Qualitative analysis of representative standard compounds in modified LWDHW-1217A and 1217B
The major active ingredients in modified LWDHW-1217A and 1217B were identified by HPLC analysis. The active compound of catalpol in Rehmannia glutinosa and loganin in Cornus officinalis were used as standards, which their retention time were at 10.5 minute and 4.0 minute, respectively (Fig. 2A). The active ingredient of allantoin in Dioscorea opposite and alisol acetate B in Alisma orientalis were listed, their retention time of these standards were at 2.75 minute and 13.5 minute (Fig. 2B). The retention time of active compound-pachymic acid in Poria cocos was at 48.5-minute, the retention time of active compound-methyl gallate in Paeonia suffruticosa was at 15.5-minute, and the saponin in Radix Dioscorea was at 31.5-minute (Fig. 2C). These three active compounds of baicalin, baicalein, and wogonin in Scutellaria baicalensis were listed, their retention time of these standards was at 11.5 minute, 20.5 minute, and 27.5 minute, respectively (Fig. 2D). In the designed chromatogram condition, the primary active compounds were identified in the formula of modified LWDHW- 1217A and 1217B that contained at least ten active ingredients order by the retention time including 1-allantoin, 2-loganin, 3-catalpol, 4-baicalin, 5- alisol acetate B, 6-methyl gallate, 7-baicalein, 8-wogonin, 9-saponin, and 10-pachymic acid (Fig. 2E and 2F). The main difference between the formula of modified LWDHW-1217A and 1217B was the active ingredient of 5-alisol acetate B from Alismatis Rhizoma at a retention time of 13.5 (Fig. 2F).
Effects of Modified LWDHW-1217A and 1217B on the Productions of Specific IgG1, IgG2a, and IgE in the Sera of Der p 2-sensitized mice
To determine the effect of 1217A and 1217B on immunoglobulin production, the mite allergen nDer p 2 was used for intraperitoneal sensitization. The Der p 2-specific IgE, IgG1, and IgG2a in the sera were measured after immunotherapy with 1217A or 1217B. After the IP sensitization and immunotherapy with saline, the Der p 2-specific IgE and IgG1 were significantly higher in the sera when compared with the naive group (Fig. 3A; p < 0.01 and Fig. 3B; p < 0.01). The differences between groups were analyzed with Mann-Whitney U-test for non-parametric analysis. Data are presented as mean ± SD and the p < 0.05 was considered statistically significant. In the immunotherapy with 1217A or 1217B, the Der p 2-specific IgE and IgG1 were significantly reduced when compared to that of the saline group (Fig. 3A and Fig. 3B; p < 0.05). However, there were no relevant changes in IgG2a compared between the naïve group and the saline group, the same as in the comparison between the saline group and other therapeutic groups (Fig. 3C). Results indicated that the productions of mite allergen-specific IgE and IgG1 could be down-regulated after immunotherapy with 1217A or 1217B in the mite-sensitized mice.
Effects of Modified LWDHW-1217A and 1217B on the Productions of Cytokines in the Sera and BALF of Der p 2-sensitized mice
To determine the effect of modified LWDHW-1217A or 1217B on cytokine productions, the cytokines in the sera and BALF were measured by ELISA after immunotherapy with 1217A or 1217B. In the group of mice treated with IP sensitization and immunotherapy with saline, the levels of inflammatory cytokine of IL-5 in the sera and BLAF, and the IL-13 in the BALF were significantly increased compared with the naïve control (Fig. 4; p < 0.05). When the mice were treated with modified LWDHW-1217A or1217B after the allergen Der p 2 IP sensitization, the results showed there were significant reductions of inflammatory cytokine IL-5 in sera and BALF and the IL-13 in the BALF; whereas, the Th1-cytokine IFN-γ in sera, and the IL-12 and IFN-γ in the BALF were significantly increased in comparison with the saline group (Fig. 4, p < 0.05). The inhibitory effects of the inflammatory cytokine IL-5 in the sera and inflammatory cytokines of IL-5 and IL-13 in BALF have also been observed in the mice treated with Dexamethasone. Although there was also a significant change in Th2-cytokines (IL-5 and IL-13) in the group treated with Dexamethasone, it did not increase Th1 cytokines. The immunotherapy with modified LWDHW-1217A or 1217B was also able to cause a decrease in inflammatory cytokines (IL-5 and IL-13) and an increase in Th1-cytokine (IL-12 and IFN-γ) in the airway BALF. Results indicated the Modified LWDHW-1217A or 1217B has the potential to down-regulate the inflammatory tendency caused by the mite allergen Der p 2-induced asthmatic model. Overall, the Th2 inhibitory effect of 1217B is slightly higher than that of 1217A, and the effect of activating Th1 of 1217B is also slightly higher than that of 1217A.
Effects of Modified LWDHW-1217A and 1217B on the Inflammatory cell infiltrations in the Airway of Der p 2-sensitized mice
The inflammatory cell infiltrations in the airway were compared in all experimental groups of mice by analyzing the leukocytes subpopulation in the BALF. After the IP sensitization with mite allergen Der p 2, the mice were immunotherapy with different experimental designs. Next, the mice were IT challenged with Der p 2 twice after the therapy, and airway inflammatory cell infiltration was collected and analyzed. After the allergen challenge, the airway infiltrations of inflammatory cells including macrophages, eosinophils, neutrophils, and lymphocytes were significantly increased in the group of mice that IP sensitization with mite allergen then immunotherapy with saline when compared to the naïve group (Fig. 5; p < 0.05). There were significant reductions in inflammatory cell infiltrations including macrophages, eosinophils, neutrophils, and lymphocytes after immunotherapy with modified LWDHW-1217A or 1217B in comparison with the saline group (Fig. 5; p < 0.05). There was also a significant decrease in the total cell numbers in mice treated with 1217A, 1217B, or Dexamethasone in comparison with the saline group (Fig. 5; p < 0.05). Results indicated that immunotherapy with modified LWDHW-1217A or 1217B had anti-inflammatory effects on inflammatory cell infiltration in the airway, especially in 1217B has better inhibitory effects.
Effects of Modified LWDHW-1217A and 1217B on the gene expressions in Lung Tissue of Der p 2-sensitized mice
The lung tissue from mice was acquired for real-time quantitative-PCR assay (Q-PCR) to investigate gene expressions related to allergy after the IP sensitization, immunotherapy with Dexamethasone, Modified LWDHW, and IT challenges with mite allergen. The TH2-related genes (IL4, IL5, and IL13), TH2-related transcription factor (GATA-3), neutrophil chemotactic chemokine (IL8), and the TH1-related transcription factor (T-bet) were used to evaluate the gene variations in asthmatic mice after the immunotherapy with modified LWDHW1217A or 1217B. GAPDH was used as the control gene for normalizing the target gene expression of each sample. After the IP sensitization of mite allergen and immunotherapy with saline, the Q-PCR results revealed the TH2-related genes of IL4, IL5, and IL13 (4.4-fold, 3.5-fold, and 3.0-fold relative to baseline GAPDH, respectively) were significant upregulation when compared to the naïve group (p < 0.05; Fig. 6). The group immunotherapy with saline also had a 3.8-fold increase in expression of neutrophil chemotactic chemokine (IL8) (p < 0.05), and a 3.1-fold increase in expression of TH2-related transcription factor (GATA-3) (p < 0.05). In the group of mice treated with IP sensitization and immunotherapy with modified LWDHW1217A or 1217B, the TH2-related genes of IL4, IL5, and IL13, the neutrophil chemotactic chemokine of IL8, and TH2-related transcription factor of GATA-3 were significantly decreased in the lung tissue when compared with the mice immunotherapy with saline (Fig. 6; p < 0.05). Similar findings were also observed that these TH2-related genes (IL4, IL5, and IL13), chemokine gene (IL8), and TH2-related transcription factor (GATA-3) were significantly down-regulated in the group of mice that received immunotherapy with Dexamethasone (Fig. 6; p < 0.05). Moreover, the TH1-related transcription factor (T-bet) was significantly increased in the group of mice immunotherapy with modified LWDHW1217A or 1217B in comparison with the saline group (Fig. 6; p < 0.05). Overall, immunotherapy modified LWDHW1217A or 1217B was also able to cause a decrease in expressions of inflammatory genes (IL4, IL5, and IL13), neutrophil chemotactic chemokine (IL8), and TH2-related transcription factor (GATA-3); on the other hand, an increase in the TH1-related transcription factor (T-bet) in the lung tissue of asthmatic mice.
Effects of Modified LWDHW-1217A and 1217B on the phosphorylation of STAT6, STAT1, and ERK and activation of GATA3
The STAT6 is mainly activated by cytokines including IL-4 and IL-13. The STAT6-mediated signaling pathway is required for the development of T-helper type 2 (Th2) cells and Th2 immune response. STAT6 plays a critical role in Th2 lung inflammatory responses and the pathogenesis of asthma. STAT1 can be activated by several ligands like IFN-γ, and then the IFN-γ signaling has been further shown to inhibit IL-4 expression by inhibiting STAT6 phosphorylation. The STAT1 signaling mechanism upregulates T-bet and mediates repression of IL4 gene in TH1 cells that subsequently represses GATA3 and IL-4 expression and antagonizes the STAT6 signaling. The ERK signaling activation participates in the release of the inflammatory mediators. The anti-inflammatory mechanisms of modified LWDHW-1217A and 1217B on the phosphorylation of STAT6, STAT1, and ERK and activation of GATA3 were investigated in the following experiment. After the IP sensitization and immunotherapy with saline, the phosphorylation of STAT6 and ERK and the expression of GATA3 were significantly enhanced compared with the naïve group (Fig. 7; p < 0.05). The expression levels of total ERK1/2 protein and GAPDH showed very similar among all groups. After immunotherapy with modified LWDHW1217A or 1217B, the expressions of phosphorylated-STAT6, phosphorylated-ERK, and GATA3 were significantly decreased when compared to the group which immunotherapy with saline (Fig. 7; ༊༊:p < 0.01; ༊: p < 0.05). The inhibitory effects of phosphorylated-STAT6, phosphorylated-ERK, and GATA3 could be also shown in the immunotherapy with Dexamethasone (Fig. 7; p < 0.05). The phosphorylation of TH1-related transcription factor- STAT1- was significantly increased in the group immunotherapy with modified LWDHW-1217A or 1217B when compared to the saline group (Fig. 7; p < 0.05). The phosphorylation of STAT1 seemed not to change significantly after immunotherapy with Dexamethasone when compared with those of saline (Fig. 7; p < 0.05). Results indicated that immunotherapy with modified LWDHW1217A or 1217B can enhance the TH1 cell-related activities or responses and suppress the TH2 cell-related activities or responses. However, the TH1 cell-related and TH2 cell-related responses were both suppressed in the group immunotherapy with Dexamethasone.
Effects of Modified LWDHW-1217A and 1217B on the Th1/Th2 Cytokine Expression of CD4 + Leukocytes in the Der p 2-sensitized mice
After the treatment or immunotherapy with 1217A or 1217B, the peripheral blood leukocytes from mice of each group were collected and treated with PMA and ionomycin, and then the expression portion of the Th1-type cytokine (IFN-γ) and Th2-type cytokine (IL-4) producing in the CD4+ cells were analyzed. After the cells were stained with monoclonal antibodies, the cell subpopulation and fluorescence quantification were analyzed by the flow cytometer. The lymphocytes were gated on an FSC (forward scatter) vs. SSC (side scatter) dot plot as gate region-1 (R1) shown in Fig. 8A. The expression of IFN-γ and IL-4 among the CD4+ T cells were gated as region-2 (R2) and region-3 (R3), respectively (Fig. 8A). The percentages of IFN-γ+/CD4+ and IL-4+/CD4+ T cells after immunotherapy among different groups were compared and presented as a histogram (Fig. 8B). The results revealed that the IL-4+/CD4+ T cells (4.17 ± 0.25%) could be significantly upregulated and the IFN-γ+/CD4+ T cells (2.23 ± 0.25%) could be significantly downregulated in the group of mice immunotherapy with saline after Der p 2 sensitization when compared to the those of naïve group. The ratio of Th2/Th1 was 0.488 in the naïve group; furthermore, there were obvious changes in the Th2/Th1 ratio of the saline group (1.527) which was sensitized with Der p 2 mite allergen and immunotherapy with saline (Fig. 8C). After immunotherapy with modified LWDHW-1217A, the population of IL-4+/CD4+ T cells could be downregulated to 2.45 ± 0.10%, and the IFN-γ+/CD4+ T cells upregulated to 3.36 ± 0.29%, whereas the ratio of Th2/Th1 was 0.729 (Fig. 8C). Similar results can also be found in the immunotherapy with the modified LWDHW-1217B that the population of IL-4+/CD4+ T cells downregulated to 2.50 ± 0.13%, and the IFN-γ+/CD4+ T cells upregulated to 3.41 ± 0.26%, whereas the ratio of Th2/Th1 was 0.733 (Fig. 8C). After immunotherapy with the Dexamethasone, not only the cell population of IL-4+/CD4+ T cells was inhibited to 2.69 ± 0.17%, but also the IFN-γ+/CD4+ T cells was inhibited and decreased to 2.31 ± 0.31%, whereas the ratio of Th2/Th1 was 1.165. Results indicated that immunotherapy with modified LWDHW-1217A or 1217B can enhance the TH1 cell population and suppress the TH2 cell population. The ratio of Th2/Th1 was also noted to have decreased much less than treatment with saline.
Effects of Modified LWDHW-1217A and 1217B on Pulmonary Function of Airway Hyperresponsiveness (AHR)
The pulmonary function of mice was evaluated by airway hypersensitivity to methacholine of whole-body plethysmography, which was measured one hour after nDer p 2 allergen IT challenges. The responses exposed to rising doses of methacholine (PBS, 6.25, 12.5, and 25.00 mg/ml) in five groups of mice were generated. Respiratory parameters such as enhanced pause (Penh), pause, peak inspiratory flow, peak expiratory flow, tidal volume, breathing frequency, end-inspiratory pause, and end-expiratory pause were recorded. Since airway constriction was mainly related to the change in Penh, the Penh value was used to evaluate the repression effects of 1217A and 1217B on pulmonary function. In the group of immunotherapy with saline after nDer p 2 sensitization, the Penh values were significantly increased after the methacholine challenge at the dose of 6.25, 12.5, and 25 mg/ml compared with those of naïve mice (Fig. 9; p < 0.05). When mice were immunotherapy with modified LWDHW-1217A or 1217B, the Penh significantly decreased after the methacholine challenge at the dose of 12.5, and 25 mg/ml compared to those with saline (Fig. 9; p < 0.05). There was also a significant reduction of Penh in airway hypersensitivity after methacholine challenge at the dose of 12.5 mg and 25 mg in the group treated with Dexamethasone compared to those with saline. Although the effect was also observed in the group of modified LWDHW-1217A, it was less than those treated with modified LWDHW-1217B. Overall, the Penh values were the lowest in the group when immunotherapy with Dexamethasone at the 25 mg/ml methacholine evocation. But there is no obvious difference between the group of 1217A, 1217B, and Dexamethasone. Results showed the Penh was significantly upregulated in the saline group at 25mg/ml methacholine, but the Penh value was significantly decreased in the 1217B groups (Fig. 9; p < 0.05). Results indicated that immunotherapy with modified LWDHW-1217A or 1217B can alleviate the inflammatory response of pulmonary function triggered by mite allergen.
Effects of Modified LWDHW-1217A and 1217B on Bronchus Pathology
The lung histopathology with hematoxylin-eosin staining was performed to determine whether immunotherapy with modified LWDHW-1217A or 1217B affected bronchus pathology. The epithelium damage in the airway and inflammatory cell infiltration were compared among the naïve group and all experiment groups after immunotherapy with modified LWDHW-1217A, 1217B, or Dexamethasone. Representative photomicrographs among the naïve group and all experiment groups are presented respectively in Fig. 10A. The histological scores for each perspective were based on tracheal thickness (µm), inflammatory cell count (cell units), and tracheal rupture (%)(Fig. 10B). The intratracheal challenge with allergen Der p 2 was performed after the IP sensitization and treatment with saline, the results of pulmonary physiology showed that the bronchus pathology with the obvious infiltration of granulocytes (Fig. 10A). Both tracheal thickness and tracheal rupture were raised in the group treated with saline after allergen sensitization when compared to the naïve group (Fig. 10A). The lung histopathology was calculated by tracheal thickness, inflammatory cell count, and tracheal rupture; therefore the histological scores were significantly higher in the saline group than in the naïve group (Fig. 10A; p < 0.05). There was a reduction of airway inflammatory cell infiltration and a decreased level of tracheal damage in the group of immunotherapy with modified LWDHW-1217A or 1217B when compared with those of saline (Fig. 10A; p < 0.05). There was also a reduction of airway inflammatory cell infiltration and epithelium damage in the tracheal after immunotherapy with Dexamethasone when compared with those of saline (Fig. 10A; p < 0.05). There was a trend of significant improvement in the bronchus pathology from mouse lungs in these immunotherapy groups, including modified LWDHW-1217A, 1217B, and Dexamethasone. Results indicated that immunotherapy with modified LWDHW-1217A or 1217B can abate the bronchus inflammation triggered by mite allergen.
Mite Allergy pathogenesis
Allergic diseases, including asthma, allergic rhinitis, and atopic dermatitis, occur worldwide and are special prevalent in westernized countries. These chronic immunological diseases are a serious issue affecting public health and the prevalence has risen in recent years. The major reasons for allergic diseases are caused by allergen-induced unfavorable immune responses leading to pathogenesis and clinical pictures, including sensitization and development of memory T and B cell responses, IgE production, and releases of inflammatory mediators. Allergic symptoms are primarily triggered by allergen-mediated cross-linking of specific IgE on the surface of mast cells and basophils, causing the release of histamine and other inflammatory mediators 27. Sensitization and provocation of indoor allergens such as mites are considerable triggers for the development of allergic diseases 28. The inhaled allergens derived from mites may change the integrity and function of airway epithelium, and intrinsic biochemical activity, thus leading to allergic symptoms and developing airway allergy 29. Airway inflammation after chronic and prolonged exposure to dust mite allergens leads to inflammatory mediator release, airway trachea remodeling, and irreversible damage. These allergic symptoms caused by mite allergens often cannot be completely controlled by current medicine.
More than 36 different allergic components that induce IgE in allergic patients and approximately 32 allergens in HDM have been identified based on IgE-binding frequency, protein sequence homology, and biochemical function 30. The major IgE frequencies of mite allergens in allergic patients are different due to the discrepancies in countries, regions, human genomes, and survey seasons. The group 2 mite allergens, such as Der p 2, have been identified as one of the most predominant sources of mite allergens and have been known as a major causative of respiratory disorders including allergic asthma, airway hypersensitiveness, and exacerbation of lung function 7. Der p 2 is a major mite allergen processing non-proteolytic characteristics whose structure is very stable so that it does not easily decompose and exists in the surrounding environment for a long time even after mite death 6. The Der p 2 exhibits molecular mimicry of structural homology and similar biological activity with Myeloid Differentiation-2 (MD-2), which is a lipopolysaccharide(LPS)-binding member of the Toll-like receptor-4 (TLR-4) signaling complex, resulting in Der p 2 can facilitate TLR-4 signaling through direct interactions to trigger innate immunity and its allergenicity 11. The major mite allergen Der p 2 significantly elevated nerve growth factor (NGF) production in the BALF, activation of alveolar macrophages and mast cells, eosinophilic infiltration, and airway inflammation through the p38 mitogen-activated protein kinase (MAPK) pathway and increasing ROS production 31. Der p 2 induced secretions of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, IL-8, monocyte-chemotactic protein (MCP)-1, and macrophage inflammatory protein-3alpha (MIP-3alpha), and intercellular adhesion molecule (ICAM)-1 in human bronchial epithelial cells resulting in it causes the inflammation in lung epithelium and aggravates respiratory airway diseases 32.
Strategies of allergy prevention
The stratagem of allergy prevention includes allergen avoidance, pharmacotherapy, and allergen-specific immunotherapy. Allergen-specific immunotherapy is an effective treatment used for allergic diseases which alleviates immoderate immune responses and relieves the pathogenesis progression. The traditional form of immunotherapy incorporates the administration of progressively raised quantities of relevant allergens until the dose approaches an effective degree for inducing immunologic tolerance. There are still many adventures in allergen immunotherapy with crude extracts such as unexpectedly anaphylactic reactions derived from the tricky standardization of allergen levels in extracts 33. The compositions of allergen crude extracts are not easy consistently due to diverse manufacturers, different batches, and extraction methods. Especially in the mite crude extracts are composed of mite bodies, fecal pellets, mite allergens, eggs, surface microbes, intracellular bacterial symbionts, and other potential immunogens. Modified strategies based on gene recombination technology of relevant recombinant allergens are used to minimize side effects caused by crude extracts and improve the therapy efficiency. Two mutants of the clinically important dust mite allergen Der p 2 with consistent low IgE binding have good potential as prophylactic hypoallergenic vaccine candidates for immunotherapy in a murine model 34. The Der p 2-based peptide vaccine shows reduced allergenicity of IgE-related and basophil activation when compared with the wild-type allergen and modified Der p 2 allergen may be used for prophylactic vaccination 35. The local nasal immunotherapy with recombinant Der p 2 (rDer p 2) in conjunction with fungal immunomodulatory protein (FIP) can downregulate the airway inflammation in both D. pteronyssinus- and rDer p 2-sensitization mice. This combination also even can enhance the Th1-biased immunogenicity and reduceTh2-biased allergenicity, it suggests the major allergen Der p 2 and in conjunction with immune adjuvant FIP might be a good alternative therapy for allergy which cause by dust mite 36. In this study, we demonstrated that the mite major allergen-Der p 2 induces airway inflammation in the asthmatic murine model and that hypersensitivity to methacholine can be modulated by TCM of modified LWDHW-1217A and 1217B. The immunoglobulin productions of Der p 2-specific IgE and IgG1 in sera and inflammatory cytokine productions of IL-5 and IL-13 in the airway BALF were significantly decreased, the results overall in this study indicated the modified LWDHW-1217A or 1217B has the potential to down-regulate the inflammatory tendency caused by the mite allergen.
TCMs used in the treatment of allergic diseases
The TCMs have been used to treat allergic diseases, especially bronchial asthma by Chinese clinicians for several centuries and significant efficiencies of clinical improvement have been noticed. Administration of LWDHW in the asthmatic guinea pigs was shown to prevent airway bronchoconstriction, decrease eosinophil infiltrations in BALF and decrease the production of inflammatory mediators (histamine and LTC4) 37. However, the LWDHW administration only partly improved clinical parameters in terms of symptom score, medication score, and peak expiratory flow rate measurement in asthmatic children, this study showed that this disposal could increase total T-cells and decrease B-cells 37. The lymphocyte differentiation subsets of T-cells and specificity immunoglobulin productions of B-cells weren’t paid special attention to observe the alternations in the previous study. The expression portion of the Th1-type cytokine (IFN-γ) and Th2-type cytokine (IL-4) produced in the CD4+ cells were analyzed in this study, results indicated immunotherapy with modified LWDHW-1217A or 1217B can enhance the TH1 cell population and suppress the TH2 cell population. The expressions of Th2-type cytokines (IL-4, IL-5, IL-10, and IL-13) and Th1-type cytokines (IL-2 and IFN-γ) were significantly inhibited both at RNA and protein levels in the treatment of traditional LWDHW with human peripheral blood lymphocytes in vitro 26. The Th1 and Th2 responses were investigated more through in vivo which focused on the cytokine productions in the sera and BALF (Fig. 4), gene expression of lung tissue (Fig. 6), phosphorylation of STAT6, STAT1, and ERK, activation of GATA3 (Fig. 7), and the percentages of IFN-γ+/CD4+ and IL-4+/CD4+ in the T cells (Fig. 8) after immunotherapy from asthmatic mice in this study. Overall, the TH1 cell-related activities or responses were enhanced; the TH2 cell-related activities or responses were significantly suppressed after immunotherapy with modified LWDHW-1217A or 1217B. Several possibilities may result in better therapeutic effects on the enhancement of TH1-related responses and inhibition of TH2-related responses. The first reason may be caused by the continuously oral administration for three weeks in vivo with high efficiency of therapeutic effects than the treatment with traditional LWDHW for one day in vitro, there is enough time for immunity adjustment of oral administration in vivo. The second reason is attributed to three Chinese traditional medicines including Radix Dioscorea, Alismatis Rhizoma, and Scutellaria baicalensis which possess anti-inflammatory effects are added in the modified LWDHW-1217A or 1217B as shown in Table-1. The combination of these Chinese traditional medicines is mainly based on other research that had found these ingredients have the potential for inhibiting inflammatory responses and informative recommendations of Chinese medical physicians.
Advantageous therapeutic effects of previous findings
Several TCMs are considered to be valuable and easily available resources from conventional medicine as a result of their advantageous therapeutic effects and fewer adverse or side effects, such as Anti-asthma Herbal Medicine Intervention (ASHMI), Modified Mai-Men-Dong-Tang (mMMDT), Ding Chuan Tang (DCT), and STA-1 (combination of mMMDT and Lui-Wei-Di-Huang Wan (LWDHW)) have been used to treat asthmatic patients 38–41. More and more evidence demonstrates that these formulas of TCMs are provided with substantial pharmacological activities, abundant ingredients, and structural diversity suitable for dealing with asthma in a multi-physiological, multi-target, and complex course manner. The asthmatics have noticeable improvements including lung function (Forced expiratory volume in 1s: FEV1), serum total IgE, dust mite specific IgE, and symptom scores after 4 months of mMMDT therapy in the persistent, mild-to-moderate asthmatic patients 39. The Chinese herbal formula mMMDT may improve lung function and ameliorate clinical symptoms, also no drug-related adverse effects were reported 39. Although the detailed mechanisms of these Chinese herbal combinations are not well understood and which compounds belong to active ingredients are still unclear, it suggests the TCM of mMMDT can be considered an effective alternative to Western medicine for mild-to-moderate asthma in addition to current therapies due to its efficacy, safety, low cost, and favorable compliance characteristics 39. The STA-1 formula (combination of mMMDT and LWDHW) could effectively suppress clinical symptoms, improve pulmonary function (FEV1), and inhibit the synthesis of total and mite-specific IgE in mild-to-moderate asthmatic patients, and there were no obvious adverse side effects during the intervention period 40. It was just that the detailed mechanism of pharmacological actions and the curative ingredients of the STA-1 formula were not very clear in this clinical trial 40. In a comprehensive summary, therapy with TCM in asthmatic patients has several advantages including clinically effective, cost inexpensive, and no life-threatening or severe side effects. These formulae of TCMs used for anti-asthma treatment are provided improvement in efficacy and safety, especially in special populations such as children with mild-to-moderate asthma.
Brief review of anti- inflammatory activities of modified LWDHW in this study
In this study, the modified LWDHW formula of 1217A (four herbs added, including Poria cocos, Radix Dioscorea, 3g of Alismatis Rhizoma, and Scutellaria baicalensis) and 1217B (four herbs added, including Poria cocos, Radix Dioscorea, 9g of Alismatis Rhizoma, and Scutellaria baicalensis) were used (Table-1). The main difference between formula 1217A and 1217B is that content of the component-Alismatis Rhizoma is different. The ethanol extract of TCM Poria cocos has been reported to reduce the production of inflammatory mediators including nitric oxide (NO), prostaglandin E2 (PGE2), interleukin (IL)-1β, and tumor necrosis factor (TNF)-α by suppressing the NF-kappaB signaling pathway in lipopolysaccharide (LPS)-stimulated macrophages 42. Both modified LWDHW formulas of 1217A and 1217B had increased the content of Poria cocos in this study, its purpose is to strengthen the pharmacological actions of anti-inflammation after the mite allergen trigger. The Yam (Dioscorea) were selected to add in the formula of 1217A or1217B due to it has been confirmed anti-inflammatory effects and to modulate inflammatory mediators by inhibiting the NF-κB pathway. Inhibition of NF-κB subsequently down-regulates inflammatory markers such as COX-2, iNOS, TNF-α, and IL-1β, therefore these effects may be attributed to the antioxidant and anti-inflammatory nature of Yam (Dioscorea)22. The pretreatment with Alismatis ethanol extracts can effectively prevent the development of neutrophil lung infiltration in an LPS-induced lung inflammation of mouse models with acute lung injury 23. The Alismatis Rhizoma extract is involved in the inhibition of pro-inflammatory transcription factor NF-kB and activation of anti-inflammatory transcription factor Nrf2 so that it can attenuate lung inflammation 23. The Alismatis Rhizoma extract represents anti-inflammatory effects that can be developed as a potential therapeutic for acute or chronic lung inflammations caused by pathogens or allergens. The active compound wogonin from Scutellaria baicalensis (Skullcap) can suppress IL-4 production ex vivo and down-regulate IgE and IL-5 production on OVA-induced Th2 immune responses in vivo, it suggests the wogonin from Scutellaria baicalensis possesses anti-inflammatory properties so that can be applied as a therapeutic agent for IgE- or IL-5 mediated allergic disorders 24.
The formula 1217A and 1217B used in this study notably included two effective ingredients, Poria cocos, and Scutellaria baicalensis, the composition of herbal complexes Saiboku-to (TJ-96)43. The herbal medicine TJ-96 has an anti-inflammatory effect on bronchial eosinophilic infiltration in the clinical trial with asthmatic patients. In the double-blind and randomized trial, results suggest that TJ-96 possessing protective activity is associated with a reduction in eosinophilic inflammation, and this formula presents therapeutic possibilities for the treatment of asthma 43. Three triterpenoids from the phenolic compounds including magnolol, dihydroxy-dihydromagnolol, and liquiritigenin which are contained in Saiboku-to have been confirmed to exert a marked inhibition on leukotriene C4 for the treatment of bronchial asthma 44. These three active components can inhibit an asthmatic effect through anti-inflammatory responses, results revealed the formula of Saiboku-to possess clinical efficacy and act as an anti-allergic agent 44. The pharmacological efficacy has been demonstrated that the Baicalein from extracts of Scutellaria root can suppress human eotaxin mRNA expression in human fibroblasts which is associated recruitment of eosinophils 45. The previous study demonstrated the extracts of yam Dioscorea could downregulate ovalbumin-induced allergic response in mice and reduce inflammatory parameters including IgE, intestinal edema, and mucus production, suggesting the yam Dioscorea has the potential to prevent and treat allergies associated with food allergens 46. The methanol extracts from Alismatis Rhizoma not only inhibit antibody-mediated allergic reactions but also influence immune cell reactions, it exhibits the inhibitory effect on contact dermatitis in mice which serves as a useful agent for the treatment of allergic reactions 47. The yam Dioscorea and Alismatis Rhizoma had been added to the formula 1217A and 1217B in this study, and the productions of mite allergen-specific immunoglobulins (IgE and IgG1), Th2-cytokines (IL-5 and IL-13), and expressions of inflammatory genes (IL4, IL5, and IL13), neutrophil chemotactic chemokine (IL8), and TH2-related transcription factor (GATA-3) can be down-regulated after immunotherapy with 1217A or 1217B. And results of this study indicated immunotherapy with modified LWDHW-1217A or 1217B can enhance the TH1 cell population and suppress the TH2 cell population, the ratio of Th2/Th1 was also noted to have decreased much less than with the saline group. The pulmonary function of Penh and bronchus inflammation of lung histopathology were significantly alleviated after the immunotherapy with 1217A or 1217B. Whether there is a difference between formula 1217A and 1217B in the asthmatic model of the therapeutic or anti-inflammatory effects was evaluated in this study. Comprehensively observing the overall results in this study, the therapeutic efficacy of 1217B appears to be better than 1217A in the asthmatic animal model, it just showed no statistical difference between them. The actual efficacy on therapeutic or anti-inflammatory effects of TCM formula 1217A or 1217B still needs more research to explore and confirm the detailed mechanism of pharmacological actions and the curative ingredients.
Administering methacholine to the subcarinal airways via the bronchial artery caused dose-dependent bronchoconstriction and contraction of the tracheal segment. Methacholine inhalation is usually used to diagnose patients with bronchial airway hyperresponsiveness or asthma severity clinician or the degree of airway inflammation and tracheal obstruction in the asthmatic animal. Methacholine inhalation may increase the sensitivity of bronchi to inflammatory mediators such as histamine and leukotrienes then cause bronchial contraction, it could significantly downregulate FEV1 and enhance Penh 48. Bronchial asthma is characterized by airway inflammation with clinical features of airway hypersensitivity. Most allergic patients with asthma symptoms have bronchial hyperresponsiveness; therefore, the Methacholine provocation test has very excellent sensitivity for the diagnosis of asthma severity. However, bronchial hyperresponsiveness may also be observed in other pulmonary diseases, including chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchitis, allergic rhinitis, or congestive heart failure. So, it is necessary to explore other physiological variations in the asthmatic animal model such as immunoglobulin generations, inflammatory cytokine productions, Th1/Th2 polarization, gene expressions in lung tissue, and bronchus pathology for the more complete and comprehensive observations in this study. The airway hyperresponsiveness to methacholine inhalation was decreased after immunotherapy with modified LWDHW-1217A or 1217B and in association with reduction of airway inflammation, suggesting that formula of 1217A or 1217B may be used as therapeutic agents in the treatment of allergic bronchial asthma.
In allergic reactions, allergens are presented to T cells by antigen-presenting cells, which causes naïve T cells to differentiate into Th2 cells and secrete IL-4, IL-5, and IL-13(Duramad et al. 2006). IL-4 secreted by Th2 cells could further stimulate B cells to produce allergen-specific IgE (Wisniewski et al. 2015). In this study, the concentration of Der p 2-specific IgE and IL-5 in BAL fluid and sera were significantly decreased after 1217A or 1217B treatment. Furthermore, the Th2/Th1 ratio was also downregulated. A similar finding was observed in the cells' differential counts of BAL fluids. Total cell numbers, macrophage, lymphocyte, eosinophil, and neutrophil were downregulated after 1217A and 1217B treatment. Decreased Th2 cytokine could inhibit Der p 2-specific IgE production, and could further lessen binding to its receptor, FcεRI, on basophil or mast cells. Less IgE binding to FcεRI could minimize the allergic reaction and further downregulate the proinflammatory cytokine expression from immune cells and prevent inflammatory cell infiltration. Therefore, 1217A and 1217B appear to be beneficial in the treatment of allergic asthma.