In Vivo Sepsis and In Vitro HUVEC Assays

In vivo sepsis model was constructed using cecal ligation and puncture (CLP) method reported previously (Zhao et al., 2016 (link)). Firstly, the mice were anesthetized by isoflurane, local analgesine by 1% bupivacaine, and an incision was made in the midline. Secondly, the cecum was eviscerated, ligated using 5–0 silk suture, and punctured through-and-through using a 20-gauge needle. Thirdly, the abdomen was closed layer by layer. Finally, the mice in sham groups were manipulated in the same way, without the CLP component. All animal experiments were performed following the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
For in vitro analysis, the Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC, Manassas, United States). They were cultured in dulbecco’s modified eagle medium (DMEM) (HyClone, Logan, Utah, United States) containing 10% fetal bovine serum (FBS) (Biological Industries, Israel) and 1% penicillin-streptomycin (HyClone, United States), and then cultured at 37°C, 5% CO₂. The HUVECs were treated with lipopolysaccharide (LPS) (E. coli O111:B4, Sigma, St Louis, MO), HFn, GF9-HFn, HFn/Str, and GF9-HFn/Str.