七叶苷抑制PERK/eIF2A/ATF4信号通路改善肝细胞脂质堆积的研究  

Study on esculin improve lipid accumulation in hepatocytes by inhibiting the PERK/eIF2A/ATF4 signaling pathway

作　　者：徐霜 洪亮 潘安娜 吴杨荷 叶晓婷 XU Shuang;HONG Liang;PAN Anna;WU Yanghe;YE Xiaoting(Department of Infectious Disease,the Third Affiliated Hospital of Wenzhou Medical University,Wenzhou 325200,Zhejiang,China)

机构地区：[1]温州医科大学附属第三医院感染科,浙江温州325200

出　　处：《中国现代医生》2024年第33期64-69,共6页China Modern Doctor

基　　金：浙江省温州市基础性科研项目(Y2023401)。

摘　　要：目的探讨七叶苷通过抑制蛋白激酶RNA样内质网激酶(protein kinase RNA-like endoplasmic reticulum kinase,PERK)/真核翻译起始因子2A(eukaryotic translation initiation factor 2A,eIF2A)/激活转录因子4(activating transcription factor 4,ATF4)信号通路改善肝细胞脂肪变性的治疗作用和机制。方法采用人源正常肝细胞系HL-7702,利用0.5mmol/L游离脂肪酸(free fatty acid,FFA)(油酸:棕榈酸=2:1)诱导体外肝细胞脂肪变性模型,经50μmol/L、200μmol/L七叶苷处理24h。测定细胞内生化指标丙氨酸转氨酶(alanine transaminase,ALT)、天冬氨酸转氨酶(aspartate transaminase,AST)、丙二醛(malondialdehyde,MDA)、还原型谷胱甘肽(glutathione,GSH)和三酰甘油(triacylglycerol,TG)含量。采用尼罗红脂肪荧光染色法检测细胞内脂滴;采用定量反转录聚合酶链反应(quantitative reverse transcriptase-mediated polymerase chain reaction,qRT-PCR)检测细胞内脂质代谢过程相关基因的转录水平。采用蛋白质印迹(Westernblot,WB)检测细胞内促凋亡因子Caspase-3和Bax的蛋白表达量及PERK/eIF2A/ATF4信号通路相关蛋白和磷酸化水平。结果生化指标检测结果证实,50μmol/L和200μmol/L的七叶苷可显著降低FFA诱导肝细胞内MDA、ALT和AST水平(P<0.05),并显著增加细胞内GSH水平(P<0.05)。WB结果显示七叶苷可显著降低细胞内促凋亡因子Caspase-3和Bax的蛋白表达量(P<0.01)。尼罗红染色和TG含量检测结果证实七叶苷可显著减少细胞内脂滴堆积和TG含量(P<0.05)。qRT-PCR结果显示七叶苷处理后肝细胞内PPARγ、FASN、Srebf1、Dgat2、Mvk、Acaca的表达量均显著降低(P<0.05)。在机制方面,七叶苷可显著降低肝细胞中PERK、eIF2A和ATF4的磷酸化水平(P<0.05)。结论七叶苷可通过调控PERK/eIF2A/ATF4信号通路改善肝细胞脂质堆积,对维护肝细胞健康状态起到积极作用。Objective To explore the effect and mechanism of esculin on hepatocyte steatosis by inhibiting protein kinase RNA-like endoplasmic reticulum kinase(PERK)/eukaryotic translation initiation factor 2A(eIF2A)/activating transcription factor 4(ATF4)signaling pathway.Methods Human normal liver cell line HL-7702 was used to induce a fatty degeneration model of hepatocytes in vitro with 0.5mmol/L free fatty acid(FFA)(oleic acid:palmitic acid=2:1)and treated with 50μmol/L,200μmol/L esculin for 24h.After the cell samples were broken by ultrasound,the supernatant was collected and the contents of alanine transaminase(ALT),aspartate transaminase(AST),malondialdehyde(MDA),glutathione(GSH)and triacylglycerol(TG)were detected.Using Nile red fat fluorescence staining to detect intracellular lipid droplets;Quantitative reverse transcriptase-mediated polymerase chain reaction(qRT-PCR)was used to detect the transcription levels of genes related to intracellular lipid metabolism processes.Western blot(WB)was used to detect the protein expression levels of pro apoptotic factors Caspase-3 and Bax,as well as PERK/eIF2A/ATF4 signaling pathway related proteins and phosphorylation levels in cells.Results The results confirmed that treatments of 50μmol/L and 200μmol/L of esculin significantly decreased the levels of FFA induced MDA,ALT and AST in hepatocytes(P<0.05),and significantly increased the levels of intracellular GSH(P<0.05).WB results showed that esculin treatment could significantly reduce the protein expression levels of Caspase-3 and Bax(P<0.01).The results of Nile red staining and TG content detection confirmed that esculin treatment could significantly reduce the accumulation of intracellular lipid droplets and TG content(P<0.05).The results of qRT-PCR showed that the expression levels of PPARγ,FASN,Srebf1,Dgat2,Mvk and Acaca in hepatocytes were significantly decreased after esculin treatment(P<0.05).In terms of mechanism,the phosphorylation levels of PERK,eIF2A and ATF4 in hepatocytes were significantly reduced by escul

关 键 词：肝细胞 脂肪变性 脂质代谢 蛋白激酶RNA样内质网激酶/真核翻译起始因子2A/激活转录因子4 

分 类 号：R575.5[医药卫生—消化系统]