中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2011年
9期
684-687
,共4页
肌细胞,平滑肌%细胞凋亡%膜电位,线粒体%半胱氨酸天冬氨酸蛋白酶3%莫西沙星
肌細胞,平滑肌%細胞凋亡%膜電位,線粒體%半胱氨痠天鼕氨痠蛋白酶3%莫西沙星
기세포,평활기%세포조망%막전위,선립체%반광안산천동안산단백매3%막서사성
Myocytes,smooth muscle%Apoptosis%Membrane potential,mitochondrial%Caspase 3%Moxifloxacin
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。
目的 觀察大鼠氣道平滑肌細胞(ASMC)在不同濃度的莫西沙星作用下，其線粒體膜電位（△Ψm）和半胱氨痠天鼕氨酰蛋白酶-3（Caspase-3）錶達及細胞凋亡的變化，探討莫西沙星促進ASMC凋亡的可能機製。方法體外原代培養大鼠ASMC，按隨機數字錶法分為空白對照組、莫西沙星40 mg/L組（M40組）、80 mg/L組（M80組）、120 mg/L組（M120組）和200 mg/L組（M200組），均孵育48 h後，Annexin-V/PI雙標記流式細胞儀分析法檢測細胞凋亡率，用JC-1熒光探針在免疫熒光顯微鏡下檢測細胞△Ψm的變化，Western blot法檢測凋亡蛋白Caspase-3的錶達。結果各莫西沙星濃度組（M40組、M80組、M120組及M200組）的細胞凋亡率分彆為(2.95±0.21)％、(7.39±0.63)％、(13.39±0.40)％和(21.20±1.42)％，均明顯高于空白對照組的(0.94±0.05)％(均P＜0.01)，併存在濃度依賴性(r=0.974,P＜0.01)；熒光顯微鏡檢測顯示空白對照組以紅色熒光為主，隨著莫西沙星濃度增大，紅色/綠色熒光彊度值比率(分彆為6.54±0.15、4.48±0.14、2.25±0.10和1.99±0.12)逐漸降低，與空白對照組(10.02±0.20)相比差異有統計學意義（均P＜0.01），併存在藥物濃度依賴性（r=-0.946,P＜0.01）；Western blot檢測顯示，空白對照組幾乎無Caspase-3蛋白錶達(0.31±0.03)，各莫西沙星濃度組Caspase-3蛋白錶達(分彆為0.45±0.05、0.59±0.04、0.69±0.06和0.84±0.04)均明顯高于空白對照組(0.31±0.03)（均P＜0.01），同時也存在濃度依賴性(r=0.979,P＜0.01)。各組細胞凋亡率與△Ψm水平呈負相關（r=-0.887,P＜0.01），與凋亡蛋白Caspase-3水平呈正相關(r=0.955，P＜0.01)，各組細胞△Ψm水平與Caspase-3水平呈負相關(r=-0.951，P＜0.01)。結論 莫西沙星可能通過影響大鼠AΨm促進ASMC凋亡。
목적 관찰대서기도평활기세포(ASMC)재불동농도적막서사성작용하，기선립체막전위（△Ψm）화반광안산천동안선단백매-3（Caspase-3）표체급세포조망적변화，탐토막서사성촉진ASMC조망적가능궤제。방법체외원대배양대서ASMC，안수궤수자표법분위공백대조조、막서사성40 mg/L조（M40조）、80 mg/L조（M80조）、120 mg/L조（M120조）화200 mg/L조（M200조），균부육48 h후，Annexin-V/PI쌍표기류식세포의분석법검측세포조망솔，용JC-1형광탐침재면역형광현미경하검측세포△Ψm적변화，Western blot법검측조망단백Caspase-3적표체。결과각막서사성농도조（M40조、M80조、M120조급M200조）적세포조망솔분별위(2.95±0.21)％、(7.39±0.63)％、(13.39±0.40)％화(21.20±1.42)％，균명현고우공백대조조적(0.94±0.05)％(균P＜0.01)，병존재농도의뢰성(r=0.974,P＜0.01)；형광현미경검측현시공백대조조이홍색형광위주，수착막서사성농도증대，홍색/록색형광강도치비솔(분별위6.54±0.15、4.48±0.14、2.25±0.10화1.99±0.12)축점강저，여공백대조조(10.02±0.20)상비차이유통계학의의（균P＜0.01），병존재약물농도의뢰성（r=-0.946,P＜0.01）；Western blot검측현시，공백대조조궤호무Caspase-3단백표체(0.31±0.03)，각막서사성농도조Caspase-3단백표체(분별위0.45±0.05、0.59±0.04、0.69±0.06화0.84±0.04)균명현고우공백대조조(0.31±0.03)（균P＜0.01），동시야존재농도의뢰성(r=0.979,P＜0.01)。각조세포조망솔여△Ψm수평정부상관（r=-0.887,P＜0.01），여조망단백Caspase-3수평정정상관(r=0.955，P＜0.01)，각조세포△Ψm수평여Caspase-3수평정부상관(r=-0.951，P＜0.01)。결론 막서사성가능통과영향대서AΨm촉진ASMC조망。
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.
