Clinical Effects And Mechanism Research Of Acupoint Application For "Winter-disease-treated-in-summer" On Stable Chronic Bronchitis: Based On Plasma Metabonomics

Chronic bronchitis, a commonly encountered clinical disease of respiratory system, is a chronic non-specific inflammation in the trachea, bronchial mucosa and their surrounding tissues caused by infection, allergies and other factors. It is characterized by cough, expectoration, or accompanied by wheezing. In recent years, with the aging of population and aggravating of environmental pollution, the number of chronic bronchitis patients is increasing in our coutry. The typical feature of this disease is long course, easy to reoccur, et al. With the progression of the disease, it may be complicated by emphysema, pulmonary hypertension or pulmonary heart disease, which may bring serious harms to human health, even affect the ability to work and quality of life. Chronic obstructive pulmonary disease(COPD) is the fourth serious cause of death in the current world, and the morbidity is increasing. So, the effective prevention and treatment of chronic bronchitis shows its important social significance. Because of the obvious effects, easy operation and low side effects, acupoint application for “Winter-Disease-Treatedin-Summer” is increasingly being recognized and accepted by chronic bronchitis patients. The mechanism of acupoint application was studied by many researchers, the dual effect composed of percutaneous absorption of drugs and meridian adjustment was considered as the main mechanism. But the medicinal ingredient of traditional Chinese medicine and meridian regulatory mechanism is not clear yet. It is important to explore other ways to reveal the mechanism of acupoint application. By observing the changes of scores in main symptoms and quality of life before and after treatment, this research confirmed the clinical efficacy of treating chronic bronchitis by acupoint application for "Winter-Disease-Treated-in-Summer". Secondly, the efficacy of acupoint application on chronic bronchitis was supported and the mechanism was observed through neurotransmitter-inflammation factorimmunoglobulin. Thirdly, the metabonomics technology was applied to estimate the overall efficacy of acupoint application and study the mechanism based on the liquid-mass spectrometry(LC/MS) technology of metabonomics analysis. The patients, plasma metabolic patterns were analysised before and after the treatment. By observing the changes of the endogenous metabolite caused by the therapy, the biochemical state changes were speculated, and then to discuss the mechanism further. Based on that, to look for the metabolic markers which reflect the body changes before and after the treatment, to prepare basic of looking for or clarifying the drug targets or receptors by means of the metabolite terminal information.This paper mainly includes the following three parts:Part one Clinical Observation of Acupoint Application for "Winter-Disease-Treated-in-Summer" in Stable Chronic BronchitisObjective: To observe the clinical effect of acupoint application for “Winter-Disease-Treated-in-Summer” in treating stable chronic bronchitis.Method: Fifty chronic bronchitis patients in stable phase were selected as CB group according to the selective criterion. The patients received acupoint application in dog days. One treatment was given in each dog day, 3 times altogether, 30 days are regarded as a course of treatment. The samples were contrasted between before and after the treatment. The symptoms of the 1 year before treatment(July 2013~June 2014) were compared with those after treatment(July 2014~June 2015). On the bases of Guiding Principle of Clinical Research in New Medicine of Chinese Traditional Medicine, the hierarchical quantitative table of symptoms of chronic bronchitis were scored, the severity and duration(days as the recording unit) of each single syndrome before and after the treatment were recorded, which included cough, expectoration and wheeze, the curative effect was evaluated at the same time. “St George’s Hospital Respiratory Questionnaire” was used to analysis patients quality of life before and after the therapy in three parts: The frequency and severity of the symptoms(part of symptoms), the activity that can trigger shortness of breath or may be restricted by the disease(part of activities), damage to social ability and psychological problems(part of impact on daily life).Result: Acupoint application for “Winter-Disease-Treated-in-Summer” significantly relieved the clinical symptoms of chronic bronchitis patients: cough integral of patients 1 year after treatment was significantly lower than the ones before treatment(P<0.05); Statistical significance was found in expectoration integral between before and after treatment group(P<0.05); The wheeze score reduced obviously than that in before the treatment group(P<0.05); Clinical effect was obvious and the total symptom effective rate was 72%; The therapy could obviously improved the quality of life of patients: part of symptoms score, part of activity evaluation, part of impact on daily life and the total SGRQ score 1 year after the treatment all reduced obviously than the ones 1 year before treatment(P <0.05).Part two Effect of Acupoint Application for "Winter-DiseaseTreated-in-Summer" on Nerve-Inflammation-Immunity System in Patient with Stable Chronic BronchitisObjective: To observe the immunoglobulin, inflammation cytokines and the neurotransmitters, to confirm the effect of acupoint application for “Winter-Disease-Treated-in-Summer” in treating chronic bronchitis, then to explore the mechanism of this therapy, and to seek the targets and pathways synthetically from the point of nerve-inflammation-immunity.Method: Fifty chronic bronchitis patients in stable phase were selected as chronic bronchitis group(CB), receiving acupoint application in dog days. One treatment was given in each dog day, 3 times altogether, 30 days are regarded as a course of treatment. Fifty healthy volunteers were enrolled as control group without any intervention(control). The level of serum immunological indexes such as immunoglobulin A(Ig A), immunoglobulin G(Ig G), immunoglobulin M(Ig M), immunoglobulin E(Ig E), inflammatory cytokines such as tumor necrosis factor-α(TNF-α), interleukin-4(IL-4), interleukin-8(IL-8), interleukin-10(IL-10)were detected before and after treatment in both CB(pre-treatment, Pre. Post-treatment, Post. winter)and control group, plasma substance P(SP), vasoactive intestinal peptide(VIP) were also detected. Compared all the indicators of Post and winter with those in Pre group. The indicators were also analyzed between each group of CB and control group. The mechanism of acupoint application for “Winter-Disease-Treated-in-Summer” in relieving of the inflammation and protecting of the body from the perspective of nerve-inflammation-immunity were explored.Result: Compared with control group, level of Ig A, Ig G, Ig M, IL-10, VIP in Pre, Post, winter group decreased obviously before the treatments, the difference was statistically significant(P<0.05); Level of Ig E, IL-4, IL-8, TNF-α, SP in Pre, Post, winter group elevated significantly, the difference was statistically significant except IL-8 in Post group(P<0.05); Compared with those before the treatments, Ig A, Ig G, Ig M, IL-10, VIP in Post group elevated obviously after the treatments, the difference was statistically significant(P<0.05); Ig E, IL-4, IL-8, SP in Post group decreased significantly, the difference was statistically significant(P<0.05); TNF-αshowed a trend of decline, but there was no statistical significance between before and after the treatments(P>0.05). Ig A, Ig G, Ig M, IL-10, VIP in winter group elevated obviously(P<0.05); Ig E, IL-4, IL-8, SP in winter group decreased significantly(P<0.05); TNF-αshowed a trend of decline, but there was no statistical significance compared with that before the treatments(P>0.05). Compared with those after the treatments, Ig M, IL-10, VIP in winter group decreased, the difference was statistically significant(P<0.05); IL-8, SP elevated, the difference was statistically significant(P<0.05); Ig A, Ig G, TNF-α in w group showed a trend of decline, but there was no statistical significance(P>0.05). Ig E, IL-4 showed a trend of elevation, but there was no statistical significance compared with that after the treatments(P>0.05).Part three Mechanism of Treating Stable Chronic Bronchitis by Acupoint Application for "Winter-Disease-Treated-in-Summer" based on Plasma MetabonomicsObjective: To research the plasma metabonomics characteristics of chronic bronchitis patients before and after acupoint application for "Winter-Disease-Treated-in-Summer" based on the modern biological mass spectrometry technology; To explore the mechanism of this therapy, and to prepare basic of looking for the targets or receptors of drugs by means of the metabolites terminal information, through the changes of potential biomarker before and after the treatments.Method: Fifty chronic bronchitis patients in stable phase were selected as chronic bronchitis group, receiving acupoint application in dog days.One treatment was given in each dog day, 3 times altogether, 30 days are regarded as a course of treatment. Fifty healthy volunteers were enrolled as control group(control) without any intervention. The blood samples of chronic bronchitis group were collected before the treatments(Pre), after the treatments(Post)and in the winter after the treatments(winter), respectively. The samples were also collected in control group. All the samples were tested by high performance liquid chromatography-mass spectrometry detector(HPLC-MS). Statistical data were analyzed by principal component analysis(PCA) and orthlgonal partial least squares discriminant analysis(OPLS-DA). The original spectrum and data of plasma metabolites were compared among the four groups.Result: Each plasma metabolites presented distribution of mass type,the metabolic spectrum in Pre group was significantly different from that in control group. The metabolism spectral of Post group and winter group regressed to that in the control group, and presenting the same metabolic components with control group. Choline, histidine, vitamin B6, methionine, biopterin were inferred as possibly potential syndrome biomarkers that made Pre group different from control group at first, they played an important role in the onset and recurrence of CB. Histidine and methionine played an important role in each comparation, histidine declined after treatment, but methionine expressed a rising trend.Conclusions: 1 Acupoint application for “Winter-Disease-Treated-in-Summer” shows a positive therapeutic effect in treating chronic bronchitis, which is worth being popularized in clinic sequentially. This observation provides a solid clinical support to further study on our paper. 2 Acupoint application for “Winter-Disease-Treated-in-Summer” can improve the body’s immune status, coordinate the immune balance, strengthen the body resistance. It can reduce the degree of inflammation by adjusting the release of inflammatory cytokines; It plays a positive role in adjusting the immune and inflammatory reaction by regulating neurotransmitters. This may be the synergetic mechanism in preventing and treating chronic bronchitis. 3 Through the study of human plasma metabonomics, it is found that the metabolism spectrum in CB patients is obvious different from that of the control group. The regression of metabolism spectrum of CB group to that of the control group after the treatments, confirms the positive effects of acupoint application for “Winter-Disease-Treated-in-Summer” on CB. The mechanism may focus on correcting the disorders in amino acids and vitamins metabolic, adjusting the secretion and release of cytokines. The results brought some enlightenment in using modern biological technology to explore traditional Chinese medicine theory.