非酒精性脂肪性肝病铁死亡机制的基础研究

Basic study on mechanism of ferroptosis in non-alcoholic fatty liver disease

目的:研究非酒精性脂肪性肝病(NAFLD)铁死亡机制的相关差异表达基因和调控信号通路。方法:从GEO数据库下载GSE89632数据集,包含正常组和非酒精性脂肪性肝炎(NASH)患者肝脏组织的芯片基因集,根据|log2(FC)|>1&р.adj<0.05条件,筛选表达差异基因(DEGS)与铁死亡数据库(FerrDb)中的259个铁死亡基因取交集得出NASH铁死亡关键基因(Fe-DEGS),并进行GO-KEGG功能及通路富集分析、PPI网络构建。体外NAFLD模型将HepG2细胞分为正常组(只含无血清培养基)、对照组(含FFA对照液的培养基)、FFA组(含1 mmol/L FFA的培养基),采用油红染色检测细胞脂质分布,甘油三脂测定试剂盒检测细胞内甘油三脂含量及western blotting检测c-jun、ZFP36、ATF3、IL-6蛋白表达。结果:生物信息学分析得出9个NASH铁死亡基因(Fe-DEGS),PPI网络具有关联基因为ZFP36、ATF3、SOCS1、IL-6、PTGS2、JUN。GO富集分析生物学过程中主要涉及脂肪细胞分化、氧化应激反应等;分子功能主要有氧化还原酶活性、三价铁结合等;通路富集有IL-17信号通路、TNF信号通路、JAK-STAT信号通路等。FFA组油红O染色可见细胞内有大量的棕红色颗粒,在正常组及对照组内只有少量棕红色颗粒;细胞甘油三酯含量测定可见,与正常组和对照组比较,FFA组细胞内甘油三脂含量升高(P<0.05);western blotting显示,与正常组和对照组比较,FFA组c-jun、ZFP36、ATF3、IL-6蛋白表达升高(P<0.05)。结论:ZFP36、JUN、ATF3、IL-6基因可能是NAFLD发生铁死亡的关键基因。

Objective:To study the differentially expressed genes and regulatory signaling pathways related to ferroptosis mechanism of non-alcoholic fatty liver disease(NAFLD).Methods:The GSE89632 dataset was downloaded from the GEO database containing a microarray gene set of liver tissue from both normal and non-alcoholic steatohepatitis(NASH)patients,and according to|log2(FC)|>1&р.adj<0.05,expression differential genes(DEGS)were screened and crossed with 259 ferroptosis genes in ferroptosis database(FerrDb)to generate NASH ferroptosis key genes(Fe-DEGS).GO-KEGG function and pathway enrichment analysis was made,and PPI network was constructed.In vitro NAFLD model,HepG2 cells were divided into normal group(serum-free medium only),control group(medium containing FFA control solution)and FFA group(medium containing 1 mmol/L FFA).Lipid distribution of cells was detected by oil red staining,triglyceride assay kit was used to detect intracellular triglyceride content,and western blotting was used to detect c-jun,ZFP36,ATF3 and IL-6 protein expression.Results:Nine ferroptosis genes of NASH(Fe-DEGs)were found by bioinformatic analysis and PPI network associated genes were ZFP36,ATF3,SOCS1,IL-6,PTGS2 and JUN.The biological process of GO enrichment analysis mainly involved adipocyte differentiation,oxidative stress response,etc.Molecular functions mainly included redox enzyme activity,ferric binding,etc.Enrichment pathways included IL-17 signaling pathway,TNF signaling pathway,JAK-STAT signaling pathway,etc.Oil-red O staining showed large brown-red particles in FFA group and only small brown-red particles in normal and control groups.Compared with normal group and control group,the intracellular triglyceride content and the protein levels of c-jun,ZFP36,ATF3 and IL-6 in FFA group were significantly increased(P<0.05).Conclusion:ZFP36,JUN,ATF3 and IL-6 genes may be the key genes of ferroptosis in NAFLD.