Evaluating Barrier Function of Herb Compounds

Puerarin (PubChem compound identifier [CID]: 5281807), peoniflorin (PubChem CID: 442534), and menthol (PubChem CID: 16666) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Polyethylene terephthalate (PET) cell culture inserts and 12-well plates (12-mm diameter, 0.4-μm pore size) were purchased from Corning Corporation (Corning, NY, USA). The rabbit anti-zonula occludens 1 (ZO-1) antibody (61-7300) and the mouse anti-claudin-1 antibody (2H10D10) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Tetramethyl rhodamine isothiocyanate (TRITC, red)-conjugated anti-rabbit IgG antibody was purchased from Beijing Zhongshan Golden Bridge Biotechnology Co, Ltd (Beijing, China). Fluorescein isothiocyanate (FITC, green)-conjugated anti-mouse IgG antibody was purchased from Kangwei Century Biotechnology Co, Ltd (Beijing, China). Acti-stain 488 (green) fluorescent phalloidin was purchased from Cytoskeleton Inc (Denver, CO, USA). 2-(6-(7-nitrobenz-2-oxa-,3-diazol-4-yl)amino) hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-C6-HPC) was purchased from Thermo Fisher Scientific. A total protein bicinchoninic acid assay kit, a Na⁺-K⁺-ATPase assay kit, and a Ca²⁺-ATPase assay kit were purchased from NanJing JianCheng Bioengineering Institute (NanJing, China). In this study, all experimental protocols were approved by the Review Committee for the Use of Human or Animal Subjects of Beijing University of Chinese Medicine.