本文選題：小檗堿 + 非酒精性脂肪性肝炎�。� 參考：《北京中醫(yī)藥大學(xué)》2014年博士論文

【摘要】：研究目的 非酒精性脂肪性肝炎(nonalcoholic steatohepatitis, NASH)的病理機制及治療方案尚未完全明確。本研究在文獻(xiàn)調(diào)研、分析的基礎(chǔ)上,探討非酒精性脂肪性肝病的中醫(yī)證治規(guī)律及病機特征�；�2種動物模型(蛋氨酸-膽堿缺乏飲食誘導(dǎo)小鼠NASH模型及高脂飼料誘導(dǎo)大鼠NASH模型),評價小檗堿干預(yù)NASH的療效。進(jìn)而以肝組織巨噬細(xì)胞表型轉(zhuǎn)化、胰島素抵抗及代謝紊亂為切入點,探討小檗堿干預(yù)NASH的分子機制。 研究方法 1基于SinoMed和PubMed數(shù)據(jù)庫,采用文本挖掘方法,通過數(shù)據(jù)檢索、數(shù)據(jù)處理、數(shù)據(jù)分析及可視化呈現(xiàn)技術(shù),挖掘非酒精性脂肪性肝病(non-alcoholic fatty liver disease, NAFLD)的中醫(yī)證候特點及用藥規(guī)律并構(gòu)建NAFLD相關(guān)的生物學(xué)網(wǎng)絡(luò)�；谥嗅t(yī)“方-證-病”相關(guān)聯(lián)理論,分析和理解NAFLD中醫(yī)病機對應(yīng)的物質(zhì)基礎(chǔ)及可能的干預(yù)靶標(biāo)。 2小檗堿干預(yù)NASH的藥效學(xué)研究：C57BL/6小鼠和SD大鼠隨機分成5組：正常對照組、模型對照組、陽性藥(羅格列酮治療)對照組、小檗堿高劑量干預(yù)組和小檗堿低劑量干預(yù)組。采用常規(guī)方法制備蛋氨酸-膽堿缺乏飲食(MCD)誘導(dǎo)的NASH小鼠模型(2W)及高脂飲食(HFD)誘導(dǎo)的大鼠NASH模型(8W)。正常對照組喂予普通飼料,其余各組喂予MCD或HFD。小檗堿和羅格列酮以預(yù)防性、灌胃給藥的方式連續(xù)干預(yù)。大鼠腹主動脈取血、小鼠摘眼球取血分離血清,肝組織分別液氮固定冰凍切片、多聚甲醛固定制作石蠟切片。采用血清生化、組織病理學(xué)、血清炎癥因子酶聯(lián)免疫法(ELISA)等檢測方法評價小檗堿干預(yù)NASH的療效。 3基于MCD誘導(dǎo)NASH小鼠模型,生理鹽水注入左心室進(jìn)行肝臟灌流,提取肝臟非實質(zhì)細(xì)胞,采用流式細(xì)胞術(shù)方法檢測肝組織中巨噬細(xì)胞(F4/80)及M1(F4/80+CD11c+)、M2(F4/80+CD206)亞型的數(shù)量,分析M1/M2比值。 4基于HFD誘導(dǎo)大鼠NASH模型,采用實時熒光定量聚合酶鏈反應(yīng)(RTFQ-PCR)及免疫組化法(IHC)檢測肝組織PPAR-γ mRNA及蛋白表達(dá)；采用超高效液相色譜-四級桿飛行時間質(zhì)譜法(UPLC-Q-TOF-MS)檢測大鼠血清中內(nèi)源性小分子代謝物(metabolites)輪廓,結(jié)合主成分分析(PCA)、偏最小二乘-判別分析(PLS-DA)等代謝組學(xué)技術(shù)分析NASH相關(guān)的代謝標(biāo)志物及代謝通路。 5全部定量數(shù)據(jù)經(jīng)SPSS17.0統(tǒng)計軟件進(jìn)行分析及統(tǒng)計學(xué)處理,數(shù)值采用X±S表示,組間均數(shù)差異比較采用單因素方差分析(one-way ANOVA), P0.05表示差異有統(tǒng)計學(xué)意義。 研究結(jié)果 1文本挖掘結(jié)果顯示,NAFLD中醫(yī)臨床最主要的證候包括：肝郁脾虛、濕熱內(nèi)蘊和痰瘀互結(jié)；治療NAFLD常用的中藥包括：丹參、柴胡、何首烏、澤瀉、山楂、白術(shù)和郁金；NAFLD中醫(yī)病機對應(yīng)的生物學(xué)過程是胰島素抵抗、脂代謝調(diào)節(jié)紊亂及慢性炎癥。 2NAFLD病理評分結(jié)果表明,HFD喂養(yǎng)SD大鼠8周后NAS病理評分6.6分,MCD飲食喂養(yǎng)C57BL/6小鼠2周NAS評分6.8分,表明NASH模型制備成功。在兩種NASH模型中,高、低劑量小檗堿預(yù)防給藥均能顯著改善NASH病理改變程度；降低血清ALT、CHO水平(P0.05)；下調(diào)促炎因子TNF-a而上調(diào)抗炎因子IL-10水平(P0.05)。結(jié)果提示：小檗堿干預(yù)NASH的療效與陽性藥(羅格列酮)相當(dāng),且高、低劑量組間差異不顯著(P0.05) 3流式細(xì)胞術(shù)結(jié)果表明,與正常對照組比較,MCD誘導(dǎo)NASH小鼠肝組織中M1型巨噬細(xì)胞數(shù)量顯著增高,M2型巨噬細(xì)胞數(shù)量顯著降低,M1/M2比值顯著增加(P0.01)；與模型組比較,小檗堿干預(yù)組小鼠肝組織中M1數(shù)量減少,M2數(shù)量顯著增多(P0.05),M1/M2比值顯著降低(P0.01)。結(jié)果提示：小檗堿和羅格列酮均能改善MCD飲食誘導(dǎo)的NASH病理過程,其作用可能是通過調(diào)節(jié)巨噬細(xì)胞的表型轉(zhuǎn)化,即升高肝組織中的M2、降低M1的比例。 4免疫組織化學(xué)結(jié)果表明,與正常對照組比較,HFD誘導(dǎo)NASH大鼠肝組織PPAR-y蛋白水平顯著下調(diào)(P0.01)。小檗堿和羅格列酮均能顯著上調(diào)肝組織中PPAR-γ蛋白的表達(dá)(P0.01)。而RTFQ-PCR結(jié)果表明,HFD誘導(dǎo)NASH大鼠肝組織PPAR-γmRNA水平顯著上調(diào)(P0.01)。與之相應(yīng),小檗堿和羅格列酮均能顯著下調(diào)肝組織中PPAR-γmRNA的表達(dá)(P0.01)。結(jié)果提示：小檗堿對HFD誘導(dǎo)的NASH大鼠改善IR、減輕病理程度可能是通過某種途徑糾正了PPAR-γ基因轉(zhuǎn)錄至翻譯的環(huán)節(jié)。 5UPLC-Q-TOF/MS檢測結(jié)果表明：大鼠血清中可獲取1494個離子峰(正離子模式下870個,負(fù)離子模式下624個)。PCA及PLS-DA結(jié)果表明正常對照組、NASH模型組、羅格列酮干預(yù)組及小檗堿干預(yù)組呈明顯的聚成3簇,羅格列酮和小檗堿組的代謝產(chǎn)物呈現(xiàn)明顯的重疊模式。多元分析(VIP1)結(jié)果表明,與正常對照組比較,NASH大鼠血清中可篩選獲得57個潛在的生物標(biāo)志物：包括下調(diào)的肌酐、乙酰肉堿、苯丙氨酸、酪氨酸、硫酸吲哚酚、膽酸、熊去氧膽酸、花生四烯酸、15羥基二十碳四烯酸、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)、磷脂酰膽堿(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)上調(diào)的13過氧氫十八碳二烯酸(13-HpODE)、11,12亞甲基十九碳酸、二十碳烯酸、二十碳二烯酸、二十碳三烯酸、二十二碳烯酸、溶血磷脂酰膽堿(LPC14:0,16:1,17:1,18:1,18:2,20:2)、磷脂酰膽堿(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)。代謝通路分析結(jié)果顯示：NASH中脂肪酸p氧化、磷脂-花生四烯酸代謝網(wǎng)絡(luò)以及苯丙氨酸-酪氨酸-甲狀腺激素合成代謝、膽固醇-膽汁酸代謝被擾動；小檗堿與羅格列酮使NASH病理狀態(tài)下紊亂的代謝輪廓向正常狀態(tài)偏移。 研究結(jié)論 現(xiàn)代中醫(yī)對NAFLD已有系統(tǒng)的研究,基于中醫(yī)“方-證-病”相關(guān)聯(lián)理論,該病的物質(zhì)基礎(chǔ)與胰島素抵抗、炎癥及代謝紊亂等生物學(xué)過程相關(guān)。小檗堿能有效抵抗HFD誘導(dǎo)大鼠及MCD飲食誘導(dǎo)小鼠NASH的形成,其初步藥理機制包括以下3個方面： 1小檗堿可調(diào)節(jié)肝組織中巨噬細(xì)胞表型轉(zhuǎn)化,抑制M1型巨噬細(xì)胞、增加M2型巨噬細(xì)胞的比例,下調(diào)促炎細(xì)胞因子、上調(diào)抗炎細(xì)胞因子的合成和分泌,從而抵抗NASH的發(fā)生、發(fā)展。 2小檗堿顯著改善機體的胰島素抵抗,其部分藥理機制通過調(diào)節(jié)PPAR-γ基因的轉(zhuǎn)錄和表達(dá)。 3代謝組學(xué)結(jié)果提示：小檗堿干預(yù)NASH,通過影響脂類和蛋白質(zhì)多個代謝通路調(diào)節(jié)代謝紊亂,而且其對代謝譜的調(diào)控與羅格列酮相近。 該研究結(jié)果區(qū)分于以往的“單—藥物-單—靶點-單—信號通路”的藥理機制研究模式,從系統(tǒng)和整體水平揭示了中藥單體化合物小檗堿的分子藥理機制。小檗堿干預(yù)NASH的靶點呈非線性特點,通過影響多個通路如胰島素抵抗、炎性因子、代謝紊亂等改善NASH的多個病理環(huán)節(jié)。該研究為小檗堿深度開發(fā)為抗脂肪性肝炎藥物提供了實驗資料和新的研究策略。
[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.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R575.5

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