Macrophage-related oxidative stress plays an important role in the inflammatory process in atherosclerosis. Recently, dextromethorphan (DXM), a common cough-suppressing ingredient with a high safety profile, was found to inhibit the activation of microglia, the resident macrophage in the nervous system. We investigated whether DXM could reduce macrophage production of cytokines and superoxide and the resultant influence on atherosclerosis formation in mice.
We used in vitro and in vivo studies to evaluate the DXM inhibitory effect on oxidative stress. Dextromethorphan pretreatment significantly suppressed the production of tumour necrosis factor-α, monocyte chemoattractant protein-1, interleukin-6, interleukin-10, and superoxide in macrophage cell culture after stimulation. Indeed, DXM reduced macrophage nicotinamide adenine dinucleotide phosphate oxidase activity by decreasing membrane translocation of p47phox and p67phox through the inhibition of protein kinase C and extracellular signal-regulated kinase activation. The anti-atherosclerosis effect of DXM was tested using two animal models, apolipoprotein E (apoE)-deficient mice and a mouse carotid ligation model. Dextromethorphan treatment (10–40 mg/kg/day) for 10 weeks in apoE-deficient mice significantly reduced superoxide production in their polymorphonuclear leukocytes and aortas. It significantly decreased the severity of aortic atherosclerosis in the apoE-deficient mice and decreased carotid neointima formation after ligation in C57BL/6 mice.
Our data show that DXM, with its novel effect in reducing oxidative stress, significantly reduces atherosclerosis and neointima formation in mice.
Atherosclerosis is a chronic inflammatory disease with monocyte extravasation into the arterial wall being the critical step in its pathogenesis.1 Initially, the activated endothelium expresses chemokines and adhesion molecules leading to monocyte recruitment and infiltration into the subendothelium with the formation of early fatty streaks.2 Inflammation and oxidative stress are central in the pathogenesis of atherosclerosis. Monocyte-derived superoxide contributes to the oxidative stress in the vessel wall inflammation, which is required for low-density lipoprotein (LDL)-cholesterol oxidation.3 Superoxide also stimulates monocytes to secrete a variety of pro-inflammatory factors such as tumour necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and interleukin-10 (IL-10), all of which are important regulators or modulators of the inflammatory reaction in the vessel wall.4 Recent advance in the understanding of atherosclerosis mechanisms has provided evidence that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme complex of the vascular cells plays an important role in the production of superoxide in atherosclerotic lesions.5 Several studies have demonstrated that atherosclerosis and neointima formation are associated with an increased vascular superoxide release, and NADPH oxidase-deficient mice develop significantly less atherosclerosis.6 Therefore, efforts have been devoted to develop NADPH oxidase inhibitors that may have therapeutic potential in the treatment of human vascular diseases.7
Dextromethorphan (DXM) is the d-isomer of the codeine analogue levophanol, a dextrorotatory morphinan. It is widely used as a cough-suppressant in cold and cough medications with a high safety profile. Recent studies have demonstrated that DXM has a novel anti-oxidative effect. It reduces lipopolysaccharide (LPS)-induced damage to dopaminergic neurons in rat neuron/glial cultures by inhibiting the microglial activation with subsequent decreased microglial production of cytokines and superoxide.8 The DXM effect in reducing LPS-induced neurotoxicity is evinced through the inhibition of microglial NADPH oxidase, the major enzyme system in microglia for the production of superoxide, because the DXM protective effect was observed only in the wild-type but not in the NADPH oxidase-deficient mice.9,10 Dextromethorphan also has beneficial effect in animal model of endotoxemia. Dextromethorphan pretreatment could significantly attenuate hypotension and tachycardia induced by LPS injection and improves animal survival rates.11 Dextromethorphan treatment in mice reduced the elevation of serum TNF-α levels and the production of TNF-α and superoxide in Kupffer cells and neutrophils. However, it only inhibited TNF-α production in the Kupffer cells isolated from the wild-type mice but not from the NADPH oxidase-deficient mice.11 We sought to investigate the influence of DXM on macrophage activation and determine the treatment effect of DXM on atherosclerosis, because macrophage-related inflammation and oxidative stress play a pivotal role in the pathogenesis of atherosclerosis. In the current study, we report that DXM treatment can reduce cytokine and superoxide production in macrophage cell culture after stimulation through the direct inhibition of NADPH oxidase. It also decreases superoxide production in the aorta and carotid artery of apolipoprotein E (apoE)-deficient mice. Dextromethorphan via oral administration decreases the severity of atherosclerotic lesion development in apoE-deficient mice and the neointima formation in the mouse carotid ligation model.
RPMI-1640 medium, phorbol 12-myristate-13-acetate (PMA), LPS (Escherichia coli 0111:B4), and DXM were purchased from the Sigma-Aldrich (St Louis, MO, USA). The human acute monocytic leukemia cell line (THP-1) was purchased from the Food Industry Research and Development Institute, Hsin Chu, Taiwan. Levels of TNF-α, MCP-1, IL-6, and IL-10 in the medium were determined with monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) kits purchased from the R&D Systems (Minneapolis, MN, USA). Apolipoprotein E-deficient mice were obtained from the Jackson Laboratories (Bar Harbor, ME, USA). All animal experiments were approved by the Institutional Animal Care and Use Committee, National Cheng Kung University; the investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996).
Dextromethorphan effect on cytokine production in macrophages
The THP-1 cells were maintained in the RPMI-1640 medium with 10% foetal bovine serum at 37°C in 5% CO2. The cells were treated with 100 nM PMA for 24 h to induce the differentiation of monocytes into macrophages. For each experiment, DXM was prepared immediately before use. Because previous study showed that DXM at micromolar concentrations can decrease LPS-induced dopaminergic neurotoxicity,12 we used 1 µM DXM as the initial concentration for all the following experiments.
For cytokine study, THP-1 cell culture was pretreated for 1 h with DXM (1 µM, 1 nM, or 1 pM) prior to treatment with 100 ng/mL LPS for up to 24 h. The supernatants were harvested, and TNF-α, MCP-1, IL-6, and IL-10 levels were determined by ELISA (R&D Systems). Lipopolysaccharide-treated samples were diluted 1:10 for the detection of TNF-α, MCP-1, and IL-6. The samples in the control group and IL-10 were measured without dilution. The minimal detectable concentration of the ELISA assays was 0.001 ng/mL. Cell viability was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay as described previously.13
Dextromethorphan effect on superoxide production and NADPH oxidase activity
Superoxide production in the THP-1 cell culture was measured by lucigenin-enhanced chemiluminescence as described previously.13 The THP-1 cell culture was pretreated for 1 h with DXM (1 µM, 1 nM, or 1 pM), apocynin (500 µM, NADPH oxidase inhibitor), or allopurinol (100 µM, xanthine oxidase inhibitor) prior to 100 ng/mL LPS stimulation. Then, the cells (1 × 106/mL) were treated with PBS containing 1.25 mM lucigenin, and counts were obtained for a 15 min period in a chamber as described previously.13
The influence of DXM on macrophage NADPH oxidase activity was measured by lucigenin-enhanced chemiluminescence.14 Briefly, the membrane fraction (15 µg protein) of the cell homogenate after centrifugation was added to glass scintillation vials containing 5 µM lucigenin in 1 mL PBS. Superoxide production was measured after adding NADPH (100 µM) into the incubation medium. Nicotinamide adenine dinucleotide phosphate oxidase activity was calculated on the basis of the amount of superoxide produced in the reaction mixture. The chemiluminescence was measured for 15 min and the integrals over this period were expressed as RLU/15 min/mL.
Dextromethorphan effect on membrane translocation of p47phox and p67 phox
The THP-1 cell culture was pretreated with DXM and LPS as described previously. Then the cells were lysed in lysis buffer (50 mM Tris–HCl, pH 7.4, 0.1 mM EDTA, and protease-inhibitor mixture). The membrane fraction of cell lysate was prepared by ultracentrifugation. Western blot analysis was used to determine the membrane fraction levels of NADPH oxidase components p47phox and p67phox (Upstate Biotechnology). Immunofluorescence and confocal microscopy were used to examine the p47phox (Santa Cruz) localization in the THP-1 cells. In brief, THP-1 cells were pretreated for 1 h with DXM (1 µM) prior to treatment with 100 ng/mL LPS for up to 24 h. Cells were immunostained with a rabbit polyclonal antibody against p47phox, then washed, and incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody. Fluorescence was detected with a laser scanning confocal microscope (Leica Microsystems, Germany) with excitation at 488 nm and detection at 585 nm using a long-pass filter. Because protein kinase C (PKC) and extracellular signal-regulated kinase 1/2 (ERK1/2) are essential for the phosphorylation and translocation of p47phox and p67phox to plasma membrane, we tested the effect of DXM on PKC or ERK activity. The THP-1 cell culture was pretreated for 1 h with DXM (1 µM, 1 nM, or 1 pM) prior to treatment with 100 ng/mL LPS for 30 min. Cell lysates were separated by SDS–PAGE, and the levels of phospho-ERK1/2 (Tyr 204) (Zymed Laboratories) and total ERK1/2, p-PKCδ (Thr505) (Cell Signalling Technology) and α-tubulin were detected by western blotting with specific antibodies.
Dextromethorphan effect in apolipoprotein E-deficient mice
Apolipoprotein E-deficient mouse fed with high-fat diet produces excessive superoxide in its aorta and carotid artery and causes endothelial dysfunction.15,16 We examined the DXM effect on the superoxide production in apoE-deficient mice. Apolipoprotein E-deficient mice (8–12 weeks old) were fed with a high-cholesterol diet containing 21% fat and 0.15% cholesterol (rodent diet 40097, Richmond, Inc.). In DXM treatment group, the mice were fed daily with DXM (5, 10, 20, and 40 mg/kg/day) dissolved in water for 10 weeks. In control group, the mice were fed with only water in the same manner. After completing the experiment, the blood sample, thoracic aorta, and left carotid artery were taken from the controls and the DXM-treated mice. First, we measured the superoxide production from polymorphonuclear leukocyte (PMN) of apoE-deficient mice. Polymorphonuclear leukocytes were isolated with Ficoll-Hypaque solution of 1.114 g/mL Polyprep (Nycomed, Birmingham, UK). The PMN suspension (1 × 106 cells/mL) and 50 µL of 1.25 mM lucigenin were added to a microplate. The chemiluminescence was measured for 15 min and the integrals over this period were expressed as RLU.
Second, superoxide production in the aorta of apoE-deficient mice was determined by lucigenin-enhanced chemiluminescence as described previously.17 After clearing the adherent periadventitial fat, aorta was cut-off into 6–8 mm segment and then incubated in warmed (37°C), oxygenated (95% O2, 5% CO2), and equilibrated modified Krebs–Hepes solution containing 20 mM Hepes (pH 7.4) for 60 min. Samples were then placed into a microplate containing 200 µL of the modified Krebs–Hepes solution with 50 µL of 1.25 mM lucigenin. The chemiluminescence of the sample was measured. All aortic rings were dried and dry weight was determined. Superoxide levels were reported as RLU per 15 min after background chemiluminescence subtraction and were normalized to milligram dry tissue weight (i.e. RLU/15 min/mg). Finally, oxidative fluorescent microtopography with the oxidative fluorescent dye dihydroethidium (Sigma) was used to evaluate the in situ production of superoxide in carotid artery as described previously.13
Quantitative analysis with MetaMorph version 6.2 software was performed by using the mean fluorescence intensity from each arterial segment and was standardized with that of the carotid arteries in control mice without DXM treatment.
Dextromethorphan effect on atherosclerosis and neointima formation
Apolipoprotein E-deficient mice were fed with a high-cholesterol diet as described previously. The mice were fed daily with DXM (5, 10, 20, and 40 mg/kg/day) dissolved in water for 10 weeks. In the control group, the mice were fed with only water daily in the same manner. After completing the treatment, blood samples were collected by direct heart puncture when sacrificing the animals. The serum levels of total cholesterol, LDL-cholesterol, high-density lipoprotein (HDL)-cholesterol, and triglyceride were measured by enzymatic methods using an automatic analyser (Model 747, Hitachi Ltd. Co., Tokyo, Japan). The aorta was dissected from the aortic valve to the iliac bifurcation. The lipid-rich atherosclerotic lesions in the whole aorta and aortic sinus were identified as described previously.13 The composition of atherosclerotic lesions at the level of the aortic sinus was specifically analysed. Collagen was detected with Masson's trichrome stain. Macrophages and smooth muscle cells were stained with anti-CD68 (Serotec) and anti-α-smooth muscle actin antibodies (Sigma). In aortic sinus, collagen content, macrophages, or smooth muscle cells were expressed as an area of positive staining. The average stained area of aortic root is reported as the mean area from the three sections of each mouse. For neointima study, mouse carotid ligation model was used. Adult C57BL/6 mice (8–12 weeks) were anesthetized by intraperitoneal injection of sodium pentobarbital. The left common carotid artery was isolated and ligated completely with a 5-0 silk suture near the carotid bifurcation as described previously.18 Then the mice were fed daily with DXM (5, 10, 20, and 40 mg/kg/day) dissolved in water for 4 weeks. In the control group, the mice were fed with only water in the same manner. After completing the treatment, the animals were sacrificed and the segment of the left common carotid artery just proximal to the ligation was excised, fixed in 4% paraformaldehyde, and embedded in paraffin. Five transverse sections per animal were cut at 100 µm intervals, stained with hematoxylin–eosin and measured as described previously.13 At 3 days after carotid ligation, three transverse sections per animal were cut and stained with hematoxylin–eosin. There was leukocyte adhesion on the luminal surface.19 The average leukocyte number around the whole luminal surface of the three sections in each artery was counted.
Data are expressed as mean ± SD. Statistical significance was analysed by unpaired Student t-test. Multiple comparisons were done by one-way ANOVA with Bonferroni corrections where appropriate using GraphPad Prism version 4.00 (GraphPad Software, San Diego, CA, USA). A P-value <0.05 was considered statistically significant.
Dextromethorphan treatment reduces macrophage cytokine production
We first explored the DXM effect on macrophage cytokine production after LPS stimulation. Lipopolysaccharide treatment induced a dramatic increase of TNF-α, MCP-1, IL-6, and IL-10 in the medium of the THP-1 cell culture when compared with the PBS treatment. Dextromethorphan pretreatment (1 µM or 1 nM) of the THP-1 cell culture significantly reduced the macrophage production of TNF-α, MCP-1, IL-6, and IL-10 in a dose-dependent fashion in the medium of THP-1 cells after LPS stimulation (Figure 1). MTT assays demonstrated that DXM treatment had no obvious effect on the cell viability.
Dextromethorphan treatment reduces macrophage superoxide production and NADPH oxidase activity
We investigated the DXM effect on macrophage superoxide production and NADPH oxidase activity after LPS stimulation. Figure 2A shows the production of superoxide in the THP-1 cells was significantly increased after LPS stimulation when compared with control group treated with only PBS. Dextromethorphan pretreatment significantly suppressed the elevation of macrophage superoxide production after LPS stimulation. Lipopolysaccharide-stimulated superoxide production in macrophage was inhibited by pretreatment of NADPH oxidase inhibitor apocynin, but not by xanthine oxidase inhibitor allopurinol indicating that LPS-stimulated superoxide production in macrophage was almost from NADPH oxidase (Figure 2A). We next examined whether DXM could directly inhibit the macrophage NADPH oxidase activity. The macrophage NADPH oxidase activity was significantly increased after LPS stimulation when compared with control group treated with only PBS. Dextromethorphan pretreatment significantly inhibited the NADPH oxidase activity after LPS stimulation (Figure 2B).
Dextromethorphan reduces membrane translocation of p47phox and p67phox in macrophage
Nicotinamide adenine dinucleotide phosphate oxidase in macrophage consists of membrane-bound (gp91phox and p22phox) and cytosolic components (p47phox, p67phox, p40phox, and Rac proteins).20 Nicotinamide adenine dinucleotide phosphate oxidase activation requires the membrane translocation of cytosolic components to associate with membrane-bound components and assemble into an active enzyme. Previous study has shown that LPS treatment rapidly activates NADPH oxidase and induces cell membrane translocation of the cytosolic p47phox and p67phox.21 We sought to determine whether DXM reduces NADPH oxidase activity by preventing p47phox and p67phox membrane translocation after LPS stimulation. Western blot assay demonstrated that DXM pretreatment (1 µM or 1 nM) of the THP-1 cell culture significantly reduced the membrane expression of p47phox and p67phox after LPS stimulation in a dose-dependent fashion (Figure 2C). Protein kinase C and ERK1/2 are essential for the phosphorylation and translocation of p47phox and p67phox to plasma membrane. Dextromethorphan pretreatment (1 µM or 1 nM) directly reduced the PKCδ and ERK1/2 activities (Figure 2D). Consistent with the western blot assay, confocal microscopical study showed that LPS treatment induced a significant membrane translocation of p47phox and DXM (1 µM) pretreatment prevented this LPS-induced p47phox membrane translocation (Figure 2E).
Dextromethorphan treatment reduces superoxide production in apolipoprotein E-deficient mice
Having established the DXM inhibitory effect on macrophage activation and NADPH oxidase, we next examined the functional consequences of which in vivo. Figure 3 shows the DXM effect in the apoE-deficient mice with excessive superoxide production after feeding high-cholesterol diet. Evidence indicates that PMN produces a more immediate and pronounce response of superoxide production than monocyte does.22 We first examined DXM effect on the superoxide production of PMN isolated from apoE-deficient mice. Treatment of the apoE-deficient mice with 10, 20, or 40 mg/kg/day DXM for 10 weeks effectively reduced the PMN production of superoxide (Figure 3A), whereas 5 mg/kg/day DXM had no significant effect. The superoxide in the thoracic aorta was also significantly reduced in apoE-deficient mice treated with 10, 20, or 40 mg/kg/day DXM for 10 weeks (Figure 3B). The apoE-deficient mice showed a marked increase in fluorescence intensity in the sections of left carotid arteries, reflecting an increase in superoxide production in the artery (Figure 3Ca). Dextromethorphan treatment (10, 20, and 40 mg/kg/day) for 10 weeks significantly reduced the fluorescence intensity indicating the decrease of superoxide production in the carotid arteries of the apoE-deficient mice (Figure 3Cc–e and D); whereas 5 mg/kg/day DXM had no significant effect (Figure 3Cb).
Dextromethorphan treatment inhibits atherosclerosis and neointima formation
We used two animal models, apoE-deficient mice and carotid ligation model, to study the DXM effect in atherosclerosis and neointima formation. In apoE-deficient mice study, there were no significant differences of the total cholesterol, triglyceride, LDL-cholesterol, and HDL-cholesterol levels between the apoE-deficient mice with and without DXM treatment. Dextromethorphan treatment (10, 20, and 40 mg/kg/day) for 10 weeks significantly reduced the severity of aortic sinus atherosclerotic lesions in apoE-deficient mice (Figure 4A and B). In composition analysis, the areas of CD68-positive, α-smooth muscle cell, and collagen were significantly reduced after DXM treatment (Figure 4A and B). Dextromethorphan treatment also reduced the severity of atherosclerotic lesions in the whole aorta (Figure 4C and D). In carotid ligation model, there was a progressively decreased lumen area and increased neointima formation in C57BL/6 mice after carotid artery ligation for 4 weeks (Figure 5A). The ratio of neointima to media was decreased in the DXM treatment group (10, 20, and 40 mg/kg/day) at 28 days after surgery indicating DXM treatment for 4 weeks significantly reduced the severity of neointima formation in the mice (Figure 5B). At 3 days after ligation, there were leukocytes adhering on the carotid luminal surface (Figure 5C). The number of adhering leukocytes was significantly reduced in mice receiving DXM treatment (Figure 5D).
Monocytes/macrophages are the major source of superoxide anions causing cellular damage in vasculature and contributing to atherosclerosis.23 Nicotinamide adenine dinucleotide phosphate oxidase is the major enzyme that is responsible for the production of superoxide in activated macrophages.23 Previous studies demonstrated that DXM, a well-known antitussive agent, significantly reduced the LPS-stimulated production of TNF-α and superoxide from the microglial cells,8,9 and DXM treatment protected mice from septic shock induced by endotoxin.11 Further evidence indicates that NADPH oxidase is the target of DXM action because the DXM-mediated effect disappears in NADPH oxidase-deficient mice.9 In the present study, our data reconfirmed that NADPH oxidase is responsible for LPS-induced elevation of superoxide levels in macrophage. There was a significant increase in superoxide and NADPH oxidase activity in THP-1 cells treated with LPS. Addition of apocynin, a specific NADPH oxidase inhibitor, to cultured cells almost blocked LPS-induced superoxide levels to that of the control. Dextromethorphan (1 µM) treatment has the similar effect as that of apocynin by reducing the superoxide level and NADPH oxidase activity in THP-1 cells after LPS stimulation. In addition to in vitro study, the DXM effect could be observed in apoE-deficient mice with excessive superoxide production. We found that DXM also reduced superoxide production in the aorta and carotid artery in apoE-deficient mice.
Decreased superoxide may further prevent cytokine production in macrophage. In our study, 1 µM DXM pretreatment could decrease ∼40% TNF-α production from the macrophage after LPS stimulation. This result accords with the previous report of a 45% reduction in TNF-α mRNA expression in microglia from NADPH oxidase-deficient mice in response to LPS stimulation when compared with the wild-type mice.24 It is likely that DXM blocks the NADPH oxidase-dependent superoxide production, accounting for ∼40–45% of the macrophage TNF-α production after LPS stimulation. In this study, we tried to elucidate the possible mechanism of the DXM inhibitory effect on NADPH oxidase. Our data show that DXM directly targets NADPH oxidase and decreases its activity by preventing membrane translocation of the cytosolic components, p47phox and p67 phox through inhibiting PKC and ERK activation.
In our study, DXM treatment with 20 and 40 mg/kg/day for 10 weeks could decrease by ∼30–36% superoxide production in the aorta of apoE-deficient mice. Because DXM had potent anti-oxidative effect, it is likely that DXM might reduce the expression of inflammation promoting factors like MCP-1 and thus inhibit inflammatory changes in the atherosclerotic lesions. The novel inhibitory effect on NADPH oxidase and superoxide production of DXM prompted us to look at the role of DXM in the treatment of atherosclerosis, a disease with increased vascular wall inflammation and oxidative stress. In our study, we found that DXM reduced the aortic atherosclerotic lesion in apoE-deficient mice. The CD68-positive area was significantly reduced after DXM treatment indicated that the reduced oxidative stress, cytokine production, and decreased inflammatory cells infiltration were the major mechanisms of DXM's anti-atherosclerotic effect. Interestingly, the severity of the neointima formation after carotid ligation was also decreased after DXM treatment. Carotid ligation causes blood stasis and inflammation, resulting in a smooth muscle cell-rich neointima formation mimicking the arterial restenosis lesion after angioplasty.18 Previous study has demonstrated that NADPH oxidase-deficient mice have less vascular inflammation, cellular proliferation, and neointimal thickening after angioplasty.25 Our data indicate that DXM blockage of NADPH oxidase activity and macrophage activation with reduced oxidative stress and inflammation in the vasculature also prevents the deterioration of neointima formation. Although much effort has been devoted to the development of NADPH oxidase inhibitor, there is still limited potential for clinical therapeutics because some cannot be administered orally and others have severe toxicities.7 Our study results may have important clinical implications because DXM has a high safety profile in clinical use. Further studies are necessary to determine the DXM treatment on vascular disease and restenosis in humans.
On the basis of these findings, we propose that DXM can inhibit macrophage activation after LPS stimulation with reduced oxidative stress. It can serve as a protective anti-oxidative agent in the prevention of atherosclerotic plaque and neointima formation in the vascular system.
Conflict of interest: none declared.
This work was supported by the National Science Council, Taipei, Taiwan, Republic of China (Grants NSC 95-2752-B-006-003-PAE, NSC95-2752-B-006-004-PAE, and NSC 95-2752-B-006-005-PAE).