Il 1β

DSS (36–50 kDa) was purchased from MP Biomedicals (Canada). Phospho–NF–κB p65 Rabbit mAb (cat. no. 3033) and NF-κB p65 Rabbit mAb (cat. no. 8242) were obtained from Cell Signaling Technology, Inc. TLR4 (19811-1-AP), zonula occludens-1 (ZO-1) (21773-1-AP), Occludin (27260-1-AP), and GAPDH (10494-1-AP) were obtained from Proteintech (USA). MyD88 (AF5195) was purchased from Affinity Biosciences (USA). COX-2 (bs-0732R) was obtained from BIOSS. Jat Standard (purity ≥98%, Cat. No. 6681-15-8) was purchased from Chroma Biotechnology Co. Ltd. (Chengdu, China). Mesalazine (5-ASA) was purchased from Losan Pharma GmbH (lot. no. L20235A). Jat and 5-ASA were dissolved in water to make an oral suspension. IL-1β, TNF-α, MPO, IL-10, and TGF-β enzyme-linked immunosorbent assay (ELISA) kits were purchased from Shanghai Enzyme-Linked Biotechnology (Shanghai, China) Co., Ltd.

The serum of mice was collected, and the serum TNF-α, IL-6, and IL-1β (Shanghai Enzyme-linked Biotechnology Co., Ltd.) levels were measured according to the manufacturer’s instructions.

CBD was purchased from Xi’a Lvruquan Biotechnology Co., LTD. (Lot number: LRQ221103-1); ELISA kits for TNF-α (ml002859), IL-1β (ml037361) and IL-6 (ml064292) were purchased from Shanghai Enzyme-Linked Biotechnology Co., LTD. Bleomycin (lot number: Z8020) was purchased from Solebol Biotechnology Co., LTD. Prednisone acetate tablets (lot number: LA22255) were purchased from Zhejiang Sienju Pharmaceutical Co., LTD. Acetonitrile (chromatographic grade) (batch number: A996-4), methanol (chromatographic grade) (batch number: 67-52-1, Dikma Technology Company); Ultra high liquid chromatography-time-of-flight Tandem Mass Spectrometer (Waters, United States); KDC-160HR high-speed refrigerated centrifuge (China University of Science and Technology Innovation Co., LTD.).
Preparation of cannabidiol (CBD): Preparation of 0.5% sodium carboxymethyl cellulose (CMC-Na) three-steam aqueous solution, take 108 mg cannabidiol dissolved in 10 mL of the above solution to make a suspension, the concentration is 10.8 mg/mL, diluted during use.

Sodium butyrate (Figure 1(a); HPLC ≥ 98.5% purity) was obtained from Sigma-Aldrich (Shanghai) Trading Co. Ltd. (China). TNF-α, IL-1β, IL-6, IL-10, TGF-β, and IL-17 assay kits were purchased from Shanghai Enzyme-linked Biotechnology Co. Ltd. (Shanghai, China). Primary antibodies, including anti-iNOS (Cell Signaling, 13120S), anti-Arg-1 (Cell Signaling, 93668S), anti-ROR gamma(t) (Invitrogen, 14-6988-82), anti-FoxP3 (Beijing Biosynthesis Biotechnology Co. Ltd., Beijing, China, bs-10211R), anti-β-actin (Proteintech, 20536-1-AP), and anti-insulin (Abcam, ab181547). Goat anti-rabbit secondary antibodies (cat. no. 10285-1-AP) were obtained from ProteinTech Group, Inc. (Chicago, IL, USA).

The ELISA kits of TNF-α (1102589), IL-1β (1102590), and IL-6 (1099199) were obtained from Shanghai Enzyme-Linked Biotechnology Co., Ltd. (Shanghai, China). The enzyme plate was sealed after adding the sample and incubated at 37°C for 30 min, washed 5 times with detergent, and incubated at 37°C for 30 min with the HRP labeled enzyme labeled reagent. The plate was washed 5 times again, and the color reagent was applied 20 min later and the reaction was stopped by adding the termination solution. OD at 450 nm was analyzed.

According to the manufacturer’s protocol, the levels of IL-1β (Shanghai Enzyme-Linked Biotechnology, cat#ml301814), TNF-α (Cayman Chemical, cat#500850), and IL-10 (Shanghai EnzymeLinked Biotechnology, cat#ml037873) were detected by indicated ELISA assay kits (Cayman Chemical, Michigan, USA). The absorbance value of each well was detected at 450 nm using the Microplate Reader (Thermo Scientific, NY, USA).

ELISA kit detected the concentration of IL-1β, IL-6, MPO and TNF-α (Shanghai Enzyme-linked Biotechnology Co., Ltd., China). According to the instruction of ELISA kit, the standard (50 μL) and sample (40 μL Sample diluent and 10 μL sample) were added to the standard holes and sample holes of microtiter plate, respectively. The culture plates were incubated at 37°C in 5% CO₂ for 30 min and then rinsed with washing solution. Then 50 μL enzyme-labeled reagent was added into each well, and the samples were incubated for 30 min and washed. Then 50 μL color developer A and color developer B were added, mixed, and stained in the dark for 10 min. After the reaction was stopped by adding stop solution, the absorbance density (OD) was measured at 450 nm by enzyme-labeled instrument (Molecular, CA, USA).