摘要
目的探讨非酒精性脂肪性肝病(NAFLD)小鼠中肠源性及肝源性高迁移率族蛋白B1(HMGB1)经外泌体的释放情况及其调控途径。方法15只雄性8周龄野生型C57BL/6J小鼠及10只合并肠道菌群紊乱的NAFLD小鼠(ASC-/-)模型。10只野生型小鼠喂食正常饲料(ND)为正常对照组、喂养高脂饮食(HFD)为高脂饮食组,每组5只,另外5只用于了解造模情况。10只ASC-/-小鼠分别喂养ND(菌群紊乱组)及HFD(高脂菌群紊乱组),每组5只。共喂养8周。采用共聚焦显微镜检测肝脏及肠黏膜外泌体标志物与HMGB1共定位情况;采用Western blotting及PCR等方法检测外泌体中HMGB1含量。通过棕榈酸(PA)及脂多糖(LPS)处理AML12细胞24 h构建体外模型,采用Western blotting检测HMGB1/CD63水平,并通过siRNA干预探讨经外泌体释放的调控机制。结果高脂饮食组的外泌体分泌较正常对照组增多[(3.5±0.2)ng/ml vs.(1.1±0.3)ng/ml,P<0.05],高脂菌群紊乱组的外泌体亦高于菌群紊乱组[(3.2±0.2)ng/ml vs.(1.9±0.4)ng/ml,P<0.05]。免疫荧光检测结果显示,体内实验高脂饮食组与正常对照组比较,体外实验高脂内毒素组与空白对照组比较,外泌体标记物(ALP或VPS16)与HMGB1的共定位增加。PCR检测发现,高脂饮食组肝细胞外泌体中的HMGB1较正常对照组升高(41.5±10.2 vs.1.3±0.3,P<0.05),高脂菌群紊乱组较菌群紊乱组升高(48.6±7.2 vs.1.5±0.5,P<0.05);高脂饮食组TLR4较正常对照组升高(13.8±6.2 vs.2.8±0.9,P<0.05),高脂菌群紊乱组较菌群紊乱组升高(22.6±4.1 vs.2.5±1.5,P<0.05)。在肠黏膜细胞中,高脂饮食组较正常对照组(0.6±0.2 vs.0.4±0.1,P<0.05)及高脂菌群紊乱组较菌群紊乱组(0.9±0.2 vs.0.5±0.1,P<0.05)中HMGB1与外泌体标记物(CD63)共显影增加。在体外实验中,内毒素组、高脂内毒素组外泌体的HMGB1(5.1±0.8,5.5±0.7)均较空白对照组(3.8±0.6)增加(P<0.05),而siRNA干预组与空白对照组比较无明显差异(3.7±0.6 vs.3.8±0.6,P>0.05)。结论在高脂饮食及肠道菌群紊乱环境下,HMGB1在肝细胞及肠细胞中经外泌体释放,并受TLR4调控,可能是促进脂肪性肝炎发展的因素之一。
Objective To investigate the release of enterogenic and hepatogenic high mobility group protein B1(HMGB1)through exosomes and its regulatory pathway.Methods We used wild-type(WT)and ASC-/-mice for this study.We randomly selected five mice per group from each strain and fed them either a normal diet(ND)or a high-fat diet(HFD)for eight weeks.The control group consisted of WT mice fed with the normal diet;the HFD group were WT mice with the HFD;the microflora disturbance(MD)group were ASC-/-mice fed with the normal diet;the high-lipid microflora disturbance(HLMD)group were ASC-/-mice with HFD.We used confocal microscopy to detect the co-localization of liver and intestinal exosome markers with HMGB1.We then measured the expression level of HMGB1 content in exosomes by Western blotting and PCR.The AML12 cells were treated with palmitic acid(PA)and lipopolysaccharide(LPS)for 24 h to build an in vitro model.We also detected HMGB1/CD63 levels using Western blotting.To understand the regulatory mechanism of exosome release,we employed siRNA intervention.Results The secretion of exosomes increased significantly in HFD group compared with control group[(3.5±0.2)ng/ml vs.(1.1±0.3)ng/ml,P<0.05],HLMD group compared with those in MD group[(3.2±0.2)ng/ml vs.(1.9±0.4)ng/ml,P<0.05].Using immunofluorescence detection,we observed increased co-localization of exosome markers(ALP or VPS16)with HMGB1 in HFD group compared with control group.We also observed this in AML12 cells treated with PA and LPS compared with blank control.The PCR data showed that HMGB1 in hepatocyte exosomes was higher in HFD group compared with control group(41.5±10.2 vs.1.3±0.3,P<0.05),HLMD group was significantly higher than that in MD group(48.6±7.2 vs.1.5±0.5,P<0.05).TLR4 expression was higher in HFD group compared with control group(13.8±6.2 vs.2.8±0.9,P<0.05),HLMD group compared with MD group(22.6±4.1 vs.2.5±1.5,P<0.05).In intestinal mucosal cells,the co-location of HMGB1 and exosome marker CD63 was significantly higher in HFD group compared with control group(0.6±0.2 vs.0.4±0.1,P<0.05),and HLMD group compared with MD group(0.9±0.2 vs.0.5±0.1,P<0.05).In vitro,the HMGB1 of exosomes was increased in endotoxin group(5.1±0.8)and high lipid endotoxin group(5.5±0.7)compared with control group(3.8±0.6,P<0.05).On the other hand,the HMGB1 of exosomes in the cell siRNA intervention group was not increased compared with control group(3.7±0.6 vs.3.8±0.6,P>0.05).Conclusion HMGB1 is released by exosomes in hepatocytes and intestinal cells,and regulated by Toll-like receptor 4(TLR4)under a high-fat diet and intestinal flora disorder,which may be one of the contributing factors in promoting the development of steatohepatitis.
作者
孙焕焕
李培杰
冯云
刘梦莹
师文
马富权
和水祥
Sun Huan-Huan;Li Pei-Jie;Feng Yun;Liu Meng-Ying;Shi Wen;Ma Fu-Quan;He Shui-Xiang(Department of Gastroenterology,the First Affiliated Hospital of Xi'an Jiaotong University,Xi'an,Shaanxi 710061,China)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2023年第11期1314-1320,共7页
Medical Journal of Chinese People's Liberation Army
基金
陕西省重点研发计划(2019SF-171,2018SF-051)。
