Abstract
Backgroundα/β-Hydrolase domain 6 (ABHD6) is one of the major enzymes for endocannabinoid 2-arachidonoylglycerol (2-AG) hydrolysis in microglia cells. Our recent studies have shown that a selective ABHD6 inhibitor WWL70 has anti-inflammatory and neuroprotective effects in animal models of traumatic brain injury and multiple sclerosis. However, the role of ABHD6 in the neuroinflammatory response and the mechanisms by which WWL70 suppresses inflammation has not yet been elucidated in reactive microglia.MethodsThe hydrolytic activity and the levels of 2-AG in BV2 cells were measured by radioactivity assay and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) synthases in microglia treated with lipopolysaccharide (LPS) with/without WWL70 was determined by western blot and quantitative RT-PCR. The conversion of 2-AG to PGE2 or PGE2-glyceryl ester (PGE2-G) was assessed by enzyme-linked immunoassay (EIA) or LC-MS/MS. The involvement of ABHD6 in PGE2 production was assessed using pharmacological inhibitors and small interfering RNA (siRNA). The effect of WWL70 on PGE2 biosynthesis activity in the microsome fraction from BV2 cells and experimental autoimmune encephalopathy (EAE) mouse brain was also examined.ResultsWe found that WWL70 suppressed PGE2 production in LPS-activated microglia via cannabinoid receptor-independent mechanisms, although intracellular levels of 2-AG were elevated by WWL70 treatment. This reduction was not attributable to WWL70 inhibition of ABHD6, given the fact that downregulation of ABHD6 by siRNA or use of KT182, an alternative ABHD6 inhibitor failed to suppress PGE2 production. WWL70 attenuated the expression of COX-2 and PGES-1/2 leading to the downregulation of the biosynthetic pathways of PGE2 and PGE2-G. Moreover, PGE2 production from arachidonic acid was reduced in the microsome fraction, indicating that WWL70 also targets PGE2 biosynthetic enzymes, which are likely to contribute to the therapeutic mechanisms of WWL70 in the EAE mouse model.ConclusionsWWL70 is an anti-inflammatory therapeutic agent capable of inhibiting PGE2 and PGE2-G production, primarily due to its reduction of COX-2 and microsomal PGES-1/2 expression and their PGE2 biosynthesis activity in microglia cells, as well as in the EAE mouse brain.
Highlights
We found that WWL70 suppressed prostaglandin E2 (PGE2) production in LPS-activated microglia via cannabinoid receptorindependent mechanisms, intracellular levels of 2-AG were elevated by WWL70 treatment
WWL70 inhibited 2-AG hydrolysis and elevated 2-AG level in BV2 cells via inhibition of a specific inhibitor for α/β-hydrolase domain 6 (ABHD6) 2-AG hydrolysis to arachidonic acid (AA) in BV2 cell membrane was confirmed by deuterated AA generation using LC-MS/MS (Additional file 1: Figure S1)
The reduction of PGE2 by WWL70 was similar in both control and ABHD6 small interfering RNA (siRNA)-transfected cells (Fig. 6c). These results suggest that the inhibition of PGE2 production by WWL70 is independent of its inhibition of ABHD6
Summary
We found that WWL70 suppressed PGE2 production in LPS-activated microglia via cannabinoid receptorindependent mechanisms, intracellular levels of 2-AG were elevated by WWL70 treatment. This reduction was not attributable to WWL70 inhibition of ABHD6, given the fact that downregulation of ABHD6 by siRNA or use of KT182, an alternative ABHD6 inhibitor failed to suppress PGE2 production. WWL70 attenuated the expression of COX-2 and PGES-1/2 leading to the downregulation of the biosynthetic pathways of PGE2 and PGE2-G. PGE2 production from arachidonic acid was reduced in the microsome fraction, indicating that WWL70 targets PGE2 biosynthetic enzymes, which are likely to contribute to the therapeutic mechanisms of WWL70 in the EAE mouse model
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