Occludin

CaCO₂ and HT‐29 cells were obtained from American Type Cell Collection (Manassas, VA). Cell culture medium and foetal bovine serum were purchased from Life Technologies, and α‐cyano‐4‐hydroxycinnamate (C2020) was supplied by Sigma. Antibody against MCT4 (sc‐50329) and CBP (sc‐7300 X) were from Santa Cruz Biotechnology. TJP1 (A0659), IL‐6 (A2447), IL‐10 (A2171), TNF‐α (A0277), phospho‐CREB (Ser133) (AP0333), CREB (A10826), E‐cadherin (A3044), Occludin (A2601), TJP2 (A0594), Claudin1 (A2196), Claudin2 (A11843), Claudin5 (A10207), Claudin7 (A2035), Claudin8 (A8174), Claudin14 (A2948), JAMA (A1241), Histone 3 (A2348) and alpha‐tubulin (AC013) were purchased from Abclonal. Phospho‐p65 (Ser536) (No.3033) and p65 (No.8242) were purchased from Cell Signaling Technology. Other reagents used in this study were purchased from Sigma.

The colonic tissues were homogenized in lysis buffer for 30 min, followed by centrifugation. The supernatant was collected, and the protein concentration was determined with BCA method. Then, the supernatant containing 50 μg of protein was boiled for 5 min, and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein separation. Then, the proteins were transferred onto nitrocellulose membranes which were then treated with 5% non-fat milk in TBST containing 10 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, and 0.05% Tween-20 at room temperature to block nonspecific reactivity for 1 h. Following primary antibodies were used: occludin (1:1,000, ABclonal, no: A12621, Wuhan, China), claudin-1(1:1000, ABclonal, no: A2196, Wuhan, China) and anti-GAPDH (1:1,000, Boster Biotechnology, BM1985, Wuhan, China).

Western blotting was performed as previously described [25 (link)]. Lysis buffer with added protease and phosphatase inhibitors were used for extracting total protein from intestinal tissues in each group (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). After grinding tissue, the supernatant protein was collected and quantified via the bicinchoninic acid method. PVDF membranes were incubated with specific primary antibodies, which included rabbit monoclonals against occludin (1:1000; Abclonal), claudin-1 (1:1000; Abclonal), COX-2 (1:1000; Abcam), caspase-3 (1:1000; CST), Bcl2 (1:1000; Abclonal), Bax (1:1000; Abclonal), HMGB1 (1:1000; NOVUS), Nrf2 (1:1000; NOVUS), HO-1 (1:1000; Abclonal), and mouse polyclonals against GAPDH (1:1000; Abcam) and β-actin (1:1000; Abcam) at 4 °C overnight. After incubation with the primaries, the membrane was washed three times, then incubated with goat anti-rabbit (1:5000; Zhongshan Golden Bridge, China) and goat anti-mouse (1:5000; Zhongshan Golden Bridge, China) IgG antibodies at room temperature for 1 h, and then washed three times. Lastly, the membranes incubated with enhanced chemiluminescence were exposed to the ChemiDoc Touch Imaging System. The grayscale values of protein bands were analyzed using Image J.

Frozen 6 μm thick tissue sections were prepared as described above [24 (link)]. Tissue sections were blocked by five percent bovine serum albumin in phosphate-buffered saline (PBS) with 0.1% Triton X-100, which were then incubated at 4 °C overnight with primary antibodies against matrix metalloproteinase-9 (MMP-9) (1:500, rabbit IgG; CST), MPO (1:500, rabbit IgG; CST, Danvers, MA, USA), occludin (1:200, rabbit IgG; Abclonal, Woburn, MA, USA), claudin-1 (1:200, rabbit IgG; Abclonal), COX-2 (1:500, rabbit IgG; Abcam, Cambridge, UK), 4-HNE (1:200, rabbit IgG; Abcam), and 8-OHDG (1:200, rabbit IgG; Santa Cruz, Dallas, TX, USA). After being rinsed four times with PBS for 5 min, the sections were then incubated with secondary antibody (1:500, Dylight488 goat anti-rabbit IgG, Abcam) for 1 h. After being washed three times with PBS, a mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) was added to the sections, which were then sealed with a coverslip. Fluorescent signals were observed under a fluorescence microscope (Olympus BX61, Tokyo, Japan). Three areas were selected from each slide for analysis, and mean values were calculated.

DSS (molecular weight 36−50 kDa) was purchased from Aladdin–Holdings Group (Shanghai, China). Naringin (purity > 98%) were provided by Energy Chemical (Shanghai, China). The ELISA kits of IL-6 and TNF-α, superoxide dismutase (SOD), glutathione (GSH) and malondialdehyde (MDA) assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Rabbit polyclonal antibodies for TNF-α, IL-6, IL-1β, COX-2, iNOS, Occludin, ZO-1, goat anti-rabbit IgG secondary antibodies and Texas Red conjugated goat anti-rabbit antibody were obtained from ABclonal Technology (Wuhan, China). Rabbit polyclonal antibodies GAPDH was purchased from Cell Signaling Technology (Boston, MA, USA). All other reagents were commercially available and of reagent grade.

Total proteins were extracted from colonic tissues and HT-29 cells using radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, China) containing 1% protease and phosphatase inhibitors (MedChemExpress, USA). Total protein concentrations were quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). Protein lysates were separated using a 10% or 7.5% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride membrane. Membranes were closed using a closure buffer consisting of 5% bovine serum albumin (BSA; Antgene Biotechnology, China) in tris-buffered saline with 0.1% Tween-20 at room temperature for 1 h before incubation with primary and secondary antibodies (Antgene Biotechnology, China). Enhanced chemoluminescence western blotting substrate (ECL; Thermo Fisher Scientific, USA) was added to the membrane with target protein for 1 min. Exposure was controlled by an automatic exposure imaging system (Bio-Rad, USA). Primary antibodies against cGAS, STING, p-STING, TBK1, p-TBK1, IRF-3, p-IRF-3, LC3, P62, Beclin1, ATG12, BiP, eIF2α, P- eIF2α, JNK1, p-JNK (Cell Signaling Technology, USA), occludin (ABClonal Technology, China), and ZO-1 (Thermo Fisher Scientific, USA) were used.

Brain tissue was taken, the tissue was lysed on ice with RIPA lysis buffer, and the lysed material was blown with 1 mL pipette to destroy the DNA. After centrifuge, 3μL supernant was collected for BCA assay to quantify, and the remaining samples were incubated at 95°C for 5–10 min for denaturation and stored at −20°C. Prepare 10% SDS-PAGE gel, add 40 g protein per well, after electrophoresis separation of protein, 200 mA constant current for 1 hour to transfer protein to PVDF membrane, then blocked with 5% skim milk for 1 hour at room temperate. Then the primary antibody was incubated overnight at 4°C,including ZO-1, claudin-1, claudin-5, Occludin, eNOS were all purchased from ABclonal (Wuhan, China), iNOS (Cat:189885-1-AP, Proteintech, USA) and the internal reference antibody β-actin (Cat:R1102-1, Huabio, Hangzhou, China). Then membrane was washed with TBST for three times, and incubated with secondary antibody at room temperature for 1 h, and then membrane was washed with TBST for three times. TBST was discarded, ECL luminescent solution was added, and developed in the protein imager. Image J was used to quantify the gray value of the band.