【摘要】：目的：經(jīng)濟(jì)的迅速發(fā)展給人們的飲食及生活習(xí)慣帶來了巨大轉(zhuǎn)變，非酒精性脂肪性肝�。∟AFLD）患者隨之增加。研究發(fā)現(xiàn)胰島素抵抗、代謝綜合征、氧化應(yīng)激、凋亡易感性增加、脂質(zhì)代謝異常等參與了NAFLD的發(fā)病，但仍不能完全解釋其發(fā)病機(jī)制。解偶聯(lián)蛋白2（UCP2）及葡萄糖轉(zhuǎn)運(yùn)蛋白4(GLUT4)與糖類和脂質(zhì)代謝、氧化應(yīng)激、胰島素抵抗及能量代謝密切相關(guān)，但在NAFLD發(fā)病中的作用尚缺乏進(jìn)一步研究。NAFLD的治療目前尚無特效藥物，大部分臨床藥物在治療同時存在一定副作用，療效也無法肯定。小檗堿（BBR）是一種低毒副作用、物美價廉的藥物，能夠降糖調(diào)脂、改善胰島素抵抗，因此是否可以用于NAFLD的治療？本研究旨在探討UCP2、GLUT4在NAFLD發(fā)病中作用，以及小檗堿對NAFLD的療效和作用機(jī)制，從而為NAFLD發(fā)病機(jī)制提供新的理論依據(jù)，為NAFLD臨床治療提供新方法。方法：雄性SD大鼠56只，常規(guī)飼養(yǎng)1周，隨機(jī)分為正常組(24只，基礎(chǔ)喂養(yǎng))和模型組(32只，高脂喂養(yǎng))。第5、9周末分別隨機(jī)處死兩組實(shí)驗(yàn)動物各8只，并將剩余模型組隨機(jī)分為兩組（模型組8只，干預(yù)組8只），干預(yù)組予以鹽酸小檗堿灌胃，模型組及剩余正常組用等量的蒸餾水灌胃，干預(yù)4周后處死全部大鼠。各階段各組處死大鼠均經(jīng)下腔靜脈采血，測定肝功(AST、ALT）、血脂（TC、TG）、血糖代謝指標(biāo)（FBG、FINS），并計算HOMA-IR。此外，采集部分肝組織制成石蠟切片后行HE染色，光鏡下觀察病理學(xué)改變，并經(jīng)免疫組化法觀察UCP2、GLUT4蛋白表達(dá)情況；部分肝組織經(jīng)液氮凍存后于-70℃下保存，用于RT-PCR檢測GLUT4mRNA表達(dá)。結(jié)果：1.模型組血AST、ALT、TC、TG、FBG、FINS、HOMA-IR較正常組升高，除第5周兩組間AST、ALT無統(tǒng)計學(xué)差異性(P＞0.05)，其余各階段兩組間的差異性均有統(tǒng)計學(xué)意義(P＜0.01)。2.隨高脂飲食時間的延長，模型組血AST、ALT、TC、TG、FBG、FINS、HOMA-IR呈升高趨勢(P＜0.05)。3.干預(yù)組較同期模型組血AST、ALT、TC、TG、FBG、FINS、HOMA-IR降低(P＜0.01)，但高于正常組(P＜0.01)。4.模型組較正常組肝組織GLUT4表達(dá)降低(P＜0.01)，UCP2表達(dá)增加(P＜0.01)。5.隨高脂飲食時間的延長，模型組肝組織GLUT4表達(dá)逐步下調(diào)(P＜0.01)，UCP2表達(dá)逐步上調(diào)(P＜0.01)。6.干預(yù)組較同期模型組肝組織GLUT4表達(dá)增加(P＜0.01)，UCP2表達(dá)下降(P＜0.01)，但沒有恢復(fù)正常(P＜0.01)。結(jié)論：1.高脂飲食喂養(yǎng)一定時間后可以成功建立大鼠NAFLD模型，模型大鼠可見到明顯肝細(xì)胞脂肪變、脂肪性肝炎、胰島素抵抗、高胰島素血癥等特征；2.隨造模周期的延長，GLUT4mRNA及蛋白表達(dá)明顯降低，UCP2蛋白表達(dá)則明顯升高，，大鼠表現(xiàn)出明顯的脂質(zhì)代謝紊亂、胰島素抵抗，肝組織可見明顯脂肪變、炎變、壞死。因此，GLUT4和UCP2可能通過參與胰島素抵抗、脂質(zhì)代謝、氧化應(yīng)激、能量代謝等在NAFLD的發(fā)生發(fā)展中發(fā)揮作用；3.小檗堿可通過降糖、調(diào)節(jié)脂質(zhì)代謝、改善胰島素抵抗、減輕炎癥反應(yīng)等途徑減輕肝臟病理病變程度及改善肝功能，發(fā)揮治療NAFLD的作用；4.小檗堿可顯著促進(jìn)肝組織GLUT4mRNA及蛋白的表達(dá)，并明顯降低肝組織UCP2蛋白的表達(dá)，這可能是其防治NAFLD的重要作用機(jī)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.
【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R575.5

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