CAJ | 학술논문

目的探讨线粒体损伤在脓毒症大鼠心肌细胞凋亡中的作用及其可能机制.方法 72只SD大鼠随机(随机数字法)分为2组:(1)阴性对照组18只,腹腔注射无菌生理盐水；(2)脓毒症组,按大肠杆菌内毒素注射后6、12、24 h分为3个亚组,各18只.取心脏组织,光镜和电镜下观察组织病理学和超微结构的变化,TUNEL法原位检测心肌细胞凋亡并计算凋亡指数,免疫印迹法检测核转录因子-κB (NF-κB) p65的表达,黄嘌呤氧化酶法测定线粒体超氧化物歧化酶(SOD)活性,比色法检测线粒体还原型谷胱甘肽(GPx)活性,硫代巴比妥酸法测定线粒体脂质过氧化产物丙二醛(MDA)含量,OxyBlot TMas蛋白氧化检测试剂盒观察线粒体蛋白氧化.结果脓毒症大鼠心肌有炎症细胞浸润,细胞水肿及空泡形成,并且在24 h最明显；心肌细胞凋亡指数和核蛋白NF-κB p65活化均显著增加(P＜0.05).脓毒症大鼠心肌线粒体损伤明显,至24 h病变最严重,线粒体膜破碎,线粒体嵴消失,大量空泡形成.脓毒症大鼠心肌线粒体抗氧化酶SOD和GPx活性显著下降(均P＜0.05),线粒体脂质过氧化和蛋白氧化均显著增加(均P＜0.05),并呈时间依赖性.结论线粒体损伤在脓毒症心肌细胞凋亡中发挥重要作用,其作用机制可能与线粒体抗氧化物酶活性降低,活性氧产生过多和聚集有关.
목적 탐토선립체손상재농독증대서심기세포조망중적작용급기가능궤제.방법 72지SD대서수궤(수궤수자법)분위2조:(1)음성대조조18지,복강주사무균생리염수；(2)농독증조,안대장간균내독소주사후6、12、24 h분위3개아조,각18지.취심장조직,광경화전경하관찰조직병이학화초미결구적변화,TUNEL법원위검측심기세포조망병계산조망지수,면역인적법검측핵전록인자-κB (NF-κB) p65적표체,황표령양화매법측정선립체초양화물기화매(SOD)활성,비색법검측선립체환원형곡광감태(GPx)활성,류대파비타산법측정선립체지질과양화산물병이철(MDA)함량,OxyBlot TMas단백양화검측시제합관찰선립체단백양화.결과 농독증대서심기유염증세포침윤,세포수종급공포형성,병차재24 h최명현；심기세포조망지수화핵단백NF-κB p65활화균현저증가(P＜0.05).농독증대서심기선립체손상명현,지24 h병변최엄중,선립체막파쇄,선립체척소실,대량공포형성.농독증대서심기선립체항양화매SOD화GPx활성현저하강(균P＜0.05),선립체지질과양화화단백양화균현저증가(균P＜0.05),병정시간의뢰성.결론 선립체손상재농독증심기세포조망중발휘중요작용,기작용궤제가능여선립체항양화물매활성강저,활성양산생과다화취집유관.
Objective To investigate the role of damaged mitochondria in cardiac cell apoptosis in septic rats and the possible mechanism involved.Methods Seventy-two Sprague-Dawley rats were randomly (random number) divided into negative control group (n =18) and septic group (further divided into three groups as per rats sacrificed 6 h,12 h,and 24 h after endotoxin injection intra-peritoneally,n =18).The hearts of rats were taken.The changes of cardiac morphology were observed under light microscope and scanning electron microscope.Cell apoptosis in situ were examined by using terminal transferase-mediated dUTP nick end-labeling assay and nuclear factor-kappa B (NF-κB) activation in myocardium was detected by using Western blotting to estimate myocardial cell apoptosis.Mitochondrial lipid and protein oxidation were measured to assess oxidative stress,and mitochondrial superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were determined to estimate antioxidant defense.Results Septic induced inflammatory cells infiltration,myocardium degeneration and effusion in a time-dependent manner.A remarkable expansion of capillaries could be observed in the hearts of infected rats at post-challenge of 24 h.Compared with sham-treated rats,the percentage of apoptosis increased in a time-dependent manner in the hearts of infected rats at 6 h,12 h,24 h of post-injection (P ＜0.05).The concentration of NF-κB p65 in the cytosol decreased gradually and increased in the nucleus during sepsis in a time-dependent manner (P ＜0.05),indicating that septic challenge provoked progressive activation of NF-κB.Mitochondrial cristae disappeared in 6 h of challenge,and significant mitochondrial cristae disappearance,vacuolization,and rupture of mitochondria membrane became markedly obvious 12 and 24 h later.Both SOD and GPx activities decreased,while mitochondrial lipid and protein oxidation increased in a time-dependent manner after 6-24 h of challenge (P ＜ 0.05).Conclusions Septic challenge induced myocardial apoptosis and mitochondrial damage.Further,damaged mitochondria might play an important role by means of alteration of defenses against reactive oxygen species in myocardial cell apoptosis during sepsis.