N‑acetylcysteine inhibits atherosclerosis by correcting glutathione‑dependent methylglyoxal elimination and dicarbonyl/oxidative stress in the aorta of diabetic mice

Abstract

In diabetic animal models, high plasma/tissue levels of methylglyoxal (MG) are implicated in atherosclerosis. N‑acetylcysteine (NAC) is a cysteine prodrug that replenishes intracellular glutathione (GSH) levels, which can increase the elimination of MG in diabetes mellitus (DM). The present study investigated the anti‑atherosclerotic role of NAC in DM and aimed to determine whether the mechanism involved GSH‑dependent MG elimination in the aorta. Apolipoprotein‑E knockdown (ApoE‑/‑) mice injected with streptozotocin for 5 days exhibited enhanced atherosclerotic plaque size in the aortic root; notably, a high‑lipid diet aggravated this alteration. NAC treatment in the drinking water for 12 weeks decreased the size of the atherosclerotic lesion, which was associated with a reduction in MG‑dicarbonyl stress and oxidative stress, as indicated by decreased serum malondialdehyde levels, and increased superoxide dismutase‑1 and glutathione peroxidase‑1 levels in the diabetic aorta. Endothelial damage was also corrected by NAC, as indicated by an increase in the expression levels of phosphorylated (p‑)Akt and p‑endothelial nitric oxide synthase (eNOS) in the aorta, as well as nitric oxide (NO) in the serum. In addition, MG‑treated human umbilical vein endothelial cells (HUVECs) exhibited increased reactive oxygen species and decreased antioxidant enzyme expression levels. NAC treatment corrected the alteration in HUVECs induced by MG, whereas the protective role of NAC was blocked via inhibition of GSH. These findings indicated that the diabetic aorta was more susceptible to atherosclerotic lesions compared with non‑diabetic ApoE‑/‑ mice. Furthermore, NAC may offer protection against atherosclerotic development in DM by altering aortic and systemic responses via correcting GSH‑dependent MG elimination, leading to decreased oxidative stress and restoration of the p‑Akt/p‑eNOS pathway in the aorta.

Keywords: N‑acetylcysteine; diabetes; glutathione; methylglyoxal; dicarbonyl stress; atherosclerosis.

Figure 1.

Atherosclerotic lesion areas in the aortic root were stained with oil red O (magnification, ×100) in ApoE^−/− mice (Con group), STZ-injected ApoE^−/− mice (DM group), STZ-injected ApoE^−/− mice fed a HLD (DM + HLD group), and STZ-injected ApoE^−/− mice fed a HLD and administered NAC-containing water (DM + HLD + NAC group). *P<0.05 vs. Con group; ^#P<0.05 vs. DM group; ^&P<0.05 vs. DM + HLD group (n=5/group). ApoE, apolipoprotein E; Con, control; DM, diabetes mellitus; HLD, high-lipid diet; NAC, N-acetylcysteine; STZ, streptozotocin.

Figure 2.

Blood lipid levels (total cholesterol, LDL-C, HDL-C and triglyceride) were assessed using an automatic biochemistry analyzer in ApoE^−/− mice (Con group), STZ-injected ApoE^−/− mice (DM group), STZ-injected ApoE^−/− mice fed a HLD (DM + HLD group), and STZ-injected ApoE^−/− mice fed a HLD and administered NAC-containing water (DM + HLD + NAC group). *P<0.05 vs. Con group; ^#P<0.05 vs. DM group (n=8/group). LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; ApoE, apolipoprotein E; Con, control; DM, diabetes mellitus; HLD, high-lipid diet; NAC, N-acetylcysteine; STZ, streptozotocin.

Figure 3.

Effects of NAC on oxidative stress, p-Akt/p-eNOS protein expression and serum NO levels in diabetic ApoE^−/− mice fed a HLD. (A) Aortic protein expression of GSH, MG, protein carbonyl contents and serum MDA levels (n=5 repeats/group). (B) Aortic protein expression levels of antioxidant enzymes (SOD-1 and GPX-1) (n=4 repeats/group). (C) Aortic protein expression levels of p-Akt and p-eNOS (n=4 repeats/group). (D) Serum levels of NO (n=4 repeats/group) in ApoE^−/− mice (Con group), STZ-injected ApoE^−/− mice (DM group), STZ-injected ApoE^−/− mice fed a HLD (DM + HLD group), and STZ-injected ApoE^−/− mice fed a HLD and administered NAC-containing water (DM + HLD + NAC group). *P<0.05 vs. Con group; ^#P<0.05 vs. DM group; ^&P<0.05 vs. DM + HLD group. ApoE, apolipoprotein E; Con, control; DM, diabetes mellitus; HLD, high-lipid diet; NAC, N-acetylcysteine; STZ, streptozotocin; GSH, glutathione; MG, methylglyoxal; p-, phosphorylated; eNOS, endothelial nitric oxide synthase; NO, nitric oxide.

Figure 4.

Effects of NAC on ROS levels, SOD-1, GPX-1 and p-Akt/p-eNOS protein expression in HUVECs. (A) Intracellular ROS levels (magnification, ×100), and (B) protein expression levels of antioxidant enzymes (SOD-1 and GPX-1), p-Akt and p-eNOS in untreated HUVECs, or in HUVECs exposed to 1 mM MG, with or without NAC, or with NAC + BSO (glutathione inhibitor). Intracellular ROS levels were measured using 2′,7′-dichlorofluorescein diacetate. Protein expression levels were assessed by western blotting (n=4 repeats/group). *P<0.05 vs. control group; ^#P<0.05 vs. MG group; ^&P<0.05 vs. MG + NAC group. HUVECs, human umbilical vein endothelial cells; NAC, N-acetylcysteine; MG, methylglyoxal; BSO, buthionine sulfoximine; ROS, reactive oxygen species; SOD-1, superoxide dismutase 1; GPX-1, glutathione peroxidase 1; p-, phosphorylated; eNOS, endothelial nitric oxide synthase.