非诺贝特保护非酒精性脂肪性肝病小鼠肝脏损伤机制的初步探讨

发布时间：2018-08-06 17:03

【摘要】：目的观察高热量高胆固醇饮食（high-calorie and high-cholesterol diet, HCD）对C57BL/6小鼠肝脏的影响，初步探讨非诺贝特（fenofibrate）对非酒精性脂肪性肝病（non-alcoholic fatty liver disease, NAFLD）小鼠肝脏损伤的保护作用机制。方法将C57BL/6小鼠随机分为正常饮食（SCD）组、高热量高胆固醇饮食（HCD）组和非诺贝特治疗（HCF）组。用高热量高胆固醇饮食喂养3月，建立非酒精性脂肪性肝病小鼠模型，3月后，用非诺贝特（40mg/kg/d）治疗4周。葡萄糖耐量试验（GTT）和胰岛素耐量试验（ITT）测定胰岛素敏感性，检测TG、TC、LDL-C、HDL-C、ALT、AST等血清生化指标以及TNF-α、IL-6、MCP-1等炎性指标水平，HE、油红及Masson染色观察肝脏组织的病理学变化，制备10%肝组织匀浆测定MDA、GSH-Px、T-AOC和SOD等氧化应激指标及TG含量情况，免疫组化检测肝脏组织中TNF-α、CD68炎性分子表达，TUNEL法检测小鼠肝细胞的原位凋亡，Real-time RT-PCR和RT-PCR测定肝组织PPARα、SREBP1-c、PTP1B、IRE-α、CHOP和GRP78mRNA表达，Western-blot检测肝组织GRP78、JNK、ERK蛋白表达量。结果①GTT试验和ITT试验表明，HCD组小鼠胰岛素敏感性降低，存在胰岛素抵抗，而非诺贝特治疗后能够增加胰岛素敏感性，改善胰岛素抵抗。②与SCD组小鼠相比，HCD组小鼠血清TG、TC、LDL-C水平显著升高，HDL-C水平明显下降（P0.01或P0.05）。非诺贝特治疗后能够显著降低血清TG水平（P0.05）。③HCD组小鼠肝细胞内可见脂滴形成，气球样变的肝细胞及炎性细胞浸润，但尚未进展至纤维化阶段，而HCF组肝脏脂肪变性程度及浸润的炎细胞明显减少。④HCD组小鼠血清TNF-α、MCP-1等促炎因子明显升高，而IL-6等抗炎因子降低（P0.05），HCF组小鼠血清促炎因子MCP-1明显降低（P0.05）。免疫组化结果表明，与SCD组相比，HCD组小鼠肝脏TNF-α和CD68的表达量均增加。非诺贝特治疗后能够降低炎性分子表达量，减轻炎症反应。⑤TUNEL结果显示HCD组小鼠肝细胞出现明显凋亡，而HCF组小鼠体内发生凋亡的肝细胞明显减少。⑥与SCD组相比，HCD组小鼠肝组织内MDA明显增加，T-SOD和GSH-Px明显降低（P0.05）。与HCD组相比，HCF组小鼠肝组织内T-SOD、T-AOC明显增加，，MDA明显降低（P0.05）。⑦HCD组小鼠肝组织内SREBP-1c、PTP1B、IRE-α、XBP-1s和CHOP mRNA的表达增加，而PPARα、GRP78mRNA的表达降低，非诺贝特治疗后能降低PTP1B、IRE-α、XBP-1s和CHOP mRNA的表达量，增加PPARα、GRP78mRNA的表达量；此外，HCD组小鼠肝组织内GRP78、JNK蛋白表达量明显降低，而ERK蛋白表达量明显升高，非诺贝特治疗后，下调的GRP78、JNK蛋白表达量显著升高，而上调的ERK蛋白表达量显著降低。结论①高热量高胆固醇饮食能够成功构建NAFLD小鼠模型；②非诺贝特治疗后可降低模型小鼠血脂水平、增加胰岛素敏感性、减轻炎症反应、减少氧化应激和内质网应激水平，改善肝脏病理损伤；③非诺贝特可能是通过减轻内质网应激中的IRE-α—XBP-1通路发挥肝脏保护作用的。
[Abstract]:Objective to observe the effect of high calorie and high cholesterol diet (high-calorie and high-cholesterol diet, HCD) on liver of C57BL/6 mice and to explore the protective mechanism of fenofibrate (fenofibrate) on liver injury in non-alcoholic fatty liver disease, NAFLD) mice. Methods C57BL/6 mice were randomly divided into normal diet (SCD) group, high calorie high cholesterol diet (HCD) group and fenofibrate treated (HCF) group. Mice were fed with high-calorie and high-cholesterol diet for 3 months to establish a model of non-alcoholic fatty liver disease. After 3 months, the mice were treated with fenofibrate (40mg/kg/d) for 4 weeks. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were used to detect insulin sensitivity, serum biochemical indexes such as TGG TCU, LDL-C, HDL-C, and inflammatory indexes such as TNF- 伪, IL-6 and MCP-1, oil red and Masson staining were used to observe the pathological changes of liver tissue. 10% liver tissue homogenate was prepared to determine the oxidative stress indexes such as GSH-PxCT-AOC and SOD and the content of TG in the liver tissue homogenate. Expression of TNF- 伪 CD68 in liver tissue was detected by immunohistochemistry and Tunel method was used to detect in situ apoptosis of mouse hepatocytes. Real-time RT-PCR and RT-PCR were used to detect the expression of PPAR 伪 -SREBP1-cPTP1-PTP1BnIRE- 伪 and Western-blot to detect the expression of GRP78-JNKERK protein in liver tissue. Results 1GTT test and ITT test showed that insulin sensitivity was decreased and insulin resistance was found in HCD group, but fenofibrate could increase insulin sensitivity after treatment. Compared with SCD group, the improvement of insulin resistance was significantly higher than that of SCD group (P0.01 or P0.05). After treatment with fenofibrate, serum TG level (P0.05) .3HCD group could significantly reduce lipid droplet formation, balloon like hepatocyte and inflammatory cell infiltration, but did not progress to fibrosis stage. However, the degree of hepatic steatosis and the infiltration of inflammatory cells in HCF group were significantly decreased. The serum TNF- 伪, MCP-1 and other anti-inflammatory factors such as TNF- 伪 MCP-1 in HCF group were significantly increased, while the anti-inflammatory factors such as IL-6 were significantly decreased (P0.05). The immunohistochemical results showed that the expression of TNF- 伪 and CD68 in liver of SCD group was higher than that of SCD group. After fenofibrate treatment, the expression of inflammatory molecules was decreased, and the inflammatory response. 5 Tunel showed that the hepatocytes in HCD group showed obvious apoptosis. The number of apoptotic hepatocytes in HCF group was significantly lower than that in SCD group (P0.05). Compared with HCD group, T-SOD T-AOC in liver tissue of HCF group increased and MDA decreased significantly (P0.05). 7 HCD group increased the expression of SREBP-1cPTP1BnPTP1- 伪 IRE- 伪 XBP-1s and CHOP mRNA, and decreased the expression of PPAR 伪 -pGRP78mRNA. After fenofibrate treatment, the expression levels of PTP1BU IRE- 伪 XBP-1s and CHOP mRNA were decreased. In addition, the expression of GRP78 JNK protein in liver tissue of PPAR group decreased significantly, while the expression of ERK protein increased significantly. After fenofibrate treatment, the down-regulated expression of GRP78 JNK protein increased significantly in the liver tissue of the mice treated with fenofibrate, and the expression of GRP78 JNK protein decreased significantly after fenofibrate treatment, and the expression of GRP78 JNK protein increased significantly after fenofibrate treatment. The up-regulated expression of ERK protein decreased significantly. Conclusion 1 High calorie and high cholesterol diet can successfully construct NAFLD mice model of fenofibrate, which can decrease the level of blood lipids, increase insulin sensitivity, alleviate inflammatory reaction and decrease the levels of oxidative stress and endoplasmic reticulum stress after treatment with fenofibrate. The improvement of liver pathological injury by fenofibrate may play a protective role by alleviating the IRE- 伪 -XBP-1 pathway in endoplasmic reticulum stress.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.5

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