化痰通络方对急性脑梗死大鼠rt-PA溶栓后MMP-9转录活化途径中AP-1与NF-κB基因表达的影响

Effect of Huatan Tongluo Formulae on Expression of AP-1 and NF-κB on Gene Transcription Activation Pathway of MMP-9 in Rat Cortex Accepted Thrombolysis Pherapy after Acute Cerebral Infarction

目的:观察化痰通络方对急性脑梗死大鼠重组组织纤溶酶原激活剂(rt-PA)溶栓后基质金属蛋白酶-9(MMP-9)及其基因转录活化途径中激活蛋白-1(AP-1)与核因子κB(NF-κB)基因表达的影响。方法:选择240只健康SD大鼠,随机分为假手术组、模型组、rt-PA组、化痰通络方联合rt-PA组(简称中药组,中药组分低、中、高剂量组),采用自身栓子法制备大鼠大脑中动脉栓塞模型(MCAO),rt-PA组与中药组分别给予尾静脉注入rt-PA(5.67 mg·kg-1)及联合3种不同剂量化痰通络方中药(3.6,7.2,14.4 g·kg-1)干预,每日2次。于相应时间点采用Western blot检测各组大鼠大脑皮质梗死区组织中MMP-9蛋白的表达,采用逆转录聚合酶链反应(Real Time PCR)方法检测MMP-9基因转录活化途径中AP-1与NF-κB基因的表达。结果:Western blot结果显示:模型组内各个时相大鼠MMP-9含量较假手术组均显著增加(P<0.05);加入中药干预后,MMP-9蛋白表达明显减少,其中以中、高剂量效果显著(P<0.05)。Real Time PCR结果显示:与假手术组比较,模型组AP-1与NF-κB基因相对表达量均明显增加(P<0.05);与模型组相比,加入中药干预后,2种基因均有所降低,以中药中、高剂量作用明显,同一时相内与rt-PA组相比,在6 h和24 h时,中药高剂量作用优于rt-PA,在72 h和7 d时,中药中剂量作用优于rt-PA。结论:化痰通络方可通过调控MMP-9上游基因转录途径中AP-1与NF-κB基因的表达,部分抑制其活化程度,进而保护血脑屏障完整性,从而更好的防治急性脑梗死溶栓后转化出血的发生与发展。

Objective：To observe the effect of Huatan Tongluo formulae on express of motrix metallo proteinase-9 （MMP-9） and activator protein-1 （AP-1） and nuclear factor kappa B （NF-κB） on gene transcription activation pathway in rat cortex accepted thrombolysis therapy after the acute cerebral infarction.Method：Two handred and forty healthy male SD rats were randomly divided into sham group,model group,recombinant tissue plasminogen activator （rt-PA） group,rt-PA-Huatan Tongluo group （Chinese medicine group for short）,Chinese medicine group were divided high dose group,middle dose group and low dose group,drug respectively as：3.6,7.2,14.4 g ·kg-1）,each group was divided into four time phases：6,24,72 h,7 days.In the corresponding time points prepares the middle cerebral artery occulation （MCAO） rat model,the rats in rtPA group were injected rt-PA （5.67 mg ·kg-1）,and those of Chinese medicine group were given rt-PA and three different doses of Huatan Tongluo formulae,2 times a day.Then respectively observe the protein expression of MMP-9 by Western blot observe the gene expression of AP-1 and NF-κB on gene transcription activation pathway by RT PCR.Result：The result of Western blot showed：compared with sham group,the expression of MMP-9 increased significantly （P ＜0.05）,compared with model group in the same time phases,the expression of MMP-9 decreased in Chinese medicine group,especially in high dose group and middle dose group.RT PCR showed that compared with sham group,the expression of AP-1 and NF-κB increased significantly in model group （P ＜ 0.05）,the expression of two genes in Chinese medicine group,especially in high dose group and middle dose group.Compared with rt-PA group,the expression of AP-1 and NF-κB decreased in high dose group at 6,24 h and in middle dose group at 72 h and 3 d.Conclusion：Huatan Tongluo can inhibit the activation of MMP-9,protect the blood brain barrier by regulating AP-1 and NF-κB on gene transcription activation pathway of MMP-9.