[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 mumol/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-1beta (IL-1beta) were detected by quantitative real-time PCR (qPCR) after 6 h, protein expression of NALP3 and IL-1beta 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 mumol/L AC-YVAD-CMK did not significantly affect the viability of RAW264.7 cells. qPCR showed that mRNA expression of IL-1beta 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-1beta 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-1beta (P<0.01, P<0.01) and NALP3 (P<0.01, P<0.01) significantly increased; but the expression of IL-1beta (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.