异丙酚对内毒素诱导小鼠RAW264.7巨噬细胞P2X7受活化和IL-1β蛋白合成的影响

摘要

目的 探讨异丙酚对内毒素诱导小鼠RAW264.7巨噬细胞P2X7受体活化和IL-1β蛋白合成的影响.方法 RAW264.7巨噬细胞经1 μmol/L亮蓝G(BBG)或1～100 μmol/L异丙酚孵育20 min,随后经1 μg/ml LPS孵育4 h,采用ELISA法测定IL-1β的释放量,采用Western blot法测定胞内IL-1β前体蛋白和成熟体蛋白含量,并计算异丙酚抑制IL-1β释放的半数有效浓度(IC_(50)).采用全细胞膜片钳记录模式,RAW264.7巨噬细胞经1 mmol/L ATP孵育5s,以诱发P2X7受体门控离子通道电流,分别经1～1 000μmol/L异丙酚孵育4 min后记录电流峰值,计算异丙酚抑制P2X7受体门控离子通道电流峰值的IC_(50).结果 异丙酚可抑制LPS诱导的IL-1β的释放,其IC_(50)为(24±3)μmol/L.异丙酚可抑制P2X7受体门控离子通道电流峰值,其IC_(50)为(33±5)μmol/L.LPS可上调胞内IL-1β前体蛋白表达(P<0.01),而3～100μmol/L异丙酚可抑制LPS介导的胞内IL-1β前体蛋白表达上调.结论 异丙酚抑制LPS诱导的小鼠RAW264.7巨噬细胞IL-1β的释放可能与抑制P2X7受体的活化和胞内IL-1β前体蛋白的合成有关.%Objective To investigate the effects of propofol on P2X7 receptor activition and IL-1β production induced by endotoxin in murine RAW264.7 macrophages. Methods RAW264.7 macruphages were treated with LPS (1 μg/ml) for 4 h to induce the production and release of IL-1β, and pretreated with BBG (specific P2X7 receptor antagonist) 1 μmol/L or propofol 1-100 μmol/L for 20 min before LPS stimulation, and IL-1β release was measured using ELISA kit. Whole-cell patch clamp technique was used to record the P2X7-gated currents induced by 1 mmol/L ATP, the cells were exposed to propofol with 1-1 000 -μmol/L for 4 min, and the IC_(50) level of propofol was achieved. Western blot technique was used to measure the production of pro-lL-1β protein and IL-1β protein intracellularly after LPS treatment for 4 h under different concentrations of propofol. Results IL-1β was released from RAW264.7 macrophages after LPS stimulation, which was decreased by propofol, and the IC_(50) level of propefol was (24±3) μmol/L. P2XT-gated currents were inhibited by propofol, and the IC_(50) level was (33±5) μmol/L. Pro-IL-1β protein intracellularly was up-regulated after LPS stimulation, and propofol with 3-100 μmol/L decreased the up-regulation of pro-IL-1β intracellularly induced by LPS. Conclusion Propefol could inhibit IL-1β release from RAW264.7 macrophages treated by LPS, which is mediated by inhibiting P2X7 receptor activition and decreasing the production of pro-IL-1β intracellularly.