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银杏内酯 K 通过 PI3K/Akt/mTOR 通路促进 血管生成改善缺血性卒中
赵茜米克拉依·阿不来提
0
()
摘要:
目的 探究银杏内酯 K(Ginkgolide K ,GK)通过磷脂酰肌醇 -3- 激酶(PI3K)/ 蛋白激酶 B (Akt)/ 哺乳动物雷帕霉素靶蛋白(mTOR)信号通路对缺血性卒中小鼠血管内皮生长因子(VEGF)表达 和脑血管生成的作用。方法 将 40 只 C57BL/6 小鼠随机分为对照组、大脑中动脉闭塞(MCAO)组、低 剂量 GK(GK-L)组(3.5 mg/kg)、中剂量 GK(GK-M)组(7 mg/kg)和高剂量(GK-H)组(14 mg/kg),每组各 8 只。建立 MCAO 模型和氧葡萄糖剥夺发(OGD)体外模型;采用改良后的神经系统严重程度评分(mNSS) 评估法检测小鼠神经功能缺损;2,3,5- 三苯基氯化四氮唑(TTC)染色检测小鼠脑缺血面积;免疫荧 光染色检测小鼠梗死灶周围皮质血管生成和星形胶质细胞覆盖。培养 hCMEC/D3 细胞,分为对照组, OGD 组、OGD+CK 组、OGD+LY 组(LY 为 PI3K 信号通路抑制剂)和 OGD+GK+LY 组,采用细胞计数试 剂盒 -8(CCK-8)检测内皮细胞活性;Western blot 检测内皮细胞缺氧诱导因子 1α(HIF-1α)、VEGF 和 PI3K/Akt/mTOR 通路相关基因蛋白表达;细胞划痕实验检测内皮细胞迁移能力;血管形成实验检测内 皮细胞管腔形成能力。结果 与 MCAO 组[(5.37±1.25)分、(11.99±1.72)%]比较,GK-M 组和 GK-H 组 小鼠 mNSS 评分[分别为(3.37±1.32)、(2.23±0.38)分]和脑缺血面积[(4.75±0.89)%、(2.42±0.42)%] 均 显 著 降 低(P< 0.05)。 与 MCAO 组 EdU+ /CD31+ 细 胞 数、EdU+ /GFAP+ 细胞数和星形胶质细胞覆盖 率[分 别 为(3.33±0.58)个、(4.33±1.53)个、(69.20±5.60)%]比 较,GK 组 小 鼠 EdU+ /CD31+ 细胞数 [(13.67±2.08)个,t=3.576]、EdU+ /GFAP+ 细 胞 数[(8.33±1.53)个,t=6.008]和 星 形 胶 质 细 胞 覆 盖 率 [(82.26±7.77)%]显著升高(均P< 0.05)。与对照组细胞活力、HIF-1α、VEGF 蛋白表达、Akt 和 mTOR 磷酸化水平[分别为(100.31±3.01)%、(0.09±0.03)、(0.13±0.03)、(0.20±0.04)、(0.18±0.03)]比较,OGD 组细胞活力[(52.37±9.06)%]、HIF-1α(0.17±0.02)和 VEGF 蛋白表达(0.18±0.03)、Akt 和 mTOR 磷酸 化水平[(0.28±0.06),(0.38±0.05)]均显著升高(均P< 0.05);与 OGD 组比较,OGD+GK 组细胞 HIF-1α (0.22±0.03)和 VEGF 蛋白表达(0.23±0.03)、Akt 和 mTOR 磷酸化水平[(0.48±0.09),(0.52±0.05)]、细胞 活力[(61.07±3.48)%]、迁移率[(85.26±11.03)%]和管状结构数量[(81.97±5.79)%]均显著升高(均 P<0.05),OGD+LY组则表现出相反变化;PI3K信号通路抑制剂LY294002可逆转GK对OGD细胞的影响。 结论 GK 通过 PI3K/Akt/mTOR 信号通路上调 VEGF 表达促进脑血管生成,改善小鼠缺血性卒中。
关键词:  银杏内酯 K  脑缺血  血管生成  磷脂酰肌醇 -3- 激酶 / 蛋白激酶 B/ 哺乳动物雷帕 霉素靶蛋白信号通路  血管内皮生长因子
DOI:10.3969/j.issn.1009-6574.2020.03.004
基金项目:新疆维吾尔自治区自然科学基金项目(2019D01C032)
Ginkgolide K promotes angiogenesis and improves ischemic stroke through PI3K/Akt/mTOR pathway
Zhao Xi, Mikelayi Abulaiti
()
Abstract:
Objective To investigate the effects of ginkgolide K on vascular endothelial growth factor (VEGF) expression and cerebral angiogenesis in mice with cerebral ischemia via the PI3K/Akt/mTOR signaling pathway. Methods A total of 40 C57BL/6 mice were randomly divided into five groups with 8 mice in each group: control group, MCAO group, GK-L group (3.5 mg/kg), GK-M group (7 mg/kg) and GK-H group (14 mg/kg), with 8 cases in each. MCAO rat models and oxygen glucose deprivation (OGD) in vitro models were established.The modified Neurological Severity Score (mNSS) was utilized to detect neurological deficits in mice. TTC staining was utilized to detect cerebral ischemic area of mice; immunofluorescence staining was utilized to detect cortical angiogenesis and astrocyte coverage in the peri-infarct cortex of ischemic mice. Hcmec / D3 cells were cultured and divided into control group, OGD group, OGD + CK group, OGD + LY group (LY as PI3K Signal pathway inhibitor) and OGD + GK + LY group. Endothelial cell activity was detected by CCK-8. Western blot was used to detected the protein expression of HIF-1α, VEGF and PI3K/Akt/mTOR pathwayrelated gene in endothelial cell; cell scratch test was used to detect the migration ability of endothelial cell; angiogenesis test was used to detect tube formation ability of endothelial cell. Results Compared with MCAO group [(5.37±1.25), (11.99±1.72)%], the mNSS scores [respectively (3.37±1.32), (2.23±0.38)]and the area of cerebral ischemia [(4.75±0.89)%, (2.42±0.42)%]of mice in GK-M group and GK-H group were significantly reduced (all P < 0.05). Compared with the number of EdU+/CD31+cells, the number of EdU +/ GFAP+cells and the astrocyte coverage of MCAO group [(3.33±0.58), (4.33±1.53), (69.20±5.60)%], GK Mouse EdU+/CD31+cell count [(13.67±2.08),t=3.576], EdU+/GFAP+cell count [(8.33±1.53), t=6.008], and astrocyte coverage [(82.26±7.77)%]increased significantly (P< 0.05). Compared with the control group (100.31±3.01)%, the cell viability (52.37±9.06)% of the OGD group was significantly reduced (F=19.914, P< 0.05), HIF-1α (0.17±0.02) and VEGF protein expression (0.18±0.03), Akt and mTOR phosphorylation levels [(0.28±0.06, 0.38±0.05)] were significantly increased (P < 0.05); Compared with OGD group (0.28±0.06), OGD+GK group cells HIF-1α (0.22±0.03) and VEGF protein expression (0.23±0.03), Akt and mTOR phosphorylation levels [(0.48±0.09),(0.52±0.05)], cell viability [(61.07±3.48)%], mobility [(85.26±11.03)%] and the number of tubular structures [(81.97±5.79)%] were significantly increased (P < 0.05). The OGD+LY group showed the opposite change; PI3K signaling pathway inhibitor LY294002 could reverse the effect of GK on OGD cells. Conclusions Ginkgolide K up-regulates the expression of VEGF through PI3K/Akt/mTOR signaling pathway to promote cerebral angiogenesis and improve ischemic stroke in mice.
Key words:  Ginkgolide K  Cerebral ischemia  Angiogenesis  PI3K/Akt/mTOR signaling pathway  Vascular endothelial growth factor

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