-
血脂代谢异常在动脉粥样硬化中的重要作用已被证实。研究[1]表明,血脂水平异常升高促进脂质沉积,并在动脉粥样硬化的形成及粥样斑块发展进程具有重要作用。载脂蛋白E(apolipoprotein E,ApoE)缺陷小鼠是目前通过基因敲除手段研究血脂代谢紊乱形成动脉硬化动物模型,最新研究[2]发现糖尿病可进一步诱发ApoE-/-小鼠血脂代谢紊乱,加重动脉硬化发生。TR3受体作为孤核受体超家族的成员,介导了许多种激素、维生素和药物细胞生物学效应[3]。有研究[4]显示TR3受体激活后增加糖尿病病人对胰岛素敏感性,加速血糖代谢,降低血糖水平,减轻糖尿病糖代谢性疾病;也有研究发现TR3受体可降低细胞增殖核转录因子-κB(NF-κB)/细胞周期蛋白D1(cylinD1)蛋白表达,抑制细胞增殖;在动脉粥样硬化斑块中发现斑块组织NF-κB/cylinD1蛋白表达水平增高,同时动脉硬化斑块周围平滑肌层细胞增生明显,降低NF-κB p65/CylinD1表达,斑块周围增生细胞明显减少,减缓动脉粥样斑块的形成[5]。因此我们认为TR3受体对糖尿病ApoE-/-小鼠血脂代谢、细胞增殖调控NF-κB/CylinD1通路与动脉硬化斑块形成与发展有密切相关,本研究将观察TR3受体激动剂(6-mercaptopurine,6-MP)对糖尿病ApoE-/-小鼠糖脂代谢及NF-κB p65/CylinD1通路的影响,探讨TR3受体激动剂改善糖尿病动脉粥样硬化可能机制。
-
6-MP对糖尿病ApoE-/-小鼠血糖的影响与ApoE-/-组相比,STZ-ApoE-/-组小鼠空腹血糖水平显著升高(P<0.01);TR3受体激动剂6-MP可呈剂量依赖性降低糖尿病ApoE-/-小鼠空腹血糖水平(P<0.01),与STZ-ApoE-/-组相比,STZ-ApoE-/-+6-MP5组小鼠空腹血糖水平显著降低(P<0.01),STZ-ApoE-/-+6-MP 10组空腹血糖水平进一步降低(P<0.01)(见表 1)。
分组 n 血糖/(mmol/L) ApoE-/-组 10 6.91±0.69 STZ-ApoE-/-组 10 18.68±2.30** STZ-ApoE-/-+6-MP5组 10 15.42±2.68**# STZ-ApoE-/-+6-MP10组 10 13.64±1.95**## F — 40.07 P — <0.01 MS组内 — 3.090 q检验:与ApoE-/-组比较**P<0.01;与STZ-ApoE组比较#P<0.05, ##P<0.01 表 1 TR3受体激动剂6-MP对糖尿病ApoE-/-小鼠血糖的影响(x±s)
-
与ApoE-/-组相比,STZ-ApoE-/-组小鼠空腹总胆固醇(TC)和低密度脂蛋白胆固醇(LDL-C)显著升高(P<0.01),2组间高密度脂蛋白胆固醇(HDL-C)和三酰甘油(TG)差异无统计学意义(P>0.05),TR3受体激动剂6-MP呈剂量依赖性降低糖尿病ApoE-/-组小鼠空腹TC、LDL-C、TG水平;与STZ-ApoE-/-组相比,STZ-ApoE-/-+6-MP 5组小鼠空腹血脂TC、LDL-C、TG水平均显著降低(P<0.01),STZ-ApoE-/-+6-MP10组空腹血脂TC、LDL-C、TG进一步降低(P<0.01);STZ-ApoE-/-组、STZ-ApoE-/-+6-MP5及STZ-ApoE-/-+6-MP10三组HDL-C差异无统计学意义(P>0.05)(见表 2)。
分组 TG TC HDL-C LDL-C ApoE-/-组 5.28±0.52 41.40±3.31 14.04±2.21 32.92±3.83 STZ-ApoE-/-组 5.60±0.43 62.04±7.12** 16.20± 4.83 50.96±6.83** STZ-ApoE-/-+6-MP5组 5.05±1.34 47.70±6.46# 15.95±0.98 42.50±4.92**## STZ-ApoE-/-+6-MP10组 4.57±1.45 40.82±5.19## 16.18±1.76 38.45±5.11**## F 1.73 29.91 1.34 20.67 P >0.05 <0.01 >0.05 <0.01 MS组内 1.088 32.580 8.068 27.909 q检验:与ApoE-/-组比较*P<0.05,**P<0.01;与STZ-ApoE-/-组比较#P<0.05,##P<0.01 表 2 TR3受体激动剂6-MP对糖尿病ApoE-/-小鼠血糖和血脂的影响(ni=10;x±s;mmol/L)
-
与C57组相比,ApoE-/-组小鼠肝脏FGF21 mRNA及蛋白表达水平均差异无统计学意义(P>0.05);与ApoE-/-组相比,STZ-ApoE-/-组小鼠肝脏FGF21 mRNA、蛋白及血清FGF21水平均明显增高(P<0.01);FGF21 miRNA可显著降低糖尿病ApoE-/-小鼠肝脏FGF21 mRNA、蛋白及血清FGF21水平(P<0.05)(见图 1、表 3)。
分组 n NF-κB p65 Cylin D1 ApoE-/-组 10 0.053±0.008 0.234±0.026 STZ-ApoE-/-组 10 0.358±0.046** 0.617±0.107** STZ-ApoE-/-+6-MP5组 10 0.213±0.025**## 0.226±0.031## STZ-ApoE-/-+6-MP10组 10 0.022±0.003**## 0.109±0.013**## F — 343.51 147.62 P — <0.01 <0.01 MS组内 — 0.001 0.003 注:蛋白印迹电泳胶片条带光密度(OD值)分析,NF-κB p65或CylinD1/β-actin光密度比值表示目的蛋白表达水平。q检验:与ApoE-/-组比较*P<0.05,**P<0.01;与STZ-ApoE-/-组比较#P<0.05,##P<0.01 表 3 TR3受体激动剂6-MP对糖尿病ApoE-/-小鼠胸主动脉NF-κB p65/CylinD1蛋白表达的影响(x±s)
-
6-MP对糖尿病ApoE-/-小鼠胸主动脉内膜脂质沉积的影响ApoE-/-组胸主动脉血管内膜有少量小团块状红染脂质,STZ-ApoE-/-组胸主动脉血管内膜有片状团块状红染脂质;STZ-ApoE-/-+6-MP5组或STZ-ApoE-/-+6-MP10组胸主动脉血管内膜少量小片状红染脂质(见图 2)。
-
6-MP对糖尿病ApoE-/-小鼠胸主动脉内膜粥样斑块形成的影响ApoE-/-组主动脉血管内膜可见小硬化斑块,斑块底部血管增厚;STZ-ApoE-/-组胸主动脉内膜可见巨大斑块突起,斑块中央有空洞形成,斑块底部血管增厚且不规则,斑块空腔周围可见大量细胞增生;STZ-ApoE-/-+6-MP5组主动脉血管内膜硬化斑块斑块较小,斑块中央空洞较小,斑块底部及周围有少量增生组织;STZ-ApoE-/-+6-MP5组胸主动脉内膜只见小硬化斑块隆起,斑块底部及周围有少量增生组织(见图 3)。
斑块面积分析:与ApoE-/-组相比,STZ-ApoE-/-组胸主动脉血管内膜斑块面积明显增加(P<0.05);TR3受体激动剂6-MP可呈剂量依赖的方式抑制STZ诱导的糖尿病ApoE-/-小鼠胸主动脉硬化斑块增加,与STZ-ApoE-/-组相比STZ-ApoE-/-+6-MP 5组胸主动脉内膜斑块面积明显降低(P<0.05);STZ-ApoE-/-+6-MP10组与STZ-ApoE-/-+6-MP 5组之间斑块面积差异有统计学意义(P<0.05)(见表 4)。
分组 n 斑块面积/μm2 ApoE-/-组 10 123.56±15.38 STZ-ApoE-/-组 10 245.16±30.29** STZ-ApoE-/-+6-MP5组 10 172.43±20.55**## STZ-ApoE-/-+6-MP10组 10 154.09±18.73**## F — 55.40 P — <0.01 MS组内 — 481.786 q检验:与ApoE-/-组比较*P<0.05,**P<0.01;STZ-ApoE-/-组比较#P<0.05,##P<0.01 表 4 TR3受体激动剂6-MP对糖尿病ApoE-/-小鼠胸主动脉内膜内膜粥样斑块面积的影响(x±s)
TR3受体激动剂6-mercaptopurine对糖尿病ApoE-/-小鼠NF-κB p65/CylinD1通路影响及其与抗动脉硬化关系
Effect of TR3 receptor agonist 6-mercaptopurine on NF-κB p65/CylinD1 pathway in diabetic ApoE-/- mice, and its relationship with anti-arteriosclerosis
-
摘要:
目的观察TR3受体激动剂(6-mercaptopurine,6-MP)对链脲佐菌素(streptozotocin,STZ)诱导的载脂蛋白E(apolipoprotein E,ApoE-/-)敲除糖尿病小鼠糖脂代谢及NF-κB p65/CylinD1通路的影响及其与抗动脉硬化的关系,探讨TR3受体在改善糖尿病动脉粥样硬化可能机制。 方法40只雄性ApoE-/-小鼠随机分为4组:ApoE-/-组、STZ-ApoE-/-组、STZ-ApoE-/-+6-MP5(5 mg·kg-1·d-1)组、STZ-ApoE-/-+6-MP10(10 mg·kg-1·d-1)组,每组10只;6-MP腹腔注射法给药,每天1次,连续8周;STZ 60 mg/kg腹腔注射ApoE-/-小鼠建立糖尿病动脉粥样硬化模型,血糖试纸法测血糖水平;酶法或匀相法测血脂水平;蛋白免疫印迹试验(Western-blotting)测胸主动脉组织NF-κB p65/CylinD1蛋白水平;油红O染色观察胸主动脉内膜动脉脂质沉积;HE染色测胸主动脉内膜粥样斑块面积。 结果与ApoE-/-组相比,STZ-ApoE-/-组血糖及血清TG、TCHO、LDL-C均显著增高(P < 0.05);TR3受体激动剂6-MP呈剂量依赖性降低糖尿病ApoE-/-小鼠血糖、血脂水平,与STZ-ApoE-/-组相比,STZ-ApoE-/-+6-MP5组血糖、TG、LDL-C、TC均显著降低(P < 0.05),与ApoE-/-组相比,STZ-ApoE-/-组胸主动脉NF-κBp65、CylinD1蛋白表达水平均显著增加(P < 0.05);6-MP呈剂量依赖性降低糖尿病ApoE-/-小鼠胸主动脉NF-κBp65/CylinD1蛋白表达,STZ-ApoE-/-+6-MP5组NF-κB p65/CylinD1蛋白表达水平均明显低于STZ-ApoE-/-组(P < 0.05);STZ-ApoE-/-+6-MP10组与STZ-ApoE-/-+6-MP5组之间胸主动脉NF-κB p65/CylinD1蛋白表达水平差异均有统计学意义(P < 0.05)。油红O染色显示:TR3受体激动剂6-MP可呈剂量依赖性抑制STZ-ApoE-/-小鼠胸主动脉内膜脂质沉积;HE染色显示:TR3受体激动剂6-MP可呈剂量依赖性降低STZ-ApoE-/-小鼠胸主动脉内膜斑块面积,减少斑块内部有空洞(脂质)形成,抑制斑块周围组织及斑块基底部平滑肌层增生,与STZ-ApoE-/-组相比,STZ-ApoE-/-+6-MP5组胸主动脉内膜斑块面积明显减小(P < 0.05),STZ-ApoE-/-6-MP10组斑块面积进一步减小(P < 0.01)。 结论TR3激动剂6-MP可明显降低糖尿病ApoE-/-小鼠动脉粥样硬化的形成,其机制可能与其改善糖脂代谢,抑制NF-κB p65/CylinD1信号通路有关。 Abstract:ObjectiveTo observe the effects of TR3 receptor agonist 6-mercaptopurine(6-MP) on the glucose and lipid metabolism and NF-κB p65/CylinD1 pathway in streptozotocin (STZ)-induced diabetic apolipoprotein E knockout(ApoE-/-) mice, and explore the possible mechanism of TR3 receptors in improving diabetic atherosclerosis. MethodsForty ApoE-/- mice were randomly divided into the ApoE-/- group, STZ-ApoE-/- group, STZ-ApoE-/- +6-MP5(5 mg·kg-1·d-1) and STZ-ApoE-/- +6-MP10(10 mg·kg-1·d-1) group(10 rats each group).The 6-MP intraperitoneal injection was implemented, once a day, for 8 weeks.The diabetic animal model was established using intraperitoneal injection of STZ.The levels of blood glucose and blood lipid were determined using the blood glucose test strip and the enzymic method or selective homogeneous method, respectively.The Western-blotting was used to detect the protein levels of NF-κB p65/CylinD1 in thoracic aorta tissue, the lipid deposition in the thoracic aortic intima was observed by oil red O staining, and the patch area in the thoracic aorta was measured using HE staining. ResultsCompared with the ApoE-/- mice group, the levels of blood glucose, triglyceride(TG), total cholesterol(TCHO) and low-density lipoprotein-cholesterol(LDL-C) significantly increased in STZ-ApoE-/- group(P < 0.05), and the level of TR3 receptor agonist 6-MP decreased the levels of plasma glucose and lipid with dose-dependently.Comapred the STZ-ApoE-/- group, the levels of the blood glucose and TG, LDL-A and TC significantly decreased in the STZ-ApoE-/- +6-MP5 group(P < 0.05).Compared with the ApoE-/- group, the levels of NF-κB p65 and CylinD1 remarkably increased in the STZ-ApoE-/- group(P < 0.05).The 6-MP reduced the expression level of NF-κB p65/CylinD1 protein in thoracic aorta of diabetic ApoE-/- mice in a dose-dependent manner, and the protein level of NF-κB p65/CylinD1 in STZ-ApoE-/- +6-MP5 group were significantly lower than that in STZ-ApoE-/- group(P < 0.05).The differences of the levels of NF-κB p65/CylinD1 in thoracic aorta between STZ-ApoE-/- +6-MP10 group and STZ-ApoE-/- +6-MP5 group were statistically significnat(P < 0.05).The results of oil red O staining showed that 6-MP inhibited the intima lipid deposition in the thoracic aorta of STZ-ApoE-/- mice in a dose-dependent manner.The results of HE staining showed that 6-MP reduced the area of intima plaque and formation of vacuoles(lipids) inside plaque, and inhibited the proliferation of smooth muscle layer around plaque and plaque base in the thoracic aorta of STZ-ApoE-/- mice in a dose-dependent manner.Compared with the STZ-ApoE-/- group, the plaque area in the STZ-ApoE-/- +6-MP5 group significantly reduced(P < 0.05), and further reduced in the STZ-ApoE-/- 6-MP10 group(P < 0.01). ConclusionsThe TR3 receptor agonist 6-MP can significantly decrease the formation of atherosclerosis in diabetic ApoE-/- mice, which may be related to the improvement of glycolipid metabolism and inhibition of NF-κB p65/CylinD1 signaling pathway. -
Key words:
- diabetes mellitus /
- ApoE-/- mice /
- 6-mercaptopurine /
- atherosclerosis /
- blood lipid /
- TR3 /
- NF-κB p65/CylinD1
-
表 1 TR3受体激动剂6-MP对糖尿病ApoE-/-小鼠血糖的影响(x±s)
分组 n 血糖/(mmol/L) ApoE-/-组 10 6.91±0.69 STZ-ApoE-/-组 10 18.68±2.30** STZ-ApoE-/-+6-MP5组 10 15.42±2.68**# STZ-ApoE-/-+6-MP10组 10 13.64±1.95**## F — 40.07 P — <0.01 MS组内 — 3.090 q检验:与ApoE-/-组比较**P<0.01;与STZ-ApoE组比较#P<0.05, ##P<0.01 表 2 TR3受体激动剂6-MP对糖尿病ApoE-/-小鼠血糖和血脂的影响(ni=10;x±s;mmol/L)
分组 TG TC HDL-C LDL-C ApoE-/-组 5.28±0.52 41.40±3.31 14.04±2.21 32.92±3.83 STZ-ApoE-/-组 5.60±0.43 62.04±7.12** 16.20± 4.83 50.96±6.83** STZ-ApoE-/-+6-MP5组 5.05±1.34 47.70±6.46# 15.95±0.98 42.50±4.92**## STZ-ApoE-/-+6-MP10组 4.57±1.45 40.82±5.19## 16.18±1.76 38.45±5.11**## F 1.73 29.91 1.34 20.67 P >0.05 <0.01 >0.05 <0.01 MS组内 1.088 32.580 8.068 27.909 q检验:与ApoE-/-组比较*P<0.05,**P<0.01;与STZ-ApoE-/-组比较#P<0.05,##P<0.01 表 3 TR3受体激动剂6-MP对糖尿病ApoE-/-小鼠胸主动脉NF-κB p65/CylinD1蛋白表达的影响(x±s)
分组 n NF-κB p65 Cylin D1 ApoE-/-组 10 0.053±0.008 0.234±0.026 STZ-ApoE-/-组 10 0.358±0.046** 0.617±0.107** STZ-ApoE-/-+6-MP5组 10 0.213±0.025**## 0.226±0.031## STZ-ApoE-/-+6-MP10组 10 0.022±0.003**## 0.109±0.013**## F — 343.51 147.62 P — <0.01 <0.01 MS组内 — 0.001 0.003 注:蛋白印迹电泳胶片条带光密度(OD值)分析,NF-κB p65或CylinD1/β-actin光密度比值表示目的蛋白表达水平。q检验:与ApoE-/-组比较*P<0.05,**P<0.01;与STZ-ApoE-/-组比较#P<0.05,##P<0.01 表 4 TR3受体激动剂6-MP对糖尿病ApoE-/-小鼠胸主动脉内膜内膜粥样斑块面积的影响(x±s)
分组 n 斑块面积/μm2 ApoE-/-组 10 123.56±15.38 STZ-ApoE-/-组 10 245.16±30.29** STZ-ApoE-/-+6-MP5组 10 172.43±20.55**## STZ-ApoE-/-+6-MP10组 10 154.09±18.73**## F — 55.40 P — <0.01 MS组内 — 481.786 q检验:与ApoE-/-组比较*P<0.05,**P<0.01;STZ-ApoE-/-组比较#P<0.05,##P<0.01 -
[1] 赵冬.中国人群的血脂流行病学研究[J].临床荟萃, 2006, 21(8):533. doi: 10.3969/j.issn.1004-583X.2006.08.001 [2] JAISWAL M, SCHINSKE A, POP-BUSUI R.Lipids and lipid management in diabetes[J].Best Pract Res Clin Endocrinol Metab, 2014, 28(3):325. doi: 10.1016/j.beem.2013.12.001 [3] LI Y, BOURBON PM, GRANT MA, et al.Requirement of novel amino acid fragments of orphan nuclear receptor TR3/Nur77 for its functions in angiogenesis[J].Oncotarget, 2015, 6(27):24261. [4] HARANT H.Negative cross-talk between the human orphan nuclear receptor Nur77/NAK-1/TR3 and nuclear factor-κB[J].Nucleic Acids Res, 2004, 32(17):5280. doi: 10.1093/nar/gkh856 [5] WU L, CHEN L.Characteristics of Nur77 and its ligands as potential anticancer compounds(Review)[J].Mol Med Rep, 2018, 18(6):4793. [6] FEODOROFF M, HARJUTSALO V, FORSBLOM C, et al.Dose-dependent effect of smoking on risk of coronary heart disease, heart failure and stroke in individuals with type 1 diabetes[J].Diabetologia, 2018, 61(12):2580. doi: 10.1007/s00125-018-4725-9 [7] NEWMAN JD, ROCKMAN CB, KOSIBOROD M, et al.Diabetes mellitus is a coronary heart disease risk equivalent for peripheral vascular disease[J].Am Heart J, 2017, 184:114. doi: 10.1016/j.ahj.2016.09.002 [8] WAARD VD, ARKENBOUT EK, VOS M, et al.TR3 nuclear orphan receptor prevents cyclic stretch-induced proliferation of venous smooth muscle cells[J].Am J Pathol, 2006, 168(6):2027. doi: 10.2353/ajpath.2006.050932 [9] ZHAO Y, BRUEMMER D.NR4A orphan nuclear receptors:transcriptional regulators of gene expression in metabolism and vascular biology[J].Arterioscler Thromb Vasc Biol, 2010, 30(8):1535. doi: 10.1161/ATVBAHA.109.191163 [10] MIN AK, BAE KH, JUNG YA, et al.Orphan nuclear receptor Nur77 mediates fasting-induced hepatic fibroblast growth factor 21 expression[J].Endocrinology, 2014, 155(8):2924. doi: 10.1210/en.2013-1758 [11] CHAO LC, WROBLEWSKI K, ILKAYEVA OR, et al.Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization[J].J Lipid Res, 2012, 53(12):2610. doi: 10.1194/jlr.M029355 [12] DE VERA IM, GIRI PK, MUNOZ-TELLO P, et al.Identification of a Binding Site for Unsaturated Fatty Acids in the Orphan Nuclear Receptor Nurr1[J].ACS Chem Biol, 2016, 11(7):1795. doi: 10.1021/acschembio.6b00037 [13] FENG M, KONG SZ, WANG ZX, et al.The protective effect of coptisine on experimental atherosclerosis ApoE-/- mice is mediated by MAPK/NF-κB-dependent pathway[J].Biomed Pharmacother, 2017, 93:721. doi: 10.1016/j.biopha.2017.07.002 [14] AL-SHOBAILI HA, FARHAN J, ZAFAR U, et al.Functional role of human interleukin-32 and nuclear transcription factor-kB in patients with psoriasis and psoriatic arthritis[J].Int J Health Sci, 2018, 12(3):29. [15] EID AH, GAD AM, FIKRY EM, et al.Venlafaxine and carvedilol ameliorate testicular impairment and disrupted spermatogenesis in rheumatoid arthritis by targeting AMPK/ERK and PI3K/AKT/mTOR pathways[J].Toxicol Appl Pharmacol, 2019, 364:83. doi: 10.1016/j.taap.2018.12.014 [16] GUILLERMIER C, DOHERTY SP, WHITNEY AG, et al.Imaging mass spectrometry reveals heterogeneity of proliferation and metabolism in atherosclerosis[J].JCI Insight, 2019, 4(11):12858. [17] 袁帅, 廖思聪, 王大新.血管平滑肌细胞与动脉粥样硬化关系的研究进展[J].实用心脑肺血管病杂志, 2018(2):6. doi: 10.3969/j.issn.1008-5971.2018.02.002 [18] 刘宇宏, 王莎莎, 沈凌汛, 等.IL-32γ对大鼠血管平滑肌细胞增殖与细胞周期的影响[J].中国病理生理杂志, 2012, 28(2):239. doi: 10.3969/j.issn.1000-4718.2012.02.009