丹灯通脑软胶囊通过激活 HIF-1α-VEGFA-Notch1 信号通路促进脑微血管血管生成，保护脑缺血再灌注损伤。
Dan-Deng-Tong-Nao softgel capsule promotes angiogenesis of cerebral microvasculature to protect cerebral ischemia reperfusion injury via activating HIF-1α-VEGFA-Notch1 signaling pathway.
机构信息
Anhui Province Key Laboratory of Chinese Medicinal Formula, Anhui University of Chinese Medicine, Hefei, Anhui, 230012, PR China; Anhui Province Key Laboratory of Research & Development of Chinese Medicine, Anhui University of Chinese Medicine, Hefei, Anhui(,) 230012, China.
Anhui Province Key Laboratory of Chinese Medicinal Formula, Anhui University of Chinese Medicine, Hefei, Anhui, 230012, PR China; Anhui Province Key Laboratory of Research & Development of Chinese Medicine, Anhui University of Chinese Medicine, Hefei, Anhui(,) 230012, China.
出版信息
Phytomedicine. 2023 Sep;118:154966. doi: 10.1016/j.phymed.2023.154966. Epub 2023 Jul 13.
BACKGROUND
A proprietary Chinese herbal product called Dan-Deng-Tong-Nao softgel capsule (DDTNC) is used to treat ischemic stroke. However, the preventive mechanisms of DDTNC against cerebral ischemia reperfusion injury (CIRI) haven not been characterized.
OBJECTIVE
To explore the mechanisms of protective effects of DDTNC against CIRI from both internal and external levels.
METHODS
Chemical characterization was performed using UPLC. The potential protective mechanisms of DDTNC against CIRI were predicted using network pharmacology. Model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established in rats. An model of brain microvascular endothelial cells (BMECs) induced by oxygen-glucose deprivation/reoxygenation (OGD/R) was also established. We evaluated neurological deficits, cerebral infarct volume, cortical neuron damage, and mitochondrial swelling in vivo. We evaluated the expression of VEGFR2, VEGFA, HIF-1α, CD31, and CD34 in ischemic cortex, and VEGF, bFGF, BDNF, angiostatin, and endostatin in serum of rats and in BMEC supernatants. We also evaluated cell viability, cytotoxicity, intracellular ROS, apoptosis, and migration ability in vitro.
RESULTS
Seven components were detected in DDTNC. KEGG enrichment analysis showed that DDTNC may modulate angiogenesis via the HIF-1 signaling pathway. DDTNC treatment reduced neurological score and infarct volume, and improved cell morphology of damaged neurons. Transmission electron microscopy showed that DDTNC reduced mitochondria swelling in cortical neurons. Furthermore, DDTNC reduced intracellular ROS and inhibited apoptosis. DDTNC boosted the expression of CD31, CD34, VEGFR2, VEGFA and HIF-1α, highlighting its involvement in angiogenesis, according to immunofluorescence studies. Furthermore, DDTNC enhanced tube formation and migration of BMECs in vitro. ELISA and western blotting indicated that DDTNCCSF induced the expression of VEGF, BDNF and bFGF, reduced the level of angiostatin and endostatin, increased the protein expression of VEGFA, Notch1 and HIF-1α in vitro and in vivo.
CONCLUSIONS
DDTNC promoted angiogenesis to protect brain tissue against MCAO/R, and exerted protective effects against OGD/R in BMECs via activating HIF-1α-VEGFA-NOTCH1 signal transduction pathway.
背景
一种名为丹灯通脑软胶囊（DDTNC）的中药专利产品用于治疗缺血性中风。然而，DDTNC 防治脑缺血再灌注损伤（CIRI）的预防机制尚未明确。
目的
从内、外两个层面探讨 DDTNC 对 CIRI 的保护作用机制。
方法
采用 UPLC 进行化学特征分析。采用网络药理学预测 DDTNC 防治 CIRI 的潜在保护机制。建立大鼠大脑中动脉阻塞/再灌注（MCAO/R）模型。建立氧葡萄糖剥夺/复氧（OGD/R）诱导的脑微血管内皮细胞（BMEC）模型。在体内评估神经功能缺损、脑梗死体积、皮质神经元损伤和线粒体肿胀。评估缺血皮质中 VEGFR2、VEGFA、HIF-1α、CD31 和 CD34 的表达，以及大鼠血清和 BMEC 上清液中 VEGF、bFGF、BDNF、血管抑素和内皮抑素的表达。还评估了细胞活力、细胞毒性、细胞内 ROS、细胞凋亡和迁移能力。
结果
在 DDTNC 中检测到 7 种成分。KEGG 富集分析表明，DDTNC 可能通过 HIF-1 信号通路调节血管生成。DDTNC 治疗可降低神经评分和梗死体积，并改善受损神经元的细胞形态。透射电镜显示 DDTNC 可减轻皮质神经元中线粒体肿胀。此外，DDTNC 可减少细胞内 ROS 并抑制细胞凋亡。免疫荧光研究显示，DDTNC 可促进 CD31、CD34、VEGFR2、VEGFA 和 HIF-1α 的表达，提示其参与血管生成。此外，DDTNC 可增强体外 BMEC 的管形成和迁移。ELISA 和 Western blot 表明，DDTNC 可诱导 VEGF、BDNF 和 bFGF 的表达，降低血管抑素和内皮抑素的水平，增加体内外 VEGFA、Notch1 和 HIF-1α 的蛋白表达。
结论
DDTNC 通过激活 HIF-1α-VEGFA-Notch1 信号转导通路，促进血管生成，保护脑组织免受 MCAO/R 损伤，并对 BMEC 中的 OGD/R 发挥保护作用。
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