高产乙醇克雷伯菌通过诱导肝细胞脂质过氧化上调乳酸化修饰的体外实验研究  

In vitro experimental study on the upregulation of cellular lactylation modification caused by HiAlc Kpn metabolites via the initiation of cell lipid peroxidation in liver cells

作　　者：徐子瑛 唐智杰 高亚钢 袁静[1] Xu Ziying;Tang Zhijie;Gao Yagang;Yuan Jing(Department of Bacteriology,Capital Institute of Pediatrics,Beijing 100020,China)

机构地区：[1]首都儿科研究所细菌学研究室,北京100020

出　　处：《中华预防医学杂志》2025年第2期222-229,共8页Chinese Journal of Preventive Medicine

基　　金：国家自然科学基金重点项目(82130065)。

摘　　要：目的:研究高产乙醇克雷伯菌(HiAlc Kpn)对肝细胞功能的影响,并从表观修饰入手探究其调控机制。 方法:以HepG2细胞系为研究对象,通过在体外分别给予酒精和三种HiAlc Kpn菌株刺激,分为对照组、酒精处理组,W8组、3-24组、4-26组。利用Incucyte活细胞成像仪观察HiAlc Kpn对肝细胞增殖的影响,并通过流式细胞仪分析肝细胞凋亡水平;采用荧光共聚焦显微镜结合活细胞探针检测肝细胞中脂肪累积情况,和细胞内ROS水平;利用流式细胞技术结合seahorse细胞代谢仪检测线粒体损伤程度,通过Western blot检测组蛋白水平泛乳酸化修饰的变化。 结果:与对照组相比,HiAlc Kpn菌株W8、3-24、4-26菌株可引起肝细胞HepG2细胞增殖速度下降和细胞凋亡数量的增加。高内涵细胞成像实验结果显示:HiAlc Kpn作用后HepG2细胞内富集ROS荧光点增高,油红O和BODIPY染色都显示脂肪累积明显增加,油滴数目和荧光点数高于对照组和酒精处理组。流式细胞分析结果显示:酒精、3种HiAlc Kpn处理后,细胞的JC-1单体/聚合体比例明显增高,W8处理组相较于对照组增高约6倍( P<0.05)。seahorse能量代谢系统对线粒体压力检测结果显示:HiAlc Kpn 4-26菌株所导致的胞外酸化程度和氧消耗率下降明显。western blot分析结果显示:单纯高浓度酒精处理后泛乳酸修饰水平有所上升,其中1 000 mmol/L酒精组泛乳酸化修饰升高倍数鱼约为对照组的3倍;HiAlc Kpn W8、3-24菌株所导致的泛乳酸化修饰升高倍数约为对照组的4、2倍,差异均有统计学意义(均 P<0.05)。 结论:HiAlc Kpn可通过调控组蛋白泛乳酸化修饰水平上升,引起肝细胞脂质过氧化,造成线粒体损伤,导致细胞增殖能力受损、凋亡水平增加。ObjectiveTo investigate the impact of High Alcohol-Producing Klebsiella pneumoniae(HiAlc Kpn)on hepatocyte function and explore its regulatory mechanism from the perspective of epigenetic modifications.MethodsUsing the HepG2 cell line as the research model,the study involved exposing the cells to alcohol and three different HiAlc Kpn strains in vitro,dividing them into a control group,alcohol-treated group,W8 group,3-24 group,and 4-26 group.The effect of HiAlc Kpn on liver cell proliferation was investigated using the Incucyte live cell imaging system,and the apoptotic level of liver cells was determined using flow cytometry.The fluorescence confocal microscopy combined with live cell probes was used to detect lipid accumulation and intracellular ROS levels in liver cells.The amount of mitochondrial damage was determined using flow cytometry combined with the seahorse cell metabolism analyzer,and changes in protein levels undergoing global lactylation modification were investigated using Western blotting.ResultsCompared with the control group,HiAlc Kpn strains W8,3-24 and 4-26 could decrease the proliferation rate and increase the ratio of apoptosis of hepatocyte HepG2 cells.The results of high-content cell imaging showed that the fluorescence points of ROS enrichment in HepG2 cells were increased after HiAlc Kpn treatment.The lipid accumulation was significantly increased by oil red O and BODIPY staining.The number of oil droplets and fluorescence points was higher than those in the control group and alcohol treatment group.The results of flow cytometry showed that the ratio of JC-1 monomer/polymer was significantly increased after alcohol and three kinds of HiAlc Kpn were treated and the W8 treatment group was about six times higher than the control group(P<0.05).Seahorse Energy Metabolism System′s mitochondrial pressure test results showed that the extracellular acidification degree and oxygen consumption rate were significantly reduced by the HiAlc Kpn 4-26 strain.Western blot analysis showed that the pan-l

关 键 词：克雷伯菌 产酸 非酒精性脂肪肝 乳酸化修饰 脂质过氧化 

分 类 号：R575.5[医药卫生—消化系统]