摘要
[目的]
探明健脾益肺口服液对慢性阻塞性肺疾病稳定期大鼠免疫功能、气道炎症的影响,求证健脾益肺口服液是否通过NF-κB信号传导途径来调节慢阻肺大鼠免疫功能及抑制其气道炎症,为健脾益肺口服液临床治疗慢阻肺病人提供实验依据,从而进一步论证将“健脾益肺法”作为慢阻肺稳定期中医药治疗手段的可行性和重要性；深入理解“肺脾气虚”这一慢阻肺稳定期中医证型和客观指标“免疫功能低下”及“气道炎症指标”等的相关性,加深对慢阻肺稳定期肺脾气虚证本质的认识,促进中医证型客观化,对指导中医药治疗慢阻肺稳定期的基础研究具有重要意义。
[方法]
1.理论探讨方面：对慢阻肺中医病机进行深入探讨；深入分析健脾益肺法治疗慢阻肺的依据；探析健脾益肺口服液的方义,分析其用于调节慢阻肺免疫功能、抑制气道炎症的可行性。
2.实验研究方面：将65只SD成年雄性大鼠随机分为6组,空白对照组10只、其余各组每组11只,分别为模型组、健脾益肺口服液高、中、低剂量组及地塞米松阳性药对照组；用香烟烟熏加脂多糖复合因素复制慢阻肺“肺气虚证”动物模型,然后用大黄煎液灌胃复制“脾气虚证”模型；造模28天结束,在14天开始按照实验设计给药,28天后测定指标；观察各组大鼠整体情况、HE染色后观察各组基本病理变化; MASSON染色后观察各组胶原含量、平滑肌厚度等；应用图像分析系统测定支气管平滑肌厚度、气管内径等,计算支气管平滑肌指数、胶原纤维面积；取动脉血,抗凝后用动脉血气分析仪测定大鼠PaCO2、PaO2、PH及SaO2等动脉血气指标；取血后剥离大鼠胸腺、脾脏,根据体质量计算大鼠胸腺指数、脾脏指数；麻醉后取血,抗凝后用流式细胞仪测定血液中CD3+、CD4+、CD8+的表达率,并计算CD4+/CD8+比值；部分血液离心后取血清,用ELISA试剂盒测定IgG、IgA、IgM水平及IL-8、TNF-α活性；取大鼠肺部组织,液氮速冻研磨用trizol提取组织mRNA,逆转录后,设计引物进行real-time PCR扩增,以内参为对照,观察各组肺组织IL-8、TNF-α mRNA的基因表达；肺部组织用试剂盒提取组织蛋白质,利用蛋白印迹(WB, Western blotting)方法,测定各组肺组织NF-κB和I-κB的表达。
[结果]
1.通过对动物模型的整体观察及肺组织病理学结果,证实了慢阻肺“肺脾气虚证”动物模型的复制是成功的,从而为实验的顺利进行提供了依据；
2.健脾益肺口服液能显著改善慢阻肺实验大鼠的整体情况,如咳嗽、体重、精神状态等；
3.健脾益肺口服液能显著抑制大鼠气道炎症,降低胶原含量、胶原纤维面积、支气管平滑肌指数,改善气道重塑,作用类似于或优于地塞米松,以高、中剂量组为佳；
4.健脾益肺口服液能改善大鼠缺氧状态,由此提高大鼠的存活率,改善生存质量,但对PC02、PH无明显改善作用;
5.健脾益肺口服液能显著提高慢阻肺大鼠细胞及体液免疫功能,降低其急性发作次数；
6.健脾益肺口服液能抑制IL-8、TNF-α炎性标志物的浓度及IL-8、TNF-αmRNA在肺组织的表达,疗效以高、中剂量组为佳；
7.健脾益肺口服可增加慢阻肺大鼠体质量、胸腺指数、脾脏指数,提高机体免疫力,增强防病抗病能力；
8.健脾益肺口服液可降低NF-κB在肺组织的表达,提高了I-κB的表达。而NF-κB信号传导途径在慢阻肺气道炎症反应、免疫调节中起着重要作用,本实验健脾益肺口服液提高了免疫功能、抑制了气道炎症,同时下调NF-κB在肺组织的表达,上调I-κB的表达,故推测健脾益肺口服液可能通过NF-κB信号传导途径调节COPD免疫功能、抑制气道炎症。
[结论]
1.肺脾气虚直接关系到慢阻肺的发生、发展,为慢阻肺稳定期的病机关键。从“肺脾气虚证”的病机出发,立法健脾益肺,通过健脾而达到补益肺气的目的。
2.健脾益肺口服液能增强慢阻肺实验大鼠免疫功能、抑制其气道炎症,改善气道重塑,且一般以高、中剂量组为佳,对部分指标的疗效呈剂量依赖性；其机制可能为通过NF-κB信号传导途径抑制慢阻肺大鼠气道炎症、调控其免疫功能；并有可能通过下调炎症因子调节慢阻肺免疫功能。
3.慢阻肺“肺脾气虚证”病证结合动物模型的成功复制使研究更有针对性,在中医基础研究与临床研究之间架起了桥梁,有助于中医药作用机制的研究,且对于深层次探讨中西医理论的内在联系有重要意义。
4.本研究加深了对慢阻肺稳定期“肺脾气虚证”本质的认识,在促进中医药疗效机制的阐明及中药新药的研发中具有重要意义,为探索出中医药治疗慢阻肺的优势环节、作用靶点提供了依据。
[Objective]
This study aims to explore the effect of Spleen-Strengthening and Lung-Supplementing Liquids on immune function and airway inflammation in rats during stationary COPD phage, and to demonstrate whether through the NF-κB signaling transduction pathway Spleen-Strengthening and Lung-Supplementing Liquid regulates immune function and inhibit airway inflammation in rats with COPD, and to provide the experimental basis for the clinical treatment of Spleen-Strengthening and Lung-Supplementing Liquids to patients with COPD. Further more, this paper is to demonstrate the feasibility and significance of the treating method of Strengthening Spleen and Supplementing Lung as the treatment of stable COPD phage, and to have a further understanding of the relationship between TCM syndrome and objective indicators (immune dysfunction, and airway inflammation indicators, etc).In addition, the study is aimed to deepen the understanding of lung-spleen deficiency syndrome as the nature of the stable COPD phage, and to make TCM syndromes objective. All above is going to have great significance to basic research of guiding Chinese medical therapy for stable COPD phage.
[Methods]
1. Theoretic exploring aspect:TCM pathogenesis of chronic obstructive pulmonary disease was in-depth explored. In addition, the basis of spleen-strengthening and lung-supplementing treatment was deeply analyzed. And furthermore, the description explanation of Spleen-Strengthening and Lung-Supplementing Liquids was discussed. What's more, the feasibility of Spleen-Strengthening and Lung-Supplementing Liquids on regulating immune function and inhibiting airway inflammation in patients with chronic obstructive pulmonary disease.
2. experimental research aspect:65SD adult male rats were randomly divided into six groups(10of the blank control group,11of every other groups), i.e. the model group, Spleen-Strengthening and Lung-Supplementing Liquids group respectively in large dose, middle dose and small dose, and positive drug (Dexamethasone) control group. Rats was treated with cigarette smoke and LPS complex factors to build the model of COPD in syndrome pattern of lung-qi deficiency. And then the model of spleen-qi deficiency was derived from gavaging rats with rhubarb liquid. Model-making lasted for28days. Rats were administrated in accordance with the experimental design for14days.28days later, the indicators were determined. Afterwards, the whole situation of the rats in each group was observd, and the basic pathological changes were observed after HE staining. And then after MASSON staining, collagen content and smooth muscle thickness in each group was observed. The bronchial smooth muscle thickness, internal diameter of the trachea was determined by application image analysis system, while the index of bronchial smooth muscle and area of collagen fibers were calculated. With arterial blood treated of anticoagulation, PaCO2, PaO2, PH and SaO2and other arterial blood gas indicator in rats were measured by arterial blood gas analysis. After the thymus, spleen of rat peeled, and thymus index, spleen index was calculated according to body mass. Next, blood was taken after anesthesia, then treated with anticoagulation, and expression rate of CD3+, CD4+, CD8+in the blood were determined by flow cytometry. Meanwhile CD4+/CD8+ratio was calculated. In addition, partial blood dealt with centrifugeserum, keeping serum, IgG, IgA, and IgM levels and IL-8and TNF-alpha activity were measured by ELISA. After blood taken, mRNA was extracted by trizol grinding after rat lung tissue frozen in liquid nitrogen. After reverse transcription, designed primers were dealt with real-time PCR amplification. With the internal reference for the gene expression of control, IL-8, TNF-alpha mRNA in the lung tissue were observed. tissue proteins of lung tissue were extracted by the kit, and with Western blot (Western blot) method, expression of NF-κB and I-κB in the lung tissue were determined.
[Results]
1.Though overall observation of animal models and pulmonary histopathologic findings, we confirmed the replication of animal models of patients with COPD of lung and spleen Qi deficiency syndrome was successful, and thus provided a basis for the smooth progress of the experiment.
2.Spleen-Strengthening and Lung-Supplementing Liquids was able to significantly improve the overall situation of experimental rats with COPD, such as weight, cough, mental status.
3.Spleen-Strengthening and Lung-Supplementing Liquids apparently inhibited the airway inflammation in rats, reduced collagen content, collagen fiber area index of bronchial smooth muscle, improve airway remodeling, with the efficacy similar to or superior to Dexamethasone, especially the efficacy in Large and middle dosage groups was better.
4.Spleen-Strengthening and Lung-Supplementing Liquids was able to improve the hypoxic state of the rats, thus improving the survival rate of rats as well as the life quality. However, there was no significant improvement in PaCO2, PH.
5.Spleen-Strengthening and Lung-Supplementing Liquids can significantly improve cellular and humoral immune function of the rats with COPD, and reduce the number of acute COPD attack.
6.Spleen-Strengthening and Lung-Supplementing Liquids was able to inhibit the concentration of inflammatory markers such as IL-8and TNF-alpha and the expression of TNF-amRNA,IL-8and TNF-alphain in the lung tissue, with the efficacy in Large and middle dosage groups better.
7. Spleen-Strengthening and Lung-Supplementing Liquids increased body weight of the COPD rats, thymus index, spleen index,and enhanced immunity, and disease prevention and disease resistance.
8. Spleen-Strengthening and Lung-Supplementing Liquids reduced expression of NF-κB in lung tissue, but increased I-κB expression. NF-κB signaling transduction pathway plays an important role in airway inflammation reaction and immune regulation during COPD, and Spleen-Strengthening and Lung-Supplementing Liquids improved immune function, inhibited airway inflammation, at the same time downregulated NF-κB in lung tissue expression, upregulated expression of I-κB, from which we would conclude that Spleen-Strengthening and Lung-Supplementing Liquids might adjust immune function and inhibit airway inflammation through the NF-κB signaling pathway in COPD.
[Conclusions]
1.Lung and spleen Qi deficiency is directly related to the occurrence and development of COPD, which is the critical pathogenesis of chronic obstructive pulmonary disease stable period. Starting from the pathogenesis of Lung and spleen Qi deficiency syndrome, we established the strengthening spleen and supplementing lung treatment, so as to achieve the purpose of supplementing lung Qi.
2.Spleen-Strengthening and Lung-Supplementing Liquids, with its description available, selected pathogesis and significant efficacy, enhanced the immune function in rats with COPD, inhibited airway inflammation, and improved the airway remodeling, especially the efficacy in Large and middle dosage groups was better. And some indicators of efficacy were dose-dependent. The mechanism is that Spleen-Strengthening and Lung-Supplementing Liquids may via NF-κB signaling transduction pathway inhibit airway inflammation in rats with COPD, regulate the immune function; and be possible to regulate immune function in rats with by down-regulating inflammatory factors.
3.It is Lung and spleen Qi deficiency syndrome combined with animal models of chronic obstructive pulmonary disease to make this study more targeted, and what's more, it builds a bridge between chinese basic study and clinic study, and promotes study of Chinese medical efficacy mechanism. In addition, this study plays an important role in exploring the deep inner link between medical theory and Chinese medicine theory.
4. This study deepens the understanding of lung and spleen Qi deficiency syndrome in COPD stable period, and plays an important role in promoting clarification of Chinese medical efficacy mechanism and research and development of new Chinese medicine. In addition, this study provides basis for exploring the super link and target of Chinese medicine treatment of chronic obstructive pulmonary disease.
引文
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