摘要
目的：
探讨翘芩清肺冲剂对卵清白蛋白诱发大鼠哮喘的平喘作用及其机制。
方法：
采用卵清白蛋白致敏并反复多次激发建立大鼠哮喘模型。91只SD大鼠随机分为正常对照组、模型组、地塞米松(0.27 mg·kg-1.d)组、氨茶碱(0.05 g·kg-1.d)组和翘芩清肺冲剂高(26g·kg-1)、中(13 g·kg-1)、低(6.5 g·kg-1)剂量组。除正常对照组外,其余大鼠在第1天和第8天时,腹腔注射10%卵清白蛋白(OVA)1ml致敏,在第15天时开始雾化吸入1%OVA(每天1次,每次20min),连续雾化吸入14天,正常对照大鼠给予同量的生理盐水替代OVA。在致敏的第2天,开始灌胃给予上述药物,正常组和模型组大鼠灌胃等容量的生理盐水,每天1次,连续给药4周。诱喘过程中,测定哮喘潜伏期和哮喘程度；末次雾化24h后处理动物,取支气管和肺组织进行HE染色,切片观察其形态学变化；采用Elisa方法分别检测大鼠血清中血小板活化因子(PAF)的含量,测定支气管肺泡灌洗液(BALF)中内皮素(ET)、IL-4、IL-5及IFN-r的含量；取血检测血中白细胞总数及嗜酸性粒细胞的含量；流式细胞技术检测血中CD4+、CD25+的表达情况。
结果：
(1)哮喘模型大鼠喘息明显,哮喘潜伏期缩短；与正常大鼠比较,哮喘模型大鼠血清血小板活化因子(PAF)含量明显增加(p<0.01),肺泡灌洗液中ET、IL-4和IL-5含量明显增加(p<0.01,p<0.05),IFN-r含量明显降低(p<0.01)；外周血白细胞总数及EOS比例增加(p<0.05)；CD4+含量降低,但差异无显著性(p>0.05)
(2)翘芩清肺冲剂高、中、低剂量和地塞米松及氨茶碱处理组大鼠,与哮喘模型组比较其哮喘程度降低,哮喘潜伏期明显延长(p<0.01)；并均有不同程度的降低PAF含量(p<0.01)。
(3)与哮喘模型组比较,地塞米松组和氨茶碱组及翘芩清肺冲剂高、中剂量组明显降低ET含量(p<0.01),而低剂量组则无明显影响(p>0.05)。
(4)与哮喘模型组比较,翘芩清肺冲剂高、中、低剂量和地塞米松及氨茶碱处理组均有不同程度的降低IL-4含量(p<0.01,p<0.05)；地塞米松及翘芩清肺冲剂高剂量组降低IL-5的含量(p<0.05)；地塞米松、氨茶碱及翘芩清肺冲剂高、中剂量组则明显增加IFN-r的含量(p<0.01)
(5)与哮喘模型组比较,翘芩清肺冲剂低剂量组对外周血嗜酸性粒细胞影响不明显(p>0.05),其余组不同程度降低白细胞总数与嗜酸性粒细胞的比例(p<0.01,p<0.05)。
(6)地塞米松组、氨茶碱组和翘芩清肺各剂量组对大鼠血中CD4+、CD25+的表达均无明显影响(p>0.05)。
(7)镜下观察哮喘模型组大鼠支气管粘膜上皮细胞及杯状细胞增生,肺泡内有分泌物及炎性细胞浸润。药物干预后支气管粘膜上皮细胞及杯状细胞增生减少,肺泡内少有分泌物及细胞浸润,平滑肌增厚不明显。
结论：
1.翘芩清肺冲剂对OVA诱致的大鼠哮喘具有改善和延缓哮喘发作的作用；
2.翘芩清肺冲剂对OVA诱致的大鼠哮喘的防治作用可能与降低外周血白细胞总数及嗜酸性粒细胞所占比例以及血小板活化因子(PAF)的含量,降低了大鼠肺泡灌洗液中内皮素(ET)的含量,调节哮喘时Th1/Th2比例失衡有关。
Objective:
To investigate Qiao-qin-qing-fei granule relieving asthma effect of the asthmatic rats induced by ovalbumin and its mechanism.
Methods:
Bronchial asthmatical rats were established by OVA-sensitized and repeated-inspiring method.91 rats were randomly divided into the control group, asthma group, dexamethasone group, aminophylline group and Qiao-qin-qing-fei (26g/kg、13g/k、6.5g/kg) group. Every rat except the control group was sensitized by injecting 10% of ovalbumin through intraperitoneal in the first day and the eighth day, while begin from the fifteenth day 1% of OVA aerosol inhalation(once a day, every time 20 minutes) for 14 days continuously, and the normal control rats were given the same amount of saline. In the second day of sensitization, the rats were administered with above-mentioned drugs by intragastric administration respectively, and the rats in normal group and model group were given the same volume of saline(once a day) for 4 weeks continuously. The degree and latency of asthma were tested in the process of inducing asthma; After 24h of the last nebulized inhalation, HE staining method was applied to observe the morphological changes of histologic lung tissues; Elisa was used to detect the contents of serum PAF and measur the levels of ET, IL-4, IL-5, IFN-r in bronchoalveolar lavage fluid; Detected total white blood cells and the content of eosinophils by blood drawing; Measured the expression of CD4+、CD25+ in peripheral blood was detected by flow cytometry.
Results:
(1) Asthmatic rats gasped significantly, shortened the latency of asthma; Compaed to control group, the content of serum PAF in model group had increased markedly (p<0.01), the level of ET, IL-4, IL-5 in bronchoalveolar lavage fluid had increased obviously (p<0.01,p<0.05) and the level of IFN-r had decreased significantly (p<0.01);Total white blood cells and the proportion of eosinophils in rat peripheral blood were higher than normal model(p<0.05); the presence of CD4+ in model group lower than normal group, but there was had no significant difference(p>0.05).
(2) Compared to model group, the degree of asthma in high-dose qiao-qin-qing-fei group, middle-dose qiao-qin-qing-fei group, low-dose qiao-qin-qing-fei group, dexamethasone group and aminophylline group had decreased markedly and significantly extended the latency of asthma (p<0.01), while the levels of PAF had decreased in varying degrees (p<0.01).
(3) Compared to model group, the content of ET had reduced greatly in dexamethasone group, aminophylline group and high-dose Qiao-qin-qing-fei group and middle-dose group (p<0.01), but there is had no significant effect in low-dose Qiao-qin-qing-fei group (p>0.05).
(4) Compared to model group, the level of IL-4 had decreased obviously in high-dose Qiao-qin-qing-fei group, middle-dose Qiao-qin-qing-fei group, low-dose Qiao-qin-qing-fei group, dexamethasone group and aminophylline group at different degrees (p<0.01,p<0.05); the content of IL-5 had reduced only in dexamethasone group and high-dose Qiao-qin-qing-fei group (p<0.05); the level of IFN-r had increased significantly in all the treatment groups except low-dose Qiao-qin-qing-fei group (p<0.01).
(5) Compared with model group, low-dose Qiao-qin-qing-fei group had no significant effect on eosinophils (p>0.05), but the other groups reduced total white blood cells and the proportion of eosinophils with different degrees(p<0.01,p<0.05).
(6) Dexamethasone, aminophylline and different dosages of qiao-qin-qing-fei granule had no obviously effect on the expression of CD4+、CD25+.
(7) Both of the bronchial epithelial cells and goblet cells hyperplasia of asthmatic rats were observed under microscopy, and there were secretion and inflammatory cells in pulmonary alveolar. After drugs treatment, bronchial epithelial cells and goblet cells hyperplasia had reduced. Furthermore, there were less secretions and cell infiltration within alveolar and the smooth muscle thickening was not obvious.
Conclusions:
1. Qiao-qin-qing-fei granule has the role of improving and delaying the onset of asthma induced by ovalbumin.
2. The preventive and therapeutic effect of qiao-qin-qing-fei may be related with reducing total white blood cells and the proportion of eosinophils in peripheral blood, as well as the contents of serum PAF, decrease ET levels of lavage fluid, regulating the balancing of Thl/Th2 in asthmatic rats.
引文
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