固本平喘汤对哮喘豚鼠气道ECP、MBP影响的研究
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摘要
【目的】在临床应用固本平喘汤防治支气管哮喘并取得良好疗效的基础上,进一步研究该方对哮喘动物模型气道嗜酸性阳离子蛋白(ECP)、碱性蛋白(MBP)以及嗜酸性粒细胞(EOS)计数的影响,探讨固本平喘汤治疗支气管哮喘的作用机制,为临床应用提供理论依据。
【方法】体重250-300g豚鼠60只,雌雄各30只,随机分为正常对照组(A组)、模型组(B组)、西药对照(地塞米松)组(C组)、中药小剂量治疗组(D1组)、中药大剂量治疗组(D2组),每组12只。造模采用Underwood法加以改进,在实验第1天,给B、C、D1、D2每只豚鼠腹腔注射临时配制OVA10mg加氢氧化铝干粉100mg与灭活百日咳菌苗5×10~9个配制而成的混悬液1mL注射液以致敏,A组则以等量生理盐水进行腹腔注射;第15天将致敏豚鼠置于14升透明玻璃钟罩内,给予1%OVA对豚鼠进行超声雾化吸入(雾化量由小到大)激发15-20秒,然后自然吸入30min,每天1次,在激发前60min给予苯海拉明10mg/kg腹腔注射,预防豚鼠因过敏反应而死亡。A组则以等量生理盐水进行超声雾化;即在每次激发时记录其豚鼠一般情况。哮喘造模成功标准为:头、面部搔痒,呼吸加深、加快、少动,弓背,前肢缩抬,点头呼吸,咳嗽,鼻煽,抽搐,呈哮喘样表现。一般情况包括:精神、活动、饮食、体重、排便、皮毛、病情以及死亡情况。此后,每天以1%OVA雾化诱喘,按最大雾化量,自然吸入30min,使哮喘反复发作,连续3天;在第一次诱喘后24小时,即从第16天开始分别用软管胃饲给药,A组及B组均予等体积胃饲生理盐水,C组予地塞米松注射液(1mg/kg)胃饲;D1、D2组分别予固本平喘汤(炙麻黄、杏仁、甘草、苏子、白芥子、莱菔子、黄芩、地龙、仙灵脾等组成)浓缩液(每毫升含生药2g)D1组(2.5g/kg)及D2组(7.5g/kg)胃饲治疗7天。观察造模前后及药物治疗后豚鼠一般情况;疗程结束后,处死动物,收集各组动物BALF、颈动脉血及肺组织标本,观察BALF中细胞总数及细胞分类含量变化、肺组织形态及病理学改变,采用酶联免疫吸附测试法测定血清ECP含量,用HE染色观察EOS情况,进行支气管平滑肌的MBPmRNART-PCR半定量分析。结果采用统计软件SPSS13.0进行数据统计分析处理;计数资料以(?)±s表示,用t检验比较。计量资料用x~2检验。
【结果】A组豚鼠体重自然增加,一般情况良好,无死亡。致敏后的豚鼠普遍精神较差,活动减少,进食及饮水量明显下降。1-2天后逐渐恢复正常。第16天哮喘B组3只豚鼠,C组4只豚鼠,D1组2只豚鼠亦出现明显皮毛蓬松现象。第二次诱喘时,B组3只豚鼠哮喘发作严重后死亡。用药后各组豚鼠情况均较哮喘模型组明显改善,但不能恢复到A组的水平。D2组豚鼠肺组织血管损伤修复,肺泡间隔变窄,上皮增生明显改善,炎症细胞明显减少,肺泡腔清晰。与B组比较,气道炎性细胞侵润显著减少,与C组相当;BALF中细胞总数及嗜酸性粒细胞以及淋巴细胞侵润明显减少,与B组比较有显著性差异(P＜0.01),与C组相当(P＞0.05);哮喘豚鼠血清ECP的分布明显降低,与B组比较亦有显著性差异(P＜0.05)。治疗前后MBPmRNA,与B组比较有显著性差异(P＜0.01)。
【结论】固本平喘汤能诱导或促进哮喘豚鼠气道EOS减少,从而减轻气道炎性细胞侵润,控制气道慢性炎症,以达到防治哮喘的目的。其作用机制可能是通过改变ECP及MBP在哮喘豚鼠肺组织的表达,及减少肺组织中ECP及MBP的含量,从而诱导EOS的凋亡,使BALF中及肺组织侵润的嗜酸性粒细胞数量显著减少、淋巴细胞亦显著减少,以达到防治哮喘的目的。
【Objectives】At the base of good therapeutic effect of gubenpingchuantang in preventing and curing bronchial asthma.We did further research to observe therapeutic influence of gubenpingchuantang on animal model of asthma in some indicators such as ECP、MBP and EOS etc.In order to discuss mechanism of action of this compound on treatment of asthma and provide theoretical evidence for clinical application.
【Methods】60 guinea pigs(Half male and female)weight 250-300g were randomly divided into normal control group(A group),model group(B group),western medicine control group(dexamethasone) group(C group),Chinese medicine treatment of low-dose group(01 group),Chinese high-dose treatment group(D2 group),12 in each group,we used the model Underwood made and and improved it.In the first day of experiment,each guinea pig in group B,C,D1,02 was given intraperitoneal injection of 10ml suspl,consists of OVA10mg、100mg dry powder aluminum hydroxide and inactivated pertussis vaccine 5×10~9mL to allergize,guinea pig in group was injected equal volume of normal saline for instead;In the 15th day we seted guinea pigs needed to be allergized in a 14 liters transparent glass Bell,and gave them 1%OVA ultrasonic atomizing inhalation(aerosol volume from small to big) about 15-20 seconds,then made them natural inhalation 30min,once every day,60min before excitation every guinea pig was given intraperitoneal injection of 10mg/kg of diphenhydramine to prevent allergic reactions which would cause death,guinea pigs in group A was ultrasonic atomized equal volume of normal saline for instead;The standard of successful asthma model is asthma-like performance emerge such as:head,facial pruritus,breathing deepen,less moving,camponotus,reduced forelimb lift,head to breathe,cough,nasal fan,convulsions and so on.From the Beginning of the 16th day guinea pigs in treatment groups were administration by gastric tube,guinea pigs group A and group B were feed with equal volume of normal saline,which in group C were feed with injection of dexamethasone(1mg/kg);which in group D1,D2 were feed with concentrated liquid of gubenpingchuantang(SunburnEphedra,Almond,Licorice,erillaseed, White mustard seed,Raphani,semen,Scutellaria,HERBA EPIMEDII etc.)(2g crude drug per ml) D1 group(2.5g / kg) and the D2 group (7.5g/kg) the treatment of administration last about 7 days.After the treatment,animals in every group were killed to collect BALF, carotid artery and lung tissue samples in order to observe the total number of BALF cells and quantity changes in various types of it, we observed morphology and pathological changes in lung tissue, used enzyme-linked immunosorbent assay test for determining content of serum ECP,we observed EOS after HE staining and did MBPmRNA RT-PCR semi-quantitative analysis of bronchial smooth muscle,we used statistical software of SPSS13.0 for for data statistical analysis;count data expressed with(?)±s,compared with test.measurement data used X~2 test for statistical analysis.
【Results】Guinea pigs in group A were in good condition,no one was died.Sensitized guinea pigs were poor-spirited,less moving, food and water intake decreased significantly,and returned to normal in 1-2 days.In the 16th day,3 guinea pigs in group B,4 guinea pigs in group C,2 guinea pigs in group D1 emerge the phenomenon of fluffying fur.When the second Induced asthma,3 guinea pigs in group B died after a serious asthma attack.After treating by administration the situation of Guinea pigs improved, but it can not restore to the level of the group A,lung tissue blood vessels damage of guinea pigs in group D2 repaired,alveolar septal became narrow,epithelial hyperplasia improved and reduced inflammatory cells,alveolar space is clear.Compare With the group B,airway inflammatory cell infiltration was significantly reduced, just like group C;The total number of BALF cells,eosinophils and lymphocyte infiltration decreased,compared with group B there were significantly different(P＜0.01);The distribution of serum gcP in asthmatic guinea pigs were significantly decreased compared with the group B there were also significant differences(P＜0.05).so was MBPmRNA(P＜0.01).
【Conclusions】Gubenpingchuantang can induce or promote reducing airway EOS of asthmatic guinea pigs which reduce the airway inflammatory cell infiltration and control of chronic airway inflammation,and achieve the purpose of prevention and treatment of asthma.Its mechanism are possibly by changing the expression of the ECP and MBP in lung tissue of asthmatic guinea pigs,and reduce content of ECP and MBP in lung tissue,and induce EOS apoptosis, so that infiltration of eosinophils cells in BALF of lung tissue significantly reducedand the number of lymphocytes is significantly reduced,which reached the purpose of prevention and treatment of asthma.
【方法】体重250-300g豚鼠60只,雌雄各30只,随机分为正常对照组(A组)、模型组(B组)、西药对照(地塞米松)组(C组)、中药小剂量治疗组(D1组)、中药大剂量治疗组(D2组),每组12只。造模采用Underwood法加以改进,在实验第1天,给B、C、D1、D2每只豚鼠腹腔注射临时配制OVA10mg加氢氧化铝干粉100mg与灭活百日咳菌苗5×10~9个配制而成的混悬液1mL注射液以致敏,A组则以等量生理盐水进行腹腔注射;第15天将致敏豚鼠置于14升透明玻璃钟罩内,给予1%OVA对豚鼠进行超声雾化吸入(雾化量由小到大)激发15-20秒,然后自然吸入30min,每天1次,在激发前60min给予苯海拉明10mg/kg腹腔注射,预防豚鼠因过敏反应而死亡。A组则以等量生理盐水进行超声雾化;即在每次激发时记录其豚鼠一般情况。哮喘造模成功标准为:头、面部搔痒,呼吸加深、加快、少动,弓背,前肢缩抬,点头呼吸,咳嗽,鼻煽,抽搐,呈哮喘样表现。一般情况包括:精神、活动、饮食、体重、排便、皮毛、病情以及死亡情况。此后,每天以1%OVA雾化诱喘,按最大雾化量,自然吸入30min,使哮喘反复发作,连续3天;在第一次诱喘后24小时,即从第16天开始分别用软管胃饲给药,A组及B组均予等体积胃饲生理盐水,C组予地塞米松注射液(1mg/kg)胃饲;D1、D2组分别予固本平喘汤(炙麻黄、杏仁、甘草、苏子、白芥子、莱菔子、黄芩、地龙、仙灵脾等组成)浓缩液(每毫升含生药2g)D1组(2.5g/kg)及D2组(7.5g/kg)胃饲治疗7天。观察造模前后及药物治疗后豚鼠一般情况;疗程结束后,处死动物,收集各组动物BALF、颈动脉血及肺组织标本,观察BALF中细胞总数及细胞分类含量变化、肺组织形态及病理学改变,采用酶联免疫吸附测试法测定血清ECP含量,用HE染色观察EOS情况,进行支气管平滑肌的MBPmRNART-PCR半定量分析。结果采用统计软件SPSS13.0进行数据统计分析处理;计数资料以(?)±s表示,用t检验比较。计量资料用x~2检验。
【结果】A组豚鼠体重自然增加,一般情况良好,无死亡。致敏后的豚鼠普遍精神较差,活动减少,进食及饮水量明显下降。1-2天后逐渐恢复正常。第16天哮喘B组3只豚鼠,C组4只豚鼠,D1组2只豚鼠亦出现明显皮毛蓬松现象。第二次诱喘时,B组3只豚鼠哮喘发作严重后死亡。用药后各组豚鼠情况均较哮喘模型组明显改善,但不能恢复到A组的水平。D2组豚鼠肺组织血管损伤修复,肺泡间隔变窄,上皮增生明显改善,炎症细胞明显减少,肺泡腔清晰。与B组比较,气道炎性细胞侵润显著减少,与C组相当;BALF中细胞总数及嗜酸性粒细胞以及淋巴细胞侵润明显减少,与B组比较有显著性差异(P＜0.01),与C组相当(P＞0.05);哮喘豚鼠血清ECP的分布明显降低,与B组比较亦有显著性差异(P＜0.05)。治疗前后MBPmRNA,与B组比较有显著性差异(P＜0.01)。
【结论】固本平喘汤能诱导或促进哮喘豚鼠气道EOS减少,从而减轻气道炎性细胞侵润,控制气道慢性炎症,以达到防治哮喘的目的。其作用机制可能是通过改变ECP及MBP在哮喘豚鼠肺组织的表达,及减少肺组织中ECP及MBP的含量,从而诱导EOS的凋亡,使BALF中及肺组织侵润的嗜酸性粒细胞数量显著减少、淋巴细胞亦显著减少,以达到防治哮喘的目的。
【Objectives】At the base of good therapeutic effect of gubenpingchuantang in preventing and curing bronchial asthma.We did further research to observe therapeutic influence of gubenpingchuantang on animal model of asthma in some indicators such as ECP、MBP and EOS etc.In order to discuss mechanism of action of this compound on treatment of asthma and provide theoretical evidence for clinical application.
【Methods】60 guinea pigs(Half male and female)weight 250-300g were randomly divided into normal control group(A group),model group(B group),western medicine control group(dexamethasone) group(C group),Chinese medicine treatment of low-dose group(01 group),Chinese high-dose treatment group(D2 group),12 in each group,we used the model Underwood made and and improved it.In the first day of experiment,each guinea pig in group B,C,D1,02 was given intraperitoneal injection of 10ml suspl,consists of OVA10mg、100mg dry powder aluminum hydroxide and inactivated pertussis vaccine 5×10~9mL to allergize,guinea pig in group was injected equal volume of normal saline for instead;In the 15th day we seted guinea pigs needed to be allergized in a 14 liters transparent glass Bell,and gave them 1%OVA ultrasonic atomizing inhalation(aerosol volume from small to big) about 15-20 seconds,then made them natural inhalation 30min,once every day,60min before excitation every guinea pig was given intraperitoneal injection of 10mg/kg of diphenhydramine to prevent allergic reactions which would cause death,guinea pigs in group A was ultrasonic atomized equal volume of normal saline for instead;The standard of successful asthma model is asthma-like performance emerge such as:head,facial pruritus,breathing deepen,less moving,camponotus,reduced forelimb lift,head to breathe,cough,nasal fan,convulsions and so on.From the Beginning of the 16th day guinea pigs in treatment groups were administration by gastric tube,guinea pigs group A and group B were feed with equal volume of normal saline,which in group C were feed with injection of dexamethasone(1mg/kg);which in group D1,D2 were feed with concentrated liquid of gubenpingchuantang(SunburnEphedra,Almond,Licorice,erillaseed, White mustard seed,Raphani,semen,Scutellaria,HERBA EPIMEDII etc.)(2g crude drug per ml) D1 group(2.5g / kg) and the D2 group (7.5g/kg) the treatment of administration last about 7 days.After the treatment,animals in every group were killed to collect BALF, carotid artery and lung tissue samples in order to observe the total number of BALF cells and quantity changes in various types of it, we observed morphology and pathological changes in lung tissue, used enzyme-linked immunosorbent assay test for determining content of serum ECP,we observed EOS after HE staining and did MBPmRNA RT-PCR semi-quantitative analysis of bronchial smooth muscle,we used statistical software of SPSS13.0 for for data statistical analysis;count data expressed with(?)±s,compared with test.measurement data used X~2 test for statistical analysis.
【Results】Guinea pigs in group A were in good condition,no one was died.Sensitized guinea pigs were poor-spirited,less moving, food and water intake decreased significantly,and returned to normal in 1-2 days.In the 16th day,3 guinea pigs in group B,4 guinea pigs in group C,2 guinea pigs in group D1 emerge the phenomenon of fluffying fur.When the second Induced asthma,3 guinea pigs in group B died after a serious asthma attack.After treating by administration the situation of Guinea pigs improved, but it can not restore to the level of the group A,lung tissue blood vessels damage of guinea pigs in group D2 repaired,alveolar septal became narrow,epithelial hyperplasia improved and reduced inflammatory cells,alveolar space is clear.Compare With the group B,airway inflammatory cell infiltration was significantly reduced, just like group C;The total number of BALF cells,eosinophils and lymphocyte infiltration decreased,compared with group B there were significantly different(P＜0.01);The distribution of serum gcP in asthmatic guinea pigs were significantly decreased compared with the group B there were also significant differences(P＜0.05).so was MBPmRNA(P＜0.01).
【Conclusions】Gubenpingchuantang can induce or promote reducing airway EOS of asthmatic guinea pigs which reduce the airway inflammatory cell infiltration and control of chronic airway inflammation,and achieve the purpose of prevention and treatment of asthma.Its mechanism are possibly by changing the expression of the ECP and MBP in lung tissue of asthmatic guinea pigs,and reduce content of ECP and MBP in lung tissue,and induce EOS apoptosis, so that infiltration of eosinophils cells in BALF of lung tissue significantly reducedand the number of lymphocytes is significantly reduced,which reached the purpose of prevention and treatment of asthma.
引文
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