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

Myriocin reduces atherosclerosis in ApoE mice by inhibiting lipidted stress response-induced integra

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300μm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-1βmRNA(t=3.968,P=0.005)、TNF-αmRNA(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-10mRNA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)、eIF2αmRNA(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。

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-1βand 6(IL-1βand 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 2α(eIF2α),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 eIF2α,ATF4,inositol-requiring enzyme 1α(IRE1α)and phosphorylated IRE1α,p65 nuclear factor-κB(p65 NF-κB)and phosphorylated p65 NF-κB,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 300μm 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-1βmRNA(t=3.968,P=0.005),TNFαmRNA(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),eIF2αmRNA(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-κB(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.