Amine Oxidase (Copper-Containing)

The broilers were weighed, and feed intake was recorded on day 1, 21, and 35. ADGs, average daily feed intake, and feed conversion ratio (FCR) were determined for each study phase. At the end of day 21 and 35, six birds (one bird from each replicate cage) were randomly selected from each treatment, and 3 ml blood samples per bird were collected from the wing vein by using a sterilized syringe with a needle (5 ml, 0.7 mm). The blood samples were then transferred into the vacuum tubes (5 ml, anticoagulant free, Xiangyuan Medical Ltd., Hebei, China). For serum analysis, the blood samples were centrifuged at 2,000 g at 4°C for 10 min to separate the serum and stored at −80°C to determine the antioxidant parameters and intestinal permeability biomarkers. The activities of the total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), and the malondialdehyde (MDA) contents in serum were measured using commercial analysis kits (Jiancheng Bioengineering Institute, Nanjing, China) according to manufacturer's instructions. The levels of D-lactic acid and diamine oxidase (DAO) activities in serum were determined using enzyme-linked immunosorbent assay kits (96T, Yubo Biotechnology Co., Ltd. Shanghai, China) according to the manufacturer's instructions.
After blood sampling, the same birds were killed (by cutting carotid arteries) to collect the intestinal samples. Subsequently, the duodenum, jejunum, and ileum were separated, and ~2 cm segments of each at the middle location were collected and fixed in 10% buffered formalin until morphology analysis, stained with hematoxylin–eosin using standard histological techniques. The liver and remaining duodenum, jejunum, and ileum were rinsed in ice-cold phosphate-buffered saline (pH 7.0). Approximately 2 g liver tissues and mucosa of the duodenum, jejunum, and ileum samples were collected immediately for antioxidant parameter analysis. The activities of T-SOD, GSH-Px, CAT, and the MDA contents in the liver, mucosa of duodenum, jejunum, and ileum were measured using commercial kits (Jiancheng Bioengineering Institute, Nanjing, China) following manufacturer's instructions. Villus height, crypt depth, and villus width were measured at 40 × magnification using computer software (Olympus, DP72), then villus height-to-crypt depth ratio was calculated. The villus surface area (VSA) was calculated by using the formula (18 (link)):

MAO-A, MAO-B, DAO, LOXL2 and LOX enzymatic assays followed the same protocol as for SSAO, with slight modifications.
For recombinant human monoamine oxidases A and B (rhMAO-A and rhMAO-B, Sigma-Aldrich) the following substrates were used: 200 μM tyramine (Sigma-Aldrich) for rhMAO-A, and 100 μM benzylamine for rhMAO-B. The low controls for rhMAO-A and rhMAO-B assay were 1.5 μM clorgiline (Sigma-Aldrich) and 1 μM mofegiline, respectively.
Recombinant human diamine oxidase (rhDAO, kindly provided by Prof. Mitchell Guss, University of Sydney, Australia), inhibition assay utilized 200 μM putrescine and 10 μM of inhibitor aminoguanidine (both from Sigma-Aldrich) as substrate and low control, respectively.
rhLOXL2 (lysyl oxidase like 2) was purchased from R&D systems and assay was performed in 1.2 M urea, 50 mM sodium borate buffer, pH 8.2. The substrate used was 10 mM putrescine and 100 μM of β-aminopropionitrile (βAPN, Sigma-Aldrich) was added to the low controls.
Bovine Lysyl Oxidase (LOX) was extracted by adapting the methodology from Rucker et al. [26 (link)] from calf aorta: the homogenized tissue was washed extensively from readily soluble proteins and salts with PBS, and then extracted in 4 M urea, 50 mM sodium borate buffer, pH 8.2. The supernatant underwent buffer exchange and was concentrated by means of Amicon 10 kDa centrifuge filters (Millipore) in 1.2 M urea, 50 mM sodium borate buffer, pH 8.2 and in the presence of protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM N-ethlylmaleimide, 0.5 mM p-aminobenzoic acid, 20 mg/mL soybean trypsin inhibitor, all from Sigma-Aldrich). LOX enzyme mixture was pre-treated with 0.5 mM pargyline and 1 μM mofegiline (to inhibit any potential endogenous monoamine oxidase A and B as well as SSAO), in 1.2 M Urea buffer, 50 mM sodium borate, pH 8.2. The substrate used was 10 mM putrescine and 100 μM of β-aminopropionitrile (βAPN, Sigma-Aldrich) was added to the low control. Reaction mix was made up in 1.2 M urea, 50 mM sodium borate buffer, pH 8.2.

Phosphate buffer solutions (0.1 M, pH 6.0, 7.0, and 8.0) were prepared from Na₂HPO₄ and NaH₂PO₄ solids (Sigma S9638 and S9763). Carbonate buffer solution (0.1 M, pH 9.0) was prepared from Na₂CO₃ (Sigma 222,321) and NaHCO₃ (Sigma S5761).
Hydrogen peroxide stock solution (33% w/v) was supplied by Panreac (131,077.1211); the exact concentration was established and periodically checked by titration using potassium permanganate (oxalic acid as primary standard). Peroxidase from Horseradish (HRP EC 1.11.1.7) was obtained from Sigma (P8125 88.6 U mg⁻¹). Diamine oxidase from Lathirus cicera 280 U mL⁻¹ (DAO EC 1.4.3.22) was purchased from Molirom P021. Tyramine oxidase (TAO EC 1.4.3.9) from Arthrobacter sp. (T-25) 4600 U mg⁻¹ was purchased from Asahi Kasei Pharma Corporation.
Cadaverine (C8561), putrescine (P7505), histamine (53,300), tyramine (T287998), 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (A1888), 10-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red™, AR) (90,101), and 3,3′,5,5′-Tetramethylbenzidine (TMB) (860,336) were supplied by Sigma. All solutions were daily prepared by weighing and dissolving in the buffer solution (minus TMB and AR, which was dissolved in dimethyl sulfoxide (Panreac131954.1611)). TMB, ABTS, and AR solutions were stored in darkness.
Cellulose microcrystalline of 20 μm of particle size and average degree of polymerization minor than 350 (Aldrich 310,697) was used to develop the biosensors.

BJO was provided by Ming Xing Pharmaceutical Co. Ltd. (Guangzhou, Guangdong, China) with the number (Lot: 20180902), stored in 4°C refrigerator of room A204, laboratory of the School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, China. To obtain the emulsion, BJO was prepared as previously reported (Wang et al., 2020 (link)). We mixed the appropriate amount of soybean lecithin and water to obtain the blank emulsion. After mixing the proper amount of BJO with the blank emulsion for 9 min, we obtained the milky white oil emulsion like cream with high-pressure homogenization. All processes were followed the standard of the ministry of Health of the people’s Republic of China (WS3-B-3646-98).
5-FU was purchased from Sigma Corporation (United States). LO was produced by Yang Senlin Pharmaceutical (Xi’an, China). Malondialdehyde (MDA) and superoxide dismutase (SOD) assay kits were bought from Jiancheng Biotechnology Company (Nanjing, Jiangsu, China). The ELISA (enzyme-linked-immunosorbent serologic assay) kits (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-4 (IL-4), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and diamine oxidase (DAO)) were obtained from Shanghai MLBIO Biotechnology (Shanghai, China). Primary antibodies against proliferating cell nuclear antigen (PCNA), cyclooxygenase-2 (COX-2), zonula occludens-1 (ZO-1), occludin, claudin-1, and β-actin were purchased from Affinity Biosciences (OH, United States). TRIzol^® reagent was provided by Thermo Fisher (Waltham, MA, United States). Mice primers for occludin, claudin-1, CXC chemokine ligand 1 (CXCL1) and CXC chemokine ligand 2 (CXCL2) were purchased from (Sangon Biotech, Shanghai, China). Other required materials were bought from Vazyme Biotech (Nanjing, China).