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目的观察补阳还五汤对气虚血瘀证小鼠局灶性脑缺血损伤的保护作用及其机制。方法采用饥饿、劳累、高脂饮食等多因素复合制作出气虚血瘀证模型,然后用线栓法在左侧大脑中动脉栓塞2h复灌24h诱导小鼠的脑缺血再灌注损伤模型。实验动物随机分为正常对照组、假手术组、模型组、Poly I:C(Polyinosinic polycytidylic acid)组、补阳还五汤低剂量组、补阳还五汤中剂量组、补阳还五汤高剂量组。脑组织切片TTC染色测定脑梗死体积;ELISA检测小鼠脑组织匀浆炎性因子TNF-ɑ、IL-6、IFN-β、IL-10的含量。Western-blot方法检测缺血侧半脑脑组织TRIF蛋白表达。结果采用饥饿、劳累、高脂、饮食控制多因素复合制作出气虚血瘀证模型,不同组别动物造模后出现不同程度气虚血瘀证症状,补阳还五汤能改善气虚血瘀症状。左侧大脑中动脉栓塞2h复灌24h能稳定地制作小鼠气虚血瘀型脑缺血再灌注损伤模型,小鼠出现明显的脑组织梗死,缺血复灌24h缺血侧脑组织TNF-ɑ、IL-6表达升高。予0.3mg/kg Poly I:C及补阳还五汤均能减少梗死面积,改善神经缺陷,提高小鼠缺血侧脑组织TRIF蛋白的表达,降低缺血再灌24h缺血侧脑组织TNF-ɑ和IL-6的含量并升高IFN-β及IL-10水平。结论补阳还五汤对气虚血瘀证小鼠脑缺血有保护作用,保护机制与激动TLR3下游TRIF-IFNβ信号通路,进而增加抗炎因子分泌及减少致炎因子产生及有关。
Objective To observe the protective effect of Buyang Huanwu decoction on focal cerebral ischemia injury in mice with deficiency of Qi and blood stasis and its mechanism. Methods The Qi-deficiency and blood-stasis syndrome model was made by the combination of starvation, exertion, high-fat diet and so on. Then the model of cerebral ischemia-reperfusion injury was induced by reperfusion of the left middle cerebral artery embolization for 2h with thread occlusion method. The experimental animals were randomly divided into normal control group, sham operation group, model group, Poly I: C (polyinosinic polycytidylic acid) group, Buyang Huanwu Decoction low dose group, Buyang Huanwu Decoction dose group, Buyang Huanwu Decoction High-dose group. TTC staining of brain tissue was used to measure the volume of cerebral infarction. The content of inflammatory cytokines TNF-ɑ, IL-6, IFN-β and IL-10 in brain tissues of mice were detected by ELISA. Western-blot method was used to detect the expression of TRIF protein in ischemic hemispheres. Results The model of qi deficiency and blood stasis syndrome was made by the combination of starvation, exertion, hyperlipidemia and diet control. The symptoms of qi deficiency and blood stasis syndrome were observed in different groups of animals after modeling, and Buyang Huanwu Decoction could improve the symptoms of qi deficiency and blood stasis. The rats in the left middle cerebral artery embolization 2h 24h reperfusion can stably produce mouse model of Qi-deficiency and blood-stasis cerebral ischemia-reperfusion injury, the mice showed obvious infarction of brain tissue, ischemic reperfusion 24h 24h ischemia of the brain tissue TNF- , IL-6 expression increased. To 0.3mg / kg Poly I: C and Buyang Huanwu decoction can reduce the infarct size, improve nerve defects, increase the expression of TRIF protein in ischemic brain tissue of mice and reduce the ischemic brain ischemia-reperfusion 24h TNF -ɑ and IL-6 and increase the levels of IFN-β and IL-10. Conclusion Buyang Huanwu decoction has protective effect on cerebral ischemia in mice with qi deficiency and blood stasis syndrome. The protective mechanism is related to the activation of TRIF-IFNβ signaling pathway downstream of TLR3, thereby increasing the secretion of anti-inflammatory cytokines and reducing the production of inflammatory cytokines.