ZAG在NAFLD小鼠中的表达变化及其对肝脏炎症的影响

发布时间：2018-08-25 08:51

【摘要】：目的：建立高脂饲料喂养的非酒精性脂肪性肝病（Nonalcoholic fatty liverdisease，NAFLD）小鼠模型，动态观察锌alpha2糖蛋白（Zinc alpha2glycoprotein,ZAG）在NAFLD发展中的表达变化，初步探讨ZAG与NAFLD的关系。方法：48只8周龄雄性C57BL/6小鼠随机分成高脂饮食组(HFD)和标准饮食组(SD)，分别饲养4周、8周、12周和16周。HE染色分析肝脏组织病理学改变；ELISA法检测血清ZAG以及炎症因子TNF-α、IL-6、IL-8和IL-1β水平；新鲜肝脏匀浆检测其甘油三酯（Triglyceride，，TG）含量；实时荧光定量PCR检测肝脏ZAG的mRNA表达水平，Western-blotting技术及免疫组化法检测肝脏ZAG的蛋白表达水平。结果：4周开始，HFD组小鼠血清TNF-α较SD组小鼠明显升高(P0.05)，且随NAFLD的发生而持续增加。8周起HFD组小鼠的IL-6、IL-8以及IL-1β逐渐升高(P0.05)。4周起，HFD组小鼠肝脏ZAG的mRNA及蛋白水平逐渐下降(P0.05)，而血清ZAG浓度从8周开始明显降低(P0.05)。结论：高脂饮食诱导C57BL/6小鼠发生NAFLD，其机制可能与肝脏ZAG表达水平的降低有关。目的：建立MCD饲料喂养的非酒精性脂肪性肝炎（Nonalcoholic steatohepatitis，NASH）小鼠模型，尾静脉注射rAAV2-ZAG-CMV-EGFP诱导其肝脏ZAG过表达，探讨ZAG在NAFLD小鼠肝脏脂质代谢及炎症反应中的作用。方法：54只8周龄雄性C57BL/6小鼠适应性喂养1周，按体重随机分为Control组（12只）、MCD+PBS组（14只）、MCD+ZAG组（14只）以及MCD+GFP组（14只）。 Control组小鼠给予标准饲料喂养8周，其余3组首先给予MCD饲料喂养4周，MCD+ZAG组小鼠给予尾静脉注射rAAV2-ZAG-CMV-EGFP2×1011vg/只,MCD+GFP组给予同剂量rAAV2-CMV-EGFP，MCD+PBS组给予同体积的PBS，继续MCD饲料喂养4周。8周末处死所有小鼠，收集血清和肝脏组织。HE及油红O染色观察肝脏组织病理学改变；生化及ELISA法检测空腹血糖、血脂、炎症因子TNF-α、IL-6、IL-8、IL-1β水平以及肝脏TG含量；RT-qPCR、Western-blotting及免疫组化分别检测肝脏ZAG、脂代谢相关基因以及炎症因子的mRNA和蛋白水平表达。结果：8周末，MCD饲料喂养的小鼠，其饮食量、体重、肝湿重、血清TG、TC、LDL、HDL、FPG明显降低（P0.05），肝脏指数、ALT、AST、血清TNF-α、IL-6、IL-8、IL-1β以及肝脏TG含量明显升高（P0.05），肝脏脂肪分解相关基因HSL、PPARα、CPT1A的mRNA和蛋白表达水平明显降低（P0.05），肝脏相关炎症因子TNF-α、IL-1β、ICAM-1、MCP-1、CD68、F4/80的mRNA和蛋白表达增加（P0.05），同时，IL-6、IL-8的蛋白表达水平也明显增加（P0.05）。ZAG过表达的NASH小鼠，其肝湿重、血清TG、TC、肝脏指数、ALT、AST、血清TNF-α、IL-6、IL-1β水平也有所降低（P0.05），血清LDL、HDL、FPG、IL-8无明显差异（P0.05）。ZAG过表达组小鼠，其肝脏脂肪分解相关基因HSL、PPARα、CPT1A的mRNA、蛋白水平增加（P0.05），而炎症因子TNF-α、IL-1β、ICAM-1、MCP-1、CD68、F4/80的mRNA和蛋白表达水平降低（P0.05），且IL-6、IL-8的蛋白水平也明显降低（P0.05）。结论：ZAG能够改善MCD饲料诱导的NAFLD小鼠肝脏脂肪变及炎症反应，可能与ZAG调节肝脏脂代谢相关基因的表达以及抑制炎症因子的表达有关。
[Abstract]:AIM: To establish a mouse model of non-alcoholic fatty liver disease (NAFLD) fed with high-fat diet and observe the expression of Zinc alpha2 glycoprotein (ZAG) in the development of NAFLD.
Methods: 48 8-week-old male C57BL/6 mice were randomly divided into high-fat diet group (HFD) and standard diet group (SD) and fed for 4 weeks, 8 weeks, 12 weeks and 16 weeks respectively. The expression of ZAG mRNA in liver was detected by real-time fluorescence quantitative PCR, and the expression of ZAG protein in liver was detected by Western-blotting and immunohistochemistry.
Results: The serum TNF-alpha level of HFD mice was significantly higher than that of SD mice (P 0.05), and increased with the occurrence of NAFLD. The levels of IL-6, IL-8 and IL-1 beta in HFD mice increased gradually (P 0.05).
CONCLUSION: High fat diet induces NAFLD in C57BL/6 mice, which may be related to the decrease of ZAG expression in liver.
AIM: To establish a non-alcoholic steatohepatitis (NASH) mouse model fed with MCD diet and to investigate the role of ZAG in lipid metabolism and inflammation of NAFLD mice by tail vein injection of rAAV2-ZAG-CMV-EGFP.
Methods: Fifty-four eight-week-old male C57BL/6 mice were randomly divided into control group (12 mice), MCD+PBS group (14 mice), MCD+ZAG group (14 mice) and MCD+GFP group (14 mice). Control group was fed with standard diet for 8 weeks. The other three groups were fed with MCD for 4 weeks. MCD+ZAG group was given rAAV2-ZAG-CMV-EG by tail vein injection. FP2 *1011vg/mouse, MCD+GFP group was given the same dose of rAAV2-CMV-EGFP, MCD+PBS group was given the same volume of PBS, MCD+PBS group was fed for 4 weeks. At the end of 8 weeks, all the mice were killed and serum and liver tissues were collected. The expression of ZAG, lipid metabolism related genes and inflammatory factors mRNA and protein were detected by RT-qPCR, Western-blotting and immunohistochemistry.
Results: At the end of 8 weeks, the diet, body weight, liver wet weight, serum TG, TC, LDL, HDL, FPG decreased significantly (P 0.05), liver index, ALT, AST, serum TNF-a, IL-6, IL-8, IL-1 beta and liver TG content increased significantly (P 0.05), hepatic fat decomposition related genes HSL, PPARa, CPT1A mRNA and protein expression levels decreased significantly (P 0.05). The mRNA and protein expression of liver-related inflammatory factors TNF-a, IL-1beta, ICAM-1, MCP-1, CD68, F4/80 were increased (P 0.05). At the same time, the protein expression of IL-6 and IL-8 was also significantly increased (P 0.05). The liver wet weight, serum TG, TC, liver index, ALT, AST, serum TNF-a, IL-6, IL-1beta levels were also decreased in NASH mice with ZAG overexpression (P 0.05). There was no significant difference between ZAG overexpression group and control group (P 0.05). The mRNA and protein levels of hepatic fat-decomposition-related genes HSL, PPAR-a, CPT1A increased (P 0.05), while the mRNA and protein levels of inflammatory factors TNF-a, IL-1beta, ICAM-1, MCP-1, CD68, F4/80 decreased (P 0.05), and the protein levels of IL-6 and IL-8 decreased significantly (P 0.05).
CONCLUSION: ZAG can improve hepatic steatosis and inflammation induced by MCD in NAFLD mice, which may be related to ZAG regulating the expression of lipid metabolism related genes and inhibiting the expression of inflammatory factors.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575.5

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