大豆异黄酮通过抑制Wnt/Ca2+信号通路减轻大鼠脑缺血再灌注引起的钙超载  

Soy isoflavones alleviates calcium overload in rats with cerebral ischemia-reperfusion by inhibiting the Wnt/Ca2+signaling pathway

作　　者：李丽 汪梦哲 刘赛赛 张小楠 陈洁 陶伟婷 李晒 秦志文 陶泉坊 刘奕 黄丽 赵士弟 LI Li;WANG Mengzhe;LIU Saisai;ZHANG Xiaonan;CHEN Jie;TAO Weiting;LI Shai;QING Zhiwen;TAO Quanfang;LIU Yi;Huang Li;ZHAO Shidi(Department of Pathophysiology,Bengbu Medical University,Bengbu 233000,China;Key Laboratory of Basic and Clinical Cardiovascular and Cerebrovascular Diseases,Bengbu Medical University,Bengbu 233000,China;Department of Clinical Medicine,Bengbu Medical University,Bengbu 233000,China;Department of Neurology,Nanjing First Hospital,Nanjing 210000,China)

机构地区：[1]蚌埠医科大学病理生理学教研室,安徽蚌埠233000 [2]蚌埠医科大学心脑血管疾病基础与临床重点实验室,安徽蚌埠233000 [3]蚌埠医科大学临床医学院,安徽蚌埠233000 [4]南京市第一医院神经内科,江苏南京210000

出　　处：《南方医科大学学报》2024年第6期1048-1058,共11页Journal of Southern Medical University

基　　金：安徽省教育厅自然科学重大研究项目(KJ2021ZD0091);安徽省大学生创新项目(S202210367110);蚌埠医科大学“512人才培育计划”(by51202204);安徽省大学生创新项目(S202310367066)。

摘　　要：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。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.

关 键 词：大豆异黄酮 脑缺血再灌注 钙超载 Wnt/Ca2+ 

分 类 号：R743.3[医药卫生—神经病学与精神病学]