Regulation of M1/M2 Polarization in LPS-Stimulated Macrophages by 1,25(OH)2D3

Objective center dot This study aims to investigate the impact of 1,25(OH)(2)D3 on the polarization of LPS-stimulated macrophages and the underlying regulatory mechanisms. Methods center dot Primary macrophages were isolated and identified using immunofluorescence assays to detect macrophage biomarker expression levels. RT-PCR was employed to measure the expression of Arginase 1 (Arg-1), Interleukin-10 (IL-10), Inducible isoform of nitric oxide synthase (iNOS), and Tumor necrosis factor-alpha (TNF-alpha) in macrophages treated with various strategies. Western blotting assessed the protein expression levels of AKT1, p-AKT1, NF-kappa B p65, p-NF-kappa B p65, STAT3, and p-STAT3 in LPS- stimulated macrophages exposed to different concentrations of 1,25(OH)(2)D3. Results center dot As the LPS concentration increased from 0 to 0.5 mg/L, Arg-1, IL-10, iNOS, and TNF-alpha expression levels significantly increased. However, at LPS concentrations ranging from 1 mg/L to 10 mg/L, the expression of Arg-1, IL-10, iNOS, and TNF-alpha displayed a trend from increase to decline. The highest M2 polarization (Arg-1 and IL-10) was observed in macrophages stimulated with 0.5 mg/L LPS among the lower concentrations, while the highest M1 polarization (iNOS and TNF-alpha) was observed in macrophages stimulated with 5 mg/L LPS among the higher concentrations. Subsequent experiments utilized 0.5 mg/L and 5 mg/L LPS as incubation concentrations. Under LPS stimulation, iNOS was significantly upregulated, surpassing the expression level of IL-10, a marker of M2 macrophages. The introduction of 1,25(OH)(2)D3 facilitated M2 polarization, with 50 nM as the incubation concentration of 1,25(OH)(2)D3. Furthermore, 1,25(OH)(2)D3 reversed the elevated expression of p-AKT1, p-NF-kappa B p65, and p-STAT3 in macrophages stimulated with 5 mg/L LPS. Conclusions center dot 1,25(OH)(2)D3 effectively regulates the M1/M2 polarization in LPS-stimulated macrophages.