MyD88抑制剂ST2825对丝状支原体山羊亚种感染宿主细胞的影响

Effect of MyD88 inhibitor ST2825 on host cells infected with mycoplasma mycoides subspecies capri

为分析MyD88在丝状支原体山羊亚种(Mmc)感染肺泡上皮细胞中的分子机制。以山羊肺泡上皮细胞为材料,用不同浓度的MyD88抑制剂(ST2825)与细胞孵育,MTT法检测细胞活性,试剂盒检测caspase-3的活性,以及实时定量PCR检测MyD88和TNF-a mRNA的表达水平,试剂盒检测H_2O_2浓度和NO浓度。当ST2825浓度为5、10和20μmol·L^(-1)不影响细胞活性,而浓度为40μmol·L^(-1)时细胞活性显著降低;感染比例(MOI)为10,感染时间为8、12和24 h,Mmc可以显著提高MyD88 mRNA的表达水平(P<0.01);可显著提高caspase-3的活性,而ST2825能够显著降低caspase-3(P<0.05);感染时间为24 h,Mmc可显著提高TNF-a mRNA的表达水平、H_2O_2浓度和NO浓度(P<0.05),而ST2825在10和20μmol·L^(-1)的浓度时,可显著降低Mmc介导的caspase-3活性、H_2O_2浓度以及LDH水平(P<0.05),ST2825在5、10和20μmol·L^(-1)浓度时可显著降低TNF-a mRNA的表达水平和NO浓度(P<0.05)。

To study the molecular mechanism of MyD88 in alveolar epithelial cells infected by filamentous mycoplasma goat subspecies, the goat alveolar epithelial cells were treated with different concentrations of inhibitor ST2825. The cell activity was detected by MTTAssayKit and the caspase-3 activity was measured using Caspase-3Colorimetric Assay Kit from APExBIO Company. Expression levelsofTNF-amRNAwere analyzed by real-time quantitative PCR,H2O2 concentrationwas measured usingH2O2Assay Kit,and NO concentrationwas measured using NO Assay Kit. The cell activity was not affected by ST2825at the concentrations of 5, 10 and 20 μmol·L-1, but it was significantly decreased on the concentration of 40 μmol·L-1. When the infection ratio （MOI） was 10, the expression ofMyD88mRNA was increased at 8 h, 12 h and 24 h post-infection, and the activity of caspase-3 was also significantly increased by the subspecies of mycelial carcinoma （P〈0.01）, while the expression of caspase-3 was significantly decreasedby ST2825 （P〈0.05）.Expression levels of TNF-a mRNA and the concentrations of H2O2and NOweresignificantly enhanced byMycoplasma at 24 h post-infection