[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.