In the control cells, the mRNA expression of ZO-1 and occludin genes did not change over time, whereas in the -chaconine treated cells, the effects on the mRNA expression were concentration- and time-dependent

In the control cells, the mRNA expression of ZO-1 and occludin genes did not change over time, whereas in the -chaconine treated cells, the effects on the mRNA expression were concentration- and time-dependent. that -chaconine significantly decreased cell proliferation rate, increased apoptosis rate, decreased transepithelial electrical resistance (TEER) value, and increased alkaline phosphatase (AKP) and lactate dehydrogenase (LDH) activities, and there were interactions between -chaconine concentration and Bumetanide incubation time. -Chaconine significantly reduced the relative and mRNA expressions of genes coding Bumetanide tight junction proteins zonula occludens-1 (ZO-1) and occludin, increased malondialdehyde (MDA) content, decreased total glutathione (T-GSH) content, reduced the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and -glutamylcysteine synthetase (-GCS) and the mRNA expressions of SOD, CAT, GSH-Px, and -GCS genes. In conclusion, -chaconine disrupts the cell cycle, destroys the mechanical barrier and permeability of mucosal epithelium, inhibits cell proliferation, and accelerates cell apoptosis. cell culture. The exploration of the toxicity of -chaconine to digestive system cells at the molecular level would provide solid scientific data for revealing the mechanisms of how -chaconine affects intestinal health. Materials and Methods Cell Culture The murine intestinal epithelial cell line MODE-K (Shanghai Jining Industrial Co., Ltd., Shanghai, China) was adopted for the study. The cells were washed with a cell culture solution that was comprised of the 1640 medium (PM150910; Procell, Wuhan, China) +10% fetal bovine serum (FBS, Gibco, Carlsbad, NM, United States) solution (pH 7.2C7.4) +1% penicillin-streptomycin (Sigma, St. Louis, MO, United States) to remove DMSO. The resuscitated cells were cultured in a fresh cell culture solution and under 5% CO2 saturated humidity at 37C. The cell culture experiments were performed in triplicate with -chaconine treatment at concentrations of 0, 0.4, and 0.8 g/mL. -Chaconine stock Bumetanide solution (16 mg/100 mL) was made and diluted with the cell culture solution. The choice of the concentrations was based on pilot trials. Measurements were performed in triplicate after incubation for 24, 48, and 72 h. Measurements of Cell Proliferation, Cell Cycle, and Apoptosis Cell Proliferation The MODE-K cells are regarded as well grown when their morphology becomes oval or polygonal in shape, in a monolayer adherent to the plate wall without overlapping, and in the arrangement of paving stones. Cells from the same generation were seeded on 96-well cell culture plates. When the cells reached confluence, -chaconine (Shanghai Yuanye Bio-Technology Co., Ltd., Shanghai, China) was added at the designated concentrations. Cell proliferation rate was measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method (Kumar et al., 2018) when cells were harvested. The cells harvested in the logarithmic growth phase were adjusted to a cell density of 5 104/ml using the 1640 medium. The cell suspension (100 L) together with sterile phosphate buffered saline (PBS; 100 L) was transferred to a 96-well cell culture plate and incubated overnight at 37C. Then 10 L of MTT (Hefei Labgic Technology Co., Ltd., Hefei, China) was added to the cells and incubated at 37C for further 4 h. After the medium was removed and 150 L DMSO (dimethyl sulfoxide; Solarbio, Shanghai, China) was added, the plate was shaken for 10 min and the absorbance was measured at OD 568. Cell Cycle MODE-K cells in their logarithmic growth phase were adjusted to a cell density of 1.5 105/mL with the 1640 IMPA2 antibody medium. The cell suspension solution was transferred into six-well plates with 2 mL each well and cultured overnight at 37C. At the end of incubation, the cells were digested with 2 mL of 0.25% trypsin (without EDTA) for 1C2 min. Once the cells were separated from each other, centrifugation was performed at 221 for 5 min (Eppendorf model 5702R, Hamburg, Germany) to remove the supernatant. Then the cells were resuspended with the PBS buffer and centrifuged as described above. This procedure was repeated and 700 L of pre-cooled 80% ethanol was slowly added to the pellet to make the final ethanol concentration of 70%. After fixing in ethanol at 4C for at least 4 h, the cells were centrifuged at 221 for 5 min and washed with pre-cooled PBS buffer and collected by centrifugation twice. The cells were incubated at 37C for 30 min in 100 L of RNase (50 g/mL) and then stained with 100 L of propidium iodide (50 g/mL) at 4C for 30 min in the dark. The stained cells were tested.