丝裂原活化蛋白激酶信号转导通路在脑缺血再灌注损伤中的作用及机制

Role and mechanism of mitogen activated protein kinase signal transduction pathway in cerebral ischemia / reperfusion injury

目的探讨丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)信号转导通路在脑缺血再灌注损伤(cerebral ischemia/reperfusion,I/R)中的作用及机制。方法将大鼠随机分为6组:未使用p38MAPK抑制剂SB203580的假手术组(Sham组)、使用抑制剂的假手术组(Sham/SB组)、未使用抑制剂再灌注24 h组(I/R 24 h组)、未使用抑制剂再灌注48 h组(I/R 48 h组)、使用抑制剂再灌注24 h组(SB 24 h组)和使用抑制剂再灌注48 h组(SB 48 h组)。采用大脑中动脉线栓法(middle cerebral artery occlusion,MCAO)复制大鼠局灶性脑缺血再灌注模型,使用抑制剂的各组于造模前30 min经腹腔注射SB203580(5 mg/kg,溶于5 mg/ml DMSO)。采用免疫荧光双标法检测大鼠大脑中动脉(middle cerebral artery,MCA)和脑微血管(microvascular,Mic.V)周围IgG的渗出;Western blot和RT-PCR法检测大鼠脑组织中p38、一氧化氮合酶(inducible nitric oxide synthase,iNOS)、基质金属蛋白酶-9(matrix metalloproteinase,MMP-9)、胶原Ⅳ型(collagenⅣ)蛋白和基因表达的变化。结果随着再灌注时间的延长,大鼠脑血管内IgG渗出量增加(P<0.01);经p38MAPK抑制剂预处理后,IgG的渗出程度有所减轻。脑缺血再灌注后,大鼠脑组织中p38、iNOS、MMP-9蛋白的表达水平和基因mRNA的转录水平均明显升高(P<0.05),经p38MAPK抑制剂预处理后,能显著抑制p38、iNos、MMP-9蛋白的表达水平和基因mRNA的转录水平(P<0.05);collagenⅣ蛋白的表达水平和基因mRNA的转录水平随着缺血时间的延长而逐渐下降(P<0.05),经p38MAPK抑制剂预处理后,其下降程度有所减轻(P<0.05)。结论 MAPK通路中的关键蛋白p38、iNOS、MMP-9在脑缺血时表达显著增加,其过度表达直接导致血脑屏障的破坏,抑制其表达,从而遏制对血脑屏障的破坏,有望为I/R的治疗提供新的途径。

Objective To investigate the role and mechanism of mitogen activated protein kinase （MAPK） signal trans-duction pathway in cerebral ischemia/repeffusion injury （I/R）. Methods The rats were randomly divided into six groups, 8 for each, i. e. Sham, Sham/SB, I/R 24 h, I/R 48 h, SB 24 h and SB 48 h groups. The rats in Sham and Sham/SB groups received sham operation, while I/R model rats were used in the other four groups. The rat model of I/R was established by middle cerebral artery occlusion （MCAO）. The rats in Sham /SB, SB 24 h and SB 4-8 h groups were injected i. p. with p38MAPK inhibitor SB203580 （5 mg/kg, dissolved in 5 mg/ml DMSO） 30 min before sham op-eration or I/R. The local exudations of IgG around middle cerebral artery （MCA） and mierovessel （Mie. V） were deter-mined by IFA, while the expressions of p38, inducible nitric oxide synthase （iNOS）, matrix metalloproteinase-9 （MMP-9） and collagen IV proteins and genes by Western blot and RT-PCR respectively. The above-mentioned indexes in I/R 24 h and SB 24 h groups were determined 24 h, while those in I/R 48 h and SB 48 h groups 48 h, after I/R. Results The exudation of IgG increased with the increasing time after I/R （P 〈 0. 01 ）. After pretreatment with p38MAPK inhibitor,the exudation of IgG decreased. Both the mRNA transcription and protein expression levels of p38, iNOS and MMP-9 in brain tissues of rats after I/R increased significantly. However, the pretreatment with p38MAPK inhibitor inhibited the transcription and expression significantly （P 〈 O. 05）. Both the mRNA transcription and protein expression levels of colla-gen Ⅳ decreased gradually with the increasing time after I/R （P 〈 0. 05 ）. After pretreatment with p38MAPK inhibitor, the decrease was relieved （P 〈 0. 05）. Conclusion The expressions of key proteins such as p38, iNOS and MMP-9 in MAPK pathway increased significantly after I/R, while the overexpressions leaded to the break-down of blood brain bar-rier （BBB） directly. The inhibition of break-down of BBB by inhibiting the expressions of above-mentioned proteins may provide a novel route fro therapy of I/R.