Tudca

Male C57BL/6 mice (6 weeks, weighing 19–20 g) were obtained from Jinan Pengyue Experimental Animal Breeding Center (Shandong, China) and housed in the Southern Medical University Animal Experiment Center (Guangzhou, China). AOPPs were prepared in vitro by mixing Rabbit Serum Albumin (Sigma, St Louis, MO) with sodium hypochlorite (Macklin, Shanghai, China) for 30 min, as described previously.¹² (link)^,13 (link)^,14 (link)^,15 (link) All mice were randomly divided into three groups (n = 5 in each group) and given intraperitoneal injections of the following: (i) vehicle group: daily injection of phosphate-buffered saline (PBS) for 28 days (pH 7.4); (ii) AOPP group: daily injection of AOPP (50 mg/kg for 28 days); (iii) AOPP plus TUDCA (Selleck, Shanghai, China)-treated group: daily injection of TUDCA (250 mg/kg) for 2 h, before injection of AOPP (50 mg/kg) for 28 days. Mice were anesthetized and exsanguinated after treatment as indicated, and the entire intestinal tissues were removed for further investigation. The experimental procedures with animals complied with ARRIVE guidelines and were performed in accordance with the U.K. Animals (Scientific Procedures) Act, 1986. In addition, all animal experiments were approved by the Laboratory Animal Care and Use Committee of Southern Medical University (Number: L2017138).

Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA). Primary human umbilical vein endothelial cell (HUVEC) was purchased from Thermo Fisher Scientific (C0035C, Waltham, MA, USA) and grown in M200 medium (Thermo Fisher Scientific, M200500, Waltham, MA, USA) containing large vessel endothelial supplement (LVES) (Thermo Fisher Scientific, A1460801, Waltham, MA, USA). For transduction, Lenti-X HEK 293T cells (Clontech, 632180, CA, USA) were cultured using DMEM high glucose medium (Gibco, USA) which was supplemented with 10% FBS and 1X Antibiotic-Antimycotic. All the cells were maintained in a humidified incubation chamber with 5% CO₂ at 37 °C. Cells were treated with freshly prepared and filter sterilized Hcy (Sigma-Aldrich, H4628, USA), Cys (Sigma-Aldrich, C9768, St. Louis, MO, USA), 4-PBA (Sigma-Aldrich, P21005, USA) and TUDCA (Selleck Chemicals, S3654, Houston, TX, USA) at indicated doses. For rescue experiments, 4-PBA and TUDCA pretreatments were performed for 2 h and 14 h, respectively.