Pingchuan formula (PCF) was created by Professor Yu Jianer. The purpose of this study was to investigate the effect of PCF on dendritic cells (DCs) and toll-like receptors (TLRs) in initiating immunity. A bronchial asthma BALB/c mouse model was established using an OVA excitation method. PCF was immediately administered by gavage after the first excitation. After 7 d, hematoxylin and eosin (HE) staining was used to observe the pathological changes in the asthma model. Eosinophil infiltration and concentrations of IL-4, IFN-r, IL-12, and IFN-
Asthma is a common lung disease in childhood, which is associated with phlegm and wheezing. The prevalence of asthma is currently increasing worldwide at a rate of 50% every decade, and the number of asthma patients will reach 400 million by 2025 [
With the research progress, various pathogenesis and treatment methods of asthma have been discovered. Although several treatment methods have been well established in western medicine, the problems of repeated attacks of wheezing and the long-term existence of phlegm remain. Hence, the application of traditional Chinese medicine is critical.
Pingchuan formula (PCF) is a special herbal prescription for asthma that was created by Professor Yu Jianer. PCF is composed of roasted ephedra, bitter almond, purple Perilla, peach kernel, radish, prickly ash, dried earthworm, Scutellaria, and roasted licorice. The formula can modulate the QI movement in the lung, clear the phlegm, and remove the blood stasis, thereby relieving asthma. Immune mechanism has become the classic pathogenesis of asthma. PCF could significantly improve the imbalance of Th1/Th2 in a mouse model [
Experimental design.
A total of 40 male BALB/c mice were provided by Shanghai Super B&K Laboratory Animal Corp. Ltd. (grade SPF, 4-6 weeks old, 18-22 g in weight, production license number: SCXK2013-0016). The mice were housed in the animal experimental center of Shanghai Traditional Chinese Medicine University (room temperature: 25°C, humidity: 60-70%). All mice were provided free access to water and food.
Forty mice were randomly divided into four groups: control group (CON,
Based on previously published modeling methods, combined with established modeling conditions and doses explored by our research group in the preliminary stage, the mouse asthma model was established. First, the mice received intraperitoneal (ip) injection of sensitizing solution on day 1 and day 15, except the CON group. The sensitizer consisted of 10 g OVA (Shanghai Jun Hui Chemical Co.), 1 g aluminum hydroxide, and 100 ml distilled water. Next, 24 hours after the last sensitization, the sensitized groups were challenged with 5% OVA (100 ml water containing 5g OVA) atomization excitation, for 40 min each time, once a day for one week. The CON group were ip injected and inhaled an equal volume of distilled water.
PCF (provided by Shanghai Hospital of TCM) was prepared at 5.33 g/ml concentration as previously described [
Mice were sacrificed, the chest was opened, and the right main bronchi was clamped with hemostatic forceps. A No. 12 blunt needle was inserted into the left main bronchus and then douched two times with 1 ml phosphate-buffered saline (PBS) at 4°C [French PBS buffer solution (pH 7.2-7.4)]. BALF was centrifuged (Germany Eppendorf centrifuge Model: 5804R), and eosinophils (EOS) were assayed using an ELISA kit as per the manufacturer’s instructions (mouse Eotaxin ELISA kit RD, USA).
The concentrations of IL-12, IL-4, IFN-r, and IFN-
Paraffin-embedded lung sections (Microtome Leica RM2235, 5
Lung sections (0.05 g) were cut and placed into a Ribozyme test tube to extract RNA, followed by amplification of the prepared cDNA. The total reaction volume was 50
Lung fragments (0.05 g) were homogenized in ice-cold RIPA lysis buffer (Beyotime, China), centrifuged at 14,000 rpm for 15 min, and the supernatant was collected. Protein concentrations were determined by the BCA protein assay kit (Beyotime, China). Polyacrylamide gel (10%) electrophoresis was used to isolate the proteins. The proteins were then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA). The nitrocellulose membrane was probed with anti-Toll-2, anti-ERK, anti-Toll-9, or anti-IDO monoclonal antibodies at 4°C overnight. The next day, the membrane was incubated with horseradish peroxidase-labelled goat anti-rabbit IgG antibody (1 : 1000) at room temperature, followed by alkaline phosphatase-conjugated secondary antibodies (BD, USA) for 2 h at room temperature on a shaker. The protein bands were displayed by ECL™ western blotting detection (GE healthcare, No. RPN2106) and quantified by Gel-Pro imaging software.
Statistical analysis was conducted using SPSS 18.0 software. If the data followed a normal distribution and homogeneity of variance, a one-way analysis of variance (ANOVA) and least significant difference (LSD) multiple comparison were used. Otherwise, the data were converted. A
The mice in the MDL group showed clustering phenomenon, different degrees of anxiety, muscle twitching, urinary and fecal incontinence, hair fluffing, lower activity, appetite reduction, irritability, and other symptoms. The DEX and PCF groups were better.
Mice in the CON group were normal. The bronchial structure was smooth and intact. The cells were arranged in neat rows, and no obvious abnormality was found in the lumen and peritubule. In the MDL group, the bronchus was deformed, the diameter of the bronchus was narrowed, and the wall structure was damaged, with extensive inflammatory exudates in the lumen and numerous inflammatory cell infiltration and accumulation in the perivasculature. The DEX and PCF groups were better than the MDL group. They showed bronchial structure integrity. The cells were disordered but cell diameter was normal. There was some inflammatory cell infiltration and accumulation in the perivasculature (Figure
Pathological changes detected using HE staining. (a) CON group, (b) MDL group, (c) DEX group, and (d) PCF group.
The mice in the MDL group had extremely high levels of Eotaxin as compared to the CON group. In contrast, the DEX and PCF groups had reduced Eotaxin in BALF (Figure
Eotaxin content in BALF:
As shown in Figure
Comparison of Toll-2/Toll-9/DC cytokines IL-12, IL-4, IFN-r, and IFN-
The data showed that the MDL group had lower levels of all cytokines than the CON, DEX, and PCF groups (
Comparison of IL-12and IFN-
As shown in Figure
Expression of ERK, Toll-2, IDO, and Toll-9 proteins in asthmatic airways. (a) Immunohistochemistry assay of ERK, Toll-2, IDO, and Toll-9 in lung tissues of the CON, MDL, DEX, and PCF groups; (b) quantitative analysis of (a);
Dendritic cells (DCs) are specialized antigen-presenting cells that are the main promoters of immune response. They activate unsensitized T cells participate in the differentiation and development of T cells and induce and maintain immune tolerance. Traditional DCs (cDCs) stimulate T primordial cells to Th2, differentiate Th17 cells, develop immune response, and promote airway inflammation. Plasmacytoid DCs (pDC) can stimulate the production of Treg cells and regulate pulmonary immune tolerance formation [
TLR is a pattern recognition receptor (PRR), which recognizes pathogen-associated molecular patterns (PAMP), activates the corresponding activation signal, participates in the activation of innate immunity and adaptive immunity, and establishes a bridge between innate immune response and adaptive immune response. TLR is mainly expressed on DCs and plays an important role in regulating the immune response of antigen-specific T cells. [
Studies have shown that TLR-2, which is mainly expressed on cDCs, is the main TLR [
TLR-9 can activate plasma-like DCs by recognizing CpG motifs of foreign antigens. pDC can secrete large amounts of IFN-
In this study, MDL was a successful model, with extensive intraluminal inflammatory infiltration, perivascular inflammatory cell infiltration, and accumulation. There was more eosinophil infiltration than in the CON group. The level of IL-4 in BALF was higher in the MDL group than in the CON group, and the levels of IL-12, IFN-
This study revealed that PCF can downregulate ERK and Toll-2 and upregulate IDO and Toll-9 expression, reduce IL-4 level, and increase IL-12, IFN-
The current study had several limitations. The mechanism of TCM treatment is very complex. However, this study uncovered the novel role of PCF in initiating the immune response. It would be beneficial to determine the effectiveness of PCF in the long-term treatment of asthma.
Pingchuan formula
Dendritic cells
Toll-like receptors
Hematoxylin and eosin
Enzyme-linked immunosorbent assay
Control group
Model group
Dexamethasone group
Intraperitoneal
Phosphate-buffered saline
Eosinophil
Analysis of variance
Traditional DCs
Plasmacytoid DCs
Immature DCs
Pattern recognition receptor
Pathogen-associated molecular patterns
Extracellular regulated kinase.
The data used and analyzed in the figures of this study are available from the corresponding author on reasonable request.
All authors declare they have no conflict of interest.
This study was supported by the National Natural Science Foundation of China (No. 81874488 and No. 81603662), Wuxi Health Committee Young Medical Talents Project (No. QNRC037), and Wuxi Health Committee Research Project (No. MS201946).