脂联素对脂肪变肝细胞内C反应蛋白表达的影响及作用机制

Effect of adiponectin on the expression of CRP in cultured steatotic hepatocytes and its possible mechanism

目的探讨脂联素对肝细胞内C反应蛋白(CRP)的影响及可能的作用机制。方法给予HL-7702细胞不同浓度和比例的油酸软脂酸混合液干预不同时间后,CCK-8法测定细胞活力,Nile red染色荧光显微镜拍照,测定荧光强度。实时定量PCR检测细胞内CRP、CREBH、BIP和CHOP mRNA表达水平变化。Western印迹检测细胞核内活化的CREBH蛋白含量。根据是否加入脂联素分组,每组加入不同比例的脂肪酸干预24 h后,实时定量PCR检测CRP、CREBH、BIP和CHOP的表达水平变化。结果相比于正常组,混合脂肪酸干预12 h后,仅OP03(1.0mmol)组CRP表达升高(P<0.05),24 h后,OP12(1.0mmol)、OP03(1.0mmol)、OP03(0.5 mmol)组CRP表达均升高(P<0.05)。混合脂肪酸干预24 h后,CREBH、BIP和CHOP mRNA均有不同程度的增高,OP12(1.0mmol)、OP03(1.0mmol)组核内活化CREBH蛋白增加。加入脂联素后,各组CRP、CREBH、BIP和CHOP表达水平下降(P<0.05)。结论软脂酸可上调肝细胞CRP表达水平,其机制可能与活化的CREBH蛋白有关。脂联素可能通过抑制CREBH蛋白相关的炎症通路而下调CRP表达。

Objective To establish a steatotic hepatocyte model in vitro and explore the effect of adiponectin on the expression of CRP in cultured steatotic hepatocytes as well as its possible mechanism .Methods HL-7702 cells （L02 cells） were cultured with free fatty acid （FFA） mixtures at different ratios of oleic acid and palmitic acid （2∶1 ,1∶2 ,0∶3） for 12 or 24 hours to induce fat-overloading .Cell viability was investigated by CCK-8 ,and levels of CRP ,CREBH ,CHOP and BIP mRNA were detected by real-time PCR .Level of CREBH in nuclear was detected by western blot .Then L02 cells were co-incubated with FFA mixtures and adiponectin or FFA mixtures only for 24 hours .Levels of CRP ,CREBH ,CHOP and BIP mRNA were detected by real-time PCR .Results 1 .Cells cultured with FFA mixtures showed significantly increased Fat droplets were significantly increased from cells cultured with FFA mixtures when compared to control group 2 .Level of CRP increased only in OP03（1 .0mM ） group with stimulation for 24 hours ,but increased in OP12（1 .0mM ）、,OP03（1 . 0mM）、and OP03（0 .5mM） groups with stimulation forafter 24 hours （P〈0 .05） .3 .Stimulated by FFA mixtures for 24 hours ,levels of CREBH ,BIP and CHOP mRNA were increased in varying degrees ,and the level of activated CREBH from nulclear was also increased in OP12（1 .0mM ） and OP03（1 .0mM ） groups （P〈0 .05） .4 .Levels of CRP ,CREBH ,BIP and CHOP were decreased by when L02 cells were co-incubated with FFA mixtures and adiponectin （P〈0 .05） .Conclusion Palmitate can up-regulate the expression of CRP in steatotic hepatocytes and the mechanism may be associated with the activated CREBH .Adiponectin could down-regulate the expression of CRP via inflammatory pathway of CREBH inhibition .