1,25-Dihydroxyvitamin D₃ Promotes High Glucose Induced M1 Macrophage Switching To M2 Via VDR-PPAR? Pathway

Objectives:Macrophages, besides their quantities in renal tissue, especially their activation state, are closely related to the progression of diabetic nephropathy (DN). Classically activated macrophages (Ml) are pro-inflammatory effectors, while alternatively activated macrophages (M2) exhibit anti-inflammatory properties. Recent studies demonstrate that PPARy, a nuclear receptor, is essential for macrophage polarization and 1,25-Dihydroxyvitamin D3 has renoprotective roles that extend beyond the regulation of mineral metabolism. But the exact mechanism has not been elucidated. This study tries to investigate the effect of 1,25(OH)2D3 on glucose induced macrophage activation state and its underlying mechanism in RAW264.7 cells.Methods:RAW264.7 cells were incubated in a glucose-dose (11.1mM,20mM, 25mM,30mM) and a time (Oh,6h,12h,24h,36h,48h) dependent manner, the activity of intracellular iNOS was measured. VDR siRNA and PPARy antagonist pre-treatment with macrophages were done before using 10-8 mol/L1,25(OH)2D3 to intervene high glucose pre-incubated macrophages. The M1 marker iNOS, M2 markers including MR?Arg-1 and nuclear receptors, VDR and PPARy were detected by RT-PCR and Western blot. Cytokines including TNF-??IL-12 and IL-10 in supernate were examined by ELISA. Immunofluorescence Staining was used to further observe their expressions.Results:(1) The iNOS activity was increased in a glucose-dose and time dependent manner. Particularly,25mmol/L glucose at 24h gave the maximum response. After being treated with 25mmol/L glucose for 24h, not only inflammatory cytokines of TNF-a, IL-12 in the supernatant were increased, but RT-PCR and Western blot analysis showed Ml marker iNOS was also up-regulated (P<0.05). However, M2 markers, i.e. MR and Arg-1 were significantly decreased (P<0.05). (2) When in the presence of 1,25(OH)2D3, the trends were reversed: the markers of M1, including TNFa, IL-12 and iNOS were obviously reduced, while M2 markers, IL-10, Arg-1 and MR were increased. In addition, VDR and PPARy were also increased (P<0.05). (3) However, the above effects of 1,25(OH)2D3 were abolished when further inhibited the expression of VDR and PPARy by VDR siRNA and PPARy antagonist. Besides, accompanied by VDR, PPARy was also decreased upon the treatment with VDR siRNA (VD versus VDR siRNA:1.16±0.08 vs 0.48±0.05; P<0.05). Immunofluorescence Staining also showed enhanced iNOS but weak MR and PPARy fluorescent expression after inhibition the expression of VDR and PPARy when compared with VD group.Conclusions:1. High glucose induces macrophages toward a M1 phenotype.2.1,25(OH)2D3 can regulate macrophage activation state, that is polarizes high glucose-induced M1 macrophages toward a M2 phenotype.3.VDR-PPARy pathway may play an important role in 1,25-dihydroxyvitamin D3 regulating macrophage activation state.