基于血浆代谢组学的“冬病夏治”穴位贴敷法治疗缓解期慢性支气管炎的临床疗效及机制研究

发布时间：2018-09-06 11:28

【摘要】：慢性支气管炎是由感染、过敏等因素引起的气管、支气管黏膜及其周围组织的慢性非特异性炎症,临床以咳嗽、咳痰或伴喘息为主要特征,是临床常见的一种呼吸系统疾病。近年来,伴随人口老龄化进程和环境污染的加剧,我国慢性支气管炎患病人数日趋增多。本病具有典型的病程长、易复发等特征,伴随疾病的进展可并发阻塞性肺气肿、肺动脉高压、肺源性心脏病等,严重危害人类健康,甚至影响患者的劳动能力和生存质量。当前,慢性阻塞性肺疾病已居世界死亡原因第四位,且发病率仍呈上升趋势。因此,对慢性支气管炎的有效防治具有非常重要的社会意义。“冬病夏治”穴位贴敷疗法防治慢性支气管炎以其疗效明显、操作简便、副作用小等优势越来越被患者接受和认可。诸多学者对中药穴位贴敷疗法的机制进行了初步研究,一般认为是药物的透皮吸收与经络系统对人体调节的双重效应。但目前中药的药效成分及经络调节机制尚未清楚,寻找其它途径的研究方法对于揭示穴位贴敷疗法的机制意义重大。故本研究首先通过观察“冬病夏治”穴位贴敷治疗前后慢性支气管炎患者发作时主要症状单项积分、生存质量等指标的变化,明确“冬病夏治”穴位贴敷法治疗慢性支气管炎的临床疗效;其次,通过观察免疫球蛋白、炎症细胞因子、神经递质的变化,一方面佐证穴位贴敷法治疗慢支的疗效,另一方面从神经-炎症-免疫的角度,综合解释“冬病夏治”穴位贴敷法治疗慢支的作用机制;第三,利用基于液相-质谱联用(LC/MS)技术的代谢组学研究方法,将代谢组学技术应用到“冬病夏治”穴位贴敷疗法的整体疗效评价以及作用机制研究,分析穴位贴敷治疗前后患者的血浆代谢图谱,并通过观察治疗所引起的内源性代谢物的变化,推测体内生化状态的变化,进而探讨该疗法的作用机制;寻找反映治疗前后机体变化的代谢标志物,利用代谢物的终端性信息,为寻找或阐明药物作用的靶点或受体奠定前期基础。本论文的研究内容主要包括以下三个部分:观察第一部分“冬病夏治”穴位贴敷法治疗缓解期慢性支气管炎的临床疗效目的:观察“冬病夏治”穴位贴敷法治疗缓解期慢性支气管炎患者的临床疗效。方法:选择符合纳入标准的缓解期慢性支气管炎患者50例作为慢支组,分别于头伏、二伏、末伏的第一天给予穴位贴敷治疗,共3次。30天为1个疗程。采取样本治疗前后自身对照的方法进行对比分析。将患者治疗前1年(2013年7月~2014年6月)的症状,与治疗后1年(2014年7月~2015年6月)的症状做比较。参照《中药新药临床研究指导原则》对慢支症状分级量化表评分,记录治疗前后观察期内咳嗽、咳痰、咳喘等各单项症状的严重程度及持续时间(以天数为记录单位),并评定疗效;采用圣乔治医院呼吸问卷(SGRQ),从症状的频率和严重程度(症状部分)、会造成气促或受疾病限制的活动(活动部分)以及疾病导致的社会能力损害和心理问题(对日常生活的影响)三个方面综合评价穴位贴敷治疗前后慢支患者的生存质量。结果:“冬病夏治”穴位贴敷法可显著改善慢支患者临床症状:治疗后1年患者咳嗽症状积分明显低于治疗前(P0.05);咳痰症状积分与治疗前比较差异有统计学意义(P0.05);咳喘症状积分较治疗前明显降低(P0.05);临床中医证候疗效明显,总有效率72%;该疗法可明显改善慢支患者的生存质量,治疗后1年症状部分评分、活动部分评分、影响部分评分及SGRQ总评分较治疗前均明显降低,差异有统计学意义(P0.05)。第二部分“冬病夏治”穴位贴敷法对缓解期慢性支气管炎患者神经-炎症-免疫系统调控影响的研究目的:通过免疫球蛋白、炎症细胞因子、神经递质的观察,在明确“冬病夏治”穴位贴敷疗法治疗慢支疗效的基础上,探讨该疗法的作用机制,从神经-炎症-免疫整体的角度探讨其作用靶点和作用途径。方法:选择符合纳入标准的缓解期慢性支气管炎患者50例,设为慢支组(chronic bronchitis,CB)。给予“三伏天”穴位贴敷治疗,30天作为一个疗程。同时设50例健康志愿者作为对照组(control)。检测对照组以及慢支病人治疗前(pre-treatment,Pre)、治疗后(post-treatment,Post)、治疗后冬季(winter)血清免疫球蛋白(Ig A、Ig G、Ig M、Ig E),炎症细胞因子(TNF-α、IL-4、IL-8、IL-10),血浆P物质(SP),血管活性肠肽(VIP)等指标。比较慢支组治疗后、治疗后冬季免疫球蛋白、炎症细胞因子以及神经递质与治疗前的差异,并将慢支组治疗前、治疗后、治疗后冬季的相应指标与对照组进行比较,从神经-炎症-免疫角度,探讨“冬病夏治”穴位贴敷疗法对慢支患者炎症的改善以及对机体的保护机制。结果:与对照组比较,慢支组治疗前、治疗后以及治疗后冬季Ig A、Ig G、Ig M、IL-10、VIP水平下降,差异有统计学意义(P0.05);Ig E、IL-4、IL-8、TNF-α、SP水平升高,除慢支组治疗后IL-8之外,与对照组比较差异均有统计学意义(P0.05);慢支组治疗后Ig A、Ig G、Ig M、IL-10、VIP水平较治疗前升高,差异有统计学意义(P0.05);Ig E、IL-4、IL-8、SP水平较治疗前下降,差异有统计学意义(P0.05);治疗后TNF-α较治疗前有下降趋势,但差异无统计学意义(P0.05)。慢支组治疗后冬季Ig A、Ig G、Ig M、IL-10、VIP水平较治疗前升高,差异有统计学意义(P0.05);Ig E、IL-4、IL-8、SP水平较治疗前下降,差异有统计学意义(P0.05);治疗后冬季TNF-α较治疗前有下降趋势,但差异无统计学意义(P0.05)。与慢支组治疗后比较,治疗后冬季Ig M、IL-10、VIP水平下降,差异有统计学意义(P0.05);IL-8、SP水平升高,差异有统计学意义(P0.05);治疗后冬季Ig A、Ig G、TNF-α水平有下降趋势,但差异无统计学意义(P0.05);Ig E、IL-4水平有上升趋势,但差异无统计学意义(P0.05)。第三部分基于血浆代谢组学的“冬病夏治”穴位贴敷法治疗缓解期慢性支气管炎的机制研究目的:基于现代生物质谱技术,运用代谢组学方法研究“冬病夏治”穴位贴敷治疗前后慢支患者的血浆代谢组学特征,通过治疗前后潜在生物标志物变化,探讨“冬病夏治”穴位贴敷疗法防治慢支的机理,并利用代谢物的终端性信息,为寻找或阐明药物作用的靶点或受体奠定前期基础。方法:选择符合纳入标准的缓解期慢支患者50例,设为慢支组,给予“三伏天”穴位贴敷疗法,30天作为一个疗程。同时设50例健康志愿者作为对照组(control)。分别采集对照组及慢支组治疗前(Pre)、治疗后(Post)、治疗后冬季(winter)血液样品,并采用高效液相色谱-质谱联用仪(HPLC-MS)进行检测。利用主成分分析法(principal component analysis,PCA)和正交偏最小二乘法判别分析(orthogonal partial least squares-discriminant analysis,OPLS-DA)进行数据统计学处理,比较各组血浆代谢组学原始图谱和数据。结果:各组血浆代谢物图谱均呈现聚类型分布,慢支组在治疗前与对照组的代谢谱存在明显不同。穴位贴敷治疗后,慢支组治疗后以及治疗后冬季的代谢谱均向对照组回归,与对照组代谢成分接近。初步推测胆碱、组氨酸、维生素B6、甲硫氨酸、生物喋呤等为慢支治疗前区分对照组的潜在标志物,在发病和复发中发挥重要作用。其中,组氨酸、甲硫氨酸在各组比较中均发挥重要作用,且呈现治疗后组氨酸降低、甲硫氨酸升高的趋势。结论:1“冬病夏治”穴位贴敷法治疗缓解期慢性支气管炎疗效确切,值得在临床继续深化和推广。该观察结果也为深入研究“冬病夏治”穴位贴敷疗法的作用机制提供了坚实的临床数据支持。2“冬病夏治”穴位贴敷法能改善机体的免疫状态,协调免疫平衡,增强机体防病抗病能力;能调节炎症细胞因子的释放,降低炎症发生的程度;能通过神经递质的调节,对免疫和炎症反应发挥良性调节作用。这可能是其预防慢支复发、减轻症状的协同机制。3通过对人体血浆的代谢组学研究,发现慢支患者存在与对照组明显不同的代谢图谱。慢支组治疗后及治疗后冬季代谢图谱向对照组的回归,证实了“冬病夏治”穴位贴敷法治疗慢支的疗效;初步阐释了“冬病夏治”穴位贴敷疗法防治慢支的代谢组学机制,可能集中在纠正氨基酸、维生素代谢紊乱、调节细胞因子的产生和释放等方面。研究结果对采用现代生物科学技术探讨传统中医药理论有一定的启发作用。
[Abstract]:Chronic bronchitis is a chronic nonspecific inflammation of trachea, bronchial mucosa and its surrounding tissues caused by infection, allergy and other factors. It is a common respiratory disease clinically characterized by cough, sputum or wheezing. Chronic obstructive pulmonary disease (COPD) is the leading cause of death in the world. With the development of COPD, obstructive pulmonary emphysema, pulmonary hypertension, pulmonary heart disease and so on, it seriously endangers human health and even affects the working ability and quality of life of patients. Therefore, it is of great social significance for the effective prevention and treatment of chronic bronchitis. Acupoint application therapy of "treating winter diseases in summer" has been accepted and recognized by patients more and more for its obvious curative effect, simple operation and less side effects. The mechanism of this method has been preliminarily studied, generally believed to be the dual effects of drug transdermal absorption and meridian system on human body regulation. However, the pharmacodynamic components of traditional Chinese medicine and the mechanism of meridian regulation are still unclear. It is of great significance to find other ways to reveal the mechanism of acupoint application therapy. The clinical effect of acupoint sticking therapy of "treating winter disease in summer" on chronic bronchitis was clarified. Secondly, the changes of immunoglobulin, inflammatory cytokines and neurotransmitters were observed, which supported the evidence on the one hand. On the other hand, the mechanism of Acupoint Application in treating chronic bronchitis is comprehensively explained from the angle of nerve-inflammation-immunity. Thirdly, the metabolomics research method based on liquid chromatography-mass spectrometry (LC/MS) technology is applied to acupoint application in treating chronic bronchitis. Methods To evaluate the overall efficacy and study the mechanism of action, analyze the plasma metabolic map of patients before and after acupoint sticking therapy, and by observing the changes of endogenous metabolites caused by treatment, infer the changes of biochemical state in vivo, and then explore the mechanism of action of this therapy; to find metabolic markers reflecting the changes of the body before and after treatment. The research contents of this paper mainly include the following three parts: the first part observes the clinical effect of acupoint sticking therapy of treating chronic bronchitis in remission period; the second part observes the acupoint sticking therapy of treating chronic bronchitis in winter and summer Methods: Fifty patients with chronic bronchitis in remission period were selected as the chronic bronchitis group. They were treated with acupoint sticking therapy on the first day of headrest, second volt and last volt respectively. The treatment course was 3 times and 30 days. The symptoms of the patients in the first year before treatment (July 2013-June 2014) were compared with those in the first year after treatment (July 2014-June 2015). The SGRQ was used to evaluate acupoint patches comprehensively from three aspects: frequency and severity of symptoms (symptoms), activities (activities) that may cause shortness of breath or be restricted by disease, social impairment and psychological problems (effects on daily life) caused by disease. Results: Acupoint sticking therapy of "treating winter disease in summer" can significantly improve the clinical symptoms of patients with chronic bronchitis: one year after treatment, the score of cough symptoms was significantly lower than that before treatment (P 0.05); the score of cough symptoms was significantly lower than that before treatment (P 0.05); the score of cough and asthma symptoms was significantly lower than that before treatment (P 0.05); the score of cough and asthma symptoms was (P 0.05); the curative effect of TCM syndromes was obvious, the total effective rate was 72%; the treatment can significantly improve the quality of life of patients with chronic bronchitis, one year after treatment symptoms partial score, activity partial score, influence partial score and SGRQ total score were significantly lower than before treatment, the difference was statistically significant (P 0.05). The second part of the "winter disease summer treatment" acupoint application method. Objective: To investigate the effect of acupoint sticking therapy on chronic bronchitis in remission stage by observing immunoglobulin, inflammatory cytokines and neurotransmitters, and to explore the mechanism of acupoint sticking therapy on chronic bronchitis from the perspective of nerve-inflammation-immunity. Methods: Fifty patients with chronic bronchitis in remission period were selected and assigned to the chronic bronchitis group (CB) according to the inclusion criteria. Serum immunoglobulin (Ig A, Ig G, Ig M, Ig E), inflammatory cytokines (TNF-alpha, IL-4, IL-8, IL-10), plasma substance P (SP), vasoactive intestinal peptide (VIP) were measured before and after treatment, and in winter after treatment. Compared with the control group, the corresponding indexes in winter after treatment were compared. From the angle of nerve-inflammation-immunity, the improvement of inflammation and the protective mechanism of acupoint sticking therapy for treating chronic bronchitis in winter were discussed. The levels of Ig A, Ig G, Ig M, IL-10 and VIP were significantly lower before and after treatment than before treatment (P 0.05), while the levels of Ig E, IL-4, IL-8, TNF-a and SP were significantly higher than those before treatment (P 0.05), except for IL-8 in the chronic bronchitis group, the levels of Ig A, Ig G, Ig M, IL-10 and VIP were significantly higher than those before treatment. The levels of Ig E, IL-4, IL-8 and SP were significantly lower than those before treatment (P 0.05). After treatment, the levels of TNF-alpha decreased, but there was no significant difference (P 0.05). After treatment, the levels of Ig A, Ig G, Ig M, IL-10 and VIP in the chronic bronchitis group were significantly higher than those before treatment (P 0.05). The levels of IL-8 and SP were significantly lower than those before treatment (P 0.05). After treatment, the levels of TNF-a in winter had a downward trend, but there was no significant difference (P 0.05). After treatment, the levels of Ig M, IL-10 and VIP in winter were significantly lower than those in CMB group (P 0.05). The levels of IL-8 and SP were significantly higher than those before treatment (P 0.0). 5) After treatment, the levels of Ig A, Ig G and TNF-alpha decreased in winter, but there was no significant difference (P 0.05); Ig E and IL-4 levels increased, but there was no significant difference (P 0.05). The third part based on plasma metabolomics, "winter disease summer treatment" acupoint sticking therapy for chronic bronchitis in remission mechanism research purposes: based on modern health Material spectrum technique was used to study the plasma metabolomic characteristics of patients with chronic bronchitis before and after acupoint sticking therapy of "Winter Disease Treated in Summer". The mechanism of acupoint sticking therapy of "Winter Disease Treated in Summer" for prevention and treatment of chronic bronchitis was discussed through the changes of potential biomarkers before and after treatment, and the terminal information of metabolites was used to find or elucidate drugs. Methods: Fifty patients with chronic bronchitis in remission period were selected as the chronic bronchitis group, and treated with Acupoint Application Therapy of "three-dog days" for 30 days as a course of treatment. T. Blood samples in winter after treatment were analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS). Data were analyzed by principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). Results: The plasma metabolite profiles of each group showed clustering distribution, and the metabolite profiles of the CMB group were significantly different from those of the control group before treatment. It is speculated that choline, histidine, vitamin B6, methionine and biopterin are the potential markers to distinguish the control group before CB treatment and play an important role in the pathogenesis and recurrence. The observation also provides a solid clinical data support for the further study of the mechanism of acupoint application therapy. 2 The acupoint application method of "treating winter diseases in summer" can improve the body's immune symptoms. It can regulate the release of inflammatory cytokines and reduce the degree of inflammation. It can play a benign role in regulating immune and inflammation through the regulation of neurotransmitters. This may be the synergistic mechanism of preventing relapse of chronic bronchitis and alleviating symptoms. The results showed that the metabolic Atlas of patients with chronic bronchitis were significantly different from those of the control group. The regression of the metabolic Atlas of patients with chronic bronchitis in winter after treatment and after treatment to the control group confirmed the curative effect of acupoint sticking therapy for treating chronic bronchitis by "treating winter diseases in summer"; and preliminarily explained the metabolomic mechanism of acupoint sticking therapy for treating chronic bronchitis by "treating winter diseases in summer", which might be a collection. The results of this study will be helpful to the application of modern biotechnology in the study of traditional Chinese medicine.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R246.1

【参考文献】