1,25-Dihydroxyvitamin D3 attenuates endotoxin-induced production of inflammatory mediators by inhibiting MAPK activation in primary cortical neuron-glia cultures.

Ya-Ni Huang,Chien-Tsai Chiu,Chien-Cheng Lai,Jia-Yi Wang,Yi-Jung Ho

doi:10.1186/s12974-015-0370-0

Abstract

BackgroundNeuroinflammation occurs in insulted regions of the brain and may be due to reactive oxygen species (ROS), nitric oxide (NO), cytokines, and chemokines produced by activated glia. Excessive production of neurotoxic molecules causes further neuronal damage. Low levels of vitamin D3 are a risk factor for various brain diseases.MethodsUsing the bacterial endotoxin, lipopolysaccharide (LPS), to induce neuroinflammation in primary cortical neuron-glia cultures, we investigated how 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) affected neuroinflammation.ResultsLPS (100 ng/ml) induced the accumulation of nitrite and the production of ROS, interleukin (IL)-6, and macrophage inflammatory protein (MIP)-2 in time-dependent manners. Inhibition of p38 and extracellular signal-regulated kinase (ERK) but not c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) by 20 μM of SB203580, PD98059, and SP600125, significantly reduced LPS-induced ROS production, NO accumulation, and inducible NO synthase (iNOS) expression, respectively. LPS-induced IL-6 and MIP-2 were significantly attenuated by inhibition of p38, ERK, and JNK MAPK. Cotreatment with 1,25(OH)2D3 attenuated LPS-induced ROS production, NO accumulation, and iNOS expression in concentration-dependent manners. 1,25(OH)2D3 also reduced LPS-induced production of IL-6 and MIP-2. Similarly, iNOS, IL-6, and MIP-2 mRNA expression in cells treated with LPS significantly increased, whereas this effect was attenuated by 1,25(OH)2D3. Moreover, LPS-induced phosphorylation of p38, ERK, and JNK MAPK was significantly inhibited by 1,25(OH)2D3.ConclusionsOur findings indicate that 1,25(OH)2D3 reduced the LPS-stimulated production of inflammatory molecules in neuron-glia cultures by inhibiting MAPK pathways and the production of downstream inflammatory molecules. We suggest that 1,25(OH)2D3 can be used to alleviate neuroinflammation in various brain injuries.

Highlights

Neuroinflammation occurs in insulted regions of the brain and may be due to reactive oxygen species (ROS), nitric oxide (NO), cytokines, and chemokines produced by activated glia
Our findings indicate that 1,25(OH)2D3 reduced the LPS-stimulated production of inflammatory molecules in neuron-glia cultures by inhibiting mitogen-activated protein kinase (MAPK) pathways and the production of downstream inflammatory molecules
These results suggest that activation of p38, extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK) MAPKs is involved in LPS-induced ROS production, nitrite accumulation, and proinflammatory mediator release

Summary

Results

LPS-induced ROS production, nitrite accumulation, and the release of proinflammatory mediators To examine whether LPS elicited neuroinflammatory responses in cultured neurons/glia, we first examined the time course of LPS-induced ROS production, nitrite accumulation, and the release of IL-6 (a cytokine) and MIP-2 (a chemokine). Western blot analyses indicated that cotreatment with SB203580 or PD98059 and LPS for 24 h significantly attenuated the level of LPS-induced iNOS expression (p < 0.001 and p < 0.01, respectively), whereas cells cotreated with the JNK inhibitor and LPS exhibited no effect (Fig. 2c). Levels of IL-6 and MIP-2 production in cultures treated with SB203580, PD98059, or SP600125 alone did not significantly differ from those produced in control cultures (data not shown) These results suggest that activation of p38, ERK, and JNK MAPKs is involved in LPS-induced ROS production, nitrite accumulation, and proinflammatory mediator release. Activation of the phosphorylation of p38, ERK, and JNK did not significantly differ among control cultures and those treated with MAPK inhibitors or 1,25(OH)2D3 alone (data not shown) These results suggest that 1,25(OH)2D3 suppressed the production of LPS-induced inflammatory mediators by inhibiting the p38, ERK, and JNK MAPK pathways

Conclusions

Materials and methods

Discussion

Conclusion