小檗碱干预非酒精性脂肪性肝炎（NASH）的分子机制研究

发布时间：2018-04-30 04:36

本文选题：小檗碱 + 非酒精性脂肪性肝炎　；参考：《北京中医药大学》2014年博士论文

【摘要】：研究目的非酒精性脂肪性肝炎(nonalcoholic steatohepatitis, NASH)的病理机制及治疗方案尚未完全明确。本研究在文献调研、分析的基础上,探讨非酒精性脂肪性肝病的中医证治规律及病机特征。基于2种动物模型(蛋氨酸-胆碱缺乏饮食诱导小鼠NASH模型及高脂饲料诱导大鼠NASH模型),评价小檗碱干预NASH的疗效。进而以肝组织巨噬细胞表型转化、胰岛素抵抗及代谢紊乱为切入点,探讨小檗碱干预NASH的分子机制。研究方法 1基于SinoMed和PubMed数据库,采用文本挖掘方法,通过数据检索、数据处理、数据分析及可视化呈现技术,挖掘非酒精性脂肪性肝病(non-alcoholic fatty liver disease, NAFLD)的中医证候特点及用药规律并构建NAFLD相关的生物学网络。基于中医“方-证-病”相关联理论,分析和理解NAFLD中医病机对应的物质基础及可能的干预靶标。 2小檗碱干预NASH的药效学研究：C57BL/6小鼠和SD大鼠随机分成5组：正常对照组、模型对照组、阳性药(罗格列酮治疗)对照组、小檗碱高剂量干预组和小檗碱低剂量干预组。采用常规方法制备蛋氨酸-胆碱缺乏饮食(MCD)诱导的NASH小鼠模型(2W)及高脂饮食(HFD)诱导的大鼠NASH模型(8W)。正常对照组喂予普通饲料,其余各组喂予MCD或HFD。小檗碱和罗格列酮以预防性、灌胃给药的方式连续干预。大鼠腹主动脉取血、小鼠摘眼球取血分离血清,肝组织分别液氮固定冰冻切片、多聚甲醛固定制作石蜡切片。采用血清生化、组织病理学、血清炎症因子酶联免疫法(ELISA)等检测方法评价小檗碱干预NASH的疗效。 3基于MCD诱导NASH小鼠模型,生理盐水注入左心室进行肝脏灌流,提取肝脏非实质细胞,采用流式细胞术方法检测肝组织中巨噬细胞(F4/80)及M1(F4/80+CD11c+)、M2(F4/80+CD206)亚型的数量,分析M1/M2比值。 4基于HFD诱导大鼠NASH模型,采用实时荧光定量聚合酶链反应(RTFQ-PCR)及免疫组化法(IHC)检测肝组织PPAR-γ mRNA及蛋白表达；采用超高效液相色谱-四级杆飞行时间质谱法(UPLC-Q-TOF-MS)检测大鼠血清中内源性小分子代谢物(metabolites)轮廓,结合主成分分析(PCA)、偏最小二乘-判别分析(PLS-DA)等代谢组学技术分析NASH相关的代谢标志物及代谢通路。 5全部定量数据经SPSS17.0统计软件进行分析及统计学处理,数值采用X±S表示,组间均数差异比较采用单因素方差分析(one-way ANOVA), P0.05表示差异有统计学意义。研究结果 1文本挖掘结果显示,NAFLD中医临床最主要的证候包括：肝郁脾虚、湿热内蕴和痰瘀互结；治疗NAFLD常用的中药包括：丹参、柴胡、何首乌、泽泻、山楂、白术和郁金；NAFLD中医病机对应的生物学过程是胰岛素抵抗、脂代谢调节紊乱及慢性炎症。 2NAFLD病理评分结果表明,HFD喂养SD大鼠8周后NAS病理评分6.6分,MCD饮食喂养C57BL/6小鼠2周NAS评分6.8分,表明NASH模型制备成功。在两种NASH模型中,高、低剂量小檗碱预防给药均能显著改善NASH病理改变程度；降低血清ALT、CHO水平(P0.05)；下调促炎因子TNF-a而上调抗炎因子IL-10水平(P0.05)。结果提示：小檗碱干预NASH的疗效与阳性药(罗格列酮)相当,且高、低剂量组间差异不显著(P0.05) 3流式细胞术结果表明,与正常对照组比较,MCD诱导NASH小鼠肝组织中M1型巨噬细胞数量显著增高,M2型巨噬细胞数量显著降低,M1/M2比值显著增加(P0.01)；与模型组比较,小檗碱干预组小鼠肝组织中M1数量减少,M2数量显著增多(P0.05),M1/M2比值显著降低(P0.01)。结果提示：小檗碱和罗格列酮均能改善MCD饮食诱导的NASH病理过程,其作用可能是通过调节巨噬细胞的表型转化,即升高肝组织中的M2、降低M1的比例。 4免疫组织化学结果表明,与正常对照组比较,HFD诱导NASH大鼠肝组织PPAR-y蛋白水平显著下调(P0.01)。小檗碱和罗格列酮均能显著上调肝组织中PPAR-γ蛋白的表达(P0.01)。而RTFQ-PCR结果表明,HFD诱导NASH大鼠肝组织PPAR-γmRNA水平显著上调(P0.01)。与之相应,小檗碱和罗格列酮均能显著下调肝组织中PPAR-γmRNA的表达(P0.01)。结果提示：小檗碱对HFD诱导的NASH大鼠改善IR、减轻病理程度可能是通过某种途径纠正了PPAR-γ基因转录至翻译的环节。 5UPLC-Q-TOF/MS检测结果表明：大鼠血清中可获取1494个离子峰(正离子模式下870个,负离子模式下624个)。PCA及PLS-DA结果表明正常对照组、NASH模型组、罗格列酮干预组及小檗碱干预组呈明显的聚成3簇,罗格列酮和小檗碱组的代谢产物呈现明显的重叠模式。多元分析(VIP1)结果表明,与正常对照组比较,NASH大鼠血清中可筛选获得57个潜在的生物标志物：包括下调的肌酐、乙酰肉碱、苯丙氨酸、酪氨酸、硫酸吲哚酚、胆酸、熊去氧胆酸、花生四烯酸、15羟基二十碳四烯酸、3-Hydroxychola-7,22-dien-24-oic acid、鞘磷脂(SM34:1,34:2,36:1,40:1,42:1,42:2,42:3)、磷脂酰胆碱(PC32:0,34:0,36:3,37:4,38:4,38:6,40:4,40:5,40:6,40:8)、溶血卵磷脂(LPC16:0,17:0,18:0,20:4)和二酰甘油(DG38:6)上调的13过氧氢十八碳二烯酸(13-HpODE)、11,12亚甲基十九碳酸、二十碳烯酸、二十碳二烯酸、二十碳三烯酸、二十二碳烯酸、溶血磷脂酰胆碱(LPC14:0,16:1,17:1,18:1,18:2,20:2)、磷脂酰胆碱(PC34:1,34:3,34:4,35:2,36:1,36:2,36:4,36:5,38:2,38:3,38:4)和二酰甘油(DG34:2,36:3)。代谢通路分析结果显示：NASH中脂肪酸p氧化、磷脂-花生四烯酸代谢网络以及苯丙氨酸-酪氨酸-甲状腺激素合成代谢、胆固醇-胆汁酸代谢被扰动；小檗碱与罗格列酮使NASH病理状态下紊乱的代谢轮廓向正常状态偏移。研究结论现代中医对NAFLD已有系统的研究,基于中医“方-证-病”相关联理论,该病的物质基础与胰岛素抵抗、炎症及代谢紊乱等生物学过程相关。小檗碱能有效抵抗HFD诱导大鼠及MCD饮食诱导小鼠NASH的形成,其初步药理机制包括以下3个方面： 1小檗碱可调节肝组织中巨噬细胞表型转化,抑制M1型巨噬细胞、增加M2型巨噬细胞的比例,下调促炎细胞因子、上调抗炎细胞因子的合成和分泌,从而抵抗NASH的发生、发展。 2小檗碱显著改善机体的胰岛素抵抗,其部分药理机制通过调节PPAR-γ基因的转录和表达。 3代谢组学结果提示：小檗碱干预NASH,通过影响脂类和蛋白质多个代谢通路调节代谢紊乱,而且其对代谢谱的调控与罗格列酮相近。该研究结果区分于以往的“单—药物-单—靶点-单—信号通路”的药理机制研究模式,从系统和整体水平揭示了中药单体化合物小檗碱的分子药理机制。小檗碱干预NASH的靶点呈非线性特点,通过影响多个通路如胰岛素抵抗、炎性因子、代谢紊乱等改善NASH的多个病理环节。该研究为小檗碱深度开发为抗脂肪性肝炎药物提供了实验资料和新的研究策略。
[Abstract]:research objective
The pathological mechanism and treatment scheme of nonalcoholic steatohepatitis (NASH) is not completely clear. On the basis of literature investigation and analysis, this study explored the rule of TCM treatment and pathogenesis of non-alcoholic fatty liver disease. Based on 2 animal models (methionine choline deficiency diet induced NASH model in mice) The effect of berberine on NASH was evaluated by the NASH model of rats induced by high fat diet. The effect of berberine on the phenotype transformation of macrophage, insulin resistance and metabolic disorder was discussed, and the molecular mechanism of berberine intervention in NASH was discussed.
research method
1 based on the SinoMed and PubMed database, using the method of text mining, through data retrieval, data processing, data analysis and visual presentation technology, we excavate the characteristics of TCM syndrome of non-alcoholic fatty liver disease (non-alcoholic fatty liver disease, NAFLD) and construct the law of drug use and construct the biological network related to NAFLD. Based on TCM "Fang - Certificate" The theory of "disease" is used to analyze and understand the material basis and possible intervention targets of NAFLD TCM pathogenesis.
2 the pharmacodynamic study of berberine intervention on NASH: C57BL/6 mice and SD rats were randomly divided into 5 groups: normal control group, model control group, positive drug (rosiglitazone) control group, berberine high dose intervention group and berberine low dose intervention group. The NASH mouse model (2W) induced by methanine choline deficiency diet (MCD) was prepared by routine method. The rat NASH model (8W) induced by high fat diet (HFD). The normal control group was fed with ordinary diet, and the other groups were fed with MCD or HFD. berberine and rosiglitazone for prophylactic treatment. The rat abdominal aorta was taken blood, the mice picked the eyeball to separate the blood serum, the liver tissue was fixed with frozen section and polyformaldehyde. Paraffin sections were made. Serum biochemistry, histopathology, serum inflammatory factors and enzyme-linked immunosorbent assay (ELISA) were used to evaluate the efficacy of berberine in NASH intervention.
3 based on the MCD induced NASH mouse model, the normal saline was injected into the left ventricle for liver perfusion, and the liver non parenchymal cells were extracted. The number of macrophages (F4/80), M1 (F4/80+CD11c+), M2 (F4/80+CD206) subtype in the liver tissue was detected by flow cytometry, and the M1/M2 ratio was analyzed.
4 based on HFD induced rat NASH model, real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and immunohistochemistry (IHC) were used to detect the expression of PPAR- gamma mRNA and protein in liver tissue. The endogenous small molecule metabolites (metabolites) profiles in rat blood were detected by ultra high performance liquid chromatography and four grade rod time of flight mass spectrometry (UPLC-Q-TOF-MS). PCA and partial least squares discriminant analysis (PLS-DA) and other metabonomics techniques were used to analyze NASH related metabolic markers and metabolic pathways.
5 all quantitative data were analyzed and statistically treated by SPSS17.0 statistical software. The numerical value was expressed by X + S, and the difference of average number between groups was compared with single factor analysis of variance (one-way ANOVA), and P0.05 indicated that the difference was statistically significant.
Research results
1 the results of the 1 text mining show that the main syndromes of the clinical Chinese medicine include liver depression and spleen deficiency, damp heat accumulation and phlegm stasis. The common Chinese medicine for the treatment of NAFLD includes Salvia miltiorrhiza, bupleurum, Radix Polygoni Multiflori, Alisma orientalis, hawthorn, Atractylodes and tulip, and the biological process of NAFLD TCM disease machine is insulin resistance, lipid metabolism regulation disorder and chronic inflammation. Disease.
2NAFLD pathological score showed that the pathological score of NAS was 6.6 points after 8 weeks of feeding in SD rats, and the NAS score of C57BL/6 mice fed with MCD diet was 6.8 points at 2 weeks, indicating that the NASH model was successfully prepared. In the two NASH models, high and low dose berberine prevention administration could significantly improve the pathological changes of NASH, decrease the ALT, CHO level, and decrease the level of NASH. Inflammatory factor TNF-a increased the anti inflammatory factor IL-10 level (P0.05). The results showed that the effect of berberine on NASH was similar to that of positive drug (rosiglitazone), and the difference was not significant (P0.05).
The results of 3 flow cytometry showed that, compared with the normal control group, the number of M1 macrophages in the liver tissue of NASH mice induced by MCD was significantly increased, the number of M2 type macrophages decreased significantly, and the M1/M2 ratio increased significantly (P0.01). Compared with the model group, the number of M1 in the berberine intervention group was reduced, the M2 quantity increased significantly (P0.05), M1/M2 ratio. Significantly lower (P0.01). The results suggest that both berberine and rosiglitazone can improve the pathological process of NASH induced by MCD diet, which may be mediated by regulating the phenotypic transformation of macrophages, that is, to increase the M2 in the liver tissue and to reduce the proportion of M1.
4 immuno histochemical results showed that the level of PPAR-y protein in liver tissue of NASH rats was significantly down regulated by HFD (P0.01). Both berberine and rosiglitazone significantly increased the expression of PPAR- gamma protein in liver tissues (P0.01). RTFQ-PCR results showed that HFD induced PPAR- gamma mRNA levels in the liver tissues of NASH rats were significantly up (P0.01). Correspondingly, both berberine and rosiglitazone can significantly reduce the expression of PPAR- gamma mRNA in liver tissues (P0.01). The results suggest that berberine may improve the IR and reduce the pathological degree of HFD induced NASH rats. It may be a way to correct the transcription of PPAR- gamma gene to the link of translation through some way.
The results of 5UPLC-Q-TOF/MS test showed that 1494 ion peaks were obtained in the serum of rats (870 in positive ion mode and 624 in negative ion mode), and the results of.PCA and PLS-DA showed that the normal control group, the NASH model group, the rosiglitazone intervention group and the berberine intervention group were obviously clustered into 3 clusters, and the metabolites of the rosiglitazone and berberine groups were bright. The VIP1 results showed that, compared with the normal control group, 57 potential biomarkers were screened in the serum of NASH rats, including the down-regulated creatinine, acetyl carnitine, phenylalanine, tyrosine, indolyl sulfate, cholic acid, ursodeoxycholic acid, peanut four enoic acid, 15 hydroxyl twenty - twenty - four enoic acid, 3-Hydroxychola-7,2 2-dien-24-oic acid, sphingomyelin (SM34:1,34:2,36:1,40:1,42:1,42:2,42:3), phosphatidylcholine (PC32:0,34:0,36:3,37:4,38:4,38:6,40:4,40:5,40:6,40:8), hemolytic lecithin (LPC16:0,17:0,18:0,20:4) and two acyl glycerol (DG38:6) up regulation of 13 peroxy hydrogen eighteen carbenadienoic acid (13-HpODE), 11,12 methylene nineteen carbonate, twenty carbenoic acid, twenty carbon Two eNIC acid, twenty carbon three enoic acid, twenty-two carbenic acid, lysophosphatidylcholine (LPC14:0,16:1,17:1,18:1,18:2,20:2), phosphatidylcholine (PC34:1,34:3,34:4,35:2,36:1,36:2,36:4,36:5,38:2,38:3,38:4) and two acylglycerol (DG34:2,36:3). Metabolic pathway analysis shows: P oxidation of fatty acids in NASH, phosphatidyl four enoic acid metabolism network As well as phenylalanine tyrosine thyroid hormone synthesis and metabolism, the cholesterol - bile acid metabolism is disturbed; berberine and rosiglitazone make the metabolic profile of the disorder in the pathological state of NASH shift to the normal state.
research conclusion
Modern traditional Chinese medicine has a systematic study of NAFLD, based on the theory of "Fang syndrome disease" associated with Chinese medicine. The material basis of the disease is related to biological processes such as insulin resistance, inflammation and metabolic disorders. Berberine can effectively resist the formation of HFD induced rat and MCD diet induced mouse NASH. Its preliminary pharmacological mechanisms include the following 3 aspects:
1 berberine can regulate the phenotypic transformation of macrophages in liver tissue, inhibit M1 macrophages, increase the proportion of M2 type macrophages, down regulate the proinflammatory cytokines, up regulate the synthesis and secretion of anti-inflammatory cytokines, so as to resist the occurrence and development of NASH.
2 berberine significantly improves insulin resistance in the body. Part of its pharmacological mechanism regulates transcription and expression of PPAR- gamma gene.
3 the results of metabolomics suggest that berberine interfered with NASH and regulates metabolic disorders by affecting many metabolic pathways of lipids and proteins, and the regulation of metabolic profiles is similar to that of rosiglitazone.
The results of the study are distinguished from the previous pharmacological mechanisms of "single drug single target single target single signal pathway". The molecular pharmacological mechanism of berberine, a single compound of Chinese medicine, is revealed from the system and the overall level. The target of berberine intervention in NASH is nonlinear and affects multiple pathways such as insulin resistance and inflammatory factors. Metabolic disorders can improve many pathological links of NASH. This study provides experimental data and new research strategies for berberine to further develop anti fatty hepatitis drugs.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R575.5

【参考文献】