丙酮酸乙酯抑制脓毒症时HMGB1释放的分子机制

【目的】 探讨丙酮酸乙酯(EP)抑制脓毒症巨噬细胞表达和释放HMGB1的相关分子机制?【方法】将小鼠腹腔巨噬细胞株RAW264.7分为LPS和LPS+ EP组,分别采用100 ng/mL LPS和100 ng/mL LPS+ 5 mmol/L EP刺激,于刺激后不同的时间点,Western blot检测细胞总蛋白内p-p38MAPK?CBP的含量变化以及胞质和胞核内NF-κB的含量;用免疫细胞化学?激光共聚焦显微镜观察培养细胞内p-p38MAPK?NF-κB和CBP的变化;Real-time PCR检测培养细胞HMGB1的mRNA水平,ELISA检测培养上清HMGB1的蛋白含量?【结果】 LPS和LPS+ EP刺激后2~6 h,细胞内p-p38MAPK蛋白含量逐渐增加,但LPS+ EP组p-p38MAPK蛋白含量明显低于LPS组;NF-κB在细胞质内的含量逐渐减少,而在细胞核内的含量逐渐增多,而LPS+ EP组NF-κB从胞质到胞核的移位明显弱于LPS组;细胞内CBP的蛋白含量逐渐增加,但LPS+ EP组明显低于LPS组?随着p-p38MAPK?NF-κB和CBP等蛋白的变化,LPS和LPS+ EP刺激后24?36和48 h,LPS+ EP组细胞内HMGB1 mRNA表达比LPS组明显减少;在LPS和LPS+ EP刺激后18~48h,LPS+ EP组培养上清中HMGB1蛋白含量明显低于LPS组?【结论】 EP通过抑制单核-巨噬细胞内的信号分子p-p38MAPK?NF-κB?和 CBP的表达,从而抑制LPS诱导单核-巨噬细胞表达和释放HMGB1?

Research Molecular Mechanisms of Inhibition Releasing of HMGB1 by Ethyl Pyruvate in Sepsis

【Objective】 To investigate the molecular mechanisms about ethyl pyruvate (EP) inhibit the expression and releasing of HMGB1 by macrophages in sepsis. 【Methods】 The murine macrophage-like cell line RAW264.7 cultured in vitro divided into LPS group and LPS +EP group. These groups were stimulated with 100 ng/mL LPS and 100 ng/mL LPS mixed with 5 mmol/L EP respectively. Western blot was used to detect the expression of protein in the cells about p-p38MAPK, CBP, NF-κB in nucleus and NF-κB in cytoplasm at different time-points. Immunocytochemistry and confocal laser scanning microscopy were used to confirm the change of p38-MAPK,NF-κB, and CBP in cultured cells. The expression of HMGB1 mRNA in cultured cells was determined by Real-time PCR. The content of HMGB1 protein in cultured cells supernatant were detected by ELISA. 【Results】 After stimulated by LPS and LPS+EP respectively from 2 to 6 hour, the protein level of p-p38MAPK in cells while this increase group of LPS+EP was obviously slower than the group of LPS. The expression of NF-κB protein in cytoplasm reduce while the same factor in nuclear increase gradually and this phenomenon was more weaker in LPS+EP group than that in LPS group. The protein of CBP in cells gradually increase while the protein of CBP in LPS+EP group was lower than that in LPS group. With variation of p38-MAPK, NF-κB, and CBP, the expression of HMGB1 mRNA in the cells of LPS group was decreased significantly than the ones in LPS group after each group stimulated with LPS and LPS+EP 24 h,36 h,and 48 h,respectively. The content of HMGB1 protein in cultured cells supernatant of LPS+EP group was obviously lower than that of LPS group at the same time points after separately stimulated by LPS+EP and LPS from 18 h to 48 h. 【Conclusion】 EP inhibits the expression of signal molecules in monocyte-macrophages including p38-MAPK, NF-κB, and CBP so as to inhibit the expression and releasing of HMGB1 by macrophages which induced by LPS in sepsis.