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. 2021 Dec 20;12:759040. doi: 10.3389/fphar.2021.759040

FIGURE 4.

FIGURE 4

Methyl gallate specifically inhibits activation of NLRP3 by blocking the oligomerization of NLRP3 inflammasome in BMDMs. (A,B) BMDMs were treated with different doses of MG for 0.5 h and then stimulated with LPS for 3 h. After that, the cells were stimulated with nigericin. Medium supernatants (SN) were analyzed by ELISA for IL-1β and TNF-α release. (C,D) BMDMs were primed with LPS for 3 h and then stimulated with different doses of MG 0.5 h, then the cells were stimulated with nigericin. SN were analyzed by ELISA for IL-1β and TNF-α release. (E) The release of IL-1β in BMDMs stimulated with the TLR2/4 agonist Pam3CSK4, and then transfected Wild type and NLRP3 effective cells with LPS. Treatment with MG (10, 50 µM) can reduce the release of IL-1β in BMDMs cells. Cells lacking NLRP3 showed low or no IL-1β production. (F) ELISA of IL-1β in SN of LPS-primed BMDMs treated with MG (50 µM) and then stimulated with MSU, nigericin, ATP, Alum, poly A:T and Salmonella. (G) Immunoprecipitation (IP, Up) and analysis (Down, mean ± SEM) of the NLRP3-ASC association in BMDMs cells stimulated with LPS/nigericin. (H) ATPase activity of recombinant NLRP3 in the presence of MG (n = 3), MCC950 (1 μM) (n = 3). MG: methyl gallate, Veh: Vehicle, Con: Control. Data are expressed as mean ± S.D. (n = 6). Statistical differences were calculated by single-factor ANOVA with Tukey HSD test (A–D,G,H), and unpaired Student’s t-test (E,F): * p < 0.05, *** p < 0.001.