Department of Medicine, University of Hong Kong, Hong Kong.
J Atheroscler Thromb. 2012;19(12):1083-92. doi: 10.5551/jat.11742. Epub 2012 Aug 1.
Lectin-like oxidized LDL receptor-1 (LOX-1) is a class E oxidized LDL specific scavenger receptor that recognizes multiple ligands. Advanced glycation end products (AGEs) have been recently identified as other ligands to LOX-1 and shown to increase LOX-1 expressions in diabetes; therefore, we investigated the underlying mechanism involved.
Confluent human aortic endothelial cells were treated with a fixed concentration of AGE-BSA or BSA as a control in the presence or absence of either antibody of the receptor for advanced glycation end products, mammalian target of rapamycin (mTOR) inhibitor rapamycin, NF-kB inhibitor, phosphoinositide 3-kinases (PI3K) inhibitor or anti-diabetic drug metformin. After stimulation, cells were lysed and Western blot protein expression on LOX-1, rapamycin-insensitive companion of mTOR (RICTOR), the phosphorylation status of p-mTOR, p-P70S6 kinase and p-Akt were determined.
AGEs induced LOX-1 expression in endothelial cells. Pretreatment either with anti-RAGE antibody or LY294002 prior to AGE-BSA decreases LOX-1 and p-mTOR expressions. Incubating endothelial cells with AGE-BSA in the presence of rapamycin down-regulated the protein expression-level of p-mTOR by 41% (p<0.05) and LOX-1 expression by 61.5% (p<0.01). Knockdown of RICTOR by RNA silencing showed a 41.5% (p<0.01) and 71.2% (p<0.01) reduction in LOX-1 and p-Akt expressions, respectively. Preincubation of endothelial cells with AGE-BSA and metformin, an anti-diabetic drug known to have an mTOR inhibition effect, significantly reduced AGE-stimulated LOX-1 expression.
Our results indicated that LOX-1 up-regulation induced by AGE-BSA was a receptor mediated through RAGE and is via the PI3K/PDK1/mTORC2 pathway. Metformincan reduce AGE-stimulated LOX-1 expression in endothelial cells in vitro.
凝集素样氧化型 LDL 受体-1（LOX-1）是一种 E 类氧化型 LDL 特异性清道夫受体，可识别多种配体。晚期糖基化终产物（AGEs）最近被鉴定为 LOX-1 的其他配体，并显示在糖尿病中增加 LOX-1 的表达；因此，我们研究了涉及的潜在机制。
在存在或不存在晚期糖基化终产物受体抗体、雷帕霉素（mTOR）抑制剂雷帕霉素、核因子-κB（NF-κB）抑制剂、磷酸肌醇 3-激酶（PI3K）抑制剂或抗糖尿病药物二甲双胍的情况下，用固定浓度的 AGE-BSA 或 BSA 处理汇合的人主动脉内皮细胞作为对照。刺激后，裂解细胞，并用 Western blot 测定 LOX-1、mTOR 无雷帕霉素敏感伴侣（RICTOR）、p-mTOR、p-P70S6 激酶和 p-Akt 的磷酸化状态的蛋白表达。
AGEs 诱导内皮细胞中 LOX-1 的表达。在用 AGE-BSA 预处理之前，用抗 RAGE 抗体或 LY294002 预处理可降低 LOX-1 和 p-mTOR 的表达。在雷帕霉素存在下孵育内皮细胞可使 p-mTOR 的蛋白表达水平降低 41%（p<0.05），LOX-1 表达降低 61.5%（p<0.01）。用 RNA 沉默敲低 RICTOR 可使 LOX-1 和 p-Akt 的表达分别降低 41.5%（p<0.01）和 71.2%（p<0.01）。在用抗糖尿病药物二甲双胍预孵育内皮细胞后，该药物已知具有 mTOR 抑制作用，可显著降低 AGE 刺激的 LOX-1 表达。
我们的结果表明，AGE-BSA 诱导的 LOX-1 上调是一种通过 RAGE 介导的受体，并且是通过 PI3K/PDK1/mTORC2 途径。二甲双胍可减少体外内皮细胞中 AGE 刺激的 LOX-1 表达。
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