体内外高脂对肝脏NLRP3炎性小体相关基因表达的影响

背景:炎症-免疫系统活化是非酒精性脂肪性肝病(NAFLD)发生、发展的重要机制。炎性小体介导的促炎细胞因子活化在NAFLD中的作用日益受到重视。目的:探讨体内外高脂处理对肝脏NLRP3炎性小体相关基因表达的影响。方法:30只C57BL/6J小鼠随机分为高脂饮食组和正常饮食组(对照组),喂饲16周后处死小鼠,光学显微镜下观察肝组织病理学表现。以胶原酶原位灌注法分离正常饮食组小鼠肝细胞,分别以含饱和脂肪酸棕榈酸(PA)]、单不饱和脂肪酸油酸(OA)]、多不饱和脂肪酸二十二碳六烯酸(DHA)]的培养液培养,以油红O染色检测肝细胞内脂质沉积。以real-time PCR法和蛋白质印迹法检测肝组织和肝细胞中的NLRP3、caspase-1、白细胞介素(IL)-1βmRNA和NLRF3蛋白表达。结果:高脂饮食组小鼠肝组织内可见空泡样脂肪变性。PA、OA和DHA组肝细胞内可见中-大量脂质沉积。与对照组相比,高脂饮食组小鼠肝组织内NLRF3、caspase-1、IL-1βmRNA表达显著升高(P0.05)。PA组肝细胞NLRP3和IL-IβmRNA表达显著高于对照组(P0.05),DHA组NLRP3和IL-1βmRNA表达显著低于对照组(P0.05),PA、OA、DHA组caspase-1 mRNA表达与对照组相比差异无统计学意义(P0.05)。PA、OA组NLRP3蛋白表达较脂多糖(LPS)组升高,DHA组NLRP3蛋白表达较LPS组降低。结论:肝内脂质尤其是饱和脂肪酸沉积可引起NLRP3炎性小体相关基因表达升高,促进肝脏局部炎症反应和NAFLD进展,而多不饱和脂肪酸可降低NLRP3炎性小体相关基因表达,可能具有抗炎、保护肝细胞的作用。

Effect of High Fat on Expressions of Hepatic NLRP3 Inflammasome-related Genes in vivo and in vitro

Background： Activation of inflammatory-immune system is an important mechanism in the development of non- alcoholic fatty liver disease （NAFLD）. Inflammasome-mediated activation of proinflammatory cytokines plays a key role in NAFLD. Aims： To investigate the effect of high fat on expressions of hepatic NLRP3 inflammasome-related genes in vivo and in vitro. Methods： Thirty C57BL/6J mice were randomly assigned into high fat diet group and normal diet group （control group）. Animals were sacrificed 16 weeks later, the histopathology of liver tissue was examined under light microscope. Hepatocytes were isolated from normal diet mice by in situ perfusion of collagenase and then were incubated in medium containing saturated fatty acid palmitic acid （ PA ） ], monounsaturated fatty acid oleic acid （ OA ） I and polyunsaturated fatty acid docosahexaenoic acid （DHA） ] , respectively. Accumulation of fat in hepatocytes was assayed by oil red O staining. Expressions of NLRP3, caspase-1 and interleukin （IL） -1 β mRNA and NLRP3 protein in liver tissue and hepatocyte were detected by real-time PCR and Western blotting. Results： Liver tissue in high fat diet group revealed vacuolar steatosis. There were moderate to severe accumulation of fat drops in hepatocytes in PA, OA and DHA groups. Comparing with control group, expressions of NLRP3, caspase-1 and IL-113 mRNA in liver tissue were significantly increased in high fat diet group （P 〈 0.05）. Expressions of NLRP3 and IL-1 β mRNA in hepatocytes in PA group were significantly higher than those in control group （P 〈 0.05 ） , and those in DHA group were significantly lower than those in control group （P 〈 0.05 ）. No significant difference in expression of caspase-1 mRNA was found among PA, OA, DHAgroups and control group （ P〈 0. 05 ）. Expression of NLRP3 protein in PA and OA groups was higher than that in lipopolysaccharide （LPS） group, and that in DHA group was lower than that in LPS group. Conclusions： Accumulation of fat especially saturated fatty acid in liver up-regulates the expressions of NLRP3-related genes, which may promote local hepatic inflammation and development of NAFLD. Polyunsaturated fatty acid may down-regulate the expressions of NLRP3- related genes and plays a role in hepatocytes protection and anti-inflammation.