Effect Of Digoxin On The Development And Stability Of Atherosclerotic Plaques

PartⅠ:Effect of Digoxin on the Development of Atherosclerotic PlaquesPurpose:Numerous in vitro studies have suggested that digoxin suppressed inflammation and altered lipid metabolism. However, the impact of dioxin on atherosclerosis is poorly understood. The present study was conducted to determine whether digoxin affects the development of atherosclerosis in a murine atherosclerotic disease model.Methods:Animals (8 weeks old,19 to 22 g) were randomly separated into 3 groups (n= 8 mice/group). Each mouse in the first (control) group was injected with the same volume (0.1 ml) of PBS containing 0.1% DMSO. The second and third groups received intraperitoneal (i.p.) injections of approximately 20μg (1 mg/kg per day; low dose) or 40 μg of digoxin (2 mg/kg per day; high dose) and were fed with atherogenic Western-type diet (WD) containing 0.15% cholesterol and 21% fat for 12 weeks. At the end of the 12-week treatment period, on the day of sacrificing, all animals were anaesthetized via i.p. injection of sodium pentobarbital (50 mg/kg) and were exsanguinated via retro-orbital venous puncture under general anaesthesia. The mice were subsequently euthanized via cervical dislocation. The blood samples obtained from the animals were stored at -80℃ for further analysis. To investigate the potential effects of digoxin on atherosclerosis, we evaluated the severity of atherosclerosis based on the aortas and aortic valves by staining with Oil Red O. The plasma levels of total cholesterol, triglycerides, low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol were measured using chemically modified enzyme-based assay. The levels of mouse IL-17A, IL-10 and IL-6 were measured using a mouse multi-cytokine detection kit.Results:Digoxin dose-dependently reduced atherosclerotic lesion formation and plasma lipid levels (reductions of 41% in total cholesterol,54% in triglycerides, and 20% in low-density lipoprotein cholesterol in the high-dose digoxin-treated group). Moreover, treatment with digoxin markedly attenuated interleukin (IL)-17A expression and IL-17A-related inflammatory responses and increased the abundance of Tregs.Conclusions:Our data demonstrate that digoxin acts as a specific antagonist of retinoid-related orphan receptor-y to suppress atherosclerosis by decreasing lipid levels and inhibiting IL-17A-related inflammatory responses.Part Ⅱ:Effect of Digoxin on the Stability of Atherosclerotic PlaquesPurpose:Stability of atherosclerotic plaques play an important role in coronary heart disease events, its stability vulnerability associated with related-inflammation and immune response.Preliminary studies have confirmed that treatment with digoxin markedly attenuated interleukin (IL)-17A expression and IL-17A-related inflammatory responses, therefore, the purpose of this study is to dectect the stability of digoxin on atherosclerotic plaque.Methods:Apolipoprotein E-deficient mice were randomly separated into 3 groups (n= 8 mice/group) and maintained on an atherogenic Western-type diet (WD) containing 0.15% cholesterol and 21% fat for 12 weeks. Each mouse in the first (control) group were administered with PBS (control). The second and third groups received intraperitoneal (i.p.) low-dose digoxin (1 mg/kg per day) or high-dose digoxin (2 mg/kg per day) via intraperitoneal injection for 12 weeks.For histological analysis, the aortic roots were sliced into 5μm serial cryostat sections in the aortic valve plane.Macrophages and smooth muscle cells were quantified by assessing the percentages of the total plaque area that was positive for each marker. CD4+ T cells and Tregs were assessed by counting the number of positively stained cells.RT-PCR was used to determine the mRNA expressions of TNF-α, IL-1β, MCP-1, IFN-γ, IL-1β, MMP-2, and MMP-9 in the aorta samples.Results:We discovered that digoxin dose-dependentlyaccelerated the stability of atherosclerotic lesions. Moreover, the proportion of lesion cross-sectional area containing CD68+ macrophages was reduced in both digoxin-treated groups compared with the PBS-treated group. More importantly, the abundance of α-SMA+ vascular smooth muscle cells (VSMCs) at the fibrous cap was significantly increased in a digoxin dose-dependent manner. Furthermore, the collagen+ area was increased in the atherosclerotic lesions of mice that received digoxin compared with those that received PBS. Additionally, the abundance of CD4+ cells was significantly decreased in the mice administered with digoxin (especially high-dose digoxin) compared with the mice treated with PBS. RT-PCR was used to determine the mRNA expressions of TNF-α, IL-1β, MCP-1, IFN-γ, IL-1β, MMP-2, and MMP-9. Digoxin treatment dose-dependently reduced the expression of TNF-α, IL-1β, MCP-1, IFN-γ, MMP-2, and MMP-9.Conclusions:Taken together, our results provide convincing evidence that digoxin dose-dependently accelerates the stability of atherosclerotic lesions, which were composed of fewer macrophages and CD4+ T cells and more frequently expressed α-SMA at the fibrous cap and collagen due to digoxin treatment.