Abstract
The endocannabinoid system has recently been attracted interest for its anti-inflammatory and anti-oxidative properties. In this study, we investigated the role of the endocannabinoid system in regulating the oxidized low-density lipoprotein (oxLDL)-induced inflammatory response in macrophages. RAW264.7 mouse macrophages and peritoneal macrophages isolated from Sprague-Dawley (SD) rats were exposed to oxLDL with or without the synthetic cannabinoid WIN55,212-2. To assess the inflammatory response, reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF- alpha) levels were determined, and activation of the mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappa B signaling pathways were assessed. We observed that: i) oxLDL strongly induced ROS generation and TNF- alpha secretion in murine macrophages; ii) oxLDL-induced TNF- alpha and ROS levels could be lowered considerably by WIN55,212-2 via inhibition of MAPK (ERK1/2) signaling and NF-kappa B activity; and iii) the effects of WIN55212-2 were attenuated by the selective CB2 receptor antagonist AM630. These results demonstrate the involvement of the endocannabinoid system in regulating the oxLDL-induced inflammatory response in macrophages, and indicate that the CB2 receptor may offer a novel pharmaceutical target for treating atherosclerosis.
Highlights
The endocannabinoid system has recently been attracted interest for its anti-inflammatory and anti-oxidative properties
After oxidized low-density lipoprotein (oxLDL) treatment with or without receptor agonist/ antagonists or signaling pathway inhibitors, intracellular reactive oxygen species (ROS) was determined in macrophages by flow cytometry using the nonfluorescent probe DCFH-diacetate, which is converted to highly fluorescent dichlorofluorescein (DCF) by ROS
A similar phenomenon was observed in peritoneal macrophages isolated from SD rats (Fig. 1D–F), higher doses of oxLDL (10–50 g/ml) were required to achieve similar intracellular ROS generation in these cells
Summary
The endocannabinoid system has recently been attracted interest for its anti-inflammatory and anti-oxidative properties. The endocannabinoid system has recently attracted interest for its anti-inflammatory and anti-oxidative effects in Abbreviations: ABCA1, ATP-binding cassette transporter; AEA, anandamide; 2-AG, 2-arachidonoylglycerol; AM251, N-(piperidin-1-yl)5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM630, 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol3-yl](4-methoxyphenyl) methanone; DCF, dichlorofluorescein; DCFH, 2′,7′-dichlorodihydrofluorescein; ERK, extracellular signal-regulated kinase; FAAH, fatty acid amide hydrolase; GAPDH, glyceraldehydes phosphate dehydrogenase; I/R, ischemia/reperfusion; ICAM, intercellular adhesion molecule; LDL, low-density lipoprotein; LOX-1, lectin-like oxidized low-density lipoprotein receptor-1; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; NF, nuclear factor; oxLDL, oxidized low-density lipoprotein; PD98059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; ROS, reactive oxygen species; SD rats, Sprague-Dawley rats; THC, ⌬9-tetrahydrocannabinol; TNF-␣, tumor necrosis factor-␣; U0126, 1,4-diamino-2,3-dicyano-1,4-bis (o-aminophenylmercapto) butadiene monoethanolate; WIN55,212-2, (R)-(+)-2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmeth anone mesylate
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