Lps pg ultrapure

The cell line of RAW 264.7 macrophages was purchased from the ATCC (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle medium (Wako, Tokyo, Japan) with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin under 5% CO₂ at 37°C in a humidified environment. The cells were incubated for 24 hours in the presence or absence of 10 µg/mL P. gingivalis LPS (LPS-PG Ultrapure, InvivoGen, San Diego, CA, USA) to induce an inflammatory response.

For inducing NLRP3, NLRC4 and AIM2 inflammasomes activation, BMDMs at 1 × 10⁶ cells/mL were seeded 0.5 mL in 24-well plates overnight. Then, 50 ng/mL LPS-PG Ultrapure (InvivoGen, San Diego, CA, USA) or 1 μg/mL Pam3CSK4 (InvivoGen) was used to stimulate BMDMs for 4 h, after which the medium was changed to Opti-MEM (Gibco) containing the main components from EF (epmedin A, epmedin A1, epmedin B, epmedin C, icariin, icaritin, icariside Ⅰ, ICS Ⅱ, anhydroicaritin; TargetMol, Boston, MA, USA), hydrogen peroxide (H₂O₂, Sigma, Darmstadt, Germany) or N-acetyl-l-cysteine (NAC, MedChemExpress). One hour later, the cells were stimulated for 1 h with ATP (5 mmol/L; Sigma) and nigericin (7.5 μmol/L; InvivoGen) or for 6 h with monosodium urate (MSU, 200 μg/mL; InvivoGen), silicon dioxide (SiO₂, 250 μg/mL; InvivoGen) or Lfn-Flic (200 ng/mL, supplied by Dr. Tao Li). Cells were transfected with poly (I:C) (2 μg/mL; InvivoGen), poly(dA:dT) (2 μg/mL; InvivoGen) or LPS (1 μg/mL; InvivoGen) for 6 h through the use of Lipofectamine 2000 (Thermo Fisher Scientific, Rockford, MI, USA) according to the manufacturer's protocols. PMA-primed THP-1 cells were seeded at 1 × 10⁶ cells/mL in 24-well plates overnight. Then, the cells were pretreated with ICS Ⅱ. One hour later, the cells were stimulated with nigericin (7.5 μmol/L) for 1 h. Cell extracts and precipitated supernatants were analyzed by immunoblot.