Objective: To explore the mechanism by which soybean isoflavone (SI) reduces calcium overload induced by cerebral ischemia-reperfusion (I/R).
Methods: Forty-eight SD rats were randomized into 4 groups to receive sham operation, cerebral middle artery occlusion for 2 h followed by 24 h of reperfusion (I/R model group), or injection of adeno-associated virus carrying Frizzled-2 siRNA or empty viral vector into the lateral cerebral ventricle after modeling.Western blotting was used to examine Frizzled-2 knockdown efficiency and changes in protein expressions in the Wnt/Ca2+ signaling pathway.Calcium levels and pathological changes in the ischemic penumbra (IP) were measured using calcium chromogenic assay and HE staining, respectively.Another 72 SD randomly allocated for sham operation, I/R modeling, or soy isoflavones pretreatment before modeling were examined for regional cerebral blood flow using a Doppler flowmeter, and the cerebral infarct volume was assessed using TTC staining.Pathologies in the IP area were evaluated using HE and Nissl staining, and ROS level, Ca2+ level, cell apoptosis, and intracellular calcium concentration were analyzed using immunofluorescence assay or flow cytometry; the protein expressions of Wnt5a, Frizzled-2, and P-CaMK Ⅱ in the IP were detected with Western blotting and immunohistochemistry.
Results: In rats with cerebral I/R, Frizzled-2 knockdown significantly lowered calcium concentration (P < 0.001) and the expression levels of Wnt5a, Frizzled-2, and P-CaMK Ⅱ in the IP area.In soy isoflavones-pretreated rats, calcium concentration, ROS and MDA levels, cell apoptosis rate, cerebral infarct volume, and expression levels of Wnt/Ca2+ signaling pathway-related proteins were all significantly lower while SOD level was higher than those in rats in I/R model group.
Conclusion: Soy isoflavones can mitigate calcium overload in rats with cerebral I/R by inhibiting the Wnt/Ca2+ signaling pathway.
目的: 探讨大豆异黄酮（SI）减轻脑缺血再灌注（I/R）引起钙超载的作用机制。
方法: 将48只SD大鼠采用随机数法分为4组：假手术组（Sham组）、脑缺血再灌注模型组（I/R组）、病毒空载组（NC 组）、Frizzled-2敲低组（Knock down组），12只/组。采用线栓法堵塞大脑中动脉2 h，再灌注24 h构建 I/R模型。采用Western blot验证病毒敲低效率并检测Wnt/Ca2+信号通路相关蛋白Wnt5a、Frizzled-2和P-CaMKⅡ的变化。采用钙含量显色法检测各组缺血半暗带（IP）区钙离子浓度变化，HE检测各组IP区组织结构变化。另将72只SD大鼠采用随机数法分为3组：Sham组、I/R组、大豆异黄酮预处理组（SI组），24只/组。采用多普勒血流仪检测局部脑血流变化，TTC染色检测脑梗死体积，HE和尼氏染色检测IP区组织变化、免疫荧光检测ROS、Ca2+和细胞凋亡水平、流式检测细胞钙离子浓度，试剂盒检测血清MDA和SOD水平，Western blotting和免疫组化检测IP区Wnt5a、Frizzled-2和P-CaMKⅡ蛋白表达。
结果: 与I/R组比较，Knock down组钙离子浓度（P<0.001）、Wnt5a（P<0.05）、Frizzled-2（P<0.05）和P-CaMKⅡ（P<0.001）表达水平均降低；与I/R组比较，SI组钙离子浓度（P<0.05）、ROS和MDA水平（P<0.001）、细胞凋亡程度（P<0.001）、脑梗死体积（P<0.001）、Wnt5a、Frizzled-2和P-CaMKⅡ表达水平（P<0.05）均降低，SOD水平升高（P<0.001）。
结论: 大豆异黄酮可能通过抑制Wnt/Ca2+信号通路减轻大鼠脑缺血再灌注引起的钙超载
Keywords: Wnt/Ca2+ signaling pathway; calcium overload; cerebral ischemia-reperfusion; soy isoflavones.