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

To illuminate the effect of NALP3 inflammasome on regulating the expression of cytokines of macrophages in periodontitis. 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 (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. 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 (0.01, 0.01) and NALP3 (0.01, 0.01) significantly increased; but the expression of IL-1β (0.002, 0.027) and NALP3 (0.01, 0.01) were decreased when pretreated with AC-YVAD-CMK. 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.