脂多糖诱导Toll样受体4对载脂蛋白E基因敲除小鼠动脉粥样硬化斑块内质网应激的影响

Effects of lipopolysaccharide-induced toll-like receptor 4 on endoplasmic reticulum stress in atherosclerotic plaques of polipoprotein E gene knockout mice

目的:通过观察脂多糖(LPS)诱导的Toll样受体4(TLR4)对载脂蛋白E基因敲除(ApoE-/-)小鼠动脉粥样硬化斑块内质网应激通路蛋白表达水平的影响,探讨内质网应激对小鼠动脉粥样硬化斑块稳定性的影响。方法:2015年10月至2016年2月选择ApoE-/-小鼠24只,高脂喂养10周后数字抽签随机分为对照组、LPS组、TLR4的特异性抑制剂TAK(TAK组),各8只。干预10周后取眼球血检测总胆固醇(TC)、三酰甘油(TG)、氧化型低密度脂蛋白(ox-LDL)。处死小鼠留取颈动脉和主动脉标本。免疫组化法检测颈动脉斑块巨噬细胞(MOMA-2)、平滑肌肌动蛋白(α-actin)、TLR4、白细胞介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNFα)及κ基因结合核因子(NFκB)的表达。免疫印迹法检测PKR样真核起始因子2α激酶(PERK)、C/EBP同源蛋白(CHOP)、糖调节蛋白78(GRP78)的水平。结果:LPS组小鼠与对照组和TAK组小鼠比较,TC(25.0±2.3)mmol/L比(20.2±1.6)mmol/L、(20.8±2.6)mmol/L、TG(1.3±0.1)mmol/L比(1.3±0.1)mmol/L、(1.0±0.1)mmol/L、ox-LDL(17.4±1.3)mmol/L比(15.8±1.6)mmol/L、(12.1±1.1)mmol/L水平升高(P<0.05);斑块形态学及病理学比较,LPS组小鼠动脉粥样硬化斑块范围大,巨噬细胞含量增多(P<0.05),平滑肌细胞含量减少(P<0.05),斑块TLR4、IL-1β、IL-6、TNFα及NFκB的表达水平较其他两组增加(P<0.05);PERK、CHOP、GRP78的表达水平增加(P<0.05)。与对照组比较,TAK组PERK、CHOP、GRP78的表达水平减少(P<0.05)。LPS组TLR4,质网应激通路蛋白PERK、CHOP、GRP78的表达水平增加。结论:LPS诱导的TLR4可上调内质网应激通路蛋白的表达,且引起内质网应激下游炎性细胞因子分泌增加,加重脂质代谢紊乱,增加动脉硬化斑块的不稳定性。

Objective To investigate the effects of endoplasmic reticulum(ER)stress on the stability of atherosclerotic plaques in mice by examining the action of lipopolysaccharide(LPS)-induced Toll-like receptor 4(TLR4)on the protein expression levels in the ER stress pathway in atherosclerotic plaques of polipoprotein E gene knockout(ApoE-/-)mice.Methods From October 2015 to February 2016,24 ApoE-/-mice were randomly divided into the control group,the LPS group and the TAK group after 10 weeks of high-fat feeding(n=8,each group).After 10 weeks of intervention,peripheral blood was extracted by removing the eyeballs for the measurement of total cholesterol(TC),triglycerides(TG)and oxidized low density lipoprotein(ox-LDL).Then mice were sacrificed to obtain carotid and aortic specimens.Immunohistochemistry was used to detect the expression of carotid plaque macrophages(MOMA-2),smooth muscle actin(α-actin),TLR4,interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNFα)and nuclear factor-κ-gene binding(NFκB).Western blotting was used to determine the expression of PKR-like eukaryotic initiation factor 2αkinase(PERK),C/EBP-homologous protein(CHOP)and glucose-regulated protein 78(GRP78).Results The levels of TC,TG and ox-LDL were elevated in the LPS group,compared with the control and TAK groups[(25.0±2.3)mmol/L vs.(20.2±1.6)mmol/L and(20.8±2.6)mmol/L,(1.3±0.1)mmol/L vs.(1.3±0.1)mmol/L and(1.0±0.1)mmol/L,(17.4±1.3)mmol/L vs.(15.8±1.6)mmol/L and(12.1±1.1)mmol/L,P<0.05].The comparison of plaque morphology and pathology showed that the LPS group had a wider range of atherosclerotic plaques,more macrophages and fewer vascular smooth muscle cells than the control and TAK groups(P<0.05).The expression of TLR4,IL-1β,IL-6,TNFα,NFκB,PERK,CHOP and GRP78 was higher in the LPS group than in the control and TAK groups(P<0.05).Compared with the control group,the expression of PERK,CHOP and GRP78 was lower in the TAK group(P<0.05).The expression of TLR4,PERK,CHOP and GRP78 was higher in the LPS group.Conclusions LPS-induced TLR4 can up-regulate the expression of proteins in the ER stress pathway,increase the secretion of inflammatory cytokines downstream of the ER stress pathway,aggravate lipid metabolism disorders and increase the instability of atherosclerotic plaques.