3-硝基-N-甲基水杨酰胺对肢体缺血再灌注损伤大鼠骨骼肌的保护作用及机制

Protective effect and mechanism of 3-nitro-N-methyl salicylamide on the skeletal muscle of rats with limb ischemia-reperfusion injury

背景:线粒体活性氧爆发已被证明在骨骼肌缺血再灌注中起着关键作用。3-硝基-N-甲基水杨酰胺(3-nitro-N-methyl salicylamide,3-NNMS)可以有效降低电子传递速度,对肢体缺血再灌注损伤具有潜在的保护作用,但目前尚无明确的研究和临床应用。目的:探讨3-NNMS对肢体缺血再灌注损伤大鼠骨骼肌的保护作用及机制。方法:40只健康8周龄SD大鼠随机分为对照组及3-NNMS的0μg/mL组、25μg/mL组、125μg/mL组,每组10只。除对照组外,其余各组制备肢体缺血再灌注损伤大鼠模型,于再灌注前30 min,向损伤部位注射相应浓度的3-NNMS。再灌注2 h后,心尖取血,取大鼠右下肢股直肌组织进行检测。苏木精-伊红染色观察大鼠股直肌组织病理形态;ELISA检测血清骨骼肌损伤因子肌酸激酶(Creatine Kinase found in the skeletal muscle,CK-MM)、乳酸脱氢酶、髓过氧化物酶水平,并检测股直肌核因子κB、肿瘤坏死因子α、白细胞介素1β、环氧合酶2、丙二醛、活性氧、超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶水平,以及股直肌ATP水平、ATPase活性、线粒体呼吸控制率(RCR)水平。结果与结论:①与对照组相比,缺血再灌注模型大鼠血清CK-MM、乳酸脱氢酶、髓过氧化物酶水平升高,股直肌核因子κB、肿瘤坏死因子α、白细胞介素1β、环氧合酶2、丙二醛及活性氧水平升高,过氧化氢酶、谷胱甘肽过氧化物酶水平下降,ATPase活性、线粒体呼吸控制率水平降低;细胞形态不规则,炎性细胞浸润明显,细胞出现肿胀。②与0μg/mL组相比,25μg/mL组大鼠血清CK-MM、乳酸脱氢酶水平降低,股直肌核因子κB、环氧合酶2水平降低,活性氧减少,超氧化物歧化酶活性升高;细胞形态较规则,炎性细胞浸润较轻,细胞肿胀现象缓解。③与0μg/mL组相比,125μg/mL组大鼠血清CK-MM、乳酸脱氢酶、髓过氧化物酶水平降低,股直肌核因子κB、肿瘤坏死因子α、环氧合酶2量减少,丙二醛、活性氧水平降低,超氧化物歧化酶、谷胱甘肽过氧化物酶活性升高,线粒体呼吸控制率水平升高;细胞排列较整齐,轮廓较清晰完整,炎性细胞浸润较轻。④结果说明:3-NNMS可以减轻肢体缺血再灌注引起的骨骼肌功能损伤,其作用机制可能是通过改善线粒体功能、减少活性氧产生、降低氧化应激和炎症反应,进而减轻组织损伤,修复骨骼肌功能。

BACKGROUND:Mitochondrial reactive oxygen bursts have been shown to play a key role in skeletal muscle ischemia-reperfusion injury.3-Nitro-Nmethylsalicylamide(3-NNMS)can effectively reduce the electron transport rate and has a potential protective effect on limb ischemia-reperfusion injury,but there is no clear research and clinical application.OBJECTIVE:To investigate the protective effect of 3-NNMS on the skeletal muscle after limb ischemia-reperfusion injury in rats and its mechanism.METHODS:Forty healthy 8-week-old Sprague-Dawley rats were randomly divided into control group,0,25 and 125μg/mL 3-NNMS groups,with 10 rats in each group.Animal models of limb ischemia-reperfusion injury were prepared in the latter three groups.3-NNMS was injected into the injury site 30 minutes before reperfusion.The animals were sacrificed 2 hours after reperfusion.Blood from the apical part of the heart,and the tissue of the rectus femoris muscle of the right lower limb were taken for testing.The pathological morphology of the rectus femoris muscle was detected by hematoxylin-eosin staining.Serum levels of creatine kinase found in the skeletal muscle(CK-MM),lactate dehydrogenase,and myeloperoxidase were detected using ELISA;the levels of nuclear factorκB,tumor necrosis factorα,interleukin 1β,cyclooxygenase 2,malondialdehyde,reactive oxygen species,superoxide dismutase,catalase and glutathione peroxidase in the rectus femoris muscle were measured;and adenosine triphosphate(ATP)level,ATPase activity,and mitochondrial respiratory control rate were tested.RESULTS AND CONCLUSION:Compared with the control group,the model rats with ischemia-reperfusion injury had increased serum levels of CK-MM,lactate dehydrogenase,and myeloperoxidase,increased levels of nuclear factorκB,tumor necrosis factorα,interleukin 1β,cyclooxygenase 2,malondialdehyde and reactive oxygen species in the rectus femoris muscle,decreased levels of catalase and glutathione peroxidase in the rectus femoris muscle,and reduced ATPase activity and mitochondrial respiratory control rate.Moreover,cell morphology was irregular,inflammatory cell infiltration was obvious,and the cells were swollen in rats after ischemia-reperfusion injury.Compared with the 0μg/mL group,the serum CK-MM and lactate dehydrogenase levels decreased,the levels of nuclear factorκB and cyclooxygenase 2 in the rectus femoris muscle decreased,reactive oxygen species level decreased,and superoxide dismutase activity increased in the 25μg/mL group;cell morphology was more regular,inflammatory cell infiltration was lighter,and cell swelling was alleviated.Compared with the 0μg/mL group,the 125μg/mL group had a reduction in the serum levels of CK-MM,lactate dehydrogenase,and myeloperoxidase and the levels of nuclear factorκB,tumor necrosis factorα,cyclooxygenase 2,malondialdehyde and reactive oxygen species in the rectus femoris muscle,as well as an increase in the levels of superoxide dismutase and glutathione peroxidase in the rectus femoris muscle,and mitochondrial respiratory control rate.Moreover,the cells were arranged neatly,the outline was clear and complete,and the inflammatory cell infiltration was light.To conclude,3-NNMS can alleviate the functional impairment of the skeletal muscle caused by limb ischemia-reperfusion,and its mechanism of action may be through improving mitochondrial function,reducing reactive oxygen species production,decreasing oxidative stress and inflammatory response,and thus reducing tissue damage and repairing skeletal muscle function.