| BackgroundAsthma is a chronic airway inflammation disease, but the dysfunction of immune system can not explain the whole picture of asthma. Previous studies show that it is a close relationship between hypothalamus-pituitary-adrenal (HPA) axis function and asthma. In order to observe inflammation and HPA axis function in asthma patients with Lung and Kidney Qi (Yang) Deficiency Syndrome, we compare the inflammation and HPA axis function in asthma patients with different Syndromes.Glucocorticoids (GCs) are currently the most effective anti-inflammation therapy, but there are still some patients poorly controlled and long time use of GC has side effects. Traditional Chinese medicine (TCM) has accumulated lots of experience for prevention and treatment for asthma. Based on the theory of TCM and our previous achievement for over half a century, we have extracted the methods that Tonifying Kidney and Benifiting Qi to improve overall physical fitness in asthma patients, control asthma, enhance the efficacy and reduce the side effects of modern medicine. However, due to the historical limitations, there is still lack of evidence-based trial to verify its efficacy and its mechanism is also not chear. Therefore, it is necessary to strictly design a trial to verify and compare the efficacy of Bushen Fangchuan Tablet (BSFCT) with Tonifying Kidney and Bushen Yiqi recipe (BSYQR) with Tonifying Kidney and Benifiting Qi on asthma patients with Lung and Kidney Qi (Yang) Deficiency Syndrome, and reveal its mechanism from the points of inflammation and HPA axis function. Thus, it could provide evidence to use Tonifying Kidney and Benifiting Qi formulae for treating asthma.Animal studies can compensate for the lack of clinical research. In order to clarify active components in Tonifying Kidney and Benifiting Qi formulae, we first attempt to establish animal models of combined asthma with Lung and Kidney Qi (Yang) Deficiency Syndrome. Meanwhile, we use the method of high performance liquid chromatography electrospray/quadrupole time-of-fight tandem mass spectrometry (HPLC-ESI/Q-TOF-MS/MS) to rapidly identify chemical components in BSFCT. According to our previous studies, we speculate that icariin might be the major active component in BSFCT for treating asthma. We study the effects of icariin and its mechanism on a murine model of combining asthma with Lung and Kidney Qi Deficiency Syndrome in order to comfirm our hypothesis.Part I Observation and correlation about inflammation and HPA axis function in asthma patients with Lung and Kidney Qi (Yang) Deficiency SyndromeObjective:By detecting and comparison levels of serum and induce sputum cytokines reflect asthma inflammation, and plasma, saliva and urine hormones reflect HPA axis function in asthma patients with different syndromes and normal healthy people. We aim to find out the situation about inflammation and HPA axis function in asthma patients with Lung and Kidney Qi (Yang) Deficiency Syndrome, and this will provide evidence to illustrate the mechanism of Tonifying Kidney and Benefiting Qi formulae for treating asthma.Methods:We selected asthma patients with Lung and Kidney Qi (Yang) Deficiency Syndrome, Lung Qi Deficiency Syndrome and Syndrome of hyperactivity of fire due to deficiency of Yin, aged 18-65 years, male or female. Besides, we selected normal healthy people matched age and sex as our normal control (NC) group. Patients with other pulmonary diseases and used GCs within one month were excluded. Sputum was induced and serum samples were collected for measurement of cytokines like Interleukin (IL)-6, Tumor Necrosis Factor (TNF-a), Transforming Growth Factor-β(TGF-β), and Interferon-y (IFN-y) by using Bio-Plex.24-hour collection of urine was performed for measurement of urinary free cortisol (UFC), 17-OH,17-keto-steroid (KS), plasma and salivary samples were obtained for assessment of cortisol by using radioimmunoassay.Results:The study population consist 71 asthma patients:28 in Lung and Kidney Qi (Yang) Deficiency Syndrome group,23 in Lung Qi Deficiency Syndrome group,20 in Syndrome of hyperactivity of fire due to deficiency of Yin group and 41 in NC group. There is no difference in demographic features including age, sex, BMI, smoking index, the severity of diseases among the groups (P>0.05). Compared with the NC and Lung Qi Deficiency Syndrome group, the level of serum TNF-a was significantly increased and IFN-y decreased in Lung and Kidney Qi (Yang) Deficiency Syndrome and Syndrome of hyperactivity of fire due to deficiency of Yin group (P<0.05). The level of IL-6 in Lung and Kidney Qi (Yang) Deficiency Syndrome group was higher than other groups (P<0.05). Compared with the NC and Lung Qi Deficiency Syndrome group, the levels of TNF-a and TGF-β in induced sputum were significantly increased in Lung and Kidney Qi (Yang) Deficiency Syndrome group (P<0.05). IFN-y in induced sputum were significantly lower in Syndrome of hyperactivity of fire due to deficiency of Yin group than other groups (P<0.05). The levels of UFC,17-OH and 17-KS in urine were lower in Lung and Kidney Qi (Yang) Deficiency Syndrome group than those in other group (P<0.05). Compared to the NC or Syndrome of hyperactivity of fire due to deficiency of Yin group, the cortisol level in plasma at the day and the next day 8:00AM was significantly lower in Lung and Kidney Qi (Yang) Deficiency Syndrome group (P<0.05). The cortisol level in salivary at the day and the next day 8:00AM was also lower in Lung and Kidney Qi (Yang) Deficiency Syndrome group than the NC group (P<0.05). And the cortisol level in salivary at the next day 8:00AM was also lower in Lung and Kidney Qi (Yang) Deficiency Syndrome group than the Syndrome of hyperactivity of fire due to deficiency of Yin group (P<0.05). The cortisol level in plasma and salivary at the day and the next day 8:00AM in Lung Qi Deficiency Syndrome group was also lower than those in NC or Syndrome of hyperactivity of fire due to deficiency of Yin group, but there were no significantly differences among them. Serum levels of TNF-a and IL-6 showed a significant negative correlation with plasma cortisol (r=-0.26, P=0.03), (r=-0.25, P=0.03). The level of IL-6 in induced sputum showed a significant negative correlation with salivary cortisol (r=-0.26, P=0.02). The level of IFN-y in serum showed a significant positive correlation with urine 17-OH (r=0.21, P=0.03).Conclusion:There were excessive pro-inflammatory cytokines secretion and anti-inflammatory cytokines deficiency in asthma patients, particularly in Lung and Kidney Qi (Yang) Deficiency Syndrome group, which might be relevant to the dysfunction of HPA axis.Part II Efficacy and its mechanism of Tonifying Kidney and Benefiting Qi formulae on asthma patients with Lung and Kidney Qi (Yang) Deficiency SyndromeObjective:By conducting a multi-center, randomized, double-blind trial, we aim to assess and compare the efficacy of BSFCT and BSYQR on asthma patients with Lung and Kidney Qi (Yang) Deficiency Syndrome and reveal its mechanism from the points of inflammation and HPA axis function in order to provide evidence for clinical use.Methods:We conducted a prospective, multi-centers, randomized, double-blind, placebo-controlled trial. Using the same inclusion and exclusion standard, outpatients and inpatients from five centers who suffered from asthma that was inadequately controlled despite inhaled GC therapy were randomly assigned to groups given BSFCT or BSYQR or placebo. The BSFCT group intervention was BSFCT plus BSYQR placebo plus basic inhaled GC. The treatment of BSYQR group was given BSYQR plus BSFCT placebo plus basic inhaled GC. The control intervention was BSYQR placebo plus BSFCT placebo plus basic inhaled GC. Participants received three courses of medication treatment each lasting 12 weeks. (1) The primary efficacy outcomes were the rate of asthma exacerbations through 12weeks and the relative change in lung function from baseline to week 12. Among the secondary outcomes were the scores of asthma control test (ACT), scores of Asthma Quality Life Questionnaire (AQLQ), changes of Asthma Daily Diary Questionnaire (ADDQ), and relative syndrome symptoms of TCM. (2) Safety monitoring contains routine blood and urine test, the function of liver and kidney, blood sugar and electrocardiogram. Adverse eventa and adverse responsea were monitored throughout the trial. (3) The levels of EOS and tIg E were detected before and after treatments.The levels of cytokines in serum like matrix metalloproteinase 9(MMP-9), IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, and IFN-y were assessed before and after treatments. The levels of corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and cortisol in plasma were evaluated before and after treatments.Results:(1) A total of 295 patients were recruited and finished the observation. There was no significant difference between three groups in baseline data before treatment (P>0.05). There was no significant trend toward lower rates of asthma exacerbations among patients in the BSFCT or BSYQR group than among patients in the placebo group (P=0.84). FEV1 of predicted value showed a significantly greater improvement among patients receiving inhaled GC and BSFCT than among those receiving inhaled GC and placebo (P<0.05). Compared with the placebo group, BSFCT or BSYQR treatment could improve FEV1/Forced Vital Capacity (FVC). Improvement in ACT score from baseline to the end of treatment was 5.4 points in the BSFCT group,4.99 points in the BSYQR group, as compared with 4.9 points in the Placebo group (P=0.74). There was a trend toward more symptom-free days in the BSFCT (6.42 days) or BSYQR (5.9 days) groups than in the placebo group (5.35 days) (P=0.33). At week 12, there was a trend for the average score of AQLQ to be lower in the BSFCT or BSYQR groups than in the placebo group (P= 0.25). BSFCT and BSYQR treatment could improve Lung and Kidney Qi (Yang) Deficiency Syndrome symptoms especially spontaneous, waist and knees aches, and chills. (2) There were no significantly differences in the safety assessments, the rates of adverse events and adverse responses among three groups (P>0.05). There were no serious adverse events reported during the study. (3) Compared with the Placebo group, serum EOS level decreased after BSFCT or BSYQR treatment, but there was no significant difference among them (P>0.05). The level of tIg E decreased in the BSYQR group, but increased in the Placebo group and BSFCT group, but there was also no significant difference among them (P>0.05). Thl cytokine IL-2 in the BSYQR group were significantly higher than that in the BSFCT group or the Placebo group at the end of the treatment (P=0.006), as well as Treg type cytokine IL-10 (P=0.04). The serum IFN-y concentrations in the BSFCT or BSYQR group at week 12 were significantly lower than that in the Placebo group (P=0.01). Serum cytokines like MMP-9, IL-4, IL-5, IL-13 and IL-17 levels in the BSFCT or BSYQR were lower than those in the Placebo group, but there was also no significant difference among them (P>0.05). The CRH, ACTH and Cortisol levels in plasma were all increased after BSYQR treatment compared to the baseline and the level of ACTH was also increased after BSFCT treatment, but a reduction in the Placebo group.Conclusion:BSFCT treatment was associated with significantly improved lung function FEV1 of predicted value and BSYQR treatment also had the trend to improve lung function. On the basis of inhaled GC, BSFCT and BSYQR treatment could not reduce asthma exacerbations. But, BSFCT and BSYQR treatments could increase asthma control score, and decrease daily asthma symptoms. They also could improve asthma life quality and ease symptoms associated with Lung and Kidney Qi (Yang) Deficiency Syndrome; the mechanism research showed that decreased pro-inflammatory mediators and increased anti-inflammatory mediators, and regulated the equilibrium of pro-inflammatory and anti-inflammatory system, recovered the HPA axis function might be the underlying mechanism for BSFCT or BSYQR treatment to asthma; BSFCT and BSYQR were safe and effective drugs to treat asthma and worth promotin in clinic application.Part III Studies about establishing animal models of combining asthma with Lung and Kidney Qi (Yang) Deficiency SyndromeObjective:To find out the active compoments, we established and evaluated animal models of combining asthma with Lung and Kidney Qi (Yang) Deficiency Syndrome.Methods:1. Comparison of two animal models of Kidney Yang Deficiency Syndrome (KYDS) induced by corticosterone (CORT) or multiple stressors and animal models of combining asthma with Lung and Kidney Qi (Yang) Deficiency Syndrome:Sixty SD rats were randomly assigned to six groups (n=10 per group): Normal group, CORT-induced KYDS group, Stress-induced KYDS group, Asthma with Lung Qi Deficiency Syndrome group (ALQDS), CORT-induced Asthma with Lung and Kidney Qi (Yang) Deficiency Syndrome (CORT-induced ALKQDS) group, Stress-induced Asthma with Lung and Kidney Qi (Yang) Deficiency Syndrome (Stress-induced ALKQDS)group. Using subcutaneous injection CORT to establish CORT-induced KYDS group; Stress-induced KYDS group was established by multiple stressors like cold, forced swimming, chronic intermittent electrical stimulation of the foot, fixed; ALQDS was built by OVA-exposed; CORT-induced ALKQDS group was established by combining CORT-induced and OVA-exposed; Stress-induced ALKQDS group was established by combining stress and OVA-exposed. General state, weight, food and water intake were observed; Using Open-field test and Sucrose Preference Test to assess animals’behaviors; Measurement of Thymus, Spleen, Adrenal, Thyroid, and Testicular Organ Index; CORT, ACTH,3,5,3’-triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH), estradiol (E2), testosterone (T), luteinizing hormone (LH), follicle stimulating hormone (FSH), cyclic adenosine monophosphate (c AMP), and cyclic guanosine monophosphate (c GMP) levels were measured by ELISA kits. Measurement of AHR by Buxco’s Modular and Invasive System; The levels of cytokines in BALF were detected using Bio-Plex Suspension Array System; Hematoxylin and Eosin (H&E) staining, Periodic acid-Schiff (PAS) staining and Masson’s Trichrome (M-T) staining were respectively used to identify general morphology, goblet cells in the epithelium, and collagen deposition in lung tissues.2. Establishment and evaluation a murine model of combined asthma with Lung and Kidney Qi Deficiency induced by psychosocial stress:Thirty-six Balb/c mice were randomly assigned to four groups (n=9 per group,3 per cage):Normal group, Kidney Qi Deficiency Syndrome group (KQDS), ALQDS, Asthma with Lung and Kidney Qi Deficiency Syndrome group (ALKQDS). By using attack due to occupied territory between mice to imitate shock harm to Kidney Qi and combined OVA to establish the model of ALKQDS. The open field video tracking system was used to assess animal behaviors; The invasive pulmonary resistance (RL) test system from Buxco was applied to detect pulmonary function; ELISA was utilized to determine IL-4, IL-5, IL-13, IL-6, and TNF-a in BALF, levels of OVA-IgE, IL-4, IL-5, IL-13 and CORT in mouse serum; H&E staining was used to assess airway inflammation in lung histology, The CCK-8 was applied to evaluate the inhibitory effect of CORT on splenocyte proliferation induced by lipopolysaccharide (LPS). ELISA was utilized to detect the levels of IL-6 and TNF-a in the supernatant of splenocytes cultured in vitro; Real time-PCR and Western blot were utilized to determine glucocorticoid receptor (GR) mRNA and GR protein expression in lungs.Results:1. The KYDS groups performed crouching, reduced locomotor activity, and increased urine, decreased weight and changes of food and water intake. When OVA challenged, rats appeared coughing, sneezing, wheezing and other symptoms similar human asthma with Lung Qi Deficiency Syndrome. Compared with Normal group, food and water intake was markedly increased in the Stress-induced KYDS with or without allergy exposure groups but decreased in the CORT-induced KYDS with or without allergy exposure groups compared to the normal group (P<0.05). The total cross counts, horizontal and vertical counts, were significantly diminished in the KYDS and ALKQDS groups (P<0.05) compared to the normal group. However, rats in the ALQDS group sharply increased the activities (P<0.05). Average sucrose intake sharply decreased in the both KYDS and ALKQDS groups (P<0.05), especially Stress-induced groups. Rats in the CORT-induced KYDS and Stress-induced KYDS groups with induced thymus atrophy (P<0.05); however, rats with OVA exposure had induced thymus and spleen hyperplasia compared to those without OVA exposure (P<0.05). The CORT-induced KYDS with or without OVA exposure groups manifested adrenal and thyroid atrophy (P<0.05), but testicular hyperplasia (P<0.05), and above three glands had hyperplasia in the Stress-induced KYDS with or without OVA exposure groups. The adrenal in ALQDS group was atrophy (P<0.05). The levels of CORT, T4, TSH, LH, and FSH that notably increased in plasma in the CORT-induced KYDS with or without OVA-exposure (P<0.05), but the level of ACTH significantly decreased (P<0.05) compared to the normal group. However, above hormones were significantly increased in the Stress-induced KYDS with or without OVA-exposure (P<0.05). The ratio between c AMP and c GMP was remarkably reduced in the other five groups compared with the normal group (P<0.05). The CORT-induced ALKQDS group had higher RL than the ALQDS group (P<0.05) but was lower than the Stress-induced ALKQDS group (P<0.05) at the methacholine (Mch) concentrations of 12.5mg/mL and 25mg/mL. The levels of pro-inflammatory cytokines like IL-6 and IL-13 in serum and BALF were sharply increased in the OVA-exposed groups (P<0.05), especially in the Stress-induced ALKQDS group, but like anti-inflammatory cytokines IL-2 and IL-10 were significantly decreased compared to non-OVA-exposed groups, especially in the CORT-induced ALKQDS group (P<0.05). The degree of inflammatory cells infiltration, mucus production and collagen deposition was higher in both ALKQDS groups than those in the ALQDS group (P<0.05).2. Mice in KQDS group induced by psychosocial stress performed crouching, reduced locomotor activity, decreased diet and water, and increased urine similar to Kidney Qi Deficiency performance. When OVA exposed, mice in the ALKQDS group performed coughing, increased respiratory rate, sneezing, and dry hair like asthma patients with Lung and Kidney Qi Deficiency Syndrome. The opern field test showed that mice in the KQDS with or without OVA-exposure significantly shortened the time the mice spent in the center of the open field (P<0.05), increased ambulatory activity (P<0.05) and the count of fecal boli (P<0.05), but deceased vertical activity (P<0.05). Results from pulmonary function demonstrated that AHR was significantly enhanced in the OVA-exposure groups (P<0.05). Moreover, R1 was higher in the ALKQDS group than that in ALQDS group at the highest Mch concentration, but there was no significant difference between them.The ELISA results showed that cytokines like IL-4, IL-5, IL-6, IL-13 and TNF-a in BALF and IL-4, IL-5, IL-13, OVA-Ig E in serum were significantly increased in the ALQDS group (P<0.05). Moreover, the levels of IL-4, IL-5, IL-13, TNF-a in BALF and IL-4, IL-5 in serum in the ALKQDS group were higher than those in the ALQDS group (P<0.05). The level of CORT significantly increased in the KQDS with or without OVA-exposed groups (P<0.05).The lung histology showed there were more severe perivascular eosinophilia, perubronchiolar eosinophilia, epithelial damage and edema in the ALKQDS group than those in ALQDS group (P<0.05). The inhibition effect of CORT on splenocyte proliferation induced by LPS was significantly impaired in the KQDS with or without OVA-exposed, especially the KQDS group.CORT could not inhibit the levels of IL-6 and TNF-a in the supernatant of splenocytes from the ALKQDS group induced by LPS. Expression of GR mRNA and GR protein were significantly reduced in the lung tissues of the KQDS with or without OVA-exposure group (P<0.05), compared to the Normal group and the ALQDS group.Conclusion:1. The KYDS rat models by usage of the high dose of CORT injection or multiple stressors had similar changes in biological characterization, abnormal behaviors, dysfunction of hypothalamic-pituitary-target organ axes, and sympathetic/parasympathetic nerve system but varied in different degrees. However, the rat model of KYDS induced by high dose of CORT injection was more near to human beings. By syndrome differentiation of disease models, we found it could establish the Lung Qi Deficiency Syndrome after repeated OVA-exposure. Combined with KYDS, we established the ALKQDS rat model. Compared to the ALQDS group, AHR, airway inflammation and airway remodeling were more serious in the ALKQDS groups, especially the Stress-induced ALKQDS group.2. We used social disruption procedure between two different rodents to imitate stress for human being and establish Kidney Qi Deficiency murine model. This model not only had the appearance of crouching, reduced locomotor activity, and increased urine similar to Kidney Qi Deficiency Syndrome, but also the activated HPA axis function. Combined with OVA challenged, we built a murine model of combining Asthma with Lung and Kidney Qi Deficiency Syndrome. Besides the performance of Lung and Kidney Qi Deficiency Syndrome, this model also had the characteristic of asthma like AHR and airway inflammation. Thus, there was feasibility to establish this model. Moreover, we found that AHR and airway inflammation were more serious in the ALKQDS group than ALQDS group, which was not consistent with the high CORT level in serum. The underlying mechanism study illustrated that GR mRNA and GR protein expression might be relevant to GC insensitivity, which resulted in serious airway inflammation.Part Ⅳ Identification active components in Tonifying Kidney and Benefiting Qi formulae for treating asthma with Lung and Kidney Qi (Yang) Deficiency SyndromeObjective:By using the method of HPLC-ESI/Q-TOF-MS/MS to identify the main chemical components in BSFCT, combined animal experiments, in order to comfirm the active components in Tonifying Kidney and Benefiting Qi formulae for treating asthma with Lung and Kidney Qi (Yang) Deficiency Syndrome.Methods:1. Using Agilent Poroshell 120-C18 reverse phase column with acetonitrile and 0.1% formic acid as the mobile phase gradient elution; MS using ESI, collecting data in the mode of positive ion, negative ion mode and UV.2. Effects and its mechanism of Icariin on the murine model of ALKQDS induced by psychosocial stress:Forty-five Balb/c mice were randomly assigned to five groups (n=9 per group,3 per cage):Normal group, ALKQDS induced by psychosocial stress group, DEX intervention group, Icariin low dose intervention group, Icariin high dose intervention group. The intervention groups were given respectively icariin or DEX administration (orally) 30 minutes before each OVA inhalation for two weeks. Observations were the same with Part III the method 2.Results:1.Through positive and negative MS, elemental composition analysis and comparison the reference and relevant literature data,18 compounds including icariin were identified in BSFCT.2. Compared to the ALKQDS group, treatment of icariin prolonged the amount of time the ALKQDS mice spent in the center of the open field (P<0.05), decreased ambulatory activity (P<0.05), increased vertical activity (P<0.05) and reduced the count of fecal boli (P<0.05). DEX also had similar effects, but the high dose of icariin was more efficient.AHR in the ALKQDS mice were significantly suppressed by icariin and DEX at the highest concentration of Mch (P<0.05), especially the high dose of icariin.Icariin and DEX treatment could significantly decrease the levels of cytokines in BALF and serum, compared with ALKQDS mice (P<0.05). Moreover, the level of CORT in serum also reduced in the treatment groups (P<0.05). The high dose of icariin was more efficient.Icariin and DEX treatment could inhibit airway inflammation in the lung histology (P<0.05), especially the high dose of Icariin treatment. Splenocytes and IL-6, TNF-α released by splenocytes from the Icariin treatment groups stimulated by LPS were inhibited by CORT. But splenocytes from the DEX group could not been inhibited by high dose of CORT. Icariin markedly increased GR mRNA and protein expression (P<0.05), especially the high dose of Icariin, but there was no difference after DEX treatment.Conclusion:1.By using HPLC-ESI/Q-TOF-MS/MS technology and analysis of relative molecular mass data, we confirmed eighteen compounds in BSFCT. Five of them including icariin were from herb Epimedium, which provided evidence for elucidating active components in Tonfying Kidney and Benefiting Qi formulae to treat asthma.2. Effects of icariin on improving abnormal behavior, AHR and airway inflammation in a murine model of combining asthma with Lung and Kidney Qi Deficiency Syndrome. The high dose of icariin was more efficient than DEX and the lose dose of icariin. The potential mechanisms might be that icariin could regulate HPA function and immune system, and in part recover GC insensitivity by GR up-regulation. Thus, we speculated that icariin might be the major active component in BSFCT for treating asthma. |
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