内质网应激PERK途径在高糖诱导血管平滑肌细胞向成骨样细胞转化中的作用

PERK pathway in transformation of smooth muscle cells to osteoblast-like cells induced by high glucose

摘要:

目的 研究高糖刺激SD大鼠胸主动脉血管平滑肌细胞（VSMCs）向成骨样细胞转分化中内质网应激蛋白激酶R样内质网激酶（PERK）途径的作用。方法 原代培养VSMCs，分为正常对照组（5mmol/L D-葡萄糖），甘露醇组（5mmol/L D-葡萄糖+25mmol/L甘露醇），高糖组（30mmol/L D-葡萄糖），高糖+PERK-小干扰RNA（siRNA）转染组（30mmol/L D-葡萄糖+PERK-siRNA转染），高糖+RNA干扰阴性对照组（30mmol/L D-葡萄糖+乱序RNA转染）。分别作用48和72h。逆转录-聚合酶链反应及Western blot分别从信使RNA（mRNA）和蛋白质水平检测不同时间点内质网应激相关分子GRP78、PERK、p-PERK、eIF2α、p-eIF2α、ATF4的改变，以及成骨样细胞标志性分子核心转录因子（Cbfα-1）、骨钙素以及平滑肌细胞标志性分子α-SMA的改变情况。应用碱性磷酸酶（ALP）活性检测试剂盒检测ALP的活性。结果 与正常对照组和甘露醇组比较，在mRNA水平，高糖组和高糖+RNA干扰阴性对照组PERK和eIF2α表达升高（P＜0.05），PERK-siRNA转染后其表达水平均降低（P＜0.05），在蛋白质水平，PERK、p-PERK、eIF2α、p-eIF2α表达升高（P＜0.05），PERK-siRNA干预后其表达均降低（P＜0.05）。高糖刺激VSMCs后ALP活性升高（P＜0.05），PERK-siRNA转染后其表达均降低（P＜0.05）。结论 内质网应激PERK途径在高糖诱导VSMCs由收缩表型向成骨样细胞表型转化中发挥作用，抑制PERK途径可以部分阻止这一转化过程。

Abstract:

Objective To investigate the role of endoplasmic reticulum stress?protein kinase R-like endoplasmic reticulum kinase (PERK) pathway in the transdifferentiation of SD rat aortic vascular smooth muscle cells (VSMCs) to osteoblast-like cells by high glucose. Methods The primary cultured VSMCs from rat’s aortic segments were divided into 5 groups, including normal control group (5mmol/L D-glucose), mannitol group (5mmol/L D-glucose plus 25mmol/L mannitol), high glucose group (30mmol/L D-glucose), high glucose＋PERK-small interference RNA (siRNA) group (30mmol/L D-glucose plus PERK siRNA transfection), and high glucose＋scramble RNA group (30mmol/L D-glucose plus scramble RNA transfection). At 48 and 72h after the corresponding treatment, the expression of endoplasmic reticulum stress markers, including GRP78, PERK, p-PERK, eukaryotic initiation factor (eIF)2α, and p-eIF2α, osteo inductive factors such as Cbfα-1, osteocalcin, and smooth muscle cells marker α-smooth muscle antibody (α-SMA) at messenger RNA (mRNA) and protein levels were detected by RT-PCR and Western blotting, respectively. The activity of alkaline phosphatase (ALP) was measured by ALP activity testing kit. Results When compared with the normal group and the mannitol group, high glucose in presence or absence of scramble RNA transfection resulted significant increases in the mRNA levels of PERK and eIF2α (P＜0.05), while PERK siRNA transfection decreased the expression (P＜0.05). High glucose also induced high protein expression of PERK, p-PERK, eIF2α, and p-eIF2α (P＜0.05), but PERK siRNA transfection decreased the expression (P＜0.05). The activity of ALP was enhanced by high glucose (P＜0.05), but partially blocked by PERK siRNA (P＜0.05). Conclusion Endoplasmic reticulum stress PERK pathway plays a role in the transdifferentiation of VSMCs from the contractile phenotype to osteoblast phenotype induced by high glucose, and the transformation process can partially blunted by suppressing the PERK pathway.