CAJ | 학술논문

目的探讨线粒体分裂抑制剂(mdivi-1)在脑缺血再灌注损伤中对能量代谢的影响.方法将体外培养8d的Wistar大鼠海马神经元分为4组:正常对照组、赋形剂组、mdivi-1组、缺血再灌注损伤组,后两组利用海马神经元氧糖剥夺法建立缺血再灌注损伤模型,mdivi-1组建模前给予mdivi-1预处理40 min.缺血6h再灌注20 h后应用Western blotting检测各组海马神经元线粒体分裂蛋白1(Drp1)、B细胞淋巴癌/白血病-2(Bcl-2)和Bcl-2相关X蛋白(Bax)的表达水平,应用流式细胞术检测线粒体膜电位,应用酶标仪法检测三磷酸腺苷(ATP)含量及Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性以及线粒体呼吸链酶复合体Ⅰ、Ⅱ、Ⅲ、Ⅳ活性. 结果与正常对照组比较,mdivi-1组和缺血再灌注损伤组Drp1 (1.001±0.276; 1.985±0.301)、Bax(2.752±0.786;4.225±1.107)表达水平明显升高,Bcl-2 (0.749±0.128; 0.336±0.109)表达水平明显降低,线粒体膜电位降低的细胞数(72.5％;92.7％)均升高,ATP含量[(74.129±5.773) μmoL/g蛋白;(36.986±5.945) μmoL/g蛋白]及Na+-K+-ATP酶活性[(4.348±0.451) U/mg蛋白;(1.709±0.477) U/mg蛋白]、CaV-Mg2+-ATP酶活性[(1.955±0.287) U/mg蛋白;(1.123±0.181) U/rag蛋白]以及线粒体呼吸链酶复合体Ⅰ、Ⅱ、Ⅲ、Ⅳ活性[(15.445±1.699) nmol/(min· mg)、(17.065±1.070) nmol/(min·mg)、(32.123±1.652) nmol/(min ·rag)、(2.814±0.180) nmol/(min· mg); (6.810±1.725) nmol/(min· mg)、(9.473±0.751) nmol/(min· mg)、(23.010±1.716) nmol/(min· mg)、(1.598±0.181) nmol/(min· mg)]均明显降低,差异均有统计学意义(P＜0.05).与缺血再灌注损伤组比较,mdivi-1组Drpl、Bax表达水平明显减少,Bcl-2表达水平明显升高,线粒体膜电位降低的细胞数减少,ATP含量及Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性以及线粒体呼吸链酶复合体Ⅰ、Ⅱ、Ⅲ、Ⅳ活性均明显升高,差异均有统计学意义(P＜0.05). 结论 mdivi-1可通过抑制线粒体分裂,有效地改善能量代谢障碍,从而减轻脑缺血再灌注损伤. 目的探討線粒體分裂抑製劑(mdivi-1)在腦缺血再灌註損傷中對能量代謝的影響.方法將體外培養8d的Wistar大鼠海馬神經元分為4組:正常對照組、賦形劑組、mdivi-1組、缺血再灌註損傷組,後兩組利用海馬神經元氧糖剝奪法建立缺血再灌註損傷模型,mdivi-1組建模前給予mdivi-1預處理40 min.缺血6h再灌註20 h後應用Western blotting檢測各組海馬神經元線粒體分裂蛋白1(Drp1)、B細胞淋巴癌/白血病-2(Bcl-2)和Bcl-2相關X蛋白(Bax)的錶達水平,應用流式細胞術檢測線粒體膜電位,應用酶標儀法檢測三燐痠腺苷(ATP)含量及Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性以及線粒體呼吸鏈酶複閤體Ⅰ、Ⅱ、Ⅲ、Ⅳ活性. 結果與正常對照組比較,mdivi-1組和缺血再灌註損傷組Drp1 (1.001±0.276; 1.985±0.301)、Bax(2.752±0.786;4.225±1.107)錶達水平明顯升高,Bcl-2 (0.749±0.128; 0.336±0.109)錶達水平明顯降低,線粒體膜電位降低的細胞數(72.5％;92.7％)均升高,ATP含量[(74.129±5.773) μmoL/g蛋白;(36.986±5.945) μmoL/g蛋白]及Na+-K+-ATP酶活性[(4.348±0.451) U/mg蛋白;(1.709±0.477) U/mg蛋白]、CaV-Mg2+-ATP酶活性[(1.955±0.287) U/mg蛋白;(1.123±0.181) U/rag蛋白]以及線粒體呼吸鏈酶複閤體Ⅰ、Ⅱ、Ⅲ、Ⅳ活性[(15.445±1.699) nmol/(min· mg)、(17.065±1.070) nmol/(min·mg)、(32.123±1.652) nmol/(min ·rag)、(2.814±0.180) nmol/(min· mg); (6.810±1.725) nmol/(min· mg)、(9.473±0.751) nmol/(min· mg)、(23.010±1.716) nmol/(min· mg)、(1.598±0.181) nmol/(min· mg)]均明顯降低,差異均有統計學意義(P＜0.05).與缺血再灌註損傷組比較,mdivi-1組Drpl、Bax錶達水平明顯減少,Bcl-2錶達水平明顯升高,線粒體膜電位降低的細胞數減少,ATP含量及Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性以及線粒體呼吸鏈酶複閤體Ⅰ、Ⅱ、Ⅲ、Ⅳ活性均明顯升高,差異均有統計學意義(P＜0.05). 結論 mdivi-1可通過抑製線粒體分裂,有效地改善能量代謝障礙,從而減輕腦缺血再灌註損傷.
목적 탐토선립체분렬억제제(mdivi-1)재뇌결혈재관주손상중대능량대사적영향.방법 장체외배양8d적Wistar대서해마신경원분위4조:정상대조조、부형제조、mdivi-1조、결혈재관주손상조,후량조이용해마신경원양당박탈법건립결혈재관주손상모형,mdivi-1조건모전급여mdivi-1예처리40 min.결혈6h재관주20 h후응용Western blotting검측각조해마신경원선립체분렬단백1(Drp1)、B세포림파암/백혈병-2(Bcl-2)화Bcl-2상관X단백(Bax)적표체수평,응용류식세포술검측선립체막전위,응용매표의법검측삼린산선감(ATP)함량급Na+-K+-ATP매、Ca2+-Mg2+-ATP매활성이급선립체호흡련매복합체Ⅰ、Ⅱ、Ⅲ、Ⅳ활성. 결과 여정상대조조비교,mdivi-1조화결혈재관주손상조Drp1 (1.001±0.276; 1.985±0.301)、Bax(2.752±0.786;4.225±1.107)표체수평명현승고,Bcl-2 (0.749±0.128; 0.336±0.109)표체수평명현강저,선립체막전위강저적세포수(72.5％;92.7％)균승고,ATP함량[(74.129±5.773) μmoL/g단백;(36.986±5.945) μmoL/g단백]급Na+-K+-ATP매활성[(4.348±0.451) U/mg단백;(1.709±0.477) U/mg단백]、CaV-Mg2+-ATP매활성[(1.955±0.287) U/mg단백;(1.123±0.181) U/rag단백]이급선립체호흡련매복합체Ⅰ、Ⅱ、Ⅲ、Ⅳ활성[(15.445±1.699) nmol/(min· mg)、(17.065±1.070) nmol/(min·mg)、(32.123±1.652) nmol/(min ·rag)、(2.814±0.180) nmol/(min· mg); (6.810±1.725) nmol/(min· mg)、(9.473±0.751) nmol/(min· mg)、(23.010±1.716) nmol/(min· mg)、(1.598±0.181) nmol/(min· mg)]균명현강저,차이균유통계학의의(P＜0.05).여결혈재관주손상조비교,mdivi-1조Drpl、Bax표체수평명현감소,Bcl-2표체수평명현승고,선립체막전위강저적세포수감소,ATP함량급Na+-K+-ATP매、Ca2+-Mg2+-ATP매활성이급선립체호흡련매복합체Ⅰ、Ⅱ、Ⅲ、Ⅳ활성균명현승고,차이균유통계학의의(P＜0.05). 결론 mdivi-1가통과억제선립체분렬,유효지개선능량대사장애,종이감경뇌결혈재관주손상.
Objective To investigate the effect of mitochondrial division inhibitor (mdivi-1) on energy metabolism in hippocampus neurons of rats after ischemia reperfusion injury (IRI).Methods The hippocampus neurons fiom Wistar rats were in vitro cultured for 8 d and then,divided into four groups:normal control group,vehicle group,IRI+mdivi-1 treatment group and IRI group.IRI models in the later two groups were established by method of oxygen glucose deprivation; and pretreatment with mdivi-1 for 40 min was given to the IRI+mdivi-1 treatment group before IRI.After ischemia for 6 h and reperfusion for 20 h of hippocampus neurons,Western blotting was employed to examine the protein expressions of dynamin-related protein 1 (Drp1),B cell lymphoma/lewkmia-2 (Bcl-2) and Bcl-2 associated X protein (Bax); mitochondrial membrane potential (△Ψm) was examined by flow cytometry;enzyme standard instrument was used to examine the ATP content,mitochondrial complex activity,Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity.Results As compared with those in the normal control group,the expressions of Drp1 (1.001±0.276 and 1.985±0.301),Bax (2.752±0.786 and 4.225±1.107)were up-regulated markedly,those of Bcl-2 (0.749 ±0.128 and 0.336 ±0.109) were significantly down-regulated,percentage of low mitochondrial membrane potential △Ψm cells was significantly increased (72.5％ and 92.7％),ATP content ([74.129±5.773] and [36.986±5.945] μmol/gprot),Na+-K+-ATPaseactivity ([4.348±0.451] and [1.709±0.477] U/mgprot),Ca2+-Mg2+-ATPaseactivity([1.955±0.287] and [1.123 ±0.181] U/mgprot) and activities of mitochondrial complex Ⅰ,Ⅱ,Ⅲ and Ⅳ [(15.445±1.699),(17.065±1.070),(32.123±1.652),(2.814±0.180) and (6.810±1.725),(9.473±0.751),(23.010±1.716),(1.598±0.181)] nmol/(min ·mg) decreased in the IRI+mdivi-1 treatment group and IRI group,with significant differences (P＜ 0.05).IRI+mdivi-1 treatment group had markedly reduced Drp1 and Bax protein expressions,significantly improved Bcl-2 protein expression,significantly reduced percentage of low mitochondrial membrane potential △Ψm cells,and significantly increased brain ATP content,Na+-K+-ATPase and Ca2+-Mg2+-ATPase activity,mitochondrial complex Ⅰ-Ⅳ activity as compared with IRI group (P＜0.05).Conclusion Mdivi-1,by inhibitting mitochondrial fission,can significantly improve mitochondrial energy metabolism and ameliorate cerebral ischemia/reperfusion injury.