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目的:探讨腺苷酸蛋白激酶(AMPK)活化在红景天苷(Sal)抑制同型半胱氨酸(Hcy)诱导的脐静脉内皮细胞(HUVECs)内质网应激中的作用。方法:将AMPK基因的siRNA载体(AMPK-si-RNA)转染HUVECs后,用红景天苷或AMPK的选择性激动剂5-氨-4-甲酰胺咪唑核糖核苷酸(AICAR)预孵育2 h,再加入Hcy处理24 h,实时荧光定量PCR(Realtime-PCR)检测免疫球蛋白结合蛋白(Bi P)、C/EBP同源蛋白(CHOP)的mRNA表达水平,Western Blot检测Bi P、CHOP、AMPK的蛋白表达变化,双链RNA依赖的PKR内质网激酶(PERK)、翻译起始因子(e IF2α)和AMPK的磷酸化水平。结果:与空白对照组相比,单用Hcy处理组的Bi P和CHOP基因表达和蛋白表达都显著升高(P<0.01),PERK和e IF2α的磷酸化水平升高(P<0.01),与单用Hcy处理组相比,红景天苷和AICAR预处理组均能抑制Hcy导致的Bi P和CHOP的基因和蛋白表达(P<0.05),降低PERK和e IF2α的磷酸化水平(P<0.05),而AMPK-si-RNA转染细胞后,红景天苷抑制Bi P和CHOP基因和蛋白表达的能力下降,抑制PERK和e IF2α的磷酸化水平的能力也相应降低。结论:红景天苷抑制Hcy诱导的内质网应激是由AMPK活化介导的,从而发挥内皮保护的效应。
AIM: To investigate the role of adenylate kinase (AMPK) activation in endoplasmic reticulum stress induced by homosysteine (Hcy) in human umbilical vein endothelial cells (HUVECs). Methods: The siRNA vector (AMPK-si-RNA) of AMPK gene was transfected into HUVECs and preincubated with Salmonella or AMPK selective agonist 5-Amino-4-carboxamide imidazole ribonucleotides 2 h, and then treated with Hcy for 24 h. Real-time PCR was used to detect mRNA expression of BiP and CHOP. Western Blot was used to detect Bi P, CHOP, AMPK protein expression changes, double-stranded RNA-dependent PKR ERK (PERK), translation initiation factor (e IF2α) and AMPK phosphorylation levels. Results: Compared with the blank control group, the expression of Bi P and CHOP mRNA and protein were significantly increased (P <0.01) and the phosphorylation levels of PERK and e IF2α were significantly increased (P <0.01) Both salidroside and AICAR pretreatment inhibited the gene and protein expression of Bi P and CHOP induced by Hcy (P <0.05) and decreased the phosphorylation of PERK and e IF2α (P <0.05). However, the ability of salidroside to inhibit the expression of Bi P and CHOP gene and protein decreased after AMPK-si-RNA transfection, and the ability of inhibiting the phosphorylation of PERK and e IF2α was also decreased. CONCLUSIONS: Salidroside inhibits Hcy-induced endoplasmic reticulum stress mediated by AMPK activation, thereby exerting the effect of endothelial protection.