亚低温对糖尿病合并脑梗死大鼠缺血半暗带凋亡的作用及机制
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摘要
目的探讨亚低温对糖尿病合并脑梗死(CI)大鼠缺血半暗带凋亡的作用及机制。方法健康雄性SD大鼠随机分为糖尿病组(n=42)和非糖尿病组(n=14),通过高脂饲料喂食联合链脲霉素(STZ)诱导2型糖尿病。制备大鼠永久性大脑中动脉栓塞模型,造模成功2周后随机分为糖尿病CI常温组(n=14)、糖尿病CI亚低温组(n=14)、糖尿病假手术组(n=14)、非糖尿病CI常温组(n=14)。建立永久性大脑中动脉栓塞CI模型后2 h开始对亚低温组进行亚低温干预6 h。于术后不同时间点进行神经行为学评估,术后3 d检测各组大鼠缺血半暗带的细胞凋亡程度以及基质金属蛋白酶9(MMP-9)表达量。结果①与非糖尿病CI常温组比较,糖尿病CI常温组神经功能评分降低(P <0. 05),缺血半暗带凋亡水平升高(P <0. 05),cleaved caspase-3和MMP-9蛋白表达量增加(P <0. 05),Bcl-2基因表达量降低(P <0. 05),Bim基因表达量增加(P <0. 05)。②与糖尿病CI常温组比较,糖尿病CI亚低温组神经功能评分增加(P <0. 05),缺血半暗带凋亡水平降低(P <0. 05),cleaved caspase-3和MMP-9蛋白表达量降低(P <0. 05),Bcl-2基因表达量增加(P <0. 05),Bim基因表达量降低(P <0. 05)。结论糖尿病加重CI大鼠的神经功能损伤,亚低温干预可以减轻糖尿病CI大鼠的神经功能损伤,减轻血脑屏障受损从而抑制缺血半暗带神经细胞凋亡可能是其保护机制。
Aim To explore the effect and mechanism of mild hypothermia on cell apoptosis in diabetic rats with cerebral infarction. Methods Male healthy SD rats were divided into a diabetic group( n = 42) and a non-diabetic group( n = 14) randomly. Type 2 diabetes was induced by high-fat diet combined with STZ. The rats were divided into a diabetic cerebral ischemic normothermic group( DNT group,n = 14),a diabetic cerebral ischemic hypothermia group( DHT group)( n = 14),a diabetic sham group( DS group,n = 14) and a non-diabetic cerebral ischemic normothermic group( NNT group,n = 14)randomly after 2 weeks of successful modeling. The hypothermia group was treated with hypothermia for 6 hours starting from 2 hours after pMCAO. The neurological deficit in each group was tested at several time points after cerebral ischemia,and the level of apoptosis,the expression of matrix metalloprotein 9( MMP-9)were detected in the ischemic penumbra in each group. Results ①Compared with the NNT group,the neurological function score was lower in DNT group after cerebral ischemia( P < 0. 05),and the level of apoptosis was higher in DNT group( P < 0. 05). Meanwhile,the expression of MMP-9 and cleaved caspase-3 was increased in DNT group( P < 0. 05). The gene expression of Bcl-2 was decreased and Bim was increased( P < 0. 05). ②Compared with the DNT group,the neurological function score was higher in DHT group after cerebral ischemia( P < 0. 05),and the level of apoptosis was lower in DHT group( P < 0. 05).At the meantime,the expression of MMP-9 and cleaved caspase-3 was reduced in DHT group( P < 0. 05).The gane expression of Bcl-2 was increased and Bim was decreased( P < 0. 05). Conclusion Diabetes aggravated the neurological deficit in rats with cerebral infarction,while mild hypothermia alleviated neurological deficit in rats with diabetic cerebral infarction. The underlying neuroprotective mechanisms of mild hypothermia may be related to inhibiting apoptosis in ischemic penumbra by reducing the damage of the blood-brain barrier.
Aim To explore the effect and mechanism of mild hypothermia on cell apoptosis in diabetic rats with cerebral infarction. Methods Male healthy SD rats were divided into a diabetic group( n = 42) and a non-diabetic group( n = 14) randomly. Type 2 diabetes was induced by high-fat diet combined with STZ. The rats were divided into a diabetic cerebral ischemic normothermic group( DNT group,n = 14),a diabetic cerebral ischemic hypothermia group( DHT group)( n = 14),a diabetic sham group( DS group,n = 14) and a non-diabetic cerebral ischemic normothermic group( NNT group,n = 14)randomly after 2 weeks of successful modeling. The hypothermia group was treated with hypothermia for 6 hours starting from 2 hours after pMCAO. The neurological deficit in each group was tested at several time points after cerebral ischemia,and the level of apoptosis,the expression of matrix metalloprotein 9( MMP-9)were detected in the ischemic penumbra in each group. Results ①Compared with the NNT group,the neurological function score was lower in DNT group after cerebral ischemia( P < 0. 05),and the level of apoptosis was higher in DNT group( P < 0. 05). Meanwhile,the expression of MMP-9 and cleaved caspase-3 was increased in DNT group( P < 0. 05). The gene expression of Bcl-2 was decreased and Bim was increased( P < 0. 05). ②Compared with the DNT group,the neurological function score was higher in DHT group after cerebral ischemia( P < 0. 05),and the level of apoptosis was lower in DHT group( P < 0. 05).At the meantime,the expression of MMP-9 and cleaved caspase-3 was reduced in DHT group( P < 0. 05).The gane expression of Bcl-2 was increased and Bim was decreased( P < 0. 05). Conclusion Diabetes aggravated the neurological deficit in rats with cerebral infarction,while mild hypothermia alleviated neurological deficit in rats with diabetic cerebral infarction. The underlying neuroprotective mechanisms of mild hypothermia may be related to inhibiting apoptosis in ischemic penumbra by reducing the damage of the blood-brain barrier.
引文
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[25]Gong C,Boulis N,Qian J,et al.Intracerebral hemorrhageinduced neuronal death[J].Neurosurgery,2001,48:875-882
[26]耿阳,郭岑,霍亚静,等.4-苯基丁酸对大鼠脑出血模型脑水肿的作用及机制探讨[J].中国临床神经科学,2016,24:376-382
[27]高素玲,刘国荣,陈瑞英,等.影响糖尿病并发急性脑梗死近期预后的相关因素分析[J].中华老年心脑血管病杂志,2015,17:1206-1207
[28]Liu X,Wen S,Zhao S,et al.Mild Therapeutic Hypothermia Protects the Brain from Ischemia/Reperfusion Injury through Upregulation of i ASPP[J].Aging Dis,2018,9:401-411
[29]Nakka VP,Gusain A,Mehta SL,et al.Molecular mechanisms of apoptosis in cerebral ischemia:multiple neuroprotective opportunities[J].Mol Neurobiol,2008,37:7-38
[30]Zeng G,Ding W,Li Y,et al.Morroniside protects against cerebral ischemia/reperfusion injury by inhibiting neuron apoptosis and MMP2/9 expression[J].Exp Ther Med,2018,16:2229-2234
[31]Chen XJ,Zhang JG,Wu L.Plumbagin inhibits neuronal apoptosis,intimal hyperplasia and also suppresses TNF-alpha/NF-kappaB pathway induced inflammation and matrix metalloproteinase-2/9 expression in rat cerebral ischemia[J].Saudi J Biol Sci,2018,25:1033-1039
[32]Roach DM,Fitridge RA,Laws PE,et al.Up-regulation of MMP-2 and MMP-9 leads to degradation of type IV collagen during skeletal muscle reperfusion injury;protection by the MMPinhibitor,doxycycline[J].Eur J Vasc Endovasc Surg,2002,23:260-269
[33]Qi Z,Liang J,Pan R,et al.Zinc contributes to acute cerebral ischemia-induced blood-brain barrier disruption[J].Neurobiol Dis,2016,95:12-21
[34]Kumari R,Willing LB,Patel SD,et al.Increased cerebral matrix metalloprotease-9 activity is associated with compromised recovery in the diabetic db/db mouse following a stroke[J].JNeurochem,2011,119:1029-1040
[2]Yang W,Lu J,Weng J,et al.Prevalence of diabetes among men and women in China[J].N Engl J Med,2010,362:1090-1101
[3]Luitse MJ,Biessels GJ,Rutten GE,et al.Diabetes,hyperglycaemia,and acute ischaemic stroke[J].Lancet Neurol,2012,11:261-271
[4]Powers WJ,Rabinstein AA,Ackerson T,et al.2018 Guidelines for the Early Management of Patients With Acute Ischemic Stroke:A Guideline for Healthcare Professionals From the American Heart Association/American Stroke Association[J].Stroke,2018,49:e46-e110
[5]Ramos-Cabrer P,Campos F,Sobrino T,et al.Targeting the ischemic penumbra[J].Stroke,2011,42:S7-S11
[6]Broughton BR,Reutens DC,Sobey CG.Apoptotic mechanisms after cerebral ischemia[J].Stroke,2009,40:e331-e339
[7]Gong L,Tang Y,An R,et al.RTN1-C mediates cerebral ischemia/reperfusion injury via ER stress and mitochondriaassociated apoptosis pathways[J].Cell Death Dis,2017,8:e3080
[8]Gu Z,Cui J,Brown S,et al.A highly specific inhibitor of matrix metalloproteinase-9 rescues laminin from proteolysis and neurons from apoptosis in transient focal cerebral ischemia[J].JNeurosci,2005,25:6401-6408
[9]Choi HA,Badjatia N,Mayer SA.Hypothermia for acute brain injury-mechanisms and practical aspects[J].Nat Rev Neurol,2012,8:214-222
[10]Polderman KH.Induced hypothermia and fever control for prevention and treatment of neurological injuries[J].Lancet,2008,371:1955-1969
[11]葛安岩,丁晶,郭景春,等.延迟亚低温对自发性高血压大鼠永久性大脑中动脉闭塞作用的研究[J].中国临床神经科学,2013,21:37-43
[12]韩锁成,吕佳宁,郭景春,等.延迟亚低温对永久性大脑中动脉阻塞大鼠的神经保护效应[J].中华老年心脑血管病杂志,2008,10:856-858
[13]吕佳宁,韩锁成,郭景春,等.延迟亚低温治疗对大鼠永久性大脑中动脉阻塞后凋亡相关蛋白表达的影响[J].中风与神经疾病杂志,2008,25:67-70
[14]Song F,Guo C,Geng Y,et al.Therapeutic time window and regulation of autophagy by mild hypothermia after intracerebral hemorrhage in rats[J].Brain Res,2018,1690:12-22
[15]Guo C,Geng Y,Song F,et al.Mild hypothermia protects rat neuronal injury after intracerebral hemorrhage via attenuating endoplasmic reticulum response induced neuron apoptosis[J].Neurosci Lett,2016,635:17-23
[16]宋斐斐,郭岑,鲍海峰,等.亚低温对脑出血后炎症反应的作用及机制研究[J].中国临床神经科学,2018,26:197-205
[17]Liu H,Ou S,Xiao X,et al.Diabetes Worsens IschemiaReperfusion Brain Injury in Rats Through GSK-3beta[J].Am JMed Sci,2015,350:204-211
[18]Srinivasan K,Viswanad B,Asrat L,et al.Combination of highfat diet-fed and low-dose streptozotocin-treated rat:a model for type 2 diabetes and pharmacological screening[J].Pharmacol Res,2005,52:313-320
[19]Xie Z,Hao H,Tong C,et al.Human umbilical cord-derived mesenchymal stem cells elicit macrophages into an antiinflammatory phenotype to alleviate insulin resistance in type 2diabetic rats[J].Stem Cells,2016,34:627-639
[20]Longa EZ,Weinstein PR,Carlson S,et al.Reversible middle cerebral artery occlusion without craniectomy in rats[J].Stroke,1989,20:84-91
[21]Uluc K,Miranpuri A,Kujoth GC,et al.Focal cerebral ischemia model by endovascular suture occlusion of the middle cerebral artery in the rat[J].J Vis Exp,2011 Feb 5;(48)
[22]霍亚静,丁晶,卓丰昊,等.自发性高血压大鼠永久性脑缺血模型建立的研究[J].中国临床神经科学,2015,23:241-248
[23]Maclellan CL,Girgis J,Colbourne F.Delayed onset of prolonged hypothermia improves outcome after intracerebral hemorrhage in rats[J].J Cereb Blood Flow Metab,2004,24:432-440
[24]Garcia JH,Wagner S,Liu KF,et al.Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats.Statistical validation[J].Stroke,1995,26:627-634
[25]Gong C,Boulis N,Qian J,et al.Intracerebral hemorrhageinduced neuronal death[J].Neurosurgery,2001,48:875-882
[26]耿阳,郭岑,霍亚静,等.4-苯基丁酸对大鼠脑出血模型脑水肿的作用及机制探讨[J].中国临床神经科学,2016,24:376-382
[27]高素玲,刘国荣,陈瑞英,等.影响糖尿病并发急性脑梗死近期预后的相关因素分析[J].中华老年心脑血管病杂志,2015,17:1206-1207
[28]Liu X,Wen S,Zhao S,et al.Mild Therapeutic Hypothermia Protects the Brain from Ischemia/Reperfusion Injury through Upregulation of i ASPP[J].Aging Dis,2018,9:401-411
[29]Nakka VP,Gusain A,Mehta SL,et al.Molecular mechanisms of apoptosis in cerebral ischemia:multiple neuroprotective opportunities[J].Mol Neurobiol,2008,37:7-38
[30]Zeng G,Ding W,Li Y,et al.Morroniside protects against cerebral ischemia/reperfusion injury by inhibiting neuron apoptosis and MMP2/9 expression[J].Exp Ther Med,2018,16:2229-2234
[31]Chen XJ,Zhang JG,Wu L.Plumbagin inhibits neuronal apoptosis,intimal hyperplasia and also suppresses TNF-alpha/NF-kappaB pathway induced inflammation and matrix metalloproteinase-2/9 expression in rat cerebral ischemia[J].Saudi J Biol Sci,2018,25:1033-1039
[32]Roach DM,Fitridge RA,Laws PE,et al.Up-regulation of MMP-2 and MMP-9 leads to degradation of type IV collagen during skeletal muscle reperfusion injury;protection by the MMPinhibitor,doxycycline[J].Eur J Vasc Endovasc Surg,2002,23:260-269
[33]Qi Z,Liang J,Pan R,et al.Zinc contributes to acute cerebral ischemia-induced blood-brain barrier disruption[J].Neurobiol Dis,2016,95:12-21
[34]Kumari R,Willing LB,Patel SD,et al.Increased cerebral matrix metalloprotease-9 activity is associated with compromised recovery in the diabetic db/db mouse following a stroke[J].JNeurochem,2011,119:1029-1040