Tnf α

Serum levels of various cytokines were measured using ELISA kits according to the manufacturer’s instructions. The cytokines tested included IFN-γ, TNF-α, and IL-17 (Neobioscience Technology, Shenzhen, China) and KLK12, MMP-1, and MMP-9 (Jonln, Shanghai, China).

Assays for proinflammatory markers were carried out by means of commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions; ELISA kits for the following molecules were used: human IL-1β, IL-6, and TNF-α (all from Neobioscience, Shenzhen, China). First, the cell supernatants were added to microplate wells, which were precoated with specific antibodies in advance. After 1.5 h of incubation at 37°C, the unbound supernatants were washed away, and enzyme-linked antibodies were added to each well, followed by incubation for 1 h at 37°C. Then, a second washing step was performed to remove the unbound reagents. Finally, the enzyme-binding diluent was pipetted into each well, followed by incubation for 30 min, and the stop solution was added to end the color development reaction. The absorbance of the samples at 450 nm was measured using the microplate reader. A standard curve based on the results obtained using eight human standard solutions was plotted, and the sample concentrations were confirmed. All assays were performed in triplicate for each experiment.

Intracellular AGE levels and inflammatory cytokines were assessed by ELISA kits specific to AGEs (Sigma‐Aldrich), TNF‐α, IL‐1β and IL‐6 (Neobioscience, Shenzhen, China) according to the manufacturer's instructions.

ELISA kits were used to detect mouse serum IL-6, TNFα (NeoBioscience Technology, Shenzhen, China) according to the manufacturer’s protocols.

The levels of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) in intestinal tissue supernatants were determined using commercially available kits (Mouse TNF-α and IL-10 ELISA Kits; Neobioscience, Shenzhen, China) according to the manufacturer's instructions.

(R)-sal, (S)-sal, and (RS)-sal were supplied by Dongguan Key-Pharma Biomedical Company (Guangdong, China). DSS (MW 36-50 kDa) was obtained from MP Biomedicals Co., Ltd. (California, USA). The positive drug 5-ASA was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The reagent for detecting fecal occult blood was supplied by Baso Diagnostics, Inc. (Zhuhai, China). The enzyme-linked immunosorbent assay (ELISA) kits for detecting the inflammatory cytokines interferon-γ (IFN-γ), interleukin- (IL-) 1β, IL-6, and tumor necrosis factor-α (TNF-α) were offered by Neobioscience Technology Company (Shenzhen, China). The antibodies of β-actin (ab227387), occludin (ab216327), and CD4 (ab183685) were obtained from Abcam Inc. (Burlingame, USA). The antibodies of zonula occludens-1 (ZO-1) (bs-1329R) and Nrf-2 (bs-1074R) were obtained from Bioss (Beijing, China). The antibodies of NF-κB p65 (10745-1-AP) and heme oxygenase-1 (HO-1) (10701-1-AP) were purchased from Proteintech (Rosemont, USA). The antibody of phospho-NF-κB p65 (p-NF-κB p65) was purchased from Cell Signaling Technology (Danvers, USA). The antibodies of transforming growth factor-β1 (TGF-β1) (BA0290) and alpha smooth muscle actin (α-SMA) (BM0002) were offered by Boster Biological Technology Co., Ltd. (Wuhan, China). All chemical reagents employed were of analytical grade.

After BMDMs were stimulated to polarize for 24 h as previously described, the supernatant was collected and centrifuged at 3000 rpm/min for 10 min to remove dead cells and debris. ELISA analyses for tumor necrosis factor-α (TNF-α, Neobioscience, China) were then performed following the manufacturer’s instructions.