朝医麻黄定喘汤对支气管哮喘模型小鼠p38MAPK/Nrf2/HO-1信号通路的影响
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摘要
探讨朝医麻黄定喘汤治疗支气管哮喘的可能作用机制。方法将40只BABL/c小鼠随机分成空白组、模型组、麻黄定喘汤低剂量组、麻黄定喘汤高剂量组,每组10只。除空白组外,其余各组用卵清蛋白致敏、激发制备小鼠哮喘模型。麻黄定喘汤低剂量组每日给予4 ml/100 g麻黄定喘汤(浓度20 mg/ml)灌胃,麻黄定喘汤高剂量组每日给予12 ml/100 g麻黄定喘汤灌胃,空白组和模型组每日给予12 ml 0. 9%氯化钠溶液灌胃,共计8周。在末次激发24 h后取肺泡灌洗液(BALF)及肺组织。观察肺组织病理学变化;检测BALF中炎症细胞计数,活性氧(ROS)及丙二醛(MDA)含量,抗氧化酶超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性; Western Blot法检测肺组织中磷酸化p38核丝裂原活化蛋白激酶(p-p38 MAPK)、核因子E2相关因子2 (Nrf2)、血红素加氧酶1 (HO-1)蛋白水平;肺组织中Nrf2、HO-1 mRNA水平。结果与空白组比较,模型组BALF中炎症细胞总数、中性粒细胞和嗜酸性粒细胞及ROS、MDA含量,肺组织中p-p38 MAPK、Nrf2、HO-1蛋白水平及Nrf2、HO-1 mRNA表达水平明显升高,SOD、CAT活性明显降低(P <0. 05);与模型组比较,中药高、低剂量组小鼠BALF中炎症细胞总数、中性粒细胞和嗜酸性粒细胞显著降低,BALF中ROS、MDA含量,肺组织中p-p38 MAPK蛋白水平降低,BALF中SOD、CAT的活性及肺组织中Nrf2、HO-1蛋白表达,Nrf2、HO-1 mRNA表达升高(P <0. 05)。与麻黄定喘汤低剂量组比较,麻黄定喘汤高剂量组BALF中炎症细胞总数、中性粒细胞、嗜酸性粒细胞数及ROS、MDA含量,肺组织p-p38MAPK水平明显减少,SOD、CAT活性及Nrf2、HO-1蛋白和mRNA表达升高(P <0. 05)。结论朝医麻黄定喘汤可抑制支气管哮喘小鼠气道炎症反应,通过抗氧化机制影响p38 MAPK/Nrf2/HO-1信号通路可能是其机制之一。
Objective To investigate the possible mechanism of traditional Korean medicine Mahuang Dingchuan Decoction( 麻黄定喘汤) in treating bronchial asthma. Methods A total of 40 BABL/c mice were randomly divided into blank group,model group,Mahuang Dingchuan Decoction low dose group and high dose group,with 10 mice in each group. Except the blank group,asthma models were established by sensitization and challenge with ovalbumin( OVA). The low dose group was given 4 ml/100 g Mahuang Dingchuan Decoction( concentration 20 mg/ml) daily by gavage,and the high dose group was given 12 ml/100 g Mahuang Dingchuan Decoction daily by gavage.The mice in the blank group and the model group were given intraperitoneal injection of 0. 9% sodium chloride solution for a total of 8 weeks. The brocho-alveolar larage fluid( BALF) and lung tissue were collected 24 h after the last challenge. The pathological changes were observed. The contents of inflammatory cytokines,reactive oxygen species( ROS),malondialdehyde( MDA) content,superoxide dismutase( SOD),and catalase( CAT) activity in the BALF were detected. The phosphorylation of p38 nuclear mitogen-activated protein kinase( p-p38 MAPK),nuclear factor E2 related factor( Nrf2),heme oxygenase 1( HO-1) levels in the lung tissue were detected by Western Blot.Results Compared with the blank group,the inflammatory cells count,neutrophils and eosinophils,ROS and MDA in the BALF,p-p38 MAPK,Nrf2,HO-1 protein levels and the expression level of Nrf2,HO-1 mRNA in lung tissue of the model group were significantly increased,and SOD and CAT activities were significantly decreased( P <0. 05). Compared with the model group,the inflammatory cells count,neutrophils and eosinophils in BALF of the high-and low-dose group were significantly decreased. The levels of ROS,MDA in the BALF,the level of p-p38 MAPK protein in lung tissue decreased. The activity of SOD and CAT in BALF and the expression of Nrf2 and HO-1 protein in lung tissue,and the expression of Nrf2 and HO-1 mRNA increased( P < 0. 05). Compared with the low dose group of Mahuang Dingchuan Decoction,the inflammatory cells count,neutrophils,eosinophils,ROS and MDA in BALF,p-p38 MAPK level in lung tissue of high dose group of Mahuang Dingchuan Decoction group was significantly reduced,and SOD CAT activity and Nrf2,HO-1 protein and mRNA expression were increased( P < 0. 05).Conclusion Traditional Korean medicine Mahuang Dingchuan Decoction can inhibit the airway inflammation in mice with bronchial asthma,of which affecting p38 MAPK/Nrf2/HO-1 signaling pathways by anti-oxidation might be one of the mechanisms.
Objective To investigate the possible mechanism of traditional Korean medicine Mahuang Dingchuan Decoction( 麻黄定喘汤) in treating bronchial asthma. Methods A total of 40 BABL/c mice were randomly divided into blank group,model group,Mahuang Dingchuan Decoction low dose group and high dose group,with 10 mice in each group. Except the blank group,asthma models were established by sensitization and challenge with ovalbumin( OVA). The low dose group was given 4 ml/100 g Mahuang Dingchuan Decoction( concentration 20 mg/ml) daily by gavage,and the high dose group was given 12 ml/100 g Mahuang Dingchuan Decoction daily by gavage.The mice in the blank group and the model group were given intraperitoneal injection of 0. 9% sodium chloride solution for a total of 8 weeks. The brocho-alveolar larage fluid( BALF) and lung tissue were collected 24 h after the last challenge. The pathological changes were observed. The contents of inflammatory cytokines,reactive oxygen species( ROS),malondialdehyde( MDA) content,superoxide dismutase( SOD),and catalase( CAT) activity in the BALF were detected. The phosphorylation of p38 nuclear mitogen-activated protein kinase( p-p38 MAPK),nuclear factor E2 related factor( Nrf2),heme oxygenase 1( HO-1) levels in the lung tissue were detected by Western Blot.Results Compared with the blank group,the inflammatory cells count,neutrophils and eosinophils,ROS and MDA in the BALF,p-p38 MAPK,Nrf2,HO-1 protein levels and the expression level of Nrf2,HO-1 mRNA in lung tissue of the model group were significantly increased,and SOD and CAT activities were significantly decreased( P <0. 05). Compared with the model group,the inflammatory cells count,neutrophils and eosinophils in BALF of the high-and low-dose group were significantly decreased. The levels of ROS,MDA in the BALF,the level of p-p38 MAPK protein in lung tissue decreased. The activity of SOD and CAT in BALF and the expression of Nrf2 and HO-1 protein in lung tissue,and the expression of Nrf2 and HO-1 mRNA increased( P < 0. 05). Compared with the low dose group of Mahuang Dingchuan Decoction,the inflammatory cells count,neutrophils,eosinophils,ROS and MDA in BALF,p-p38 MAPK level in lung tissue of high dose group of Mahuang Dingchuan Decoction group was significantly reduced,and SOD CAT activity and Nrf2,HO-1 protein and mRNA expression were increased( P < 0. 05).Conclusion Traditional Korean medicine Mahuang Dingchuan Decoction can inhibit the airway inflammation in mice with bronchial asthma,of which affecting p38 MAPK/Nrf2/HO-1 signaling pathways by anti-oxidation might be one of the mechanisms.
引文
[1]LI Z,WANG H,LIU L,et al.Interleukin-25 Enhances allergic inflammation through p38MAPK and NF-kappaBpathways in mouse models of allergic rhinitis[J].Iran JAllergy Asthma Immunol,2014,13(6):412-419.
[2]LI LC,PIAO HM,ZHENG MY,et al.Ginsenoside Rh2attenuates allergic airway inflammation by modulating nuclear factorκB activation in a murine model of asthma[J].Mol Med Rep,2015,12(5):6946-6954.
[3]秦钰,郝华,洪天一,等.麻黄定喘汤对咳嗽变异性哮喘模型小鼠Th1/Th2类细胞因子水平的影响[J].延边大学医学学报,2011,34(4):262-264.
[4]李京玉,冯晓纯,金京丽,等.朝医加味麻黄定喘汤在小鼠哮喘模型中对ERK信号通路的影响[J].中华中医药杂志,2013,28(12):3744-3747.
[5]张鑫,朴宇,郑明昱.鹿茸大补汤对太阴人哮喘缓解期的疗效[J].长春中医药大学学报,2013,29(5):871-872.
[6]周鑫,王志强.Nrf2-ARE信号通路与哮喘的关系[J].广东医学,2011,32(17):2348-2349.
[7]谢建林,林明宝,侯琦.核转录因子Nrf2与肺部炎症疾病研究进展[J].药学学报,2015,50(9):1080-1087.
[8]宋冬梅,牛英豪,于磊,等.王宝山乌司他丁通过Nrf2/HO-1抗氧化途径在OVA诱导的支气管哮喘小鼠中发挥治疗作用[J].中国药理学通报,2014,30(12):1713-1720.
[9]阮晓琳,周星星,彭来君,等.气阴双补中药对哮喘大鼠肺组织TGF-β1及Smad2/3表达的影响[J].中华中医药杂志,2017,32(3):1271-1275.
[10]NADEEM A,MASOOD A,SIDDIQUI N.Oxidant-antioxidant imbalance in asthma:scientific evidence,epidemiological data and possible therapeutic options[J].Ther Adv Respir Dis,2008,2(4):215-235.
[11]王金磊,李承德,孙宏伟,等.黄芪多糖抑制NF-κB/MAPK信号通路和改善哮喘大鼠气道炎症的作用[J].中国药理学通报,2016,32(4):489-493.
[12]DONOVAN CE,MARK DA,HE HZ,et al.NF-κB/Rel transcription factors:c-Rel promotes airway hyperresponsiveness and allergic pulmonary inflammation[J].J Immunol,1999,163(12):6827-6833.
[13]KASPAR JW,NITURE SK,JAISWAL AK.Nrf2:INrf2(Keap1)signaling in oxidative stress[J].Free Radic Biol Med,2009,47(9):1304-1309.
[14]MANEECHOTESUWAN K,XIN Y,ITO K,et al.Regulation of Th2 cytokine genes by p38 MAPK-mediated phosphory lation of GATA-3[J].J Immunol,2007,178(4):2491-2498.
[15]万晓红,王雁,赵国良,等.转导血红素氧合酶-1蛋白减轻脑缺血/再灌注沙土鼠海马神经元的损伤[J].中国药理学通报,2014,30(5):628-632.
[16]SONG DM,LIU G,NIU YH,et al.Anti-oxidative effect of Ulinastatin and potential role of heme oxygenase-1 in an ovalbumin-induced murine asthma model[J].Chin J Tubercul Respir Dis,2013,36(3):225-226.
[17]KARNAD DR,BHADADE R,VERMA PK,et al.Intravenous administration of ulinastatin(human urinary trypsin inhibitor)in severe sepsis:a multicenter randomized controlled study[J].Intensive Care Med,2014,40(6):830-835.
[18]袁红艳,张晔,王丹,等.柞树皮对脂多糖和D-氨基半乳糖诱导败血症休克小鼠的保护作用[J].时珍国医国药,2011,22(12):2886-2887.
[19]WANG Z,ZHANG H,SUN X,et al.The protective role of vitamin D3 in a murine model of asthma via the suppression of TGF-beta/Smad signaling and activation of the Nrf2/HO-1 pathway[J].Mol Med Rep,2016,14(3):2389-2396.
[20]宋冬梅,刘刚,牛英豪,等.乌司他丁对支气管哮喘小鼠氧化应激及血红素加氧酶-1的影响[J].中华结核和呼吸杂志,2013,36(3):225-226.
[21]JAISWAL AK.Nrf2 signaling in coordinated activation of antioxidant gene expression[J].Free Radic Biol Med,2004,36(10):1199-1207.
[2]LI LC,PIAO HM,ZHENG MY,et al.Ginsenoside Rh2attenuates allergic airway inflammation by modulating nuclear factorκB activation in a murine model of asthma[J].Mol Med Rep,2015,12(5):6946-6954.
[3]秦钰,郝华,洪天一,等.麻黄定喘汤对咳嗽变异性哮喘模型小鼠Th1/Th2类细胞因子水平的影响[J].延边大学医学学报,2011,34(4):262-264.
[4]李京玉,冯晓纯,金京丽,等.朝医加味麻黄定喘汤在小鼠哮喘模型中对ERK信号通路的影响[J].中华中医药杂志,2013,28(12):3744-3747.
[5]张鑫,朴宇,郑明昱.鹿茸大补汤对太阴人哮喘缓解期的疗效[J].长春中医药大学学报,2013,29(5):871-872.
[6]周鑫,王志强.Nrf2-ARE信号通路与哮喘的关系[J].广东医学,2011,32(17):2348-2349.
[7]谢建林,林明宝,侯琦.核转录因子Nrf2与肺部炎症疾病研究进展[J].药学学报,2015,50(9):1080-1087.
[8]宋冬梅,牛英豪,于磊,等.王宝山乌司他丁通过Nrf2/HO-1抗氧化途径在OVA诱导的支气管哮喘小鼠中发挥治疗作用[J].中国药理学通报,2014,30(12):1713-1720.
[9]阮晓琳,周星星,彭来君,等.气阴双补中药对哮喘大鼠肺组织TGF-β1及Smad2/3表达的影响[J].中华中医药杂志,2017,32(3):1271-1275.
[10]NADEEM A,MASOOD A,SIDDIQUI N.Oxidant-antioxidant imbalance in asthma:scientific evidence,epidemiological data and possible therapeutic options[J].Ther Adv Respir Dis,2008,2(4):215-235.
[11]王金磊,李承德,孙宏伟,等.黄芪多糖抑制NF-κB/MAPK信号通路和改善哮喘大鼠气道炎症的作用[J].中国药理学通报,2016,32(4):489-493.
[12]DONOVAN CE,MARK DA,HE HZ,et al.NF-κB/Rel transcription factors:c-Rel promotes airway hyperresponsiveness and allergic pulmonary inflammation[J].J Immunol,1999,163(12):6827-6833.
[13]KASPAR JW,NITURE SK,JAISWAL AK.Nrf2:INrf2(Keap1)signaling in oxidative stress[J].Free Radic Biol Med,2009,47(9):1304-1309.
[14]MANEECHOTESUWAN K,XIN Y,ITO K,et al.Regulation of Th2 cytokine genes by p38 MAPK-mediated phosphory lation of GATA-3[J].J Immunol,2007,178(4):2491-2498.
[15]万晓红,王雁,赵国良,等.转导血红素氧合酶-1蛋白减轻脑缺血/再灌注沙土鼠海马神经元的损伤[J].中国药理学通报,2014,30(5):628-632.
[16]SONG DM,LIU G,NIU YH,et al.Anti-oxidative effect of Ulinastatin and potential role of heme oxygenase-1 in an ovalbumin-induced murine asthma model[J].Chin J Tubercul Respir Dis,2013,36(3):225-226.
[17]KARNAD DR,BHADADE R,VERMA PK,et al.Intravenous administration of ulinastatin(human urinary trypsin inhibitor)in severe sepsis:a multicenter randomized controlled study[J].Intensive Care Med,2014,40(6):830-835.
[18]袁红艳,张晔,王丹,等.柞树皮对脂多糖和D-氨基半乳糖诱导败血症休克小鼠的保护作用[J].时珍国医国药,2011,22(12):2886-2887.
[19]WANG Z,ZHANG H,SUN X,et al.The protective role of vitamin D3 in a murine model of asthma via the suppression of TGF-beta/Smad signaling and activation of the Nrf2/HO-1 pathway[J].Mol Med Rep,2016,14(3):2389-2396.
[20]宋冬梅,刘刚,牛英豪,等.乌司他丁对支气管哮喘小鼠氧化应激及血红素加氧酶-1的影响[J].中华结核和呼吸杂志,2013,36(3):225-226.
[21]JAISWAL AK.Nrf2 signaling in coordinated activation of antioxidant gene expression[J].Free Radic Biol Med,2004,36(10):1199-1207.
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