肝细胞脂肪变性影响巨噬细胞炎症反应加速动脉粥样硬化形成

Hepatocyte steatosis activates macrophage inflammatory response accelerating atherosclerosis development

目的:基于代谢组学和网络药理学探讨非酒精性脂肪性肝病(NAFLD)与动脉粥样硬化共病的发病机制。方法:野生型C57BL/6J小鼠和ApoE^(-/-)小鼠各6只,分别给予普通饮食和高脂饮食16周,构建NAFLD和动脉粥样硬化共病模型,取血清标本进行非靶向代谢组学检测,鉴定差异代谢产物。运用网络药理学探讨差异代谢物对动脉粥样硬化和NAFLD的可能作用机制,并采用小鼠NCTC1469细胞和RAW264.7细胞构建体外共病模型,油红O染色检测细胞脂质积蓄情况,细胞计数试剂盒检测细胞活性,透射电镜和JC-1染色检测线粒体形态和功能,并验证共病模型的可能病理机制。结果:代谢组学分析共鉴定出85种共病差异代谢产物。将前二十种差异代谢物进行网络药理学分析,结果显示差异代谢产物影响动脉粥样硬化和NAFLD的核心靶点为STAT3、EGFR、MAPK14、PPARG、NFKB1、PTGS2、ESR1、PPARA、PTPN1、SCD;基因组百科全书富集分析显示前十位的信号通路为PPAR信号通路、糖尿病并发症中的AGE-RAGE信号通路、酒精性肝病、催乳素信号通路、胰岛素抵抗、TNF信号通路、乙型肝炎、松弛素信号通路、IL-17信号通路、非酒精性脂肪肝。体外实验验证结果显示,脂代谢相关基因PPARG、PPARA、PTPN1和SCD在肝细胞模型中变化明显,且脂肪变性的肝细胞影响巨噬细胞炎症相关基因STAT3、NFKB1和PTGS2的表达;脂肪变性的肝细胞促进泡沫细胞的形成,加重泡沫细胞的脂质积蓄;脂肪变性后的肝细胞线粒体受损,形态破坏,功能受损,活性氧产生增加,而泡沫细胞形成后加重线粒体损伤。结论:脂代谢异常和炎症反应是动脉粥样硬化和NAFLD共病模型的显著特点,肝细胞脂肪变性后线粒体受损,导致线粒体功能障碍,活性氧增多,激活巨噬细胞炎症反应,加速动脉粥样硬化的发展。

Objective:To investigate the mechanism of comorbidity between non-alcoholic fatty liver disease(NAFLD)and atherosclerosis(AS)based on metabolomics and network pharmacology.Methods:Six ApoE^(-/-)mice were fed with a high-fat diet for 16 weeks as a comorbid model of NAFLD and AS(model group).Normal diet was given to 6 wildtype C57BL/6J mice(control group).Serum samples were taken from both groups for a non-targeted metabolomics assay to identify differential metabolites.Network pharmacology was applied to explore the possible mechanistic effects of differential metabolites on AS and NAFLD.An in vitro comorbid cell model was constructed using NCTC1469 cells and RAW264.7 macrophage.Cellular lipid accumulation,cell viability,morphology and function of mitochondria were detected with oil red O staining,CCK-8 assay,transmission electron microscopy and JC-1 staining,respectively.Results:A total of 85 differential metabolites associated with comorbidity of NAFLD and AS were identified.The top 20 differential metabolites were subjected to network pharmacology analysis,which showed that the core targets of differential metabolites related to AS and NAFLD were STAT3,EGFR,MAPK14,PPARG,NFKB1,PTGS2,ESR1,PPARA,PTPN1 and SCD.The Kyoto Encyclopedia of Genes and Genomes showed the top 10 signaling pathways were PPAR signaling pathway,AGE-RAGE signaling pathway in diabetic complications,alcoholic liver disease,prolactin signaling pathway,insulin resistance,TNF signaling pathway,hepatitis B,the relax in signaling pathway,IL-17 signaling pathway and NAFLD.Experimental validation showed that lipid metabolism-related genes PPARG,PPARA,PTPN1,and SCD were significantly changed in hepatocyte models,and steatotic hepatocytes affected the expression of macrophage inflammation-related genes STAT3,NFKB1 and PTGS2;steatotic hepatocytes promoted the formation of foam cells and exacerbated the accumulation of lipids in foam cells;the disrupted morphology,impaired function,and increased reactive oxygen species production were observed in steatotic hepatocyte mitochondria,while the formation of foam cells aggravated mitochondrial damage.Conclusions:Abnormal lipid metabolism and inflammatory response are distinctive features of comorbid AS and NAFLD.Hepatocyte steatosis causes mitochondrial damage,which leads to mitochondrial dysfunction,increased reactive oxygen species and activation of macrophage inflammatory response,resulting in the acceleration of AS development.