【作者】 苗宇船；

【导师】 韩德五；

【作者基本信息】 山西医科大学 ， 生理学， 2008， 博士

【摘要】 非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)是指肝组织病理学改变与酒精性肝病相类似,但无过量饮酒史的临床病理综合征,包括单纯性脂肪肝、脂肪性肝炎和脂肪性肝纤维化和肝硬化三种病理类型。NAFLD由于发病率逐年攀升而起病渐趋低龄化,已日益成为医学界关注和研究的热点。然而迄今为止它的确切发病机制尚未完全阐明。目前公认的理论是“二次打击学说”,认为胰岛素抵抗(insulin resistance ,IR)参与肝脏形成单纯性脂肪肝的第一次打击,并通过氧化应激及脂质过氧化,对肝脏进行第二次打击,导致脂肪性肝炎的发生并进一步发展。目前已有足够的证据表明肠源性内毒素血症(intestinal endotoxemia,IETM)在NAFLD的发生发展中起着非常重要的作用。为了探讨IETM与IR在NAFLD的发生发展中的机制以及二者之间的关系,我们进行了以下实验。实验共分三个部分:第一部分,肠源性内毒素血症合并胰岛素抵抗的非酒精性脂肪肝大鼠模型的建立。实验一、实验二采用含20%玉米油的饲料喂养大鼠。从8周末开始大鼠的体重、肝指数、肝组织TG和Tch、血浆ALT、ET已经有明显升高;12周末开始出现HA的增高;14周末胰岛素抵抗指数明显升高。结合形态学观察得出结论:模型大鼠8周末至10周末形成了单纯性脂肪肝,12周末至14周末发展为脂肪性肝炎,16周末发展为脂肪性肝纤维化。14周末形成IETM合并IR的非酒精性脂肪肝大鼠模型。而以甘氨酸干预后各项指标均比16周组减轻,提示随着IETM的减轻,IR亦随之改善,NAFLD得到了有效控制,在NAFLD的发生发展中IETM与IR之间有密切关系。第二部分,脂多糖对脂肪细胞及肝细胞胰岛素抵抗的影响。实验三以油红染色证实以特异性诱导剂8天后3T3-L1脂肪前体细胞诱导分化为成熟的脂肪细胞;观察培养液的糖、胰岛素和胰岛素抵抗指数水平,葡萄糖摄取以及IRS-1mRNA与PPARγmRNA的表达,结果提示以高浓度TNFα(20ng/ml)诱导培养24h,可成功复制出胰岛素抵抗模型;在此基础上,以具有抑制内毒素血症的甘氨酸干预,并以胰岛素增敏剂罗格列酮作为阳性对照,结果发现脂肪细胞的IR得到了有效控制,进一步证实了实验二动物实验的结果,即IETM与IR之间有密切关系,并可能在NAFLD的发生发展中起重要作用。而且,Gly对NAFLD的防治作用,很可能是通过拮抗内毒素血症而控制脂肪组织的IR来发挥作用的。实验四运用脂多糖处理后的脂肪细胞糖、胰岛素和胰岛素抵抗指数水平均有不同程度的增高,两种状态下的葡萄糖摄取和IRS-1mRNA与PPARγmRNA表达的相对含量均有不同程度的降低,其中少量多次LPS组的胰岛素抵抗结果更接近于TNFα组。结果提示,少量多次给予LPS模拟了体内IETM的作用形式,刺激脂肪细胞可以产生类似高浓度TNFα的效果,诱导脂肪细胞发生IR。实验五、六培养HepG2细胞,观察各组培养液的糖、胰岛素和胰岛素抵抗指数水平,葡萄糖摄取、糖原含量,PAS染色观察糖原颗粒,结果提示以高浓度(5×10-7mol/L)的胰岛素作用于HepG2细胞24h后可成功复制胰岛素抵抗模型,胰岛素抵抗状态从24h开始持续到48h;以甘氨酸干预发现HepG2细胞的IR得到了一定的控制,进一步反证了第一、二部分实验的结果,即IETM与IR之间有密切关系,并可能在NAFLD的发生发展中起重要作用;当模拟体内IETM形成的特点,少量多次给予LPS和与实验三诱导脂肪细胞IR同等剂量的TNFα直接作用于肝细胞,对HepG2细胞IR的发生作用并不明显,而当以上刺激作用于同时给予高胰岛素刺激产生IR的HepG2细胞时,细胞发生的IR比单纯高胰岛素刺激更为强烈,提示在NAFLD的发生发展中,IETM可能与IR互为因果并形成恶性循环,对肝细胞IR有促进作用。第三部分肠源性内毒素血症与胰岛素抵抗在非酒精性脂肪性肝病发病中关系的研究。实验七、八,以高不饱和脂肪酸、高饱和脂肪酸及高糖高脂饮食分别喂养大鼠9周,观察各模型组大鼠各周体重曲线、肝指数、血清与肝组织匀浆中TG、血浆FFA含量、ALT活性、空腹血糖、FINS和FIRI、组织学检查、肝脂变结果、炎症活动度计分、苏丹IV染色并进行超微结构观察,结果表明4周末随机抽查活检的三组模型组大鼠肝脏已呈明显的气球样变,轻度脂肪变;9周末大鼠分别形成了单纯性脂肪肝及脂肪性肝炎,同时合并肠源性内毒素血症与胰岛素抵抗。各模型组共同特点是腹部脂肪丰富,脂肪体积肥大,周围巨噬细胞浸润,脂肪细胞TNFα强阳性表达且以上表现随NAFLD的发展依次加强。实验提示三种膳食均可建立伴有IEETM、IR的NAFLD的动物模型,不同的内毒素水平影响了IR并进一步影响NAFLD的严重程度。腹部脂肪及其间质中以巨噬细胞为主的炎症细胞浸润是NAFLD发病中TNFα的起始来源。综上得出结论:在NAFLD的发生发展过程中,IR做为NAFLD的始动环节,IETM则是不可或缺的触发因子,二者以TNFα为纽带,并互相作用、互为因果,伴随NAFLD发生发展的全过程。通过上述实验,得出以下结论:1、高不饱和脂肪酸、高饱和脂肪酸与高糖高脂膳食均可建立伴有肠源性内毒素血症、胰岛素抵抗的非酒精性脂肪性肝病的动物模型。2、脂多糖可在体外培养的3T3-L1脂肪细胞与HepG2肝细胞中诱发胰岛素抵抗。3、脂多糖可诱导巨噬细胞(包括脂肪细胞)释放TNFα等细胞因子,使之不但引起胰岛素抵抗,同时加剧氧应激。故脂多糖是非酒精性脂肪性肝病的触发因子。4、本研究证实甘氨酸可以通过拮抗内毒素控制脂肪肝的发生发展,为非酒精性脂肪性肝病的预防和治疗提供了新的思路。更多还原

【Abstract】 Nonalcoholic fatty liver disease (NAFLD) is the clinic-pathology syndrome that hepatic tissue pathological change is similar to alcoholic fatty liver disease without chronic alcohol abuse. It includes pure fatty liver, fatty hepatitis, fatty fibrosis and hepatic cirrhosis. NAFLD has become a hot topic in medical research due to its higher morbility and lower age.However, further studies is necessary on how and why it occurs. The publicly-accepted‘two hits’theory holds that insulin resistance (IR) involved in the‘first hit’during the formation of pure fatty liver; and the‘second hit’through oxidative stress and lipid peroxidation, which further worsen fatty hepatitis.Now sufficient evidences indicate intestinal endotoxemia (IETM) plays a key role during the occurrence and development of NAFLD. Thus, the following experiments were carried out so as to investigate the mechanism and relationship between IETM and IR during the course of NAFLD.The experiments were divided into three parts:PartⅠInducing NAFLD rat model complicated with IETM and IRRats were fed with special stuff containing 20% corn oil in Experiment 1, 2. The body weight, liver index, liver tissue Tch and TG, blood plasma ALT and ET increased obviously at the end of the 8th week. HA started to elevate at the end of 12th week. FIRI rised obviously at the end of the 14th week. The conclusion combined with observation of histomorphology show that pure fatty liver was induced from the end of the 8th week to the end of the 10th week, and developed into fatty hepatitis from the end of the 12th week to the end of the 14th week and formed fatty fibrosis at the end of the 16th week. NAFLD rat model combining with IETM and IR was induced at the end of the 14th week. But each indicator in the group intervened with GLY were lower than those in the 16 week group. It indicated that NAFLD was alleviated obviously along with relief of IETM and IR. IETM and IR are closely related with each other during the course of NAFLD.PartⅡEffect of LPS on IR of adipose cell and liver cellThe 3T3-L1 adipose precursor cell differentiated into ripe adipose cell induced by specificity inductor after 8 days, which had been proved by oil red stained in experiment 3. Sugar, insulin and IRI in medium and expression of IRS-1mRNA and PPARγmRNA were observed. It indicated that cell IR model may be induced successfully by high concentration of TNFα(20ng/ml)for 24 hours. IR of adipose cell was controlled obviously by Gly compared with euglycemic agent rosiglitazone. This further provided evidence to the result of experiment 2. In other words, there was close relationship between IETM and IR, and they may play significant role in the occurrence and development of NAFLD. Then preventive and therapeutic effect of Gly on NAFLD may be realized by IETM antagonisis to control IR of fatty tissue through rivaliry.Adipose cell sugar, insulin and IRI incresed after being treated by LPS. Glucose uptake in two conditions and expression of IRS-1mRNA and PPARγmRNA lowered in experiment 4. The result of small dosage and frequent LPS fed group was similar to the TNFαgroup. It indicate small dosage and frequent LPS fed may imitate the pattern of IETM in vivo and excite adipose cell to create the similar enviroment of high concentration TNFαwhich can induce adipose cell to form IR.The content of sugar, insulin, IRI, glycogen, glycogen particle dyed with PAS and glucose uptake of medium in every group in experiment 5,6 were observed. The result indicated that IR model was copied successfully with high concentration insulin(5×10-7mol/L)on HepG2 cell for 24 hours, it continued to 48 hours. IR of HepG2 cell intervend with Gly was controlled, which confirmed the results in PartⅠand PartⅡ, that is there was close relationship between IETM and IR and they played important role during development of NAFLD. IR of HepG2 cell was not obvious in small dosage and frequent LPS administration group and the group administrated with equal amount TNFαin experiment 3. IR was obvious when administrated with high concentration insulin simultaneously. It indicated that IETM and IR affectd each other in a vicious circle and promoted IR of liver cell during the development of NAFLD.PartⅢStudy relation of IETM and IR during the development of NAFLDRats were fed with a great quantity of unsaturated fat acid, saturated fat acid and high glucose and fat diet for 9 weeks, and were observed BW plot, liver index, TG in blood serum and hepatic tissue homogenate, plasma FFA, ALT, FBS,FINS and FIRI, histology observation, liver steatosis comparison, inflammation activity counts comparison, dyed with sudanⅣultramicrostructurely. The result indicated that rat liver has become ballooning degenerationat the end of the 4th week, then became pure fatty liver and fatty hepatitis complicated with IETM and IR at the end of the 9th week. Abundant abdomen fat, large fat area, surrounding macrophage infiltrating, strong positive expression of TNFαin adipose cell were proceeded with the development of NAFLD. It indicated that original resource of TNFαwas abdomen fat and inflammation cell during the development of NAFLD.The study indicated that: IR is a beginning factor, and IETM is a indispensible trigger factor, the two accompany during the course of NAFLD with TNFαas a connection.The main conclusions are as follows:1. NAFLD animal model complicated with IETM and IR can be induced by unsaturated fat acid diet, saturated fat acid or high glucose and fat diet.2. IR can be induced by LPS in 3T3-L1 lipocyte and HepG2 hepatocyte in vitro culture.3. The cytokine such as TNFαcan be released by macrophagus (including lipocyte) induced by LPS , which can cause IR and aggravate oxidative stress simultaneously. So, we consider that LPS is the trigger factor to NAFLD.4. Gly is proved to be the effective antagonists for endotoxin in this study which provided something new for the prevention and therapy of NAFLD.更多还原