【作者】 罗文政；

【导师】 吕志平；

【作者基本信息】 南方医科大学 ， 中西医结合临床， 2009， 博士

【摘要】 研究背景非酒精性脂肪肝(non-alcoholic fatty liver disease,NAFLD)仅次于病毒性肝炎的第二大肝病,已经成为我国常见的慢性肝病之一,其发病率大约为14～20%,严重危害人民生活健康。NAFLD以弥漫性肝细胞大泡性脂肪变为主要特征的临床病理综合征,包括单纯性脂肪肝及其演变的脂肪性肝炎(NASH)和肝硬化。研究发现遗传易感性、胰岛素抵抗、肠源性内毒素血症以及PPARα与其发病关系密切。其中胰岛素抵抗是关键的始动因素,肠源性内毒素血症以及PPARα的表达下调作为“二次打击”,最终造成脂肪在肝脏大量蓄积出现脂肪肝的病理改变。祖国医学虽无“脂肪肝”的病名,《内经》中有“肝满”、“肝胀”、“胁痛”等记载,符合脂肪肝的临床表现。其病位在肝,与胆、脾、胃、肾均有关。多因过食肥甘,或饮酒过度,或感受湿热疫毒,或情志失调,或久病体虚,导致痰湿瘀阻互结,痹阻肝脏脉络而成。红曲性味甘、温、无毒,归肝、脾胃、大肠经,具有健脾消食、活血化淤之功效;现代药理研究发现其有效成份之一的洛伐他汀,具有较为理想的抗炎、降胆固醇的功效,已在临床广泛利用。因此,本课题组推测,红曲可能具有较好的抗NAFLD的效应,对红曲抗NAFLD的分子机制深入研究,是中药创新的切入点。本项目在中医药理论指导下,结合现代医学研究进展,通过高脂饮食诱导胰岛素抵抗NAFLD大鼠模型,继而予以不同剂量红曲干预8周,以绞股蓝为阳性对照组,采用HE染色、全自动生化分析、胰岛素-血糖钳夹、RT-PCR、Western-blot、ELISA等实验技术检测各组大鼠血脂、肝脏生化、胰岛素抵抗以及TNF-α、IL-6、IL-10、PPARα等指标的变化,探讨红曲治疗NAFLD的效应及其作用机制,为提高中医药防治水平提供实验依据。目的观察红曲对胰岛素抵抗,肝脏生化、血脂改变以及TNF-α、IL-6、IL-10、PPARα等指标的影响,探讨胰岛素抵抗、肠源性内毒素血症以及过氧化物增殖激活受体在大鼠NAFLD发病中的作用及红曲拮抗NAFLD的分子机制。方法:48只SD大鼠,雌雄各半,适应性喂养1周后,按体重随机分为6组,正常组、模型组、绞股蓝组、红曲低剂量组、红曲中剂量组、红曲高剂量组,每组8只。正常组予普通饲料,同步饲养8周;其余各组予高脂饲养喂养,并分别予等剂量生理盐水、绞股蓝水溶液、低、中、高剂量红曲水溶液灌胃,连续8周。第9周,处死所有大鼠,取肝脏及腹主动脉血备用。HE染色、光镜下对各组大鼠肝脏组织病理学评分,从形态学上分析NAFLD大鼠及红曲干预后肝脏组织结构的变化;全自动生化分析仪分析血清ALT、AST、及血脂变化;放免法检测肝纤四项;正常葡萄糖-高胰岛素钳夹技术评估胰岛素抵抗变化,以观察红曲改善胰岛素抵抗的效应;ELSIA及RT-PCR技术检测各组大鼠TNF-α、IL-6、IL-10水平变化,分析红曲对TNF-α、IL-6、IL-10的影响,以观察红曲对上述细胞因子的调控机制;Western-blot、RT-PCR技术检测各组大鼠肝脏PPARαmRNA和蛋白水平变化,从蛋白表达水平和从基因转录水平分析NAFLD大鼠肝脏PPARα的情况及红曲对其进行治疗后的肝脏PPARα的影响。统计学处理:计量资料用平均值±标准差((?)±s)表示,数据处理采用SPSS11.5统计软件包进行单因素方差分析(One-way ANOVA);计数资料采用秩和检验。P＜0.05有统计学意义。结果1红曲对NAFLD大鼠的症状、体征的影响正常组大鼠表现为体重自然增加,反应灵活,活动敏捷,皮毛密集而有光泽,紧贴皮肤,鼻、唇湿润清洁,眼睛明亮,大便正常,在饲养过程中未发现死亡。模型组动物表现饮食减少,生长缓慢,皮毛干枯、蓬松且易脱落,活动迟缓,反应迟钝,眼睛、鼻、唇、耳、尾部色淡少泽。各治疗组行为学表现、体重和营养状况等方面与模型组比较有一定改善,无统计学意义P=0.487。以红曲高剂量组改善情况为佳。2红曲对NAFLD大鼠肝组织形态学的影响从光镜下肝脏病理组织学变化看,模型组大鼠成模率100%,肝脏全小叶脂肪变性,大囊泡性,并有大囊泡融合形成脂囊。基本存在小叶内炎症,以单核细胞、淋巴细胞浸润为主,并可见点状坏死、灶状坏死、碎屑样坏死。绞股蓝组可见部分肝脂肪细胞变性、仅见少许炎性细胞浸润,无汇管区纤维组织增生;红曲高剂量组仅见少许肝细胞脂肪变性,肝小叶内仅有少量炎细胞浸润,无灶状及碎屑样坏死,汇管区纤维组织增生、淋巴细胞浸润不明显。红曲低、中剂量组脂肪变性及炎症程度介于模型组和红曲高剂量组之间。3红曲对NAFLD大鼠肝功能、血脂的影响①肝脏酶学变化提示:模型组与各组比较,大鼠存在肝脏酶学异常改变(P=0.000),以ALT、AST升高为主,ALT/AST＜1,这表明存在脂肪性肝炎肝细胞坏死的生化改变;予红曲、绞股蓝干预后,ALT、AST呈下降改变。红曲各治疗组与绞股蓝组之间比较,无统计学差异(P=0.337)。②血脂变化表明:模型组与正常组比较,大鼠血脂紊乱,存在总胆固醇以及低密度脂蛋白增高、高密度脂蛋白降低变化,有统计学差异(P=0.001);经红曲干预后,大鼠血脂紊乱无改善;但绞股蓝组出现TG、LDL-C下降以及HDL-C上调的趋势,红曲高剂量组与绞股蓝组比较在CHOL及HDL-C,有统计学差异(P=0.013,P=0.006)。4.红曲对NAFLD大鼠肝纤维化的影响NAFLD大鼠肝纤四项变化提示,模型组LN、HA、PCⅢ、CⅣ均呈上升趋势;除红曲低剂量组肝纤四项变化呈下降趋势;其余各组肝纤四项变化不明显。模型组与红曲各剂量组、绞股蓝组比较,无统计学差异(P=0.057);红曲无改善大鼠肝纤维化的作用。5红曲对NAFLD大鼠胰岛素抵抗的影响实验前(即钳夹开始前60min)各组大鼠血糖差别较大,模型组大鼠空腹血糖与正常对照组相比升高(P=0.016),表明NAFLD大鼠存在血糖代谢的障碍。实验开始60min后(即钳夹开始0min)各组血糖均达到稳态(5.3mmol/l),表明葡萄糖钳夹形成。实验过程中各组血糖保持稳定,符合高胰岛素葡萄糖钳夹实验要求。高脂饮食组(包括模型组及各治疗组)和正常对照组的空腹血糖水平相近。空腹胰岛素水平高脂饮食组高于正常对照组(P=0.030)。通过钳夹实验发现高脂饮食大鼠的GIR低于普通饮食,说明高脂饮食大鼠对胰岛素敏感性降低,存在胰岛素抵抗。予红曲治疗后,空腹胰岛素水平呈下降趋势,表明红曲具有一定改善胰岛素抵抗的作用。6红曲对NAFLD大鼠肠源性内毒素血症的影响NAFLD大鼠炎性细胞因子变化结果提示。大鼠TNF-α(pg/ml):正常组38.34±4.57、模型组50.05±3.74、绞股蓝组34.99±7.99、红曲低、中、高剂量组分别为49.40±6.61、44.90±12.04和35.68±7.29,模型组与各治疗组比较,有统计学意义(P=0.000);红曲高剂量组与绞股蓝组比较,无统计学意义(P＞0.05);红曲高剂量组与低、中剂量组比较,有统计学意义(P＜0.001);大鼠IL-6(pg/ml):正常组26.65±3.67、模型组34.35±2.36、绞股蓝组30.79±6.50、红曲低、中、高剂量组分别为34.89±7.27、30.75±1.81和28.61±9.51,模型组与各治疗组比较,无统计学意义(P=0.879);红曲高剂量组与低、中剂量组比较,无统计学意义(P=0.929);红曲高剂量组与绞股蓝组比较,无统计学意义(P=1.000)。大鼠IL-10(pg/ml):正常组为36.87±4.58、模型组20.20±5.79、绞股蓝组20.61±7.44、红曲低、中、高剂量组分别为27.65±4.28、25.18±9.82和24.00±11.15,模型组与各治疗组比较,无统计学意义(P=0.326);红曲高剂量组与低、中剂量组比较,有统计学意义(P=0.344);红曲高剂量组与绞股蓝组比较,无统计学意义(P=0.388)。②大鼠TNF-αmRNA表达:正常组0.51±0.50、模型组1.21±0.55、绞股蓝组0.54±0.24、红曲低、中、高剂量组分别为0.63±0.28、0.65±0.50、0.73±0.39,模型组与各治疗组比较,无统计学意义(P=0.143);红曲高剂量组与低、中剂量组比较,无统计学意义(P=0.546);红曲高剂量组与绞股蓝组比较,无统计学意义(P=0.774)。大鼠IL-6mRNA表达:正常组0.29±0.16、模型组0.53±0.18、绞股蓝组0.41±0.22、红曲低、中、高剂量组分别为0.29±0.27、0.50±0.18和0.50±0.14,模型组与各治疗组比较,无统计学意义(P=0.130);红曲高剂量组与低、中剂量组比较,无统计学意义(P=0.390);红曲高剂量组与绞股蓝组比较,无统计学意义(P=0.433)。大鼠IL-10mRNA表达:正常组分别为0.55±0.07、模型组0.32±0.16、绞股蓝组0.40±0.13、红曲低、中、高剂量组分别为0.40±0.13、0.32±0.12和0.33±0.12,模型组与各治疗组比较,无统计学意义(P=0.399);红曲高剂量组与低、中剂量组比较,无统计学意义(P=0.942);红曲高剂量组与绞股蓝组比较,无统计学意义(P=0.809)。7红曲对NAFLD大鼠肝脏PPARα的影响肝脏PPARαmRNA及蛋白质定性及半定量表达。大鼠PPARαmRNA:正常组1.13±0.01、绞股蓝组1.04±0.01、模型组0.56±0.01、红曲低、中、高剂量组分别为1.03±0.01、1.06±0.01和1.07±0.02,模型组与各治疗组比较,有统计学意义(P=0.000);红曲高剂量组与绞股蓝组比较,无统计学意义(P=0.073);红曲高剂量与低剂量组比较,有统计学意义(P=0.013);红曲高剂量与中剂量组比较,无统计学意义(P=0.275);大鼠PPARα蛋白质:正常组1.01±0.04、绞股蓝组1.42±0.04、模型组0.76±0.01、红曲低、中、高剂量组分别为1.36±0.02、1.42±0.06和1.43±0.07,模型组与各治疗组比较,有统计学意义(P=0.000);红曲高剂量组与绞股蓝组比较,无统计学意义(P=0.965);红曲高剂量与低剂量组比较,无统计学意义(P=0.276);红曲高剂量与中剂量组比较,无统计学意义(P=0.948);结论1连续8周高脂饮食成功建立了大鼠脂肪肝胰岛素抵抗模型。该模型的病理特征为肝细胞呈大泡性脂肪变性,伴小叶内炎症细胞浸润、一定程度的肝纤维化和线粒体结构的异常;代谢特征为胰岛素抵抗、高胰岛素血症、高脂血症、和血清转氨酶升高。该模型与人类肥胖所致NAFLD的病理和代谢改变极其相似,造模简单、模型成功率高,为研究NAFLD的发病机制,筛选有效的防治药物提供了良好的工具。2红曲能有效的拮抗大鼠NAFLD的进程,其作用机制为:①红曲能够改善NAFLD模型大鼠的一般情况:如改善大鼠模型的精神状况、耗食量、以及营养状况。②肝脏病理学观察表明红曲能改善肝脏脂肪变性,减少肝细胞坏死以及炎性细胞浸润。③红曲降低NAFLD模型大鼠血清ALT、AST含量,但无明显降低血甘油三脂的效应。提示红曲能有效减轻炎症反应,保护肝细胞,减轻脂肪性肝炎改变。④红曲降低NAFLD模型大鼠胰岛素抵抗及高胰岛素血症,增加胰岛素敏感性,从而阻断“初次打击”。⑤红曲降低NAFLD模型大鼠组织中TNF-α的表达,改善肠原性内毒素血症,从而抑制选择性胰岛素抵抗,阻断“二次打击”,抑制脂肪肝的发生。⑥红曲能升高NAFLD模型大鼠PPARα的mRNA及其蛋白质的表达,促进脂肪β氧化,减少脂肪肝毒性损害,发挥缓解脂肪肝的效应。综上所述,红曲可能通过改善胰岛素抵抗,抑制促炎因子TNF-α表达,发挥抗炎效应;诱导肝细胞PPARα的mRNA和蛋白水平增加,维持体内脂质代谢的动态平衡,发挥多环节、多靶点拮抗NAFLD的药理效应。更多还原

【Abstract】 BackgroundNon-alcoholic fatty liver disease(NAFLD) is the most frequent cause of abnormal liver function tests in China,with an estimated prevalence of 14-20%.It is caused by triglyceride(TG) accumulation within the liver and can either be a benign self-limiting state or a condition associated with steatohepatitis,which may progress to end-stage liver disease requiring liver transplantation.The most common disorder associated with NAFLD is genetic susceptibility,insulin resistance,intestinal endotoxemia,PPARα,an association which holds true in both lean and obese subjects. Insulin resistance is in turn a key element in the pathogenesis of NAFLD,However, the changes of IE and PPARαare "second-hit".Overexpressing lipoprotein lipase (LPL) in the liver in mice results in fatty liver and liver-specific insulin resistance.There is no similar title of NAFLD in traditional Chinese medicine.But there is similar manifestation of NAFLD in Internal Classic of Huang Di,such as’hepatic frllness’,`liver swelling’,’hypochondriac pain’,and so on.The disease located in the liver,which has a close relationship with gallbladder,spleen,stomach,kidney.The causes of NAFLD are overeating high-fat diet,excessive drinking,suffered from dampness-heat,imbalance of emotional,etc.These factors lead to Phlegm and Blood Stagnation in the liver.The effective components of Monascus are Lovastatin,which has an exact effect in anti-inflammatory and lipid-lowering.The studying team speculate that the Monascus maybe has an effect of anti-NAFLD.So,as a matter of fact,to discuss the mechanism of Monascus is an innovation in studying Chinese herb. Based on the theory of TCM and the research progress of modern medicine,We use high-fat diet to induce an experimental of NAFLD in rats.Each model is intervened by different dose of Monascus for eight weeks.the changes of blood lipid,hepatic biochemical,insulinre sistance,TNF-α、IL-6、IL-10、PPARαin rats are detected by HE stains,automatic biochemistry analyzer,RT-PCR、Western-blot、ELISA.To discuss the mechanism of Monascus is to improving the research level of TCM.ObjectiveTo observe the effect of monascus on insulin resistance,hepatic biochemical, blood lipid and the expression of TNF-α、IL-6、IL-10、PPARαin an experimental rat model of NAFLD induced by high-fat diet.The purpose is to discuss the pathogenic mechanism of NAFLD and the intervention effect of monascus.MethodAfter one week adaptive feed 48 SD rats were randomly divided into 6 groups with 8 rats in each group:(1)Normal group,(2)Blank group,(3)gynostemae group, (4)low-dose intervention group,(5)medium-dose intervention group,(6)high-dose intervention group.Group 1 was fed by common diet for 8 weeks,the other group were fed by high-fat diet for 8 weeks.Meanwhile,except normal group,the other group were treated by intragastric administration respectively using normal saline,gynostemae and different dose of Monascus once a day.In the ninth week,all the rats are sacrificed.The levels of blood lipid,hepatic biochemical,insulinre sistance,TNF-α、IL-6、IL-10、PPARαin rats are detected by HE stains,automatic biochemistry analyzer, RT-PCR、Western-blot、ELISA.Statistical analysis:All the data are represented as mean+SE;the statistical analysis was carried out using SPSS 11.5 software followed by One-way ANOVA-test and p-values less than 0.05 were considered as statistical significance.Result1.To study the influence of monascus on symptoms,physical signs in NAFLD rat model.The normal group expressed weigh increased,nose humid and clean,normal stool,no death in experiment.However,Rats in blank group displayed lassitude,no appetite,grew slowly,fur withered,retarded,and so on.Compared with blank group,there is an improvement in the expression of behavior,weight and nutrition in each intervention group(P=0.487).Especial in high-dosage monascus.2.The influence ofmonascus on liver morphology in NAFLD rat modelAfter the animal experiment gross manifestations such as the changes of liver histomorphology,the successful rate of NAFLD is 100 percent.The liver lobular is characterized by fatty degeneration,monocytes infiltration,lymphocyte infiltration and spotty necrosis in blank group.However,there is little fatty degeneration and a few inflammatory cells infiltration in gynostemae group and high-dose Monascus group.The changes in medium-dose and low-dose Monascus groups lie between the blank group and the high-dose group.3.The effects of monascus on serum hepatic function and lipid in NAFLD rat modelThe blank group shown an high levels of ALT and AST from the changes in serum hepatic function.This means that there is a pathology manifestation of hepatic cells apoptosis in NAFLD.After treatment with monascus or gynostemae,the levels of ALT and AST are on the decline.Compared with the intervention group,there is a significant difference in the levels of ALT,AST(P=0.000).There is no significant difference between Monascus and gynostemae group(P=0.337).There is an obvious lipid disorder in the blank group.Such changes are characterized by a rise of TG or LDL and fall of HDL.Compared with normal groups, there is a significant difference in blank groups(P=0.001).After treatment with monascus the changes of blood lipid are not obvious.There is no significant difference between Monascus group and Blank group(P=0.256).There is a significant difference in the expression of CHOL and HDL-C between high-dosage monascus group and gynostemae group(P=0.013,P=0.006).4.The influence of monascus on liver fibrosis in NAFLD rat modelFrom the changes of laboratory examination of liver cirrhosis,We can see clearly that the expression of LN,HA,PCⅢ、CⅣare on the increased in blank group.Except for low-dosage monascusmonasucs,there are no significant changes of above indexes in the other intervention group.There is no significant difference between blank group and high-dosage、medium-dosage、low-dosage monascusgroup and gynostemae(P=0.057).The monasucs has no effect on NAFLD.5.The effects of monascus on insulin resistance in NAFLD rat modelThe difference in the changes of blood sugar is great before the experiment began.The BS in blank is higher than that in normal group(P=0.030).This means there is a dysmetabloism of blood sugar in NAFLD rat models.Sixty minutes after the experiment,the changes of blood sugar show a homeostasis(5.3mmol/l),which means the formation of glucose clamp,is success.The changes of BS are still in an state of equilibrium.There is an similar change of blood sugar in high-fat diet group(including blank group and intervention group) and normal group.After the the glucose clamp experiment,we found that the GIR in high-fatty diet group is lower than that in the ordinary diet group.This means there is an insulin resistance in high-fat diet group. After treatments with monascus,the levels of insulin are on the decline.The monascus has an exact effect of decrease insulin.6.The influence of monascus on intestinal endotoxemia in NAFLD rat model Judgement from the changes in intestinal endotoxemia,The levels in the TNF-α,IL-6 and IL-10 are as follows.In blank group,the expression of above indexes are respectively 50.05±3.74,34.35±2.36 and 20.20±5.79;In the gynostemae group,the levels of above indexes are respectively 34.99±7.99,30.79±6.50 and 20.61±7.44;In the Monascus intervention group,the expression of above indexes are respectively 49.40±6.61、34.89±7.27 and 27.65±4.28;44.90±7.27,30.75±1.81 and 25.18±9.82; 35.68±7.29、28.61±9.51 and 24.00±11.15.Compared with blank group,the difference is obvious(P=0.000) in the changes of TNF-αin different intervention group.There is no significant difference between the blank group and other intervention group in the changes of IL-6 and IL-10(P=0.879,P=0.326).The expression of TNF-α,IL-6 and IL-10 mRNA in the liver are as follows.In normal group,the expression of above indexes are 0.51±0.50、0.29±0.16 and 0.55±0.07;In blank group are 1.21±0.55、0.53±0.18 and 0.32±0.16;In gynostema group are 0.54±0.24,0.41±0.22 and 0.40±0.13;In different monascus group are 0.63±0.28、0.65±0.50 and 0.73±0.39;0.29±0.27、0.50±0.18 and 0.50±0.14; 0.32±0.12,0.33±0.12 and 0.47±0.23.There is no significant difference between blank group and other groups in the changes of TNF-α,IL-6 and IL-10 mRNA(P=0.143, P=0.130,P=0.399).7.The influence of monascus on PPAR-alpha in NAFLD rat modelFrom the qualitative and quantitative expression of PPARαand protein in the liver,we can see clearly the sign that the PPARαmRNA in each group is 1.13±0.01、0.56±0.01、1.04±0.01、1.03±0.01、1.06±0.02、1.07±0.02 respectively.The PPARαprotein is 1.01±0.04、0.76±0.02、1.42±0.04、1.36±0.02、1.42±0.06、1.43±0.07.There is a significant difference between blank group and other groups(P=0.000).But there is no obvious difference between monasucs and gynostemae group(P=0.965).Conclusion1.NAFLD rat model was induced for eight weeks high-fatty diet.This model is similar to human NAFLD.The pathology was characterized by hyperlipidemia and hepatocytic macrovesicular steatosis,lobular inflammatory cell infiltration and necrosis.The metabolism is characterized by insulin-resistance,hyperinsulinemia, hyperlipoidemia and high levels in serum transaminase.This model is easy to establish,which provide a way to study the mechanism of NAFLD and screen a medicine on treating NAFLD. 2.The monascus maybe exert an effect on treating NAFLD.The mechanisms are as follows.①The monascus can improve the common condition in NAFLD rat model:For example,improving mentally condition,the consumption of food,nutrition.②The monascus can improve liver adipose degeneration and reduce the hepatic cells apoptosis and inflammatory cell infiltration according to the pathology manifestation of liver.③The monascus can decline the levels of serum ALT and AST in NAFLD rat model.The monascus restrain the changes of NASH by alleviate the inflammatory reaction.However,the monascus has no obvious effect on regulating the changes of blood lipid.④The monascus can block the "first attack"by declining the insulin resistance and hyperinsulinemia and increasing the sensitivity of insulin.⑤The monasucs can improve IE and alleviate selectivity IR by decreasing the levels of TNF-αin NAFLD rat model.So,as a matter of fact;the monascus can block the onset of NAFLD.⑥The monascus can exert an effect on alleviating fatty liver by up-regulating the expression of PPARαmRNA and protein in NAFLD rat model.Furthermore,the monascus can accelerate adiposeβ-oxidation in order to protect liver.ALL in all,The monascus may exert an exact effect on alleviating NAFLD by improving insulin r(?)sistance,inhibiting the expression of TNF-αas well as inducing the levels of mRNA and proteins of PPARαand maintaining the balance of lipid metabolism.The high-dose of monascus worth to be recommended.更多还原