摘要：

目的 探讨Myriocin对高脂饮食诱导的整合应激反应的影响及作用机制。方法 18只ApoE^-/-小鼠随机分为高脂饮食+磷酸盐缓冲液（对照组，9只）或高脂饮食+磷酸盐缓冲液+Myriocin（Myriocin组，9只）两组，12周后测定血清脂质［总胆固醇、甘油三酯、低密度脂蛋白胆固醇（LDL-C）、极低密度脂蛋白胆固醇和高密度脂蛋白胆固醇］，流式细胞术测定淋巴细胞抗原6复合体（Ly-6c）^high亚型单核细胞比例，HE染色测定主动脉窦斑块和脂质坏死核相对面积，免疫荧光染色观察单核细胞趋化蛋白-1（MCP-1）表达，实时荧光定量聚合酶链反应（PCR）测定炎症反应相关分子［炎性因子白细胞介素-1β和6（IL-1β和IL-6）、肿瘤坏死因子-α（TNF-α）、细胞间黏附分子-1（ICAM-1）、血管内皮生长因子（VEGF）、血管细胞黏附分子-1（VCAM-1）和抗炎性因子IL-10］和整合应激反应相关分子［葡萄糖调节蛋白78（GRP78）、蛋白激酶R样内质网激酶（PERK）、真核翻译起始因子2α（eIF2α）、活化转录激活因子4和6（ATF4和ATF6）、内质网应激相关促凋亡蛋白C/EBP同源蛋白（CHOP）和Caspase12］mRNA表达，Western blotting法测定整合应激反应相关蛋白［eIF2α、ATF4、肌醇依赖酶1α（IRE1α）和磷酸化IRE1α、p65核因子-κB（NF-κB）和磷酸化p65 NF-κB、Caspase12和活化型Caspase12］表达水平。结果（1）经Myriocin灌胃12周后，小鼠血清LDL-C水平降低（t=2.830，P=0.012）。（2）流式细胞术分析，经Myriocin灌胃8周后，小鼠Ly-6c^high亚群单核细胞比例下降（t=2.866，P=0.011）。（3）HE染色观察，Myriocin组小鼠斑块面积小于对照组，在距主动脉瓣200和300 μm处，Myriocin组斑块面积小于对照组（t=2.281，P=0.045；t=3.506，P=0.003），Myriocin组小鼠脂质坏死核相对面积亦小于对照组（Z=-2.870，P=0.004）。（4）免疫荧光染色观察，Myriocin组小鼠MCP-1水平低于对照组。实时荧光定量PCR显示，Myriocin组小鼠IL-1β mRNA（t=3.968，P=0.005）、TNF-α mRNA（t=7.696，P=0.000）、ICAM-1 mRNA（t=3.294，P=0.013）、VCAM-1 mRNA（t=5.449，P=0.001）和VEGF mRNA（t=2.574，P=0.037）表达均降低，IL-10 mRNA升高（t=-3.132，P=0.017）。（5）实时荧光定量PCR显示，Myriocin组小鼠PERK mRNA（t=4.174，P=0.004）、eIF2α mRNA（Z=-2.692，P=0.007）、ATF4 mRNA（t=3.342，P=0.012）、ATF6 mRNA（t=5.841，P=0.001）和Caspase12 mRNA（t=7.270，P=0.000）表达降低。（6）Western blotting检测，Myriocin组小鼠eIF2α（t=2.175，P=0.047）、ATF4（t=2.923，P=0.011）和磷酸化p65 NF-κB/总p65 NF-κB比值（t=2.909，P=0.011）下降。结论 鞘脂抑制剂Myriocin可以抑制高脂饮食诱导的整合应激反应和炎症反应，延缓ApoE^-/-小鼠动脉粥样硬化进展。

关键词: 动脉粥样硬化, 髓磷脂蛋白质类, 炎症, 流式细胞术, 聚合酶链反应, 免疫印迹法, 疾病模型, 动物

Abstract:

Objective To investigate the effect of Myriocin on high fat diet induced integrated stress response and further explore the mechanism. Methods A total of 18 ApoE^-/- mice were fed a high-fat diet and randomly divided into control group (n=9, phosphate buffer solution) and Myriocin group (n=9, phosphate buffer solution + Myriocin). The drugs were administered orally for 12 weeks. Serum lipids[total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C), very low density lipoprotein-cholesterol (VLDL-C) and high density lipoprotein-cholesterol (HDL-C)] were measured. Flow-cytometric analysis was used to determine the proportion of lymphocyte antigen 6 complex (Ly-6c)^high phenotype monocytes. HE staining was performed to compare the size and detailed composition of atherosclerotic plaques and immunofluorescence staining was used to observe the expression of monocyte chemotactic protein-1 (MCP-1). Real-time fluorescence quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression levels of inflammation related molecules[including pro-inflammatory factors, interleukin-1β and 6 (IL-1β and IL-6), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), vascular endothelial growth factor (VEGF) and anti-inflammatory factor, IL-10]. In addition, the mRNA expression levels of integrated stress response related molecules[including glucose regulated protein 78 (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2 α (eIF2 α), activating transcription factor 4 and 6 (ATF4 and ATF6), endoplasmic reticulum stress-related apoptosis protein C/EBP homologous protein (CHOP) and Caspase12] were tested. The expression levels of integrated stress response related protein[including eIF2 α, ATF4, inositol-requiring enzyme 1 α (IRE1 α) and phosphorylated IRE1 α, p65 nuclear factor-κB (p65 NF-κB) and phosphorylated p65 NF-κ B, Caspase12 and cleaved Caspase12] were explored by Western blotting. Results 1) Treatment with Myriocin led to lower level of serum LDL-C (t=2.830, P=0.012). 2) Myriocin suppressed monocytes differentiating toward a Ly-6c^high phenotype (t=2.866, P=0.011). 3) HE staining showed less atherosclerotic lesions at 200 and 300 μ m distance away from the aortic valve (t=2.281, P=0.045; t=3.506, P=0.003) and less necrotic core areas (Z=-2.870, P=0.004) in the Myriocin group. 4) Immunofluorescence staining showed the reduction of MCP-1 protein expression in the Myriocin group; real-time fluorescence quantitative PCR showed that IL-1β mRNA (t=3.968, P=0.005), TNFα mRNA (t=7.696, P=0.000), ICAM mRNA (t=3.294, P=0.013), VCAM mRNA (t=5.449, P=0.001) and VEGF mRNA (t=2.574, P=0.037) were generally decreased in the Myriocin group, while IL-10 mRNA was increased (t=-3.132, P=0.017) in the Myriocin group. 5) Myriocin downregulated PERK mRNA (t=4.174, P=0.004), eIF2α mRNA (Z=-2.692, P=0.007), ATF4 mRNA (t=3.342, P=0.012), ATF6 mRNA (t=5.841, P=0.001) and Caspase12 mRNA (t=7.270, P=0.000). 6) Western blotting showed that Myriocin suppressed the protein expression of eIF2α (t=2.175, P=0.047) and ATF4 (t=2.923, P=0.011), and the phosphorylation of p65 NF-κ B (t=2.909, P=0.011). Conclusions Myriocin could alleviate atherosclerosis progression of ApoE^-/- mice by reducing integrated stress response and inflammatory response in the arterial walls.

Key words: Atherosclerosis, Myelin proteins, Inflammation, Flow cytometry, Polymerase chain reaction, Immunoblotting, Disease models,animal