Nalmefene, an opioid receptor modulator, aggravates atherosclerotic plaque formation in apolipoprotein E knockout mice by enhancing oxidized low-density lipoprotein uptake in macrophages

Abstract

Nalmefene, an antagonist of mu- and delta-opioid receptors and a partial agonist of kappa-opioid receptors, has shown promise in reducing alcohol consumption among patients with alcohol dependence. Opioid receptors play pivotal roles in various physiological processes, including those related to peripheral inflammatory diseases such as colitis and arthritis, as well as functions in the immune system and phagocytosis. Atherosclerosis, a chronic inflammatory disease, progresses through the phagocytosis and uptake of oxidized low-density lipoprotein (oxLDL) by macrophages in atherosclerotic plaques. Despite this knowledge, it remains unclear whether nalmefene influences the formation of atherosclerotic plaques and increases the risk of serious cardiovascular events. This study aims to elucidate the impact of nalmefene on atherosclerosis in apolipoprotein E knockout (ApoE KO) mice and peritoneal macrophages in vitro. In this experiment, 8-week-old male ApoE KO mice were fed a high-fat diet intraperitoneally administered either vehicle (saline) or nalmefene (1 mg and 3 mg kg^-1 day^-1) for 21 days. Oil red O-staining and immunohistochemistry with an anti-MOMA2 (monocyte/macrophage) antibody showed that a dose-dependent increase in atherosclerotic plaque formation and augmentation of macrophage-rich plaque formation in ApoE-KO mice. Further investigations focused on the effects of nalmefene on the expression of scavenger receptor CD36 in RAW264.7 cells, conducted through western blotting analysis. Nalmefene demonstrated a significant increase in CD36 protein expression in RAW264.7 cells. To explore the impact on oxidized LDL uptake in peritoneal macrophages, cells were treated with nalmefene (300 μg/mL) for 24 h, followed by the addition of DiI-labeled oxLDL (DiI-oxLDL) for 4 h. Nalmefene significantly enhanced DiI-oxLDL uptake in macrophages. Additionally, treatment with nalmefene (300 μg/mL) for 24 h decreased the mRNA expression of mu-, delta-, and kappa-opioid receptors in RAW264.7 cells. In conclusion, nalmefene may augment oxLDL uptake by macrophages through increased CD36 expression and decreased opioid receptor, thereby contributing to atherosclerotic plaque formation and vulnerability. Consequently, the use of nalmefene may be associated with an elevated risk of cardiovascular events.

Keywords: Atherosclerosis; Nalmefene; Opioid receptors; Side effect.

Fig. 1

Atherosclerotic plaque formation in the aorta of nalmefene-treated apolipoprotein E knockout mice. Representative en face photographs of the aorta showing oil red O-stained atherosclerotic lesions (red) (A). Quantitative analysis of the Oil Red O-positive area in the whole aorta (B) and aortic arch (C). Each dot shows data obtained from each mouse. Data represent the means ± SD (n = 8–9). *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Atherosclerotic plaque formation in the aortic root of apolipoprotein E knockout mice treated with vehicle and nalmefene (1 mg or 3 mg kg⁻¹ day⁻¹) for 21 days. (A) Representative photomicrographs of oil red O (red color in the upper panels) and MOMA2 (monocyte/macrophage)-stained (brownish red color in the lower panels). Bar; 100 μm. (B) The oil red O-positive and (C) the MOMA2-positive areas (mm²) in the aortic root were measured in 5 separate sections (7 μm). Each dot shows data obtained from each mouse. Data represent means ± SD (n = 8–9). **P < 0.01, ***P < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Effect of nalmefene on the protein expression levels of CD36 in RAW264.7 cells. Cells were treated with nalmefene (0–300 μg/mL) for 24 h. (A) Representative immunoblots showing the protein expression levels of CD36. (B) Quantitative analysis of CD36 expression GAPDH was used as a loading control. Each dot shows data obtained from individual experiment. Data represent means ± SD (n = 4) *P < 0.05, ***P < 0.001.

Fig. 4

Effect of nalmefene on oxLDL uptake in peritoneal macrophages. Peritoneal macrophages were treated with nalmefene (300 μg/mL) for 24 h and then 5 μg/mL DiI-labeled oxLDL was added to the culture medium for 4 h. (A) Representative images showing incorporated oxLDL in macrophages (red). Bar; 100 μm. (B) Quantitative analysis of DiI-positive and oxLDL-incorporated areas. The results are expressed as the DiI-labeled area per cell. Each dot represents data obtained from individual experiments. Data represent means ± SD (n = 7). **P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Effect of nalmefene on mRNA expression of opioid receptors in RAW264.7 cells. Cells were treated with nalmefene (300 μg/mL) for 24 h. Dot graph shows quantitative real-time RT-PCR analyses of mRNA expression of mu- (A), delta- (B), and kappa-opioid receptors (C). Data represent means ± SD (n = 6). *P < 0.05.