脑络欣通通过激活HIF-1α/VEGF信号通路促进氧糖剥夺/复氧复糖损伤后大鼠的脑微血管内皮细胞增殖

doi:10.12122/j.issn.1673-4254.2025.09.17

摘要/Abstract

摘要：

目的探讨脑络欣通（NLXTD）靶向缺氧诱导因子/血管内皮生长因子（HIF-1α/VEGF）信号通路对大鼠脑微血管内皮细胞（BMECs）氧糖剥夺/复氧复糖（OGD/R）损伤后增殖的影响及其作用机制。方法构建OGD/R损伤BMECs模型，随机分为5组：正常细胞组（Normal）、模型组（OGD/R）、脑络欣通血清组（OGD/R+10%NLXTD）、脑络欣通血清+HIF-1α通路阻断剂组（OGD/R+10%NLXTD+2ME2），脑安颗粒血清组（OGD/R+10%NAKL），并分别给予相应的药物干预24 h。利用CCK-8法评估NLXTD对BMECs增殖以及对BMECs通透性的影响；采用Transwell实验、管腔形成实验检测NLXTD对BMECs迁移和管腔形成的影响；采用ELISA法检测VEGF含量的表达，采用激光共聚焦免疫荧光法检测VEGF、Notch含量的表达。采用Western blotting法检测HIF-1α、VEGFR2及其下游信号通路中Notch1以及ERK、P-ERK1/2蛋白的表达。结果 BMECs经OGD/R诱导后细胞活力出现明显下降（P<0.01），相较于OGD/R组，NLXTD能显著促进BMECs增殖、促进其迁移和管腔形成能力（P<0.01），而NLXTD+2ME2组相较NLXTD组，细胞的增殖、迁移、成管能力下降（P<0.01）。ELISA实验结果显示，与OGD/R组比较，NLXTD可促进BMECs上清VEGF表达（P<0.01），激光共聚焦免疫荧光实验结果同样显示，相较于OGD/R组，NLXTD组VEGF与Notch阳性细胞数占比提升（P<0.01），Western blotting实验结果进一步显示，NLXTD提高了HIF-1α、VEGFR2、及VEGF下游Notch1、P-ERK1/2蛋白表达量（P<0.01）。结论 NLXTD能够促进OGD/R损伤后BMECs增殖，影响血管新生，其机制可能与调控HIF-1α/VEGF信号通路有关。

关键词: 缺血性脑卒中, 脑络欣通, HIF-1α/VEGF信号通路, 氧糖剥夺/复氧复糖, 脑微血管内皮细胞

Abstract:

Objective To investigate the effects of Naoluo Xintong Decoction (NLXTD) on proliferation of rat brain microvascular endothelial cells (BMECs) after oxygen-glucose deprivation/reoxygenation (OGD/R) injury and role of the HIF-1α/VEGF pathway in mediating its effect. Methods Using a BMEC model of OGD/R, we tested the effects of 10% NLXTD-medicated rat serum, alone or in combination with 2ME2 or 10% NAKL, on cell proliferation, migration, tube-forming ability and permeability using CCK-8 assay, Transwell chamber assay, tube formation assay and permeability assay. Cellular expressions of VEGF and Notch were detected using ELISA and laser confocal immunofluorescence analysis, and the expressions of HIF-1α, VEGFR2, Notch1, ERK and P-ERK1/2 proteins were detected with Western blotting. Results OGD/R injury significantly decreased viability of BMECs. NLXTD treatment of the cells with OGD/R could significantly promoted cell proliferation, migration and tube formation ability, but these effects were strongly attenuated by application of 2ME2. NLXTD treatment also significantly increased the percentages of VEGF- and Notch-positive cells in the cell models and obviously enhanced the expression levels of HIF-1α, VEGFR2, Notch1 and P-ERK1/2. Conclusion NLXTD promotes proliferation, migration, and tube formation of rat BMECs after OGD/R injury possibly by activating the HIF-1α/VEGF signaling pathway.

Key words: ischemic stroke, Naoluo Xintong Decoction, HIF-1α/VEGF signaling pathway, oxygen-glucose deprivation/reoxygenation injury, cerebral microvascular endothelial cells

张玉, 胡音琦, 李佩佩, 时潇, 徐伟, 胡建鹏. 脑络欣通通过激活HIF-1α/VEGF信号通路促进氧糖剥夺/复氧复糖损伤后大鼠的脑微血管内皮细胞增殖[J]. 南方医科大学学报, 2025, 45(9): 1980-1988.

Yu ZHANG, Yinqi HU, Peipei LI, Xiao SHI, Wei XU, Jianpeng HU. Naoluo Xintong Decoction promotes proliferation of rat brain microvascular endothelial cells after oxygen-glucose deprivation by activating the HIF-1α/VEGF signaling pathway[J]. Journal of Southern Medical University, 2025, 45(9): 1980-1988.

图/表 6

图1 NLXTD含药血清对OGD/R大鼠BMECs活力的影响

Fig.1 Effect of NLXTD-medicated serum on viability of BMECs with OGD/R injury (n=6). ##P<0.01 vs normal group; &&P<0.01 vs OGD/R group.

图2 NLXTD含药血清对OGD/R大鼠BMECs迁移能力的影响

Fig.2 Effect of NLXTD-medicated serum on migration capacity of BMECs with OGD/R injury (Original magnification: ×100, n=6). ##P<0.01 vs normal group; &&P<0.01 vs OGD/R group; *P<0.01 vs OGD/R+10%NLXTD group.

图4 NLXTD含药血清对OGD/R大鼠BMECs渗透率的影响

Fig.4 Effect of NLXTD-medicated serum on permeability of BMECs after OGD/R injury (n=6). ##P<0.01 vs normal group; &&P<0.01 vs OGD/R group;*P<0.05 vs OGD/R+10%NLXTD+2ME2 group.

图5 NLXTD含药血清对OGD/R大鼠BMECs上清VEGF的影响

Fig.5 Effect of NLXTD-medicated serum on supernatant VEGF level in BMECs after OGD/R injury. ##P<0.01 vs normal group; &&P<0.01 vs OGD/R group;*P<0.05 vs OGD/R+10%NLXTD group.

图6 NLXTD含药血清对OGD/R损伤大鼠BMECs VEGF、Notch荧光蛋白表达的影响

Fig.6 Effect of NLXTD-medicated serum on expressions of VEGF and Notch fluorescent protein in BMECs after OGD/R injury ( n=6). ##P<0.01 vs normal group; &&P<0.01 vs OGD/R group;*P<0.01 vs OGD/R+10%NLXTD group.

图7 NLXTD含药血清对OGD/R损伤大鼠BMECs HIF-1a、VEGFR2、Notch1、ERK1/2、P-ERK1/2蛋白表达的影响

Fig.7 Effect of NLXTD-medicated serum on expressions of HIF-1a, VEGFR2, Notch1, ERK1/2, and P-ERK1/2 proteins in BMECs after OGD/R injury (n=4). ##P<0.05 vs normal group; &&P<0.01 vs OGD/R group; *P<0.01 vs OGD/R+10%NLXTD+2ME2 group.

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