The attenuating effects of 1,2,3,4,6 penta-O-galloyl-β-d-glucose on pro-inflammatory responses of LPS/IFNγ-activated BV-2 microglial cells through NFƙB and MAPK signaling pathways

Abstract

Background: Overactivated microglial cells exhibit chronic inflammatory response and can lead to the continuous production of pro-inflammatory cytokines, perpetuating inflammation, and ultimately resulting in neuronal injury. 1,2,3,4,6-Penta-O-Galloyl-β-d-Glucose (PGG), which is a naturally occurring polyphenolic compound, has exhibited anti-inflammatory effect through the inhibition of many cytokines in different experimental models, but its effect on activated microglia cells was never described. In the present study, we investigated PGG effect in proteins involved in the NFƙB and MAPK signaling pathways, which play a central role in inflammation through their ability to induce transcription of pro-inflammatory genes.

Methods: PCR arrays and RT-PCR with individual primers were used to determine the effect of PGG on mRNA expression of genes involved in NFƙB and MAPK signaling pathways. Western blots were performed to confirm PCR results.

Results: The data obtained showed that PGG modulated the expression of 5 genes from the NFƙB (BIRC3, CHUK, IRAK1, NFƙB1, NOD1) and 2 genes from MAPK signaling pathway (CDK2 and MYC) when tested in RT-PCR assays. Western blots confirmed the PCR results at the protein level, showing that PGG attenuated the expression of total and phosphorylated proteins (CDK2, CHUK, IRAK1, and NFƙB1) involved in NFƙB and MAPK signaling.

Conclusion: These findings show that PGG could modulate the expression of genes and proteins involved in the production of pro-inflammatory cytokines in microglia cells.

Keywords: 1,2,3,4,6-penta-O-galloyl-β-d-glucose; Microglia cells; NFƙB and MAP kinase signaling pathways.

Figure 1:. NFƙB (A) and MAPK (B) PCR array layout.

Figures A and B show the tested genes in each one of the arrays. The red circles show the genes down or up-regulated after PGG pre-treatment (1 h before) in LPS/IFNγ activated BV-2 microglia cells.

Figure 2:. RT-PCR Assay (individual primers): Proteins from NFƙB Signaling.

The effect of PGG on the expression of genes involved in the NFƙB signaling pathway: BIRC3 (A), CHUK (B), IRAK1 (C), NFƙB1 (D), and NOD1 (E). Graph bars show control, PGG (25µM), LPS/IFNy, and PGG pre-treatment (PGG (1 h before) + LPS/IFNγ). Data represent protein expression as the mean ± S.E.M (n=3).). Statistical significance of the difference between control and different treatments was determined by a one-way ANOVA, followed by a Dunnett multiple comparison test. *p≤0.05, **p≤0.01, ***p≤0.001and ****p≤0.0001, ns: not significant.

Figure 3:. RT-PCR Assay (individual primers): Proteins from MAPK Signaling.

The effect of PGG on the expression of genes involved in the MAPK signaling pathway: CDK2 (A) and MYC (B). Graph bars show control, PGG (25µM), LPS/IFNy, and PGG pre-treatment (PGG (1 h before) + LPS/IFNγ). Data represent protein expression as the mean ± S.E.M (n=3).). Statistical significance of the difference between control and different treatments was determined by a one-way ANOVA, followed by a Dunnett multiple comparison test. *p≤0.05, **p≤0.01, ***p≤0.001and ****p≤0.0001, ns: not significant.

Figure 4:. Western Blot Assay.

The effect of PGG on the expression of total proteins involved in the NFƙB (CHUK, IRAK1, NFƙB1, and NOD1) and MAPK signaling pathways (CDK2 and MYC). Bands (A) represent control, PGG (25µM), LPS/IFNγ, and PGG pre-treatment (PGG (1 h before) + LPS/IFNγ) after 24 hours of treatment. B shows the protein expression of normalized intensity, expressed as % of control of the proteins inhibited by PGG treatment (mean ± S.E.M. n=3).

Figure 5:. ProteinSimple Western Assay.

The effect of PGG in the expression of the phosphorylated proteins: IRAK1, CHUK, NFƙB1, and CDK2. Bands represent control, PGG (25µM), LPS/IFNy, and PGG pre-treatment (PGG (1 h before) + LPS/IFNγ) after 24 hours of treatment (n=3).

Figure 6:. Protein Association Network.

A- Interactions among IRAK1, CHUK, NFƙB1, and CDK2. B- Interaction among IRAK1, CHUK, NFƙB1, and CDK2 and other proteins in the same network. C- Table showing the protein interactions and the suggested score for the functional link between them, according to STRING database.

Figure 7:. Chemokine (A) and Toll-like Receptor (B) Signaling Pathways.

The figure shows the activation pathway for cytokine production, including the steps of receptor activation, gene transcription, and cytokine production. Red stars show where CHUK, IRAK1, and NFƙB1 play their role (KEGG Pathway).

Figure 8:

Proposed effects of PGG on the NFƙB and MAPK signaling proteins involved in the release of MCP-5 and Pro MMP-9 cytokines.