组织学变化(HE染色)；HBV DNA、HBsAg；肝组织MDA及SOD水平；肝组织内淋巴细胞亚群情况变化(CD4+、CD8+、CD4+／CD8+);细胞凋亡相关基因(Fas、FasL、Bax、bcl-2);肝组织中TNF-α、INF-γ、IL-4、IL-10等细胞因子的变化。(2)慢性肝损伤实验：HBV转基因小鼠69只,随机分为正常组10只(尾静脉注射PBS 0.3ml)和ConA造模组59只,采用ConA6μg/g剂量尾静脉注射制作慢性免疫性肝损伤模型。造模结束后,将所有ConA造模组小鼠随机分为模型组、荣肝合剂组、茵陈组、茵陈蒿汤组及联苯双酯五组。各组小鼠每日灌胃给予相应药物,正常组与模型组给予生理盐水灌胃,共给药28天。末次灌胃给药后24h处死动物,取血或组织标本检测指标(指标同急性肝损伤实验)。4.采用随机对照研究。区组随机,以荣肝合剂为治疗药物,与西药口服抗病毒药核苷(酸)类似物(NUCs)为对照,治疗期为6个月,以3个月为一访视点,考核患者血清ALT、AST、HBV DNA水平变化,结合普适性量表SF-36及慢性肝病问卷(CLDQ)观察荣肝合剂对于CHB患者生活质量的影响。结果：1.CHB属本虚标实之证,湿热、血瘀为标,脾虚为本。或见肝郁,久则及肾。2.CHB的保肝降酶未真正做到降低酶学指标与肝组织学改善并重,未结合抗病毒、提高免疫力、防止纤维化的治疗、少有关注患者生存质量。3.(1)急性肝损伤实验：①ALT、AST、TBiL情况：与正常组比较,模型组小鼠血清ALT、AST、TBiL均显著升高(P<0.01)。荣肝合剂、联苯双酯均可显著降低血清ALT、AST (P<0.01),荣肝合剂可降低TBiL (P<0.01)。②病理组织学变化：与模型组比较,荣肝合剂组肝细胞变性、坏死及炎细胞浸润程度较轻,显示其对肝组织的病理损伤有较好的改善作用(P<0.05)；而联苯双酯组、茵陈组与模型组的病损程度相似。③HBsAg、HBV DNA变化：荣肝合剂组HBVDNA与茵陈组比较有显著性差异(P<0.05),其余指标各组比较无显著性差异。④MDA、SOD变化：与正常组相比较,模型组肝组织中SOD水平明显低于正常组,MDA水平则明显高于正常组(P<0.05)。荣肝合剂组可明显提高肝组织中SOD水平,且与各干预组比较均有显著性差异(P<0.01)。荣肝合剂、茵陈汤可降低肝组织中MDA水平(P<0.05)。⑤淋巴细胞亚群变化：CD4+CD8-T淋巴细胞所占比例,各药物干预组与模型组比较均有升高,以荣肝合剂最著,比较有显著性差异(P<0.05)。CD4-CD8+T细胞所占比例,荣肝合剂组与模型组、联苯双酯组比较有显著性差异(P<0.05)。CD4+/CD8+T细胞比例变化,与模型组相比,各中药组均可见较大幅度升高,荣肝合剂组与茵陈蒿汤组比较有显著性差异(P<0.05)。⑥Fas、FasL、Bax、bcl-2的变化：正常组中,FasL和Bax没有表达,模型组Fas和bcl-2的表达水平与正常组比较,均有显著差异(P<0.05)。与模型组比较,荣肝合剂组Fas、FasL、Bax、bcl-2、bcl-2/Bax比值等差异有统计学意义(P<0.05)。⑦TNF-α、INF-γ、IL-4、IL-10的表达：模型组TNF-α、IFN-γ水平明显高于正常组,而IL-4, IL-10水平明显低于正常组(P<0.05)。荣肝合剂组TNF-α、INF-γ、IL-4、IL-10均较模型组有统计学差异(P<0.05)。(2)慢性肝损伤实验：①ALT、AST、TBiL情况：与正常组比较,模型组小鼠血清ALT、AST、TBiL均显著升高(P<0.01)。荣肝合剂、联苯双酯均可显著降低血清ALT、AST及TBiL (P<0.01)。②病理组织学变化：荣肝合剂组、茵陈蒿汤组与模型组比较,在减轻肝细胞坏死、减少变性、减轻炎细胞浸润以及减轻肝纤维化方面有统计学差异,荣肝合剂的作用优于茵陈蒿汤(P<0.05)。③HBsAg、HBV DNA变化：荣肝合剂组HBV DNA水平与模型组比较有显著性差异(P<0.05)；HBsAg方面,荣肝合剂组与其余各组比较均有显著性差异(P<0.05)。④MDA、SOD变化：模型组肝组织中SOD含量明显低于正常对照组,MDA水平明显高于正常对照组,并有显著性差异(P<0.01)。荣肝合剂组MDA、SOD均较模型组有统计学差异,较其余组比较部分有显著差异(P<0.05)。⑤肝组织中淋巴细胞亚群变化：CD4+CD8-T淋巴细胞,各药物干预组与模型组比较,均有升高(P<0.05)；荣肝合剂与茵陈、茵陈蒿汤、联苯双酯比较,均有显著性差异(P<0.05)；CD4-CD8+T细胞,荣肝合剂组、联苯双酯组与模型组比较,有显著性差异(P<0.05)；CD4+/CD8+T细胞比例变化,与模型组相比,各中药组均可见较大幅升高,荣肝合剂组与茵陈蒿汤组、联苯双酯组比较有显著性差异(P<0.05)。⑥Fas、FasL、Bax、bcl-2的变化：正常组中,FasL和Bax没有表达,模型组Fas和bcl-2的表达与正常组比较,均有显著差异(P<0.05)。荣肝合剂组Fas、FasL、Bax、bcl-2、bcl-2/Bax比值均较模型组有统计学差异,较其余组比较部分指标有显著差异(P<0.05)。⑦TNF-α、INF-γ、IL-4、IL-10的表达：模型组TNF-α、IFN-γ水平明显高于正常组,而IL-4、IL-10水平明显低于正常组(P<0.05)。TNF-α,荣肝合剂、茵陈组与模型组比较,均有显著性差异(P<0.05)。IFN-γ,各药物干预组与模型比较,均有显著性差异(P<0.01),荣肝合剂组与茵陈组、联苯双酯组比较,均有显著性差异(P<0.05)。IL-4,各药物干预组与模型比较,均有显著性差异(P<0.05)。IL-10,荣肝合剂、茵陈组与模型组比较,均有显著性差异(P<0.05)。4.治疗3个月,两组患者的ALT、AST的复常率均在80%以上,组间比较无统计学差异(P>0.05)。荣肝合剂与NUCs对HBV DNA均有降低作用,但NUCs则明显优于荣肝合剂(P<0.05)。治疗6个月后,两组患者的ALT、AST水平均已降至正常值上限以下,组间比较无统计学差异(P>0.05)；HBV DNA方面,荣肝合剂和NUCs对HBV均持续发挥抑制作用(P<0.05),但NUCs的HBV DNA阴转率明显高于荣肝合剂(P<0.05)。生活质量改善方面：①治疗3个月,荣肝合剂组患者生活质量各个维度均有改善；而服用NUCs在生理职能、情感职能维度无明显改善；组间比较显示,荣肝合剂三个月治疗后,患者的生理机能、生理职能、精力、生理内容综合测量、心理内容综合测量、SF-36总分、CLDQ各维度均较NUCs有明显的改观(P<0.05)。②治疗6个月：荣肝合剂对于患者生活质量的改善持续起效,较基线与治疗3月均有明显好转(P<0.05)；NUCs对CHB患者生活质量亦有改善,但与基线比较,生理职能、情感职能和躯体疼痛维度无统计学差异；较治疗3月,躯体疼痛、社会功能、一般健康状况及情感职能、生理内容综合测量维度无统计学差异(P>0.05)；组间比较显示,荣肝合剂与NUCs对患者生存质量的影响,除腹部症状维度外,荣肝合剂均优于NUCs (P<0.05)。结论：1.CHB治则当以清热利湿、活血化瘀、健脾扶正为大法,荣肝合剂立法与此一致,与慢性乙型肝炎病机相契合。2.CHB的保肝降酶治疗要真正做到对肝脏组织学的改善与降低酶学指标并重,并应该结合抗病毒、提高免疫力、防止纤维化发生、提高患者生存质量,并要加强临床研究。3.通过本研究的进行,可得出以下结论：①荣肝合剂的保肝降酶作用不仅是对于外周血中酶的水平的降低,同时有伴有病理组织学的改善：减轻细胞炎症、坏死及炎细胞浸润、甚至逆转纤维化的作用。②中医药复方荣肝合剂的保肝降酶作用要明显优于单味中药(茵陈汤)和联苯双酯,清利活血健脾的治则对于乙肝的干预是有效的。③中医药保肝降酶作用起效的病理机制是多方面的：可减轻细胞膜脂质过氧化反应,提高淋巴细胞亚群功能,减少炎症因子的刺激、抑制肝细胞的凋亡。4.中药荣肝合剂可使CHB患者的ALT、AST复常并降低HBV DNA的载量,但其抗病毒能力弱于NUCs。荣肝合剂可提高患者的生活质量,其作用优于NUCs的治疗。更多还原

【Abstract】 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.更多还原