12/15-LOX花生四烯酸代谢通路在脑梗死继发性脑损伤中的作用及氧化苦参碱治疗对脑梗死12/15-LOX通路的影响
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摘要
第一部分12/15-LOX在脑梗死继发性脑损伤中的作用
目的：缺血性脑血管病是引起人类死亡及残疾的主要原因之一。脑梗死后继发性脑组织损伤是病情加重及影响预后的重要原因。由于神经组织的易损难复特性，局部损伤将导致永久性的神经结构的丢失及功能障碍。虽然大量的动物实验证实，许多针对不同作用机制的神经保护药物可以明显减轻缺血后脑组织损伤，但迄今为止，无一证据证明神经保护治疗的临床有效性。随着对脑损伤后病理机制的研究进展，科学家发现，脑缺血后炎症反应在继发性脑组织损伤病理发展过程中起非常重要的作用。脑梗死急性期，外周循环炎症细胞向脑组织迁移聚集、小胶质细胞迅速激活增殖、缺血脑组织中炎症相关信号通路过度激活、炎症介质持续高度表达，引起血脑屏障功能破坏、导致神经元凋亡坏死。因此，炎症损伤机制为临床治疗脑梗死提供新的治疗策略。
花生四烯酸（Arachidonic acid）代谢通路为炎性介质及过氧化产物的主要来源之一。促炎细胞因子白介素1β（interleukin-1β）及肿瘤坏死因子α （tumor necrosis factor alpha, TNFα）,通过NF-κB及AP-2等信号通路调节包括花生四烯酸系列代谢酶在内的多种因子的表达。缺血脑组织中，解离的花生四烯酸及炎性代谢产物影响信号通路的转导、基因转录、神经元活性、细胞凋亡、及其他生物学过程。12/15-脂氧合酶（12/15-lipoxygenase,12/15-LOX）通路是花生四烯酸的主要代谢途径之一，催化花生四烯酸形成白三烯及二十碳烯酸类活性代谢产物。前期的研究发现，脑梗死后24~48h，缺血脑组织中12/15-LOX高度表达。12/15-LOX基因敲除小鼠，血脑屏障的渗透性明显减轻降低、脑损伤减轻，提示12/15-LOX在缺血脑损伤过程中起重要作用。本研究通过应用12/15-LOX抑制剂baicalein研究花生四烯酸代谢酶12/15-LOX在缺血后脑损伤中的作用。
方法：成年健康雄性Sprague-Dawley大鼠，应用改良线栓法建立大鼠右侧大脑中动脉闭塞模型（middle cerebral artery occlusion, MCAO）。实验动物随机分为假手术组（Sham）、假手术+安慰剂组（Sham+DMSO）、MCAO组、MCAO+安慰剂组（vehicle, MCAO+DMSO）及baicalein组（MCAO+baicalein）。脑梗死后24h，进行神经功能评分，采用2，3，5—氯化三苯基四氮唑染色（2,3,5-Triphenyltetrazolium chloride, TTC）测定脑梗死体积，干湿重法测定脑组织含水量，western blot、逆转录多聚酶链反应（Reverse transcription–polymerase chain reaction, RT-PCR）测定缺血脑组织12/15-LOX蛋白及基因表达。同时行为学评分后，4%多聚甲醛固定脑组织，采用石蜡包埋、免疫组织化学测定缺血侧脑组织中12/15-LOX阳性细胞数。Baicalein溶于DMSO，MCAO模型成功建立后，即刻颈静脉注射30mg/kg baicalein，安慰剂组给予同等体积的DMSO治疗。
结果：
1脑缺血24h后，缺血脑组织中12/15-LOX蛋白及基因表达明显升高（MCAO vs. Sham, P <0.05），12/15-LOX阳性细胞数明显增多（MCAOvs. Sham, P <0.05）。
212/15-LOX抑制剂baicalein明显降低缺血脑组织中12/15-LOX蛋白及基因的表达（Bai vs. MCAO, P <0.05），减少12/15-LOX阳性细胞数（Baivs. MCAO, P <0.05）。
3脑缺血24h后，MCAO组、安慰剂组、baicalein组大鼠均出现不同程度的左侧肢体偏瘫。Baicalein抑制12/15-LOX表达后神经功能评分明显改善。
4脑缺血24h后，MCAO组缺血侧脑组织含水量明显升高（MCAOvs. Sham,85.94%±1.17%vs.77.5%±2.28%）。Baicalein抑制12/15-LOX治疗明显减轻脑组织含水量（Bai,82.93%±0.67%, P <0.05）。
5脑缺血24h后，与MCAO组相比，baicalein抑制12/15-LOX治疗组明显减小脑梗死体积（Bai vs. MCAO,43.10%±2.81%vs.52.51%±2.10%; P <0.05）。
6安慰剂治疗组与MCAO相比，神经功能评分、脑组织含水量及脑梗死体积无明显改变（P>0.05）。
结论：Baicalein抑制12/15-LOX表达后，明显改善脑梗死后神经功能损伤、降低脑水肿及梗死体积，有效保护缺血脑组织。结果提示12/15-LOX在缺血脑组织中的过度表达促进缺血性脑损伤。
第二部分12/15-LOX对脑梗死p38MAPK及cPLA2表达的影响
目的：研究表明12/15-LOX通路通过激活p38丝裂原激活的蛋白激酶（p38mitogen-activated protein kinase, p38MAPK）在病理过程中起重要作用。胞质型磷脂酶A2（cytosolic phospholipaseA2, cPLA2）催化膜磷脂
释放游离的花生四烯酸，为花生四烯酸代谢的限速步骤，并且cPLA2为催化花生四烯酸生成的主要蛋白酶之一。研究发现12/15-LOX促进cPLA2的磷酸化，进而激活cPLA2，诱导花生四烯酸的大量产生。并且cPLA2的活化与转录因子p38MAPK的活性密切相关。据此推测，12/15-LOX在缺血脑组织中的过度表达可能与调节促炎转录因子p38MAPK及花生四烯酸代谢通路主要蛋白酶cPLA2的活性有关。本研究应用baicalein抑制缺血脑组织中12/15-LOX的表达，观察12/15-LOX是影响缺血脑组织中其他花生四烯酸代谢相关蛋白酶的活性，是否影响缺血脑组织中炎症介质的表达。
方法：成年健康雄性Sprague-Dawley大鼠，应用改良线栓法建立MCAO动物模型。将baicalein溶于DMSO，MCAO模型成功建立后，即刻颈静脉注射30mg/kg baicalein。实验动物随机分为假手术组（Sham）、假手术组+安慰剂组（Sham+DMSO）、MCAO组、MCAO+安慰剂组（vehicle, MCAO+DMSO）及baicalein组（MCAO+baicalein）。脑梗死后24h，采用石蜡切片免疫组织化学染色测定缺血脑组织中磷酸化p38MAPK （phosphorylated p38mitogen-activated protein kinase,p-p38MAPK）、cPLA2阳性细胞数。Western blot及RT-PCR测定缺血脑组织p-p38MAPK、cPLA2蛋白的表达及cPLA2基因表达。
结果：
1脑缺血24h后，缺血脑组织中p-p38MAPK、cPLA2蛋白及cPLA2基因表达明显升高（MCAO vs. Sham, P <0.05），p-p38MAPK及cPLA2阳性细胞数明显增多（MCAO vs. Sham, P <0.05）。
2脑缺血24h后， baicalein明显降低缺血脑组织中p-p38MAPK蛋白的表达（Bai vs. MCAO,1.95±0.191.21±0.09; P <0.05），减少p-p38MAPK阳性细胞数（Bai vs. MCAO, P <0.05）。
3脑缺血24h后， baicalein明显降低缺血脑组织中cPLA2蛋白及基因的表达（P <0.05），减少缺血脑组织中cPLA2阳性细胞数（P <0.05）。
结论：Baicalein治疗明显抑制缺血脑组织p38MAPK的活性，降低cPLA2的表达，提示12/15-LOX为缺血脑损伤过程中重要的促炎因子，在炎症损伤过程中起重要作用。
第三部分氧化苦参碱对脑梗死缺血脑组织中12/15-LOX花生四烯酸代谢通路的影响
目的：氧化苦参碱（oxymatrine）为中国传统中药苦参的主要成分。我们前期的研究结果证实，氧化苦参碱治疗后，MCAO大鼠于术后24h及72h神经功能缺损明显减轻、局部脑梗死体积明显减小，同时缺血脑组织中NF-κB、TLR4、TLR2等炎症因子的表达明显降低，证明氧化苦参碱对缺血脑组织具有明显的神经保护作用。因此本研究通过系统应用氧化苦参碱治疗，检测12/15-LOX花生四烯酸代谢通路是否参与脑梗死后氧化苦参碱的神经保护机制。从而验证，12/15-LOX花生四烯酸代谢通路为脑梗死继发性脑损伤的新治疗靶点。
方法：成年健康雄性Sprague-Dawley大鼠，应用改良线栓法建立MCAO动物模型。MCAO模型成功建立后，即刻腹腔注射120mg/kg氧化苦参碱。实验动物随机分为假手术组（Sham）、MCAO组（MCAO+vehicle）及氧化苦参碱组（MCAO+oxymatrine）。脑梗死后24h，进行神经功能评分，采用干湿重法测定脑组织含水量， TTC测定脑梗死体积，免疫组织化学染色测定缺血脑组织中12/15-LOX、p-p38MAPK、cPLA2阳性细胞数，western blot和RT-PCR分别测定缺血脑组织12/15-LOX、p-p38MAPK、cPLA2蛋白的表达及12/15-LOX、cPLA2基因表达。
结果：
1与MCAO组相比，缺血后24h，氧化苦参碱治疗明显减小脑梗死体积（MCAO vs. OMT,53.54±1.7%vs.42.37±2.1%）。
2与MCAO组相比，缺血后24h，氧化苦参碱治疗明显减轻缺血侧脑组织含水量（MCAO vs. OMT,86%±1%vs.82%±1%; P <0.05），但不影响损伤对侧组织含水量。
3与MCAO组相比，缺血后24h，氧化苦参碱治疗明显降低缺血侧脑组织12/15-LOX阳性细胞数（P <0.05）、蛋白及基因的表达（MCAO vs.OMT,1.62±0.21vs.1.2±0.05; P <0.05）。
4与MCAO组相比，缺血后24h，氧化苦参碱治疗明显降低缺血侧脑组织p-p38MAPK阳性细胞数（P <0.05）及蛋白的表达（MCAO vs. OMT,0.71±0.06vs.0.56±0.03; P <0.05）。
5与MCAO组相比，缺血后24h,氧化苦参碱治疗明显降低缺血侧脑组织cPLA2阳性细胞数（P <0.05）、蛋白和基因的表达（MCAO vs. OMT,2.85±0.73vs.1.18±0.32; P <0.05）。
结论：氧化苦参碱治疗明显降低缺血侧脑组织中12/15-LOX通路花生四烯酸代谢酶的表达激活，减轻缺血后脑组织损伤。12/15-LOX花生四烯酸代谢途径为脑梗死后继发性脑损伤的治疗提供新的作用靶点。
Part I The role of12/15-LOX in cerebral ischemia
Objective: Cerebral ischemia is one of the leading causes of death andadult disability wordwide. The secondary injury after cerebral ischemiaaggravated the progress of stroke and influenced the recovery of strokepatients. Despite the significant efficiency of neuroprotective reagents in alarge growing of experimental stroke models, none of these drugs was provedavailable in stroke patients. With the advanced understanding ofpathophysiology of cerebral ischemia in recent years, it has been found thatinflammation plays an important role in the progress of ischemic injury afterstroke. The production of inflammatory mediators induced by the recruitmentof blood-derived leukocytes and the activation of resident inmmune cells inischemic brain will damage the blood-brain barrier and promoted neuronalnecrosis after stroke. Because of the susceptibility of neurons to variousstimuli, local brain injury will cause permanent neuronal dysfunction afterstroke. Thereafter, inflammaroty response provides a new therapeutic strategyfor stroke therapy. Pro-inflammatory cytokines including interleukin-1β (IL-1β) and tumornecrosis factor α (TNFα) regulates the expression of arachidonicacid cascades enzymes through NF-κB and AP-2signaling pathways. In thebrain, arachidonic acid and its metabolites influence gene transcription, signaltransduction, neuronal activity, aptosis and other process.12/15-LOX is one ofthe major enzymes that catalyze the metabolism of arachidonic acid. Ourprevious studies have demonstrated that the expression of12/15-LOX wasup-regulated after stroke. Deficiency of12/15-LOX improved blood-brainbarrier permeability and functional recovery after stroke. In this study, weinvestigated the potential effect of12/15-LOX in cerebral ischemia by using the specific inhibitor, bacalain.
Methods: Male and healthy Sprague-Dawley rats were subjected tomodified permanent middle cerebral artery occlusion (MCAO). Baicalein wasadministered at a dose of30mg/ml by jugular vein injection immediately afterMCAO. Rats were randomly assigned to four groups: Sham operated group(Sham), MCAO, vehicle (MCAO+DMSO), and baicalein group (MCAO+baicalein). At24h after ischemia neurological deficit was evaluated, brainwater content was measured by wet-dry method, infarct size were analyzedwith2,3,5-triphenyltetrazolium chloride (TTC) staining.Immunohistochemistry, reverse transcription–polymerase chain reaction(RT-PCR) and western blot were used to analyze the expression of12/15-LOX.
Results:
1The expression of12/15-LOX protein and gene as well as the12/15-LOX positive cells was significantly increased at24h after cerebralischemia.
2The inhibitor of12/15-LOX, baicalein, efficiently suppressed theexpression of12/15-LOX in ischemic brain.
3Rats in MCAO group, vehicle group and baicalein group all showedsome degree of neurological deficit at24h after stroke. The12/15-LOXinhibitor, baicalein, significantly improved functional recovery after stroke.
4The brain water content of ischemic hemisphere was increased at24hafter stroke. Compared with vehicle treatment, baicalein significantlydecreased brain water content after stroke.
5Compared with vehicle treatment, baicalein significantly decreasedinfarct size at24h after stroke.
6Vehicle did not have effect on behavior test, brain edema and infarctsize.
Conclusion: Inhibition of12/15-LOX significantly improvedneurological deficit, decreased brain water content and infarct size at24hafter stroke.12/15-LOX contributes to the brain injury after stroke.
Part II The influence of12/15-LOX in the expression p38MAPK andcPLA2in cerebral ischemia
Objective: It has been demonstrated that activation of p38mitogen-activated protein kinase (p38MAPK) is one of the most importantmechanism for12/15-LOX pathway. Release of AA induced by cytosolicphospholipase A2(cPLA2) is the rate-limiting step in the12/15-LOX pathway.Several reports have shown that12/15-LOX derivatives from AA can directlyactivate p38MAPK and cPLA2, induce more release of AA. The activatedp38MAPK is also linked to activation and phosphorylation of cPLA2and AArelease. However, little is known about the relationship between12/15-LOXexpression and the activity of p38MAPK and cPLA2in ischemic stroke. Inthis study, we investigated whether inhibition of12/15-LOX influences thesefactors’ expression after stroke.
Methods: Male and healthy Sprague-Dawley rats were subjected tomodified permanent middle cerebral artery occlusion (MCAO). Baicalein wasadministered at a dose of30mg/ml by jugular vein injection immediately afterMCAO. Rats were randomly assigned to four groups: Sham operated group(Sham), MCAO, vehicle (MCAO+DMSO), and baicalein group (MCAO+baicalein). At24h after ischemia the phosphorylated p38mitogen-activatedprotein kinase (p-p38MAPK) and cytosolic phospholipase A2(cPLA2)positive cells were detected using immunohistochemistry. And the expressionof p-p38MAPK and cPLA2protein was measured using western blot. RT-PCRwas used to analyze the gene expression of cPLA2.
Results:
1The number of p-p38MAPK and cPLA2positive cells was significantlyincreased after cerebral ischemia, and the expression of p-p38MAPK andcPLA2showed increase in western blot and RT-PCR.
2Baicalein significantly decreased the expression of p-p38MAPK and p-p38MAPK positive cells in ischemic brain at24h after MCAO.
3Baicalein significantly decreased the expression of cPLA2and cPLA2positive cells in ischemic brain at24h after MCAO.
Conclusion: Bacalein protected the brain against cerebral ischemicdamage by inhibiting the activation of p38MAPK and down-regulating theexpression of cPLA2. Inhibition of AA metabolism contributes to theneuroprotection induced by baicalein after stroke.
Part III The role of oxymatrine in the expression of12/15-LOX pathwayin cerebral ischemia
Objective: Oxymatrine is an alkaloid extracted from the root of Sophoraflavescent Arr (Kushen). Our previous studies have demonstrated thatoxymatrine protects the brain against ischemic injury by inhibitinginflammatory responses after stroke. In this study, we investigated whetheroxymatrine treatment has influence in the expression and activity ofarachidonic acid cascade enzymes after stroke, and demonstrated thatinhibition of arachidonic acid metabolism is an efficient therapeutic target forstroke.
Methods: Male and healthy Sprague-Dawley rats were subjected topermanent MCAO. Oxymatrine was administered at a dose of120mg/ml byperitoneal injection immediately after MCAO. Rats were randomly assignedto three groups: Sham operated group (Sham), MCAO (MCAO+vehicle), andoxymatrine group (MCAO+oxymatrine). At24h after ischemia, neurologicaldeficit was evaluated, brain water content was measured by wet-dry method,and infarct size was analyzed with TTC staining. The expression of12/15-LOX, p-p38MAPK, and cPLA2was respectively detected usingimmunohistochemistry, western blot and RT-PCR at24h after ischemia.
Results:
1Oxymatrine significantly decreased infarct size at24h after stroke.
2Oxymatrine significantly decreased ipsilesional but not contralesionalbrain water content at24h afte stroke.
3Compared with MCAO group, the number of12/15-LOX positive cellsas well as the protein and gene expression of12/15-LOX in ischemic brainwas significantly decreased by oxymatrine at24h after stroke.
4Compared with MCAO group, the number of p-p38MAPK positivecells as well as the protein expression in ischemic brain was significantlydecreased by oxymatrine at24h after stroke.
5Compared with MCAO group, the number of cPLA2positive cells aswell as the protein and gene expression of cPLA2in ischemic brain wassignificantly decreased by oxymatrine at24h after stroke.
Conclusion: Oxymatrine protected the brain against cerebral ischemicdamage by inhibiting the activation and expression of arachidonic acidcascade enzymes after stroke. Inhibition of arachidonic acid metabolism istherapeutic target for anti-inflammatory threatment after stroke.
目的：缺血性脑血管病是引起人类死亡及残疾的主要原因之一。脑梗死后继发性脑组织损伤是病情加重及影响预后的重要原因。由于神经组织的易损难复特性，局部损伤将导致永久性的神经结构的丢失及功能障碍。虽然大量的动物实验证实，许多针对不同作用机制的神经保护药物可以明显减轻缺血后脑组织损伤，但迄今为止，无一证据证明神经保护治疗的临床有效性。随着对脑损伤后病理机制的研究进展，科学家发现，脑缺血后炎症反应在继发性脑组织损伤病理发展过程中起非常重要的作用。脑梗死急性期，外周循环炎症细胞向脑组织迁移聚集、小胶质细胞迅速激活增殖、缺血脑组织中炎症相关信号通路过度激活、炎症介质持续高度表达，引起血脑屏障功能破坏、导致神经元凋亡坏死。因此，炎症损伤机制为临床治疗脑梗死提供新的治疗策略。
花生四烯酸（Arachidonic acid）代谢通路为炎性介质及过氧化产物的主要来源之一。促炎细胞因子白介素1β（interleukin-1β）及肿瘤坏死因子α （tumor necrosis factor alpha, TNFα）,通过NF-κB及AP-2等信号通路调节包括花生四烯酸系列代谢酶在内的多种因子的表达。缺血脑组织中，解离的花生四烯酸及炎性代谢产物影响信号通路的转导、基因转录、神经元活性、细胞凋亡、及其他生物学过程。12/15-脂氧合酶（12/15-lipoxygenase,12/15-LOX）通路是花生四烯酸的主要代谢途径之一，催化花生四烯酸形成白三烯及二十碳烯酸类活性代谢产物。前期的研究发现，脑梗死后24~48h，缺血脑组织中12/15-LOX高度表达。12/15-LOX基因敲除小鼠，血脑屏障的渗透性明显减轻降低、脑损伤减轻，提示12/15-LOX在缺血脑损伤过程中起重要作用。本研究通过应用12/15-LOX抑制剂baicalein研究花生四烯酸代谢酶12/15-LOX在缺血后脑损伤中的作用。
方法：成年健康雄性Sprague-Dawley大鼠，应用改良线栓法建立大鼠右侧大脑中动脉闭塞模型（middle cerebral artery occlusion, MCAO）。实验动物随机分为假手术组（Sham）、假手术+安慰剂组（Sham+DMSO）、MCAO组、MCAO+安慰剂组（vehicle, MCAO+DMSO）及baicalein组（MCAO+baicalein）。脑梗死后24h，进行神经功能评分，采用2，3，5—氯化三苯基四氮唑染色（2,3,5-Triphenyltetrazolium chloride, TTC）测定脑梗死体积，干湿重法测定脑组织含水量，western blot、逆转录多聚酶链反应（Reverse transcription–polymerase chain reaction, RT-PCR）测定缺血脑组织12/15-LOX蛋白及基因表达。同时行为学评分后，4%多聚甲醛固定脑组织，采用石蜡包埋、免疫组织化学测定缺血侧脑组织中12/15-LOX阳性细胞数。Baicalein溶于DMSO，MCAO模型成功建立后，即刻颈静脉注射30mg/kg baicalein，安慰剂组给予同等体积的DMSO治疗。
结果：
1脑缺血24h后，缺血脑组织中12/15-LOX蛋白及基因表达明显升高（MCAO vs. Sham, P <0.05），12/15-LOX阳性细胞数明显增多（MCAOvs. Sham, P <0.05）。
212/15-LOX抑制剂baicalein明显降低缺血脑组织中12/15-LOX蛋白及基因的表达（Bai vs. MCAO, P <0.05），减少12/15-LOX阳性细胞数（Baivs. MCAO, P <0.05）。
3脑缺血24h后，MCAO组、安慰剂组、baicalein组大鼠均出现不同程度的左侧肢体偏瘫。Baicalein抑制12/15-LOX表达后神经功能评分明显改善。
4脑缺血24h后，MCAO组缺血侧脑组织含水量明显升高（MCAOvs. Sham,85.94%±1.17%vs.77.5%±2.28%）。Baicalein抑制12/15-LOX治疗明显减轻脑组织含水量（Bai,82.93%±0.67%, P <0.05）。
5脑缺血24h后，与MCAO组相比，baicalein抑制12/15-LOX治疗组明显减小脑梗死体积（Bai vs. MCAO,43.10%±2.81%vs.52.51%±2.10%; P <0.05）。
6安慰剂治疗组与MCAO相比，神经功能评分、脑组织含水量及脑梗死体积无明显改变（P>0.05）。
结论：Baicalein抑制12/15-LOX表达后，明显改善脑梗死后神经功能损伤、降低脑水肿及梗死体积，有效保护缺血脑组织。结果提示12/15-LOX在缺血脑组织中的过度表达促进缺血性脑损伤。
第二部分12/15-LOX对脑梗死p38MAPK及cPLA2表达的影响
目的：研究表明12/15-LOX通路通过激活p38丝裂原激活的蛋白激酶（p38mitogen-activated protein kinase, p38MAPK）在病理过程中起重要作用。胞质型磷脂酶A2（cytosolic phospholipaseA2, cPLA2）催化膜磷脂
释放游离的花生四烯酸，为花生四烯酸代谢的限速步骤，并且cPLA2为催化花生四烯酸生成的主要蛋白酶之一。研究发现12/15-LOX促进cPLA2的磷酸化，进而激活cPLA2，诱导花生四烯酸的大量产生。并且cPLA2的活化与转录因子p38MAPK的活性密切相关。据此推测，12/15-LOX在缺血脑组织中的过度表达可能与调节促炎转录因子p38MAPK及花生四烯酸代谢通路主要蛋白酶cPLA2的活性有关。本研究应用baicalein抑制缺血脑组织中12/15-LOX的表达，观察12/15-LOX是影响缺血脑组织中其他花生四烯酸代谢相关蛋白酶的活性，是否影响缺血脑组织中炎症介质的表达。
方法：成年健康雄性Sprague-Dawley大鼠，应用改良线栓法建立MCAO动物模型。将baicalein溶于DMSO，MCAO模型成功建立后，即刻颈静脉注射30mg/kg baicalein。实验动物随机分为假手术组（Sham）、假手术组+安慰剂组（Sham+DMSO）、MCAO组、MCAO+安慰剂组（vehicle, MCAO+DMSO）及baicalein组（MCAO+baicalein）。脑梗死后24h，采用石蜡切片免疫组织化学染色测定缺血脑组织中磷酸化p38MAPK （phosphorylated p38mitogen-activated protein kinase,p-p38MAPK）、cPLA2阳性细胞数。Western blot及RT-PCR测定缺血脑组织p-p38MAPK、cPLA2蛋白的表达及cPLA2基因表达。
结果：
1脑缺血24h后，缺血脑组织中p-p38MAPK、cPLA2蛋白及cPLA2基因表达明显升高（MCAO vs. Sham, P <0.05），p-p38MAPK及cPLA2阳性细胞数明显增多（MCAO vs. Sham, P <0.05）。
2脑缺血24h后， baicalein明显降低缺血脑组织中p-p38MAPK蛋白的表达（Bai vs. MCAO,1.95±0.191.21±0.09; P <0.05），减少p-p38MAPK阳性细胞数（Bai vs. MCAO, P <0.05）。
3脑缺血24h后， baicalein明显降低缺血脑组织中cPLA2蛋白及基因的表达（P <0.05），减少缺血脑组织中cPLA2阳性细胞数（P <0.05）。
结论：Baicalein治疗明显抑制缺血脑组织p38MAPK的活性，降低cPLA2的表达，提示12/15-LOX为缺血脑损伤过程中重要的促炎因子，在炎症损伤过程中起重要作用。
第三部分氧化苦参碱对脑梗死缺血脑组织中12/15-LOX花生四烯酸代谢通路的影响
目的：氧化苦参碱（oxymatrine）为中国传统中药苦参的主要成分。我们前期的研究结果证实，氧化苦参碱治疗后，MCAO大鼠于术后24h及72h神经功能缺损明显减轻、局部脑梗死体积明显减小，同时缺血脑组织中NF-κB、TLR4、TLR2等炎症因子的表达明显降低，证明氧化苦参碱对缺血脑组织具有明显的神经保护作用。因此本研究通过系统应用氧化苦参碱治疗，检测12/15-LOX花生四烯酸代谢通路是否参与脑梗死后氧化苦参碱的神经保护机制。从而验证，12/15-LOX花生四烯酸代谢通路为脑梗死继发性脑损伤的新治疗靶点。
方法：成年健康雄性Sprague-Dawley大鼠，应用改良线栓法建立MCAO动物模型。MCAO模型成功建立后，即刻腹腔注射120mg/kg氧化苦参碱。实验动物随机分为假手术组（Sham）、MCAO组（MCAO+vehicle）及氧化苦参碱组（MCAO+oxymatrine）。脑梗死后24h，进行神经功能评分，采用干湿重法测定脑组织含水量， TTC测定脑梗死体积，免疫组织化学染色测定缺血脑组织中12/15-LOX、p-p38MAPK、cPLA2阳性细胞数，western blot和RT-PCR分别测定缺血脑组织12/15-LOX、p-p38MAPK、cPLA2蛋白的表达及12/15-LOX、cPLA2基因表达。
结果：
1与MCAO组相比，缺血后24h，氧化苦参碱治疗明显减小脑梗死体积（MCAO vs. OMT,53.54±1.7%vs.42.37±2.1%）。
2与MCAO组相比，缺血后24h，氧化苦参碱治疗明显减轻缺血侧脑组织含水量（MCAO vs. OMT,86%±1%vs.82%±1%; P <0.05），但不影响损伤对侧组织含水量。
3与MCAO组相比，缺血后24h，氧化苦参碱治疗明显降低缺血侧脑组织12/15-LOX阳性细胞数（P <0.05）、蛋白及基因的表达（MCAO vs.OMT,1.62±0.21vs.1.2±0.05; P <0.05）。
4与MCAO组相比，缺血后24h，氧化苦参碱治疗明显降低缺血侧脑组织p-p38MAPK阳性细胞数（P <0.05）及蛋白的表达（MCAO vs. OMT,0.71±0.06vs.0.56±0.03; P <0.05）。
5与MCAO组相比，缺血后24h,氧化苦参碱治疗明显降低缺血侧脑组织cPLA2阳性细胞数（P <0.05）、蛋白和基因的表达（MCAO vs. OMT,2.85±0.73vs.1.18±0.32; P <0.05）。
结论：氧化苦参碱治疗明显降低缺血侧脑组织中12/15-LOX通路花生四烯酸代谢酶的表达激活，减轻缺血后脑组织损伤。12/15-LOX花生四烯酸代谢途径为脑梗死后继发性脑损伤的治疗提供新的作用靶点。
Part I The role of12/15-LOX in cerebral ischemia
Objective: Cerebral ischemia is one of the leading causes of death andadult disability wordwide. The secondary injury after cerebral ischemiaaggravated the progress of stroke and influenced the recovery of strokepatients. Despite the significant efficiency of neuroprotective reagents in alarge growing of experimental stroke models, none of these drugs was provedavailable in stroke patients. With the advanced understanding ofpathophysiology of cerebral ischemia in recent years, it has been found thatinflammation plays an important role in the progress of ischemic injury afterstroke. The production of inflammatory mediators induced by the recruitmentof blood-derived leukocytes and the activation of resident inmmune cells inischemic brain will damage the blood-brain barrier and promoted neuronalnecrosis after stroke. Because of the susceptibility of neurons to variousstimuli, local brain injury will cause permanent neuronal dysfunction afterstroke. Thereafter, inflammaroty response provides a new therapeutic strategyfor stroke therapy. Pro-inflammatory cytokines including interleukin-1β (IL-1β) and tumornecrosis factor α (TNFα) regulates the expression of arachidonicacid cascades enzymes through NF-κB and AP-2signaling pathways. In thebrain, arachidonic acid and its metabolites influence gene transcription, signaltransduction, neuronal activity, aptosis and other process.12/15-LOX is one ofthe major enzymes that catalyze the metabolism of arachidonic acid. Ourprevious studies have demonstrated that the expression of12/15-LOX wasup-regulated after stroke. Deficiency of12/15-LOX improved blood-brainbarrier permeability and functional recovery after stroke. In this study, weinvestigated the potential effect of12/15-LOX in cerebral ischemia by using the specific inhibitor, bacalain.
Methods: Male and healthy Sprague-Dawley rats were subjected tomodified permanent middle cerebral artery occlusion (MCAO). Baicalein wasadministered at a dose of30mg/ml by jugular vein injection immediately afterMCAO. Rats were randomly assigned to four groups: Sham operated group(Sham), MCAO, vehicle (MCAO+DMSO), and baicalein group (MCAO+baicalein). At24h after ischemia neurological deficit was evaluated, brainwater content was measured by wet-dry method, infarct size were analyzedwith2,3,5-triphenyltetrazolium chloride (TTC) staining.Immunohistochemistry, reverse transcription–polymerase chain reaction(RT-PCR) and western blot were used to analyze the expression of12/15-LOX.
Results:
1The expression of12/15-LOX protein and gene as well as the12/15-LOX positive cells was significantly increased at24h after cerebralischemia.
2The inhibitor of12/15-LOX, baicalein, efficiently suppressed theexpression of12/15-LOX in ischemic brain.
3Rats in MCAO group, vehicle group and baicalein group all showedsome degree of neurological deficit at24h after stroke. The12/15-LOXinhibitor, baicalein, significantly improved functional recovery after stroke.
4The brain water content of ischemic hemisphere was increased at24hafter stroke. Compared with vehicle treatment, baicalein significantlydecreased brain water content after stroke.
5Compared with vehicle treatment, baicalein significantly decreasedinfarct size at24h after stroke.
6Vehicle did not have effect on behavior test, brain edema and infarctsize.
Conclusion: Inhibition of12/15-LOX significantly improvedneurological deficit, decreased brain water content and infarct size at24hafter stroke.12/15-LOX contributes to the brain injury after stroke.
Part II The influence of12/15-LOX in the expression p38MAPK andcPLA2in cerebral ischemia
Objective: It has been demonstrated that activation of p38mitogen-activated protein kinase (p38MAPK) is one of the most importantmechanism for12/15-LOX pathway. Release of AA induced by cytosolicphospholipase A2(cPLA2) is the rate-limiting step in the12/15-LOX pathway.Several reports have shown that12/15-LOX derivatives from AA can directlyactivate p38MAPK and cPLA2, induce more release of AA. The activatedp38MAPK is also linked to activation and phosphorylation of cPLA2and AArelease. However, little is known about the relationship between12/15-LOXexpression and the activity of p38MAPK and cPLA2in ischemic stroke. Inthis study, we investigated whether inhibition of12/15-LOX influences thesefactors’ expression after stroke.
Methods: Male and healthy Sprague-Dawley rats were subjected tomodified permanent middle cerebral artery occlusion (MCAO). Baicalein wasadministered at a dose of30mg/ml by jugular vein injection immediately afterMCAO. Rats were randomly assigned to four groups: Sham operated group(Sham), MCAO, vehicle (MCAO+DMSO), and baicalein group (MCAO+baicalein). At24h after ischemia the phosphorylated p38mitogen-activatedprotein kinase (p-p38MAPK) and cytosolic phospholipase A2(cPLA2)positive cells were detected using immunohistochemistry. And the expressionof p-p38MAPK and cPLA2protein was measured using western blot. RT-PCRwas used to analyze the gene expression of cPLA2.
Results:
1The number of p-p38MAPK and cPLA2positive cells was significantlyincreased after cerebral ischemia, and the expression of p-p38MAPK andcPLA2showed increase in western blot and RT-PCR.
2Baicalein significantly decreased the expression of p-p38MAPK and p-p38MAPK positive cells in ischemic brain at24h after MCAO.
3Baicalein significantly decreased the expression of cPLA2and cPLA2positive cells in ischemic brain at24h after MCAO.
Conclusion: Bacalein protected the brain against cerebral ischemicdamage by inhibiting the activation of p38MAPK and down-regulating theexpression of cPLA2. Inhibition of AA metabolism contributes to theneuroprotection induced by baicalein after stroke.
Part III The role of oxymatrine in the expression of12/15-LOX pathwayin cerebral ischemia
Objective: Oxymatrine is an alkaloid extracted from the root of Sophoraflavescent Arr (Kushen). Our previous studies have demonstrated thatoxymatrine protects the brain against ischemic injury by inhibitinginflammatory responses after stroke. In this study, we investigated whetheroxymatrine treatment has influence in the expression and activity ofarachidonic acid cascade enzymes after stroke, and demonstrated thatinhibition of arachidonic acid metabolism is an efficient therapeutic target forstroke.
Methods: Male and healthy Sprague-Dawley rats were subjected topermanent MCAO. Oxymatrine was administered at a dose of120mg/ml byperitoneal injection immediately after MCAO. Rats were randomly assignedto three groups: Sham operated group (Sham), MCAO (MCAO+vehicle), andoxymatrine group (MCAO+oxymatrine). At24h after ischemia, neurologicaldeficit was evaluated, brain water content was measured by wet-dry method,and infarct size was analyzed with TTC staining. The expression of12/15-LOX, p-p38MAPK, and cPLA2was respectively detected usingimmunohistochemistry, western blot and RT-PCR at24h after ischemia.
Results:
1Oxymatrine significantly decreased infarct size at24h after stroke.
2Oxymatrine significantly decreased ipsilesional but not contralesionalbrain water content at24h afte stroke.
3Compared with MCAO group, the number of12/15-LOX positive cellsas well as the protein and gene expression of12/15-LOX in ischemic brainwas significantly decreased by oxymatrine at24h after stroke.
4Compared with MCAO group, the number of p-p38MAPK positivecells as well as the protein expression in ischemic brain was significantlydecreased by oxymatrine at24h after stroke.
5Compared with MCAO group, the number of cPLA2positive cells aswell as the protein and gene expression of cPLA2in ischemic brain wassignificantly decreased by oxymatrine at24h after stroke.
Conclusion: Oxymatrine protected the brain against cerebral ischemicdamage by inhibiting the activation and expression of arachidonic acidcascade enzymes after stroke. Inhibition of arachidonic acid metabolism istherapeutic target for anti-inflammatory threatment after stroke.
引文
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