Chinese Journal of Contemporary Neurology and Neurosurgery ›› 2020, Vol. 20 ›› Issue (10): 853-861. doi: 10.3969/j.issn.1672-6731.2020.10.003

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Myriocin reduces atherosclerosis in ApoE-/- mice by inhibiting lipid-induced integrated stress response

YU Ze-mou1, LI Yong-chao2, HAO Hong-jun1, HUANG Yi-ning1, PENG Qing1   

  1. 1 Department of Neurology, Peking University First Hospital, Beijing 100034, China;
    2 Department of Cardiac Surgery, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing 102218, China
  • Received:2020-09-29 Online:2020-10-25 Published:2020-10-30
  • Supported by:

    This study was supported by the Fundamental Research Funds for the Central Universities, Peking University Medicine Seed Fund for Interdisciplinary Research (No. BMU2017MC005).

Myriocin抑制脂质诱导的整合应激反应延缓ApoE-/-小鼠动脉粥样硬化进展研究

于泽谋1, 李永超2, 郝洪军1, 黄一宁1, 彭清1   

  1. 1 100034 北京大学第一医院神经内科;
    2 102218 清华大学附属北京清华长庚医院心脏外科
  • 通讯作者: 彭清,Email:qing054@sina.com
  • 基金资助:

    北京大学医学青年科技创新平台发展基金资助项目(项目编号:BMU2017MC005)

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-6chigh 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

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-6chigh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