杜鹃素对卵蛋白和内毒素诱导气道炎症的作用及机制研究

发布时间：2018-06-13 08:12

本文选题：杜鹃素 + 气道炎症　；参考：《吉林大学》2012年博士论文

【摘要】：肺组织是一种非常复杂的免疫器官，可应答多种吸入性的物质包括常见的抗原、部分有机或无机物、特定种类的感染物或寄生物、气体及刺激物等，从而引发气道阻塞、并伴发肺间隙、肺泡和支气管的炎性堵塞。根据免疫应答模式不同将肺部疾病分为固有性肺部免疫应答（中性粒细胞参与的）和适应性肺部炎症应答（TH1和TH2淋巴细胞、嗜酸性粒细胞等参与的）。本文通过构建脂多糖（LPS）诱导的急性肺损伤（固有免疫性肺疾病）和鸡卵蛋白（OVA）诱导的过敏性哮喘（适应性免疫肺疾病）两种不同的动物模型，研究杜鹃素对不同免疫反应引起的气道炎症的治疗作用及其作用机制。急性肺损伤是以弥漫性气道炎症为特征的，包括细胞因子（如TNF-α、IL-6和IL-8）、趋化因子和致炎介质等调节的中性粒细胞进入间质组织的免疫应答。过敏性哮喘临床上是以黏液分泌过盛、气道堵塞及炎症、气道高反应性和气道重塑为特征的慢性炎症性疾病。过敏性气道炎症包括肺组织炎性细胞浸润、黏液分泌过多、过敏源特异性IgE增多、Th2型细胞因子如白细胞介素4（IL-4）、白细胞介素5（IL-5）及白细胞介素13（IL-13）和趋化因子如CCL11和CCL5的表达增多等。同时Th1细胞因子如干扰素γ（IFN-γ）可通过下调Th2应答反应抑制过敏性炎症的发生。嗜酸性粒细胞是哮喘发生时浸润到肺组织中的主要炎性细胞，它与IL-5浓度有关。对于炎症的作用机制的研究表明，TLR蛋白通过相似的信号转导通路最终可激活核转录因子（NF-κB）、激活蛋白（AP-1）、磷脂酰肌醇3激酶（PI3K）和丝裂原蛋白激酶（MAPK）等信号分子，进而在肺部感染性疾病、炎症和过敏性哮喘中发挥重要作用。植物当中提取的化合物种类最多且具有抗炎活性的是黄酮类中药单体，已成为是植物酚的最大组成部分。杜鹃素是一种从杜鹃花中提取出的2,3-二氢-黄酮类化合药物，已有研究表明，杜鹃素具有抗菌活性，但是没有关于其抗炎活性的相关报道。本实验通过构建两种小鼠的肺疾病模型、评估了杜鹃素对过敏性哮喘气道炎症和急性肺损伤气道炎症的药理作用，并进一步探讨其作用机制。研究结果如下： 1.与空白对照组相比，OVA处理组小鼠肺泡灌洗液中炎性细胞数（包括细胞总数、嗜酸性粒细胞数、中性粒细胞数、巨噬细胞数和淋巴细胞数）等显著升高。杜鹃素（20和40mg/kg）治疗组显著地抑制了过敏性哮喘肺泡灌洗液中细胞总数和嗜酸性粒细胞数，对巨噬细胞、中性粒细胞和淋巴细胞无显著抑制作用。 2.组织病理学结果显示，OVA处理组小鼠的支气管和血管周围出现炎性细胞浸润、黏液产物增多以及杯状细胞增生等。杜鹃素（20和40mg/kg）治疗组显著地降低了支气管和血管周围出现的炎性细胞浸润、黏液产物增多以及杯状细胞增生等。 3.与空白对照组相比，OVA处理组哮喘模型组肺泡灌洗液中IFN-γ、IL-10、IL-4、IL-5、IL-13和Eotaxin浓度明显升高。杜鹃素（20和40mg/kg）治疗组可显著地降低IL-4、IL-5、IL-13和嗜酸性粒细胞趋化因子的浓度并升高IFN-γ浓度，但是对IL-10无显著调节作用。此外，杜鹃素（20和40mg/kg）治疗组可显著地降低外周血中OVA-特异性IgE含量。 4.应答于不同浓度的乙酰甲胆碱，OVA处理组小鼠可形成气道高反应性，表现为高肺阻力和低肺动态顺应性。杜鹃素（20和40mg/kg）治疗组可显著的降低肺阻力并升高肺动态顺应性。 5.与空白对照组相比，OVA处理组肺组织中趋化因子CCL5和CCL11以及它们的受体CCR1和CCR3基因表达显著升高。杜鹃素（20和40mg/kg）治疗组可显著地降低趋化因子极其受体的表达。 6.杜鹃素可显著地抑制肺组织中与气道炎症和气道重塑相关的E-selectin、AMCase、Ym1、Ym2和Muc5ac的mRNA表达。 7.杜鹃素可显著地降低由OVA诱导产生的肺组织中p-Akt和p-p70S6K的蛋白的表达，对丝裂原蛋白激酶无显著抑制作用。 8.杜鹃素通过阻断OVA诱导产生的IκBα降解，抑制IκBα磷酸化，进而抑制了NF-κB p65从胞浆转移到核内。 9.在体外，杜鹃素可显著地降低anti-CD3/CD28诱导产生Th2细胞分泌的IL-4、IL-5和IL-13产物，并可抑制anti-CD3/CD28诱导产生p-Akt、p-p70S6K和p-IκBα的蛋白表达。 10.低剂量的杜鹃素可协同低剂量的地塞米松，显著地抑制OVA诱导的Th2细胞因子过表达、炎性细胞浸润和肺部病理学改变。 11.与空白对照组相比，LPS可显著升高急性肺损伤肺泡灌洗液中细胞总数、中性粒细胞数、巨噬细胞数以及细胞因子（TNF-α、IL-6和IL-8）的浓度，，并可上调NF-κB、p-AKT和MAPKs的蛋白表达。与地塞米松治疗组相比，杜鹃素治疗组对LPS诱导产生的肺损伤症状无显著地抑制作用。综上所述，本研究通过采用两种不同的肺气道炎症模型，研究杜鹃素对不同气道炎症的治疗作用，首次阐明了杜鹃素对哮喘性气道炎症的抑制作用及其作用机制，并可协同糖皮质激素类治疗过敏性气道炎症，为杜鹃素可作为糖皮质激素类联合用药用以治疗过敏性气道炎症提供理论和实验依据。
[Abstract]:Lung tissue is a very complex immune organ that respond to a variety of inhalation substances including common antigens, partially organic or inorganic substances, specific types of infection or parasites, gases, and irritants, causing airway obstruction, associated with pulmonary spaces, alveoli and bronchioled clogging. The lungs are different according to the immune response pattern. The disease is divided into intrinsic pulmonary immune response (neutrophilic granulocyte participation) and adaptive pulmonary inflammatory response (TH1 and TH2 lymphocytes, eosinophils). In this paper, an allergic asthma (adaptive immune lung) induced by lipopolysaccharide (LPS) induced acute lung injury (innate immune lung disease) and chicken ovalbumin (OVA) is constructed. Two different animal models were used to study the therapeutic effect of rhododendrin on airway inflammation induced by different immune responses and its mechanism.
Acute lung injury is characterized by diffuse airway inflammation, including cytokines (such as TNF- alpha, IL-6 and IL-8), chemokines and inflammatory mediators that regulate the immune response of neutrophils into interstitial tissue. Allergic asthma is characterized by mucus secretion, airway plugging and inflammation, airway hyperresponsiveness, and airway remodeling. Chronic inflammatory diseases. Allergic airway inflammation includes inflammatory cell infiltration of the lung tissue, excessive mucus secretion, anaphylactic specific IgE, Th2 cytokines such as interleukin 4 (IL-4), interleukin 5 (IL-5) and interleukin 13 (IL-13), and chemokine such as CCL11 and CCL5, such as Th1 cell factors such as interference. IFN- gamma can inhibit the occurrence of anaphylactic inflammation by down regulation of the Th2 response. Eosinophils are the main inflammatory cells infiltrating into the lung tissue during the occurrence of asthma. It is related to the concentration of IL-5. The study of the mechanism of the action of inflammation indicates that the TLR protein can eventually activate the nuclear transcription factor (NF- kappa B) through similar signal transduction pathways. Activated protein (AP-1), phosphatidylinositol 3 kinase (PI3K) and mitogen kinase (MAPK) signal molecules, and thus play an important role in pulmonary infectious diseases, inflammation and allergic asthma.
The most species and anti-inflammatory activity of the compounds are flavonoids, which are the largest component of the plant phenols. The azalein is a 2,3- two hydrogen flavone compound extracted from the Rhododendron. The studies have shown that rhododendrin has anti - bacterial activity but no anti - inflammatory activity. Close the report.
In this experiment, the pharmacological effects of Rhododendron on airway inflammation in allergic asthma and airway inflammation in acute lung injury were evaluated by constructing two models of lung disease in mice, and the mechanism of its action was further explored.
The results of the study are as follows:
1. compared with the blank control group, the number of inflammatory cells in the alveolar lavage fluid (including the number of cells, eosinophils, neutrophils, number of macrophages, and the number of lymphocytes) in the OVA treatment group were significantly increased. The total number of cells and the eosinophils in the alveolar lavage fluid of the allergic asthma were significantly inhibited by the azalein (20 and 40mg/kg) treatment group. The number of cells had no significant inhibitory effect on macrophages, neutrophils and lymphocytes.
2. the results of histopathology showed that inflammatory cells infiltrated, mucus products and goblet cells proliferated around the bronchi and blood vessels of the OVA treatment group. The treatment group of rhododendrin (20 and 40mg/kg) significantly reduced inflammatory cell infiltration, increased mucous products and goblet cell proliferation around the bronchi and blood vessels.
3. compared with the blank control group, the concentration of IFN- gamma, IL-10, IL-4, IL-5, IL-13 and Eotaxin in the alveolar lavage fluid of the OVA treatment group was significantly increased. The concentration of IL-4, IL-5, IL-13 and eosinophil chemotactic factors and the increase of IFN- gamma concentration were significantly reduced in the treatment group of azalein (20 and 40mg/kg), but there was no significant regulation on the concentration of IFN- gamma. In addition, rhododendrin (20 and 40mg/kg) treatment group could significantly reduce OVA- specific IgE content in peripheral blood.
4. in response to different concentrations of methacholine, OVA treated mice could form airway hyperresponsiveness, showing high lung resistance and low lung dynamic compliance. The treatment group of azalein (20 and 40mg/kg) could significantly reduce lung resistance and increase lung dynamic compliance.
5. compared with the blank control group, the expression of chemokine CCL5 and CCL11, and their receptor CCR1 and CCR3 genes in the lung tissue of the OVA treatment group were significantly increased. The expression of chemokine and extreme receptors could be significantly reduced by the azalein (20 and 40mg/kg) treatment groups.
6. rhododendrin significantly inhibited the expression of E-selectin, AMCase, Ym1, Ym2 and Muc5ac in lung tissue associated with airway inflammation and airway remodeling.
7. rhododendrin significantly decreased the expression of p-Akt and p-p70S6K in lung tissue induced by OVA, and had no significant inhibitory effect on mitogen activated protein kinase.
8. rhododendrin inhibited the degradation of I kappa B alpha induced by OVA and inhibited the phosphorylation of I kappa B alpha, thereby inhibiting the transfer of NF- B p65 from cytoplasm to the nucleus.
9. in vitro, rhododendrin can significantly reduce anti-CD3/CD28 induced IL-4, IL-5 and IL-13 products produced by Th2 cells, and inhibit the expression of p-Akt, p-p70S6K and p-I kappa B alpha induced by anti-CD3/CD28.
10. the low dose of rhododendrin can be combined with low dose of dexamethasone, which significantly inhibits the overexpression of Th2 cytokines induced by OVA, infiltration of inflammatory cells and pathological changes in the lungs.
11. compared with the blank control group, LPS could significantly increase the total number of cells, the number of neutrophils, the number of macrophages and the concentrations of cytokines (TNF-, IL-6 and IL-8) in the alveolar lavage fluid of acute lung injury, and up regulation of the protein expression of NF- kappa B, p-AKT and MAPKs. Compared with the dexamethasone treatment group, the lung damage induced by the rhododenin treatment group was induced by LPS. The symptoms of injury had no significant inhibitory effect.
To sum up, we studied the therapeutic effect of Rhododendron on different airway inflammation by using two different pulmonary airway inflammation models. It was the first time to elucidate the inhibitory effect of Rhododendron on asthmatic airway inflammation and its mechanism. It can be used in the treatment of allergic airway inflammation with glucocorticoid, and the rhododendrin can be used as glucocorticoid. Combined drug use provides theoretical and experimental evidence for the treatment of allergic airway inflammation.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R562.25

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