高迁移率族蛋白1对肝星状细胞Toll样受体4信号途径的激活作用

HMGB1-mediated activation of TLR4 signaling in hepatic stellate cells

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-KB—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（HMGBl）处理组，检测HMGBl、LPS处理后细胞NF-KB及AP-1的涪胜、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土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。

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 （HMGB1 treatment）. All groups were transfected with luciferase reporter plasmids carrying responsive elements for either the nuclear factor-kappa B （NF-r,B） or activator protein-1 AP-1 transcription factors. Following stimulation with normal saline, LPS （100 ng/mL） or HMGB 1 （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 were determined by enzyme-linked immunosorbent assay （ELISA） of the culture supematants. Results Activation of NF-r.B- and AP-l-responsive reporters was significantly up-regulated in JS1 cells treated with HMGB1 or LPS, and the activation was coincident with markedly up-regulated transcription and secretion of MCP-1. However, I-IMGB 1 and LPS treatment produced no responsive of the NF-r,B 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.