GLUT4和UCP2在非酒精性脂肪性肝病中的作用以及小檗碱对GLUT4和UCP2表达的影响

发布时间：2018-09-11 07:05

【摘要】：目的：经济的迅速发展给人们的饮食及生活习惯带来了巨大转变，非酒精性脂肪性肝病（NAFLD）患者随之增加。研究发现胰岛素抵抗、代谢综合征、氧化应激、凋亡易感性增加、脂质代谢异常等参与了NAFLD的发病，但仍不能完全解释其发病机制。解偶联蛋白2（UCP2）及葡萄糖转运蛋白4(GLUT4)与糖类和脂质代谢、氧化应激、胰岛素抵抗及能量代谢密切相关，但在NAFLD发病中的作用尚缺乏进一步研究。NAFLD的治疗目前尚无特效药物，大部分临床药物在治疗同时存在一定副作用，疗效也无法肯定。小檗碱（BBR）是一种低毒副作用、物美价廉的药物，能够降糖调脂、改善胰岛素抵抗，因此是否可以用于NAFLD的治疗？本研究旨在探讨UCP2、GLUT4在NAFLD发病中作用，以及小檗碱对NAFLD的疗效和作用机制，从而为NAFLD发病机制提供新的理论依据，为NAFLD临床治疗提供新方法。方法：雄性SD大鼠56只，常规饲养1周，随机分为正常组(24只，基础喂养)和模型组(32只，高脂喂养)。第5、9周末分别随机处死两组实验动物各8只，并将剩余模型组随机分为两组（模型组8只，干预组8只），干预组予以盐酸小檗碱灌胃，模型组及剩余正常组用等量的蒸馏水灌胃，干预4周后处死全部大鼠。各阶段各组处死大鼠均经下腔静脉采血，测定肝功(AST、ALT）、血脂（TC、TG）、血糖代谢指标（FBG、FINS），并计算HOMA-IR。此外，采集部分肝组织制成石蜡切片后行HE染色，光镜下观察病理学改变，并经免疫组化法观察UCP2、GLUT4蛋白表达情况；部分肝组织经液氮冻存后于-70℃下保存，用于RT-PCR检测GLUT4mRNA表达。结果：1.模型组血AST、ALT、TC、TG、FBG、FINS、HOMA-IR较正常组升高，除第5周两组间AST、ALT无统计学差异性(P＞0.05)，其余各阶段两组间的差异性均有统计学意义(P＜0.01)。2.随高脂饮食时间的延长，模型组血AST、ALT、TC、TG、FBG、FINS、HOMA-IR呈升高趋势(P＜0.05)。3.干预组较同期模型组血AST、ALT、TC、TG、FBG、FINS、HOMA-IR降低(P＜0.01)，但高于正常组(P＜0.01)。4.模型组较正常组肝组织GLUT4表达降低(P＜0.01)，UCP2表达增加(P＜0.01)。5.随高脂饮食时间的延长，模型组肝组织GLUT4表达逐步下调(P＜0.01)，UCP2表达逐步上调(P＜0.01)。6.干预组较同期模型组肝组织GLUT4表达增加(P＜0.01)，UCP2表达下降(P＜0.01)，但没有恢复正常(P＜0.01)。结论：1.高脂饮食喂养一定时间后可以成功建立大鼠NAFLD模型，模型大鼠可见到明显肝细胞脂肪变、脂肪性肝炎、胰岛素抵抗、高胰岛素血症等特征；2.随造模周期的延长，GLUT4mRNA及蛋白表达明显降低，UCP2蛋白表达则明显升高，，大鼠表现出明显的脂质代谢紊乱、胰岛素抵抗，肝组织可见明显脂肪变、炎变、坏死。因此，GLUT4和UCP2可能通过参与胰岛素抵抗、脂质代谢、氧化应激、能量代谢等在NAFLD的发生发展中发挥作用；3.小檗碱可通过降糖、调节脂质代谢、改善胰岛素抵抗、减轻炎症反应等途径减轻肝脏病理病变程度及改善肝功能，发挥治疗NAFLD的作用；4.小檗碱可显著促进肝组织GLUT4mRNA及蛋白的表达，并明显降低肝组织UCP2蛋白的表达，这可能是其防治NAFLD的重要作用机制之一。
[Abstract]:Objective: Rapid economic development has brought about great changes in people's diet and living habits, and the number of patients with non-alcoholic fatty liver disease (NAFLD) has increased. Insulin resistance, metabolic syndrome, oxidative stress, increased susceptibility to apoptosis, abnormal lipid metabolism and so on are involved in the pathogenesis of NAFLD, but can not fully explain the pathogenesis. Uncoupling protein 2 (UCP2) and glucose transporter 4 (GLUT4) are closely related to glucose and lipid metabolism, oxidative stress, insulin resistance and energy metabolism, but their roles in the pathogenesis of NAFLD have not been further studied. It is uncertain. Berberine (BBR) is a kind of low toxic side effects, cheap drugs, can reduce blood sugar and lipid, improve insulin resistance, so whether it can be used in the treatment of NAFLD? This study aims to explore the role of UCP2, GLUT4 in the pathogenesis of NAFLD, and the efficacy and mechanism of berberine on NAFLD, so as to provide a new mechanism for the pathogenesis of NAFLD. Methods: Fifty-six male SD rats were randomly divided into normal group (24 rats, basic feeding) and model group (32 rats, high fat feeding). Eight rats in each group were killed at the end of the fifth and ninth week, and the remaining model groups were randomly divided into two groups (8 rats in model group and 8 rats in intervention group). All rats were sacrificed after 4 weeks of intervention. Blood samples were collected from the inferior vena cava, and liver function (AST, ALT), blood lipid (TC, TG), blood glucose metabolic index (FBG, FINS) were measured. HOMA-IR was also calculated. The expression of UCP2 and GLUT4 protein was observed by HE staining and immunohistochemical staining. Some liver tissues were cryopreserved in liquid nitrogen and stored at - 70 ~C for RT-PCR to detect the expression of GLUT4 mRNA. Results: 1. AST, ALT, TC, TG, FBG, FINS, HOMA-IR in the model group were higher than those in the normal group, except AST and ALT in the fifth week. There were significant differences between the two groups at the other stages (P > 0.05). 2. With the prolongation of high-fat diet, AST, ALT, TC, TG, FBG, FINS, HOMA-IR in the model group increased (P < 0.05). 3. Compared with the model group, AST, ALT, TC, TG, FBG, FINS and HOMA-IR in the intervention group decreased (P < 0.01), but higher than that in the normal group (P < 0.01). The expression of GLUT4 and UCP2 in liver tissue of model group decreased (P < 0.01) and increased (P < 0.01). 5. With the prolongation of high-fat diet, the expression of GLUT4 and UCP2 in liver tissue of model group decreased gradually (P < 0.01) and increased gradually (P < 0.01). CONCLUSIONS: 1. NAFLD model of rats can be established successfully after feeding high-fat diet for a certain period of time, the model rats can see obvious hepatocyte steatosis, steatohepatitis, insulin resistance, hyperinsulinemia and other characteristics; 2. With the prolongation of the modeling cycle, GLUT4 mRNA and protein expression decreased significantly, UCP2 protein surface. Therefore, GLUT4 and UCP2 may play an important role in the development of NAFLD by participating in insulin resistance, lipid metabolism, oxidative stress, energy metabolism and so on. 3. Berberine can reduce blood sugar and regulate lipid metabolism. Metabolism, improvement of insulin resistance, alleviation of inflammation, alleviation of liver pathological changes and improvement of liver function, play a role in the treatment of NAFLD; 4. Berberine can significantly promote the expression of GLUT4 mRNA and protein in liver tissue, and significantly reduce the expression of UCP2 protein in liver tissue, which may be one of the important mechanisms of its prevention and treatment of NAFLD.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.5

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