salvinorin A上调MALAT1表达减轻缺血性脑卒中大鼠的脑血管内皮损伤

MALAT1‑mediated salvinorin A attenuates vascular endothelial damage in ischemic stroke

目的研究κ阿片受体(κopioid receptor,KOR)激动剂salvinorin A(SA)通过上调脑血管内皮细胞的长链非编码RNA(long noncoding RNA,lncRNA)MALAT1,抑制线粒体分裂蛋白‑1(dynamin‑related protein 1,Drp‑1)磷酸化,减轻线粒体损伤,产生对缺血性脑卒中大鼠的保护作用。方法动物实验采用雄性SD大鼠60只,体重200~250 g,按随机数字表法分为4组(每组15只):假手术组(S组),大鼠不做任何干预,只分离一侧颈内动脉;大脑中动脉栓塞模型组(MCAO组),采用线栓阻断大鼠一侧颈内动脉90 min,拔出线栓进行再灌注;salvinorin A组(SA组),线栓放入即刻经尾静脉给予大鼠SA(20μg/kg);KOR拮抗剂组(NB组),注射SA前30 min给予大鼠KOR拮抗剂norbinaltorphimine(norBIN 2 mg/kg),其余处理同SA组。大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)后24 h用Bederson评分法评估大鼠脑梗死程度。每组取5只处死取材,2,3,5‑三苯基氯化四氮唑(2,3,5‑Triphenyltertrazolium chloride,TTC)染色测定缺血区脑梗死面积、伊文蓝检测血脑屏障通透性。MCAO后1、2、5 d行运动神经功能评分。离体实验采用人脑血管内皮细胞(human brain microvascular endothelial cell,HBMEC)缺糖缺氧(oxygen‑glucose deprivation,OGD)模型,分为5组:空白对照组(Blank组),不进行缺氧及复氧处理;OGD组(A组),缺氧6 h,复氧24 h;SA+OGD组(B组),OGD模型复氧前1 h加入SA(5μmol/L);MALAT1特异性小干扰RNA(specific small interfering RNA,siRNA)+OGD组(C组),加入100 nmol/L lncRNA MALAT1 siRNA 24 h,缺氧6 h,复氧24 h;MALAT1 siRNA+OGD+SA组(D组),复氧前1 h加入SA(5μmol/L),其余处理同C组。采用Cell Counting Kit‑8(CCK‑8)测定HBMEC活力、磷酸化Drp‑1(phosphorylated Drp‑1,pDrp‑1)/Drp‑1、活性氧(reactive oxygen species,ROS)水平,电镜下观察线粒体形态。结果动物实验显示,与S组比较,MCAO组大鼠Bederson评分增高,脑梗死面积和伊文蓝渗出增加,运动神经功能下降(P<0.05);与MCAO组比较,SA组大鼠Bederson评分降低,脑梗死面积减小,伊文蓝渗出减轻,运动神经功能改善(P<0.05);与SA组比较,NB组大鼠Bederson评分增高,脑梗死面积和血管通透性增加,神经功能减退(P<0.05)。离体实验显示,与Blank组比较,A组MALAT1水平增加,HBMEC活性下降,pDrp‑1/Dpr‑1升高(P<0.05),ROS含量增加(P<0.01),电镜下可见线粒体形态破坏;与A组比较,B组MALAT1水平增加,HBMEC活性增加,pDrp‑1/Dpr‑1下降(P<0.05),ROS含量下降(P<0.01),线粒体形态趋于正常;与B组比较,C组HBMEC活性下降(P<0.05),pDrp‑1/Dpr‑1和ROS含量增高(P<0.01),线粒体形态破坏;与C组比较,D组HBMEC活性增加,pDrp‑1/Dpr‑1、ROS含量降低(P<0.05),线粒体仍有一定程度的肿胀。结论KOR激动剂SA通过上调MALAT1的表达,减轻缺血性脑卒中后脑血管内皮细胞线粒体的损伤,减轻HBMEC的氧化应激反应,减轻脑卒中后血脑屏障的渗透增加,对脑卒中后的脑功能产生保护作用。

Objective To investigate theκopioid receptor(KOR)agonist salvinorin A(SA)produced protective effects in ischemic stroke rats by upregulating long noncoding RNA(lncRNA)MALAT1 in cerebral vascular endothelial cells,inhibiting dynamin‑re‑lated protein 1(Drp‑1)phosphorylation,and mitigating mitochondrial damage.Methods Sixty male SD rats,weighed 200‒250 g,were used for animal experiments and divided into four groups(15 rats each)according to random number table method.In the Sham group(S group),rats were isolated one side of the internal carotid artery without any intervention.In the middle cerebral artery occlu‑sion model group(MCAO group),the rat carotid artery was blocked by wire embolus for 90 min,and the wire embolus was removed for reperfusion.In SA group,SA(20μg/kg)was given via the tail vein immediately after internal carotid block.In the KOR antagonist group(NB group),norbinaltorphimine(norBIN 2 mg/kg)was given to rats 30 min before SA injection,and the rest of the treatment was the same as in the SA group.24 h after MCAO,the degree of cerebral infarction in rats was assessed by Bederson score.After the execu‑tion,the rat cerebral infarct area was assessed by 2,3,5 Triphenyltertrazolium chloride(TTC),blood brain barrier permeability(Evans blue)were measured,and behavioral assessment and neural assessment(total motor score)was performed 1,2 d and 5 d after MCAO.In vitro experiments were conducted using human brain microvascular endothelial cell(HBMEC)model of oxygenglucose deprivation(OGD)and divided into five groups:blank control group(Blank group),without OGD and reoxygenation;OGD group(group A),6 h hy‑poxia,24 h reoxygenation;SA+OGD group(group B),SA(5μmol/L)was added 1 h before OGD model reoxygenation;MALAT1 specific small interfering RNA(siRNA)+OGD group(group C),100 nmol/L lncRNA MALAT1 siRNA was added for 24 h,hypoxia for 6 h,and reoxygenation for 24 h;MALAT1 siRNA+OGD+SA group(group D),100 nmol/L lncRNA MALAT1 siRNA was added for 24 h,6 h of hypoxia,24 h of reoxygenation,and SA was added 1 h before reoxygenation(5μmol/L).The Cell Counting Kit8(CCK8)was used to de‑termine the activity of HBMEC,phosphorylated Drp‑1(pDrp‑1)/Drp‑1,reactive oxygen species(ROS)levels,and mitochondrial mor‑phology was observed under electron microscope.Results Animal experiments showed that,compared with S group,rats in MCAO group had significantly higher behavioral scores,significantly increased infarct size,significantly increased Envans blue exudation,and significantly decreased motor nerve function(P<0.05).Compared with MCAO group,the behavioral scores of rats in the SA group were significantly lower,the infarct area was significantly reduced,Envans blue exudation was significantly reduced,and the motor nerve function of rats was significantly improved(P<0.05).Compared with SA group,rats in NB group had significantly higher behavioral scores,larger infarct size,increased vascular permeability,and decreased neurological function(P<0.05).In vitro experiments showed an increase in MALAT1 levels,a significant decrease in HBMEC activity,an increase in pDrp‑1/Dpr‑1(P<0.05),a significant increase in ROS content(P<0.01),and mitochondrial morphological disruption visible under electron microscopy in group A compared with the Blank group.Compared with group A,group B had increased MALAT1 level,significantly increased HBMEC activity,decreased pDrp‑1/Drp‑1(P<0.05),significantly decreased ROS content(P<0.01),and normalized mitochondrial morphology under electron micro‑scope.Compared with group B,group C had significantly decreased HBMEC activity(P<0.05),increased pDrp‑1/Drp‑1,increased ROS content(P<0.01)and disrupted mitochondrial morphology.Compared with group C,group D had significantly increased HBMEC activi‑ty and decreased pDrp‑1/Drp‑1 and ROS levels(P<0.05),and the mitochondrial still had some degree of swelling.Conclusions The KOR agonist SA protects brain function after ischemic stroke by upregulating MALAT1 expression,attenuating mitochondrial damage in ce‑rebral vascular endothelial cells after ischemic stroke,reducing oxidative stress in HBMEC,and reducing increased blood‑brain.