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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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Dose-dependent inhibition of NO and TNF-α production by α-GalCer analogs 4d and 4e in LPS-stimulated BV2 microglial cells.Cells were incubated for 16 h with LPS in the absence or presence of α-GalCer analogs 4d and 4e (5–10 µM), and the amounts of released NO and TNF-α were measured in supernatants. Treatment with α-GalCer analogs alone did not affect NO or TNF-α production. Bars indicate the mean ± S.E.M. of three independent experiments. *P<0.05; significantly different from LPS-treated microglial cells.
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pone-0087030-g006: Dose-dependent inhibition of NO and TNF-α production by α-GalCer analogs 4d and 4e in LPS-stimulated BV2 microglial cells.Cells were incubated for 16 h with LPS in the absence or presence of α-GalCer analogs 4d and 4e (5–10 µM), and the amounts of released NO and TNF-α were measured in supernatants. Treatment with α-GalCer analogs alone did not affect NO or TNF-α production. Bars indicate the mean ± S.E.M. of three independent experiments. *P<0.05; significantly different from LPS-treated microglial cells.

Mentions: To identify the best candidate for further in vitro study, we analyzed individual inhibition activities via sorting the most effective inhibitors in three independent assays. Anti-inflammatory compounds identified more than twice among the top five inhibitors of NO, ROS, or TNF-α production are highlighted in Figure 5 as dark orange, and compounds identified once among the top five inhibitors are highlighted in pale orange. This color code-based analysis revealed somewhat interesting patterns in structure-activity relationships of α-GalCer analogs in their inhibitory activity on NO, ROS, and TNF-α production. For instance, N-modifications of the pyrazole moiety with phenyl-containing alkyl chains in the sphingosine backbone (c, d, and e) showed general anti-inflammatory activity. In addition, α-GalCer analogs containing relatively short acyl chains (3 and 4) showed significant inhibitory activity on NO, ROS, and TNF-α release in LPS-induced BV2 microglial cells. In the case of α-GalCer analogs (5) containing triazole—an isostere of amide bonds—in acyl chains, we observed slight inhibitory activity on cellular production of NO, ROS, and TNF-α. Among the 25 α-GalCer analogs, 4d and 4e, which have relatively short acyl chains containing terminal phenyl rings and sphingosine backbones containing pyrazole and phenyl rings, showed the best efficacy in terms of percent inhibition of NO, ROS, and TNF-α production in LPS-induced microglia. We also confirmed the dose-dependent inhibition of 4d and 4e on NO and TNF-α production (Figure 6) and determined IC50 values; in the case of 4d, IC50s for NO and TNF-α release were 13.8 and 9.1 µM, respectively, and in case of 4e, IC50s for NO and TNF-α release were 10.2 and 6.3 µM. To exclude the possibility that the decrease in NO and cytokine levels was simply due to cell death, we assessed cell viability at various concentrations of 4d and 4e. MTT assay showed that 4d and 4e were not cytotoxic at concentrations up to 10 µM (data not shown). Furthermore, DMSO (up to 0.3%) did not affect NO, TNF-α, ROS production in LPS-stimulated BV2 cells, which confirmed that the cellular inhibitory activity is caused by the treatment of 4d and 4e, not due to DMSO (data not shown). Therefore, we further investigated the anti-inflammatory mechanism of 4d and 4e in activated microglia.


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)

Dose-dependent inhibition of NO and TNF-α production by α-GalCer analogs 4d and 4e in LPS-stimulated BV2 microglial cells.Cells were incubated for 16 h with LPS in the absence or presence of α-GalCer analogs 4d and 4e (5–10 µM), and the amounts of released NO and TNF-α were measured in supernatants. Treatment with α-GalCer analogs alone did not affect NO or TNF-α production. Bars indicate the mean ± S.E.M. of three independent experiments. *P<0.05; significantly different from LPS-treated microglial cells.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0087030-g006: Dose-dependent inhibition of NO and TNF-α production by α-GalCer analogs 4d and 4e in LPS-stimulated BV2 microglial cells.Cells were incubated for 16 h with LPS in the absence or presence of α-GalCer analogs 4d and 4e (5–10 µM), and the amounts of released NO and TNF-α were measured in supernatants. Treatment with α-GalCer analogs alone did not affect NO or TNF-α production. Bars indicate the mean ± S.E.M. of three independent experiments. *P<0.05; significantly different from LPS-treated microglial cells.
Mentions: To identify the best candidate for further in vitro study, we analyzed individual inhibition activities via sorting the most effective inhibitors in three independent assays. Anti-inflammatory compounds identified more than twice among the top five inhibitors of NO, ROS, or TNF-α production are highlighted in Figure 5 as dark orange, and compounds identified once among the top five inhibitors are highlighted in pale orange. This color code-based analysis revealed somewhat interesting patterns in structure-activity relationships of α-GalCer analogs in their inhibitory activity on NO, ROS, and TNF-α production. For instance, N-modifications of the pyrazole moiety with phenyl-containing alkyl chains in the sphingosine backbone (c, d, and e) showed general anti-inflammatory activity. In addition, α-GalCer analogs containing relatively short acyl chains (3 and 4) showed significant inhibitory activity on NO, ROS, and TNF-α release in LPS-induced BV2 microglial cells. In the case of α-GalCer analogs (5) containing triazole—an isostere of amide bonds—in acyl chains, we observed slight inhibitory activity on cellular production of NO, ROS, and TNF-α. Among the 25 α-GalCer analogs, 4d and 4e, which have relatively short acyl chains containing terminal phenyl rings and sphingosine backbones containing pyrazole and phenyl rings, showed the best efficacy in terms of percent inhibition of NO, ROS, and TNF-α production in LPS-induced microglia. We also confirmed the dose-dependent inhibition of 4d and 4e on NO and TNF-α production (Figure 6) and determined IC50 values; in the case of 4d, IC50s for NO and TNF-α release were 13.8 and 9.1 µM, respectively, and in case of 4e, IC50s for NO and TNF-α release were 10.2 and 6.3 µM. To exclude the possibility that the decrease in NO and cytokine levels was simply due to cell death, we assessed cell viability at various concentrations of 4d and 4e. MTT assay showed that 4d and 4e were not cytotoxic at concentrations up to 10 µM (data not shown). Furthermore, DMSO (up to 0.3%) did not affect NO, TNF-α, ROS production in LPS-stimulated BV2 cells, which confirmed that the cellular inhibitory activity is caused by the treatment of 4d and 4e, not due to DMSO (data not shown). Therefore, we further investigated the anti-inflammatory mechanism of 4d and 4e in activated microglia.

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