盐皮质受体对脂多糖诱导的巨噬细胞NOD样受体热蛋白结构域相关蛋白3炎症复合体激活的作用及其机制

目的探讨盐皮质受体对脂多糖（LPS）诱导巨噬细胞炎症复合体激活的作用，并探讨其机制。 方法对小鼠巨噬细胞系RAW264.7，用LPS、嘌呤受体激动剂腺苷5′-（3-硫代三磷酸盐）四锂盐（ATP-γ-s）、盐皮质激素醛固酮（Ald）干预建立NOD样受体热蛋白结构域相关蛋白3（NLRP3）炎症复合体模型；运用盐皮质受体阻滞剂螺内酯（SPI）、P₂X7嘌呤受体拮抗剂（A438）对LPS诱导的炎症复合体模型进行干预，分为生理盐水（NS）组、LPS组、LPS+SPI组、LPS+A438组、LPS+SPI+A438组，实时定量聚合酶链反应（RT-PCR）法检测各组NLRP3炎症复合体的基因表达水平；使用三磷酸腺苷（ATP）检测试剂盒，检测LPS、Ald作用于RAW264.7细胞后培养上清ATP水平，并检测SPI对上述过程的干预作用。 结果LPS、Ald、ATP-γ-s干预RAW264.7细胞，细胞NLRP3的基因表达水平分别较NS组上调（2.51±0.42）、（2.22±0.28）、（2.07±0.11）倍，差异均有统计学意义（均P＜0.05）；SPI、A438单独干预或SPI+A438同时干预，可以下调NLRP3的表达至NS组的（1.39±0.20）、（1.31±0.15）、（1.25±0.09）倍，均较LPS组显著下降（均P＜0.05）。LPS、Ald干预RAW264.7显著提高了细胞上清ATP水平，而SPI干预显著降低了LPS、Ald干预所致ATP释放，差异均有统计学意义（均P＜0.05）。 结论SPI可能是通过阻断盐皮质受体以降低胞外ATP的释放，从而抑制LPS导致的NLRP3炎症复合体的激活。

Role and mechanism of action of mineralocorticoid receptorin lipopolysaccharide induced NLRP3 inflammasomes activation

ObjectiveTo investigate the influence of spironolactone on lipopolysaccharide-induced activation of nucleotide-binding oligomerization domain NOD-like receptor 3 (NLRP3) in macrophages and explore the exact mechanism involved. MethodsThe mousemacrophage cellline RAW264.7 was treated with lipopolysaccharide (LPS), aldosterone (Ald), and adenosine 5'-O-3-thiotriphosphate (ATP-γ-s) to establish anNLRP3activation model. Then,themineralocorticoid receptor blocker spironolactone(SPI) and P₂X7 receptor antagonist A438, alone or in combination, were used to intervene the NLRP3 activation model,and the NLRP3 expression level inRAW264.7 cells was detected using real-time polymerase chain reaction (RT-PCR). The supernatant triphosadenine(ATP) concentrationsin RAW264.7 cells challenged withLPS, Ald, LPS+SPI, and Ald+SPI were tested. ResultsLPS,Ald,and ATP-γ-s can activate NLRP3 inflammasomes, and the NLRP3 gene expression levels were significantly elevated by (2.51±0.42), (2.22±0.28), and (2.07±0.11) times, respectively, compared with that inthe NS group (P＜0.05). The NLRP3 activation level in the LPS groupwassignificantly down-regulate by SPI, A438, SPI+A438 by (1.39±0.20), (1.31±0.15), and (1.25±0.09) times, respectively, compared with that inthe NS group (P＜0.05). The supernatant ATP concentration inRAW264.7 cells challenged by LPS and Ald waselevated significantly when compared with that in theNS group (P＜0.05), while SPI treatment can reduce ATP concentration significantly in LPS and Ald-challenged cell supernatant (P＜0.05). ConclusionBlocking mineralocorticoid receptor using SPI can devitalize NLRP3 activation viaextracellular ATP down-regulation.