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

doi:10.12122/j.issn.1673-4254.2024.06.05

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (6): 1048-1058.doi: 10.12122/j.issn.1673-4254.2024.06.05

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

李丽¹^,²(), 汪梦哲¹^,², 刘赛赛¹^,², 张小楠¹^,², 陈洁¹^,², 陶伟婷¹^,², 李晒²^,⁴, 秦志文²^,³, 陶泉坊²^,³, 刘奕²^,³, 黄丽¹^,²(), 赵士弟¹^,²()

^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

收稿日期:2023-11-02 出版日期:2024-06-20 发布日期:2024-07-01
通讯作者: 黄丽,赵士弟 E-mail:19556126120@163.com;zi05@163.com;zhsdi@126.com
作者简介:李丽，在读硕士研究生，E-mail:19556126120@163.com
基金资助:
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)

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

Li LI¹^,²(), Mengzhe WANG¹^,², Saisai LIU¹^,², Xiaonan ZHANG¹^,², Jie CHEN¹^,², Weiting TAO¹^,², Shai LI²^,⁴, Zhiwen QING²^,³, Quanfang TAO²^,³, Yi LIU²^,³, Li Huang¹^,²(), Shidi ZHAO¹^,²()

^1.Department of Pathophysiology, Bengbu 233000, China
^2.Key Laboratory of Basic and Clinical Cardiovascular and Cerebrovascular Diseases, Bengbu 233000, China
^3.Department of Clinical Medicine, Bengbu Medical University, Bengbu 233000, China
^4.Department of Neurology, Nanjing First Hospital, Nanjing 210000, China

Received:2023-11-02 Online:2024-06-20 Published:2024-07-01
Contact: Li Huang, Shidi ZHAO E-mail:19556126120@163.com;zi05@163.com;zhsdi@126.com

摘要/Abstract

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

关键词: 大豆异黄酮, 脑缺血再灌注, 钙超载, Wnt/Ca²⁺

Abstract:

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.

Key words: soy isoflavones, cerebral ischemia-reperfusion, calcium overload, Wnt/Ca²⁺ signaling pathway

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. 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[J]. 南方医科大学学报, 2024, 44(6): 1048-1058.

Li LI, Mengzhe WANG, Saisai LIU, Xiaonan ZHANG, Jie CHEN, Weiting TAO, Shai LI, Zhiwen QING, Quanfang TAO, Yi LIU, Li Huang, Shidi ZHAO. Soy isoflavones alleviates calcium overload in rats with cerebral ischemia-reperfusion by inhibiting the Wnt/Ca²⁺ signaling pathway[J]. Journal of Southern Medical University, 2024, 44(6): 1048-1058.

图/表 6

图1 抑制 Wnt/Ca2+ 信号通路对CIRI的影响

Fig.1 Effect of inhibition of Wnt/Ca2+ signaling pathway on cerebral ischemia-reperfusion (I/R) injury in rats. A, B: Expression of Frizzled-2 in the brain tissue of sham-operated rats detected by Western blotting (n=3). C-F: Western blotting for detecting expressions of Frizzled-2, Wnt5a and P-CaMK II in the brain tissues of rats in each group (n=3). G: Ca2+ level in the brain tissues of the rats (n=6). H: HE staining of the brain tissues of the rats. *P<0.05, **P<0.01, ***P<0.001 vs Sham group; #P<0.05, ###P<0.001 vs I/R group.

图2 局部脑血流的动态变化

Fig.2 Dynamic changes of regional cerebral blood flow (rCBF). A: Changes of rCBF in I/R group. B: Changes of rCBF in soy isoflavones (SI)-pretreated group.

图3 脑梗死体积和神经缺损评分的变化

Fig.3 Brain infarct volume and neurological deficit scores of the rats with sham operation, cerebral I/R injury and soy isoflavones (SI) pretreatment. A, B: Brain infarct volume (n=5). C: Neurological deficit scores (n=6). ***P<0.001 vs Sham group; ##P<0.01, ###P<0.001 vs I/R group.

图4 HE染色和尼氏染色的变化

Fig.4 HE staining (A) and Nissl staining (B) of the rats with sham operation, cerebral I/R injury and soy isoflavones (SI) pretreatment before modeling (scale bar=20 μm).

图6 氧化应激水平的变化

Fig.6 Changes of oxidative stress level in the brain tissue of the rats in the 3 groups. A, B: Immunofluorescence assay for detecting ROS level in the brain tissues (n=3). C: SOD activity in the brain tissue (n=6); D: Content of MDA in the brain tissue (n=6). ***P<0.001 vs Sham group; ###P<0.001 vs I/R group.

图8 Wnt/Ca2+信号通路相关蛋白的变化

Fig.8 Western blotting (A) and immunohistochemistry (B, original magnification:×400) for detecting protein expression of Wnt5a, Frizzled-2 and P-CaMK II in the brain tissues of the rats in the 3 groups. **P<0.01, ***P<0.001 vs Sham group; #P<0.05 vs I/R group.

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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

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