养阴方对HSP65攻击下ApoE~(-/-)小鼠Th17、Treg细胞失衡的作用研究
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摘要
目的考察在热休克蛋白(HSP)65攻击下养阴方对载脂蛋白E敲除(ApoE~(-/-))小鼠动脉粥样硬化(AS)的病变及辅助性T细胞17(Th17)、调节性T细胞(Treg)的影响及可能机制。方法将ApoE~(-/-)小鼠分别饲喂普通和高脂饲料,并分别sc无菌PBS或HSP65,以辛伐他汀为阳性药,研究养阴方对AS模型小鼠血脂、斑块、Th17细胞与Treg细胞比例(Th17/Treg)及相关炎症指标的影响。给药方式皆为ig给药,并以等体积生理盐水做对照。采用酯酶法检测血清总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白(LDL)及高密度脂蛋白(HDL)水平;油红O染色法测定主动脉斑块面积;流式细胞术测定抗凝血中IL-17A~+CD4~+Th17及Foxp3~+CD25~+CD4~+Treg细胞数量;ELISA法测定血清白细胞介素-6(IL-6)、IL-17、IL-2、转化生长因子-β(TGF-β)、IL-10水平;实时荧光定量PCR(q RT-PCR)法测定脾脏中Stat3、Stat5a、Foxp3和主动脉中Stat3、Stat5a mRNA表达;Western blotting法测定脾脏中磷酸化STAT3(pSTAT3)、磷酸化STAT5(pSTAT5)、FOXP3和主动脉中pSTAT3蛋白表达。结果养阴方使小鼠血清TC、TG、LDL水平显著降低(P<0.01),HDL显著升高(P<0.05);主动脉斑块面积明显减小(P<0.01);外周血IL-17A~+CD4~+Th17细胞显著减少(P<0.05),Foxp3~+CD25~+CD4~+Treg显著增加(P<0.05),Th17/Treg值大幅下降(P<0.01);血清IL-6水平显著下降(P<0.05),IL-2(P<0.01)、TGF-β(P<0.05)、IL-10(P<0.05)水平显著升高;脾脏pSTAT3蛋白水平下降(P<0.01),pSTAT5(P<0.01)和FOXP3(P<0.05)蛋白水平上升;主动脉Stat3 mRNA(P<0.01)和pSTAT3蛋白(P<0.05)水平下降。结论养阴方能够改善HSP65攻击下ApoE~(-/-)小鼠的高脂血症及AS,同时调节外周血中Th17/Treg的平衡和炎症因子的分泌从而改善炎症。养阴方对Th17、Treg的效应可能与减弱的IL-6/pSTAT3和增强的IL-2/pSTAT5信号有关。
Objective To investigate the effects of Yangyin Recipe on atherosclerosis(AS) lesions, helper T cells 17(Th17), and regulatory T cells(Treg) in apolipoprotein E knockout(ApoE~(-/-)) mice under the heat shock protein(HSP) 65 attack, and explore the potential mechanisms. Methods ApoE~(-/-)mice were fed with normal and high-fat diets respectively, and injected with sterile PBS or HSP65. Simvastatin was used as the positive drug to study the effects of Yangyin Recipe on blood lipids, plaques, Th17/Treg cells and related inflammation indicators in AS model mice. Drugs were all intragastric administrated with equal volume of normal saline as a control. Serum total cholesterol(TC), triglyceride(TG), low-density lipoprotein(LDL) and high-density lipoprotein(HDL) were detected by esterase method; The area of aortic plaque was determined by oil red O staining; The amounts of IL-17 A~+CD4~+ Th17 and Foxp3~+CD25~+CD4~+ Treg cells in anticoagulated blood were determined by flow cytometry; Serum IL-6, IL-17, IL-2, TGF-β, and IL-10 were determined by ELISA; mRNA expression of Stat3, Stat5 a, Foxp3 in spleens and Stat3, Stat5 a in aortas were detected by qPCR;Protein expression of phosphorylated STAT3(pSTAT3), phosphorylated STAT5(pSTAT5), FOXP3 in spleen, and pSTAT3 in aortas were determined by Western blotting. Results Under the influence of Yangyin Recipe, serum TC, TG, LDL levels decreased(P <0.01), and HDL increased(P < 0.05); Aortic plaque area decreased remarkably(P < 0.01); IL-17 A~+CD4~+Th17 cells in peripheral blood decreased(P < 0.05), Foxp3~+CD25~+CD4~+ Treg increased(P < 0.05), and Th17/Treg ratio decreased drastically(P < 0.01); Serum IL-6 decreased(P < 0.05), IL-2(P < 0.01), TGF-β and IL-10(P < 0.05) increased; Protein levels of pSTAT3 in spleens decreased(P < 0.01),but pSTAT5(P < 0.01) and FOXP3(P < 0.05) increased; Aortic Stat3 mRNA(P < 0.01) and pSTAT3(P < 0.05) protein decreased.Conclusion Yangyin Recipe can improve hyperlipidemia and AS in ApoE~(-/-)mice during HSP65 attack, and regulate the balance of Th17/Treg in peripheral blood and the secretion of inflammatory factors to improve inflammation, which may be related to attenuate IL-6/pSTAT3 and enhance IL-2/pSTAT5 signaling.
Objective To investigate the effects of Yangyin Recipe on atherosclerosis(AS) lesions, helper T cells 17(Th17), and regulatory T cells(Treg) in apolipoprotein E knockout(ApoE~(-/-)) mice under the heat shock protein(HSP) 65 attack, and explore the potential mechanisms. Methods ApoE~(-/-)mice were fed with normal and high-fat diets respectively, and injected with sterile PBS or HSP65. Simvastatin was used as the positive drug to study the effects of Yangyin Recipe on blood lipids, plaques, Th17/Treg cells and related inflammation indicators in AS model mice. Drugs were all intragastric administrated with equal volume of normal saline as a control. Serum total cholesterol(TC), triglyceride(TG), low-density lipoprotein(LDL) and high-density lipoprotein(HDL) were detected by esterase method; The area of aortic plaque was determined by oil red O staining; The amounts of IL-17 A~+CD4~+ Th17 and Foxp3~+CD25~+CD4~+ Treg cells in anticoagulated blood were determined by flow cytometry; Serum IL-6, IL-17, IL-2, TGF-β, and IL-10 were determined by ELISA; mRNA expression of Stat3, Stat5 a, Foxp3 in spleens and Stat3, Stat5 a in aortas were detected by qPCR;Protein expression of phosphorylated STAT3(pSTAT3), phosphorylated STAT5(pSTAT5), FOXP3 in spleen, and pSTAT3 in aortas were determined by Western blotting. Results Under the influence of Yangyin Recipe, serum TC, TG, LDL levels decreased(P <0.01), and HDL increased(P < 0.05); Aortic plaque area decreased remarkably(P < 0.01); IL-17 A~+CD4~+Th17 cells in peripheral blood decreased(P < 0.05), Foxp3~+CD25~+CD4~+ Treg increased(P < 0.05), and Th17/Treg ratio decreased drastically(P < 0.01); Serum IL-6 decreased(P < 0.05), IL-2(P < 0.01), TGF-β and IL-10(P < 0.05) increased; Protein levels of pSTAT3 in spleens decreased(P < 0.01),but pSTAT5(P < 0.01) and FOXP3(P < 0.05) increased; Aortic Stat3 mRNA(P < 0.01) and pSTAT3(P < 0.05) protein decreased.Conclusion Yangyin Recipe can improve hyperlipidemia and AS in ApoE~(-/-)mice during HSP65 attack, and regulate the balance of Th17/Treg in peripheral blood and the secretion of inflammatory factors to improve inflammation, which may be related to attenuate IL-6/pSTAT3 and enhance IL-2/pSTAT5 signaling.
引文
[1]Taleb S,Tedgui A,Mallat Z.IL-17 and Th17 cells in atherosclerosis:Subtle and Ctrlextual roles[J].Arterioscler Thromb Vasc Biol,2015,35(2):258-264.
[2]Melzer S,Ankri R,Fixler D,et al.Nanoparticle uptake by macrophages in vulnerable plaques for atherosclerosis diagnosis[J].J Biophot,2015,8(11/12):871-883.
[3]Rizza S,Copetti M,Cardellini M,et al.A score including ADAM17 substrates correlates to recurring cardiovascular event in subjects with atherosclerosis[J].Atherosclerosis,2015,239(2):459-464.
[4]田树平,李春平,宋翔,等.高血压患者前降支心肌桥与桥前段>50%狭窄的冠状动脉性心脏病发病相关性分析[J].中华医学杂志,2014,94(21):1601-1604.
[5]Horie N,Tateishi Y,Morikawa M,et al.Acute stroke with major intracranial vessel occlusion:Characteristics of cardioembolism and atherosclerosis-related in situ stenosis/occlusion[J].J Clin Neurosci,2016,32:24-29.
[6]Kim J B,Kim K,Lindsay M E.Risk of rupture or dissection in descending thoracic aortic aneurysm[J].Circulation,2015,132(17):1620-1629.
[7]Potekhina A V,Pylaeva E,Provatorov S,et al.Treg/Th17balance in stable CAD patients with different stages of coronary atherosclerosis[J].Atherosclerosis,2015,238(1):17-21.
[8]Zhu M,Mo H,Li D,et al.Th17/Treg imbalance induced by increased incidence of atherosclerosis in patients with systemic lupus erythematosus(SLE)[J].Clin Rheumatol,2013,32(7):1045-1052.
[9]Walecki M,Eisel F,Klug J,et al.Androgen receptor modulates Foxp3 expression in CD4+CD25+Foxp3+regulatory T-cells[J].Mol Biol Cell,2015,26(15):2845-2857.
[10]Dardalhon V,Awasthi A,Kwon H,et al.IL-4 inhibits TGF-beta-induced Foxp3+T cells and,together with TGF-beta,generates IL-9+IL-10+Foxp3(-)effector Tcells[J].Nat Immunol,2008,9(12):1347-1355.
[11]Bommireddy R,Ormsby I,Yin M,et al.TGF beta 1inhibits Ca2+-calcineurin-mediated activation in thymocytes[J].J Immunol,2003,170(7):3645-3652.
[12]Li M O,Wan Y Y,Flavell R A.T cell-produced transforming growth factor-beta1 controls T cell tolerance and regulates Th1-and Th17-cell differentiation[J].Immunity,2007,26(5):579-591.
[13]Ramesh R,Kozhaya L,Mc Kevitt K,et al.Pro-inflammatory human Th17 cells selectively express P-glycoprotein and are refractory to glucocorticoids[J].JExp Med,2014,211(1):89-104.
[14]Yin M,Liu Q,Yu L,et al.Downregulations of CD36 and Calpain-1,inflammation,and atherosclerosis by Simvastatin in apolipoprotein E knockout mice[J].J Vasc Res,2017,54(3):123-130.
[15]Liu M,Yu Y,Jiang H,et al.Simvastatin suppresses vascular inflammation and atherosclerosis in ApoE-/-mice by downregulating the HMGB1-RAGE axis[J].Acta Pharmacol Sin,2013,34(6):830-836.
[16]瞿文云,吴敏.糖尿病“瘀热致消”与炎症因子[J].河南中医,2014,34(9):1654-1656.
[17]张宏宁.自拟骨八方对股骨干骨折瘀热证及炎症介质影响的研究[D].广州:广州中医药大学,2010.
[18]王昀.凉血通瘀方对中风急性期瘀热阻窍证的热象炎症相关指标的研究[D].南京:南京中医药大学,2010.
[19]孙云霞.论热毒理论与动脉粥样硬化的相关性[J].辽宁中医药大学学报,2011,13(5):52-54.
[20]Grundtman C,Kreutmayer S B,Almanzar G,et al.Heat shock protein 60 and immune inflammatory responses in atherosclerosis[J].Arterioscler Thromb Vasc Biol,2011,31(5):960-968.
[21]Grundtman C,Wick G.The autoimmune concept of atherosclerosis[J].Curr Opin Lipidol,2011,22(5):327-334.
[22]Ross R.Atherosclerosis-an inflammatory disease[J].NEngl J Med,1999,340(2):115-126.
[23]Shah P K,Chyu K Y,Dimayuga P C,et al.Vaccine for atherosclerosis[J].Am Coll Cardiol,2014,64(25):2779-2791.
[24]Grundtman C,Kreutmayer S B,Almanzar G,et al.Heat shock protein 60 and immune inflammatory responses in atherosclerosis[J].Arterioscler Thromb Vasc Biol,2011,31(5):960-968.
[25]Grundtman C,Wick G.The autoimmune concept of atherosclerosis[J].Curr Opin Lipidol,2011,22(5):327-334.
[26]Jun L,Jie L,Dongping Y,et al.Effects of nasal immunization of multi-target preventive vaccines on atherosclerosis[J].Vaccine,2012,30(6):1029-1037.
[27]Xiong Q,Li J,Jin L.Nasal immunization with heat shock protein 65 attenuates atherosclerosis and reduces serum lipids in cholesterol-fed wild-type rabbits probably through different mechanisms[J].Immunol Lett,2009,125(1):40-45.
[28]Stefanadis C,Diamantopoulos L,Vlachopoulos C,et al.Thermal heterogeneity within human atherosclerotic coronary arteries detected in vivo.A new method of detection by application of a special thermography catheter[J].Circulation,1999,99(15):1965-1971.
[29]赖陈岑,王和生,赵建雄,等.制首乌提取物对兔动脉粥样硬化模型中ABCA1表达的影响[J].中药药理与临床,2015,31(6):87-89.
[30]何慧明,刘宇.黄精降脂方降血脂及抗动脉粥样硬化的实验研究[J].辽宁中医杂志,2005,32(2):168-169.
[31]张萍,刘丹,李友元.黄精多糖对动脉粥样硬化家兔血清IL-6及CRP的影响[J].医学临床研究,2006,23(7):1100-1101.
[32]Yao W,Huang C,Sun Q,et al.Tetrahydroxystilbene glucoside protects against oxidized LDL-induced endothelial dysfunction via regulating vimentin cytoskeleton and its colocalization with ICAM-1 and VCAM-1[J].Cell Physiol Biochem,2014,34(5):1442-1454.
[33]Wang Y,Xing F,Ye S,et al.Jagged-1 signaling suppresses the IL-6 and TGF-βtreatment-induced Th17cell differentiation via the reduction of RORγt/IL-17A/IL-17F/IL-23a/IL-12rb1[J].Sci Rep,2015,5:8234.
[34]Huss D J,Mehta D S,Sharma A,et al.In vivo maintenance of human regulatory T cells during CD25blockade[J].J Immunol,2015,194(1):84-92.
[35]Bailey-Bucktrout S L,Martinez-Llordella M,Zhou X,et al.Self-antigen-driven activation induces instability of regulatory T cells during an inflammatory autoimmune response[J].Immunity,2013,39(5):949-962.
[36]Jang Y N,Baik E J.JAK-STAT pathway and myogenic differentiation[J].JAKSTAT,2013,2(2):e23282.
[2]Melzer S,Ankri R,Fixler D,et al.Nanoparticle uptake by macrophages in vulnerable plaques for atherosclerosis diagnosis[J].J Biophot,2015,8(11/12):871-883.
[3]Rizza S,Copetti M,Cardellini M,et al.A score including ADAM17 substrates correlates to recurring cardiovascular event in subjects with atherosclerosis[J].Atherosclerosis,2015,239(2):459-464.
[4]田树平,李春平,宋翔,等.高血压患者前降支心肌桥与桥前段>50%狭窄的冠状动脉性心脏病发病相关性分析[J].中华医学杂志,2014,94(21):1601-1604.
[5]Horie N,Tateishi Y,Morikawa M,et al.Acute stroke with major intracranial vessel occlusion:Characteristics of cardioembolism and atherosclerosis-related in situ stenosis/occlusion[J].J Clin Neurosci,2016,32:24-29.
[6]Kim J B,Kim K,Lindsay M E.Risk of rupture or dissection in descending thoracic aortic aneurysm[J].Circulation,2015,132(17):1620-1629.
[7]Potekhina A V,Pylaeva E,Provatorov S,et al.Treg/Th17balance in stable CAD patients with different stages of coronary atherosclerosis[J].Atherosclerosis,2015,238(1):17-21.
[8]Zhu M,Mo H,Li D,et al.Th17/Treg imbalance induced by increased incidence of atherosclerosis in patients with systemic lupus erythematosus(SLE)[J].Clin Rheumatol,2013,32(7):1045-1052.
[9]Walecki M,Eisel F,Klug J,et al.Androgen receptor modulates Foxp3 expression in CD4+CD25+Foxp3+regulatory T-cells[J].Mol Biol Cell,2015,26(15):2845-2857.
[10]Dardalhon V,Awasthi A,Kwon H,et al.IL-4 inhibits TGF-beta-induced Foxp3+T cells and,together with TGF-beta,generates IL-9+IL-10+Foxp3(-)effector Tcells[J].Nat Immunol,2008,9(12):1347-1355.
[11]Bommireddy R,Ormsby I,Yin M,et al.TGF beta 1inhibits Ca2+-calcineurin-mediated activation in thymocytes[J].J Immunol,2003,170(7):3645-3652.
[12]Li M O,Wan Y Y,Flavell R A.T cell-produced transforming growth factor-beta1 controls T cell tolerance and regulates Th1-and Th17-cell differentiation[J].Immunity,2007,26(5):579-591.
[13]Ramesh R,Kozhaya L,Mc Kevitt K,et al.Pro-inflammatory human Th17 cells selectively express P-glycoprotein and are refractory to glucocorticoids[J].JExp Med,2014,211(1):89-104.
[14]Yin M,Liu Q,Yu L,et al.Downregulations of CD36 and Calpain-1,inflammation,and atherosclerosis by Simvastatin in apolipoprotein E knockout mice[J].J Vasc Res,2017,54(3):123-130.
[15]Liu M,Yu Y,Jiang H,et al.Simvastatin suppresses vascular inflammation and atherosclerosis in ApoE-/-mice by downregulating the HMGB1-RAGE axis[J].Acta Pharmacol Sin,2013,34(6):830-836.
[16]瞿文云,吴敏.糖尿病“瘀热致消”与炎症因子[J].河南中医,2014,34(9):1654-1656.
[17]张宏宁.自拟骨八方对股骨干骨折瘀热证及炎症介质影响的研究[D].广州:广州中医药大学,2010.
[18]王昀.凉血通瘀方对中风急性期瘀热阻窍证的热象炎症相关指标的研究[D].南京:南京中医药大学,2010.
[19]孙云霞.论热毒理论与动脉粥样硬化的相关性[J].辽宁中医药大学学报,2011,13(5):52-54.
[20]Grundtman C,Kreutmayer S B,Almanzar G,et al.Heat shock protein 60 and immune inflammatory responses in atherosclerosis[J].Arterioscler Thromb Vasc Biol,2011,31(5):960-968.
[21]Grundtman C,Wick G.The autoimmune concept of atherosclerosis[J].Curr Opin Lipidol,2011,22(5):327-334.
[22]Ross R.Atherosclerosis-an inflammatory disease[J].NEngl J Med,1999,340(2):115-126.
[23]Shah P K,Chyu K Y,Dimayuga P C,et al.Vaccine for atherosclerosis[J].Am Coll Cardiol,2014,64(25):2779-2791.
[24]Grundtman C,Kreutmayer S B,Almanzar G,et al.Heat shock protein 60 and immune inflammatory responses in atherosclerosis[J].Arterioscler Thromb Vasc Biol,2011,31(5):960-968.
[25]Grundtman C,Wick G.The autoimmune concept of atherosclerosis[J].Curr Opin Lipidol,2011,22(5):327-334.
[26]Jun L,Jie L,Dongping Y,et al.Effects of nasal immunization of multi-target preventive vaccines on atherosclerosis[J].Vaccine,2012,30(6):1029-1037.
[27]Xiong Q,Li J,Jin L.Nasal immunization with heat shock protein 65 attenuates atherosclerosis and reduces serum lipids in cholesterol-fed wild-type rabbits probably through different mechanisms[J].Immunol Lett,2009,125(1):40-45.
[28]Stefanadis C,Diamantopoulos L,Vlachopoulos C,et al.Thermal heterogeneity within human atherosclerotic coronary arteries detected in vivo.A new method of detection by application of a special thermography catheter[J].Circulation,1999,99(15):1965-1971.
[29]赖陈岑,王和生,赵建雄,等.制首乌提取物对兔动脉粥样硬化模型中ABCA1表达的影响[J].中药药理与临床,2015,31(6):87-89.
[30]何慧明,刘宇.黄精降脂方降血脂及抗动脉粥样硬化的实验研究[J].辽宁中医杂志,2005,32(2):168-169.
[31]张萍,刘丹,李友元.黄精多糖对动脉粥样硬化家兔血清IL-6及CRP的影响[J].医学临床研究,2006,23(7):1100-1101.
[32]Yao W,Huang C,Sun Q,et al.Tetrahydroxystilbene glucoside protects against oxidized LDL-induced endothelial dysfunction via regulating vimentin cytoskeleton and its colocalization with ICAM-1 and VCAM-1[J].Cell Physiol Biochem,2014,34(5):1442-1454.
[33]Wang Y,Xing F,Ye S,et al.Jagged-1 signaling suppresses the IL-6 and TGF-βtreatment-induced Th17cell differentiation via the reduction of RORγt/IL-17A/IL-17F/IL-23a/IL-12rb1[J].Sci Rep,2015,5:8234.
[34]Huss D J,Mehta D S,Sharma A,et al.In vivo maintenance of human regulatory T cells during CD25blockade[J].J Immunol,2015,194(1):84-92.
[35]Bailey-Bucktrout S L,Martinez-Llordella M,Zhou X,et al.Self-antigen-driven activation induces instability of regulatory T cells during an inflammatory autoimmune response[J].Immunity,2013,39(5):949-962.
[36]Jang Y N,Baik E J.JAK-STAT pathway and myogenic differentiation[J].JAKSTAT,2013,2(2):e23282.