DHA对果糖诱导的肝细胞脂质蓄积的影响及机制研究

发布时间：2018-08-20 11:17

【摘要】：目的:果糖摄入增多可引起肝脏内脂质蓄积,进而导致肝脏发生脂肪变性。有研究发现ω-3多不饱和脂肪酸(如DHA)对肝脏的脂肪变性有一定的保护作用。本研究拟以原代肝脏细胞体外培养为研究体系,探讨DHA是否能够改善果糖诱导的肝脏脂质蓄积及可能的分子机制。方法:选用正常雄性的C57 BL/6J小鼠,采用肝脏肝门静脉灌流的方法,获得肝脏细胞;得到的肝脏细胞分别用果糖、果糖和DHA、DHA、PBA和果糖、TM处理细胞24小时以后进行油红O染色观察细胞内红染脂滴蓄积的情况;Real-time PCR检测肝细胞内目标基因表达水平;Western blotting检测肝细胞内ACC、SREBP、ACOX1、GRP78/BIP、IRE、p-IRE蛋白表达水平。结果:油红O染色结果表明原代肝细胞在果糖和衣霉素作用下发生明显的脂质蓄积,在DHA的作用下细胞内脂质蓄积明显减少,而果糖和DHA复合处理组细胞内脂质蓄积也明显减轻,同时PBA预处理后再加入果糖干预肝细胞脂质蓄积明显减轻;Real-time PCR结果显示原代肝细胞在果糖和衣霉素作用下FAS、ACC、SREBP、GRP78的mRNA表达水平较对照组明显上调;DHA处理组FAS、ACC、SREBP、GRP78的mRNA表达水平较对照组无明显变化,CPT1α、ACOX1明显上调;果糖和DHA复合处理组FAS、ACC、GRP78的mRNA表达水平较果糖组明显下调,CPT1α、ACOX1明显上调;果糖和PBA复合处理组FAS、ACC、SREBP、GRP78的mRNA表达水平较果糖组明显下调,CPT1α、ACOX1无明显变化。Western blotting结果提示原代肝细胞在果糖和衣霉素作用下ACC、SREBP、GRP78/BIP、IRE和p-IRE蛋白表达较对照组显著增强;DHA处理组ACC、SREBP、GRP78/BIP、IRE和p-IRE蛋白表达较对照组无明显变化,ACOX1蛋白表达明显增强;果糖和DHA复合处理组与果糖和PBA复合处理组ACC、SREBP、GRP78/BIP、IRE和p-IRE蛋白表达较果糖组蛋白表达明显减弱,ACOX1蛋白表达明显增强。结论:果糖可通过诱导内质网应激激活脂质代谢相关基因的表达进而导致肝脏脂肪变性,DHA通过抑制内质网应激相关基因的表达进而缓解果糖诱导的肝脏脂肪变性。饮食中添加少量的DHA可以预防果糖摄入过多引发的非酒精行脂肪肝的发生,从而减少疾病的发生。
[Abstract]:Objective: increased fructose intake may lead to lipid accumulation in the liver, which leads to fatty degeneration of the liver. It has been found that 蠅 -3 polyunsaturated fatty acids (such as DHA) have a protective effect on liver steatosis. The aim of this study was to investigate whether DHA could improve hepatic lipid accumulation induced by fructose and its possible molecular mechanism with primary hepatocyte culture in vitro. Methods: normal male C57 BL/6J mice were used to obtain liver cells by hepatic portal vein perfusion, and the liver cells were obtained by fructose. After treated with fructose and DHADHAPBA and fructose TM for 24 hours, oil red O staining was performed to observe the accumulation of lipid droplets in the cells. Real-time PCR was used to detect the expression level of target genes in hepatocytes. Western blotting was used to detect the expression level of ACC-SREBPN-GRP78 / BIPIREp-IRE in hepatocytes. Results: the results of oil red O staining showed that the primary hepatocytes had obvious lipid accumulation under the action of fructose and itlamycin, and the intracellular lipid accumulation was obviously decreased under the action of DHA. The intracellular lipid accumulation in fructose and DHA treatment group was also significantly reduced. At the same time, after pretreatment with PBA, fructose was added to inhibit lipid accumulation of hepatocytes significantly. The results showed that the expression of mRNA in primary hepatocytes was significantly up-regulated in the primary hepatocytes treated with fructose and chlortetracycline as compared with the control group. The mRNA expression of FASACC-SREBPnGRP78 was up-regulated by fructose and chlortetracycline. Compared with the control group, the level of CPT1 伪 and ACOX1 was up-regulated. Compared with fructose and DHA group, the expression of mRNA in FASA ACC-GRP78 was significantly down-regulated in fructose group, and the expression of CPT1 伪 and ACOX1 was significantly up-regulated in fructose group. Compared with fructose group, fructose and PBA combined treatment group had significantly down-regulated the expression of mRNA in FASACC-SREBPnGRP78. Western blotting showed that the expression of ACC-SREBPnGRP78 / BIPIIRE and p-IRE protein in primary hepatocytes treated with fructose and chlortetracycline was significantly enhanced than that in control group. Compared with the control group, the expression of ACOX1 protein increased significantly in the expression of ACC-SREBPnGRP78 / BIPP78 and p-IRE protein. Compared with fructose and DHA groups and fructose and PBA groups, the expression of ACC-SREBPG-GRP78 / BIPII-IRE and p-IRE protein was significantly decreased compared with fructose histone group and fructose combined treatment group and fructose and PBA combined treatment group, and the expression of ACOX1 protein was significantly increased. Conclusion: fructose can induce endoplasmic reticulum stress to activate the expression of genes related to lipid metabolism, and then induce hepatic steatosis by inhibiting the expression of endoplasmic reticulum stress-related genes, thereby alleviating fructose induced hepatic steatosis. Dietary supplementation of a small amount of DHA can prevent the occurrence of nonalcoholic fatty liver caused by excessive fructose intake, thus reducing the incidence of disease.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R575

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