蓝莓促进人肝细胞mitofilin/Mic60表达抑制超氧化物生成减轻代谢功能障碍相关肝病的肝损伤

Blueberry attenuates liver injury in metabolic dysfunction-associated liver disease by promoting the expression of mitofilin/Mic60 in human hepatocytes and inhibiting the production of superoxide

目的研究蓝莓调控代谢功能障碍相关肝病(MAFLD)体外模型中线粒体内膜蛋白mitofilin/Mic60的作用及其机制.方法游离脂肪酸(FFA)诱导L02人肝细胞建立MAFLD细胞模型,设立正常组、模型组、80μg/mL蓝莓原浆处理组、Mic60短发夹RNA(Mic60 shRNA)转染组、Mic60敲低联合80μg/mL蓝莓原浆处理组.油红O染色观察细胞内的脂质沉积情况;噻唑蓝(MTT)法检测不同浓度蓝莓原浆对FFA处理的L02细胞存活率的影响;可见分光光度法检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)和脂代谢指标甘油三酯(TG)、总胆固醇(TC)水平及超氧化物歧化酶(SOD)活性、还原型谷胱甘肽(GSH)和丙二醛(MDA)含量.荧光显微镜观察肝细胞内活性氧(ROS)的情况;采用实时定量PCR及Western blot法分别检测Mic60的mRNA及蛋白表达.结果FFA诱导刺激L02细胞24 h后,L02细胞胞质内有大量红色脂滴沉积;80μg/mL蓝莓原浆处理的细胞存活率较高;蓝莓原浆处理组的ALT、AST、TG、TC、MDA检测结果和ROS的荧光强度显示均低于模型组,而SOD、GSH水平及Mic60 mRNA和蛋白水平均高于模型组.结论蓝莓促进Mic60表达,增加肝细胞SOD及GSH水平,减少ROS的生成,进而减轻MAFLD肝细胞损伤,调节脂质代谢紊乱.

Objective To study the effect and mechanism of blueberry on regulating the mitochondrial inner membrane protein mitofilin/Mic60 in an in vitro model of metabolic dysfunction-associated liver disease(MAFLD).Methods L02 human hepatocytes were induced by free fatty acids(FFA)to establish MAFLD cell model.A normal group,a model group,an 80 ug/mL blueberry treatment group,a Mic60 short hairpin RNA(Mic60 shRNA)transfection group,and Mic60 knockdown combined with an 80μg/mL blueberry treatment group were established.The intracellular lipid deposition was observed by oil red 0 staining,and the effect of different concentrations of blueberry pulp on the survival rate of L02 cells treated with FFA was measured by MTT assay.The levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),triglyceride(TG),total cholesterol(TC),superoxide dismutase(SOD)activity,glutathione(GSH)and malondialdehyde(MDA)contents were measured by visible spectrophotometry.The expression of reactive oxygen species(ROS)in hepatocytes was observed by fluorescence microscopy,and the mRNA and protein expression of Mic60 were detected by real-time quantitative PCR and Westerm blot analysis,respectively.Results After 24 hours of FFA stimulation,a large number of red lipid droplets in the cytoplasm of L02 cells was observed,and the surival rate of L02 cells treated with 80 ug/mL blueberry was higher.The results of ALT,AST,TG,TC,MDA and the fluorescence intensity of ROS in blueberry treated group were lower than those in model group,while the levels of SOD,GSH,Mic60 mRNA and protein in blueberry treated group were higher than those in model group.Conclusion Blueberry promotes the expression of Mic60,increases the levels of SOD and GSH in hepatocytes,and reduces the production of ROS,thus alleviating the injury of MAFLD hepatocytes and regulating the disorder of lipid metabolism.