莫西沙星对大鼠气道平滑肌细胞线粒体膜电位及凋亡的影响

目的 观察大鼠气道平滑肌细胞(ASMC)在不同浓度的莫西沙星作用下,其线粒体膜电位（△Ψm）和半胱氨酸天冬氨酰蛋白酶-3（Caspase-3）表达及细胞凋亡的变化,探讨莫西沙星促进ASMC凋亡的可能机制。方法体外原代培养大鼠ASMC,按随机数字表法分为空白对照组、莫西沙星40 mg/L组（M40组）、80 mg/L组（M80组）、120 mg/L组（M120组）和200 mg/L组（M200组）,均孵育48 h后,Annexin-V/PI双标记流式细胞仪分析法检测细胞凋亡率,用JC-1荧光探针在免疫荧光显微镜下检测细胞△Ψm的变化,Western blot法检测凋亡蛋白Caspase-3的表达。结果各莫西沙星浓度组（M40组、M80组、M120组及M200组）的细胞凋亡率分别为(2.95±0.21)％、(7.39±0.63)％、(13.39±0.40)％和(21.20±1.42)％,均明显高于空白对照组的(0.94±0.05)％(均P＜0.01),并存在浓度依赖性(r=0.974,P＜0.01);荧光显微镜检测显示空白对照组以红色荧光为主,随着莫西沙星浓度增大,红色/绿色荧光强度值比率(分别为6.54±0.15、4.48±0.14、2.25±0.10和1.99±0.12)逐渐降低,与空白对照组(10.02±0.20)相比差异有统计学意义（均P＜0.01）,并存在药物浓度依赖性（r=-0.946,P＜0.01）;Western blot检测显示,空白对照组几乎无Caspase-3蛋白表达(0.31±0.03),各莫西沙星浓度组Caspase-3蛋白表达(分别为0.45±0.05、0.59±0.04、0.69±0.06和0.84±0.04)均明显高于空白对照组(0.31±0.03)（均P＜0.01）,同时也存在浓度依赖性(r=0.979,P＜0.01)。各组细胞凋亡率与△Ψm水平呈负相关（r=-0.887,P＜0.01）,与凋亡蛋白Caspase-3水平呈正相关(r=0.955,P＜0.01),各组细胞△Ψm水平与Caspase-3水平呈负相关(r=-0.951,P＜0.01)。结论 莫西沙星可能通过影响大鼠AΨm促进ASMC凋亡。

The effect of moxifloxacin on apoptosis of airway smooth muscle cells and mitochondria membrane potential

ObjectiveTo observe the effects of moxifloxacin at various concentrations on the expression of Caspase-3, the alteration of mitochondria membrane potential (△Ψm) and the apoptosis of airway smooth muscle cells (ASMCs), and to explore the possible mechanisms.Methods ASMCs were derived from rat airway tissues and cultured in vitro.The cells were randomly divided into 5 groups including a control group and 4 groups to which moxifloxacin was added at different concentrations (40,80,120,200mg/L, groups M40, M80, M120 and M200 respectively).Then the cells of different groups were incubatedfor 48 h.An apoptosis detection kit was used for annexin V and PI staining, and JC-1 probe was employed to measure mitochondrial depolarization in ASMCs, and the protein of Caspase-3 was measured by Western blot.ResultsThe apoptosis rates of ASMCs in groups M40, M80, M120 and M200 were (2.95 ±0.21) ％, (7.39 ± 0.63) ％ , (13.39 ± 0.40) ％ and (21.20 ± 1.42) ％ , respectively , all of which were higher than that in the control group (0.94 ±0.05)％, F =399.77, P ＜ 0.01. Furthermore, the concentration of moxifloxacin was positively related to the apoptosis rate (r =0.974, P ＜0.01 ).Compared to the control group(the ratio of orange-red fluorescence to green fluorescence was 10.02 ±0.20), there was a shift from mitochondrial orange-red fluorescence to green fluorescence among groups with the concentrations of moxifloxacin increasing (6.54±0.15,4.48 ±0.14,2.25 ±0.10 and 1.99±0.12) ; the difference was significant (F = 1565.12, P ＜0.01), and there was a dose-dependent response (r =-0.946, P ＜0.01 ).The results of Western blot indicated that the expression of Caspase-3 increased with the concentrations of moxifloxacin increasing (0.45 ± 0.05, 0.59 ± 0.04, 0.69 ± 0.06 and 0.84 ± 0.04, respectively), and there was a very low expression of Caspase-3 in the control group (0.31± 0.03). The expression of Caspase-3 showed a positive correlation with the concentration of moxifloxacin (r =0.979, P ＜ 0.01 ).The apoptosis rate of ASMCs in the different groups had a remarkable correlation with the △Ψm and Caspase-3 (r =-0.887, P ＜ 0.01 ; r = 0.955, P＜0.01). There was also a remarkable negative correlation between △Ψm and Caspase-3 (r =-0.951, P ＜ 0.01).Conclusion Moxifloxacin was shown to promote ASMC apoptosis by altering △Ψm.