Effects of HT-2 toxin on expressions of SIRT1, autophagy and apoptosis pathway related proteins in chondrocytes / 中华地方病学杂志

ABSTRACT

Objective:To explore the effects of HT-2 toxin on expressions of silent information regulator of transcription 1 (SIRT1) and autophagy and apoptosis pathway related proteins in cultured chondrocytes in vitro. Methods:The third-generation chondrocytes of SD neonatal rats aged 1 to 2 days were cultured in vitro and identified by toluidine blue staining and type Ⅱ collagen immunofluorescence staining. CCK-8 method was used to detect the proliferation of chondrocytes. According to the cell survival rate, 2, 4 and 8 ng/ml HT-2 toxin were selected for subsequent experiments, and the exposure time was 48 h. At the same time, a negative control group and a solvent (absolute ethanol) control group were set up. Western blotting was used to detect the expressions of SIRT1 and autophagy and apoptosis pathway related proteins [microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ, LC3-Ⅰ, p62, Beclin1, Caspase-3, B-cell lymphoma-2 (Bcl-2) and Bcl-2-Associated X protein (Bax)] in each group. Results:After staining, the cells were identified as chondrocytes with high purity. The expression levels of SIRT1 protein in 2, 4, 8 ng/ml HT-2 toxin groups (0.69 ± 0.18, 0.46 ± 0.13, 0.35 ± 0.19) were significantly lower than that in negative control group (1.00 ± 0.39, P < 0.05). In 2, 4 and 8 ng/ml HT-2 toxin groups, the ratios of autophagy pathway related proteins LC3-Ⅱ and LC3-Ⅰ expressions (LC3-Ⅱ/LC3-Ⅰ, 1.47 ± 0.15, 1.37 ± 0.13, 1.81 ± 0.34) were higher than that in negative control group (1.00 ± 0.21, P < 0.05), and the expression levels of p62 protein in 4, 8 ng/ml HT-2 toxin groups (0.70 ± 0.04, 0.57 ± 0.01) were lower than that in negative control group (1.00 ± 0.15, P < 0.05). In 2, 4, 8 ng/ml HT-2 toxin groups, the expression levels of apoptosis pathway related protein Bcl-2 (0.61 ± 0.06, 0.54 ± 0.16, 0.47 ± 0.06) were significantly lower than that in negative control group (1.00 ± 0.14, P < 0.05), and the ratio of Bax to Bcl-2 protein expressions in 8 ng/ml HT-2 toxin group (Bax/Bcl-2, 3.27 ± 0.18) was higher than that in negative control group (1.00 ± 0.27, P < 0.05). The expression level of SIRT1 protein was significantly negatively correlated with the expression level of autophagy pathway related protein LC3-Ⅱ ( r = - 0.819, P < 0.01), and was significantly positively correlated with the expression level of p62 protein( r = 0.772, P < 0.01), but not with the expression level of Beclin1 protein ( r = 0.399 , P > 0.05); there was no correlation between SIRT1 protein expression and apoptosis pathway related protein Caspase-3 and Bax expressions ( r = - 0.297、- 0.284, P > 0.05), but there was a significant positive correlation with Bcl-2 protein expression ( r = 0.755, P < 0.01). Conclusion:HT-2 toxin may increase the expression of autophagy pathway related protein LC3-Ⅱ/LC3-Ⅰ, decrease the expression of p62 protein, and increase the apoptosis pathway related protein Bax/Bcl-2 by inhibiting the expression of SIRT1 protein in chondrocytes, resulting in abnormal autophagy and apoptosis, and finally leads to the injury of chondrocytes.