褪黑素腹腔注射对糖尿病合并脑缺血再灌注损伤小鼠脑组织损伤的预防作用及其机制  

Preventive effect of intraperitoneal injection of melatonin on brain injury in diabetic rats with cerebral ischemia reperfusion injury

作　　者：刘雅静[1] 李冰玉 宋文沁 黎梅[1] 刘恋[1] LIU Yajing;LI Bingyu;SONG Wenqin;LI Mei;LIU Lian(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14期14-18,共5页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81901994,82102295)。

摘　　要：目的观察褪黑素对糖尿病合并脑缺血再灌注损伤(CIRI)小鼠脑组织损伤的预防作用,并探讨其作用机制与SIRT1-BMAL1通路介导的自噬的关系。方法取健康雄性C57BL/6小鼠24只,采用腹腔注射STZ的方法建立糖尿病模型,将糖尿病小鼠随机分为假手术组、缺血再灌注组、缺血再灌注+褪黑素组、缺血再灌注+褪黑素+SIRT1抑制剂组,每组各6只。假手术组分离大脑中动脉但不阻断;缺血再灌注组采用MCAO法建立脑缺血再灌注模型;缺血再灌注+褪黑素组采用MCAO法建立脑缺血再灌注模型,于缺血即刻和再灌注前腹腔注射褪黑素10 mg/kg;缺血再灌注+褪黑素+SIRT1抑制剂组于MCAO术前5、3、1 d腹腔注射SIRT1抑制剂5 mg/kg,采用MCAO法建立脑缺血再灌注模型,于缺血即刻和再灌注前腹腔注射褪黑素10 mg/kg。于再灌注24 h后处死各组小鼠,取脑组织,采用HE染色法观察脑组织病理变化,TTC染色法检测脑梗死体积,Western blotting法检测脑组织SIRT1-BMAL1通路相关蛋白SIRT1、BAML1、ac-BMAL1和自噬相关蛋白LC3Ⅱ/Ⅰ、Beclin1、Atg14表达。结果与假手术组相比,缺血再灌注组脑组织神经元数量减少,胞质疏松并伴有空泡形成,缺血再灌注+褪黑素组上述改变较缺血再灌注组减轻,缺血再灌注+褪黑素+SIRT1抑制剂组上述改变较缺血再灌注+褪黑素组加重。与假手术组相比,缺血再灌注组脑梗死体积增加(P<0.05),缺血再灌注+褪黑素组脑梗死体积较缺血再灌注组减少(P<0.05),缺血再灌注+褪黑素+SIRT1抑制剂组脑梗死体积较缺血再灌注+褪黑素组增加(P<0.05)。与假手术组相比,缺血再灌注组脑组织LC3Ⅱ/Ⅰ、Beclin1、Atg14及SIRT1、BAML1表达降低,ac-BMAL1表达增加(P均<0.05);与缺血再灌注组相比,缺血再灌注+褪黑素组LC3Ⅱ/Ⅰ、Beclin1、Atg14及SIRT1、BAML1表达增加,ac-BMAL1表达降低(P均<0.05);与缺血再灌注+褪黑素组相比,缺血再灌注+褪黑素+SIRT1抑制剂组LC3Ⅱ/Objective To observe the preventive effect of melatonin on brain injury in diabetic mice with cerebral ischemia reperfusion injury(CIRI),and to investigate the relationship between its mechanism and autophagy mediated by SIRT1-BMAL1 pathway.Methods Twenty-four healthy male C57BL/6 mice were used to establish diabetic models by intraperitoneal injection of streptozotocin(STZ).Diabetic mice were randomly divided into four groups(n=6):sham-operation group,ischemia reperfusion group,ischemia reperfusion+melatonin group,and ischemia reperfusion+melatonin+SIRT1 inhibitor group.The middle cerebral artery was separated but not blocked in mice of the sham-operation group.In the ischemia reperfusion group,cerebral ischemia reperfusion model was established by MCAO method.Likewise,cerebral ischemia reperfusion model was established by MCAO method in the ischemia reperfusion+melatonin group with intraperitoneal injection of 10 mg/kg melatonin after ischemia immediately and before reperfusion.In the ischemia reperfusion+melatonin+SIRT1 inhibitor group,5 mg/kg SIRT1 inhibitor was injected intraperitoneally on the 5th,3rd and 1st days before MCAO,followed by intraperitoneal injection of 10 mg/kg melatonin after ischemia immediately and before reperfusion.At 24 h after reperfusion,the mice in each group were killed to extract their brain tissues.The pathological changes were observed by HE staining,the cerebral infarction volume was detected by TTC staining,and the expression levels of SIRT1-BMAL1 pathway-related proteins(SIRT1,BAML1,ac-BMAL1)and autophagy-related proteins(LC3Ⅱ/Ⅰ,Beclin1,Atg14)were detected by Western blotting.Results Compared with the sham-operation group,the number of neurons in the ischemia reperfusion group decreased,the cytoplasm was loose and vacuoles were formed.These injuries were alleviated in the ischemia reperfusion+melatonin group,but were aggravated in the ischemia reperfusion+melatonin+SIRT1 inhibitor group.Meanwhile,compared with the sham-operation group,the cerebral infarction volume expanded

关 键 词：褪黑素 糖尿病 脑缺血再灌注损伤 生物钟基因 去乙酰化酶 自噬 动物实验 

分 类 号：R587.1[医药卫生—内分泌]