大黄酸对脑缺血大鼠脑组织中AQP4和小胶质细胞介导炎症反应的影响

Effect of Rhein on AQP4 and Microglia Mediated Inflammatory Response in Cerebral Ischemia Rats

目的:本研究旨在探讨大黄酸对脑缺血损伤后水通道蛋白4(AQP4)与脑水肿的影响以及小胶质细胞介导的炎症在此过程中的作用。方法:选择改良线栓法建立大鼠右侧大脑中动脉栓塞(MCAO)的脑缺血模型,并将其分为假手术组、模型组、米诺环素组和大黄酸高、中、低剂量组(3.46,1.73,0.865 mg·kg^(-1))。通过改良神经行为学评分测量神经行为功能。采用干湿重法测定脑缺血损伤大鼠脑组织含水量变化。蛋白免疫印迹法(Western blot)检测各组大鼠脑缺血周边区γ-干扰素(IFN-γ),白细胞介素-2(IL-2)的表达变化。免疫荧光双标记法检测小胶质细标记物离子钙接头蛋白抗原-1(Iba-1),AQP4蛋白的表达和定位。结果:与假手术组比较,模型组大鼠神经功能评分、脑组织损伤侧含水量明显增高(P<0.05);与模型组比较,各药物组大鼠神经功能评分、脑组织含水量均明显降低(P<0.05,P<0.01);与假手术组比较,模型组IFN-γ,IL-2蛋白表达水平明显升高(P<0.05);与模型组比较,各药物组脑缺血周边区IFN-γ,IL-2蛋白水平均明显改善(P<0.05,P<0.01)。与假手术组比较,模型组可见激活的小胶质细胞上AQP4蛋白荧光表达明显增强;与模型组比较,各药物组可减少激活的小胶质细胞上AQP4蛋白荧光的表达,各组大鼠脑缺血周边区可见不同程度激活的小胶质细胞标记物Iba-1与AQP4共定位表达。结论:大黄酸可以减轻脑缺血损伤引起的脑水肿程度,其机制可能与其抑制小胶质细胞介导的神经炎症、下调AQP4蛋白表达有关。

Objective:To investigate the effect of rhein on aquaporin 4(AQP4)and brain edema after cerebral ischemia and the role of microglia-mediated inflammation in this process. Method: The modified thread embolization method was selected to establish the cerebral ischemia model of the right middle cerebral artery embolism(MCAO)in rats. The rats were divided into sham operation group,model group,minocycline group,and high,medium and low-dose rhein groups(3.46,1.73,0.865 mg·kg^(-1)). The neurobehavioral function was measured by a modified neurobehavioral score. Wet and dry weight methods were used to measure the changes of water content in brain tissue of rats with cerebral ischemic injury. Western blot was used to detect the expressions of interferon-γ(IFN-γ)and interleukin-2(IL-2)in the peripheral ischemic area of rats in each group. Immunofluorescence double labeling method was used to detect the expressions and localization of microglia fine markers Iba-1 and AQP4. Result: Compared with the sham operation group,neurological function score and water content on the side of brain tissue injury of the model group were significantly increased(P<0.05). Compared with the model group,the neurological function score and the water content of the brain tissue of each drug group were reduced(P<0.05,P<0.01). Compared with sham operation group,the protein expressions of IFN-γ and IL-2 in the model group increased significantly(P<0.05). Compared with the model group,the protein expressions of IFN-γ and IL-2 in the peripheral area of cerebral ischemia of each drug group were significantly improved(P<0.05, P<0.01). Immunofluorescence double staining results showed that compared with the sham operation group,the model group showed significant increase in the fluorescence expression of AQP4 protein on activated microglia,while each drug group could reduce the fluorescence expression of AQP4 protein on activated microglia,different levels of activated microglia markers Iba-1 and AQP4 were co-localized in the peripheral area of cerebral ischemia in each group. Conclusion: Rhein could reduce the degree of brain edema caused by cerebral ischemic injury,and its mechanism may be related to the inhibition of microglia-mediated neuroinflammation and the down-regulation of AQP4 expression.