P ampk

Proteins were extracted from the liver or colon tissue, separated using SDS-PAGE, then transferred to PVDF membranes. The membranes were incubated overnight at 4 °C using rabbit polyclonal antibodies against β-actin (Catalog no. AF0003), AMPKα (Catalog no. AF1627), p-AMPK (Catalog no. AF2677), PPARγ (Catalog no. AF7797), ZO-1 (Catalog no. AF8394), and Occludin (Catalog no. AF7644) (Beyotime Biotechnology, Shanghai, China). The corresponding secondary antibody and ECL reagent were used to detect the target protein immobilized on the membrane.

Bovine serum albumin (BSA) and advanced glycation end products (AGEs) were purchased from Biovision (San Francisco, USA). Recombinant irisin was obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell culture medium α-MEM and fetal bovine serum were provided by HyClone (Pittsburgh, USA) and Gibco-Invitrogen (Grand Island, USA). Osteogenic and adipogenic induction media were obtained from Cyagen (Santa Clara, CA, USA). Annexin-V fluorescein isothiocyanate (FITC) apoptosis detection kit and JC-1 probe were obtained from Beyotime (Shanghai, China). Primary antibodies for Bax, SIRT3, LC3, Parkin, p62, and β-actin were provided by Cell Signaling Technology (Danvers, MA, USA); Bcl-2 and AIF antibodies were from Santa Cruz (Dallas, Texas, USA); PINK1, acetyl-SOD2-K68, and SOD2 antibodies were from Abcam (Cambridge, UK); and p-AMPK, t-AMPK, and PGC-1α antibodies were from Beyotime. And the secondary antibodies were provided by Thermo Fisher Scientific (Waltham, MA, USA). The hydrogel scaffolds used in calvarial defects were purchased from Sigma-Aldrich (HYSHP020, St. Louis, MO, USA) and prepared following the manufacturer's instructions.

Neutrophils were seeded into the 6-well plate and treated with NaF (0.25, 0.5, and 1 mM) for 2 h. Previous studies showed that these proteins are still phosphorylated when neutrophils were stimulated by toxins, such as Ochratoxin A, Fumonisin B1, and sodium arsenic [20 (link)–22 (link)]. Meanwhile, NET formation was detected 2 h after NaF stimulation. Therefore, we chose 2 h based on these previous published articles. Total protein was extracted using the Protein Extraction Reagent Kit (Beyotime Biotechnology, China). The protein concentration was measured by the BCA protein quantitative kit (Beyotime Biotechnology, China). The extracted samples were separated by 12% SDS-PAGE and transferred to the PVDF membrane. The membrane was sealed by 5% BSA at room temperature for 2 h. Then, the membrane was incubated with primary antibodies: p38 (Cell Signaling Technology, Cat# 8690), p-p38 (Cell Signaling Technology, Cat# 9215), p-AMPK (Beyotime, Cat#AA393), AMPK (Proteintech, Cat#10929-2-AP), and β-actin (Cell Signaling Technology, Cat# 8457) at 4°C overnight and the second antibody (1 : 10000, ImmunoWay, RS0001, RS0002) at room temperature for 2 h. The reactivity of the primary antibodies used in this study is against Cyprinus carpio. The ECL Plus Western Blotting detection system was used to detect the signal. Finally, the gray level was analyzed by ImageJ software.

Dietary tannic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). Assay kits of total cholesterol (TC), triacylglycerol (TG), high-density-lipoprotein cholesterol (HDL-C), low-density-lipoprotein cholesterol (LDL-C), liver malondialdehyde (MDA), total superoxide dismutase (SOD), alkaline phosphatase (AKP), alanine transaminase (ALT), and aspartate transaminase (AST) concentrations were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). ELISA kits of insulin, leptin, lipopolysaccharides (LPS), serum inflammatory cytokines (TNF-α and IL-6), and immunoglobulins (IgA, IgG, IgE, and IgM) were purchased from Wuhan Ilerite Biotechnology Co., Ltd., (Wuhan, China). Antibodies, including β-actin, AMPKα, p-AMPK, PPARγ, ZO-1, and Occludin, were purchased from Beyotime Biotechnology (Shanghai, China).