目的：探究清热活血组分治疗急性脑梗死火毒证的协同增效作用机制。方法采用腹腔注射角叉菜胶以及线栓法致大脑中动脉闭塞( MCAO)的方法建立急性脑梗死火毒证模型,SD大鼠随机分为正常组( N)、假手术组( S)、脑缺血再灌注模型组( I)、苦碟子注射液治疗组( A)、血栓通注射液治疗组(B)、苦碟子注射液(1.8 mL/kg)+血栓通注射液(20 mg/kg)治疗组(C)、苦碟子注射液(1.8 mL/kg)+血栓通注射液(80 mg/kg)治疗组(D)、苦碟子注射液(7.2 mL/kg)+血栓通注射液(20 mg/kg)治疗组( E),共8组。通过TTC染色观测各组大鼠脑梗死面积比变化；Western Blot方法检测梗死侧大脑皮层中NF-κB p65及IKKβ的蛋白表达情况；采用Real-TimePCR方法检测梗死侧大脑皮层中NF-κB p65及IKKβ mRNA的表达情况。结果①各治疗组较模型组大鼠的脑梗死面积均有所减小(P<0.05),其中清热活血组脑梗死面积比较模型组有显著改善(P<0.01)。3个清热活血组较苦碟子组和血栓通组差异无统计学意义。②模型组与正常组相比,其NF-κB p65、IKKβ蛋白均呈高表达( P<0.01),而各个给药组NF-κB p65、IKKβ蛋白表达较模型组有所下降。C、E组大鼠患侧大脑皮层中NF-κB p65蛋白表达较模型组明显下降( P<0.01)；而苦碟子治疗组和血栓通治疗组NF-κB p65蛋白表达虽然较模型组有所减少( P<0.05),但相对C组、E组下降不明显。3个清热活血组与苦碟子治疗组、血栓通治疗组相比较NF-κB p65的蛋白表达无统计学意义(P>0.05)。 C、D、E组较苦碟治疗组IKKβ蛋白表达下降较明显(P<0.01)。另外,3个清热活血组IKKβ的蛋白表达较血栓通组有统计学意义( P<0.01)；C、E组较苦碟子治疗组IKKβ蛋白表达亦有统计学意义( P<0.01)。③模型组NF-κB p65、IKKβ mRNA的表达均较正常组上调,正常组的表达仅为模型组的0.37、0.48倍。各给药组NF-κB p65、IKKβ mRNA表达均较模型组降低,其中D组NF-κB p65、IKKβ mRNA的表达较模型组有差异( P<0.05)；E组IKKβ mRNA的表达较模型组有差异( P<0.05)；而E组NF-κB p65 mRNA的表达较模型组有明显差异( P<0.01)。结论清热活血联合应用组可通过抑制急性脑梗死炎症反应NF-κB信号通路中的关键因子IKKβ、NF-κB p65基因和蛋白的表达,对急性脑梗死的治疗发挥协同增效作用。%Objectives To study the synergetic effects of heat-clearing and blood-activating combination in the treatment of acute cerebral ischemia of heat toxin pattern. Methods Animal model of acute cerebral ischemia of heat toxin pattern was established by using middle cerebral artery occlusion ( MCAO) and carrageenan injection in rats. Rats were randomly divided into eight groups:normal group, sham group, ischemia 1. 5 h/reperfusion 72 h group, Kudiezi ( KDZ) injection group ( heat-clearing, group A), Xueshuantong(XST) injection group (blood activating, group B), KDZ1. 8 mL/kg+XST20 mg/kg (group C), KDZ1. 8 mL/kg+XST80 mg/kg (group D), and KDZ7. 2 mL/kg+XST20 mg/kg ( group E) . Infarction area ratios was measured by TTC staining; activity or content of NF-κB p65 and IKKβof cerebral tissues, by Western bogene expression of NF-κB p65 and IKKβ, by real-time PCR. Results Cerebral tissue ischemia in all treatment groups was reduced ( P<0 . 05 ) . Heat-clearing and blood-activating combined treatment improved significantly (P<0. 01) compared with the model group, yet there were no significant differences between these groups. NF-κB p65 and IKKβ in model group were highly expressed (P<0. 01) compared with the normal group. They both decreased in all treatment groups. NF-κB p65 protein expression in group C and E significantly decreased compared with the model group (P<0. 01);and they also decreased (P<0. 05) in group A and B. Expression of IKKβ protein decreased in combination treatment groups (group C, D, E and A) markedly (P<0. 01). In addition, IKKβ protein expression in groups C, D and E were lower than that in group B significantly (P<0. 01);It was markedly lower in groups C and E than in KDZ group ( P <0 . 01 ) . Expression of NF-κBp65、IKKβ mRNA in the model group was higher (0. 48%) than that in the normal group (0. 37%). NF-κB p65 and IKKβ mRNA expression in each treatment group decreased compared with the model group. Expression of NF-κB p65 and IKKβmRNA in group D and the expression of IKKβmRNA in group E was lower than the model group; the expression of NF-κB p65 mRNA in group E was significantly different compared with the model group. Conclusion Heat-clearing and blood-activating combination has synergetic effects on acute cerebral ischemia via regulating the protein and gene expression of NF-κB p65 and IKKβ in NF-κB signaling pathway.
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