【作者】 匡双玉；

【导师】 刘塔斯； 肖小河；

【作者基本信息】 湖南中医药大学 ， 方剂学， 2014， 博士

【摘要】 随着生活水平提高,非酒精性脂肪肝病(non-alcohol fatty liver disease, NAFLD)发病率快速增加,或将成为我国首要肝病。然而,世界范围内尚缺少NAFLD的特效药物。在国家自然科学基金课题(81274026；81373984)支持下,本文根据中医理论对NAFLD“湿热”和“血瘀”病机的认识,对临床常用治疗NAFLD的具有清热燥湿或活血化瘀功效的中药--黄连、大黄和姜黄及其有效成分的活性进行评价研究,并初步探讨可能的作用机制,以期为筛选和发现NAFLD的有效治疗中药和组分提供参考依据。研究方法如下：(1)采用高脂饲料构建大鼠NAFLD模型,考察黄连、大黄和姜黄单味药对NAFLD大鼠血清生化指标、肝细胞脂肪变的改善作用,并采用real-time PCR、Western blotting和组织免疫组化染色考察药物对脂质调节相关蛋白-过氧化物酶体增殖物激活受体γ(PPARy).固醇调节元件结合蛋白-lc (SREBP-lc)、硬脂酰辅酶A去饱和酶(SCD-1)表达的影响。(2)采用人肝L-02细胞脂变模型,考察黄连、大黄和姜黄的有效成分小檗碱、大黄素和姜黄素对肝细胞脂变的保护作用及其对SREBP-lc及小凹蛋白-1(caveolin-1)蛋白表达的影响。(3)采用NAFLD大鼠模型,考察小檗碱、姜黄素及其组合对NAFLD的治疗作用,以及对脂质调控和炎症相关蛋白SREBP-lc、caveolin-1、肿瘤坏死因子-a (TNF-a)、核因子-KB p65(NF-κBp65)、磷酸化c-jun氨基末端激酶(p-JNK)表达的影响。研究结果如下：(1)1g生药量.kg-1.d-1的黄连、姜黄、大黄分别治疗6周后大鼠肝细胞脂肪变程度均有显著改善,其中姜黄和黄连改善效果优于洛伐他汀(10mg·kg-1·d-1);3种中药还显著降低NAFLD模型大鼠的血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)(P<0.05或0.01),其中对ALP的改善作用均优于洛伐他汀,说明姜黄、黄连和大黄单用均可有效治疗NAFLD,且优于他汀。黄连、姜黄、大黄治疗组均可显著降低NAFLD模型大鼠的血清甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)(P<0.05或0.01),说明3种中药对血脂也有调控作用。黄连、姜黄、大黄均可显著上调NAFLD大鼠肝脏PPARγ基因和蛋白表达量(P<0.05或0.01),其中姜黄上调作用最显著；3种中药还可显著下调NAFLD大鼠肝脏SREBP-1c、SCD-1基因表达量,显著下调SREBP-1c、p-JNK蛋白表达量(P<0.05或0.01)；对SREBP-1肝脏免疫组化染色结果与Western blotting结果的规律一致。(2)在细胞水平研究表明,姜黄素、大黄素均能降低脂变人肝L-02细胞脂滴含量、TG水平下降,SREBP-lc及caveolin-1蛋白表达下调；而小檗碱对脂变人肝L-02细胞脂滴含量及TG水平影响较小,对SREBP-1c及caveolin-1蛋白表达影响不明显,说明姜黄素和大黄素对肝细胞脂质调控具有直接作用,而小檗碱不能直接调控肝细胞脂质代谢。小檗碱治疗NAFLD的间接作用机制将在后面实验中研究。(3)前述结果表明姜黄和黄连在动物水平都可有效治疗NAFLD,但其有效成分姜黄素和小檗碱在细胞水平的作用截然不同,推测姜黄素和小檗碱可能作用不同途径和靶点,可能存在协同增效作用。为此,进一步实验考察姜黄素联合小檗碱给药8周对NAFLD大鼠的治疗作用,结果表明：与NAFLD模型组相比,恢复正常饲料饮食NAFLD大鼠的肝组织病理改变无明显恢复；相对于NAFLD模型大鼠,姜黄素(100mg·kg-1·d-1)和小檗碱(100mg·kg-1·d-1)单用均可显著改善NAFLD大鼠肝细胞脂肪变程度、降低内脏脂肪组织含量,而2个组分联合给药(各50mg·kg-1·d-1)组肝脏病理改善程度明显优于小檗碱或姜黄素单独给药；2个组分联合给药组血清TG、TC、 LDL-C、ALT、AST、ALP含量降低,高密度脂蛋白胆固醇(HDL-C)水平提高(P<0.05或0.01),且基本恢复至正常组水平,与小檗碱或姜黄素单独给药组对比具有统计学差异(P<0.05)；2个组分联合给药组血清内毒素、空腹血糖水平(FBG)、胰岛素水平(INS)、MDA含量显著降低(P<0.05或0.01),GSH-Px活力增强(P<0.01),且基本恢复至正常组水平,与小檗碱或姜黄素单独给药组对比具有统计学差异(P<0.05)；2个组分联合给药组肝脏组织PPARy基因表达上调,SREBP-1c、SCD-1、NF-κB基因表达下调,SREBP-1c、TNF-α、 NF-κB p65、p-JNK蛋白表达下调,caveolin-1蛋白表达上调,联合组对相关基因和蛋白表达的调节程度与小檗碱或姜黄素单独给药组相比具有统计学差异(P<0.05)。综合说明姜黄素和小檗碱联合应用(各50mg·kg-1·d-1)对NAFLD的治疗效果优于2个组分(100mg·kg-1·d-1)单独应用的效果,显示出协同增效的作用。研究结果表明：(1)黄连、大黄、姜黄单独给药均对大鼠NAFLD模型具有较好的治疗作用,其中姜黄和黄连的治疗效果较优；(2)黄连的有效成小檗碱和姜黄的有效成分姜黄素均对大鼠NAFLD模型具有较好的治疗作用,且小檗碱与姜黄素联合应用具有协同增效作用。其中姜黄素可直接干预肝细胞脂质代谢,SREBP-1c和caveolin-1可能是其重要作用靶点；而小檗碱不能直接干预肝细胞脂质代谢,可能通过降低肠道内毒素入血、改善胰岛素抵抗发挥治疗和协同治疗作用。更多还原

【Abstract】 With the improvement of living standards, there is a rapid increase in incidence of non-alcohol fatty liver disease (NAFLD) and it may be the primary liver disease in future of China. However, effective drugs for NAFLD are still in shortage worldwidely. The study is based on understanding of "damp heat" and "blood stasis" in TCM (traditional Chinese medicines) theory, evaluated effective components of Coptis chinensis Franch., Curcuma longa L.and R. tanguticum Maxim. ex Balf.(traditional Chinese medicines which are used for clearing heat and eliminating dampness, activating blood and resolving stasis) in treatment of NAFLD, and to explore the possible effect mechanism primarily, hoping to provide reference for screening and finding effective herbs and ingredients in treatment of NAFLD.Research methods are as follows:(1) Construct NAFLD models in rats with high-fat diet, and investigate the effect of Coptis chinensis Franch., Curcuma longa L.and R. tanguticum Maxim. ex Balf. in improving serum biochemical indicators, liver steatosis and inflammation in rats. And use real-time PCR, Western blotting and immunohistochemical staining to investigate the herbs’impact on expression of lipid regulation-associated protein-peroxisome proliferator-activated receptor-y (PPARy), sterol regulatory element binding protein-1c (SREBP-1c) and stearoyl-coenzyme A desaturase (SCD-1).(2) Use human liver L-02cells steatosis model to evaluate protective activity of berberine, emodin and curcumin (the effective components of Coptis chinensis Franch., Curcuma longa L.and R. tanguticum Maxim. ex Balf.) to hepatocyte steatosis and their impact on expression of SREBP-1c and caveolin-1protein.(3) Use NAFLD models in rats to investigate therapeutic effect of berberine, curcumin and their combinations in treatment of NAFLD, and impact on lipid regulation, expression of SREBP-1c, caveolin1, tumor necrosis factor-α(TNF-α), nuclear factor-κB p65(NF-κB p65), and phosphorylated c-jun N-terminal kinase (p-JNK).Research results are as follows:(1) Coptis chinensis Franch., Curcuma longa L.and R. tanguticum Maxim. ex Balf. at a dose of1g crude drug·kg-1·d-1for6weeks showed significant improvement in hepatic steatosis of NAFLD rats, and improvement of Coptis chinensis Franch., Curcuma longa L.were better than lovastatin (10mg·kg-1·d-1). The three herbs could also reduce serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in NAFLD rats (P<0.05or0.01), and improvement of ALP was better than lovastatin, which means Coptis chinensis Franch., Curcuma longa L.and R. tanguticum Maxim. ex Balf. could treat NAFLD alone and have better effects than lovastatin. Coptis chinensis Franch., Curcuma longa L.and R. tanguticum Maxim. ex Balf. could all significantly reduce serum levels of triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) in NAFLD rats (P<0.05or0.01), which means the three herbs have an effect on regulation of blood lipids. All three herbs could significantly increase both PPARy gene and protein expression in liver of NAFLD rats, and Curcuma longa L. was the most effective (P<0.05or0.01). They could also reduce SREBP-lc, SCD-1gene and SREBP-lc p-JNK protein expression in liver of NAFLD rats. For SREBP-1, liver immunohistochemical staining results were consistent with Western blotting results.(2)The study have shown that in cellular level, both curcumin and emodin can reduce lipid droplets content, TG levels, and expression of SREBP-1c and caveolin-1protein in human hepatic cell L-02, while berberine has little effect on lipid droplets content and TG levels, no obvious effect on expression of SREBP-lc and caveolin-1protein in human hepatic cell L-02. This means that curcumin and emodin have a direct impact on hepatocellular lipid regulation, while berberine does not have direct effect on metabolism of liver cells. Indirect mechanism of berberine in treatment of NAFLD will be studied in later experiments.(3) Foregoing results showed that Coptis chinensis Franch. and Curcuma longa L. could be effective in treating NAFLD in animal level, but their effective components-berberine and curcumin had distinct roles in cellular level. It may be speculated that berberine and curcumin have different pathways and targets in treating NAFLD, and may have synergies between them. So, further experiment was carried out of8weeks observation of the effects of berberine and curcumin in treatment of NAFLD rats. Results showed that compared to NAFLD model, NAFLD rats that accepted normal diets for8weeks showed no significant recovery in pathological changes of liver tissue; compared to NAFLD model, berberine (100mg·kg-1·d-1) and curcumin (100mg·kg-1·d-1) administrated alone could significantly improve liver steatosis degree, reduce visceral adipose content in NAFLD rats, and combination therapy of both herbs (each50mg-kg-1·d-1) had a significantly better improvement in liver pathology changes. Combination therapy could reduce serum levels of TG, TC, LDL-C, ALT, AST, ALP, and increase high-density lipoprotein cholesterol (HDL-C) levels (P<0.05or0.01) to generally normal level as in normal groups. The difference was statistically significant compared to berberine and curcumin alone (P<0.05). Combination therapy could reduce serum levels of endotoxin, fasting blood glucose (FBG), insulin (INS) and MDA, and increase GSH-Px activity (P<0.01) to generally normal level as in normal groups. The difference was statistically significant compared to berberine and curcumin alone (P<0.05). PPARy gene expression was increased in liver tissue of rats in combination therapy, while gene expression of SREBP-lc, SCD-1, NF-κB and protein expression of SREBP-lc, TNF-a, NF-κB p65, p-JNK were down-regulated, caveolin-1protein expression was up-regulated to various levels. The difference was statistically significant compared to berberine and curcumin alone (P <0.05). Overall, it could be concluded that combined administration of berberine and curcumin (each50mg·kg-1·d-1) has a better effect than berberine or curcumin alone (100mg-kg-1·d-1), and a synergistic effect was shown.The results show that:(1) Coptis chinensis Franch., Curcuma longa L. and R. tanguticum Maxim. ex Balf. administrated alone all had a favorable effect on treating NAFLD rats, in which Coptis chinensis Franch. and, Curcuma longa L. had better effect.(2)10541Berberine, the effective component of Coptis chinensis Franch. and curcumin, the effective component of Curcuma longa L. all had a favorable therapeutic effect in NAFLD rats, and combination of the2components had synergistic effect. Curcumin can directly interfere with lipid metabolism in liver cells, with SREBP-lc and caveolin-1as its possible important targets; while berberine could not interfere with lipid metabolism directly, it may accomplish curative or synergistic effect by reducing endotoxin into the blood from gut and improving insulin resistance.更多还原