The cytotoxic effect and injury mechanism of deoxynivalenol on articular chondrocytes in human embryo

Abstract

This study was to explore the cytotoxic effect and the related injury mechanism of deoxynivalenol (DON) on articular chondrocytes in human embryo. Articular cartilage cells were isolated from knees of human embryo and cultured in DMEM/F12 medium. The cells of the 4th generation were divided into five groups and incubated with varying concentrations of DON as the followings: control group and group with DON of 0.1, 0.2, 0.4, 1.0 µg/ml. The effects of DON were observed 72 hours after incubation. Cell apoptosis was assayed by flow cytometry (FCM) with Annexin V-FITC/PI staining; MMP-13 and PGE2 were detected by ELISA kits; NO was measured by Griess assay with spectrophotometer. Inducible nitric oxide synthase (iNOS) and collagen II in cells were detected by FCM. The expression levels of iNOS, mRNA and collagen II mRNA were measured with RT-PCR. The rates of cell apoptosis in DON groups were 6.78% - 19.05%, which were significantly higher than that in control (1.20%, F = 174.761, P < 0.05). The levels of NO in DON groups were 20.8 - 40.7 µmol/L, which were significantly higher than that in control (10.2 µmol/L, F = 91.966, P < 0.05). The levels of MMP-13 in DON groups were 0.25 - 0.56 µmol/L, which were significantly higher than that in control (0 µmol/L, F = 78.420, P < 0.05). The levels of PGE2 in DON groups were 3.2-20.6 µmol/L, which were significantly higher than that in control (11.6 µmol/L, F = 276.453, P < 0.05). The proportions of cells with positive iNOS in DON groups were 14.8% - 56.8% which were significantly higher than that in controls (7.1%, F = 214.614, P < 0.05). The proportions of cells with positive collagen II in groups with DON of 0.4 µg/ml and 1.0 µg/ml were 56.7% and 52.7%, which were significantly lower than that in control (62.2%, F = 5.134, P < 0.05). The relative absorbance values of iNOS mRNA in DON groups were 1.07 - 1.33, which were significantly higher than that in control (0.62, F = 8.358, P < 0.05). The levels of collagen II mRNA in groups with DON of 0.4 µg/ml and 1.0 µg/ml were 0.83 and 0.82, which were significantly lower than that in control (1.14, F = 7.887, P < 0.05). DON could promote anabolism of NO in articular cartilage cells by which up-regulated the expression of PGE2 and MMP-13, which both promoted resolution of articular cartilage matrix such as collagen II. DON induced apoptosis in articular cartilage cells.