粪肠球菌脂磷壁酸对巨噬细胞自噬的影响

Effect of Enterococcus faecalis lipoteichoic acid on macrophage autophagy

目的通过研究粪肠球菌脂磷壁酸(LTA)对巨噬细胞自噬功能的影响,为后续进一步深入探讨顽固性根尖周炎的发病机制提供实验依据。方法体外培养小鼠巨噬细胞系RAW 264.7,细胞计数试剂盒(CCK-8)检测LTA的细胞毒性作用;蛋白免疫印迹法(Western blot)检测LTA作用下巨噬细胞自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ比值、Beclin1的表达水平;实时荧光定量聚合酶链反应(PCR)检测自噬相关基因LC3、Beclin1 m RNA表达水平的变化;构建稳定表达自噬双荧光慢病毒载体HBLVm RFP-GFP-LC3-PURO的巨噬细胞系,激光共聚焦显微镜观察自噬情况;SPSS 20.0统计软件进行数据分析。结果 CCK-8结果表明,选用20μg/m L LTA及其以下浓度处理巨噬细胞24 h均未见明显毒性作用(t=2.102,P=0.1106);Western blot结果表明,LTA刺激巨噬细胞后:LC3-Ⅱ/LC3-Ⅰ比值表达量(0.42±0.04)与对照组(0.04±0.02)相比显著提高,差异有统计学意义(F=14.25,P<0.001),Beclin1蛋白表达量(0.56±0.11)与对照组(0.28±0.08)相比呈上升趋势(F=3.459,P=0.0258),均存在剂量依赖效应;实时荧光定量PCR结果表明,LTA刺激巨噬细胞后,LC3基因表达水平(5.94±0.85)与对照组(1.03±0.28)相比显著提高(F=12.93,P<0.001);Beclin1基因表达水平(2.22±0.21)与对照组(1.02±0.28)相比呈上升趋势(F=6.036,P<0.001),存在剂量依赖效应;成功构建稳定表达HBLV-m RFP-GFP-LC3-PURO的巨噬细胞系,激光共聚焦显微镜下观察显示,LTA作用组自噬体形成量(58±6)与对照组(18±8)相比差异有统计学意义(F=12.36,P=0.0021)。结论粪肠球菌LTA可诱导巨噬细胞产生自噬,且存在剂量依赖效应。

Objective By investigating the effect of Enterococcus faecalis lipoteichoic acid(LTA)on macrophages autophagy,this study aimed to provide experimental evidence for further exploration of the pathogenesis of persistent periapical periodontitis. Methods RAW 264.7 was cultured and cell counting kit-8(CCK-8)was used to detect the cytotoxicity of LTA. Western blot was used to detect the expression level of autophagy-related protein LC3-Ⅱ/LC3-Ⅰ and Beclin1 and qPCR was applied to detect the expression level of LC3 and Beclin1. RAW 264.7 cell strain stably expressing HBLV-m RFP-GFP-LC3-PURO was established,and laser confocal microscopy was used to observe the formation of autophagosome.SPSS 20.0 statistical software for data analysis. Results The result of CCK-8 showed no obvious toxicity to macrophage when stimulated with 20 μg/m L and the lower concentration of LTA for 24 hours(t = 2.102,P = 0.1106). After LTA stimulated,Western blot suggested that the expression of LC3-Ⅱ/LC3-Ⅰ(0.42 ±0.04)and Beclin1(0.56 ± 0.11)significantly increased with dose-dependent effect(F = 14.25,P<0.001;F = 3.459,P = 0.0258). In the meanwhile,q PCR results showed that the expression of LC3(5.94 ± 0.85)and Beclin1(2.22 ± 0.21)significantly increased with dose-dependent effect(FLC3 = 12.93,PLC3<0.001;FBeclin1= 6.036,PBeclin1<0.001)after LTA stimulated. Laser confocal microscopy detected that LTA group(58 ± 6)had significant difference(F = 12.36,P = 0.0021)compared with control group(18 ± 8).Conclusion Enterococcus faecalis LTA may regulate macrophages autophagy with dose-dependent effect.