补阳还五汤激活PI3K<strong>-</strong>AKT通路促进氧糖剥夺再灌注损伤的脑微血管内皮细胞血管生成

doi:10.3724/zdxbyxb-2022-0324

浙江大学学报（医学版）

2022, Vol. 51

Issue (5): 544-551 DOI: 10.3724/zdxbyxb-2022-0324

专题报道

补阳还五汤激活PI3K-AKT通路促进氧糖剥夺再灌注损伤的脑微血管内皮细胞血管生成

胡小伟¹,李琳¹,龚荧荧²,方燕¹,杨琰¹,许家栋¹,储利胜^1,*(

)

1. 浙江中医药大学基础医学院，浙江杭州 310053
2. 浙江中医药大学第二临床医学院，浙江杭州 310053

Buyang Huanwu decoction promotes angiogenesis of rat brain microvascular endothelial cells after oxygen-glucose deprivation reperfusion injury via activation of PI3K-AKT signaling pathway

HU Xiaowei¹,LI Lin¹,GONG Yingying²,FANG Yan¹,YANG Yan¹,XU Jiadong¹,CHU Lisheng^1,*(

)

1. College of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China;
2. The Second Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou 310053, China

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摘要：

目的：探讨补阳还五汤促进氧糖剥夺再灌注（OGD/R）损伤的大鼠脑微血管内皮细胞（RBMEC）血管生成的机制。方法：RBMEC经补阳还五汤含药血清孵育24?h后，构建OGD/R细胞损伤模型。将细胞随机分为正常对照组、模型对照组、补阳还五汤组（给予补阳还五汤含药血清）和LY294002组[给予补阳还五汤含药血清前使用磷脂酰肌醇3激酶（PI3K）抑制剂LY294002预处理1?h]。采用CCK-8法、细胞划痕实验和Transwell迁移实验、管腔形成实验分别检测细胞增殖、迁移和形成管腔能力。蛋白质印迹法检测PI3K、蛋白激酶B（AKT）、低氧诱导因子（HIF）-1α及血管内皮生长因子（VEGF）等蛋白的表达。结果：与模型对照组比较，补阳还五汤组RBMEC存活率、迁移能力及管腔形成能力显著增强（均P<0.01），磷酸化PI3K、磷酸化AKT、HIF-1α和VEGF蛋白表达增加（均P<0.05），而LY294002可阻断上述效应。结论：补阳还五汤含药血清可促进OGD/R损伤的RBMEC血管生成，其机制可能与其激活PI3K-AKT信号通路上调HIF-1α和VEGF表达有关。

关键词： 补阳还五汤; 脑微血管内皮细胞; 氧糖剥夺再灌注; 血管生成; PI3K-AKT通路; 低氧诱导因子-1α; 血管内皮生长因子

Abstract:

Objective: To investigate the effect and mechanism of Buyang Huanwu decoction (BYHWD) on angiogenesis of rat brain microvascular endothelial cells (RBMECs) after oxygen-glucose deprivation reperfusion (OGD/R) injury. Methods: RBMECs were pretreated with BYHWD containing serum 24?h before OGD/R injury was induced. Cells were randomly divided into blank control group, model control group, BYHWD group (provided BYHWD containing serum) and LY294002 group [treated with phosphoinositide 3-kinase (PI3K) inhibitor LY294002 for 1?h before provided BYHWD containing serum]. The cell viability, migration and tube formation abilities of RBMECs were detected by CCK-8, scratch wound healing, Transwell migration and tube formation assays, respectively. The protein expression levels of PI3K, p-PI3K, protein kinase B (AKT), p-AKT, hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were determined by Western blotting. Results: Compared with model control group, cell viability, migration and tube formation abilities of RBMECs were significantly improved in BYHWD group (all P<0.01), the protein expression levels of p-PI3K, p-AKT, HIF-1α and VEGF were up-regulated (allP<0.05); while above effects were blocked by LY294002.Conclusion: BYHWD can promote angiogenesis of RBMECs after OGD/R injury, which may be related to the increased protein expression of HIF-1α and VEGF through activation of PI3K-AKT signaling pathway.

Key words: Buyang Huanwu decoction Brain microvascular endothelial cells Oxygen- glucose deprivation reperfusion Angiogenesis PI3K-AKT signaling pathway Hypoxia-inducible factor-1α Vascular endothelial growth factor

收稿日期: 2022-06-18 出版日期: 2022-12-28

CLC:

R285.5

基金资助: 国家自然科学基金（81073075）; 浙江省自然科学基金（LY22H280009）; 浙江中医药大学校级科研基金项目（2019ZY20，2021JKZC01）

通讯作者: 储利胜 E-mail: cls2004@zcmu.edu.cn

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引用本文:

胡小伟,李琳,龚荧荧,方燕,杨琰,许家栋,储利胜. 补阳还五汤激活PI3K-AKT通路促进氧糖剥夺再灌注损伤的脑微血管内皮细胞血管生成[J]. 浙江大学学报（医学版）, 2022, 51(5): 544-551.

HU Xiaowei,LI Lin,GONG Yingying,FANG Yan,YANG Yan,XU Jiadong,CHU Lisheng. Buyang Huanwu decoction promotes angiogenesis of rat brain microvascular endothelial cells after oxygen-glucose deprivation reperfusion injury via activation of PI3K-AKT signaling pathway. J Zhejiang Univ (Med Sci), 2022, 51(5): 544-551.

链接本文:

https://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2022-0324 或 https://www.zjujournals.com/med/CN/Y2022/V51/I5/544

图1 不同浓度补阳还五汤含药血清处理后各组RBMEC存活率比较(=5)n与模型对照组比较，<0.05，<0.01；与2.5%含药血清浓度比较， <0.05. RBMEC：大鼠脑微血管内皮细胞.

表1 各组RBMEC存活率、划痕愈合率、迁移细胞数及管腔形成数比较

图2 各组RBMEC划痕愈合比较24 h时，与正常对照组比较，其他各组划痕面积均增大；与模型对照组比较，补阳还五汤组划痕面积减小；而LY294002组划痕面积比补阳还五汤组增大. 标尺=200 μm. RBMEC：大鼠脑微血管内皮细胞.

图3 各组RBMEC迁移细胞数比较与正常对照组比较，其他各组RBMEC均减少；与模型对照组比较，补阳还五汤组RBMEC增加；与补阳还五汤组比较，LY294002组RBMEC减少. 标尺=100 μm. RBMEC：大鼠脑微血管内皮细胞.

图4 各组RBMEC形成管腔比较正常情况下，单个分散的RBMEC在适合的基底上互相趋近，最终形成环状结构. 与正常对照组比较，模型对照组趋近细胞数及环状结构减少；与模型对照组比较，补阳还五汤组趋近细胞数及环状结构增加；而LY294002组趋近细胞数及环状结构比补阳还五汤组减少. 标尺=100 μm. RBMEC：大鼠脑微血管内皮细胞.

图5 各组RBMEC中磷酸化PI3K、磷酸化AKT、HIF-1α、VEGF蛋白表达电泳图RBMEC：大鼠脑微血管内皮细胞；PI3K：磷脂酰肌醇3激酶；AKT：蛋白激酶B；HIF：低氧诱导因子；VEGF：血管内皮生长因子；GAPDH：甘油醛-3-磷酸脱氢酶.

表2 各组RBMEC磷酸化PI3K、磷酸化AKT、HIF-1α、VEGF蛋白表达水平比较

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