Isoorientin Inhibits Inflammation in Macrophages and Endotoxemia Mice by Regulating Glycogen Synthase Kinase 3β

Abstract

Isoorientin has anti-inflammatory effects; however, the mechanism remains unclear. We previously found isoorientin is an inhibitor of glycogen synthase kinase 3β (GSK3β) in vitro. Overactivation of GSK3β is associated with inflammatory responses. GSK3β is inactivated by phosphorylation at Ser9 (i.e., p-GSK3β). Lithium chloride (LiCl) inhibits GSK3β and also increases p-GSK3β (Ser9). The present study investigated the anti-inflammatory effect and mechanism of isoorientin via GSK3β regulation in lipopolysaccharide- (LPS-) induced RAW264.7 murine macrophage-like cells and endotoxemia mice. LiCl was used as a control. While AKT phosphorylates GSK3β, MK-2206, a selective AKT inhibitor, was used to activate GSK3β via AKT inhibition (i.e., not phosphorylate GSK3β at Ser9). The proinflammatory cytokines TNF-α, IL-6, and IL-1β were detected by ELISA or quantitative real-time PCR, while COX-2 by Western blotting. The p-GSK3β and GSK3β downstream signal molecules, including NF-κB, ERK, Nrf2, and HO-1, as well as the tight junction proteins ZO-1 and occludin were measured by Western blotting. The results showed that isoorientin decreased the production of TNF-α, IL-6, and IL-1β and increased the expression of p-GSK3β in vitro and in vivo, similar to LiCl. Coadministration of isoorientin and LiCl showed antagonistic effects. Isoorientin decreased the expression of COX-2, inhibited the activation of ERK and NF-κB, and increased the activation of Nrf2/HO-1 in LPS-induced RAW264.7 cells. Isoorientin increased the expressions of occludin and ZO-1 in the brain of endotoxemia mice. In summary, isoorientin can inhibit GSK3β by increasing p-GSK3β and regulate the downstream signal molecules to inhibit inflammation and protect the integrity of the blood-brain barrier and the homeostasis in the brain.

Anti-Neuroinflammatory Effects of a Semi-Synthetic Isoorientin-Based Glycogen Synthase Kinase-3β Inhibitor in Lipopolysaccharide-Activated Microglial Cells

“…We have shown that TFGF-18 suppressed the nuclear concentration of NF-κB/p65 and increased IκBα expression in activated microglia, indicating a mechanistic role of GSK-3β in modulating the NF-κB/p65 pathway leading to the noted reduction in levels of inflammatory mediators. Regulation of GSK-3β activity by TFGF-18 was confirmed in experiments where the pretreatment increased the phosphorylation of GSK-3β­(Ser9) and decreased phosphorylation of GSK-3β­(Tyr216) as GSK-3β activity is negatively regulated by phosphorylation on Ser9 and positively regulated by phosphorylation on Tyr216. , However, TDZD-8 pretreatment (10 μM) did not significantly change the phosphorylation of GSK-3β­(Ser9) or GSK-3β­(Tyr216). In addition, the nuclear concentration of NF-κB/p65 and expression of IκBα remained unchanged in comparison with the group treated with LPS alone.…”

“…Regulation of GSK-3β activity by TFGF-18 was confirmed in experiments where the pretreatment increased the phosphorylation of GSK-3β(Ser9) and decreased phosphorylation of GSK-3β(Tyr216) as GSK-3β activity is negatively regulated by phosphorylation on Ser9 and positively regulated by phosphorylation Tyr216. 42,43 However, TDZD-8 pretreatment (10 μM) did not significantly change the phosphorylation of GSK-3β(Ser9) or GSK-3β(Tyr216). In addition, the nuclear concentration of NF-κB/p65 and expression of IκBα remained unchanged in comparison with the group treated with LPS alone.…”

Astragalus polysaccharide improves diabetic ulcers by promoting M2-polarization of macrophages to reduce excessive inflammation via the β-catenin/ NF-κB axis at the late phase of wound-healing

“…2 D). To our knowledge, glycogen synthetic kinase-3β (GSK-3β) [ 22 ], β-catenin [ 23 ] and R-Spondin 3 (RSPO3) [ 24 ] were the upstream genes of NF-κB. Through auto-docking assay, we found that APS bound with GSK-3β ( Fig.…”

Anti-Inflammatory Effects of Phytochemical Components of Clinacanthus nutans

“…The phytochemical also decreased activation of ERK and NF-κB and expression of COX-2, and increased activation of Nrf2/HO-1, in LPS-induced RAW264.7 cells. These results suggest an effect of isoorientin in reducing inflammation and protecting the integrity of the blood–brain barrier in endotoxemic mice [ 93 ]. Isoorientin could also modulate Aβ25-35-induced increase in pro-inflammatory mediators such as IL-6, TNF-α, and COX-2, as well as ROS production in BV-2 microglial cells [ 94 ].…”