*4.9. Immunohistochemical Analysis*

The kidney slices were dewaxed and hydrated by SP immunohistochemical method, the antigens were repaired with citric acid buffer solution, catalase and nonspecific reaction sites were sealed with H2O2 and goat serum, and the slices were rinsed with PBS. According to the requirements of the kit, the expression of caspase-3 protein in renal tissue was detected. The images were observed with an optical microscope and analyzed with ImageJ software.

#### *4.10. Statistical Analysis*

Data are expressed as mean ± standard deviation. Statistical analysis was conducted through one-way ANOVA using SPSS 20.0 software (IBM, New York, NY, USA). All data are presented as the mean ± SD, and values of *p* < 0.05 and *p* < 0.01 were considered statistically significant.

### **5. Conclusions**

In this study, we demonstrated the antioxidant activity and corresponding inflammatory response of ulvan against oxidative stress induced by D-gal in mice. The results showed that ulvan could significantly decrease the contents of Scr, BUN, and Cys-C in the kidney; increase the glomerular filtration rate; improve the activities of SOD and GSH-Px and total antioxidant capacity in mice; and reduce the damage to biomacromolecules caused by oxidative damage. In addition, it also significantly improved the changes in TNF-α and IL-6 caused by oxidative stress. In the aspect of apoptosis, ulvan could protect against the D-gal-induced apoptosis of renal tubule cells. These results confirm that ulvan has a protective effect on kidney injury caused by oxidative stress. It provides a basis for the development of antioxidants and the treatment of related diseases in the future.

**Author Contributions:** Conceptualization, Q.Y. and Z.S.; methodology, Q.Y. and S.F.; formal analysis, Q.Y., Y.J. and W.Z.; data curation, Q.Y., Y.J. and Z.S.; writing, Q.Y., Y.J. and Z.S.; visualization, Q.Y., Z.S. and X.J.; supervision, X.J. and Z.S.; project administration, Z.Z. and X.J.; funding acquisition, Z.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the Qingdao Marine Biological Medicine Science and Technology Innovation Center Construction Project (2017-CXZX01-4-6); the Key Project of New and Old Energy Transformation in Shandong Province; the University Co-construction Project—Marine Health Products, Qingdao, Shandong, China (grant number HYJK1907, 2019); and Fujian Provincial Regional Development Project, 2020N3016.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethical Committee of Experimental Animal Care at Ocean University of China (certificate no. SYXK2012014).

**Conflicts of Interest:** The authors declare that they have no competing financial or nonfinancial conflict of interest.

#### **References**

