Effects of T-2 toxin on chondrocyte proliferation and cell cycle

Abstract

Objective To study the effect of T-2 toxin on proliferation and cell cycle of rat chondrocytes, in order to provide a new idea in molecular mechanism of T-2 toxin-induced chondrocyte damage. Methods Primary chondrocytes of neonatal Wistar rats were isolated and stained by toluidine blue staining and type Ⅱ collagen immunofluorescence staining. The effects of different concentrations of T-2 toxin [0 (control), 1, 5, 10, 20, 50, 100 μg/L)] on proliferation of chondrocytes for 24 h were detected by cell counting kit-8 (CCK-8) method, and control, 1 (low dose), 5 (medium dose), and 10 μg/L (high dose) T-2 toxin were selected for subsequent experiment; cell cycle changes were detected by flow cytometry; Real-time PCR and Western blotting were used to detect the effects of T-2 toxin on mRNA and protein expressions of proliferating cell nuclear antigen (PCNA) and Cyclin D1 in chondrocytes. Results With increase of T-2 toxin concentration (control, 1, 5, 10, 20, 50, 100 μg/L), the cell survival rates[(100.00 ± 0.00)%, (93.12 ± 1.66)%, (77.12 ± 1.11)%, (59.44 ± 4.09)%, (46.64 ± 3.86)%, (38.15 ± 3.37)%, (33.79 ± 0.99)%] were decreased, and the differences were statistically significant (F= 139.21, P < 0.05). The percentages of quiescent phase/pre-DNA synthesis phase (G0/G1 phase) cells in 1, 5, 10 μg/L T-2 toxin groups [(22.03 ± 0.42)%, (30.54 ± 2.61)%, (36.01 ± 1.51)%] were significantly higher than that in control group [(13.79 ± 1.65)%, P < 0.05]; the percentages of DNA synthesis phase (S phase) cells [(60.27 ± 3.53)%, (53.88 ± 4.38)%, (49.55 ± 2.49)%] were significantly lower than that in control group [(76.72 ± 4.24)%, P < 0.05]. The differences of mRNA levels of PCNA and Cyclin D1 between groups were statistically significant (F= 46.80, 17.97, P < 0.05), and 5, 10 μg/L T-2 toxin groups (0.77 ± 0.13, 0.79 ± 0.08, 0.60 ± 0.07, 0.56 ± 0.05) were lower than the control group (0.99 ± 0.02, 1.01 ± 0.01, P < 0.05). The expressions of PCNA protein in 5, 10 μg/L T-2 toxin groups (0.69 ± 0.03, 0.49 ± 0.03) were lower than that in control group (0.92 ± 0.05, P < 0.05); the expressions of Cyclin D1 protein in 1, 5, 10 μg/L T-2 toxin groups (0.80 ± 0.06, 0.60 ± 0.07, 0.33 ± 0.13) were lower than that in control group (0.95 ± 0.07, P < 0.05). Conclusion T-2 toxin can inhibit the proliferation of chondrocytes, which may be worked through influencing the expression of cell cycle protein, causing cell cycle arrest, thereby inhibiting DNA synthesis. Key words: T-2 toxin; Chondrocytes; Cell proliferation; Cell cycle