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Anti-inflammatory effects of α-galactosylceramide analogs in activated microglia: involvement of the p38 MAPK signaling pathway.

Jeong YH, Kim Y, Song H, Chung YS, Park SB, Kim HS - PLoS ONE (2014)

Bottom Line: Here, we designed and synthesized α-galactosylceramide (α-GalCer) analogs to exert anti-inflammatory effects in activated microglia.After identification of 4d and 4e as hit compounds, we further investigated the underlying mechanism of their anti-inflammatory effects using RT-PCR analysis.Taken together, these results suggest that p38 MAPK plays an important role in the anti-inflammatory effects of 4d and 4e via the modulation of NF-κB and AP-1 activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine and Global Top5 Research Program, Tissue Injury Defense Research Center, Ewha Womans University Medical School, Seoul, Republic of Korea.

ABSTRACT
Microglial activation plays a pivotal role in the development and progression of neurodegenerative diseases. Thus, anti-inflammatory agents that control microglial activation can serve as potential therapeutic agents for neurodegenerative diseases. Here, we designed and synthesized α-galactosylceramide (α-GalCer) analogs to exert anti-inflammatory effects in activated microglia. We performed biological evaluations of 25 α-GalCer analogs and observed an interesting preliminary structure-activity relationship in their inhibitory influence on NO release and TNF-α production in LPS-stimulated BV2 microglial cells. After identification of 4d and 4e as hit compounds, we further investigated the underlying mechanism of their anti-inflammatory effects using RT-PCR analysis. We confirmed that 4d and 4e regulate the expression of iNOS, COX-2, IL-1β, and IL-6 at the mRNA level and the expression of TNF-α at the post-transcriptional level. In addition, both 4d and 4e inhibited LPS-induced DNA binding activities of NF-κB and AP-1 and phosphorylation of p38 MAPK without affecting other MAP kinases. When we examined the anti-inflammatory effect of a p38 MAPK-specific inhibitor, SB203580, on microglial activation, we observed an identical inhibitory pattern as that of 4d and 4e, not only on NO and TNF-α production but also on the DNA binding activities of NF-κB and AP-1. Taken together, these results suggest that p38 MAPK plays an important role in the anti-inflammatory effects of 4d and 4e via the modulation of NF-κB and AP-1 activities.

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The p38 MAPK-specific inhibitor SB203580 inhibited NO and TNF-α production by suppressing NF-κB and AP-1 activities.(A) BV2 cells were treated with LPS for 16 h in the absence or presence of SB203580, and released NO and TNF-α were measured as previously described. Bars indicate the mean ± S.E.M. of three independent experiments. *P<0.05; significantly different from LPS-treated cells. SB indicates SB203580. (B) Effect of SB203580 on LPS-induced NF-κB and AP-1 DNA binding activities.
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pone-0087030-g010: The p38 MAPK-specific inhibitor SB203580 inhibited NO and TNF-α production by suppressing NF-κB and AP-1 activities.(A) BV2 cells were treated with LPS for 16 h in the absence or presence of SB203580, and released NO and TNF-α were measured as previously described. Bars indicate the mean ± S.E.M. of three independent experiments. *P<0.05; significantly different from LPS-treated cells. SB indicates SB203580. (B) Effect of SB203580 on LPS-induced NF-κB and AP-1 DNA binding activities.

Mentions: To analyze the molecular mechanism underlying the anti-inflammatory effects of 4d and 4e, we further examined their inhibitory effect on phosphorylation of MAP kinases, which are upstream signaling molecules in inflammatory responses. Western blot analysis revealed that 4d and 4e markedly inhibited LPS-induced p38 MAPK phosphorylation. However, 4d and 4e did not affect phosphorylation of ERK or JNK (Figure 9A). In addition, 4d and 4e inhibited the DNA binding activities of NF-κB and AP-1, which are key transcription factors for inflammatory gene expression in microglial cells (Figure 9B). To confirm an involvement of the p38 pathway in the anti-inflammatory effects of 4d and 4e, we examined the effect of a p38 MAPK-specific inhibitor, SB203580, on microglial activation. As shown in Figure 10A, SB203580, like 4d and 4e, significantly inhibited NO and TNF-α production in LPS-stimulated microglia. Furthermore, identical to 4d and 4e, SB203580 inhibited the DNA binding activities of NF-κB and AP-1 (Figure 10B). Thus, these data collectively suggest that p38 MAPK plays a key role in the anti-inflammatory effects of 4d and 4e via the modulation of NF-κB and AP-1 activities.


Anti-inflammatory effects of α-galactosylceramide analogs in activated microglia: involvement of the p38 MAPK signaling pathway.

Jeong YH, Kim Y, Song H, Chung YS, Park SB, Kim HS - PLoS ONE (2014)

The p38 MAPK-specific inhibitor SB203580 inhibited NO and TNF-α production by suppressing NF-κB and AP-1 activities.(A) BV2 cells were treated with LPS for 16 h in the absence or presence of SB203580, and released NO and TNF-α were measured as previously described. Bars indicate the mean ± S.E.M. of three independent experiments. *P<0.05; significantly different from LPS-treated cells. SB indicates SB203580. (B) Effect of SB203580 on LPS-induced NF-κB and AP-1 DNA binding activities.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3921125&req=5

pone-0087030-g010: The p38 MAPK-specific inhibitor SB203580 inhibited NO and TNF-α production by suppressing NF-κB and AP-1 activities.(A) BV2 cells were treated with LPS for 16 h in the absence or presence of SB203580, and released NO and TNF-α were measured as previously described. Bars indicate the mean ± S.E.M. of three independent experiments. *P<0.05; significantly different from LPS-treated cells. SB indicates SB203580. (B) Effect of SB203580 on LPS-induced NF-κB and AP-1 DNA binding activities.
Mentions: To analyze the molecular mechanism underlying the anti-inflammatory effects of 4d and 4e, we further examined their inhibitory effect on phosphorylation of MAP kinases, which are upstream signaling molecules in inflammatory responses. Western blot analysis revealed that 4d and 4e markedly inhibited LPS-induced p38 MAPK phosphorylation. However, 4d and 4e did not affect phosphorylation of ERK or JNK (Figure 9A). In addition, 4d and 4e inhibited the DNA binding activities of NF-κB and AP-1, which are key transcription factors for inflammatory gene expression in microglial cells (Figure 9B). To confirm an involvement of the p38 pathway in the anti-inflammatory effects of 4d and 4e, we examined the effect of a p38 MAPK-specific inhibitor, SB203580, on microglial activation. As shown in Figure 10A, SB203580, like 4d and 4e, significantly inhibited NO and TNF-α production in LPS-stimulated microglia. Furthermore, identical to 4d and 4e, SB203580 inhibited the DNA binding activities of NF-κB and AP-1 (Figure 10B). Thus, these data collectively suggest that p38 MAPK plays a key role in the anti-inflammatory effects of 4d and 4e via the modulation of NF-κB and AP-1 activities.

Bottom Line: Here, we designed and synthesized α-galactosylceramide (α-GalCer) analogs to exert anti-inflammatory effects in activated microglia.After identification of 4d and 4e as hit compounds, we further investigated the underlying mechanism of their anti-inflammatory effects using RT-PCR analysis.Taken together, these results suggest that p38 MAPK plays an important role in the anti-inflammatory effects of 4d and 4e via the modulation of NF-κB and AP-1 activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine and Global Top5 Research Program, Tissue Injury Defense Research Center, Ewha Womans University Medical School, Seoul, Republic of Korea.

ABSTRACT
Microglial activation plays a pivotal role in the development and progression of neurodegenerative diseases. Thus, anti-inflammatory agents that control microglial activation can serve as potential therapeutic agents for neurodegenerative diseases. Here, we designed and synthesized α-galactosylceramide (α-GalCer) analogs to exert anti-inflammatory effects in activated microglia. We performed biological evaluations of 25 α-GalCer analogs and observed an interesting preliminary structure-activity relationship in their inhibitory influence on NO release and TNF-α production in LPS-stimulated BV2 microglial cells. After identification of 4d and 4e as hit compounds, we further investigated the underlying mechanism of their anti-inflammatory effects using RT-PCR analysis. We confirmed that 4d and 4e regulate the expression of iNOS, COX-2, IL-1β, and IL-6 at the mRNA level and the expression of TNF-α at the post-transcriptional level. In addition, both 4d and 4e inhibited LPS-induced DNA binding activities of NF-κB and AP-1 and phosphorylation of p38 MAPK without affecting other MAP kinases. When we examined the anti-inflammatory effect of a p38 MAPK-specific inhibitor, SB203580, on microglial activation, we observed an identical inhibitory pattern as that of 4d and 4e, not only on NO and TNF-α production but also on the DNA binding activities of NF-κB and AP-1. Taken together, these results suggest that p38 MAPK plays an important role in the anti-inflammatory effects of 4d and 4e via the modulation of NF-κB and AP-1 activities.

Show MeSH
Related in: MedlinePlus