阿司匹林预处理对大鼠局灶性脑缺血/再灌注后IL-6、SOCS-3mRNA表达的影响
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摘要
目的：研究阿司匹林(Aspirin, ASA)预处理后,大鼠局灶性脑缺血／再灌注(cerebral ischemia/reperfusion,CI/RP)后缺血脑组织白细胞介素6(inter leukin-6,IL-6)、细胞因子信号转导抑制因子-3 (supressor of cytokine signaling-3,SOCS-3) mRNA动态表达变化,从而阐明阿司匹林脑保护作用的可能机制。方法：将雄性健康SD大鼠95只随机分为假手术组、缺血对照组、小剂量ASA组(10mg.kg-1)、大剂量ASA组(150mg.kg-1)。采用改良线栓法制作大鼠局灶性脑缺血／再灌注模型。每组大鼠造模前7天给予各自不同剂量ASA或溶媒灌胃,1次/d。术前1h再灌胃1次。术后分别在6h、12h、24 h、72h、7d时间点处死大鼠,立刻开颅取脑并检测相应指标,每个时间点5只大鼠。另外每组还有5只大鼠于术后24h处死,用于TTC染色计算脑梗死体积。在各时间点进行神经功能评分。取缺血侧大脑半球脑组织分别测定脑组织含水量、IL-6及SOCS-3mRNA的含量、并进行病理染色。结果：1大鼠MCA-CI/RP术后,对照组大鼠出现不同程度神经功能障碍、梗死灶周围脑组织含水量增加,两者均在24h达高峰,TTC染色可以清晰显示梗死病灶范围、HE染色显示病灶侧脑组织有缺血的表现。2脑缺血侧IL-6、SOCS-3mRNA的动态变化：缺血再灌注6h后IL-6表达较假手术组IL-6表达增多(P<0.05),24h后达高峰,以后逐渐下降,第7天仍维持稍高水平。RT-PCR测定SOCS-3 mRNA显示相同规律。3 ASA对上述各项指标的影响：(1)CI/RP术后,两个实验组大鼠神经功能评分较对照组明显升高,神经功能得以不同程度的改善,同时梗死病灶周围脑组织含水量减轻,小剂量的ASA组在术后12h与对照组比较即出现显著的差异(P<0.05),至术后7d两组与对照组比较均有显著差异(P<0.05)；(2)小、大剂量ASA组术后24h梗死病灶体积与对照组比较分别减少54.48%,30.90%。(3)HE染色ASA10mg.kg-组病灶侧脑组织神经元细胞缺血坏死程度轻、细胞间隙部分增宽；ASA150mg.kg-1组病灶侧脑组织神经元细胞缺血坏死程度较重、细胞间隙较显著增宽；均好于对照组。(4)CI/RP术后,两个实验组病灶侧脑组织IL-6含量较对照组明显下降,在术后12h到3d与对照组相比较均出现显著的差异(P<0.05)。(5)CI/RP术后,两个实验组病灶侧脑组织SOCS-3mRNA含量较对照组明显上升,在术后12h到3d与对照组相比较均出现显著的差异(P<0.05)。结论：(1)改良的大鼠局灶性脑缺血再灌注模型是一种制备简单、成功率较高、有利于药物预处理研究的模型。(2：脑缺血再灌注后脑损伤主要表现在神经功能评分下降、脑含水量升高、脑梗死及脑组织HE染色异常,而IL-6、SOCS-3mRNA在CI/RP术后升高,与上述指标存在时效关系,提示炎症反应及脑水肿的形成和发展是造成神经功能缺损的主要原因之一,而SOCS-3mRNA可能起到了内源性神经保护剂的作用。(3)ASA药物预处理可以提高MCA-CI/RP大鼠神经功能缺损评分,减轻脑含水量；减少脑梗死体积；改善脑组织HE染色的病理改变。其可能机制与抑制炎性细胞因子IL-6的产生和上调SOCS-mRNA表达有关。(4)ASA的神经保护作用并非随剂量的增加而增加,小剂量ASA作用优于大剂量的ASA。
Objective:After aspirin (ASA) pretreatment,the changes dyna-mically of the ischemic brain tissue IL-6 (IL-6) and cytokine signal transduction inhibitor-3 (SOCS-3) mRNA expression are studied after rat focal cerebral ischemia/reperfusion (CI/RP), so as to clarify the role of aspirin, the possible mechanism of brain protection. Methods:95 male healthy SD rats were randomly divided into sham-operated group, ischemic control group, low-dose ASA group (10mg.kg-1) and high-dose ASA group (150mg.kg-1). Modified suture method is used rat focal cerebral ischemia/ reperfusion model. Each rat for 7 days before modeling their different doses of ASA or vehicle gavage,1/d. Administered 1h before surgery and then 1. After at 6h,12h,24 h,72h,7d time rats were killed immediately and tested the corresponding cranial indicators of brain,5 rats at each time point. Another 5 rats in each group also were killed 24h after the operation, for TTC staining and infarct volume. At each time point neurological score. Taking ischemic cerebral hemisphere is measured in brain tissue water content in brain tissue, IL-6 and SOCS-3mRNA content, and the pathological staining.
Results:1A rat MCA-CI/RP after the control group, neurological deficits of different degrees, the water content in cerebral infarction increased, both in the 24h peak, TTC staining can clearly show the scope of infarct lesion, HE staining lesion side shows the performance of ischemic brain tissue.2 ischemic side IL-6, SOCS-3mRNA dynamic changes:6h after ischemia and reperfusion IL-6 expression compared with sham group the expression of IL increased (P<0.05),24h after the peak, then gradually decreased,7 days remains slightly high. RT-PCR determination of SOCS-3 mRNA shows the same regularity.3 ASA on the above indexes:(1) CI/RP post-operative, two experimental Neurological score is significantly higher than the control group, nerve function can be improved to varying degrees, while the surrounding brain tissue with infarct reduce the amount of water, small doses of ASA group compared with the control group after 12h a significant difference appears to 7d after the two groups compared with the control group was significantly different(P<0.05); (2) Small, high-dose ASA group after 24h infarct lesion volume decreased by 54.48%,30.90%, compared with the control group.(3)It was less that extent of ischemic necrosis of neuronal cells of HE staining ASA10mg.kg-1 group of lesions in brain tissue, intercellular part of the widened. It was severe that extent of ischemic necrosis of neuronal cells of ASA150mg.kg-1 group of lesions of neurons in brain tissue, cell gap widened more dramatically; were better than the control group. (4) CI /RP post-operative, two experimental group's side of the brain lesions IL-6 levels decreased significantly compared with the control group,12h after the operation to the 3d compared with the control group there were significant differences(P<0.05). (5) CI/RP post-operative, two experimental groups side of the brain lesions SOCS-3mRNA levels significantly increased compared with the control group,12h after the operation to the 3d compared with the control group there were significant differences(P<0.05). Conclusions:(1) improved cerebral ischemia-reperfusion model is a simple preparation, high success rate, favorable pharmacological preconditioning of the model. (2) brain injury after cerebral ischemia mainly in neurological function score decreased brain water content increased, cerebral infarction and brain abnormalities HE staining, and IL-6, SOCS-3mRNA in the CI/RP patients increased relationship with the indexes in aging, suggesting that inflammation and brain edema formation and development of neurological deficits caused by one of the main, while SOCS-3mRNA may play an endogenous neuroprotective agent. (3) ASA drugs can reduce the MCA-CI/RP pretreatment neurological deficits, reduce brain water content; reduced cerebral infarct volume; improve HE staining of brain tissue pathological changes. The possible mechanism and inhibition of inflammatory cytokines IL-6 production and increases SOCS-mRNA related. (4)With the increase of ASA's dose the neuroprotective effect is not increase; low dose of ASA is superior to high-dose ASA group
Objective:After aspirin (ASA) pretreatment,the changes dyna-mically of the ischemic brain tissue IL-6 (IL-6) and cytokine signal transduction inhibitor-3 (SOCS-3) mRNA expression are studied after rat focal cerebral ischemia/reperfusion (CI/RP), so as to clarify the role of aspirin, the possible mechanism of brain protection. Methods:95 male healthy SD rats were randomly divided into sham-operated group, ischemic control group, low-dose ASA group (10mg.kg-1) and high-dose ASA group (150mg.kg-1). Modified suture method is used rat focal cerebral ischemia/ reperfusion model. Each rat for 7 days before modeling their different doses of ASA or vehicle gavage,1/d. Administered 1h before surgery and then 1. After at 6h,12h,24 h,72h,7d time rats were killed immediately and tested the corresponding cranial indicators of brain,5 rats at each time point. Another 5 rats in each group also were killed 24h after the operation, for TTC staining and infarct volume. At each time point neurological score. Taking ischemic cerebral hemisphere is measured in brain tissue water content in brain tissue, IL-6 and SOCS-3mRNA content, and the pathological staining.
Results:1A rat MCA-CI/RP after the control group, neurological deficits of different degrees, the water content in cerebral infarction increased, both in the 24h peak, TTC staining can clearly show the scope of infarct lesion, HE staining lesion side shows the performance of ischemic brain tissue.2 ischemic side IL-6, SOCS-3mRNA dynamic changes:6h after ischemia and reperfusion IL-6 expression compared with sham group the expression of IL increased (P<0.05),24h after the peak, then gradually decreased,7 days remains slightly high. RT-PCR determination of SOCS-3 mRNA shows the same regularity.3 ASA on the above indexes:(1) CI/RP post-operative, two experimental Neurological score is significantly higher than the control group, nerve function can be improved to varying degrees, while the surrounding brain tissue with infarct reduce the amount of water, small doses of ASA group compared with the control group after 12h a significant difference appears to 7d after the two groups compared with the control group was significantly different(P<0.05); (2) Small, high-dose ASA group after 24h infarct lesion volume decreased by 54.48%,30.90%, compared with the control group.(3)It was less that extent of ischemic necrosis of neuronal cells of HE staining ASA10mg.kg-1 group of lesions in brain tissue, intercellular part of the widened. It was severe that extent of ischemic necrosis of neuronal cells of ASA150mg.kg-1 group of lesions of neurons in brain tissue, cell gap widened more dramatically; were better than the control group. (4) CI /RP post-operative, two experimental group's side of the brain lesions IL-6 levels decreased significantly compared with the control group,12h after the operation to the 3d compared with the control group there were significant differences(P<0.05). (5) CI/RP post-operative, two experimental groups side of the brain lesions SOCS-3mRNA levels significantly increased compared with the control group,12h after the operation to the 3d compared with the control group there were significant differences(P<0.05). Conclusions:(1) improved cerebral ischemia-reperfusion model is a simple preparation, high success rate, favorable pharmacological preconditioning of the model. (2) brain injury after cerebral ischemia mainly in neurological function score decreased brain water content increased, cerebral infarction and brain abnormalities HE staining, and IL-6, SOCS-3mRNA in the CI/RP patients increased relationship with the indexes in aging, suggesting that inflammation and brain edema formation and development of neurological deficits caused by one of the main, while SOCS-3mRNA may play an endogenous neuroprotective agent. (3) ASA drugs can reduce the MCA-CI/RP pretreatment neurological deficits, reduce brain water content; reduced cerebral infarct volume; improve HE staining of brain tissue pathological changes. The possible mechanism and inhibition of inflammatory cytokines IL-6 production and increases SOCS-mRNA related. (4)With the increase of ASA's dose the neuroprotective effect is not increase; low dose of ASA is superior to high-dose ASA group
引文
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1全国第四届脑血管病学术会议.脑卒中患者临床神经功能缺损程度评分标准(1995)[J].中华神经科杂志,1996,29：381
2张进,兰卫光.白细胞介素-6与脑缺血[J].亚太传统医药,2007,6：35-36
3 Buttini M,Appel K,Sauter A,et al. Expression of TNF a after focal cerebr-al ischemia in rats. Neuroscience,1996,71(1):1～4
4 Tarkowski E,Rosengren L,Blomstrand C,et al. Early intrathecal produc-tion of IL-6 predicts the size of brain lesion in stroke.Stroke,1995,26(2) 1393～1396.
5 Legos JJ,Whit more RG,Erhardt JA,et al. Quantitative changes in int-erleukin in proteins following focal stroke in the rat[J].Neurosci Lett, 2000;282 (3):189-92.
6向赞,董大翠,刘庆莹.脑缺血与白细胞介素-6的变化[J].江汉大学学报(自然科学版),2005,23(2)：83-87.
7 Tarkowski E,Rosengren L,Blomstmd C,et al.Early intrathecal production Interlekin-6 predicts the size of brain lesion in stroke[J]. Stroke,1996,27:1 553-1557
8 Starr R,Willson TA,Viney EM,et al.A family of cytokine-inducible inhibitors of signaling. Nature,1997,387:917～921.
9 Naka T,Narazaki M,Hirata M, et al. Structure and function of a new STAT-induced STAT inhibitor. Nature,1997,387:924-929.
10 Endo TA,Masuhara M,Yokouchi M,et al. A new protein containing an SH2 domain that inhibits JAK kinases. Nature,1997,387:921-924.
11 Polizzotto MN, Bartlett PF, Turnley AM, Expression of "suppressor of cyt-okine signaling"(SOCS) genes in the developing and adult mouse central nervous system. J Comp Neurol,2000,423:348-358.
12 Rosell DR,Akama KT,Nacher J.et al.Differential expression of suppressors of cytokine signaling-1,-2, and in the rat hippocampus after seizure; Implications for neuro modulation by gp130 cytokines[J].Neuro science,2003,122:349-358.
13 Tang Y,LU A, Aronow Bl,et al.Genomic responses of the brain toischemic stroke, intraceerbral haemorrhage, kainate seizures,hypoglycemia, and hypoxia[J]. J Neurosci,2002,15(12):1937-1952.
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16 Kelly JF, Elias CF, Lee CE,et al. Ciliary neurotrophic factor and leptin ind-uce distinct patterns of immediate early gene expression in the brain.Diab-etes,2004,53(4):911-920.
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18 El Kasmi KC, Holst J, Coffm M, et al.General nature of the STAT3 activat-ed anti-inflammatory response. J Immunol,2006,177(11):7880-7888.
19 Campbell IL Cytokine-mediated inflammation, tumorigenesis, and disease-associated JAK/STAT/SOCS signaling circuits in the CNS. Brain Res Rev, 48(2),166-177.
20 Chong ZZ, Kang JQ, Maiese K, et al. Hematopoietic factor erythropoietin fosters neuroprotection through novel signal traneduction cascades. J Cereb Blood Flow Metab,2002,22(5);503-514
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1全国第四届脑血管病学术会议.脑卒中患者临床神经功能缺损程度评分标准(1995)[J].中华神经科杂志,1996,29：381
2张进,兰卫光.白细胞介素-6与脑缺血[J].亚太传统医药,2007,6：35-36
3 Buttini M,Appel K,Sauter A,et al. Expression of TNF a after focal cerebr-al ischemia in rats. Neuroscience,1996,71(1):1～4
4 Tarkowski E,Rosengren L,Blomstrand C,et al. Early intrathecal produc-tion of IL-6 predicts the size of brain lesion in stroke.Stroke,1995,26(2) 1393～1396.
5 Legos JJ,Whit more RG,Erhardt JA,et al. Quantitative changes in int-erleukin in proteins following focal stroke in the rat[J].Neurosci Lett, 2000;282 (3):189-92.
6向赞,董大翠,刘庆莹.脑缺血与白细胞介素-6的变化[J].江汉大学学报(自然科学版),2005,23(2)：83-87.
7 Tarkowski E,Rosengren L,Blomstmd C,et al.Early intrathecal production Interlekin-6 predicts the size of brain lesion in stroke[J]. Stroke,1996,27:1 553-1557
8 Starr R,Willson TA,Viney EM,et al.A family of cytokine-inducible inhibitors of signaling. Nature,1997,387:917～921.
9 Naka T,Narazaki M,Hirata M, et al. Structure and function of a new STAT-induced STAT inhibitor. Nature,1997,387:924-929.
10 Endo TA,Masuhara M,Yokouchi M,et al. A new protein containing an SH2 domain that inhibits JAK kinases. Nature,1997,387:921-924.
11 Polizzotto MN, Bartlett PF, Turnley AM, Expression of "suppressor of cyt-okine signaling"(SOCS) genes in the developing and adult mouse central nervous system. J Comp Neurol,2000,423:348-358.
12 Rosell DR,Akama KT,Nacher J.et al.Differential expression of suppressors of cytokine signaling-1,-2, and in the rat hippocampus after seizure; Implications for neuro modulation by gp130 cytokines[J].Neuro science,2003,122:349-358.
13 Tang Y,LU A, Aronow Bl,et al.Genomic responses of the brain toischemic stroke, intraceerbral haemorrhage, kainate seizures,hypoglycemia, and hypoxia[J]. J Neurosci,2002,15(12):1937-1952.
14 Muramatsu H,Welsh FA, Kariku K,et al.Cerebral preconditioning using cortical application of hypertonic salt solutions:upregulation of mRNAs encoding inhibitors of inflammation[J].Brain Res,2006,1097(1):31-38.
15石献忠,赵靖,刘猛等.细胞因子信号转导抑制蛋白SOCS-3在成年大鼠脑内的基础表达[J].神经解剖学杂志,2004,20(3)301-304.
16 Kelly JF, Elias CF, Lee CE,et al. Ciliary neurotrophic factor and leptin ind-uce distinct patterns of immediate early gene expression in the brain.Diab-etes,2004,53(4):911-920.
17 Raghavendra Rao VL,Bowen KK,Dhodda VK,et al.Gene expression analys-is of spontaneously hypertensive rat cerebral cortex following transient focal cerebral ischemia.J Neurochem,2002,83(5):1072-1086.
18 El Kasmi KC, Holst J, Coffm M, et al.General nature of the STAT3 activat-ed anti-inflammatory response. J Immunol,2006,177(11):7880-7888.
19 Campbell IL Cytokine-mediated inflammation, tumorigenesis, and disease-associated JAK/STAT/SOCS signaling circuits in the CNS. Brain Res Rev, 48(2),166-177.
20 Chong ZZ, Kang JQ, Maiese K, et al. Hematopoietic factor erythropoietin fosters neuroprotection through novel signal traneduction cascades. J Cereb Blood Flow Metab,2002,22(5);503-514
21 Seki Y, Inoue H, Nagate N, et al. SOCS-3 regulates onset and maintenance of T(H)2-mediated allergic responses. Nat Med,2003,9(8):1047-1054.
22 Kinjyo I, Inoue H,Hemana S,et al.Lose of SOCS3 in T helper cells resulted in reduced immune responses and hyperproduction of intedeukin 10 and transforming growth facfor-beta 1. J Exp Med,2006,203(4):1021-1031.
23 Arumugam TV, Granger DN, Mattson MP. Stroke and Tcells.Neuromolec-ular Med,2005,7(3):229-242.
24 Cacmichael ST. Gene expression changes after focal stroke,traumatic brain and spinal cord injuries. Curt Opin Neural,2001,6(6):699-704
25黄帆,扬静,仲飞等等.脑梗死患者外周血单个核细胞SOCS-3动态变化及临床意义[J].中国神经医学杂志,2008；7(1)：46-50.
26 Xuan YT,Guo Y,Han H.Free in PMC:an essential role of the JAK-STAT Pathway in ischemic preconditioning [J].Proc Natl Acad Sci,2001;98(16) :9050-4.
27徐皓亮,冯亦璞.细胞因子、趋化细胞因子与脑缺血后炎症损伤[J].中国药理学通报1999,3(15)：1-7
28 Xuan YT,Guo Y,Han H. Free in PMC An essential role of the JAK-STAT pathway in ischemic preconditioning[J]. Proc Natl Acad Sci USA,2001, 98(16):9050-9055