Anti occludin

Corilagin (CLG) was procured from Sigma-Aldrich (St. Louis, MO). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor^® 488 donkey anti-mouse IgG (H+L) antibody and Fluor^® 594 donkey anti-rabbit IgG (H+L) antibody was obtained from Life Technologies (Grand Island, NY, USA). Anti-MnSOD(sc-137254), anti-Fibronectin(sc-6952), anti-Vimentin(sc-6260), anti-E-cadherin(sc-8426), anti-N-cadherin(sc-271386), anti-Occludin(sc-5562), anti-Twist(sc-15393), anti-MMP-2(sc-53630), anti-MMP-9(sc-393859), anti-Wnt3a(sc-136163), anti-FZD-1(sc-398082), and anti-β-actin(sc-47778) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Snail(3879S), anti-Axin-1 anti-Azin-1(3323S), anti-β-catenin(9562S), anti-p-GSK3β(9322S), and anti-GSK3β(9315S) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA).

Western blots were performed as described [41 (link)]. Primary antibody: anti-PPAR-α (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany, sc-398394), anti-TRL4 (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany, sc-293072), anti-pERK 1/2 (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany, sc-7383), anti-NLRP3 (1:500, Santa Cruz Biotechnology, Heidelberg, Germany, sc-134306), anti-transforming growth factor-beta3 (TGF-β3), (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany, sc-166833), anti-claudin-11 (1:100, Santa Cruz Bio-technology, Heidelberg, Germany, sc-271232), or anti-occludin (1:100, Santa Cruz Biotechnology, Heidelberg, Germany, sc-133255) [44 (link),45 (link)].

Anti-p-p65, anti-p65, anti-p-ERK1/2, anti-ERK1/2, anti-p-AKT, and anti-AKT were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-GAPDH antibody was purchased from the ProteinTech Group (Chicago, IL, USA). Anti-BMP2, anti-BMP4, anti-BMPRIa, anti-BMPRII, anti-BMPRIb, and anti-occludin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant BMP2, BMP4, and noggin were purchased from Peprotech (NJ, USA). The inhibitor of NF-κB, PDTC, was purchased from Beyotime (Wuhan, China).

Liver tissues were fixed in 4% paraformaldehyde, and Hematoxylin, and Eosin (H&E) staining was used for histologic analysis. Liver fibrosis was determined through Masson’s Trichrome staining. and the sizes of fibrotic septa were quantitated via densitometry. Lymphocyte infiltration in the liver was determined via anti-CD3 (Abcam, Cat. 16669, United States). Colorimetric images for H&E and Masson’s Trichrome staining were captured via Nikon eclipse Ti-U microscope. Tight junctions in the ileum were examined using immunofluorescent staining with anti-occludin (Santa Cruz Biotechnology, Cat. SC5562, United States) and all images were captured via the Leica TCS SP5 II system.

Total protein was extracted from cells using a protein lysis buffer. Total protein was quantified using a Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Inc.) and equal amounts of protein were separated via SDS/PAGE. The separated proteins were subsequently transferred to a polyvinylidene difluoride (PVDF) membrane and blocked with 5% skimmed milk for 2 h. The membranes were incubated with the following primary antibodies: anti-Tim-3 antibody (cat. no. ab241332; Abcam), anti-GAPDH (cat. no. sc-47724; Santa Cruz Biotechnology, Inc.), anti-cyclin D1 (CCND1) (cat. no. sc-8396; Santa Cruz Biotechnology, Inc.), anti-ZO2 (cat. no. sc-11448; Santa Cruz Biotechnology, Inc.) and anti-Occludin (cat. no. sc-133256; Santa Cruz Biotechnology, Inc.). Following the primary antibody incubation, the membranes were incubated with anti-mouse (cat. no. A5278; Sigma–Aldrich; Merck KGaA), anti-rabbit (cat. no. A0545; Sigma–Aldrich; Merck KGaA) and anti-goat (cat. no. A8919; Sigma–Aldrich; Merck KGaA) secondary antibodies. Protein bands were visualized using Luminata Forte (Merck KGaA) and quantified using ImageJ software (National Institutes of Health) based on the band intensities.

The sections were incubated with 5% bovine serum albumin (BSA) for 1 hr at room temperature and then incubated overnight at 4°C with primary antibodies in blocking buffer (Claudin‐5, Santa Cruz Biotechnology; Occludin, Santa Cruz Biotechnology; CD31, Santa Cruz Biotechnology). Then the cords were separately incubated with secondary antibody (Alexa Fluor 488‐conjugated anti‐IgG, Abcam; Texas red‐conjugated anti‐IgG, Santa Cruz Biotechnology). The nuclei were stained with Hoechst 33258 (0.25 mg/ml) dye (Beyotime Institute of Biotechnology, Shanghai, China). For cells, grown on 14 × 14 mm microscopic glass were washed with ice‐cold PBS, fixed with 4% paraformaldehyde for 30 min., then washed with ice‐cold PBS, and blocked in 5% BSA for 1 hr. Then cells were incubated with anti‐p120‐Catennin (Abcam), anti‐beta‐Catenin (Abcam), anti‐Occludin (Santa Cruz Biotechnology), anti‐Claudin‐5 (Santa Cruz Biotechnology) diluted in 1% BSA at 4°C overnight. Cells were washed with PBS followed by incubation with Alexa Fluor 488‐conjugated anti‐IgG or Texas red‐conjugated anti‐IgG secondary antibodies for 1 hr at room temperature. After washing with PBS, the nuclei were stained with Hoechst 33258 (0.25 mg/ml) dye for 7 min., washed with PBS. At last, cells were added with Antifade Mounting Medium (Beyotime Institute of Biotechnology).