基于Th17/Treg平衡探讨黄芩茎叶黄酮对病毒性心肌炎小鼠的保护作用
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摘要
目的探究黄芩茎叶黄酮通过辅助性T细胞17(Th17)/调节性T细胞(Treg)平衡对病毒性心肌炎(VM)小鼠的保护作用。方法将BALB/c小鼠随机分为正常组、模型组(腹腔注射CVB3病毒悬液)、黄芩茎叶黄酮低剂量组和高剂量组(200、400 mg/kg黄芩茎叶黄酮灌胃)、阳性组(腹腔注射1 mg/kg利巴韦林)。收集小鼠血液以及心脏组织,酶联免疫法检测肌酸激酶同工酶(CK-MB)、肌钙蛋白T(cTnT)、肌红蛋白(Mb)水平,心肌组织进行HE、Masson染色,细胞流式仪检测小鼠脾脏淋巴液中Th17、Treg;Western blot法测定心脏组织中白细胞介素-17(IL-17)、视黄酸相关孤儿受体(RORrt)、白细胞介素-10(IL-10)、叉头状转录因子-3(Foxp3)表达。结果与正常组相比,模型组小鼠左室射血分数(LVEF)、左心室短轴缩短率(LVFS)、左心室舒张期后壁厚度(LVPWDd)、左心室收缩期后壁厚度(LVPWDs)、Treg细胞比例、IL-10、Foxp3蛋白表达降低,左心室舒张期前壁厚度(LVAWd)、左心室收缩期前壁厚度(LVAWs)、左室舒张期内径(LVIDd)、左室收缩期内径(LVIDs)、CK-MB、c TnI、Hb水平、病理学评分、心肌胶原纤维比例、Th17细胞比例、IL-17、RORrt蛋白表达升高(P<0.05)。与模型组相比,阳性组、黄芩茎叶黄酮组LVEF、LVFS、LVPWDd、LVPWDs、Treg细胞比例、IL-10、Foxp3蛋白表达升高,LVAWd、LVAWs、LVIDd、LVIDs、CK-MB、cTnI、Hb水平、病理学评分、心肌胶原纤维比例、Th17细胞比例、IL-17、RORrt蛋白表达降低,具有剂量依赖性(P<0.05)。结论黄芩茎叶黄酮能够缓解VM小鼠心功能,降低炎症反应,其作用机制可能是通过影响Th17/Treg细胞免疫平衡。
Objective To explore the protective effect of Scutellaria baicalensis stem and leaf flavonoid on viral myocarditis(VM)mice by the T helper cells 17(Th17)/regulatory T cells(Treg)balance. Methods BALB/c mice were randomlydivided into normal group,model group(intraperitoneal injection of CVB3 virus),Scutellaria baicalensis stem and leaf lowdose group,high dose group(gavage 200,400 mg/kg Scutellaria baicalensis stem and leaf),and positive group(intraperitonealinjection of 1 mg/kg ribavirin). The blood and heart tissues of the mice were collected,and the levels of creatine kinaseisoenzyme(CK-MB),troponin T(cTnT),and myoglobin(Mb)were detected by enzyme-linked immunosorbent assay.Myocardial tissue was stained with HE and Masson. Th17 and Treg cells in mouse spleen lymph fluid were detected byflowcytomerty. Interleukin-17(IL-17),retinoic acid-related orphan receptor(RORrt),interleukin-10(IL-10),and fork-like transcription factor-3(Foxp3)in cardiac tissue were detected by Western blot analysis. Results Compared with thenormal group,the expression of left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),leftventricular posterior wall diameter diastole(LVPWDd),left ventricular posterior wall diameter at systole(LVPWDs),Tregcells,IL-10 and Foxp3 protein in model group decreased. left ventricular anterior wall diastolic(LVAWd),left ventricular endsystolic anterior wall thickness(LVAWs),left ventricular internal diastolic diameter(LVIDd),left ventricular systolic diameter(LVIDs),CK-MB,c TnI,Hb,pathology score,myocardial collagen fiber ratio,Th17 cell ratio,IL-17,RORrt proteinexpression increased(P<0.05). Compared with the model group,the expression of LVEF,LVFS,LVPWDd,LVPWDs,Treg cells,IL-10 and Foxp3 protein in the positive group and scutellaria stem leaf flavonoid group were increased. LVAWd,LVAWs,LVIDd,LVIDs,CK-MB,cTnI,Hb,pathology score,myocardial collagen fiber ratio,Th17 cell ratio,IL-17,RORrt protein expression were decreased in a dose-dependent manner(P<0.05). Conclusion The Scutellaria baicalensis stem and leaf flavonoid could relieve the heart function of VM mice and reduce the inflammatory response,the protective mechanism might be achieved by affecting the balance of Th17/Treg cells.
Objective To explore the protective effect of Scutellaria baicalensis stem and leaf flavonoid on viral myocarditis(VM)mice by the T helper cells 17(Th17)/regulatory T cells(Treg)balance. Methods BALB/c mice were randomlydivided into normal group,model group(intraperitoneal injection of CVB3 virus),Scutellaria baicalensis stem and leaf lowdose group,high dose group(gavage 200,400 mg/kg Scutellaria baicalensis stem and leaf),and positive group(intraperitonealinjection of 1 mg/kg ribavirin). The blood and heart tissues of the mice were collected,and the levels of creatine kinaseisoenzyme(CK-MB),troponin T(cTnT),and myoglobin(Mb)were detected by enzyme-linked immunosorbent assay.Myocardial tissue was stained with HE and Masson. Th17 and Treg cells in mouse spleen lymph fluid were detected byflowcytomerty. Interleukin-17(IL-17),retinoic acid-related orphan receptor(RORrt),interleukin-10(IL-10),and fork-like transcription factor-3(Foxp3)in cardiac tissue were detected by Western blot analysis. Results Compared with thenormal group,the expression of left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),leftventricular posterior wall diameter diastole(LVPWDd),left ventricular posterior wall diameter at systole(LVPWDs),Tregcells,IL-10 and Foxp3 protein in model group decreased. left ventricular anterior wall diastolic(LVAWd),left ventricular endsystolic anterior wall thickness(LVAWs),left ventricular internal diastolic diameter(LVIDd),left ventricular systolic diameter(LVIDs),CK-MB,c TnI,Hb,pathology score,myocardial collagen fiber ratio,Th17 cell ratio,IL-17,RORrt proteinexpression increased(P<0.05). Compared with the model group,the expression of LVEF,LVFS,LVPWDd,LVPWDs,Treg cells,IL-10 and Foxp3 protein in the positive group and scutellaria stem leaf flavonoid group were increased. LVAWd,LVAWs,LVIDd,LVIDs,CK-MB,cTnI,Hb,pathology score,myocardial collagen fiber ratio,Th17 cell ratio,IL-17,RORrt protein expression were decreased in a dose-dependent manner(P<0.05). Conclusion The Scutellaria baicalensis stem and leaf flavonoid could relieve the heart function of VM mice and reduce the inflammatory response,the protective mechanism might be achieved by affecting the balance of Th17/Treg cells.
引文
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[4]张凤玉,张蓓,沈若武,等.黄芩茎叶总黄酮调节EAE大鼠Th17/Treg平衡的研究[J].免疫学杂志,2016,32(10):884-889.Zhang FY,Zhang B,Shen RW,et al. The Scutellaria baicalensis stem-leaf total flavonoid regulate the balance of Th17/Treg in EAE rats[J]. Immunology Journal,2016,32(10):884-889.
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[7]付珊珊,陈琳琳,傅强,等.依那普利对小鼠心肌梗死后Th1/Th2、Treg/Th17的调节作用[J].热带医学杂志,2014,14(8):1000-1004,1029.Fu SS,Chen LL,Fu Q,et al. Study on regulatory effect of enalapril on Th1/Th2,Treg/Th17 of post myocardial infarction in mice[J]. J Trop Med,2014,14(8):1000-1004,1029.
[8]薛贻敏,林风辉,刘艳丽,等.白细胞介素17A对柯萨奇B3病毒性心肌炎小鼠病毒复制的影响及其机制[J].临床心血管病杂志,2017,33(7):688-693.Xue YM,Lin FH,Liu YL,et al.Effect and mechanism of interleukin-17A on viral replication in mice with coxsackievirus B3-induced viral myocarditis[J]. Journal of Clinical Cardiology,2017,33(7):688-693.
[9]陶莉莉,杨思进,白雪,等.心安颗粒对病毒性心肌炎小鼠IL-6,TNF-α及病毒复制的影响[J].中国实验方剂学杂志,2017,23(18):135-140.Tao LL,Yang SJ,Bai X,et al. Effect of Xin′an Granule on IL-6,TNF-αand viral replicationonrats with viral myocarditis[J].Chinese Journal of Experimental Traditional Medical Formulae,2017,23(18):135-140.
[10]彭易兰,刘云华,黄志芳,等.UV和HPLC法测定黄芩黄酮总苷元提取物中总黄酮和3种主要成分的含量[J].天然产物研究与开发,2015,27(7):1210-1214.Peng YL,Liu YH,Huang ZF,et al.Determination of total flavonoids and three main components in Scutellariae radix total Flavone aglycone extract by Ultraviolet Spectrophotometry and high-performance liquid chromatogaphy[J]. Natural Product Research and Development,2015,27(7):1210-1214.
[11]杨焱,王羽,崔晓雪,等. miRNA378对柯萨奇B3病毒感染心肌细胞凋亡、网腔钙结合蛋白、内质网应激及信号通路因子的作用[J].临床心血管病杂志,2016,32(12):1254-1257.Yang Y,Wang Y,Cui XX,et al. The effect of miRNA378 on the myocardial cells apoptosis,calumenin,endoplasmic reticulum stress and signaling pathway factor caused by Coxsackie virus[J]. Journal of Clinical Cardiology,2016,32(12):1254-1257.
[12]任应国,张保朝,贾东佩,等.黄岑苷对实验性自身免疫性脑脊髓炎小鼠的影响[J].中国比较医学杂志,2017,27(3):52-56.Ren YG,Zhang BC,Jia DP,et al. Protective effect of Baicalin on experimental autoimmune encephalomyelitis in mice[J].Chinese Journal of Comparative Medicine,2017,27(3):52-56.
[13] Patel DD,Kuchroo VK. Th17 cell pathway in human immunity:Lessons from genetics and therapeutic interventions[J].Immunity,2015,43(6):1040-1051.
[14] Choi HG, Kim WS, Back YW, et al. Mycobacterium tuberculosis RpfE promotes simultaneous Th1-and Th17-type Tcell immunity via TLR4-dependent maturation of dendritic cells[J]. Eur J Immunol,2015,45(7):1957-1971.
[15]雷茵,韦俊杰,唐玉兰,等.载脂蛋白E对实验性自身免疫脑脊髓炎IL-17、IL-6、TNF-α表达的影响[J].中华神经医学杂志,2016,15(5):433-438.Lei Y,Wei JJ,Tang YL,et al. Effect of apolipoprotein E on inflammation in mice with experimental autoimmune encephalomyelitis[J]. Chin J Neuromed,2016,15(5):433-438.
[16]钱红,刘理静.IL-17单克隆抗体对病毒性心肌炎小鼠的保护作用及其机制[J].细胞与分子免疫学杂志,2014,30(5):509-512.Qian H,Liu LJ. Protective effect and mechanism of IL-17monoclonal antibody on mice with viral myocarditis[J]. Chin J Cell Mol Immunol,2014,30(5):509-512.
[17] Takatori H,Kawashima H,Matsuki A,et al. Helios enhances Treg cell function in cooperation with Foxp3[J]. Arthritis Rheumatol,2015,67(6):1491-1502.
[18] Bao R, Hou J, Li Y, et al. Adenosine promotes Foxp3expression in Treg cells in sepsis model by activating JNK/AP-1pathway[J]. Am J Transl Res,2016,8(5):2284-2292.
[19]徐玲,吕仕超,张军平. Th17/Treg细胞失衡与自身免疫性心肌炎[J].中国免疫学杂志,2015,31(8):1129-1131,1137.Xu L, Lv SC, Zhang JP. Th17/Treg cell imbalance and autoimmune myocarditis[J]. Chinese Journal of Immunology,2015,31(8):1129-1131,1137.
[2]刘艳丽,伍伟锋.microRNA-451和辅助性T淋巴细胞17亚群在病毒性心肌炎小鼠中的变化及意义[J].广东医学,2014,35(17):2656-2658.Liu YL,Wu WF.Alteration and significance of microRNA-451and Th17 cells in mice with coxsackie virus induced myocarditis[J]. Guangdong Medical Journal,2014,35(17):2656-2658.
[3]蒋娜,廖雯婷,匡希斌.大黄素对病毒性心肌炎小鼠IL-23/IL-17炎症轴、Th17细胞及病毒复制的影响[J].南方医科大学学报,2014,34(3):373-378.Jiang N,Liao WT,Kuang XB.Effects of emodin on IL-23/IL-17inflammatory axis,Th17 cells and viral replication in mice with viral myocarditis[J]. J South Med Univ,2014,34(3):373-378.
[4]张凤玉,张蓓,沈若武,等.黄芩茎叶总黄酮调节EAE大鼠Th17/Treg平衡的研究[J].免疫学杂志,2016,32(10):884-889.Zhang FY,Zhang B,Shen RW,et al. The Scutellaria baicalensis stem-leaf total flavonoid regulate the balance of Th17/Treg in EAE rats[J]. Immunology Journal,2016,32(10):884-889.
[5]曹永,郝林,韩丛辉,等.五参二连颗粒对病毒性心肌炎小鼠免疫调节机制的研究[J].中国实验方剂学杂志,2014,20(19):142-145.Cao Y,Hao L,Han CH,et al. Immune regulation mechanism of Wushen Erlian Granules in mice with viral myocarditis[J].Chinese Journal of Experimental Traditional Medical Formulae,2014,20(19):142-145.
[6]刘振明,胡何节,方征东.树突状细胞调控Th17/Treg平衡在动脉粥样硬化中的作用[J].安徽医科大学学报,2015,50(4):562-564.Liu ZM,Hu HJ,Fang ZD. Dendritic cells regulate the role of Th17/Treg balance in atherosclerosis[J]. Acta Universitatis Medicinalis Anhui,2015,50(4):562-564.
[7]付珊珊,陈琳琳,傅强,等.依那普利对小鼠心肌梗死后Th1/Th2、Treg/Th17的调节作用[J].热带医学杂志,2014,14(8):1000-1004,1029.Fu SS,Chen LL,Fu Q,et al. Study on regulatory effect of enalapril on Th1/Th2,Treg/Th17 of post myocardial infarction in mice[J]. J Trop Med,2014,14(8):1000-1004,1029.
[8]薛贻敏,林风辉,刘艳丽,等.白细胞介素17A对柯萨奇B3病毒性心肌炎小鼠病毒复制的影响及其机制[J].临床心血管病杂志,2017,33(7):688-693.Xue YM,Lin FH,Liu YL,et al.Effect and mechanism of interleukin-17A on viral replication in mice with coxsackievirus B3-induced viral myocarditis[J]. Journal of Clinical Cardiology,2017,33(7):688-693.
[9]陶莉莉,杨思进,白雪,等.心安颗粒对病毒性心肌炎小鼠IL-6,TNF-α及病毒复制的影响[J].中国实验方剂学杂志,2017,23(18):135-140.Tao LL,Yang SJ,Bai X,et al. Effect of Xin′an Granule on IL-6,TNF-αand viral replicationonrats with viral myocarditis[J].Chinese Journal of Experimental Traditional Medical Formulae,2017,23(18):135-140.
[10]彭易兰,刘云华,黄志芳,等.UV和HPLC法测定黄芩黄酮总苷元提取物中总黄酮和3种主要成分的含量[J].天然产物研究与开发,2015,27(7):1210-1214.Peng YL,Liu YH,Huang ZF,et al.Determination of total flavonoids and three main components in Scutellariae radix total Flavone aglycone extract by Ultraviolet Spectrophotometry and high-performance liquid chromatogaphy[J]. Natural Product Research and Development,2015,27(7):1210-1214.
[11]杨焱,王羽,崔晓雪,等. miRNA378对柯萨奇B3病毒感染心肌细胞凋亡、网腔钙结合蛋白、内质网应激及信号通路因子的作用[J].临床心血管病杂志,2016,32(12):1254-1257.Yang Y,Wang Y,Cui XX,et al. The effect of miRNA378 on the myocardial cells apoptosis,calumenin,endoplasmic reticulum stress and signaling pathway factor caused by Coxsackie virus[J]. Journal of Clinical Cardiology,2016,32(12):1254-1257.
[12]任应国,张保朝,贾东佩,等.黄岑苷对实验性自身免疫性脑脊髓炎小鼠的影响[J].中国比较医学杂志,2017,27(3):52-56.Ren YG,Zhang BC,Jia DP,et al. Protective effect of Baicalin on experimental autoimmune encephalomyelitis in mice[J].Chinese Journal of Comparative Medicine,2017,27(3):52-56.
[13] Patel DD,Kuchroo VK. Th17 cell pathway in human immunity:Lessons from genetics and therapeutic interventions[J].Immunity,2015,43(6):1040-1051.
[14] Choi HG, Kim WS, Back YW, et al. Mycobacterium tuberculosis RpfE promotes simultaneous Th1-and Th17-type Tcell immunity via TLR4-dependent maturation of dendritic cells[J]. Eur J Immunol,2015,45(7):1957-1971.
[15]雷茵,韦俊杰,唐玉兰,等.载脂蛋白E对实验性自身免疫脑脊髓炎IL-17、IL-6、TNF-α表达的影响[J].中华神经医学杂志,2016,15(5):433-438.Lei Y,Wei JJ,Tang YL,et al. Effect of apolipoprotein E on inflammation in mice with experimental autoimmune encephalomyelitis[J]. Chin J Neuromed,2016,15(5):433-438.
[16]钱红,刘理静.IL-17单克隆抗体对病毒性心肌炎小鼠的保护作用及其机制[J].细胞与分子免疫学杂志,2014,30(5):509-512.Qian H,Liu LJ. Protective effect and mechanism of IL-17monoclonal antibody on mice with viral myocarditis[J]. Chin J Cell Mol Immunol,2014,30(5):509-512.
[17] Takatori H,Kawashima H,Matsuki A,et al. Helios enhances Treg cell function in cooperation with Foxp3[J]. Arthritis Rheumatol,2015,67(6):1491-1502.
[18] Bao R, Hou J, Li Y, et al. Adenosine promotes Foxp3expression in Treg cells in sepsis model by activating JNK/AP-1pathway[J]. Am J Transl Res,2016,8(5):2284-2292.
[19]徐玲,吕仕超,张军平. Th17/Treg细胞失衡与自身免疫性心肌炎[J].中国免疫学杂志,2015,31(8):1129-1131,1137.Xu L, Lv SC, Zhang JP. Th17/Treg cell imbalance and autoimmune myocarditis[J]. Chinese Journal of Immunology,2015,31(8):1129-1131,1137.