利福平诱导小鼠脂肪肝及部分作用机理

发布时间：2018-01-02 09:50

本文关键词：利福平诱导小鼠脂肪肝及部分作用机理　出处：《安徽医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：背景和目的利福平是临床广泛应用的抗结核药物,众所周知利福平具有肝毒性。以往临床关注的是利福平对肝细胞的损伤,而利福平对肝脏脂质代谢的影响及其机制鲜有报道。本课题拟在建立利福平诱导小鼠脂肪肝实验模型的基础上,观察利福平引起的小鼠肝脏脂肪变及其动态演变过程,并进一步探讨其可能的发生机制。方法为观察利福平引起的小鼠肝脂肪变,将42只健康雄性成年小鼠随机分成7组,分别为对照组,利福平处理8小时(8 h)组、24小时(24 h)组、3天(3 d)组、1周(1 wk)组、2周(2 wk)组、4周(4 wk)组,其中8 h组、24 h组于清晨9点钟灌胃给予利福平200 mg/kg一次后于对应时点剖杀取材;3 d组、1 wk组、2 wk组、4 wk组均于每日清晨9点钟连续经口灌胃给予利福平200 mg/kg,后于相应时点剖杀取材。所有小鼠剖杀前均禁食6小时。采集小鼠血及肝脏组织,称取小鼠肝脏重量。计算小鼠肝脏系数;分离血清检测小鼠血清丙氨酸氨基转移酶(ALT)的水平;HE染色法观察小鼠肝脏组织病理学改变;检测小鼠血清中胆固醇酯(TCH),甘油三酯(TG),低密度脂蛋白(VLDL),高密度脂蛋白(HDL)水平;检测肝脏组织中TG相对含量及总含量;用油红O染色法检测小鼠肝脏组织中肝脏脂质沉积情况。选取对照组、利福平处理3 d组、1 wk组、4 wk组,用RT-q PCR方法检测肝细胞中参与肝脏脂质从头合成的酶及调节因子(Acc、Fas及Scd-1),脂肪酸氧化相关酶(Cpt-1α、Cyp4a10和Cyp4a14),脂肪酸摄取相关酶及调节因子(Cd36、Fatp、L-fabp)和Pparγm RNA水平;用蛋白质免疫印迹技术检测小鼠肝细胞中SREBP-1c、LXR-α、PXR、PPARγ核蛋白及PPARγ总蛋白表达水平。结果与对照组相比,利福平处理各组小鼠肝脏重量、肝脏系数从24 h组开始均呈逐渐升高趋势,于4 wk组达峰值。利福平处理24 h,3 d及1 wk组ALT水平较对照组升高;而利福平处理4 wk组较其显著升高。HE染色结果显示,利福平处理8 h、24 h组肝脏组织与对照组未见明显变化;利福平处理3 d组可见较多小的圆形空泡;而从1 wk组开始,小鼠肝组织可观察到明显增多的大的圆形空泡。利福平处理8 h,24 h组小鼠血清TG,VLDL水平较对照组升高,3 d组时显著升高,具有统计学意义,而1 wk组开始则较对照组呈显著下降趋势。与对照组相比利福平处理各组小鼠血清中TCH,HDL则呈逐渐降低趋势。利福平处理小鼠各组织TG含量则从8 h组即升高,1 wk时最明显。肝脏组织油红O染色显示,利福平处理8 h、24 h组肝组织可见少量圆形脂滴;3 d组开始出现显著增加的大的圆形脂滴。用RT-q PCR技术检测发现,利福平处理3 d、1 wk、4 wk组与脂肪酸从头合成有关的酶Acc、Fas、Scd-1 m RNA表达水平较对照组均明显上调;进一步检测SREBP-1c核蛋白表达水平,利福平处理各组SREBP-1c核蛋白表达水平与对照组相比无明显变化,另一上游调节因子LXR-α核蛋白表达水平较对照组也无显著变化;用RT-q PCR技术检测各组与脂肪酸氧化相关酶Cpt-1α、Cyp4a10及Cyp4a14 m RNA表达水平,利福平处理各组Cpt-1α、Cyp4a10及Cyp4a14 m RNA呈逐渐上调趋势;用RT-q PCR技术检测各组与脂肪酸摄取有关酶Cd36、Fatp及L-Ffabp m RNA表达水平,利福平处理各组Cd36、Fatp m RNA表达水平较对照组是上调的,而L-Ffabp m RNA表达水平较对照组无明显变化;检测肝脏组织中PPARγm RNA、总蛋白及核蛋白表达水平,其表达较对照组均明显上调;进一步检测肝脏组织内PXR核蛋白水平及其下游目的基因Cyp3a11 m RNA表达水平,利福平处理各组PXR核蛋白表达水平均较对照组明显增多,Cyp3a11 m RNA表达水平较对照组明显上调。结论1.利福平能引起小鼠肝脏组织脂质沉积,且具有时间-效应关系;2.利福平通过非依赖SREBP-1c及LXR-α激活途径上调参与肝脏脂肪酸从头合成途径相关酶的表达进而促进脂质合成;3.在利福平诱导的小鼠肝脏脂质沉积过程中,由外周摄取进入肝细胞内游离脂肪酸的增多可能部分归因于利福平诱导的小鼠肝内PXR的激活及PPARγ的上调;4.利福平诱导的小鼠肝脂质沉积不依赖于脂肪酸氧化的减少。
[Abstract]:Background and objective: rifampin is anti tuberculosis drugs in clinical applications, as everyone knows rifampicin with liver toxicity. Previous clinical attention is rifampicin on liver cell damage, and the effect of rifampicin on hepatic lipid metabolism and its mechanism is rarely reported. This topic is based on the establishment of rifampicin induced experimental fatty liver model in mice, the evolution process of mice hepatic steatosis caused by rifampicin and dynamic observation, and further explore its possible mechanisms. Methods for observation of rifampicin induced hepatic steatosis, 42 healthy adult male mice were randomly divided into 7 groups, including control group, rifampicin treatment 8 hours (8 h) group, 24 hours (24 h) group, 3 days group (3 D), 1 weeks (1 wk) group, 2 weeks group (2 wk), 4 weeks (4 wk) group, 8 h group, 24 h group at 9 o'clock in the morning by intragastric administration of rifampicin 200 mg/kg once in the corresponding time were killed; 3 group D 1, w K group, 2 wk group, 4 wk group at 9 o'clock in the morning daily continuous oral intragastric administration of rifampicin 200 mg/kg, at the same time after killing animals. All mice were killed before the mice were fasted for 6 hours. Collection of blood and liver tissue, weighing the weight of liver in mice. Calculation of liver coefficient; serum alanine amino mouse serum transferase (ALT) level; to observe the histological change of the liver in mice with pathological HE staining; detection of serum cholesterol ester (TCH), triglyceride (TG), low density lipoprotein (VLDL), high density lipoprotein (HDL) levels in liver tissue; detection of TG relative content and total content; oil red O staining of liver lipid deposition in liver tissue of mice was detected. The control group, rifampin treated 3 D group, 1 wk group, 4 wk group, with the participation of de novo synthesis of enzymes and lipid regulating factor of RT-q PCR method in the detection of liver cells (Acc, Fas and Scd-1), fat Fatty acid oxidation enzymes (Cpt-1 alpha, Cyp4a10 and Cyp4a14), fatty acid uptake related enzymes and regulatory factors (Cd36, Fatp, L-fabp) and Ppar m RNA gamma level; SREBP-1c detection of mouse liver cell protein immunoblotting technique in LXR- alpha, PXR, PPAR protein and PPAR expression of gamma gamma nuclear total egg white level. Results compared with the control group, rifampicin treatment of mice liver weight, liver coefficient from 24 h group was significantly increased in wk group, 4 reached the peak. Rifampicin treatment for 24 h, 3 D and 1 wk group ALT level was higher than the control group; and 4 wk group compared with the treatment of rifampicin significantly increased.HE the staining results showed that rifampicin treatment for 8 h, and the control group had no obvious change in liver tissue of 24 h group; 3 rifampin treated group D showed many small round vacuoles; and from the 1 wk group, liver tissue of mice can be observed in large round vacuoles increased significantly. Rifampin treatment for 8 h, 24 h mice blood The TG and VLDL levels increased compared with the control group, 3 D group increased significantly, with statistical significance, while the 1 wk group compared with the control group decreased significantly compared with the control group. The serum TCH rifampin treated mice, HDL was gradually decreased. The content of TG fabric rifampin treated mice from 8 the H group is increased 1 wk. The most obvious liver oil red O staining showed that the rifampin treated 8 h liver tissue 24 h group showed a small circular lipid droplets; 3 D group began to appear round large lipid droplets increased significantly. By detection of RT-q PCR technology, rifampicin treatment for 3 D, 1 wk. 4 wk group and fatty acid biosynthesis related enzymes Acc, Fas, Scd-1 m expression level of RNA than in the control group were significantly increased; further detection of SREBP-1c nuclear protein expression level, there was no significant change in the treatment groups of rifampicin SREBP-1c nuclear protein expression levels compared with the control group, another upstream regulator of L Expression of XR- protein levels compared with the control group had no significant change; using RT-q PCR technology to detect fatty acid oxidation related enzymes and Cpt-1 alpha, Cyp4a10 and Cyp4a14 m expression levels of RNA were rifampin treated Cpt-1 alpha, Cyp4a10 Cyp4a14 and m RNA was gradually up-regulated; using RT-q PCR technique to detect fatty acid uptake and about Cd36 Fatp and L-Ffabp m enzyme, the expression level of RNA, rifampin treated Cd36 were Fatp m RNA expression levels than the control group was up-regulated, and the expression level of RNA L-Ffabp m compared with the control group, no significant changes in liver tissue; detection of PPAR gamma m RNA, the expression level of total protein and nuclear protein, its expression compared to the control group to investigate the liver; further detection of PXR nuclear protein levels and its downstream gene Cyp3a11 m expression levels of RNA were rifampin treated PXR nuclear protein expression levels were increased significantly compared with the control group, the expression of Cyp3a11 m RNA The level increased significantly compared with the control group. Conclusion 1. rifampicin can cause hepatic lipid deposition, and has a time effect relationship; 2. rifampicin expression through non dependent SREBP-1c and LXR- alpha activation pathway is involved in the upregulation of hepatic fatty acid biosynthesis pathway enzymes and promote lipid synthesis; 3. in mouse liver lipid deposition induced by rifampicin in the peripheral uptake into hepatocytes in free fatty acids increased and may activate PPAR gamma in mouse liver partly induced by rifampicin PXR increases; reduce the liver lipid deposition induced by rifampicin 4. does not depend on the fatty acid oxidation.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575.5

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