止哮平喘方对哮喘大鼠嗜酸性粒细胞凋亡及调控基因表达的影响
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摘要
1目的
本课题通过观察止哮平喘方对哮喘大鼠症状评分、肺溢流量、气管条张力、全血和肺泡灌洗液(BALF)中自细胞、嗜酸性粒细胞(EOS)数量、血清免疫球蛋白(Ig)E、白细胞介素(IL)-5、IL-13等的含量、支气管肺组织形态学、气道重塑参数、肺组织EOS凋亡及调控基因Fas、Bcl-2mRNA表达等的影响。围绕EOS的凋亡,从免疫反应、气道炎症、气道高反应性及气道重塑等方面探讨止哮平喘方的作用机制。
2方法
以卵蛋白致敏和激发复制大鼠哮喘模型。SD大鼠随机分为6组:正常对照组(简称正常组)、哮喘模型组(简称模型组)、地塞米松治疗组(简称地塞米松组)、止哮平喘方小剂量组(简称小剂量组)、止哮平喘方中剂量组(简称中剂量组)、止哮平喘方大剂量组(简称大剂量组),每组10只。实验第8天,即第2次致敏的同一天开始给药,共治疗14天。观察各组大鼠一般生活情况,并对末次激发10min时大鼠的症状表现进行评分。末次激发24h后,进行肺溢流实验,处死大鼠,采集标本。检测离体气管条张力,进行全血及肺泡灌洗液(BALF)中血细胞计数,ELISA法检测血清IgE、IL-5、IL-13的含量,光镜观察支气管肺组织形态学的改变,医学图像分析系统测量气道重塑参数,TUNEL法测定肺组织中EOS凋亡情况,原位杂交法测定肺组织Fas、Bcl-2mRNA的表达。
3结果
3.1大鼠激发时的一般状况
模型组大鼠在吸入OVA激发后即出现躁动不安、搔抓头面及全身皮毛、呼吸急促和明显的腹式呼吸、打喷嚏、口鼻紫绀、呼气相喘鸣音等症状。反复激发后,大鼠毛色失去光泽,精神不振,反应迟钝,体重较正常组明显减轻(P＜0.05)。应用药物干预后,各治疗组症状明显改善。
3.2症状积分比较应用药物干预后,各治疗组大鼠激发后症状积分减少,与模型组比较有显著性差异(P＜0.01或P＜0.05)。
3.3肺溢流量差值和离体气管条张力变化比较
各治疗组与模型组比较,大鼠肺溢流量变化明显减少,有显著性差异(P＜0.01或P＜0.05)。与模型组比较,正常组及各治疗组大鼠气管条张力明显降低(P＜0.0l或P＜0.05)。
3.4哮喘大鼠全血和BALF中白细胞总数、EOS百分率比较模型组大鼠全血和BALF中白细胞总数、EOS百分率显著增加,与正常组比较均有显著性差异(P＜0.01)。各治疗组大鼠全血和BALF中白细胞总数、EOS百分率均降低,与模型组比较均有显著性差异(P＜0.01或P＜0.05),其中地塞米松组和止哮平喘方大剂量组全血和BALF中白细胞总数和EOS百分率与正常组比较无显著性差异(P＞0.05),中剂量组全血白细胞总数和EOS百分率与正常组比较无显著性差异(P＞0.05)。
3.5哮喘大鼠血清IgE、IL-5、IL-13含量比较
模型组大鼠血清IgE、IL-5、IL-13含量明显升高,与正常组比较均有显著性差异(P＜0.01)。各治疗组大鼠血清IgE、IL-5、IL-13含量较模型组均明显降低(P＜0.01或P＜0.05),其中地塞米松组和止哮平喘方大剂量组血清IgE含量与正常组比较无显著性差异(P＞0.05),各治疗组血清IL-5和IL-13含量与正常组比较,无显著性差异(P＞0.05)。
3.6支气管肺组织形态学光镜观察
哮喘模型组大鼠气道炎症明显、管腔狭窄、肺泡隔明显增宽、平滑肌增厚;地塞米松组和止哮平喘方大、中剂量组均能显著减轻大、小气道及肺组织炎症情况,支气管肺组织大致正常;而小剂量组则变化小。
3.7气道重塑图像分析参数比较
模型组支气管总管壁厚度(WAt/Pbm)、平滑肌厚度(WAm/Pbm)、平滑肌细胞数(N/Pbm)均明显高于正常组(P＜0.01)。地塞米松组、止哮平喘方大、中剂量组WAt/Pbm、WAm/Pbm和N/Pbm值均较模型组降低,结果有显著性差异(P＜0.01或P＜0.05)。小剂量组WAt/Pbm和N/Pbm值较模型组降低(P＜0.05),但WAm/Pbm与模型组比较无显著性差异(P＞0.05)。
3.8大鼠肺组织EOS凋亡及Fas、Bcl-2mRNA的表达
模型组大鼠肺组织中EOS的数量较多,但EOS凋亡减少,同时FasmRNA表达明显降低、Bcl-2mRNA的表达明显增加,与正常组比较有显著性差异(P＜0.01)。各治疗组EOS凋亡增加,同时FasmRNA表达也增加、Bcl-2mRNA的表达降低,与模型组比较有显著性差异(P＜0.01或P＜0.05),且与止哮平喘方有明显的剂量依赖关系。FasmRNh表达与EOS凋亡呈正相关(P＜0.01),而Bcl-2mRNA的表达与EOS凋亡呈负相关(P＜0.01)。
4结论
以OVA致敏和激发,可以成功复制大鼠哮喘模型。止哮平喘方能通过直接松弛气管平滑肌,解除气道痉挛;降低全血和BALF中自细胞及EOS数量;降低血清IgE、IL-5、IL-13含量;促进肺组织FasmRNA表达、抑制Bcl-2mRNA表达,促进肺组织EOS凋亡,从而减轻免疫反应,减轻气道炎症,抑制气道高反应性和气道重塑,发挥平喘作用。
1 Objective:
To investigate the effects of ZhiXiaoPingChuanFang on asthmatic symptom scores,pulmonary overflow amount,tension of tracheal strips in vitro,the count of leukocyte and eosinophil in whole blood and bronchial alveolar lavage fluid(BALF),the content of IgE,IL-5 and IL-13 in serum,Histomorphology of bronchopulmonary tissue,airway remodeling,expression of Fas,Bcl-2mRNA and eosinophil apoptosis in lung tissue.The effective mechanism of ZhiXiaoPingChuanFang on immune response,airway inflammation, airway hyperresponsiveness and airway remodeling of asthma were explored.
2 Methods:
The rat asthma model was established by sensitization and challenge with ovalbumin(OVA).Sixty SD rats were evenly assigned to six groups at random:normal control group,asthma model group, dexamethasone treated group,low dosage group,middle dosage group and high dosage group of ZhiXiaoPingChuanFang.After the rat models were formed successfully,the decoction of ZhiXiaoPingChuanFang was used for treatment.The asthmatic symptom scores,pulmonary overflow amount,tension of tracheal strips,the count of leukocyte and eosinophil in whole blood and BALF were determined.The content of IgE,IL-5 and IL-13 in serum were detected by enzyme linked immuoabsorbent assay(ELISh).The histomorphology of the bronchopulmonary tissue was observed by the light microscop. Parameters of airway remodeling were determined by medical image analysis system.Apoptosis was detected by TUNEL,the expressions of FasmRNA and Bcl-2 mRNA in the lung tissue were detected in situ hybridization.
3 Results:
3.1 The general condition and symptoms of rats at the time of provocation:After inhalation of OVA,model rats came forth symptom of hypersensitiveness such as restlessness and scratching fur on their face;and were in tachypnea or dyspnea(visible abdominal breathing) with slight cyanosis.Some of model rats sounded sonorous wheeze during expiratory phase.After repeated provocation, fur of model rats lost luster,depressed and responsed slowly, the weight growth of model group was slow obviously after repeatedly provocation.There was significant difference on the weight between normal group and model group(P＜0.05).Compared with model group, after treated with medicine,the symptoms of each treatment group alleriated obviously.
3.2 Asthmatic symptom scores:Compared with model group,the asthmatic symptom scores of each treatment group decreased significantly(P＜0.01 or P＜0.05).
3.3 Determination of pulmonary overflow amount and tension of tracheal strips in vitro:Compared with model group,the pulmonary overflow amount and tracheal strips tension of each treatment group decreased significantly(P＜0.01 or P＜0.05).
3.4 Count of leukocyte and eosinophil in whole blood and BALF: Compared with normal group,the count of leukocyte and eosinophil in whole blood and BALF in model group increased significantly(P＜0.01).Compared with model group,the count of leukocyte and eosinophil in whole blood and BALF of each treatment group decreased significantly(P＜0.01 or P＜0.05).Compared with normal group,there was no significant difference on count of leukocyte and eosinophil in whole blood and BALF among dexamethasone group, high dosage group and normal group(P＞0.05).
3.5 The content of IgE,IL-5 and IL-13 in serum:Compared with normal group,the content of IgE,IL-5 and IL-13 in model group increased significantly(P＜0.01).Compared with model group,the content of IgE,IL-5 and IL-13 in each treatment group decreased significantly(P＜0.01 or P＜0.05).Compared with normal group, there was no significant difference on IgE in dexamethasone group and high dosage group(P＞0.05),and there was no significant difference on IL-5 and IL-13 between normal group and each treatment group(P＞0.05).
3.5 Histomorphology of bronchopulmonary tissue:There were obvious infiltration of inflammatory cells,severe luminal stenosis and thickening of alveolar septa in model group,significant hyperplasias of basement membrane and smooth muscle layer of central and peripheral airways were observed in the bronchopulmonary tissue too.The inflammation in the bronchopulmonary was significantly improved in dexamethasone group,high dosage group and middle dosage group.
3.7 Parameters of airway remodeling:Compared with normal group, the thickness of bronchial wall(WAt/Pbm),thickness of bronchial smooth muscle(WAm/Pbm) and the number of airway smooth muscle cells(N/Pbm) increased significantly in model group(P＜0.01). Compared with model group,the parameters of WAt/Pbm,WAm/Pbm and N/Pbm decreased significantly in dexamethasone group,high dosage group and middle dosage group(P＜0.01 or P＜0.05),the parameters of WAt/Pbm and N/Pbm decreased significantly in low dosage group(P＜0.01 or P＜0.05),there was no significant difference on parameters of WAm/Pbm between low dosage group and model group(P＞0.05).
3.8 Expression of Fas,Bcl-2mRNA and eosinophil apoptosis in lung tissue:Compared with normal group,the count of eosinophil increased and eosinophil apoptosis rates decreased,the expression of FasmRNA significantly decreased and the expression of Bcl-2mRNA significantly increased in model group(P＜0.01).Compared with model group,eosinophil apoptosis rates and the expression of FasmRNA increased significantly,while the expression Bcl-2mRNA decreased significantly in each treatment group(P＜0.01 or P＜0.05).The mechanism of ZhiXiaoPingChuanFang on the expressions of Fas,Bcl-2mRNA and eosinophil apoptosis was in a dose-dependent manner,A positively significant correlation was found between the FasmRNA expression and eosinophil apoptosis in each group(P＜0.01),and a very significant negative correlation was found between the Bcl-2mRNA expression and eosinophil apoptosis(P＜0.01).
4 Conclusion:
Sensitization and challenge with OVA could replicate asthma model in rats.ZhiXiaoPingChuanFang could relax the airway smooth muscle directly,relieve the airway spasm,could reduce the count of leukocyte and eosinophil in whole blood and BALF,decrease the content of IgE,IL-5 and IL-13 in serum,promote eosinophil apoptosis,FasmRNA expression,and inhibit Bcl-2mRNA expression. Consequently,by means of reducing immune response and airway inflammation,inhibiting airway hyperresponsiveness and airway remodeling,ZhiXiaoPingChuanFang might play a important role in relieving asthma.
本课题通过观察止哮平喘方对哮喘大鼠症状评分、肺溢流量、气管条张力、全血和肺泡灌洗液(BALF)中自细胞、嗜酸性粒细胞(EOS)数量、血清免疫球蛋白(Ig)E、白细胞介素(IL)-5、IL-13等的含量、支气管肺组织形态学、气道重塑参数、肺组织EOS凋亡及调控基因Fas、Bcl-2mRNA表达等的影响。围绕EOS的凋亡,从免疫反应、气道炎症、气道高反应性及气道重塑等方面探讨止哮平喘方的作用机制。
2方法
以卵蛋白致敏和激发复制大鼠哮喘模型。SD大鼠随机分为6组:正常对照组(简称正常组)、哮喘模型组(简称模型组)、地塞米松治疗组(简称地塞米松组)、止哮平喘方小剂量组(简称小剂量组)、止哮平喘方中剂量组(简称中剂量组)、止哮平喘方大剂量组(简称大剂量组),每组10只。实验第8天,即第2次致敏的同一天开始给药,共治疗14天。观察各组大鼠一般生活情况,并对末次激发10min时大鼠的症状表现进行评分。末次激发24h后,进行肺溢流实验,处死大鼠,采集标本。检测离体气管条张力,进行全血及肺泡灌洗液(BALF)中血细胞计数,ELISA法检测血清IgE、IL-5、IL-13的含量,光镜观察支气管肺组织形态学的改变,医学图像分析系统测量气道重塑参数,TUNEL法测定肺组织中EOS凋亡情况,原位杂交法测定肺组织Fas、Bcl-2mRNA的表达。
3结果
3.1大鼠激发时的一般状况
模型组大鼠在吸入OVA激发后即出现躁动不安、搔抓头面及全身皮毛、呼吸急促和明显的腹式呼吸、打喷嚏、口鼻紫绀、呼气相喘鸣音等症状。反复激发后,大鼠毛色失去光泽,精神不振,反应迟钝,体重较正常组明显减轻(P＜0.05)。应用药物干预后,各治疗组症状明显改善。
3.2症状积分比较应用药物干预后,各治疗组大鼠激发后症状积分减少,与模型组比较有显著性差异(P＜0.01或P＜0.05)。
3.3肺溢流量差值和离体气管条张力变化比较
各治疗组与模型组比较,大鼠肺溢流量变化明显减少,有显著性差异(P＜0.01或P＜0.05)。与模型组比较,正常组及各治疗组大鼠气管条张力明显降低(P＜0.0l或P＜0.05)。
3.4哮喘大鼠全血和BALF中白细胞总数、EOS百分率比较模型组大鼠全血和BALF中白细胞总数、EOS百分率显著增加,与正常组比较均有显著性差异(P＜0.01)。各治疗组大鼠全血和BALF中白细胞总数、EOS百分率均降低,与模型组比较均有显著性差异(P＜0.01或P＜0.05),其中地塞米松组和止哮平喘方大剂量组全血和BALF中白细胞总数和EOS百分率与正常组比较无显著性差异(P＞0.05),中剂量组全血白细胞总数和EOS百分率与正常组比较无显著性差异(P＞0.05)。
3.5哮喘大鼠血清IgE、IL-5、IL-13含量比较
模型组大鼠血清IgE、IL-5、IL-13含量明显升高,与正常组比较均有显著性差异(P＜0.01)。各治疗组大鼠血清IgE、IL-5、IL-13含量较模型组均明显降低(P＜0.01或P＜0.05),其中地塞米松组和止哮平喘方大剂量组血清IgE含量与正常组比较无显著性差异(P＞0.05),各治疗组血清IL-5和IL-13含量与正常组比较,无显著性差异(P＞0.05)。
3.6支气管肺组织形态学光镜观察
哮喘模型组大鼠气道炎症明显、管腔狭窄、肺泡隔明显增宽、平滑肌增厚;地塞米松组和止哮平喘方大、中剂量组均能显著减轻大、小气道及肺组织炎症情况,支气管肺组织大致正常;而小剂量组则变化小。
3.7气道重塑图像分析参数比较
模型组支气管总管壁厚度(WAt/Pbm)、平滑肌厚度(WAm/Pbm)、平滑肌细胞数(N/Pbm)均明显高于正常组(P＜0.01)。地塞米松组、止哮平喘方大、中剂量组WAt/Pbm、WAm/Pbm和N/Pbm值均较模型组降低,结果有显著性差异(P＜0.01或P＜0.05)。小剂量组WAt/Pbm和N/Pbm值较模型组降低(P＜0.05),但WAm/Pbm与模型组比较无显著性差异(P＞0.05)。
3.8大鼠肺组织EOS凋亡及Fas、Bcl-2mRNA的表达
模型组大鼠肺组织中EOS的数量较多,但EOS凋亡减少,同时FasmRNA表达明显降低、Bcl-2mRNA的表达明显增加,与正常组比较有显著性差异(P＜0.01)。各治疗组EOS凋亡增加,同时FasmRNA表达也增加、Bcl-2mRNA的表达降低,与模型组比较有显著性差异(P＜0.01或P＜0.05),且与止哮平喘方有明显的剂量依赖关系。FasmRNh表达与EOS凋亡呈正相关(P＜0.01),而Bcl-2mRNA的表达与EOS凋亡呈负相关(P＜0.01)。
4结论
以OVA致敏和激发,可以成功复制大鼠哮喘模型。止哮平喘方能通过直接松弛气管平滑肌,解除气道痉挛;降低全血和BALF中自细胞及EOS数量;降低血清IgE、IL-5、IL-13含量;促进肺组织FasmRNA表达、抑制Bcl-2mRNA表达,促进肺组织EOS凋亡,从而减轻免疫反应,减轻气道炎症,抑制气道高反应性和气道重塑,发挥平喘作用。
1 Objective:
To investigate the effects of ZhiXiaoPingChuanFang on asthmatic symptom scores,pulmonary overflow amount,tension of tracheal strips in vitro,the count of leukocyte and eosinophil in whole blood and bronchial alveolar lavage fluid(BALF),the content of IgE,IL-5 and IL-13 in serum,Histomorphology of bronchopulmonary tissue,airway remodeling,expression of Fas,Bcl-2mRNA and eosinophil apoptosis in lung tissue.The effective mechanism of ZhiXiaoPingChuanFang on immune response,airway inflammation, airway hyperresponsiveness and airway remodeling of asthma were explored.
2 Methods:
The rat asthma model was established by sensitization and challenge with ovalbumin(OVA).Sixty SD rats were evenly assigned to six groups at random:normal control group,asthma model group, dexamethasone treated group,low dosage group,middle dosage group and high dosage group of ZhiXiaoPingChuanFang.After the rat models were formed successfully,the decoction of ZhiXiaoPingChuanFang was used for treatment.The asthmatic symptom scores,pulmonary overflow amount,tension of tracheal strips,the count of leukocyte and eosinophil in whole blood and BALF were determined.The content of IgE,IL-5 and IL-13 in serum were detected by enzyme linked immuoabsorbent assay(ELISh).The histomorphology of the bronchopulmonary tissue was observed by the light microscop. Parameters of airway remodeling were determined by medical image analysis system.Apoptosis was detected by TUNEL,the expressions of FasmRNA and Bcl-2 mRNA in the lung tissue were detected in situ hybridization.
3 Results:
3.1 The general condition and symptoms of rats at the time of provocation:After inhalation of OVA,model rats came forth symptom of hypersensitiveness such as restlessness and scratching fur on their face;and were in tachypnea or dyspnea(visible abdominal breathing) with slight cyanosis.Some of model rats sounded sonorous wheeze during expiratory phase.After repeated provocation, fur of model rats lost luster,depressed and responsed slowly, the weight growth of model group was slow obviously after repeatedly provocation.There was significant difference on the weight between normal group and model group(P＜0.05).Compared with model group, after treated with medicine,the symptoms of each treatment group alleriated obviously.
3.2 Asthmatic symptom scores:Compared with model group,the asthmatic symptom scores of each treatment group decreased significantly(P＜0.01 or P＜0.05).
3.3 Determination of pulmonary overflow amount and tension of tracheal strips in vitro:Compared with model group,the pulmonary overflow amount and tracheal strips tension of each treatment group decreased significantly(P＜0.01 or P＜0.05).
3.4 Count of leukocyte and eosinophil in whole blood and BALF: Compared with normal group,the count of leukocyte and eosinophil in whole blood and BALF in model group increased significantly(P＜0.01).Compared with model group,the count of leukocyte and eosinophil in whole blood and BALF of each treatment group decreased significantly(P＜0.01 or P＜0.05).Compared with normal group,there was no significant difference on count of leukocyte and eosinophil in whole blood and BALF among dexamethasone group, high dosage group and normal group(P＞0.05).
3.5 The content of IgE,IL-5 and IL-13 in serum:Compared with normal group,the content of IgE,IL-5 and IL-13 in model group increased significantly(P＜0.01).Compared with model group,the content of IgE,IL-5 and IL-13 in each treatment group decreased significantly(P＜0.01 or P＜0.05).Compared with normal group, there was no significant difference on IgE in dexamethasone group and high dosage group(P＞0.05),and there was no significant difference on IL-5 and IL-13 between normal group and each treatment group(P＞0.05).
3.5 Histomorphology of bronchopulmonary tissue:There were obvious infiltration of inflammatory cells,severe luminal stenosis and thickening of alveolar septa in model group,significant hyperplasias of basement membrane and smooth muscle layer of central and peripheral airways were observed in the bronchopulmonary tissue too.The inflammation in the bronchopulmonary was significantly improved in dexamethasone group,high dosage group and middle dosage group.
3.7 Parameters of airway remodeling:Compared with normal group, the thickness of bronchial wall(WAt/Pbm),thickness of bronchial smooth muscle(WAm/Pbm) and the number of airway smooth muscle cells(N/Pbm) increased significantly in model group(P＜0.01). Compared with model group,the parameters of WAt/Pbm,WAm/Pbm and N/Pbm decreased significantly in dexamethasone group,high dosage group and middle dosage group(P＜0.01 or P＜0.05),the parameters of WAt/Pbm and N/Pbm decreased significantly in low dosage group(P＜0.01 or P＜0.05),there was no significant difference on parameters of WAm/Pbm between low dosage group and model group(P＞0.05).
3.8 Expression of Fas,Bcl-2mRNA and eosinophil apoptosis in lung tissue:Compared with normal group,the count of eosinophil increased and eosinophil apoptosis rates decreased,the expression of FasmRNA significantly decreased and the expression of Bcl-2mRNA significantly increased in model group(P＜0.01).Compared with model group,eosinophil apoptosis rates and the expression of FasmRNA increased significantly,while the expression Bcl-2mRNA decreased significantly in each treatment group(P＜0.01 or P＜0.05).The mechanism of ZhiXiaoPingChuanFang on the expressions of Fas,Bcl-2mRNA and eosinophil apoptosis was in a dose-dependent manner,A positively significant correlation was found between the FasmRNA expression and eosinophil apoptosis in each group(P＜0.01),and a very significant negative correlation was found between the Bcl-2mRNA expression and eosinophil apoptosis(P＜0.01).
4 Conclusion:
Sensitization and challenge with OVA could replicate asthma model in rats.ZhiXiaoPingChuanFang could relax the airway smooth muscle directly,relieve the airway spasm,could reduce the count of leukocyte and eosinophil in whole blood and BALF,decrease the content of IgE,IL-5 and IL-13 in serum,promote eosinophil apoptosis,FasmRNA expression,and inhibit Bcl-2mRNA expression. Consequently,by means of reducing immune response and airway inflammation,inhibiting airway hyperresponsiveness and airway remodeling,ZhiXiaoPingChuanFang might play a important role in relieving asthma.
引文
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