Gsh px

Rat skeletal muscle tissue was quickly removed, homogenized in cold PBS on ice, and preserved at -80°C. Approximately 60 mg of skeletal muscle tissue was obtained by ultrasonic grinding and centrifuged at 12,000 rpm for 15 minutes at 4°C. Before collection of serum, blood was centrifuged at 3,000 rpm for 15 minutes. All supernatants of gastrocnemius and serum were collected for oxidative stress analysis. After digesting the C2C12 cells in different groups with trypsin, the culture medium was centrifuged at 1,000 rpm for 10 minutes at room temperature and the supernatant was discarded to retain the cell precipitation. Then, SOD, GSH-Px, and CAT activity and the MDA level were measured in gastrocnemius tissue, serum, and C2C12 cells using the detection kits (SOD: A00-1; MDA: A003-1; CAT: A007-1; GSH-Px: A005, Jiancheng Bioengineering Ltd., Nanjing, China) in accordance with the manufacturer's instructions.

The expression levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in cell supernatant were measured using SOD, GSH-Px and CAT commercial kits (Nanjing Jiancheng Bioengineer Institute, China) according to the manufacturer’s instructions.

We used CAT, SOD, GSH-Px, MDA, and mouse hydroxyproline (Hyp) assay kits (Jiancheng Institute of Biotechnology, Nanjing, China), as well as ELISA kits: TNF-α, IL-6, MMP-1, MMP-3, IL-1β, COX-2 and PGE₂ (Cheng Lin Biological Technology Co., Ltd., Beijing, China). All other reagents used were of analytical grade.

The standard feed for mice was purchased from Beijing Keaoki Feed Co. Ltd. (Beijing, China). Cyclophosphamide was obtained from Sigma Aldrich Company (St Louis, USA). SOD, GSH‐Px, CAT, and MDA assay kits were purchased from the Nanjing Jiancheng Institute of Biological Engineering (Nanjing, China). The Nrf2 and Keap1 antibodies for western blot were purchased from the Abcam (Cambridge, MA, USA). Rabbit anti‐GAPDH and hematoxylin and eosin (HE) staining solutions were purchased from Beijing Solaibao Technology Co. Ltd (Beijing, China). In addition to the above reagents, all other analytical reagents are either of the highest or commercial grade.

Commercial kits obtained for SOD, GSH, GSH-Px, and MDA from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) were used to measure their activities in the hemolymph supernatant. They were measured using a UV-spectrophotometer (Beijing Purkinje General Instrument Co., Ltd) at 520, 420, 412, and 532 nm as described by the manufacturer's protocols, respectively.

Fertilized eggs were purchased from a hatchery (Changlv Native Products, Nanjing, China), and were incubated at 37.8°C and 60% relative humidity in an incubator (Photoshop Solar Energy Co., Zibo, China). Fertilized eggs were studied at day 14, 15, 16, 17, 18, 19, and 20 of incubation. SOD, GSH-PX, Peroxidase (POD) and total protein quantitative assay kits were provided by Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).

The levels of reactive oxygen species (ROS) (specifically H₂O₂) and malondialdehyde (MDA) and the activities of antioxidants (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px)) in gingival tissues were detected by corresponding assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. The optical density (OD) values were read with a microplate reader (BioTek, Winooski, VT, USA).