Amlexanox Exerts Anti-inflammatory Actions By Targeting Phosphodiesterase 4B In Lipopolysaccharide-activated Macrophages

Background:In 1996,amlexanox became the first product approved by the US FDA for the treatment of aphthous ulcers.Until now,it has been applied in more than 20 countries as a first-line anti-inflammatory drug for this indication.Although amlexanox is able to alleviate classical inflammation which is characterized by "calor,dolor,rubor and tumor",namely "hot inflammation",the precise molecular mechanism and target is still unknown.By contrast,the possibility that amlexanox might be repurposed for treating metaflammation,namely "cold inflammation",has caught extensive attention.Recent studies showed that amlexanox exhibited exact improvement on obese patients with type 2 diabetes and nonalcoholic fatty liver disease,while this effect resulted from inhibiting homologous kinases IKKε/TBK1,a couple of novel targets in treating cold inflammation.However,it is strange that in LPS-primed macrophages,the absence of IKKε cannot decrease the expression of inflammatory cytokines,or rather increase it.What makes things much more confusing is that in the same model,IKKε/TBK1 inhibitor amlexanox exerts definitely anti-inflammatory action.Objective:To explore the anti-inflammatory effect of amlexanox and the molecular mechanism(s)in LPS-induced hot inflammation models.Methods:LPS was administered intravenously to induce the in vivo hot inflammatory model,while for the in vitro one,macrophages were exposed to LPS.The effects of amlexanox on inflammation-related cytokine production were quantified with Griess method and ELISA.Multimode microplate reader was used to obtain the three-dimensional fluorescence spectra of amlexanox,while laser scanning confocal microscopy was employed to analyse its intracellular distribution.In LPS-primed macrophages,RT-qPCR,luciferase reporter gene assay and Western Blotting were performed to determine the actions of amlexanox on NF-κB and MAPK/AP-1 inflammatory signal pathways.Then molecular docking,fluorescence polarization,enzyme activity assay in cell-free system along with siRNA techniques were applied to explore its anti-inflammatory target.Results:Intravenous injection of LPS led to marked increase in TNF-α and IL-6 levels in serum,while single-dose oral administration of amlexanox significantly decreased these two cytokines,and it could also significantly reduce TNF-α level in peritoneum lavage fluid.In LPS-activated macrophages,amlexanox not only suppressed TNF-α expression,but also inhibited NO production,iNOS activity and its expression,as well as increased the secretion of the anti-inflammatory cytokine IL-10.Mechanistically,for one thing,amlexanox prevented LPS-induced phosphorylation and degradation of IκBα,suppressed subsequent p65 translocation and the transcriptional activity of NF-κB;for another,it blocked ERK1/2 phosphorylation and downstream AP-1 activation.Further,in LPS-primed macrophages,amlexanox could elevate and sustain cAMP level and activate its effector kinase PKA,along with regulating intracellular chloride concentration and nigericin-induced IL-1β secretion.Furthermore,cAMP antagonist could block the inhibitory effects of amlexanox on NO and TNF-α.Accidentally,fluorescent property of amlexanox was discovered(Ex/Em=352 nm/398 nm).By virue of this property,we found that amlexanox permeated into the cells in a dynamic process within 10 min and distributed mainly throughout the cytoplasm of macrophages.Molecular docking result suggested that amlexanox could occupy the cAMP hydrolytic pocket of PDE4B through aπ-π stacking interaction with Phe446 and electrostatic interactions with Zn2+and Mg2+.Then fluorescence polarization(FP)verified that PDE4B protein led to an increase in the FP value of amlexanox,indicating that amlexanox could directly bind to PDE4B protein.Moreover,the FP-quantified binding of amlexanox to PDE4B could be partially displaced by IBMX,a non-selective PDE inhibitor.The PDE inhibition assay showed that amlexanox directly inhibited not only the activity of recombinant human PDE4B(IC50=11 μM),but also that of recombinant human PDE1C,PDE3 A,and PDE3B,with IC50 values of 18 μM,3 μM,and 18 μM,showing that amlexanox is a non-selective PDEs inhibitor.However,the application of selective PDE3 inhibitor didn’t influence LPS-induced cytokines’production.As a contrast,knockdown of PDE4B in RAW264.7 cells abolished anti-inflammatory actions of amlexanox.Conclusion:The present study firstly identified amlexanox as a non-selective PDEs inhibitor.In LPS-primed RAW264.7 macrophages,amlexanox directly binds to cytoplasmic PDE4B and inhibits it activity to prevent the hydrolysis of cAMP,and thus activates its effector protein PKA.Activated PKA can not only stabilize IκBα and prevent subsequent p65 nuclear translocation to suppress NF-κB activation,but also block ERK1/2 phosphorylation to inhibit downstream AP-1 activation.Whereafter,inhibitory effects of amlexanox on these two pathways lead to decreased expression of iNOS and TNF-α,and ultimately mitigate hot inflammation.