TLR 4在绵羊肺炎支原体介导肺泡上皮细胞凋亡中的作用机理

Effect of TLR4 on Mycoplasma pneumoniae-mediated alveolar epithelial cell apoptosis in sheep

为了分析TLR4在绵羊肺炎支原体介导肺泡上皮细胞凋亡中的分子机制,以绵羊肺泡上皮细胞为材料,用不同浓度的抑制剂TAK-242与细胞孵育,用MTT法检测细胞活性,用试剂盒检测Caspase-3和Caspase-8的活性,以及用实时定量PCR检测TLR4和TNF-αmRNA的表达水平,接着用流式细胞仪检测细胞的凋亡率。结果:当抑制剂TAK-242浓度为1 nmol/L、5 nmol/L、10 nmol/L不影响细胞活性,而当浓度为20 nmol/L和30 nmol/L时细胞活性显著降低(P<0.05);在感染比例(MOI)为10的情况下,在12 h和24 h支原体可以提高TLR4 mRNA的表达水平;绵羊肺炎支原体可显著提高Caspase-3和Caspase-8的活性,而TAK-242能够显著降低Caspase-3和Caspase-8的活性(P<0.01);在感染时间为24 h,支原体可以提高促TNF-αmRNA的表达水平,而抑制剂TAK-242在10 nmol/L的浓度下可以降低支原体介导的TNF-αmRNA的表达水平;在24 h支原体引起的细胞的细胞凋亡率为16.7%±1.81%,而抑制剂TAK-242处理组引起的细胞凋亡率为11.7%±1.32%。表明TLR4在绵羊肺炎支原体诱导肺泡上皮细胞凋亡中发挥重要作用。

To analyze the effect of TLR4 on mycoplasma - mediated alveolar epithelial cell apoptosis. In this study, sheep alveolar epithelial cells were used as subjects and were incubated with different concentrations of inhibitor TAK -242, cell viability was measured by MTY assay, the activity of caspase - 3 and caspase - 8 was detected by a kit, levels of TLR4 and TNF - a m RNA were measured by real - time quantitative PCR, and cell apoptosis rate was measured by flow cytometry. TAK - 242 did not affect cell via- bility at the concentration of lnmol/L, 5nmol/L, and tOnmol/L, but the cell activity was significantly decreased when the concentra- tion of TAK -242 was at 20nmol/L and 30nmol/L（P 〈 0. 05 ）. There was a significant increase in the activity of caspase -3 and caspase - 8 induced by sheep mycoplasma pneumoniae, and a significant decrease induced by TAK - 242. There was a significant increase in the expression of TLR4 mRNA at 12h and 24h post - infection （ MOI 10） （P 〈0. 01 ）. At 24 h post - infection, there was a significant increase in the expression of TNFa mRNA, and the expression of TNFa mRNA was reduced by inhibitor TAK - 242 at the concentration of 10nmol/L （P 〈 0. 01 ）. The apoptosis rate of mycoplasma - induced ceils was 16.7 ＋ 1.81%, significantly higher than that of the control group （P 〈0. 05）. The apoptosis rate of TAK -242 treated group was 11.7 ：t： 1.32%, which was significantly lower than that of Mmc - infected group. TLR4 plays an important role in the apoptosis of alveolar epithelial cells induced by mycoplasma pneumoniae in sheep.