Inhibition of Extracellular Calcium Influx Results in Enhanced IL-12 Production in LPS-Treated Murine Macrophages by Downregulation of the CaMKK-AMPK-SIRT1 Signaling Pathway

Figure 6

SIRT1 is upregulated by AMPK activation to suppress IL-12 expression in the LPS-activated RAW 264.7 cells. (a) Cells were treated with 100 ng/mL LPS in normal DMEM or in DMEM without calcium or supplemented with 2 mM CaCl₂ for 1 h. NAD⁺/NADH levels were detected (). (b) Cells were treated with 1 mM AICAR, 100 ng/mL LPS, or LPS plus calcium-free DMEM for 2 h. Protein and mRNA levels of SIRT1 were detected by western blot or real-time PCR. and versus medium; or versus LPS (). (c, d) Cells were treated with LPS alone or with 2 μM EXT527 or 2 μM SRT1720 (c). Cells were also treated with LPS, calcium-free LPS, or calcium-free LPS with SRT1720 (d). Supernatants IL-12 p40 and IL-12 p70 (24 h) were detected by ELISA. and versus LPS for each cytokine (). (e) Cells were treated with NC siRNA or SIRT1 siRNA for 24 h. Protein levels of SIRT1 were detected by western blot (upper). The siRNA-treated cells were further stimulated by 100 ng/mL LPS for 24 h (lower). Supernatants IL-12 p40 and IL-12 p70 were detected. and versus LPS for each cytokine ().

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