[Study on the role of NALP3 inflammasome in Porphyromonas gingivalis lipopolysaccharide induced RAW264.7].

Objective: To illuminate the effect of NALP3 inflammasome on regulating the expression of cytokines of macrophages in periodontitis. Methods: RAW264.7 cells were cultured and divided into three groups. The first group stayed normal as control, the second group was stimulated by 1 mg/L Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS), the third group was pretreated with AC-YVAD-CMK (caspase-1 inhibitor) before stimulated with 1 mg/L Pg LPS. RAW264.7 cells pretreated with various concentrations (0, 5, 10, 25, 50, 75, 100, 200 μmol/L) of AC-YVAD-CMK for 2 h, and stimulated by 1 mg/L Pg LPS for 24 h in the third group. After that, cell survival rate were detected by cell counting kit-8. Every group cells gene transcription of NALP3 and interleukin-1β (IL-1β) were detected by quantitative real-time PCR (qPCR) after 6 h, protein expression of NALP3 and IL-1β were separately detected by Western blotting and enzyme linked immunosorbent assay (ELISA) after 24 h, respectively. Results: It is observed that treatment with 5, 10, 25, 50, 75, 100, 200 μmol/L AC-YVAD-CMK did not significantly affect the viability of RAW264.7 cells. qPCR showed that mRNA expression of IL-1β level (1.03±0.08, 5.48±0.22, 4.31±0.20) and NALP3 level (0.96±0.05, 2.62±0.44, 1.73±0.09). Western blotting showed that protein expression of NALP3 level (1.00±0.10, 2.34±0.04, 1.64±0.04), ELISA showed protein secretion of IL-1β level ([40.20±0.25], [61.50±1.81], [52.40±1.91] ng/L). After stimulated by Pg LPS, mRNA and protein expression of IL-1β (P<0.01, P<0.01) and NALP3 (P<0.01, P<0.01) significantly increased; but the expression of IL-1β (P=0.002, P=0.027) and NALP3 (P<0.01, P<0.01) were decreased when pretreated with AC-YVAD-CMK. Conclusions: NALP3 inflammasome signal pathway can be activated by Pg LPS in RAW264.7. Block of the pathway can inhibit Pg LPS-induced secretion of cytokines.目的： 研究NALP3（NACHT-LRR-PYD结构域蛋白3）炎性体在牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）脂多糖刺激小鼠巨噬细胞系RAW264.7细胞中调控炎症因子的作用。 方法： 对RAW264.7细胞常规培养并平均分为3组，每组4×10(5)个细胞：A组为对照组（细胞未处理）；B组为1 mg/L Pg脂多糖刺激组；C组为胱天蛋白酶1抑制剂（AC-YVAD-CMK）预处理+1 mg/L Pg脂多糖刺激组。C组RAW264.7细胞采用连续稀释（5、10、25、50、75、100、200 μmol/L）的AC-YVAD-CMK预处理2 h，随后Pg脂多糖（1 mg/L）孵育24 h，通过细胞计数试剂盒检测细胞生存率。采用实时荧光定量PCR检测3组RAW264.7细胞6 h后NALP3和白细胞介素1β（interleukin-1β，IL-1β）基因转录水平；蛋白质印迹法和酶联免疫吸附测定法检测3组细胞24 h后上述两种蛋白的表达。 结果： 不同浓度（0、5、10、25、50、75、100、200 μmol/L）AC-YVAD-CMK处理RAW264.7细胞对细胞活力无明显影响；实时荧光定量PCR结果显示，A、B、C组IL-1β mRNA表达分别为1.03±0.08、5.48±0.22和4.31±0.20；NALP3 mRNA分别为0.96±0.05、2.62±0.44、1.73±0.09；蛋白质印迹法结果显示，A、B、C组NALP3蛋白的表达量分别为1.00±0.10、2.34±0.04、1.64±0.04；酶联免疫吸附测定结果显示，A、B、C组IL-1β的分泌值分别为（40.20±0.25）、（61.50±1.81）、（52.40±1.91）ng/L。在Pg脂多糖刺激下，IL-1β基因和蛋白表达显著增加（P<0.01，P<0.01），同时NALP3基因和蛋白表达显著升高（P<0.01，P<0.01）；运用AC-YVAD-CMK预处理后IL-1β基因表达和蛋白质合成显著减少（P=0.002，P=0.027），NALP3基因表达和蛋白质合成显著降低（P<0.01，P<0.01）。 结论： Pg脂多糖可激活RAW264.7细胞中NALP3炎性体信号通路，阻断该通路可以抑制炎症因子分泌。.