Lin Han-Yu, Chang Ya-Ying, Kao Ming-Chang, Huang Chun-Jen
Department of Anesthesiology, Taipei Tzu Chi Hospital, Taipei, Taiwan.
Department of Anesthesiology, Taipei Tzu Chi Hospital, Taipei, Taiwan; School of Medicine, Tzu Chi University, Hualien, Taiwan.
J Surg Res. 2017 Nov;219:72-77. doi: 10.1016/j.jss.2017.05.119. Epub 2017 Jun 23.
Naloxone, an opioid receptor antagonist, possesses potent anti-inflammation effects. We previously confirmed the effects of naloxone on inhibiting upregulation of inflammatory cytokine interleukin-1β (IL-1β). Production of mature form IL-1β is mediated by the nod-like receptor protein 3 (NLRP3) inflammasome, a multiprotein complex composed of NLRP3, and the adaptor protein apoptosis-associated speck-like protein contains a caspase recruitment domain (ASC). We elucidated whether naloxone could inhibit the activation of NLRP3 inflammasome.
To induce IL-1β production and NLRP3 inflammasome activation, the human monocytic leukemia cell line THP-1 cells were first primed with lipopolysaccharide (LPS, 1 μg/mL) and then activated with adenosine triphosphate (ATP, 1 mM). For NLRP3 transcription, THP-1 cells were only treated with LPS priming.
Enzyme-link immunosorbent assay data revealed that the concentration of IL-1β in THP-1 cells treated with LPS plus ATP was significantly higher than that in THP-1 cells treated with LPS plus ATP plus naloxone (0.1 μM) (P < 0.001). Real-time quantitative reverse transcription and polymerase chain reaction data also revealed that NLRP3 mRNA concentration in THP-1 cells treated with LPS was significantly higher than that in THP-1 cells treated with LPS plus naloxone (P = 0.001). ASC speck formation, that is, ASC assembles into a large protein complex, is an indicator for NLRP3 inflammasome activation. Our data revealed that the percentage of cells containing ASC specks in THP-1 cells treated with LPS plus ATP was also significantly higher than that in THP-1 cells treated with LPS plus ATP plus naloxone (P < 0.001).
Naloxone inhibits NLRP3 inflammasome activation.
纳洛酮是一种阿片受体拮抗剂，具有强大的抗炎作用。我们之前证实了纳洛酮对抑制炎性细胞因子白细胞介素-1β（IL-1β）上调的作用。成熟形式的IL-1β的产生由NOD样受体蛋白3（NLRP3）炎性小体介导，NLRP3炎性小体是一种由NLRP3和衔接蛋白凋亡相关斑点样蛋白（含半胱天冬酶募集结构域，ASC）组成的多蛋白复合物。我们阐明了纳洛酮是否能抑制NLRP3炎性小体的激活。
为诱导IL-1β产生和NLRP3炎性小体激活，首先用人单核细胞白血病细胞系THP-1细胞用脂多糖（LPS，1μg/mL）进行预处理，然后用三磷酸腺苷（ATP，1mM）激活。对于NLRP3转录，THP-1细胞仅用LPS预处理。
酶联免疫吸附测定数据显示，用LPS加ATP处理的THP-1细胞中IL-1β的浓度显著高于用LPS加ATP加纳洛酮（0.1μM）处理的THP-1细胞（P<0.001）。实时定量逆转录聚合酶链反应数据还显示，用LPS处理的THP-1细胞中NLRP3 mRNA浓度显著高于用LPS加纳洛酮处理的THP-1细胞（P=0.001）。ASC斑点形成，即ASC组装成大的蛋白质复合物，是NLRP3炎性小体激活的指标。我们的数据显示，用LPS加ATP处理的THP-1细胞中含有ASC斑点的细胞百分比也显著高于用LPS加ATP加纳洛酮处理的THP-1细胞（P<0.001）。
纳洛酮抑制NLRP3炎性小体激活。
