**2. Results**

#### *2.1. Protective Effect of Fx on VIC Cell Viability from H2O2-Induced Oxidative Stress* 2.1.1. Effect of H2O2 on the VIC Cell Viability

After the cell extraction (Supplementary Figure S1), we treated the cells with H2O2 in serial doses for 15 min, 1 h and 4 h to induce high oxidative stress in VIC to measure the cell viability. The results showed that H2O2 at doses of 0.5 mM and above significantly reduced cell proliferation at all the time points. Notably, H2O2 at 0.5 mM for 15 min (min) decreased the cell viability by 30%, which is considered a moderate effect of oxidative stress to be used in following experiments (Figure 1A).

#### 2.1.2. Effect of Fx on VIC Cell Viability

To evaluate Fx's effect on the VIC cell viability, we treated the cells with different doses of Fx for 24, 48, and 72 h and measured cell viability using and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. The results showed that Fx did not significantly affect the VIC cell viability, except when the highest dosage (5 mg/mL) was used, probably due to its toxic effect (Figure 1B).

**Figure 1.** Protective effect of fucoxanthin (Fx) on VIC cell viability following H2O2-induced oxidative stress. Rat heart valve interstitial cells (3000 cells/well) were cultured in DMEM/F12 supplemented with 10% FBS for 24 h. (**A**) Treated with H2O2 (0.1–10 mM) for 15 min, 1 h and 4 h. (**B**) Treated with Fx (0.01–5 mg/mL) for 24, 48, and 72 h. (**C**, **D**) Cells were pretreated with Fx (Fx, 0.01–1 mg/mL) for 24 h, then treated with 0.5 mM of H2O for 15 min. Cell viability was analyzed by MTT assay in (**C**) or through cell counting in (**D**). \*\*\*, *p* < 0.001 compared with untreated control cells. #, *p* < 0.05; ##, *p* < 0.01; ### , *p* < 0.001 compared with the H2O2-induced group. White bar, control group; Blue bar, different dosage of H2O2-induced group; Green bar, H2O2-induced group; Yellow bar, Fx-treated group.

#### 2.1.3. Fx-Abrogated H2O2-Induced VIC Viability Change

Next, we explored the combined effect of the Fx and H2O2 on VIC viability to evaluate the protective effect of Fx. Cells were first pretreated with different doses of Fx for 24 h, then exposed to 0.5 mM of H2O2 for 15 min., which earlier was enough to inhibit cell proliferation. MTT assay was used to measure the cell viability. The results showed that Fx could alleviate the inhibitory effect of H2O2-induced oxidative stress on VIC growth (Figure 1C,D).

#### *2.2. Fx-Ameliorated H2O2-Induced DNA Damage and Apoptosis-Related Protein Expression* 2.2.1. Fx Decreased H2O2-Induced VIC Cell Morphology Changes and DNA Damage

To further explore the protective effect of Fx on VIC cells in the context of halting DNA damage, we used PI staining and microscopy to visually observe any morphological changes. Following the pretreatment of the cells with Fx and then H2O2, PI staining with

fluorescence signal increased, the result showed that Fx was able to prevent oxidative stress-induced DNA damage (Figure 2A,B).

**Figure 2.** Fucoxanthin (Fx) ameliorated H2O2-induced DNA damage and apoptosis-related protein expression. Rat heart valve interstitial cells were cultured in a 6-well plate for 24 h, pretreated with Fx for 24 h, and then treated with H2O2 for 15 min. (**A**) Cells were stained with PI solution (1 μg/mL) for 1 h and visualized by microscopy at 200× magnification. (**B**) Quantified by Image J. Western blotting was used to explore the protein expression of (**C**) total and cleaved PARP, and (**D**) apoptosis-related markers cleaved caspase 3, Bcl2, and Bax. \*\*\*, *p* < 0.001 compared with untreated control group. ### , *p* < 0.001 compared with H2O2-induced group. White bar, control group; Green bar, H2O2-induced group; Yellow bar, Fx-treated group.

#### 2.2.2. Fx-Ameliorated H2O2-Induced Apoptosis-Related Protein Expression

Along with the increase in cell damage and decrease in cell viability, we used the Western blotting technique to explore the effect of the administration of both H2O2 and Fx on the apoptosis-related protein expression. The H2O2-induced group showed a significant increase in the expression of the cleaved form of PARP, while pretreatment with Fx significantly reversed the expression of cleaved PARP (Figure 2C). Furthermore, we explored other apoptosis-related markers, such as cleaved caspase 3 and the Bax/Bcl2 ratio (Figure 2D). The results confirmed that H2O2-induced apoptosis, which was reversed after the Fx treatments. Collectively, these results highlight the anti-apoptotic effect of Fx in VIC cells.

#### *2.3. Effect of Fx on H2O2-Induced Reactive Oxygen Species*

According to DCFDA's result, treatment with H2O2 induced a high level of ROS, which in turn could lead to damage to the valve structure and subsequent calcification [17]. The results show that Fx could alleviate the high oxidative stress-induced ROS level, as shown by a reduction in the fluorescence (Figure 3A) and density (Figure 3B).

**Figure 3.** Effect of fucoxanthin (Fx) on the H2O2-induced ROS level. Rat heart valve interstitial cells were cultured in DMEM/F12 supplemented with 10% FBS for 24 h, pretreated with 0.01–1 mg/mL of Fx for 24 h, then treated with 0.5 mM of H2O2 for 15 min. ROS were visualized using DCFDA. These were assessed by microscopy at 200× magnification (**A**) and we used Image J for the density quantification (**B**). \*\*\*, *p* < 0.05 compared with untreated control group; ### , *p* < 0.001 compared with H2O2 group. White bar, control group; Green bar, H2O2-induced group; Yellow bar, Fx-treated group.

#### *2.4. Effect of Fx on Oxidative Stress-Induced Calcification and the Expression of Its Related Markers in VIC*

Oxidative stress was shown to trigger calcification in VIC [18], which involves the activation of the Akt/ERK signaling pathway [19]. Therefore, we evaluated the effect of both H2O2 and Fx on the ROS and calcification-related Akt/ERK signaling pathway. The results showed that H2O2 treatment significantly increased the phosphorylation of Akt and ERK proteins. Notably, the pre-treatment with Fx was able to decrease such activation (Figure 4A,B). Furthermore, we used Alizarin Red-S for calcification staining and the results showed that calcification was increased in response to H2O2 and alleviated by Fx pretreatment (Figure 4C,D). Consistently, the Western blotting results showed that the Fx treatment was able to partially oppose the H2O2-induced ECM remodeling of marker matrix metalloproteinase 2 (MMP-2) in VIC cells (Figure 4E).

**Figure 4.** Effect of fucoxanthin (Fx) on oxidative stress-induced calcification and the expression of its related markers in VIC. Rat heart valve interstitial cells were cultured in a 6-well plate for 24 h, pretreated with Fx for 24 h, then induced with H2O2 for 15 min. Western blotting was used to analyze the protein expression of (**A**) pAkt/Akt (**B**) pERK/ERK. (**C**) Alizarin Red-S staining was used to visualize calcification, which was assessed by microscopy at a 200× magnification and (**D**) quantified by Image J. (**E**) MMP-2 protein expression was analyzed using Western blotting. \*\*, *p* < 0.01; \*\*\*, *p* < 0.001 compared with untreated control group. ##, *p* < 0.01; ###, *p* < 0.001 compared with H2O2-induced group. White bar, control group; Green bar, H2O2-induced group; Yellow bar, Fx-treated group.

#### *2.5. Long-Term Cardioprotective Effect of Fx Treatment in Dogs*

Long-term treatment with Fx in the 26 dogs resulted in a significant decrease in their vertebral heart size (VHS) (Figure 5A). VHS score is a number that normalizes heart size to body size using the mid-thoracic vertebrae as units of measurement, reflecting compensatory cardiac enlargement [20]. In addition, treatment showed the improvement of the left atrium to aortic (LA/AO) dimension ratio (Figure 5B), Tei index (Figure 5C), and E/e value (Figure 5D). The linkage of the mitral valve and tricuspid valve showed a significant decrease in echocardiography (Figure 5E,F). Collectively, Fx supplementation could improve the overall function of ventricular contraction and relaxation, confirming the findings of previous studies [21].

**Figure 5.** Long-term cardioprotective effect of fucoxanthin (Fx) treatment in dog. After Fx treatments, echocardiography analysis showed (**A**) a significant decrease in the VHS score, (**B**) an improvement in LA/AO, (**C**) a reduction in the Tei index, and (**D**) a decrease in the E/e percentage. The linkage of (**E**) the mitral valve and (**F**) the tricuspid valve decreased. \*\*\*, *p* < 0.001 compared with the baseline.
