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目的在体外培养的人脐静脉内皮细胞(HUVEC)上,观察α-亚麻酸对高糖致凋亡效应的影响,探讨其发挥细胞内保护作用的信号转导机制。方法体外培养HUVEC,分为4组:(1)对照组(低糖处理组,NG组);(2)高糖处理组(HG组);(3)高糖+α-亚麻酸处理组(HG+ALA组);(4)高糖+α-亚麻酸+3-磷脂酰肌醇激酶(PI3K)阻断剂LY294002处理组(HG+ALA+LY组)。各组在培养终点应用TUNEL法检测细胞凋亡指数(AI);ELISA法检测培养液中NO及·O2的水平;采用Western Blot方法检测内皮细胞Akt的磷酸化程度。结果 (1)HG组、HG+ALA组及HG+ALA+LY组AI较NG组明显升高;HG+ALA组AI较HG组明显降低;HG+ALA+LY组AI较HG+ALA组明显升高(P均<0.01);(2)HG组、HG+ALA组及HG+ALA+LY组培养液NO水平较NG组明显降低,HG+ALA组培养液NO水平较HG组明显升高,HG+ALA+LY组培养液NO水平较HG+ALA组明显降低(P<0.01);而各组培养液中·O2的水平则呈相反变化;(3)HG+ALA组细胞磷酸化的Akt蛋白(pAkt)表达较HG组明显升高(P<0.05);HG+ALA+LY组细胞pAkt蛋白表达较HG+ALA组明显降低。结论α-亚麻酸能够抑制高糖诱导的人脐静脉内皮细胞凋亡,该效应可能通过促进细胞内蛋白激酶B(Akt)磷酸化激活来实现,并通过调整内皮细胞的NO/·O2生成平衡,抑制细胞凋亡、发挥细胞保护作用。
OBJECTIVE: To observe the effect of α-linolenic acid on the apoptosis induced by high glucose in cultured human umbilical vein endothelial cells (HUVECs) in vitro and to explore the signal transduction mechanism of a-linolenic acid exerting the protective effect on cells. Methods HUVECs were cultured in vitro and divided into 4 groups: (1) control group (low glucose treatment group, NG group); (2) high glucose treatment group (HG group); + ALA group); (4) High glucose + α-linolenic acid + 3-phosphatidylinositol kinase (PI3K) blocker LY294002 treatment group (HG + ALA + LY group). Cell apoptosis index (AI) was detected by TUNEL method at the end of culture, NO and O2 levels were detected by ELISA, and phosphorylation of Akt was detected by Western Blot. Results AI in HG group, HG + ALA group and HG + ALA + LY group was significantly higher than that in NG group; AI in HG + ALA group was significantly lower than that in HG group; AI in HG + ALA + LY group was significantly higher than that in HG + ALA + LY group (P <0.01). (2) The levels of NO in HG group, HG + ALA group and HG + ALA + LY group were significantly lower than those in NG group. The NO level in HG + ALA group was significantly higher than that in HG group (P <0.01), while the level of O2 in the culture medium of each group changed in opposite. (3) The phosphorylation of cell in HG + ALA + LY group was significantly lower than that in HG + ALA group The expression of Akt protein (pAkt) was significantly higher than that of HG group (P <0.05). The expression of pAkt protein in HG + ALA + LY group was significantly lower than that in HG + ALA group. Conclusion α-linolenic acid can inhibit the apoptosis of human umbilical vein endothelial cells induced by high glucose. This effect may be achieved through the activation of intracellular phosphorylation of Akt phosphorylation and by adjusting the balance of NO / · O2 production in endothelial cells , Inhibit apoptosis, play a cytoprotective effect.