褪黑素调控SIRT3对糖尿病脑缺血再灌注损伤小鼠脑组织线粒体的保护作用  

Protective effect of melatonin regulating SIRT3 on mitochondria in brain tissues of diabetic rats with cerebral ischemia-reperfusion injury

作　　者：李亚男 刘恋[1] 李冰玉 夏中元[1] 赵博[1] LI Yanan;LIU Lian;LI Bingyu;XIA Zhongyuan;ZHAO Bo(Department of Anesthesiology,Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院麻醉科,武汉430060

出　　处：《山东医药》2022年第20期20-23,共4页Shandong Medical Journal

基　　金：国家自然科学基金项目(81901994,82102295)。

摘　　要：目的观察褪黑素对糖尿病脑缺血再灌注损伤(CIRI)小鼠脑组织线粒体的保护作用,并基于去乙酰化酶3(SIRT3)探讨褪黑素的相关作用机制。方法健康雄性C57BL/6小鼠24只,随机分为糖尿病假手术组(DS组)、糖尿病缺血再灌注组(DIR组)、糖尿病缺血再灌注+褪黑素组(DIR+Mel组)、糖尿病缺血再灌注+褪黑素+3-TYP组(DIR+Mel+3-TYP组),每组6只。各组以腹腔注射链脲佐菌素建立糖尿病模型,DIR组、DIR+Mel组、DIR+Mel+3-TYP组采用tMCAO法建立脑缺血再灌注模型,DS组分离大脑中动脉但不阻断血流,DIR+Mel组、DIR+Mel+3-TYP组于缺血即刻和再灌注前腹腔注射褪黑素10 mg/kg,DIR+Mel+3-TYP组分别于术前5、3、1 d腹腔注射SIRT3特异性抑制剂3-TYP 50 mg/kg。再灌注24 h后处死各组小鼠。透射电镜观察病灶脑组织线粒体超微结构,比色法测定病灶脑组织线粒体丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)活性、线粒体ATP含量,Westernblotting检测病灶脑组织中的核呼吸因子1(NRF1)、线粒体转录因子A(TFAM)、胞质细胞色素C(Cyt-CytoC)。结果与DS组相比,DIR组线粒体严重空泡化伴肿胀,线粒体MDA含量、Cyt-CytoC表达升高,线粒体ATP含量、SOD活性、CAT活性及病灶脑组织中NRF1、TFAM表达降低(P均<0.05);与DIR组相比,DIR+Mel组线粒体空泡化和肿胀减轻,MDA含量、Cyt-CytoC表达降低,ATP含量、SOD活性、CAT活性及NRF1、TFAM表达升高(P均<0.05);与DIR+Mel组相比,DIR+Mel+3-TYP组线粒体空泡化和肿胀加重,MDA含量、Cyt-CytoC表达升高,ATP含量、SOD活性、CAT活性及NRF1、TFAM表达降低(P均<0.05)。结论褪黑素干预可维持糖尿病CIRI小鼠线粒体正常形态,降低线粒体氧化应激水平,增加ATP含量,促进线粒体生成,作用可能与调控SIRT3表达有关。Objective To observe the protective effect of melatonin(Mel)on brain mitochondria in diabetic mice with cerebral ischemia-reperfusion injury(CIRI)and to explore the specific mechanism of melatonin based on sirtuin 3(SIRT3).Methods Twenty-four healthy male C57BL/6 mice were randomly divided into four groups(n=6),the diabet-ic sham operation group(DS group),diabetic ischemia-reperfusion group(DIR group),diabetic ischemia-reperfusion+Mel group(DIR+Mel group),and diabetic ischemia-reperfusion+Mel+3-TYP group(DIR+Mel+3-TYP group).The dia-betic models were established by intraperitoneal injection of streptozotocin in each group.The cerebral ischemia reperfu-sion models in the DIR group,DIR+Mel group and DIR+Mel+3-TYP group were established by tMCAO method.The mid-dle cerebral artery was separated without blocking in the DS group.3-TYP was a kind of specific inhibitor of SIRT3.Mice in the DIR+Mel group and DIR+Mel+3-TYP group received intraperitoneal injection of melatonin 10 mg/kg immediately af-ter ischemia and before reperfusion,while mice in the DIR+Mel+3-TYP group were injected with 50 mg/kg 3-TYP intra-peritoneally on the 5th,3rd and 1st days before operation,respectively.The mice in each group were killed at 24 h after re-perfusion.The ultrastructure of mitochondria was observed by transmission electron microscope.The content of malondialdehyde(MDA),the activity of superoxide dismutase(SOD)and catalase(CAT)and the content of mitochondrial ATP were measured by colorimetry.Nuclear respiratory factor 1(NRF1),mitochondrial transcription factor A(TFAM)and cy-toplasmic cytochrome C(Cyt-Cyto C)were detected by Western blotting.Results Compared with DS group,the mito-chondria in the DIR group were seriously vacuolated with swelling,MDA,Cyt-Cyto C increased,while ATP,SOD,CAT,NRF1 and TFAM in the focal brain tissues decreased(all P<0.05).Compared with DIR group,mitochondrial vacuolation and swelling were alleviated,MDA and Cyt-Cyto C decreased,and ATP,SOD,CAT NRF1,and TFAM increased in the DIR+Mel group(all P<0.05).Compare

关 键 词：糖尿病 脑缺血再灌注损伤 褪黑素 去乙酰化酶3 线粒体保护 

分 类 号：R587.1[医药卫生—内分泌]