博来霉素不同给药方式致大鼠肺纤维化模型探讨
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摘要
目的:比较博来霉素(BLM)气管灌注、尾静脉注射及雾化吸入致大鼠肺纤维化模型的优劣,以便筛选出最佳的给药途径。方法:选择SPF级雄性SD大鼠80只,采用体重随机分组法将其分为正常组,单次气管灌注组(10 mg·kg~(-1)),多次气管灌注组(5 mg·kg~(-1)),单次静脉注射组(150 mg·kg~(-1)),多次静脉注射组(50 mg·kg~(-1)),单次雾化吸入模型组(30 min),多次雾化吸入模型组(30 min)。观察给药后7,14,28 d各组大鼠死亡情况及体质量变化。给药后28 d,观察各组大鼠肺系数,取大鼠肺组织制备石蜡切片并进行苏木素-伊红(HE)及马松(Masson)染色,并通过酶联免疫吸附测定(ELISA)检测肺组织中羟脯氨酸(HYP)和纤溶酶原激活物抑制剂-1(PAI-1)含量,评价各组大鼠肺泡炎及肺纤维化程度。结果:与正常组比较,气管灌注组的大鼠死亡率在所有药物处理组中最高。大鼠肺系数的测定结果显示,与正常组比较,多次静脉注射组和多次雾化吸入组大鼠的肺系数显著升高(P<0.05,P<0.01)。雾化造模组较高于其他模型组,多次雾化模型组高于单次雾化模型组。大鼠HE及Masson染色结果提示,气管灌注组、静脉注射组和多次雾化吸入组大鼠肺组织中肺间隔增厚和肺间质纤维化程度较高,其中多次雾化模型组较其他组肺纤维化程度最为明显。ELISA结果发现,与正常组比较,雾化吸入组和气管灌注组大鼠肺组织中HYP和PAI-1含量显著升高(P<0.05)。多次雾化吸入组与单次雾化吸入组显著高于其他造模组。结论:多次雾化吸入博来霉素制备肺纤维化模型,造模大鼠的病理损伤和生理指标相对稳定,符合肺纤维化的演变过程,是一种有效的肺纤维化模型制备手段。
Objective:Compare the effects of 3 administration methods(tracheal perfusion,tail vein injection and aerosol inhalation)with bleomycin(BLM)in inducing pulmonary fibrosis in rats,in order to find out the optimal administration methods.Method:Eighty sprague-dawley(SD)male rats with SPF were randomly divided into aerosol inhalation blank group,single tracheal perfusion group(10 mg·kg~(-1)),multiple tracheal perfusion group(5 mg·kg~(-1)),single intravenous injection group(150 mg·kg~(-1)),multiple intravenous injection group(50 mg·kg~(-1)),single aerosol inhalation group(30 min)and multiple aerosol inhalation group(30 min).The mortality and body weight of rats in each group were observed at 7 d,14 d and 28 d after the administration.And 28 days later after the administration,the lung coefficients of rats in each group were observed,paraffin sections were prepared,hematoxylin-eosin staining(HE)and Masson staining were performed,and the contents of hydroxyproline(HYP)and plasminogen activator inhibitor-1(PAI-1)in lung tissues were detected by enzymelinked immunosorbent assay(ELISA),so as to evaluate the alveoli inflammation and pulmonary fibrosis of rats in each group.Result:Compared with the aerosol inhalation blank group,the rats in the trachea perfusion group had the highest mortality among the drug treatment groups.The pulmonary coefficients of rats in the multiple intravenous injection group and the multiple inhalation group were significantly higher than those in the blank group(P<0.05,P<0.01).The multiple inhalation group was higher than the other model group and the single atomization model group.The results of HE and Masson staining showed thickening of pulmonary septum and higher degree of pulmonary interstitial fibrosis in tracheal perfusion group,intravenous injection group and multiple inhalation group.The degree of pulmonary fibrosis in the multiple inhalation group was more obvious than that in other groups.The results of ELISA showed that the levels of HYP and PAI-1 in lung tissues of rats in aerosol inhalation group and tracheal perfusion group were significantly higher than those in control group(P<0.05).The multiple inhalation group and the single atomization inhalation group were significantly higher than other modules.Conclusion:Bleomycin was inhaled repeatedly to establish pulmonary fibrosis model.The pathological injury and physiological indexes of the model rats were relatively stable,which conforms with the evolution process of pulmonary fibrosis.
Objective:Compare the effects of 3 administration methods(tracheal perfusion,tail vein injection and aerosol inhalation)with bleomycin(BLM)in inducing pulmonary fibrosis in rats,in order to find out the optimal administration methods.Method:Eighty sprague-dawley(SD)male rats with SPF were randomly divided into aerosol inhalation blank group,single tracheal perfusion group(10 mg·kg~(-1)),multiple tracheal perfusion group(5 mg·kg~(-1)),single intravenous injection group(150 mg·kg~(-1)),multiple intravenous injection group(50 mg·kg~(-1)),single aerosol inhalation group(30 min)and multiple aerosol inhalation group(30 min).The mortality and body weight of rats in each group were observed at 7 d,14 d and 28 d after the administration.And 28 days later after the administration,the lung coefficients of rats in each group were observed,paraffin sections were prepared,hematoxylin-eosin staining(HE)and Masson staining were performed,and the contents of hydroxyproline(HYP)and plasminogen activator inhibitor-1(PAI-1)in lung tissues were detected by enzymelinked immunosorbent assay(ELISA),so as to evaluate the alveoli inflammation and pulmonary fibrosis of rats in each group.Result:Compared with the aerosol inhalation blank group,the rats in the trachea perfusion group had the highest mortality among the drug treatment groups.The pulmonary coefficients of rats in the multiple intravenous injection group and the multiple inhalation group were significantly higher than those in the blank group(P<0.05,P<0.01).The multiple inhalation group was higher than the other model group and the single atomization model group.The results of HE and Masson staining showed thickening of pulmonary septum and higher degree of pulmonary interstitial fibrosis in tracheal perfusion group,intravenous injection group and multiple inhalation group.The degree of pulmonary fibrosis in the multiple inhalation group was more obvious than that in other groups.The results of ELISA showed that the levels of HYP and PAI-1 in lung tissues of rats in aerosol inhalation group and tracheal perfusion group were significantly higher than those in control group(P<0.05).The multiple inhalation group and the single atomization inhalation group were significantly higher than other modules.Conclusion:Bleomycin was inhaled repeatedly to establish pulmonary fibrosis model.The pathological injury and physiological indexes of the model rats were relatively stable,which conforms with the evolution process of pulmonary fibrosis.
引文
[1]刘晓,于文涛,靳贺超,等.补肾通络方对肺纤维化大鼠肺组织TGF-β1/Smads通路的影响[J].中国实验方剂学杂志,2019,25(2):96-102.
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[5]高云星,蒋莉莉,张倩,等.吴茱萸次碱通过抑制Notch1/e IF3a信号通路保护博莱霉素诱导的大鼠肺纤维化[J].中国中药杂志,2018,43(17):3510-3538.
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[8]周欢.气管内喷雾注入博莱霉素诱导大鼠肺纤维化模型的建立及其评价[D].重庆:重庆理工大学,2014.
[9]王燕,王真,何琪杨.博来霉素族抗生素作用机制的研究进展[J].国外医药(抗生素分册),2009,30(1):5-8,18.
[10]王淑月.博莱霉素类抗生素的构效关系[J].河北医学院学报,1995,11(16):50-51.
[11]马哈迪,叶燕青,李艳丽.内毒素与博莱霉素所致肺损伤及纤维化的病理改变[J].武汉大学学报:医学版,2009,30(3):326-329.
[12]张伟,鲁香凤,张晓梅,等.气管插管快速灌注博莱霉素复制大鼠肺间质纤维化模型[J].中西医结合学报,2008,6(1):60-67.
[13]李丽娜,王华,周蕾,等.博莱霉素诱导小鼠肺间质纤维化造模方式的选择[J].中国免疫学杂志,2010,26(3):254-257.
[14]郑金旭,夏德刚,田高润,等.白细胞介素32在博来霉素诱导的小鼠肺纤维化组织中的表达及意义[J].江苏医药,2010,36(7):799-801,869.
[15]Szapiel S V,Elson N A,Fulmer J D,et al. Bleomycininduced interstitial pulmonary disease in the nude,athymic mouse.[J]. Am Rev Respir Dis,1979,120(4):893-897.
[16]王健,黄志兰,王涛,等.平阳霉素致肺纤维化动物模型高分辨率CT的评价[J].第三军医大学学报,1998,20(5):419-422.
[17]张晓晔,志村早苗,增田辙,等.博莱霉素静脉注射制备小鼠肺间质纤维化模型[J].中国实验动物学报,2007,15(4):241-244,230.
[18]孟婕,邹叶强.小剂量多次尾静脉注射博来霉素致小鼠肺纤维化模型的研究[C]//中华医学会第三届全国间质性肺病暨弥漫性泛细支气管炎学术会议论文汇编,北京:中华医学会,2010:143.
[19]孟婕,彭张哲,陶立坚.小剂量多次尾静脉注射与气管内滴注博来霉素致小鼠肺纤维化模型的比较研究[J].中南大学学报:医学版,2013,38(12):1228-1232.
[20]杨聪颖,彭雄群,阳惠湘,等.不同剂量博莱霉素诱导小鼠肺纤维化的差异及动态变化[J].中国实验动物学报,2013,21(2):45-51,96.
[21]肖强,郑晓滨,刘香,等.静脉注射不同剂量博莱霉素诱导小鼠肺纤维化的比较研究[J].临床肺科杂志,2015,20(2):251-256.
[2]王振兴,孙中莉,王明杰,等.补阳还五汤对肺纤维化中HMGB1-RAGE信号通路的调控作用[J].中国实验方剂学杂志,2017,23(13):138-144.
[3]Williamson J D,Sadofsky L R,Hart S P. The pathogenesis of bleomycin-induced lung injury in animals and its applicability to human idiopathic pulmonary fibrosis[J]. Exp Lung Res,2015,41(2):57-73.
[4]周语平,郭宏亮.理肺化纤方对肺纤维化CTGF和FN信号转导通路影响的研究[J].中国实验方剂学杂志,2011,17(14):201-205.
[5]高云星,蒋莉莉,张倩,等.吴茱萸次碱通过抑制Notch1/e IF3a信号通路保护博莱霉素诱导的大鼠肺纤维化[J].中国中药杂志,2018,43(17):3510-3538.
[6]Safaeian L,Jafarian A,Rabbani M,et al. The effect of AT1 receptor blockade on bax and bcl-2 expression in bleomycin-induced pulmonary fibrosis[J]. DARU,2009,17(1):53-59.
[7]陈丽,钱洁,叶延,等.百草枯反复小剂量腹腔给药诱导小鼠肺纤维化模型[J].中华急诊医学杂志,2011,12(20):1285-1289.
[8]周欢.气管内喷雾注入博莱霉素诱导大鼠肺纤维化模型的建立及其评价[D].重庆:重庆理工大学,2014.
[9]王燕,王真,何琪杨.博来霉素族抗生素作用机制的研究进展[J].国外医药(抗生素分册),2009,30(1):5-8,18.
[10]王淑月.博莱霉素类抗生素的构效关系[J].河北医学院学报,1995,11(16):50-51.
[11]马哈迪,叶燕青,李艳丽.内毒素与博莱霉素所致肺损伤及纤维化的病理改变[J].武汉大学学报:医学版,2009,30(3):326-329.
[12]张伟,鲁香凤,张晓梅,等.气管插管快速灌注博莱霉素复制大鼠肺间质纤维化模型[J].中西医结合学报,2008,6(1):60-67.
[13]李丽娜,王华,周蕾,等.博莱霉素诱导小鼠肺间质纤维化造模方式的选择[J].中国免疫学杂志,2010,26(3):254-257.
[14]郑金旭,夏德刚,田高润,等.白细胞介素32在博来霉素诱导的小鼠肺纤维化组织中的表达及意义[J].江苏医药,2010,36(7):799-801,869.
[15]Szapiel S V,Elson N A,Fulmer J D,et al. Bleomycininduced interstitial pulmonary disease in the nude,athymic mouse.[J]. Am Rev Respir Dis,1979,120(4):893-897.
[16]王健,黄志兰,王涛,等.平阳霉素致肺纤维化动物模型高分辨率CT的评价[J].第三军医大学学报,1998,20(5):419-422.
[17]张晓晔,志村早苗,增田辙,等.博莱霉素静脉注射制备小鼠肺间质纤维化模型[J].中国实验动物学报,2007,15(4):241-244,230.
[18]孟婕,邹叶强.小剂量多次尾静脉注射博来霉素致小鼠肺纤维化模型的研究[C]//中华医学会第三届全国间质性肺病暨弥漫性泛细支气管炎学术会议论文汇编,北京:中华医学会,2010:143.
[19]孟婕,彭张哲,陶立坚.小剂量多次尾静脉注射与气管内滴注博来霉素致小鼠肺纤维化模型的比较研究[J].中南大学学报:医学版,2013,38(12):1228-1232.
[20]杨聪颖,彭雄群,阳惠湘,等.不同剂量博莱霉素诱导小鼠肺纤维化的差异及动态变化[J].中国实验动物学报,2013,21(2):45-51,96.
[21]肖强,郑晓滨,刘香,等.静脉注射不同剂量博莱霉素诱导小鼠肺纤维化的比较研究[J].临床肺科杂志,2015,20(2):251-256.