Korean J Physiol Pharmacol.  2014 Oct;18(5):397-402. 10.4196/kjpp.2014.18.5.397.

Regulatory Effect of 25-hydroxyvitamin D3 on Nitric Oxide Production in Activated Microglia

• Hur J
• Lee P
• Kim MJ
• Cho YW

• KMID: 2285538
• DOI: http://doi.org/10.4196/kjpp.2014.18.5.397

Abstract

Microglia are activated by inflammatory and pathophysiological stimuli in neurodegenerative diseases, and activated microglia induce neuronal damage by releasing cytotoxic factors like nitric oxide (NO). Activated microglia synthesize a significant amount of vitamin D3 in the rat brain, and vitamin D3 has an inhibitory effect on activated microglia. To investigate the possible role of vitamin D3 as a negative regulator of activated microglia, we examined the effect of 25-hydroxyvitamin D3 on NO production of lipopolysaccharide (LPS)-stimulated microglia. Treatment with LPS increased the production of NO in primary cultured and BV2 microglial cells. Treatment with 25-hydroxyvitamin D3 inhibited the generation of NO in LPS-activated primary microglia and BV2 cells. In addition to NO production, expression of 1-alpha-hydroxylase and the vitamin D receptor (VDR) was also upregulated in LPS-stimulated primary and BV2 microglia. When BV2 cells were transfected with 1-alpha-hydroxylase siRNA or VDR siRNA, the inhibitory effect of 25-hydroxyvitamin D3 on activated BV2 cells was suppressed. 25-Hydroxyvitamin D3 also inhibited the increased phosphorylation of p38 seen in LPS-activated BV2 cells, and this inhibition was blocked by VDR siRNA. The present study shows that 25-hydroxyvitamin D3 inhibits NO production in LPS-activated microglia through the mediation of LPS-induced 1-alpha-hydroxylase. This study also shows that the inhibitory effect of 25-hydroxyvitamin D3 on NO production might be exerted by inhibiting LPS-induced phosphorylation of p38 through the mediation of VDR signaling. These results suggest that vitamin D3 might have an important role in the negative regulation of microglial activation.

Keyword

25-Hydroxyvitamin D3; 1-alpha-Hydroxylase; Microglia activation; Vitamin D receptor

MeSH Terms

Animals
Brain
Calcifediol*
Cholecalciferol
Microglia*
Negotiating
Neurodegenerative Diseases
Neurons
Nitric Oxide*
Phosphorylation
Rats
Receptors, Calcitriol
RNA, Small Interfering
Calcifediol
Cholecalciferol
Nitric Oxide
RNA, Small Interfering
Receptors, Calcitriol

Figure

• 

  Fig. 1 Effect of 25(OH)D3 on the production of NO in LPS-activated BV2 cells (A) and primary microglia (B). Treatment with 100 ng/ml LPS increased the production of NO in both BV2 cells and primary microglia (Student's t-test). 25(OH)D3 dose-dependently (1, 10 and 100 µmol/L) inhibited the production of NO in 100 ng/ml LPS-stimulated BV2 cells and primary microglia (One-way ANOVA). Mean values were obtained from three independent experiments. **p<0.01, ***p<0.001 when compared to the LPS alone-treated group; ##p<0.001 when compared to the LPS-untreated control group.

• 

  Fig. 2 Effect of LPS on the expression of 1-α-hydroxylase in BV2 cells and primary microglia. The expression of 1-α-hydroxylase mRNA began to increase at 3 h of 100 ng/ml LPS treatment in BV2 cells and at 4 h in primary microglia. The mRNA expression of 1-α-hydroxylase were normalized with β-actin. The inset shows the representative RT-PCR product of 1-α-hydroxylase mRNA in BV-2 cells and microglia. Mean values were obtained from three independent experiments. *p<0.05, **p<0.01 when compared to the untreated control group (Student's t-test).

• 

  Fig. 3 Effect of 1-α-hydroxylase siRNA on the inhibitory effect of 25(OH)D3 in LPS-activated BV2 cells. Knockdown of 1-α-hydroxylase with siRNA (20 µmol/L) suppressed the inhibitory effect of 100 µmol/L 25(OH)D3 on the increased NO production in 100 ng/ml LPS-activated BV2 cells. The transfection efficiency of 1-α-hydroxylase siRNA was measured by Western blotting (inset). Mean values were obtained from three independent experiments. **p<0.01 when compared to the untreated control group (Student's t-test).

• 

  Fig. 4 Effect of VDR siRNA on the inhibitory effect of 25(OH)D3 in LPS-activated BV2 cells. Knockdown of VDR with siRNA (50 µmol/L) blocked the inhibitory effect of 100 µmol/L 25(OH)D3 on the increased NO production in 100 ng/ml LPS-activated BV2 cells. The transfection efficiency of VDR siRNA was measured by Western blotting (inset). Mean values were obtained from three independent experiments. **p<0.01 when compared to the untreated control group (Student's t-test).

• 

  Fig. 5 Effect of 25(OH)D3 and VDR siRNA on p38 phosphorylation in LPS-activated BV2 cells. BV-2 cells were treated with 25(OD)D3 in the presence or absence of VDR siRNA (50 µmol/L). Treatment with 100 µmol/L 25(OH)D3 inhibited the increased phosphorylation of p38 in 100 ng/ml LPS-activated BV2 cells. The extent of phosphorylation of p38 was measured by the ratio of phospho p38 to p38. VDR siRNA blocked the inhibitory effect of 25(OH)D3 on the LPS-induced phosphorylation of p38. The inset shows the representative Western blotting product of p38 and phospho-p38 (p-p38). Mean values were obtained from three independent experiments. *p<0.05 when compared to the untreated control group (Student's t-test).

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