作　　者： ; ; ; ; (覃兰惠）;

机构地区： 广西医科大学第一附属医院

出　　处： 《中风与神经疾病杂志》 2015年第5期388-393,共6页

摘　　要： 目的探讨在脂多糖(LPS)诱导血脑屏障(BBB)破坏中紧密连接Occludin、ZO-1表达的变化以及c-Jun氨基末端激酶(JNK)信号通路与基质金属蛋白酶-9(MMP-9)的作用机制。方法利用MTT法检测不同浓度LPS以及SP600125(JNK信号通路抑制剂)对人脑微血管内皮细胞(h CMEC/D3)活力的影响。Western blot法检测不同浓度LPS对Occludin、ZO-1蛋白表达的影响。用SP600125阻断JNK信号通路,Western blot法检测LPS诱导的JNK磷酸化水平、Occludin和ZO-1蛋白表达的变化,qRT-PCR法检测Occludin、ZO-1及MMP-9 mRNA表达的变化。用SB-3CT特异性抑制MMP-9活性,Western blot法检测LPS诱导的Occludin蛋白表达的变化。结果 LPS、SP600125浓度分别在10μg/ml(0.19±0.02)和0.22μg/ml(0.18±0.01)以下时对h CMEC/D3细胞活力无明显影响(P>0.05)。不同浓度LPS(1μg/ml、10μg/ml、50μg/ml、100μg/ml)处理h CMEC/D3后Occludin和ZO-1蛋白的表达量均下降。LPS处理h CMEC/D3后JNK磷酸化水平增高,而抑制JNK磷酸化可以明显上调LPS诱导的Occludin蛋白(0.87±0.03、P<0.001)及其mRNA(1.00±0.09、P<0.05)的表达,但对ZO-1蛋白(1.14±0.06、P>0.05)及其mRNA(0.81±0.03、P>0.05)的表达无明显影响。另外,抑制MMP-9的活性可以明显上调Occludin蛋白的表达(0.53±0.06、P<0.01),阻断JNK信号通路可显著降低LPS诱导的MMP-9的高表达(0.77±0.08、P<0.001)。结论 LPS可下调紧密连接Occludin、ZO-1蛋白及其mRNA的表达导致血脑屏障破坏;LPS可通过激活JNK信号通路,调节Occludin蛋白及其mRNA的表达,而对ZO-1蛋白及其mRNA表达的影响可能通过其他信号通路实现;LPS诱导Occludin蛋白的下调可能与MMP-9的高表达有关,JNK信号通路可能参与了此过程的调控。 Objective To evaluate the role of JNK signaling pathway and matrix-metalloproteinase-9 （ MMP-9 ） in lipopolysaccharide（LPS）-induced damage of blood-brain barrier. Methods Treated human cerebral microvascular endo- thelial cells （ hCMEC/D3 ） with different concentrations of LPS and JNK inhibitor SP600125, hCMEC/D3 viability was test- ed by MTF assay. Then hCMEC/D3 cells were pretrcated with SP600125 or SB-3CT （MMP-9 inhibitor） ,the expression of TJ proteins （Occludin and ZO-1 ） and phosphorylation of JNK were determined by Western blot, the mRNA level of TJ and MMP-9 were measured with quantitative Real Timc-PCR （qRT-PCR）. Results Treated with different concentrations of LPS（ 1 μg/ml,10 μg/ml,50 μg/ml、100 μg/ml）could reduce the protein expression of Oceludin and ZO-1. Neither LPS nor SP600125 under the concentrations of 10μg/ml （ 0.19 ± 0.02, P 〉 0.05 ） and 0.22 μg/ml （ 0.18 ± 0.01, P 〉 0.05 ） re- spectively affected cell viability as determined by the MTF assay. Treatment with LPS decreased protein and mRNA levels of Occludin（ 0.51 ± 0.01 ,P 〈 0. 001 in protein levels ;0.88 -＋ 0.02, P 〈 0.05 in mRNA levels） and ZO-1 （ 1.24 ± 0. 09, P 〈 0.05 in protein levels ,0.87 ± 0.01, P 〈 0.01 in mRNA levels, and also increased the phosphorylation of JNK （ 0.76 ± 0. 10 ,P 〈0.01 ）. These effects on Occludin（0.87 ± 0.03 ,P 〈 0. 001 in protein levels, 1.00 ± 0.09 ,P 〈 0.05 in mRNA levels） were attenuated by pretreatment with SP600125 ,hut not on LPS-induced decrease on ZO-1 protein（ 1.14 ± 0.06, P 〉 0.05 ）and mRNA（ 0.81 ± 0. 03 ,P 〉 0.05 ）levels. Furthermore, LPS could induce the overexpression of MMP-9 （0.77 ± 0.08 ,P 〈 0. 001 ） which was attenuated by pretreatment with 5P600125, while pretreatment with SB-3CT could upregulate the dysregulation expression of Occludin （0.53 ± 0. 06,P 〈 0. 01 ） induced by LPS. Conclusion These results show that LPS induces dysregulation of TJ proteins and mRNA in hCMEC/D3

关 键 词： 脂多糖 血脑屏障 紧密连接 信号通路 基质金属蛋白酶

领　　域： [生物学]