【作者】 李海丽；

【导师】 姜玲玲；

【作者基本信息】 河北医科大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 目的:外周组织转出的胆固醇主要经高密度脂蛋白(HDL)逆向转运至肝脏代谢。HDL前体是由肝细胞合成的,肝细胞中的ATP结合盒转运蛋白A1(ABCA1)表达增加时,HDL前体也随之增加。HDL前体入血后募集外周组织游离的胆固醇,并在血浆卵磷脂胆固醇脂酰转移酶(LCAT)作用下生成胆固醇酯,HDL成为成熟的HDL,成熟的HDL又被肝细胞膜上的HDL受体清道夫受体BI(SR-BI)识别结合后,将胆固醇转入肝细胞,发挥其特定的生物功能或转化成胆汁酸,随胆汁排出体外。胆固醇7α羟化酶(CYP7A1)是胆汁酸合成的限速酶,其活性的大小决定了胆汁酸合成的速度。研究报道,糖尿病时血总胆固醇升高,进入肝脏的胆固醇也随之增加,同时CYP7A1酶活性升高。那么,糖尿病肝脏胆固醇的摄入增多是否与肝脏ABCA1和SR-BI基因表达有关呢?糖尿病状态肝CYP7A1酶活性升高是不是因CYP7A1表达增加而引起的呢?未见报道。细胞内脂肪酸代谢主要由过氧化物酶体增殖物激活受体α(PPARα)调节,胆固醇代谢主要由肝X受体α(LXRα)调节。两个核受体对脂肪酸和胆固醇代谢调节存在交互作。糖尿病状态,脂肪动员加强,增高的游离脂肪酸可激活肝PPARα。那么,糖尿病状态PPARα被脂肪酸激活时,肝中LXRα是否也被激活?肝胆固醇相关基因的表达会不会随着LXRα的激活而改变呢?未见报道。非诺贝特是PPARα的人工合成配体,是临床常用的降脂药,它除了通过激活PPARα促进与脂肪酸分解代谢有关的酶基因表达,加强脂肪酸的分解达到降血脂的目的外,也有轻微的降血总胆固醇的作用。糖尿病时它是否通过激活PPARα进而激活LXRα,使其下游基因表达增加,从而调节胆固醇代谢,使血总胆固醇和肝胆固醇水平下降从而改善肝细胞的结构与功能?未见报道。为此,本研究一方面以链脲佐菌素(STZ)诱导的糖尿病大鼠为实验对象,通过肝组织形态学直接观察,和血清谷丙转氨酶(ALT)和谷草转氨酶(AST)的测定,确定糖尿病大鼠肝功能损伤的同时,观测糖尿病大鼠肝胆固醇含量;用RT-PCR方法,测定肝组织中PPARα、脂酰CoA氧化酶(ACOX1)以及LXRα、ABCA1、SR-BI、CYP7A1等与胆固醇代谢有关的基因表达情况;另一方面,观察PPARα人工合成的激活配体,降脂药-非诺贝特,对上述各项指标的影响,了解糖尿病肝胆固醇代谢情况,进一步探讨糖尿病肝脏脂代谢紊乱的分子机理,深化对糖尿病脂代谢紊乱的认识。方法:1动物分组及取材选用成年200-250g雄性清洁级Wistar大鼠,随机分为:正常对照组(CON)、糖尿病组(DM)、糖尿病+非诺贝特组(DM+F)、正常对照+非诺贝特组(CON+F)。正常大鼠一次性腹腔注射2% STZ诱导糖尿病发生。CON+F组和DM+F组在糖尿病大鼠模型建立后4周末,分别用生理盐水和非诺贝特连续两周给正常和STZ鼠灌胃(100mg/kg/day)。颈动脉采空腹血,血清用于血糖、血脂、血胆汁酸、血谷丙转氨酶(ALT)和谷草转氨酶(AST)的测定。肝脏用于组织切片、RNA及脂质提取。2光镜观察组织病理变化按常规方法将肝组织用甲醛固定、石蜡包埋、切片、HE染色后,光镜观察。3肝脏总脂提取参照文献[12]提取肝脏总脂,用于胆固醇测定。4血糖、血脂、血胆汁酸、血ALT、血AST、肝胆固醇的测定上述指标用Olympus Au2700型全自动生化分析仪测定。5肝组织有关基因mRNA相对表达量的测定用Trizol法提取总RNA。以GAPDH作为内参,用RT-PCR法测定PPARα、ACOX1、LXRα、ABCA1、SR-BI及CYP7A1 mRNA的相对表达量。结果:1正常对照和糖尿病组各项指标变化1.1血AST/ALT和肝组织形态结构变化:糖尿病组血AST/ALT明显下降(1.776±0.279 vs 2.261±0.445, p<0.01);糖尿病组肝细胞排列紊乱,放射状结构消失,血窦扩张。表明,糖尿病时肝细胞结构异常,肝功能受损。1.2血脂变化:糖尿病时,血总胆固醇(1.870±0.180mmol/L vs 1.359±0.197mmol/L, p<0.01)、血HDL-C(1.308±0.188mmol/L vs 1.011±0.209mmol/L, p<0.01 )、血胆汁酸(59.626±16.349μmol/L vs 9.334±2.011μmol/L, p<0.01)、血甘油三酯(1.346±0.393mmol/L vs 0.651±0.130mmol/L, p<0.01)都明显升高。表明,糖尿病时胆固醇和甘油三酯代谢异常。1.3肝胆固醇含量的变化:糖尿病组与正常对照组肝胆固醇含量区别( 32.974±5.526mmol/mg vs 31.977±4.782mmol/mg, p>0.05)无统计学意义。1.4肝基因表达结果1.4.1肝PPARα、ACOX1 mRNA相对表达量的改变:糖尿病组和正常组相比,PPARαmRNA相对表达量(0.921±0.123 vs 0.621±0.132, p<0.01 )以及ACOX1 mRNA相对表达量(0.801±0.128 vs 0.587±0.081, p<0.01)都明显升高。表明,糖尿病时,PPARα表达增加并且被激活。1.4.2肝LXRα、ABCA1、SR-BI、CYP7A1 mRNA相对表达量的改变:糖尿病组和正常组相比,LXRα(1.216±0.128 vs 0.702±0.097, p<0.01)、ABCA1(0.725±0.127 vs 0.177±0.042, p<0.01),SR-BI(0.672±0.105 vs 0.220±0.040, p<0.01)及CYP7A1(1.137±0.212 vs 0.697±0.141, p<0.01)的mRNA相对表达均明显升高。表明,糖尿病时肝PPARα表达增加的同时,调节胆固醇代谢的核受体LXRα的表达也协同增加。HDL前体形成增多,肝脏摄入HDL-C增加,胆汁酸生成增多,胆固醇的转运、吸收和转化增强。2糖尿病+非诺贝特组和正常对照+非诺贝特组各项指标变化2.1血AST/ALT和肝组织形态结构变化:糖尿病加药组与糖尿病组(1.772±0.193 vs 1.776±0.279, p>0.05)之间,正常加药组与正常组(2.687±0.512 vs2.261±0.445, p>0.05)之间大鼠血AST/ALT变化无统计学意义;糖尿病+非诺贝特组与糖尿病组相比以及正常对照+非诺贝特组与正常组相比,肝组织结构没有明显改变。表明,非诺贝特不改变大鼠肝组织结构和功能。2.2血脂变化:除糖尿病加药组大鼠血甘油三酯( 0.743±0.269mmol/L )明显低于糖尿病组(1.346±0.393mmol/L, p<0.01)外,两组之间血总胆固醇(1.903±0.312mmol/L vs 1.870±0.180mmol/L, p>0.05)、血HDL-C(1.332±0.249mmol/L vs 1.308±0.188mmol/L, p>0.05)、血胆汁酸(56.671±29.694μmol/L vs 59.626±16.349μmol/L, p>0.05)的变化均无统计学意义;正常加药组和正常组相比,血总胆固醇( 0.979±0.156mmol/L vs 1.359±0.197mmol/L, p<0.01 )、血HDL-C ( 0.566±0.111mmol/L vs 1.011±0.209mmol/L, p<0.01 )明显下降,血总胆汁酸(30.880±9.226μmol/L vs 9.334±2.011μmol/L, p<0.01)明显升高,血甘油三酯(0.757±0.108mmol/L vs 0.651±0.130mmol/L, p>0.05)变化无统计学意义。表明,非诺贝特降低正常鼠血总胆固醇,但不降低糖尿病鼠血总胆固醇;非诺贝特不降低正常鼠血甘油三酯,但降低糖尿病鼠血甘油三酯;糖尿病鼠和正常鼠的脂代谢受非诺贝特的影响不同。2.3肝胆固醇含量的变化:糖尿病加药组肝胆固醇含量(26.167±2.937mmol/mg)明显低于糖尿病组(32.974±5.526 mmol/mg, p<0.01 );正常加药组肝胆固醇含量( 24.417±1.429mmol/mg )明显低于正常组(31.977±4.782mmol/mg, p<0.01)。表明,非诺贝特能降低大鼠肝胆固醇水平。2.4肝基因表达结果2.4.1肝PPARα,ACOX1 mRNA相对表达量的改变:糖尿病加药组与糖尿病组(0.784±0.138 vs 0.921±0.123, p>0.05)以及正常加药组与正常组(0.651±0.133 vs 0.621±0.132, p>0.05)之间PPARαmRNA相对表达量变化均无统计学意义。糖尿病加药组ACOX1 mRNA相对表达量(1.253±0.125)明显高于糖尿病组(0.801±0.128, p<0.01),正常加药组ACOX1 mRNA相对表达量(1.273±0.194)明显高于正常组(0.587±0.081, p<0.01)。表明,非诺贝特激活了正常大鼠和糖尿病大鼠肝PPARα。2.4.2肝LXRα、ABCA1、SR-BI、CYP7A1 mRNA相对表达量的改变:糖尿病加药组与糖尿病组之间,LXRα(1.090±0.087 vs 1.216±0.128, p>0.05)、ABCA1(0.809±0.050 vs 0.725±0.127, p>0.05)和SR-BI(0.554±0.081 vs 0.672±0.105, p>0.05)的mRNA相对表达量变化均无统计学意义;糖尿病加药组CYP7A1 mRNA相对表达量(1.399±0.149)明显高于糖尿病组(1.137±0.212, p<0.01)。正常加药组和正常组相比,LXRα(0.944±0.189 vs 0.702±0.097, p<0.01)、ABCA1(0.807±0.131 vs 0.177±0.042, p<0.01)、SR-BI(0.540±0.140 vs 0.220±0.040, p<0.01)以及CYP7A1 (1.038±0.116 vs 0.697±0.141, p<0.01)的mRNA相对表达量均明显升高。表明,非诺贝特对糖尿病鼠和正常鼠基因表达的影响不同。结论:1糖尿病时,大鼠肝PPARα的激活伴随LXRα的激活。糖尿病肝脂肪酸代紊乱时,与胆固醇代谢有关的基因表达也出现异常。2非诺贝特对糖尿病鼠肝胆固醇代谢相关基因mRNA表达影响不同于正常鼠。在我们实验条件下,它虽然可以降低糖尿病鼠肝胆固醇水平,但不能降低糖尿病鼠血总胆固醇。更多还原

【Abstract】 Objective: The major function of HDL is to transfer cholesterol from peripheral tissues to the liver for biliary excretion. There is a major role for hepatocyte ATP binding cassette transporter A1 (ABCA1) in generating a critical pool of HDL precursor particles that enhance further HDL generation. HDL precursor particles are released to blood and recruit cholesterol from peripheral tissues. With the effect of lecithin cholesterol acyl transferase (LCAT), HDL precursor particles change into cholesterol esters and become mature HDL particles. The scavenger receptor class B type I (SR-BI) is the liver cell-surface HDL receptor and mediates selective HDL cholesterol uptake. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile acid biosynthesis. During diabetes course, with high blood cholesterol, more cholesterol was englobed to liver and enzyme activity of CYP7A1 become higher. Then whether the increasing of cholesterol to liver is related to the expression changes of ABCA1, SR-BI and whether the high activity of CYP7A1 is related to alter of the expression of CYP7A1 are not reported.Peroxisome proliferator activated receptorα(PPARα) is the main nuclear receptor which regulate fatty acid metabolism. Liver X receptorα(LXRα) is the main nuclear receptor which regulate cholesterol metabolism. There is an interaction between fatty acid metabolism and cholesterol metabolism. In diabetic state fat mobilization upgrade and fatty acid increase, then hepatic PPARαis activating. Whether the relative expression of LXRαand its target genes were changed accompany with PPARαactivating are not reported by present.The Fibrate group of drugs, used extensively as lipid-lowering agents, are pharmacological activators of PPARα. It can lower blood triglyceride by the way of activating some fatty acid catabolism genes, decomposing fatty acid, and it also has little effect on decreasing blood total cholesterol. In diabetic state if it can activate PPARαand affect some cholesterol metabolism gene expression to decrease the level of blood cholesterol and ameliorate the morphosis and function of hepatocyte are not reported by present.In present experiment, diabetes mellitus (DM) was induced by intraperitoneal injection with streptozocin (STZ). In the condition of injured liver structure and function, such as the decreasing of serum aspartate aminotransferase (AST)/ alanine aminotransferase (ALT) in DM rat, we observe the total cholesterol level in liver, detecte the expression of liver PPARα, ACOX1, LXRα, ABCA1, SR-BI and CYP7A1 genes by RT-PCR. We also observe the effect of Fenofibrate-the activating ligand of PPARαon the above indexes, in order to investigate the molecule mechanism of lipid metabolism in DM rat liver, deepening the recognition of lipid metabolic disorder in DM.Methods:1 Animals and materialsMale Wistar rat weighed 200-250g were divided randomly into: control group (CON), diabetes group (DM), diabetes+Fenofibrate (DM+F), control+Fenofibrate (CON+F). DM was induced by intraperitoneal injection with 2% STZ. Four weeks after DM was successfully induced, DM+F group and CON+F group were perfused with 100mg/kg/day of Fenofibrate for two weeks. CON group and DM group were perfused with 100mg/kg/day of isotonic Na chloride for two weeks. After abrosia for one night, blood was collected by carotid artery bloodletting, and the serum was used for the detection of serum glucose, lipids, bile acid, ALT, AST. The liver was used for histological section and extraction of RNA and lipids.2 Observation of the pathological changes by light microscopeThe liver tissue was routinely fixed by citromint, and prepared for light microscope observing.3 The extraction of lipids in rat hepatocyte Refer to reported methods .4 Detection of blood glucose, lipids, bile acid, ALT, AST, liver cholesterolWhat above-mentioned were detected by Olympus Au2700 automatic biochemistry analysator.5 Detection of the relative expression of related genes in the liverTotal RNA was extracted with Trizol method. The relative expression of PPARα, ACOX1, LXRα, ABCA1, SR-BI and CYP7A1 were evaluated by RT-PCR, with GAPDH as inner standard.Results:1 All results in control and diabetes group1.1 The changes of blood AST/ALT and liver morphous: DM group blood AST/ALT were singnificantly decreased (1.776±0.279 vs 2.261±0.445, p<0.01); hepatic cells structure confused, hepatic sinusoid broaden. The results showed that DM group hepatic structure and function were injured.1.2 The changes of blood lipids: In diabetic state, blood total cholesterol (1.870±0.180mmol/L vs 1.359±0.197mmol/L, p<0.01), blood HDL-C (1.308±0.188mmol/L vs 1.011±0.209mmol/L, p<0.01), blood bile acid (59.626±16.349μmol/L vs 9.334±2.011μmol/L, p<0.01), blood triglyceride (1.346±0.393mmol/L vs 0.651±0.130mmol/L, p<0.01) were singnificantly increased. The results showed that in diabetic state blood cholesterol and triglyceride metabolism abnormality.1.3 Cholesterol content in hepatic cells: DM group compared to CON group, (32.974±5.526mmol/mg vs 31.977±4.782mmol/mg, p>0.05) the changes of cholesterol content in liver were no statistical significance.1.4 The expression of liver genes 1.4.1 The relative expression of PPARαand ACOX1 mRNA . DM group compared to CON group, the relative expression of PPARα(0.921±0.123 vs 0.621±0.132, p<0.01) and ACOX1 (0.801±0.128 vs 0.587±0.081, p<0.01) were singnificantly increased. The results showed that in diabetic state PPARαwas activated and the expression of PPARαmRNA was increased.1.4.2 The relative expression of LXRα, ABCA1, SR-BI and CYP7A1 mRNA. DM group compared to CON group, the relative expression of LXRα(1.216±0.128 vs 0.702±0.097, p<0.01), ABCA1 (0.725±0.127 vs 0.177±0.042, p<0.01), SR-BI (0.672±0.105 vs 0.220±0.040, p<0.01) and CYP7A1 (1.137±0.212 vs 0.697±0.141, p<0.01) were singnificantly increased. The results showed that the expression of LXRαwas increased with the increasing of PPARαin liver of STZ-induced rats; HDL precursor particles formation, HDL-C intaking and bile acid synthesis were all increased in liver of STZ-induced rats.2 All results of Fenofibrate to DM group and CON group2.1 The changes of blood AST/ALT and liver morphous: DM+F group compared to DM group (1.772±0.193 vs 1.776±0.279, p>0.05) and CON+F group compared to CON group (2.687±0.512 vs 2.261±0.445, p>0.05) the changes of blood AST/ALT were no statistical significance; Hepatic cells structure had no singnificant change between CON+F group and CON group, DM+F group and DM group . The results showed that Fenofibrate didn’t alter the structure and function of liver. 2.2 The changes of blood lipids: DM+F group compared to DM group, the changes of blood total cholesterol (1.903±0.312mmol/L vs 1.870±0.180mmol/L, p>0.05), blood HDL-C (1.332±0.249mmol/L vs 1.308±0.188mmol/L, p>0.05), blood bile acid (56.671±29.694μmol/L vs 59.626±16.349μmol/L, p>0.05) were no statistical significance; Blood triglyceride (0.743±0.269mmol/L vs 1.346±0.393mmol/L, p<0.01) were singnificantly decreased. CON+F group compared to CON group, blood total cholesterol (0.979±0.156mmol/L vs 1.359±0.197mmol/L, p<0.01) and blood HDL-C (0.566±0.111mmol/L vs 1.011±0.209mmol/L, p<0.01) were singnificantly decreased; blood bile acid (30.880±9.226μmol/L vs 9.334±2.011μmol/L, p<0.01) was singnificantly increased; the changes of blood triglyceride (0.757±0.108mmol/L vs 0.651±0.130mmol/L, p>0.05) were no statistical significance. The results showed that Fenofibrate can decrease normal rats blood total cholesterol and diabetic rats blood triglyceride but not STZ-induced rats blood cholesterol. Fenofibrate has different regulation between normal and STZ-induced rats.2.3 Cholesterol content in hepatic cells: DM+F group compared to DM group (26.167±2.937mmol/mg vs 32.974±5.526mmol/mg, p<0.01), CON+F group compared to CON group (24.417±1.429mmol/mg vs 31.977±4.782mmol/mg, p<0.01), cholesterol content in hepatic cells were all decreased. The results showed that Fenofibrate can lower cholesterol level of liver. 2.4 The expression of liver genes2.4.1 The relative expression of PPARαand ACOX1 mRNA: DM+F group compared to DM group (0.784±0.138 vs 0.921±0.123, p>0.05), CON+F group compared to CON group (0.651±0.133 vs 0.621±0.132, p>0.05), the changes of relative expression of PPARαmRNA were no statistical significance. DM+F group compared to DM group (1.253±0.125 vs 0.801±0.128, p<0.01) and CON+F group compared to CON group (1.273±0.194 vs 0.587±0.081, p<0.01) the relative expression of ACOX1 mRNA were increased. The results showed that Fenofibrate activated PPARαof normal and STZ-induced rats liver.2.4.2 The relative expression of LXRα, ABCA1, SR-BI and CYP7A1 mRNA. DM+F group compared to DM group, the changes of LXRα(1.090±0.087 vs 1.216±0.128, p>0.05), ABCA1 (0.809±0.050 vs 0.725±0.127, p>0.05) and SR-BI (0.554±0.081 vs 0.672±0.105, p>0.05) were no statistical significance; CYP7A1 (1.399±0.149 vs 1.137±0.212, p<0.01) was increased. CON+F group compared to CON group, the relative expression of LXRα, (0.944±0.189 vs 0.702±0.097, p<0.01), ABCA1 (0.807±0.131 vs 0.177±0.042, p<0.01), SR-BI (0.540±0.140 vs 0.220±0.040,p<0.01) and CYP7A1(1.038±0.116 vs0.697±0.141, p<0.01) were all increasd. The results showed that there are some different effects of Fenofibrate on the expression of genes involved in cholesterol metabolism in liver between normal and STZ-induced rats. Conclusion:1 LXRαis activated with the activation of PPARαin liver of STZ-induced rats; disorder of fatty acid metabolism is accompanied with the abnormal expression of genes involved in cholesterol metabolism.2 There are some different effects of Fenofibrate on the expression of genes involved in cholesterol metabolism in liver between normal and STZ-induced rats. Under this experiment Fenofibrate can decreases liver cholesterol level, but not blood cholesterol level in STZ-induced rats.更多还原