解放军医学杂志2023，Vol.48Issue(11)：1314-1320,7.DOI:10.11855/j.issn.0577-7402.2484.2023.0804
经外泌体释放的高迁移率族蛋白B1在菌群紊乱非酒精性脂肪性肝病小鼠中的表达及Toll样受体4信号的调控作用
Expression of high-mobility group B1 released by exosome in nonalcoholic fatty liver disorder and regulated by TLR4 signal
摘要
Abstract
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.关键词
非酒精性脂肪性肝病/脂肪性肝炎/高迁移率族蛋白B1/外泌体/Toll样受体4Key words
nonalcoholic fatty liver disease/steatohepatitis/high mobility group protein B1/exosome/Toll-like receptor 4分类
医药卫生引用本文复制引用
孙焕焕,李培杰,冯云,刘梦莹,师文,马富权,和水祥..经外泌体释放的高迁移率族蛋白B1在菌群紊乱非酒精性脂肪性肝病小鼠中的表达及Toll样受体4信号的调控作用[J].解放军医学杂志,2023,48(11):1314-1320,7.基金项目
This work was supported by the Key Research and Development Project of Shaanxi Province(2019SF-171,2018SF-051) 陕西省重点研发计划(2019SF-171,2018SF-051) （2019SF-171,2018SF-051）
