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目的分析热应激对巨噬细胞产生白细胞介素1β(interleukin-1β,IL-1β)的影响,以及巨噬细胞内核苷酸结合寡聚化结构域样受体蛋白3(nucleotide-binding oligomerization domain receptor protein 3,NLRP3)炎性小体在该过程中的作用,并探讨NLRP3炎性小体在热应激引起的炎症反应中的作用。方法应用酶联免疫吸附测定法(enzyme-linked immuno sorbent assay,ELISA)检测热应激对巨噬细胞细胞培养液中IL-1β的浓度的影响,应用Western blot检测热应激对巨噬细胞内NLPR3表达和含半胱氨酸的天冬氨酸蛋白水解酶1(cysteinyl aspartate specific proteases-1,caspase-1)活化的影响,应用吸光度法检测试剂盒检测热应激对巨噬细胞内caspase-1活性的影响,应用逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)技术检测热应激对巨噬细胞内IL-1βmRNA转录的影响,应用siRNA干扰NLRP3表达和化学抑制剂Z-WEHD-FMK抑制caspase-1活化检测热应激对IL-1β产生影响与NLRP3炎性小体的相关性。结果热应激明显使骨髓细胞诱导来源的巨噬细胞中NLRP3表达量和IL-1β胞内mRNA含量和分泌量增加,caspase-1的活性也明显增加。经靶向NLRP3的siRNA或caspase-1抑制剂Z-WEHD-FMK处理后,热应激对巨噬细胞IL-1β的分泌量和胞中IL-1β转录水平的影响明显下降。结论热应激能够增加巨噬细胞中NLRP3炎性小体的产生,并通过该类炎性小体增加巨噬细胞IL-1β的产量。
Objective To analyze the effects of heat stress on the production of interleukin-1β (IL-1β) by macrophages and the effects of nucleotide-binding oligomerization domain 3 receptor protein 3 (NLRP3) inflammasome in the process and to explore the role of NLRP3 inflammasome in the inflammatory response induced by heat stress. Methods The effect of heat stress on the concentration of IL-1β in macrophage cell culture fluid was detected by enzyme-linked immunosorbent assay (ELISA). The effect of heat stress on the concentration of IL-1β in macrophage NLPR3 expression and cysteinyl aspartate specific proteases-1 (caspase-1) activation, the use of absorbance detection kit detection of heat stress on macrophages caspase- 1 activity. The effect of heat stress on the transcription of IL-1βmRNA in macrophages was detected by reverse transcription-polymerase chain reaction (RT-PCR). SiRNA was used to interfere with the expression of NLRP3 and chemosensitivity Agent Z-WEHD-FMK Inhibits Caspase-1 Activation The correlation between the effect of heat stress on IL-1β production and NLRP3 inflammasome was examined. Results Heat stress significantly increased the expression of NLRP3 and IL-1β mRNA and protein, and the activity of caspase-1 in bone marrow-derived macrophages. After treated with siRNA targeting NLRP3 or Z-WEHD-FMK, a caspase-1 inhibitor, the effect of heat stress on IL-1β secretion and IL-1β transcription level in macrophages was significantly decreased. Conclusion Heat stress can increase the production of NLRP3 inflammasome in macrophages and increase the production of IL-1β by macrophages.