*2.2. LOE Decreases Excessive Vascular ROS Formation by Inhibiting NADPH Oxidase Subunits in WD-Fed apoE*−*/*<sup>−</sup> *Mice*

To determine the in vivo vascular pro-oxidant effects of LOE therapy, the vascular ROS in the aortic walls of apoE−/<sup>−</sup> mice and C57BL/6 mice were evaluated using the redox-sensitive fluorescent probe dihydroethidine (DHE). Almost no fluorescence signal was observed in the aortic walls of chow diet-fed C57BL/6 mice (Figure 2). In contrast, there was a significant increase in the DHE fluorescence signal in aortic plaques of WD-fed apoE−/<sup>−</sup> mice compared with chow diet-fed C57BL/6 mice (Figure 2). Similar to losartan

treatment (18.1 ± 0.5 vs. 30.8 ± 1.8, 41.3 ± 1% reduction), LOE administration markedly reduced DHE fluorescence in WD-fed apoE−/<sup>−</sup> mouse aortas (14.6 <sup>±</sup> 1.6 vs. 30.8 <sup>±</sup> 1.8 and 52.6 ± 5% reduction) (Figure 2).

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**Figure 1.** LOE and losartan treatments improve endothelium-dependent relaxation in response to Ach in the aortas of apoE<sup>−</sup>/<sup>−</sup> mice. Aortic rings (3–4 mm in length) with endothelium derived from the indicated groups of mice were suspended in organ baths containing oxygenated Krebs solution and precontracted with phenylephrine (100 nM) before the construction of concentration–relaxation curves to Ach (1 nM–10 µM). The results are shown as mean ± SEM (n = 5–7). \* *p* < 0.05 indicates a significant difference between the apoE<sup>−</sup>/− vehicle group versus the C57BL/6 group and # *p* < 0.05 the apoE<sup>−</sup>/<sup>−</sup> LOE group or apoE<sup>−</sup>/<sup>−</sup> losartan group versus the apoE<sup>−</sup>/<sup>−</sup> vehicle group. **Figure 1.** LOE and losartan treatments improve endothelium-dependent relaxation in response to Ach in the aortas of apoE−/<sup>−</sup> mice. Aortic rings (3–4 mm in length) with endothelium derived from the indicated groups of mice were suspended in organ baths containing oxygenated Krebs solution and precontracted with phenylephrine (100 nM) before the construction of concentration–relaxation curves to Ach (1 nM–10 µM). The results are shown as mean ± SEM (*n* = 5–7). \* *p* < 0.05 indicates a significant difference between the apoE−/<sup>−</sup> vehicle group versus the C57BL/6 group and # *p* < 0.05 the apoE−/<sup>−</sup> LOE group or apoE−/<sup>−</sup> losartan group versus the apoE−/<sup>−</sup> vehicle group. sensitive fluorescent probe dihydroethidine (DHE). Almost no fluorescence signal was observed in the aortic walls of chow diet-fed C57BL/6 mice (Figure 2). In contrast, there was a significant increase in the DHE fluorescence signal in aortic plaques of WD-fed apoE−/− mice compared with chow diet-fed C57BL/6 mice (Figure 2). Similar to losartan treatment (18.1 ± 0.5 vs. 30.8 ± 1.8, 41.3 ± 1% reduction), LOE administration markedly reduced DHE fluorescence in WD-fed apoE−/− mouse aortas (14.6 ± 1.6 vs. 30.8 ± 1.8 and 52.6 ± 5% reduction) (Figure 2).

**Figure 2.** LOE and losartan treatments prevent vascular generation of reactive oxygen species in aortic sections derived from apoE−/<sup>−</sup> mice. The aortic sections were exposed to dihydroethidine (DHE, 2.5 µM), a redox-sensitive fluorescent dye, for 30 min. Subsequently, ethidium fluorescence was evaluated via confocal microscopy. (**a**) Representative images show H&E staining (**top**), DHE staining in red (**middle**), and merged images (**bottom**). (**b**) Corresponding cumulative data. The scale bar represents 100 µm. The results are shown as mean ± SEM (*n* = 4–6). \* *p* < 0.05 versus the C57BL/6 group and # *p* < 0.05 versus the apoE−/<sup>−</sup> vehicle group.

In addition, since ROS generation in apoE−/<sup>−</sup> mice has been correlated with the upregulation of NADPH oxidase activity, the activity of NADPH oxidase was assessed based on the expression of its subunits, including p22phox and p47phox [11,24]. The 0.05 versus the apoE<sup>−</sup>/<sup>−</sup> vehicle group.

fluorescence signals of p22phox and p47phox were markedly increased in WD-fed apoE−/<sup>−</sup> mice; however, LOE intake significantly reduced the levels of both NADPH oxidase subunits (p22phox: 9.5 ± 1.1 vs. 39.1 ± 1.9; p47phox: 8.6 ± 1.2 vs. 37 ± 0.3) (Figure 3). fluorescence signals of p22phox and p47phox were markedly increased in WD-fed apoE−/<sup>−</sup> mice; however, LOE intake significantly reduced the levels of both NADPH oxidase subunits (p22phox: 9.5 ± 1.1 vs. 39.1 ± 1.9; p47phox: 8.6 ± 1.2 vs. 37 ± 0.3) (Figure 3).

In addition, since ROS generation in apoE−/− mice has been correlated with the upregulation of NADPH oxidase activity, the activity of NADPH oxidase was assessed based on the expression of its subunits, including p22phox and p47phox [11,24]. The

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**Figure 2.** LOE and losartan treatments prevent vascular generation of reactive oxygen species in aortic sections derived from apoE<sup>−</sup>/<sup>−</sup> mice. The aortic sections were exposed to dihydroethidine (DHE, 2.5 µM), a redox-sensitive fluorescent dye, for 30 min. Subsequently, ethidium fluorescence was evaluated via confocal microscopy. (**a**) Representative images show H&E staining (top), DHE staining in red (middle), and merged images (bottom). (**b**) Corresponding cumulative data. The scale bar represents 100 µm. The results are shown as mean ± SEM (n = 4–6). \* *p* < 0.05 versus the C57BL/6 group and # *p* <

**Figure 3.** LOE and losartan treatments inhibit NADPH oxidase subunits p22phox and p47phox in aortic sections obtained from apoE<sup>−</sup>/<sup>−</sup> mice. The expression of the NADPH oxidase subunits p22phox and p47phox was determined by confocal microscopy using a purified polyclonal antibody and a fluorescence-tagged secondary antibody. Nuclei were stained with DAPI (blue). (**a**) Representative images show H&E staining (top), p22phox and p47phox staining in red (middle), and merged fluorescence staining and DAPI (bottom). (**b**) Corresponding cumulative data. The scale bar represents 100 µm. The results are shown as mean ± SEM (n = 4–6). \* *p* < 0.05 versus the C57BL/6 group and # *p* < 0.05 versus the apoE<sup>−</sup>/<sup>−</sup> vehicle group. **Figure 3.** LOE and losartan treatments inhibit NADPH oxidase subunits p22phox and p47phox in aortic sections obtained from apoE−/<sup>−</sup> mice. The expression of the NADPH oxidase subunits p22phox and p47phox was determined by confocal microscopy using a purified polyclonal antibody and a fluorescence-tagged secondary antibody. Nuclei were stained with DAPI (blue). (**a**) Representative images show H&E staining (**top**), p22phox and p47phox staining in red (**middle**), and merged fluorescence staining and DAPI (**bottom**). (**b**) Corresponding cumulative data. The scale bar represents 100 µm. The results are shown as mean <sup>±</sup> SEM (*<sup>n</sup>* = 4–6). \* *<sup>p</sup>* < 0.05 versus the C57BL/6 group and # *<sup>p</sup>* < 0.05 versus the apoE−/<sup>−</sup> vehicle group.

### *2.3. LOE Suppresses Inflammation in Murine Aortic Atherosclerosis 2.3. LOE Suppresses Inflammation in Murine Aortic Atherosclerosis*

(Figure 4).

WD-fed apoE−/− mice exhibited abundant atherosclerotic plaques, especially in the aortic root, arch, and abdominal aorta along the renal artery branches, while atherosclerotic plaques were rarely observed in the aortas of C57BL/6 mice. The plaque inflammation-modulating effects of LOE were assessed via an ex vivo fluorescence reflectance imaging (FRI) study performed to measure inflammation in the aortic atherosclerotic plaques. After 20 weeks of treatment, the aortas of WD-fed apoE−/− mice showed markedly increased inflammation compared with those of C57BL/6 mice (Figure 4). LOE treatment significantly reduced the degree of plaque inflammation in WD-fed apoE−/− mice (594.7 ± 429.8 vs. 1971.0 ± 99.9 AU), whereas losartan treatment did not WD-fed apoE−/<sup>−</sup> mice exhibited abundant atherosclerotic plaques, especially in the aortic root, arch, and abdominal aorta along the renal artery branches, while atherosclerotic plaques were rarely observed in the aortas of C57BL/6 mice. The plaque inflammationmodulating effects of LOE were assessed via an ex vivo fluorescence reflectance imaging (FRI) study performed to measure inflammation in the aortic atherosclerotic plaques. After 20 weeks of treatment, the aortas of WD-fed apoE−/<sup>−</sup> mice showed markedly increased inflammation compared with those of C57BL/6 mice (Figure 4). LOE treatment significantly reduced the degree of plaque inflammation in WD-fed apoE−/<sup>−</sup> mice (594.7 <sup>±</sup> 429.8 vs. 1971.0 ± 99.9 AU), whereas losartan treatment did not (Figure 4).
