蜂毒素抑制博莱霉素诱导小鼠肺纤维化的机制研究
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摘要
目的:观察蜂毒素对博莱霉素诱导小鼠肺纤维化的干预作用。方法:70只SPF级C57BL/6小鼠被随机分为正常组10只和模型组、地塞米松组、蜂毒素低剂量组、蜂毒素中剂量组、蜂毒素高剂量组各12只。除正常组外,均采用气管穿刺注入博莱霉素(BLM)制备肺纤维化小鼠模型。从术后第1天开始,地塞米松组按3 mg/kg的剂量腹腔注射,蜂毒素低、中、高剂量组分别给予5μg/(kg·d)、10μg/(kg·d)、20μg/(kg·d),对照组和模型组给予等体积生理盐水灌胃,连续2周。分别于第7、第14天处死动物,收集小鼠外周血样本,通过ELISA方法检测血清转化生长因子(TGF-β1)、胶原蛋白I(Collagen I)、胶原蛋白III(Collagen III)、基质金属蛋白酶2(MMP2)和基质金属蛋白酶9(MMP9)的水平。取肺组织进行苏木精-伊红(HE)分析,Masson染色和羟脯氨酸(HYP)评估以观察组织病理学变化和胶原沉积。采用实时荧光定量(Realime PCR)法和蛋白兔疫印迹法(Western blot)观察各组大鼠肺组织TGF-β1、Smad2、Smad3等蛋白和基因的表达变化。结果:与对照组比较,模型组大鼠肺纤维化明显,HYP、TGF-β1、Collagen I、Collagen I的含量升高(P <0. 05),肺组织TGF-β1、Smad2、Smad3蛋白和基因表达升高(P <0. 05);与模型组比较,蜂毒素中高剂量组血清HYP、TGF-β1、Collagen I、Collagen I的含量下降(P <0. 05),肺组织TGF-β1、Smad2、Smad3蛋白和基因表达降低,差异有统计学意义(P <0. 05)。结论:蜂毒素可以减轻博来霉素诱导的小鼠肺纤维化的程度,其机制可能与抑制TGF-β1/Smads通路有关。
Objective: To observe the effects of melittin on inhibiting pulmonary fibrosis induced by bleomycin in rats. Methods:Seventy SPF grade C57 BL/6 rats were randomly divided into the normal group( n = 10) and the model group,dexamethasone group and melittin low dose group,melittin middle dose group and melittin high dose group( n = 12). In addition to the normal group,a rat model of pulmonary fibrosis was prepared by injecting bleomycin( BLM) into the trachea. From the first day after surgery,the dexamethasone group was intraperitoneally injected at a dose of 3 mg/kg,while the low,medium and high doses of melittin were administered to 5,10,and 20 μg/( kg·d) respectively. The control group and the model group were given an equal volume of saline for 2 weeks. The animals were sacrificed on the 7 th and 14 th day,respectively,peripheral blood samples of which were collected.ELISA was used to detect serum transforming growth factor( TGF-β1),collagen I( Collagen I),collagen III( Collagen III),and matrix metalloproteinase 2( MMP2) and the level of matrix metalloproteinase 9( MMP9). Lung tissue was taken for hematoxylin-eosin( HE) analysis,Masson staining and hydroxyproline( HYP) evaluation to observe histopathological changes and collagen deposition.The expressions of TGF-β1,Smad2,Smad3 and other proteins and genes in lung tissue of each group were observed by Real-time PCR and Western blot. Results: Compared with the control group,the pulmonary fibrosis was significantly increased in the model group. The contents of HYP,TGF-β1,Collagen I and Collagen I were increased( P < 0. 05),and the expression of TGF-β1,Smad2,Smad3 protein and gene in lung tissue was increased( P < 0. 05); Compared with the model group,the levels of serum HYP,TGF-β1,Collagen I,Collagen I in the high dose group of melittin decreased( P < 0. 05),and TGF-β1,Smad2,Smad3 protein in lung tissue and gene expression was decreased( P < 0. 05). There was no statistically significant difference in the low-dose group. Conclusion: Melittin can effectively reduce the degeneration of pulmonary fibrosis induced by bleomycin,and its mechanism may be related to the regulation of TGF-β1/Smads pathway.
Objective: To observe the effects of melittin on inhibiting pulmonary fibrosis induced by bleomycin in rats. Methods:Seventy SPF grade C57 BL/6 rats were randomly divided into the normal group( n = 10) and the model group,dexamethasone group and melittin low dose group,melittin middle dose group and melittin high dose group( n = 12). In addition to the normal group,a rat model of pulmonary fibrosis was prepared by injecting bleomycin( BLM) into the trachea. From the first day after surgery,the dexamethasone group was intraperitoneally injected at a dose of 3 mg/kg,while the low,medium and high doses of melittin were administered to 5,10,and 20 μg/( kg·d) respectively. The control group and the model group were given an equal volume of saline for 2 weeks. The animals were sacrificed on the 7 th and 14 th day,respectively,peripheral blood samples of which were collected.ELISA was used to detect serum transforming growth factor( TGF-β1),collagen I( Collagen I),collagen III( Collagen III),and matrix metalloproteinase 2( MMP2) and the level of matrix metalloproteinase 9( MMP9). Lung tissue was taken for hematoxylin-eosin( HE) analysis,Masson staining and hydroxyproline( HYP) evaluation to observe histopathological changes and collagen deposition.The expressions of TGF-β1,Smad2,Smad3 and other proteins and genes in lung tissue of each group were observed by Real-time PCR and Western blot. Results: Compared with the control group,the pulmonary fibrosis was significantly increased in the model group. The contents of HYP,TGF-β1,Collagen I and Collagen I were increased( P < 0. 05),and the expression of TGF-β1,Smad2,Smad3 protein and gene in lung tissue was increased( P < 0. 05); Compared with the model group,the levels of serum HYP,TGF-β1,Collagen I,Collagen I in the high dose group of melittin decreased( P < 0. 05),and TGF-β1,Smad2,Smad3 protein in lung tissue and gene expression was decreased( P < 0. 05). There was no statistically significant difference in the low-dose group. Conclusion: Melittin can effectively reduce the degeneration of pulmonary fibrosis induced by bleomycin,and its mechanism may be related to the regulation of TGF-β1/Smads pathway.
引文
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[6]Oyama Y,Enomoto N,Suzuki Y,et al.Evaluation of urinary desmosines as a noninvasive diagnostic biomarker in patients with idiopathic pleuroparenchymal fibroelastosis(PPFE)[J].Respiratory Medicine,2017,123:63-70.
[7]Sambataro G,Sambataro D.Letter to editor:“Interstitial pneumonia with autoimmune features:Clinical,radiologic and histological characteristics and outcome in a series of 57 patients”[J].Respiratory Medicine,2017,127:65-66.
[8]Hirano C,Ohshimo S,Horimasu Y,et al.FAM13A polymorphism as a prognostic factor in patients with idiopathic pulmonary fibrosis[J].Respiratory Medicine,2017,123:105-109.
[9]赵存刚,黄文海,王尊元,等.转化生长因子-β1,在特发性肺纤维化中的研究新进展[J].中国药学杂志,2016,51(5):341-345.
[10]Bomhard E M.Particle-induced Pulmonary Alveolar Proteinosis and Subsequent Inflammation and Fibrosis[J].Toxicologic Pathology,2017,45(3):389-401.
[11]姜岩,于功昌,张兴国.转化生长因子尹,及其介导的信号通路在百草枯致肺上皮间质转化中的作用[J].环境与职业医学,2017,34(8):729-733.
[12]Madri J A,Furthmayr H.Collagen polymorphism in the lung.An immunochemical study of pulmonary fibrosis[J].Human Pathology,1980,11(4):353-366.
[13]Yu Q,Stamenkovic I.Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis[J].Genes Dev,2000,14(2):163-76.
[14]张冰清,刘晓波.蜂毒的主要成分及药理作用的研究进展[J].药学研究,2016,35(3):172-174.
[15]Glttli A,Chandrasekhar I,van Gunsteren WF.A molecular dynamics study of the bee venom melittin in aqueous solution,in methanol,and inserted in a phospholipid bilayer[J].Eur Biophys J,2006,35(3):255-267.
[16]Kolb M,Margetts PJ,Galt T,et al.Transient transgene expression of decorin in the lung reduces the fibrotic response to bleomycin[J].Am J Respir Crit Care Med,2001,163(3 Pt 1):770-777.
[17]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,1993:33.
[18]Untergasser G,Gander R,Lilg C,et al.Profiling molecular targets of TGF-beta1 in prostate fibroblast-to-myofibroblast transdifferentiation[J].Mech Ageing Dev,2005,126(1):59-69.
[19]Inomata M,Kamio K,Azuma A,et al.Pirfenidone inhibits fibrocyte accumulation in the lungs in bleomycin-induced murine pulmonary fibrosis[J].Respir Res,2014,15:16.
[20]Xiong S,Guo R,Yang Z,et al.Treg depletion attenuates irradiationinduced pulmonary fibrosis by reducing fibrocyte accumulation,inducing Th17 response,and shifting IFN-γ,IL-12/IL-4,IL-5 balance[J].Immunobiology,2015,220(11):1284-1291.
[21]陈宝,李惠萍,范峰,等.三氧化二砷对博莱霉素致大鼠肺纤维化的影响及其作用机制[J].中国病理生理杂志,2010,26(1):65-66.
[22]Ahn J Y,Kim M H,Lim M J,et al.The inhibitory effect of ginsan on TGF-βmediated fibrotic process[J].Journal of Cellular Physiology,2011,226(5):1241-1247.
[23]Li L,Huang W,Li K,et al.Metformin attenuates gefitinib-induced exacerbation of pulmonary fibrosis by inhibition of TGF-βsignaling pathway[J].Oncotarget,2015,6(41):43605-43619.
[24]Yu W,Liu D,Liang C,et al.Sulfur Dioxide Protects Against Collagen Accumulation in Pulmonary Artery in Association With Downregulation of the Transforming Growth Factorβ1/Smad Pathway in Pulmonary Hypertensive Rats[J].J Am Heart Assoc,2016,5(10):e003910.
[25]Sriram N,Kalayarasan S,Manikandan R,et al.Epigallocatechin gallate attenuates fibroblast proliferation and excessive collagen production by effectively intervening TGF-β1 signalling[J].Clinical&Experimental Pharmacology&Physiology,2015,42(8):849-859.
[26]蔡后荣,郑培德.肺间质纤维化前胶原和肿瘤坏死因子α基因表达[J].中华结核和呼吸杂志,1998,21(12):753-755.
[27]García-de-Alba C,Becerril C,Ruiz V,et al.Expression of matrix metalloproteases by fibrocytes:possible role in migration and homing[J].American Journal of Respiratory&Critical Care Medicine,2010,182(9):1144.
[28]Allan J A,Docherty A J P,Barker P J,et al.Binding of gelatinases A and B to type-I collagen and other matrix components[J].Biochemical Journal,1995,309(1):299-306.
[29]Yu Q,Stamenkovic I.Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis[J].Genes Dev,2000,14(2):163-176.
[30]柯善英.蜂针治疗纤维化结节[J].蜜蜂杂志,2010,30(8):34-35.
[31]刘瑞,陈宏莉,柏桦,等.蜂胶提取物CAPE对大鼠肺纤维化的干预作用研究[J].生物物理学报,2009(s1):433-434.
[32]辛绍杰,李惠敏.蜂毒对大鼠实验性肝纤维化的疗效观察[J].中华肝脏病杂志,1997,5(4):242-243.
[33]Kim S K,Park K Y,Yoon W C,et al.Melittin enhances apoptosis through suppression of IL-6/s IL-6R complex-induced NF-κB and STAT3 activation and Bcl-2 expression for human fibroblast-like synoviocytes in rheumatoid.arthritis[J].Joint Bone Spine,2011,78(5):471-477.
[34]Park J H,Kum Y S,Lee T I,et al.Melittin attenuates liver injury in thioacetamide-treated mice through modulating inflammation and fibrogenesis[J].Exp Biol Med(Maywood),2011,236(11):1306-1313.
[2]Raghu G,Rochwerg B,Zhang Y,et al.An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline:Treatment of Idiopathic Pulmonary Fibrosis:An Update of the 2011 Clinical Practice Guideline[J].A-merican Journal of Respiratory&Critical Care Medicine,2015,192(2):e3-19.
[3]Gross TJ,Hunninghake GW.Idiopathic pulmonary fibrosis.N Engl JMed,2001,345(7):517-25.
[4]Wynn TA.Cellular and molecular mechanisms of fibrosis[J].The Journal of Pathology,2010,214(2):199-210.
[5]Strieter R M,Mehrad B.New mechanisms of pulmonary fibrosis[J].Chest,2009,136(5):1364-1370.
[6]Oyama Y,Enomoto N,Suzuki Y,et al.Evaluation of urinary desmosines as a noninvasive diagnostic biomarker in patients with idiopathic pleuroparenchymal fibroelastosis(PPFE)[J].Respiratory Medicine,2017,123:63-70.
[7]Sambataro G,Sambataro D.Letter to editor:“Interstitial pneumonia with autoimmune features:Clinical,radiologic and histological characteristics and outcome in a series of 57 patients”[J].Respiratory Medicine,2017,127:65-66.
[8]Hirano C,Ohshimo S,Horimasu Y,et al.FAM13A polymorphism as a prognostic factor in patients with idiopathic pulmonary fibrosis[J].Respiratory Medicine,2017,123:105-109.
[9]赵存刚,黄文海,王尊元,等.转化生长因子-β1,在特发性肺纤维化中的研究新进展[J].中国药学杂志,2016,51(5):341-345.
[10]Bomhard E M.Particle-induced Pulmonary Alveolar Proteinosis and Subsequent Inflammation and Fibrosis[J].Toxicologic Pathology,2017,45(3):389-401.
[11]姜岩,于功昌,张兴国.转化生长因子尹,及其介导的信号通路在百草枯致肺上皮间质转化中的作用[J].环境与职业医学,2017,34(8):729-733.
[12]Madri J A,Furthmayr H.Collagen polymorphism in the lung.An immunochemical study of pulmonary fibrosis[J].Human Pathology,1980,11(4):353-366.
[13]Yu Q,Stamenkovic I.Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis[J].Genes Dev,2000,14(2):163-76.
[14]张冰清,刘晓波.蜂毒的主要成分及药理作用的研究进展[J].药学研究,2016,35(3):172-174.
[15]Glttli A,Chandrasekhar I,van Gunsteren WF.A molecular dynamics study of the bee venom melittin in aqueous solution,in methanol,and inserted in a phospholipid bilayer[J].Eur Biophys J,2006,35(3):255-267.
[16]Kolb M,Margetts PJ,Galt T,et al.Transient transgene expression of decorin in the lung reduces the fibrotic response to bleomycin[J].Am J Respir Crit Care Med,2001,163(3 Pt 1):770-777.
[17]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,1993:33.
[18]Untergasser G,Gander R,Lilg C,et al.Profiling molecular targets of TGF-beta1 in prostate fibroblast-to-myofibroblast transdifferentiation[J].Mech Ageing Dev,2005,126(1):59-69.
[19]Inomata M,Kamio K,Azuma A,et al.Pirfenidone inhibits fibrocyte accumulation in the lungs in bleomycin-induced murine pulmonary fibrosis[J].Respir Res,2014,15:16.
[20]Xiong S,Guo R,Yang Z,et al.Treg depletion attenuates irradiationinduced pulmonary fibrosis by reducing fibrocyte accumulation,inducing Th17 response,and shifting IFN-γ,IL-12/IL-4,IL-5 balance[J].Immunobiology,2015,220(11):1284-1291.
[21]陈宝,李惠萍,范峰,等.三氧化二砷对博莱霉素致大鼠肺纤维化的影响及其作用机制[J].中国病理生理杂志,2010,26(1):65-66.
[22]Ahn J Y,Kim M H,Lim M J,et al.The inhibitory effect of ginsan on TGF-βmediated fibrotic process[J].Journal of Cellular Physiology,2011,226(5):1241-1247.
[23]Li L,Huang W,Li K,et al.Metformin attenuates gefitinib-induced exacerbation of pulmonary fibrosis by inhibition of TGF-βsignaling pathway[J].Oncotarget,2015,6(41):43605-43619.
[24]Yu W,Liu D,Liang C,et al.Sulfur Dioxide Protects Against Collagen Accumulation in Pulmonary Artery in Association With Downregulation of the Transforming Growth Factorβ1/Smad Pathway in Pulmonary Hypertensive Rats[J].J Am Heart Assoc,2016,5(10):e003910.
[25]Sriram N,Kalayarasan S,Manikandan R,et al.Epigallocatechin gallate attenuates fibroblast proliferation and excessive collagen production by effectively intervening TGF-β1 signalling[J].Clinical&Experimental Pharmacology&Physiology,2015,42(8):849-859.
[26]蔡后荣,郑培德.肺间质纤维化前胶原和肿瘤坏死因子α基因表达[J].中华结核和呼吸杂志,1998,21(12):753-755.
[27]García-de-Alba C,Becerril C,Ruiz V,et al.Expression of matrix metalloproteases by fibrocytes:possible role in migration and homing[J].American Journal of Respiratory&Critical Care Medicine,2010,182(9):1144.
[28]Allan J A,Docherty A J P,Barker P J,et al.Binding of gelatinases A and B to type-I collagen and other matrix components[J].Biochemical Journal,1995,309(1):299-306.
[29]Yu Q,Stamenkovic I.Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis[J].Genes Dev,2000,14(2):163-176.
[30]柯善英.蜂针治疗纤维化结节[J].蜜蜂杂志,2010,30(8):34-35.
[31]刘瑞,陈宏莉,柏桦,等.蜂胶提取物CAPE对大鼠肺纤维化的干预作用研究[J].生物物理学报,2009(s1):433-434.
[32]辛绍杰,李惠敏.蜂毒对大鼠实验性肝纤维化的疗效观察[J].中华肝脏病杂志,1997,5(4):242-243.
[33]Kim S K,Park K Y,Yoon W C,et al.Melittin enhances apoptosis through suppression of IL-6/s IL-6R complex-induced NF-κB and STAT3 activation and Bcl-2 expression for human fibroblast-like synoviocytes in rheumatoid.arthritis[J].Joint Bone Spine,2011,78(5):471-477.
[34]Park J H,Kum Y S,Lee T I,et al.Melittin attenuates liver injury in thioacetamide-treated mice through modulating inflammation and fibrogenesis[J].Exp Biol Med(Maywood),2011,236(11):1306-1313.