【摘要】 目的:观察通心络对缺氧血管内皮细胞中缺氧诱导因子(HIF)及其上游信号转导通路PI-3K/Akt的影响,探讨PI-3K/Akt/HIF信号通路在通心络改善血管内皮细胞抗缺氧损伤中的作用。方法:将体外培养的人脐静脉血管内皮细胞(HUVECs)分为常氧对照组、通心络组、缺氧组和缺氧+通心络组。采用CCK-8试剂盒检测各组细胞的活性,Western blotting检测HIF-1α、Bcl-2、Mcl-1、Bax表达变化及Akt的磷酸化情况。利用HIF-1α的显性负性突变体(DN-HIF)瞬时转染HUVECs,流式细胞仪分析细胞的凋亡情况。利用PI-3K和Akt的显性负性突变体瞬时转染HUVECs,探讨PI-3K/Akt信号通路在通心络改善血管内皮细胞抗缺氧损伤中的作用。结果:与常氧条件下比较,通心络对缺氧内皮细胞虽有明显的促增殖作用,但程度明显减弱。通心络可显著上调HIF-1α、Bcl-2、Mcl-1蛋白表达水平及Akt磷酸化水平,且下调Bax的表达。利用DN-HIF抑制HIF-1α的活化后,通心络提高缺氧HUVECs活性的程度明显下降,但仍可一定程度上降低细胞凋亡百分率。进一步利用PI-3K显性负性突变体(Δp85)和Akt显性负性突变体(DN-Akt)阻断HIF-1α上游信号通路PI-3K/Akt后,通心络上调缺氧HUVECs HIF-1α蛋白表达的作用明显减弱,促进Akt磷酸化的作用基本消失。结论:在缺氧条件下,通心络上调HUVECs HIF-1α蛋白表达水平,促进抗凋亡因子同时抑制促凋亡因子的表达,降低细胞凋亡率,从而提高缺氧细胞的增殖率,这些作用在一定程度上依赖于PI-3K/Akt/HIF通路的活性。更多还原

【Abstract】 AIM: To investigate the effects of PI-3K/Akt/HIF pathway on anti-hypoxia ability of vascular endothelial cells influenced by Tongxinluo under hypoxic condition.METHODS: Human umbilical vein endothelial cells(HUVECs) were divided into the following groups: control group,Tongxinluo(100 mg/L) group,hypoxia group and hypoxia+Tongxinluo(100 mg/L) group.The CCK-8 assay were used to detect the viability and proliferation rate of the cells in each group.The protein levels of HIF-1α,Bcl-2,Mcl-1,Bax and phosphorylated Akt were studied by immunoblotting analysis.The HUVECs were transiently transfected with the dominant negative mutant of HIF-1α(DN-HIF).The apoptotic rates were analyzed by flow cytometry(FCM).The HUVECs were transiently transfected with the dominant negative mutant of PI-3K(Δp85) or Akt(DN-Akt) to investigate the role of PI-3K/Akt signal pathway in the anti-hypoxia ability of Tongxinluo on endothelial cells.RESULTS: Under hypoxic condition,although the proliferation rate increased significantly in Tongxinluo group compared with hypoxia group,the degree was notably weak compared with control group.The protein levels of HIF-1α,Bcl-2,Mcl-1 and phosphorylated Akt were up-regulated by Tongxinluo.Meanwhile,the expression of Bax was down-regulated.Inhibition of HIF-1α activation by DN-HIF and inhibition of the PI-3K/Akt pathway by Δp85 or DN-Akt attenuated the increase in HIF-1α expression and HUVEC viability induced by Tongxinluo.The percentage of apoptotic HUVECs was down-regulated to a certain extent by Tongxinluo.CONCLUSION: Tongxinluo improves the anti-hypoxia ability of vascular endothelial cells by up-regulating the protein level of HIF-1α,promoting the expression of anti-apoptotic factors,improving the cell viability and eventually reducing the apoptotic rate.These effects of Tongxinluo depend on PI-3K/Akt signal pathway.更多还原