咪唑克生对体外血脑屏障炎症模型通透性的影响

Effect of idazoxan on permeability of inflammatory blood-brain barrier model in vitro

目的:观察咪唑克生(idazoxan,IDA)对体外血脑屏障(blood-brain barrier,BBB)炎症模型的通透性、紧密连接蛋白ZO-1表达和分布及基质金属蛋白酶-9(matrix metalloproteinases-9,MMP-9)和MMP-9抑制物——金属蛋白酶组织抑制物-1(tissue inhibitor of metalloproteinase-1,TIMP-1)表达的影响。方法:采用培养7 d的小鼠脑微血管内皮细胞株b.End3建立体外BBB模型,采用TNF-α(10 nmol/L)处理24 h诱导BBB炎症模型,并对BBB炎症模型分别采用IDA 50、100、200μmol/L预处理6 h以观察IDA对BBB炎症模型的作用。通过检测异硫氰酸荧光标记的葡聚糖通过率来确立模型的通透性,采用Western blot法检测紧密连接蛋白ZO-1的表达,通过免疫荧光法观察ZO-1的分布情况,并通过RT-PCR法检测MMP-9/TIMP-1的表达。结果:培养7 d的b.End3细胞汇合成稳定的单层连接,有较好的屏障功能,而采用TNF-α处理24 h后诱导的BBB炎症模型的通透性明显升高,紧密连接蛋白ZO-1在膜上的表达明显减少、不连续,Western blot法显示ZO-1蛋白表达水平明显下降,MMP-9的表达明显升高;而采用IDA 50、100、200μmol/L预处理6 h能明显降低其通透性,增加ZO-1蛋白的表达和改善ZO-1的分布异常,降低MMP-9的表达,其中200μmol/L IDA作用最明显,差异有统计学意义(P<0.01)。结论:IDA能够直接作用于脑血管内皮细胞,降低TNF-α诱导的体外BBB炎症模型MMP-9的表达,增加紧密连接蛋白ZO-1的表达、修复紧密连接ZO-1的异常分布,从而改善其异常增高的通透性。

AIM： To study the effect of idazoxan on the permeability of inflammatory blood-brain barrier （ BBB） model in vitro and the expression of tight junction protein ZO-1.METHODS：In vitro BBB model was established by murine brain endothelial cell line bEnd.3 incubated for 7 d.The cells were treated with TNF-α（10 nmol/L） for addi-tional 24 h to establish the inflammatory BBB model, which was pretreated with IDA at doses of 50, 100 and 200μmol/L, respectively.The permeability was measured using fluorescein isothiocyanate-conjugated dextran （FD-40, MW 40,000）, the expression of ZO-1 was detected by Western blot analysis, the distribution of ZO-1 was observed by immunofluores-cence, and the mRNA expression of MMP-9/TIMP-1 was measured by RT-PCR.RESULTS：After incubated for 7 d, b. End3 cells converged to be confluent monolayer with low permeability.The inflammatory BBB model induced by TNF-αtreatment displayed much higher permeability with decreased expression of tight junction protein ZO-1, destroyed distribu-tion of ZO-1 and increased mRNA expression of MMP-9.When pretreated with IDA, the permeability was greatly de-creased, the expression of ZO-1 was greatly increased, the abnormal distribution of ZO-1 was greatly ameliorated and the mRNA expression of MMP-9 was obviously reduced.The effect was most significant in IDA （ 200 μmol/L ）-pretreated group （P〈0.01）.CONCLUSION：IDA directly acts on brain endothelial cells to reduce the expression of MMP-9, in-crease the expression of tight junction protein ZO-1 and ameliorate the destroyed distribution of ZO-1 in the inflammatory BBB, thus reversing the abnormally elevated permeability in a inflammatory BBB model in vitro induced by TNF-α.