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.%AIM: To invesligale 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 Lo delect the viability and proliferation rale of the cells in each group. The prolein levels of HIF - 1α, Bcl -2, Mcl - 1, Bax and phosphorylaled Akt were studied by immu-noblolling analysis. The HUVECs were transienlly transfecled with the dominanl negalive mulanl of HIF -1α ( DN - HIF) . The apoplolic rales were analyzed by flow cylomelry (FCM). The HUVECs were transienlly transfecled wilh the dominanl negalive mulanl of PI - 3K ( △p85) or Akt ( DN - Akt) lo invesligale the role of PI - 3K/Akl signal palhway in the anti -hypoxia ability of Tongxinluo on endolhelial cells. RESULTS: Under hypoxic condition, although the proliferation rale in- creased significantly in Tongxinluo group compared with hypoxia group, the degree was nolably weak compared with control group. The protein levels of HIF - 1α, Bcl - 2, Mcl - 1 and phosphorylaled Akt were up - regulaled by Tongxinluo. Meanwhile,the expression of Bax was down - regulaled. Inhibition of HIF - 1α aclivalion by DN - HIF and inhibition of the PI -3K/Akt pathway by Ap85 or DN - Akt allenualed the increase in HIF - 1α expression and HUVEC viability induced by Tongxinluo. The percentage of apoplolic HUVECs was down - regulaled to a certain exlenl by Tongxinluo. CONCLUSION : Tongxinluo improves the anti - hypoxia ability of vascular endolhelial cells by up - regulating the prolein level of HIF - 1α, promoling the expression of anli- apoplolic factors, improving the cell viability and eventually reducing the apoplolic rale. These effects of Tongxinluo depend on PI - 3K/Akt signal pathway.
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