抗支口服液对哮喘大鼠模型作用机制的实验研究
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摘要
目的：以建立支气管哮喘大鼠模型为基础,给予抗支口服液治疗,通过动物实验,从免疫组织化学、病理学和分子生物学等多方面探讨中药抗支口服液治疗哮喘大鼠模型作用机制,为临床应用抗支口服液治疗小儿支气管钳哮喘提供实验依据。
方法：
1、实验动物模型的建立及动物分组
将70只wistar未成年大鼠,体重120～150g,适应性饲养3天。参照有关文献,将大鼠于实验第1天和第8天,腹腔内注射卵蛋白0.1mg和氢氧化铝凝胶1mg,从而使实验大鼠致敏。于实验的第15天起用3%卵蛋白对实验大鼠以5ml/分钟的雾化流速进行哮喘雾化激发,6分钟／次,两天一次,持续4周。以大鼠出现躁动、喷嚏、咳嗽、呼吸急促、流泪及站立不稳等表现为激发成功。将造模成功的50只大鼠随机分为模型组、地塞米松组、抗支口服液高、中、低剂量组。另取10只为空白组,空白对照组则用生理盐水代替卵蛋白对其进行腹腔注射及雾化激发。
2、实验动物的灌胃给药及取材
从第1次哮喘激发开始(于实验第15天)至处死前各治疗组每天于雾化前0.5h灌胃给药,高剂量组20g/kg/d.中剂量组10g/kg/d.低剂量组5g/kg/d,西药(地塞米松)组,给予0.5mg/kg/d地塞米松灌胃,持续给药4周,正常组用生理盐水灌胃。造模之前对实验动物进行体重测量,此后每4天对大鼠进行体重测量,于取材之前对大鼠进行最后一次体重测量。末次给药2小时后,对实验动物进行麻醉后取材。取各组大鼠肺组织、肺泡灌洗液(BALF)及其血清以备用。光镜下观察肺组织炎性病理改变,显微镜下观察肺泡灌洗液涂片细胞数及嗜酸性粒细胞和淋巴细胞含量,以酶联免疫吸附(ELISA)法测定肺组织中的LTB4、LTC4含量及血清内ECP的含量,以逆转录聚合酶联反应(reverse transcriptase, RT-PCR)测定哮喘大鼠肺组织IL-5mRNA表达,以放射免疫法测定血清中IgE含量。
结果：
1、肺组织病理学改变。
光镜下见模型组大鼠肺组织有大量炎性细胞浸润,气道粘膜水肿伴有脱落的上皮细胞,上皮下纤维化,平滑肌增厚。中药高、中剂量组大鼠肺组织与模型组相比,以上改变明显减轻
2、各指标改变
实验结果显示抗支口服液能明显降低哮喘大鼠肺组织中炎症的主要调节因子IL-5mRNA的表达,从而减少气道内炎区的EOS的增殖、分化、聚集和活化,从而控制气道炎症；能降低哮喘大鼠血清免疫球蛋白E(IgE)的产生增加,减少肥大细胞的分化,并减慢嗜酸性粒细胞的生长、趋化和激活,从而减轻哮喘气道炎症；能降低哮喘大鼠血清中诱导气道炎症、介导气道上皮细胞损伤脱落的强碱性颗粒蛋白ECP含量；能降低肺组织中炎性介质LTB4、LTC4含量；降低哮喘大鼠肺泡灌洗液(BALF)涂片细胞数及嗜酸性粒细胞和淋巴细胞含量。
结论：
1、抗支口服液各剂量组能改善哮喘大鼠一般症状,减少哮喘大鼠肺组织炎性浸润,减少粘膜下水肿及分泌物的产生,从而减轻气道狭窄及气道高反应性,尤其是抗支口服液高、中剂量组。
2、抗支口服液高、中剂量组均能降低哮喘大鼠肺组织内嗜酸性粒细胞、淋巴细胞含量,说明抗支口服液可通过降低肺组织内嗜酸性粒细胞、淋巴细胞含量,从而控制气道炎症。
3、抗支口服液可明显降低哮喘大鼠肺组织IL-5mRNA的表达,减少炎性细胞浸润,从而减轻气道炎症,缓解哮喘气道痉挛状态。
4、抗支口服液高、中剂量组明显降低了哮喘大鼠外周血内ECP含量从而抑制EOS的活化,保护呼吸道上皮,减轻AHR,抑制变态反应,最终达到抗炎的效果。
5、抗支口服液可明显降低哮喘大鼠外周血内IgE含量,减轻哮喘的Ⅰ型变态反应的发生,从而达到控制哮喘发作的目的。
6、抗支口服液高、中剂量组可明显降低哮喘大鼠肺组织内的LTB4、LTC4含量,从而减轻气道炎症及减缓支气管平滑肌收缩来缓解哮喘症状。
Objective:The study was established on the basis of bronchial asthma rat model, while giving the anti-bronchial asthma oral liquid intervention, we explore that rat model mechanism in the anti-bronchial asthma oral liquid heal cough variant asthma about immunohistochemistry, pathology aspects and Cell Biology through animal experiments, the results of this study for which anti-bronchial asthma oral liquid healed bronchial asthma in children to provide experimental evidence.
Method:
1. Animal groups and the establishment of animal model
We will the of70wistar adult rats that its weight120to150g, these rats by the Experimental Animal Center of Heilongjiang University of Chinese Medicine.After3days, We refer to the relevant literature, the researchers used the rats in each group, except for the normal group, the rest of the rats in experiments1and8days, intraperitoneal injection of10%ovalbumin, aluminum hydroxide mixture of1ml of a total of2ml, Injection in rats on both sides of the waist, in order to make the sensitization. Since the fifteenth day, the researchers applied3%ovalbumin40ml spray excited in models of asthma, the atomization flow control in5ml per minute, for15minutes each time, once every two days, consecutive four weeks in order to stimulate. The the rat appears rapid breathing, lips cyanosis, nodded breathing and standing instability and other symptoms, these symptoms indicate that inspire success.The50sucessful rats after3days were randomly divided into control group, model group, dexamethasone group, anti-support oral high, medium and low dose group. We will the other10wistar adult rats as normal group, Group of normal saline instead of ovalbumin by intraperitoneal injection and inhalation.
2. Experimental animals gavage and drawn
Researchers begin to the first provocation (the modeling3weeks) to each treatment group before being killed daily irrigation0.5h gastric administration before the atomization high dose group40mg/kg/d, middle dose group20mg/kg/d, low dose group10mg/kg/d, western medicine (dexamethasone) group, giving0.5mg/kg/d dexamethasone orally; sustained delivery of four weeks, the normal group with normal saline.
The experimental animals were weighed before modeling; Thereafter rats were weighed every5days, the rats were drawn before the last weighing; excited after4weeks, the rats on experimental rats per unit time within the respiratory frequencyand the number of sneezing records.2hours after the last administration, the experimental animals were anesthetized, and then drawn. Researcher take each rat lung tissue, bronchoalveolar lavage fluid (BALF) and serum to spare. Associated enzyme-linked immunosorbent assay (ELISA) method for the determination of lung tissue LTB4, LTC4content and associated reverse transcriptase method for the determination of serum IL-5mRNA expression, and IgE levels in serum were measured by radio immunoassay.
3. The experimental results
(1) Lung histopathological changes.
The study found that the lung tissue of the model under a microscope to generate a large number of inflammatory cell infiltration, airway mucosal edema associated with shedding of epithelial cells, subepithelial fibrosis, smooth muscle thickening. Chinese medicine, the low-dose group lung tissue of rats with the model group, these changes significantly reduced, compared with the dexamethasone group, there was no significant difference.
(2) Each index change
The experimental results show that anti-bronchial asthma oral liquid1can significantly reduce a major regulator of inflammatory factors IL-5mRNA levels in asthmatic rats serum, then reduce airway inflammation District EOS proliferation, differentiation, aggregation and activation, to achieve the purpose of the control of airway inflammation; It can also reduce the generation of suffering from asthma rat serum immunoglobulin E (IgE), to reduce the differentiation of mast cells and slow down the growth of eosinophil chemotaxis and activation, reduce asthma eosinophilic airway inflammation. It can reduce the serum of asthma-induced airway inflammation-mediated airway epithelial cell damage off the strong alkaline granule protein ECP content; and reduce inflammatory mediators in the lung tissue LTB4, LTC4; and reduce asthma BALFeosinophils in bronchoalveolar lavage fluid (BALF) supernatant anti-bronchial asthma oral liquid through a multi-level, multi-channel, multi-target control airway inflammation of asthma rat model, it can reduce the high reactivity of the airways of asthmatic rats, so it has broad application prospects.
Conclusion:
1. Each dose group of anti-bronchial asthma oral liquid can improve the general symptoms, reduce inflammatory infiltration, submucosal edema, reduce the production of secretions in asthmatic rats, thereby reducing airway narrowing and airway hyperresponsiveness, especially high dose group and medium dose group.
2. High dose group and medium dose group of anti-bronchial asthma oral liquid can reduce the content of eosinophils and lymphocytes in lung tissue, which indicated that anti-bronchial asthma oral liquid can control airway inflammation by reducing eosinophils and lymphocytes content in the lung tissue.
3. anti-bronchial asthma oral liquid can significantly reduce the expression of IL-5mRNA in the lung tissue of asthmatic rats, reduce the infiltration of inflammatory cells, thereby to reduce airway inflammation and relieve airway spasm state.
4. ECP content in the peripheral blood of asthmatic rats had been significantly reduced by high dose group and medium dose group, thereby inhibited the activation of EOS, to protect the respiratory epithelium, reduce AHR, inhibit allergic reactions, eventually to achieve the effect of resolving phlegm to relieve cough, anti-inflammatory and relieving allergy.
5. anti-bronchial asthma oral liquid can significantly reduce IgE levels in the peripheral blood of asthmatic rats, and reduce the incidence of type I allergic reaction of asthma, so as to achieve the purpose of controlling asthma attacks.
6. High dose group and medium dose group of anti-bronchial asthma oral liquid can reduce the content of LTB4and LTC4in lung tissue, which indicated that anti-bronchial asthma oral liquid can reduce asthma airway inflammation and slow down the bronchial smooth muscle contraction to relieve asthma symptoms by reducing LTB4and LTC4.
方法：
1、实验动物模型的建立及动物分组
将70只wistar未成年大鼠,体重120～150g,适应性饲养3天。参照有关文献,将大鼠于实验第1天和第8天,腹腔内注射卵蛋白0.1mg和氢氧化铝凝胶1mg,从而使实验大鼠致敏。于实验的第15天起用3%卵蛋白对实验大鼠以5ml/分钟的雾化流速进行哮喘雾化激发,6分钟／次,两天一次,持续4周。以大鼠出现躁动、喷嚏、咳嗽、呼吸急促、流泪及站立不稳等表现为激发成功。将造模成功的50只大鼠随机分为模型组、地塞米松组、抗支口服液高、中、低剂量组。另取10只为空白组,空白对照组则用生理盐水代替卵蛋白对其进行腹腔注射及雾化激发。
2、实验动物的灌胃给药及取材
从第1次哮喘激发开始(于实验第15天)至处死前各治疗组每天于雾化前0.5h灌胃给药,高剂量组20g/kg/d.中剂量组10g/kg/d.低剂量组5g/kg/d,西药(地塞米松)组,给予0.5mg/kg/d地塞米松灌胃,持续给药4周,正常组用生理盐水灌胃。造模之前对实验动物进行体重测量,此后每4天对大鼠进行体重测量,于取材之前对大鼠进行最后一次体重测量。末次给药2小时后,对实验动物进行麻醉后取材。取各组大鼠肺组织、肺泡灌洗液(BALF)及其血清以备用。光镜下观察肺组织炎性病理改变,显微镜下观察肺泡灌洗液涂片细胞数及嗜酸性粒细胞和淋巴细胞含量,以酶联免疫吸附(ELISA)法测定肺组织中的LTB4、LTC4含量及血清内ECP的含量,以逆转录聚合酶联反应(reverse transcriptase, RT-PCR)测定哮喘大鼠肺组织IL-5mRNA表达,以放射免疫法测定血清中IgE含量。
结果：
1、肺组织病理学改变。
光镜下见模型组大鼠肺组织有大量炎性细胞浸润,气道粘膜水肿伴有脱落的上皮细胞,上皮下纤维化,平滑肌增厚。中药高、中剂量组大鼠肺组织与模型组相比,以上改变明显减轻
2、各指标改变
实验结果显示抗支口服液能明显降低哮喘大鼠肺组织中炎症的主要调节因子IL-5mRNA的表达,从而减少气道内炎区的EOS的增殖、分化、聚集和活化,从而控制气道炎症；能降低哮喘大鼠血清免疫球蛋白E(IgE)的产生增加,减少肥大细胞的分化,并减慢嗜酸性粒细胞的生长、趋化和激活,从而减轻哮喘气道炎症；能降低哮喘大鼠血清中诱导气道炎症、介导气道上皮细胞损伤脱落的强碱性颗粒蛋白ECP含量；能降低肺组织中炎性介质LTB4、LTC4含量；降低哮喘大鼠肺泡灌洗液(BALF)涂片细胞数及嗜酸性粒细胞和淋巴细胞含量。
结论：
1、抗支口服液各剂量组能改善哮喘大鼠一般症状,减少哮喘大鼠肺组织炎性浸润,减少粘膜下水肿及分泌物的产生,从而减轻气道狭窄及气道高反应性,尤其是抗支口服液高、中剂量组。
2、抗支口服液高、中剂量组均能降低哮喘大鼠肺组织内嗜酸性粒细胞、淋巴细胞含量,说明抗支口服液可通过降低肺组织内嗜酸性粒细胞、淋巴细胞含量,从而控制气道炎症。
3、抗支口服液可明显降低哮喘大鼠肺组织IL-5mRNA的表达,减少炎性细胞浸润,从而减轻气道炎症,缓解哮喘气道痉挛状态。
4、抗支口服液高、中剂量组明显降低了哮喘大鼠外周血内ECP含量从而抑制EOS的活化,保护呼吸道上皮,减轻AHR,抑制变态反应,最终达到抗炎的效果。
5、抗支口服液可明显降低哮喘大鼠外周血内IgE含量,减轻哮喘的Ⅰ型变态反应的发生,从而达到控制哮喘发作的目的。
6、抗支口服液高、中剂量组可明显降低哮喘大鼠肺组织内的LTB4、LTC4含量,从而减轻气道炎症及减缓支气管平滑肌收缩来缓解哮喘症状。
Objective:The study was established on the basis of bronchial asthma rat model, while giving the anti-bronchial asthma oral liquid intervention, we explore that rat model mechanism in the anti-bronchial asthma oral liquid heal cough variant asthma about immunohistochemistry, pathology aspects and Cell Biology through animal experiments, the results of this study for which anti-bronchial asthma oral liquid healed bronchial asthma in children to provide experimental evidence.
Method:
1. Animal groups and the establishment of animal model
We will the of70wistar adult rats that its weight120to150g, these rats by the Experimental Animal Center of Heilongjiang University of Chinese Medicine.After3days, We refer to the relevant literature, the researchers used the rats in each group, except for the normal group, the rest of the rats in experiments1and8days, intraperitoneal injection of10%ovalbumin, aluminum hydroxide mixture of1ml of a total of2ml, Injection in rats on both sides of the waist, in order to make the sensitization. Since the fifteenth day, the researchers applied3%ovalbumin40ml spray excited in models of asthma, the atomization flow control in5ml per minute, for15minutes each time, once every two days, consecutive four weeks in order to stimulate. The the rat appears rapid breathing, lips cyanosis, nodded breathing and standing instability and other symptoms, these symptoms indicate that inspire success.The50sucessful rats after3days were randomly divided into control group, model group, dexamethasone group, anti-support oral high, medium and low dose group. We will the other10wistar adult rats as normal group, Group of normal saline instead of ovalbumin by intraperitoneal injection and inhalation.
2. Experimental animals gavage and drawn
Researchers begin to the first provocation (the modeling3weeks) to each treatment group before being killed daily irrigation0.5h gastric administration before the atomization high dose group40mg/kg/d, middle dose group20mg/kg/d, low dose group10mg/kg/d, western medicine (dexamethasone) group, giving0.5mg/kg/d dexamethasone orally; sustained delivery of four weeks, the normal group with normal saline.
The experimental animals were weighed before modeling; Thereafter rats were weighed every5days, the rats were drawn before the last weighing; excited after4weeks, the rats on experimental rats per unit time within the respiratory frequencyand the number of sneezing records.2hours after the last administration, the experimental animals were anesthetized, and then drawn. Researcher take each rat lung tissue, bronchoalveolar lavage fluid (BALF) and serum to spare. Associated enzyme-linked immunosorbent assay (ELISA) method for the determination of lung tissue LTB4, LTC4content and associated reverse transcriptase method for the determination of serum IL-5mRNA expression, and IgE levels in serum were measured by radio immunoassay.
3. The experimental results
(1) Lung histopathological changes.
The study found that the lung tissue of the model under a microscope to generate a large number of inflammatory cell infiltration, airway mucosal edema associated with shedding of epithelial cells, subepithelial fibrosis, smooth muscle thickening. Chinese medicine, the low-dose group lung tissue of rats with the model group, these changes significantly reduced, compared with the dexamethasone group, there was no significant difference.
(2) Each index change
The experimental results show that anti-bronchial asthma oral liquid1can significantly reduce a major regulator of inflammatory factors IL-5mRNA levels in asthmatic rats serum, then reduce airway inflammation District EOS proliferation, differentiation, aggregation and activation, to achieve the purpose of the control of airway inflammation; It can also reduce the generation of suffering from asthma rat serum immunoglobulin E (IgE), to reduce the differentiation of mast cells and slow down the growth of eosinophil chemotaxis and activation, reduce asthma eosinophilic airway inflammation. It can reduce the serum of asthma-induced airway inflammation-mediated airway epithelial cell damage off the strong alkaline granule protein ECP content; and reduce inflammatory mediators in the lung tissue LTB4, LTC4; and reduce asthma BALFeosinophils in bronchoalveolar lavage fluid (BALF) supernatant anti-bronchial asthma oral liquid through a multi-level, multi-channel, multi-target control airway inflammation of asthma rat model, it can reduce the high reactivity of the airways of asthmatic rats, so it has broad application prospects.
Conclusion:
1. Each dose group of anti-bronchial asthma oral liquid can improve the general symptoms, reduce inflammatory infiltration, submucosal edema, reduce the production of secretions in asthmatic rats, thereby reducing airway narrowing and airway hyperresponsiveness, especially high dose group and medium dose group.
2. High dose group and medium dose group of anti-bronchial asthma oral liquid can reduce the content of eosinophils and lymphocytes in lung tissue, which indicated that anti-bronchial asthma oral liquid can control airway inflammation by reducing eosinophils and lymphocytes content in the lung tissue.
3. anti-bronchial asthma oral liquid can significantly reduce the expression of IL-5mRNA in the lung tissue of asthmatic rats, reduce the infiltration of inflammatory cells, thereby to reduce airway inflammation and relieve airway spasm state.
4. ECP content in the peripheral blood of asthmatic rats had been significantly reduced by high dose group and medium dose group, thereby inhibited the activation of EOS, to protect the respiratory epithelium, reduce AHR, inhibit allergic reactions, eventually to achieve the effect of resolving phlegm to relieve cough, anti-inflammatory and relieving allergy.
5. anti-bronchial asthma oral liquid can significantly reduce IgE levels in the peripheral blood of asthmatic rats, and reduce the incidence of type I allergic reaction of asthma, so as to achieve the purpose of controlling asthma attacks.
6. High dose group and medium dose group of anti-bronchial asthma oral liquid can reduce the content of LTB4and LTC4in lung tissue, which indicated that anti-bronchial asthma oral liquid can reduce asthma airway inflammation and slow down the bronchial smooth muscle contraction to relieve asthma symptoms by reducing LTB4and LTC4.
引文
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