WWL70 attenuates PGE₂ production derived from 2-arachidonoylglycerol in microglia by ABHD6-independent mechanism

α/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.

The 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.

We 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.

WWL70 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.