CAJ | 학술논문

目的探讨高迁移率族蛋白1 (HMGB1)对肝星状细胞Toll样受体4(TLR4)信号途径的激活作用及其下游表达产物的影响.方法培养永生化小鼠肝星状细胞株野生型(JS1)、TLR4基因敲除TLR4-/-(JS2)、MyD88基因敲除MyD88-/- (JS3),用脂质体介导虫荧光酶标记的NF-κB(NF-κB-luc)或AP-1 (AP-1-luc)反应性报告质粒与内参照质粒(R-Luc)共转染JS1、JS2和JS3细胞.每种细胞均分为阴性对照组(NC组)、脂多糖(LPS)处理组、高迁移率族蛋白1(HMGB1)处理组,检测HMGB1、LPS处理后细胞NF-κB及AP-1的活性、各组细胞单核细胞趋化蛋白(MCP-1)mRNA及蛋白的表达.数据经正态性及方差齐性检验,两组间样本均数比较采用t检验.结果 JS1细胞LPS处理组和HMGB1处理组的NF-κB活性分别为(12.72±5.06)、(1.97±0.29)与NC组的(0.85±0.08)相比,t值分别为4.06和6.27,P值均＜0.05,差异均有统计学意义; JS1细胞LPS处理组和HMGB1处理组的AP-1活性分别为(2.01±0.21)、(1.07±0.17)与NC组的(0.61±0.11)相比,t值分别为7.93和3.32,P值均＜0.05,差异均有统计学意义;JS1细胞LPS处理组和HMGB1处理组MCP-1的mRNA相对表达量分别为4.44±0.38、2.42±0.26,与NC组(值为1)相比,t值分别为15.54和9.29,P值均＜0.05,差异均有统计学意义; JS1细胞LPS处理组和HMGB1处理组的MCP-1的蛋白相对表达量分别为(765.57±10.23)、(550.46±15.97)与NC组的(437.14±3.68)相比,t值分别为52.32和11.97,P值均＜0.05,差异均有统计学意义.在JS2和JS3细胞中,LPS和HMGB1处理后上述观测指标与NC组相比,P值均＞0.05,差异无统计学意义.结论 HMGB1作为一种内源性TLR4配体,能激活肝星状细胞株JS1细胞的TLR4信号,促进TLR4介导的炎症表型.
목적 탐토고천이솔족단백1 (HMGB1)대간성상세포Toll양수체4(TLR4)신호도경적격활작용급기하유표체산물적영향.방법 배양영생화소서간성상세포주야생형(JS1)、TLR4기인고제TLR4-/-(JS2)、MyD88기인고제MyD88-/- (JS3),용지질체개도충형광매표기적NF-κB(NF-κB-luc)혹AP-1 (AP-1-luc)반응성보고질립여내삼조질립(R-Luc)공전염JS1、JS2화JS3세포.매충세포균분위음성대조조(NC조)、지다당(LPS)처리조、고천이솔족단백1(HMGB1)처리조,검측HMGB1、LPS처리후세포NF-κB급AP-1적활성、각조세포단핵세포추화단백(MCP-1)mRNA급단백적표체.수거경정태성급방차제성검험,량조간양본균수비교채용t검험.결과 JS1세포LPS처리조화HMGB1처리조적NF-κB활성분별위(12.72±5.06)、(1.97±0.29)여NC조적(0.85±0.08)상비,t치분별위4.06화6.27,P치균＜0.05,차이균유통계학의의; JS1세포LPS처리조화HMGB1처리조적AP-1활성분별위(2.01±0.21)、(1.07±0.17)여NC조적(0.61±0.11)상비,t치분별위7.93화3.32,P치균＜0.05,차이균유통계학의의;JS1세포LPS처리조화HMGB1처리조MCP-1적mRNA상대표체량분별위4.44±0.38、2.42±0.26,여NC조(치위1)상비,t치분별위15.54화9.29,P치균＜0.05,차이균유통계학의의; JS1세포LPS처리조화HMGB1처리조적MCP-1적단백상대표체량분별위(765.57±10.23)、(550.46±15.97)여NC조적(437.14±3.68)상비,t치분별위52.32화11.97,P치균＜0.05,차이균유통계학의의.재JS2화JS3세포중,LPS화HMGB1처리후상술관측지표여NC조상비,P치균＞0.05,차이무통계학의의.결론 HMGB1작위일충내원성TLR4배체,능격활간성상세포주JS1세포적TLR4신호,촉진TLR4개도적염증표형.
Objective To determine the potential of the high mobility group box-1 protein 1 (HMGB1) to activate Toll-like receptor 4 (TLR4) signaling in hepatic stellate cells (HSCs) and investigate the subsequent transition of HSC towards the inflammatory phenotype.Methods Three immortalized mouse HSC cell lines,wild-type (JS1),TLR4-/- (JS2) and MyD88-/- (JS3),were subcultured in plates and divided into groups of normal control (untreated),postive control (lipopolysaccaride,LPS treatment),and experimental (HMGB 1 treatment).All groups were transfected with luciferase reporter plasmids carrying responsive elements for either the nuclear factor-kappa B (NF-κB) or activator protein-1 AP-1 transcription factors.Following stimulation with normal saline,LPS (100ng/mL) or HMGB1 (100 ng/mL),the activation of NF-κB or AP-1 was detected by a dual-luciferase reporter assay system.The induction of monocyte chemotactic protein- 1 (MCP- 1) transcription was determined by measuring the mRNA levels using real time quantitative reverse transcription PCR (qRT-PCR).The secreted protein levels of MCP-1 wore determined by enzyme-linked immunosorbent assay (ELISA) of the culture supematants.Results Activation of NF-κB-and AP-l-responsive reporters was significantly up-regulated in JS1 cells treated with HMGBI or LPS,and the activation was coincident with markedly up-regulated transcription and section of MCP-1.However,HMGB1 and LPS treatment produced no responsive of the NF-κB and AP-1 reporters,and no increase in expression or secretion of MCP-1,in JS2 or JS3 cells.Conclusion As an endogeous ligand of TLR4,HMGB 1 may activate TLR4 signaling and the TLR4-mediated inflammatory response of HSC.