过表达pellino-1在Kupffer细胞内毒素耐受时对TRAF3泛素化及MAPK信号通路的调控

Role of Pellino-1 Overexpression on Ubiquitin of Tumor Necrosis Factor Receptor-associated Factors 3 and MAPK Signaling Pathway in Endotoxin-tolerant Kupffer Cells

【目的】探讨上调pellino-1在Kupffer细胞(KC)内毒素耐受时对肿瘤坏死因子受体相关因子3(TRAF3)泛素化、促分裂原活化蛋白激酶(MAPK)信号通路及下游细胞因子分泌情况的影响。【方法】分离、培养C57BL/6小鼠KC,随机分为2组:(1)空载对照组:转染空载质粒48 h后,先给予小剂量LPS(10 ng/mL)刺激24 h,再给予大剂量LPS(300 ng/mL)刺激。(2)过表达组:过表达pellino-1慢病毒转染48 h后,先给予小剂量LPS(10 ng/mL)刺激24 h,再给予大剂量LPS(300 ng/mL)刺激。两组分别于处理后0、5、10、30、60 min收获细胞,蛋白质免疫印迹试验(Western blot)检测pellino-1、K48泛素化(K48ubiquitin,K48-Ub)、TRAF3、JNK、p-JNK、p38、p-p38蛋白水平表达变化;酶联免疫吸附法(ELISA)检测细胞培养上清液中IL-1β、TNF-α及IL-10的分泌情况。【结果】与空载对照组相比,过表达组pellino-1蛋白表达量在各个时间点均明显升高;K48-Ub水平明显升高;TRAF3蛋白表达量明显降低;JNK、p38蛋白表达量没有明显变化,但p-JNK及p-p38蛋白表达量显著升高;过表达组细胞上清中IL-1β及TNF-α的量明显升高(P<0.05),而IL-10的量则明显降低(P<0.05)。【结论】过表达pellino-1可促进TRAF3蛋白K-48泛素化降解,导致TRAF3蛋白表达量降低,激活下游的MAPK信号,从而抑制内毒素耐受的形成。

【Objective】To investigate the effect of pellino-1 gene overexpression by lentivirus vector on the ubiquitin of tumor necrosis factor receptor-associated factors 3（TRAF3）and the activation of mitogen-activated protein kinases（MAPK）signaling pathway in endotoxin-tolerant kupffer cells（KCs）.【Methods】Isolated KCs of C57BL/6 mouse were randomly divided into two groups：control group,which transfected with control lentivirus vector for 48 h,then pretreated with 10 ng/mL lipopolysaccharide（LPS）for 24 h,and next treated with 300 ng/mL LPS;overexpression group,which transfected with pellino-1 gene overexpression lentivirus vector for 48 h,then pretreated with 10 ng/mL lipopolysaccharide（LPS）for 24 h,and next treated with 300 ng/mL LPS.The protein expressions of pellino-1,K48-Ub,TRAF3,p38,p-p38,c-Jun N-terminal kinase（JNK）and p-JNK were analyzed by Western blot. The level of interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）and interleukin-10（IL-10）in supernatants were measured by enzyme linked immunosorbent assay（ELISA）.【Results】Compared with control group,the protein expressions of pellino-1,K48-Ub,p-JNK and p-p38 and the levels of IL-1β（P〈0.05）,TNF-α（P〈0.05）in supernatants was in-creased in overexpression group,while the protein levels of TRAF3 and the levels of IL-10（P〈0.05）in supernatants were decreased.【Conclusion】Overexpression of pellino-1 can promote TRAF3 K48 ubiquitination degradation,decrease the protein expression of TRAF3,activate the downstream MAPK signaling pathway,and thus impair the formulation of endotoxin tolerance.