右美托咪定对机械拉伸A549细胞IL-8和PAI-1表达及细胞凋亡率的影响

Effects of dexmedetomidine on expression of IL-8 and PAI-1 and apoptosis rate in mechanically stretched A549 cells

目的本研究模拟机械通气诱发肺损伤,探讨不同浓度右美托咪定对机械拉伸损伤A549细胞PAI-1、IL-8表达及细胞凋亡率的影响。方法①将体外培养的肺Ⅱ型上皮细胞(A549细胞)随机分为非机械拉伸组和机械拉伸组,每组按拉伸时间分为4个亚组:0 h组、4 h组、12 h组和24 h组。机械拉伸组与体外细胞应力加载装置拉伸相连接,机械拉伸加载装置拉伸幅度为20%,频率为0.3 Hz的正弦波,非机械拉伸组不与拉伸装置连接。采用MTT比色法和ELISA测定细胞存活率和PAI-1、IL-8的表达水平,筛选A549细胞损伤最明显的时间。②将A549细胞接种于包被胶原基底膜(胶原基底蛋白Ⅰ)的BioFlex6孔培养板,分为预处理对照组和预处理拉伸组,每组分为4个亚组,并在机械拉伸前20 min滴加不同浓度右美托咪定(0,0.01,0.1,1μmol/L)进行预处理。预处理拉伸组与体外细胞应力加载装置拉伸相连接,机械拉伸加载装置拉伸幅度为20%,频率为0.3 Hz的正弦波,持续拉伸24 h;预处理对照组不与拉伸装置连接,不拉伸。采用流式细胞术测定细胞凋亡率,ELISA法测定PAI-1和IL-8的表达。结果与各自非机械拉伸组相比,机械拉伸4,12,24 h细胞存活率明显降低(P<0.05),IL-8表达水平上调(P<0.05),而PAI-1表达仅机械拉伸24 h差异有统计学意义(P<0.05),表达上升近一倍。随着拉伸时间延长,非机械拉伸组和机械拉伸组A549细胞存活率逐渐减低(P<0.05),IL-8表达水平逐渐升高(P<0.05),以24 h为著;非机械拉伸组PAI-1表达没有明显差异(P>0.05),机械拉伸4 h组和12 h组PAI-1表达差异无统计学意义(P>0.05),但24 h组PAI-1表达水平显著上调(P<0.05)。右美托咪定处理后,预处理拉伸组细胞凋亡率、PAI-1和IL-8表达水平较各自预处理对照组均升高(P<0.05);随着右美托咪定浓度增加,预处理拉伸组细胞凋亡率、PAI-1和IL-8表达水平逐渐减低,与0μmol/L比较,差异均有统计学意义(P<0.05),以1μmol/L最为明显;预处理对照组细胞凋亡率和PAI-1表达水平逐渐减低,与0μmol/L比较,差异也有统计学意义(P<0.05),但IL-8变化无统计学意义(P>0.05)。结论机械拉伸能下调A549细胞存活率,上调PAI-1和IL-8表达。右美托咪定预处理可下调PAI-1和IL-8表达,降低机械拉伸后A549细胞的凋亡率,提示右美托咪定预处理可能减轻机械拉伸肺上皮细胞损伤。

Objective To investigate the effects of different concentrations of dexmedetomidine on the expression of PAI-1,IL-8 and apoptosis of A549 cells during the mechanical stretching injury.Methods①The typeⅡlung epithelial cells(A549 cells)cultured in vitro were randomly divided into non-mechanical stretching group and mechanical stretching group.Each group was divided into 4 subgroups according to the stretching time:0 h group,4 h group,12 h group and 24 h group.The plate in mechanical stretching group was attached to the mechanical tensile loading device with a stretching amplitude of 20%and a frequency of 0.3 Hz sine wave.The plate in non-mechanical stretching group was not connected to the stretching device.MTT colorimetry and ELISA were used to determine the cell survival rate and the expression levels of PAI-1 and IL-8 in each group,and the best time for A549 cell damage was selected.②A549 cells were inoculated into BioFlex6-well culture plate coated with collagen basal membrane(collagen basal proteinⅠ)and divided into preconditioning control group and preconditioning stretching group.According to dexmedetomidine concentration,each group was divided into 4 subgroups.Then dexmedetomidine(0,0.01,0.1,1μmol/L)was added at 20 min before stretching.The plate in preconditioning stretching group was connected with the Flexercell-4000 TM loading device with a stretching amplitude of 20%and a frequency of 0.3 Hz,sine wave for 24 h,while the plate was not attached to the stretching device in preconditioning control group.Flow cytometry and ELISA were used to detect the apoptosis rate and the expression levels of PAI-1 and IL-8,respectively.Results Compared with non-mechanical stretching group,the cell survival rate was significantly decreased in mechanical stretching group at 4,12,24 h(P<0.05),the IL-8 expression was up-regulated at 4,12,24 h(P<0.05),and the expression of PAI-1 was increased by nearly twice in mechanical stretch group at 24 h(P<0.05).As the stretching time increased,the cell survival rate was gradually decreased in non-mechanical stretching group and mechanical stretching group(P<0.05),while the expression level of IL-8 was gradually increased(P<0.05),especially at 24 h.As the stretching time increased,there was no significant change in PAI-1 expression in non-mechanical stretching group(P>0.05),but the expression level of PAI-1 was significantly increased at 24 h in mechanical stretching group(P<0.05).After dexmedetomidine treatment,the apoptosis rate,PAI-1 and IL-8 expression levels in preconditioning stretching group were increased compared with preconditioning control group(P<0.05).As the concentration of dexmedetomidine increased,the apoptosis rate,PAI-1 and IL-8 expression levels in preconditioning stretching group were gradually down-regulated(P<0.05 vs 0μmol/L),especially in 1μmol/L;the apoptosis rate and PAI-1 expression were gradually down-regulated in preconditioning control group(P<0.05 vs 0μmol/L),while the expression level of IL-8 showed no statistical change(P>0.05).Conclusion Mechanical stretching can decrease the cell survival rate,and up-regulate the expression of PAI-1 and IL-8.Dexmedetomidine pretreatment treatment can down-regulate the expression of PAI-1 and IL-8 and reduce the apoptosis rate of A549 cells after mechanical stretching,suggesting that dexmedetomidine pretreatment may reduce the mechanical stretching-induced injury of human lung epithelial cells.