**5. Conclusions**

Treatment with Fx was demonstrated to effectively protect against the harmful effects of high H2O2-induced oxidative stress in heart valve interstitial cells through the antioxidant potential of Fx as follows: (1) Fx can recover H2O2-induced cell viability impairment, (2) Fx can oppose H2O2-induced apoptosis, (3) Fx can inhibit the Akt/ERK-related signaling pathway to reduce heart valve calcification, (4) long-term treatment with Fx could recover the heart valve function and leakage in dogs. These data show that Fx has the potential to protect heart valve cells from damage caused by high oxidative stress (Figure 6).

**Figure 6.** Schematic representation of the potential effects of Fx on protection against high oxidative stress-related cell apoptosis and the ROS-related calcification signaling pathway. Furthermore, in vivo experiment shows Fx's ability to protect against valve-related disease in dogs.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/md19060307/s1: Figure S1. Protein marker expression in rat valve interstitial cells (A) Used western blot Rat valve interstitial cells (VICs) were negative for the endothelial cell marker, CD31, positive with vimentin and α-SMA. (B) Used microscopy in 200× magnification to capture the morphology of VICs.

**Author Contributions:** Conceptualization, Y.-F.C., H.-Y.C. (Hsin-Yuan Chen) and S.-M.H.; experimentation, Y.-F.C. and C.-H.T.; data analysis and figure preparation, Y.-F.C. and H.-Y.C. (Hsin-Yuan Chen); methodology and resources, Y.-F.C., C.-H.T., T.-M.S., Y.-J.H., Y.-H.H., K.-L.W., H.-Y.C. (Hsin-Yi Chang), C.-I.L. and T.-C.H.; writing—original draft preparation, Y.-F.C.; writing—review and editing, Y.-F.C., M.A. and S.-M.H.; editing and approval of the final version of the manuscript, S.-M.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by grants (MOST109-2314-B-038-059, MOST 109-2628-B-038- 015, MOST 109-2320-B-254-001, MOST 109-2811-B-038-523 and MOST 109-2320-B-424-001) from the Ministry of Science and Technology, Taiwan, and grants (MOE-RSC-108RSN0005) from the Ministry of Education, Taiwan.

**Institutional Review Board Statement:** The animal studies were conducted according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Medical University (NO. LAC-2020-0149).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Ethical Approval for Experiments on Animals:** The study was approved by the Experimental Animal Care and Use Committee of Taipei Medical University (NO. LAC-2020-0149). All animals received humane care in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals, published by the National Science Council, Taiwan.
