【摘要】 目的:观察条件反射箱应激对哮喘小鼠气道上皮细胞的破坏及影响,以及气道上皮细胞凋亡的规律性变化与气道炎症的相关性,探讨疏肝理肺方治疗情志哮喘的可能机制。方法:实验于2005-03/12在江西中医学院附属医院临床检验中心完成。选择10月龄昆明种小鼠120只,采用随机数字表法随机分为4组:正常对照组、哮喘模型组和心理应激哮喘组以及心理应激哮喘中药组。每组30只。①分组后第1天,正常对照组腹腔注射生理盐水0.8mL,1次/d;哮喘模型组、心理应激哮喘组、心理应激哮喘中药组分别腹腔注射致敏液(含卵蛋白100mg、氢氧化铝100mg、百日咳杆菌5×109个)0.8mL,1次/d。②2周后哮喘模型组、心理应激哮喘组、心理应激哮喘中药组均用10g/L卵蛋白、正常对照组给予生理盐水雾化吸入1次/d。③雾化吸入5d,正常对照组、哮喘模型组、心理应激哮喘组开始给予生理盐水灌胃,心理应激哮喘中药组开始给予疏肝理肺汤(由小青龙汤与四逆散合方制成浓缩液,由江西中医学院附属医院制剂室提供)浓缩液12.5mL/(kg·d),至造模结束。④灌胃3d后,心理应激哮喘组、心理应激哮喘中药组每天1次条件反射箱加以应激,对1只鼠进行电击,电压20V,电流强度<1mA,持续时间5s,间隔时间15s,共60次,总时间为20min。同时,将一只进行情绪应激的小鼠放入另一小室中,不给电击,但电击鼠的反应如跑动、跳跃、嘶叫等给情绪应激小鼠的听觉、视觉和嗅觉以强烈刺激,条件反射箱应激1次/d,连续1周。结束取血。⑤观察小鼠的哮喘表现和呼吸频率、外周血细胞计数、血清、支气管灌洗液中白细胞介素4含量和TFAR19水平的变化。结果:120只小鼠全部进入结果分析。①各组小鼠哮喘表现及呼吸频率变化:在整个激发阶段,正常对照组无特殊,心理应激哮喘组较哮喘模型组哮喘特征表现显著,呼吸频率明显增加(P<0.05),并明显表现出不安、惊恐等的行为特点;心理应激哮喘中药组出现上述症状时间后延,程度明显减轻,呼吸频率显著减少(P<0.05)。②各组小鼠外周血嗜酸性粒细胞计数:与正常对照组相比,哮喘模型组、心理应激哮喘组嗜酸性粒细胞计数明显升高((0.15±0.16)%,(0.31±0.62)%,(0.38±1.51)%,P<0.05);与心理应激哮喘组相比,心理应激哮喘中药组嗜酸性粒细胞计数明显降低((0.22±0.58)%,P<0.05)。③各组小鼠血清、支气管肺泡灌洗液中白细胞介素4和TFAR19的含量:与正常对照组相比,哮喘模型组、心理应激哮喘组白细胞介素4和TFAR19含量明显升高(哮喘模型组P<0.05,心理应激哮喘组P<0.01);与心理应激哮喘组相比,心理应激哮喘中药组白细胞介素4和TFAR19含量明显降低(P<0.05)。结论:情绪应激通过神经内分泌免疫网络而影响气道上皮细胞的功能,且通过激活炎症细胞释放炎性介质,影响免疫状态,促发哮喘。疏肝理肺方具抗炎、抑制介质释放,改变细胞凋亡状况,调节应激反应作用,从而对气道上皮细胞的损伤起保护作用。更多还原

【Abstract】 AIM: To investigate the damage and effect of condition reflection box stress on the airway endothelial cells in mice with asthma as well as the regular changes in apoptosis of airway endothelial cells and its correlation with airway inflammation, and probe into the possible mechanism in the treatment of emotional asthma by soothing the liver and regulating the circulation of qi. METHODS: The experiment was performed in the Center of Clinical Laboratory, Affiliated Hospital of Jiangxi College of Traditional Chinese Medicine from March to December 2005. 120 Kunming mice of 10 months old were randomly divided into 4 groups: normal control group, asthma model group, psychological stress asthma group and psychological stress asthma TCM treatment group (TCM group) with 30 mice in each group. ① Mice in the normal control group were peritoneally injected with 0.8 mL of normal saline at one day after grouping once a day. Mice in the asthma model group, psychological stress asthma group and TCM group were intraperitoneally injected with 0.8 mL of sensitization fluid (containing 100 mg of egg protein, 100 mg of aluminum hydroxide, 5 ×109 bacillus pertussis respectively) once a day. ② Two weeks later, mice in the normal control group inhaled the atomization of normal saline once a day, while mice in the other 3 groups were given 10 g/L of egg protein. ③ On the 5th day of atomization-inhalation, mice in the normal control group, asthma model group and psychological stress asthma group were given gastric perfusion of normal saline, while mice in the TCM group were given soothing liver and regulating qi decoction (The concentrated solution was prepared by small qinglong decoction and prescription of powder for regulating liver and spleen, which was manufactured by the Manufacturing Laboratory of Affiliated Hospital of Jiangxi College of Traditional Chinese Medicine) at 12.5 mL/kg each day until the end of modeling. ④ Three days after gastric perfusion, mice in the psychological stress asthma group and TCM group were stimulated in the condition box for stress once a day, and one mouse was shocked at the voltage of 20 V and the current intensity below 1 mA for 5 s with an interval of 15 s for totally 60 times and 20 minutes. Meanwhile, another mouse, performing emotional stress was placed in another room without shock, but intensively stimulated by the reaction of shocked mouse such as running, jumping, yelling at auditory sensation, vision and olfactory sense. The mouse was stressed in the box once a day for 1 week. The blood was obtained at the end of stress. ⑤ The changes in asthma, respiratory frequency, number of peripheral blood cells and the levels of interleukin-4 and TEAR19 in serum and the bronchial lavage liquid were studied in mice. RESULTS: A total of 120 mice were involved in the analysis of results. ① Changes in the respiratory frequency and asthma in mice of each group: During the whole stress, there were no changes in the normal control group, while the characteristic of asthma in mice of the psychological stress asthma group was more significant than that in the asthma model group, and the respiratory frequency was significant increased (P < 0.05). Besides, the mouse was uneasy and scared. The above mentioned symptoms appeared in the TCM group were much slower, which were significant ameliorated, and the respiratory frequency was remarkably decreased (P < 0.05). ② The number of acidophilic granulocyte in the peripheral blood of cells in mice of all groups: It was significantly higher in the asthma model group and psychological stress asthma group than that in the normal control group [(0.15±0.16)%,(0.31±0.62)%,(0.38±1.51)%,P < 0.05], and it was significantly lower in the psychological stress asthma group than that in the TCM group [(0.22±0.58)%,P < 0.05]. ③ The level of interleukin-4 and TFAR19 in serum and bronchoalveolar lavage fluid: Those were significantly increased in the asthma model group and psychological stress asthma group than those in the normal control group (asthma model group: P < 0.05; the psychological stress asthma group P < 0.01), while those in the TCM group was significantly lower than those in the psychological stress asthma group (P < 0.05). CONCLUSION: The emotional stress can influence the function of airway endothelial cells by neuroendocrine immune network, and affect the immune state by activating the inflammatory cells and releasing the inflammatory medium, thus induce the asthma. Prescription of soothing liver and regulating the circulation of qi can resist the inflammation, suppress the media release, change the cell apoptosis, regulate the stress response, and thereby protect the airway endothelial cells from injury.更多还原