Occludin

Total proteins from jejunal epithelium were extracted through RIPA lysis buffer (#AR0108, BOSTER). The mitochondrial proteins were extracted using the Tissue Mitochondria Isolation Kit (Beyotime). A BCA Protein Assay Kit (Beyotime) quantified protein concentration following the manufacturer’s protocols. Certain amounts of proteins (~30 μg) were loaded onto each lane of SDS-PAGE gel to undergo electrophoresis, then transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After blocking and washing, the membranes were incubated with specific primary antibodies (below) at 4°C overnight. Membrane incubation of corresponding HRP-conjugated secondary antibodies was conducted at room temperature for 1 h. Subsequently, ECL reagents (#E170-01, GenStar) were prepared and added to the membranes for chemiluminescence, which were imaged and captured using a ChemiDoc Touch imaging system (BIO-RAD, Hercules, CA, USA). Primary antibodies used in this study were: Occludin (#AF7644, Beyotime), Claudin 1 (#28674-1-AP, Proteintech), Claudin 2 (#AF0128, Affinity, Changzhou, China), IL-6 (#500286, ZEN BIO, Chengdu, China), Caspase-3 (#19677-1-AP, Proteintech), Caspase-9 (#10380-1-AP, Proteintech), AIF (#AA306, Beyotime), Calpain-1 (#381868, ZEN BIO), Cox-IV (#200147, ZEN BIO), and β-actin (#T0022, Affinity) (internal reference for total protein).

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.

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).