Wenyang-Huayin, a Chinese Medicine formulation, inhibits in ammation in OVA-Induced asthma rats

Peizheng Yan Shandong University of Traditional Chinese Medicine Qingxiang Zhang (  1323113659@qq.com ) Shandong University of Traditional Chinese Medicine https://orcid.org/0000-0003-1171-8950 Zhu Qingjun Shandong University of Traditional Chinese Medicine Xing Chang Shandong University of Traditional Chinese Medicine Meng Qingyan Shandong University of Traditional Chinese Medicine Yan Liu Shandong University of Traditional Chinese Medicine


Introduction
Asthma has become a severe public health issue in various countries over the world 1 . As a health problem for all ages and with approximately 300 million patients worldwide, asthma leads to 250,000 annual deaths, most of which, however, are preventable 2,3 .
Bronchial asthma is an allergic reaction disease induced by a variety of factors and is a chronic airway in ammatory response involved in multiple cells and cell components, usually associated with Th2 cell mediated eosinophilic airway in ammation. Airway hyperresponsiveness and chronic airway in ammation are the main characteristics of asthma. Asthma is closely related to environmental factors, allergies, airway in ammation, airway hyperresponsiveness, nerve, and other factors 4 . However, chronic in ammation can make the aggravation of airway responsiveness, lead to a variety of reversible air ow limitations, or cause recurrent symptoms such as pu ng, chest tightness, or coughing and phlegm. Respiratory tract in ammation is the pathological basis of asthma. Therefore, the control of airway in ammation is the focus of asthma treatment in both acute and chronic remission stages of asthma.
The imbalance between Th1 and Th2 cells' immune responses is a main property of in ammatory asthma. The typical Th1 cell-derived cytokines include IFN-γ, IL-1, IL-2, and TNF-β, and the typical Th2 cell-derived cytokines are IL-4, IL-5, TNF-α and IL-13 5 . Previous studies have demonstrated that Th2 cells are vital in the pathogenesis of asthma, and Th2 cell-derived cytokines are closely related to mucus hypersecretion, eosinophil (EOS) accumulation, hyperresponsiveness (AHR) development, and lung remodeling 6,7 .
Additionally, TGF-β1, mainly produced by EOS 8 , plays a pivotal role in secreting and mediating a mass of growth factors, and cytokines cause airway in ammation and airway remodeling 9 . Expression of TGF-β1 is correlated with basement membrane thickness, broblast number, and in ammation severity 10 .
Despite inhaled corticosteroids, asthma patients still show a decrease in the level of pulmonary function 11 . In recent decades, the development of traditional Chinese medicine has made great progress. It has played a great role in the prevention and treatment of asthma, and the clinical symptoms have been clearly improved [12][13][14] .
Wenyang-Huayin (WYHY) formulation is a useful prescription for asthma in clinical. In this study, we explored the effects and properties of WYHY in the treatment of asthma induced by OVA and aim to provide support for its deeply clinical applications. and ground into powder. The extraction rate of the dry extract was 20.29% (actual dry powder/actual crude herbs). The prepared powder was kept at 0℃-4℃ and dissolved with puri ed water into a suspension of different concentrations. Astragalus granules (crude drug proportion 1:10). These herbs were from Huarun three-nine Medicine Co.,Ltd., in Jinan. Rapamycin (100 mg/bottle) were obtained from MedChem Express in America (#HY-10219)
2.5. Induction of the syndrome by accumulation of cold uid in the lung in chronic bronchitis asthma rat model Forty-eight male rats were randomly divided into six groups (8 rats per group): a normal group (group 1), a model control group (group 2), a rapamycin group (group 3), Xiaoqinglong Decoction group (group 4), WYHY group (group 5), Astragalus granules group (group 6).
Establishment of asthma models were performed according to previous reports [31][32][33] . Brie y, rats of groups 2, 3, 4, 5, and 6 were injected intraperitoneally (i.p.) with a suspension containing 100 mg OVA and 100 mg Al(OH) 3 dissolved in 1mL saline on days 1 and 8. Meanwhile, the rats in the normal saline group received 0.5 mL saline. Every two days during days 15 to 22, rats of groups other than the normal saline one were stimulated by 1% atomized OVA for 30 min; saline was used instead of OVA for rats of the normal saline group. Drink cold body cold, fatigue stimulation were performed for 20 days, except the rst and 8th day, 30 min before OVA exposure. Rats were sacri ced at day 23.
Induction of the syndrome of accumulation of cold uid in the lung in a rat model: (1) Drink cold stimulation is to feed rats with ice water mixture daily; (2) Body cold stimulation is to freeze rats in -10 ℃ refrigerator for 3 hours daily; (3) Fatigue stimulation is that rats swim in water at 24℃, until the nose will sink into the water unable to oat, then the rats get out. (Figure 1A) 2.6. Drug intervention According to the drug dosage conversion formula between rats and human, rats in the norm and model control group were given puri ed water and rats in the rapamycin group were given rapamycin suspension 0.013 mg·(kg·d) −1 . The rats in the groups of Xiaoqinglong decoction, WYHY were treated with powder suspension of modi ed Xiaoqinglong decoction, WYHY equal to the crude drug of 6.69642855 g·(kg·d) −1 and 9.82142857 g·(kg·d) −1 . the rats in Astragalus granules group were given Astragalus granules 0.223214286 g·(kg·d) −1 . Rats were weighed once a week for adjusting drug dosage in the 3 weeks' the intervention period.

Sample collection and test
At the end of the three-week treatment period, rats were anesthetized with pentobarbital sodium solution (4%, intraperitoneal injection) after 12h fasting, then the pulmonary function was measured by AniRes 2005 Animal pulmonary function Analysis system Inspiratory resistance (IR), expiratory resistance (ER) and pulmonary ventilation adaptability (Cldyn). And blood samples were collected from the abdominal aorta, and the left lung tissue was quickly removed, cleaned with saline rinse, and frozen immediately in -80℃ refrigerator for cryopreservation.

Enzyme-linked immunosorbent assay (ELISA)
The blood serum samples from the abdominal aorta were centrifuged to collect serum. IL-13, TGF-β1, TNF-αand IFN-γ were measured by an ELISA assay.

Hematoxylin-Eosin staining
Isolated lungs were xed with 4% paraformaldehyde and embedded in para n. Then we cut the specimens into a thickness of 3-4 µm sections. The sections were stained with hematoxylin and eosin (H&E). The results were visualized using a microscope. Three pathologists individually assessed all staining results.

Western blot detection
The expressions of LC-3B, beclin-1, p-mTOR, and mTOR were detected by Western blot. RIPA lysate was used to extract total protein. The samples were taken for sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis in a buffer system. Then the protein in the gel was transferred to the nitrocellulose membrane. The membrane was sealed with 5% skimmed milk at room temperature for 90 min. Then, the membrane was washed with TBST buffer for 3 times and incubated with the rst antibody overnight at 4℃. After washing with TBST buffer, the corresponding secondary antibody was added the next day and incubated at room temperature for 2 h. At last, the protein bands were visualized using Clarity™ Western ECL Substrate (Bio-Rad) and then analyzed using ImageJ v1.40 software (Hercules, CA, USA).

Transmission electron microscope (TEM)
The autophagosomes in lung sections were observed by TEM. In brief, the pathological sections of the lung in each group were xed with 2% glutaraldehyde buffered with 0.2 mmol/L cacodylate and post xed in osmium tetroxide before embedding in epoxy resin for electron microscopy.

Statistical analysis
All data were expressed as mean ± SD and analyzed with SPSS13.0 software. Differences of parametric data among several groups were determined by one-way analysis of variance (ANOVA). Any differences with < 0.05 were considered signi cant (two-tailed).

Effect of WYHY on general conditions
Rats in Group 1 had the following characteristics: body strong, normal daily food and water intake, normal excretion of urine and stool, smooth fur in good mental state, and swift action, respiration rhythm heat. Rats in Group 2 had the following characteristics: body emaciation, rough and lusterless fur, or thinning limbs, not warm, nose, ears and tail, pale, enuresis, loose stools, ecphysesis, pants, wheezing, white secretion on mouth and nose, mental fatigue, slow in reacting or agitated state, and weight loss ( Figure 1B). symptoms of ecphysesis, pants, wheezing, and weight loss ( Figure 1B), with glossy fur and swift response. The rats in Group 3 also showed improvement of symptoms and signs, although not as signi cant as those in the three herbal medicine decoction groups.

WYHY improved pulmonary function
As shown in Table 1, the baselines of inspiratory resistance (Ri), expiratory resistance (Re), and lung compliance (Cldyn) were similar between groups (P > 0.05). On the 23rd day, Ri and Re in group 2 were signi cantly higher than group 1, while Cldyn in group 2 was signi cantly lower than group 1 (P<0.01); Ri and Re in the treatment groups (including groups 3, 4, 5, 6) were signi cantly lower than Group 2, while Cldyn in the treatment groups was signi cantly higher than group 2 (P<0.01). In addition, there was no signi cant difference in Ri and Re between the treatment groups (P > 0.05), while Cldyn in group 5 was higher than group 3 (P<0.05) and group 6 (P<0.01). Compared with group 1, *P 0.05, **P 0.01;Compared with group 2, # P 0.05, ## P 0.01; Compared with group 3, Δ P 0.05; Compared with group 5, ▲▲ P 0.01; Compared with Baseline, @ P 0.05, @@ P 0.01.

WYHY alleviated the pathological injury of lung
The histopathological features of each group were shown in Figure 2. In group 1, the pulmonary alveolus structure was intact, with rare in ammatory cell in ltration around. The bronchial smooth muscle was normal, with lumen patency and cilia regularly arranged. In group 2, there was a lot of in ammatory cell in ltration around the bronchus and epithelial cells of the bronchial mucous membrane, especially lymphocytes and EOS, and there were also in ammatory cells and increased mucus content inside the bronchial lumen. The alveolar walls are thickened with blood cells and pulmonary alveolus seeped. In group 3 and group 6, although the general pathology feature of pulmonary alveolus and bronchus was better than that in group 2, but there were still a lot of in ammatory cell in ltrations around the bronchus, epithelial cells of bronchial mucous, and inside the bronchial lumen. In group 4 and group 5, the epithelial cells of the bronchial mucous membrane were slightly swelled, and the bronchial smooth muscles were slightly thickened, and there were little in ammatory cells and mucus content seeped around the bronchus and inside the bronchial lumen. In addition, group 5 got the least in ammatory cell in ltration and luminal stenosis. These results suggested that all treatment methods might slow down the damage caused by molding factors, and WYHY group was the best in both groups.

WYHY reduced the levels of circle cytokines
Compared with group 1, the levels of IL-13, TGF-β, and TNF-α in group 2 increased, while the level of INF-γ decreased (P<0.05 or P<0.01). Compared with group 2, the IL-13, TGF-β, and TNF-α in the treatment groups were all decreased, while the level of INF-γ increased (P<0.05 or P<0.01). There was no signi cant difference in IL-13, TGF-β, TNF-α, and INF-γ levels between the treatment groups (P>0.05) (Figure 3).

WYHY activated cell autophagy in lung
We noticed a decrease in the number of autophagosomes in the lung tissue of asthma rats, and all treatment groups might affect autophagy ( Figure 4). Therefore, we determined the autophagy associated proteins LC-3II, LC-3I, beclin 1, and p-mTOR ( Figure 5). Compared with group 1, the expression levels of LC-3II/ LC-3I and beclin 1 in group 2 increased, while p-mTOR/ mTOR decreased (P<0.01). Compared with group 2, the expression levels of LC-3II/ LC-3I and beclin 1 in the treatment groups decreased, while p-mTOR/ mTOR increased (P<0.05 or P<0.01).

Discussion
This study is based on the theory of Chinese medicine, according to the pathogeny doctrine of TCM and western medicine, to establish the animal model of the syndrome of accumulation of cold uid in the lung in chronic bronchitis asthma 15 . The syndrome of accumulation of cold uid in the lung in chronic bronchitis asthma re ects two aspects of the disease syndrome: yang de ciency and spleen and lung qi de ciency. Qingxiang Z, Shaohong Y 16,17 induced asthma model by sensitizing and challenging with OVA, and established the model of syndrome of accumulation of cold uid in the lung in chronic bronchitis asthma by drink, body cold and fatigue stimulation. The asthma model rats in our research show a few syndromes, such as rapid breathing, wheezing, mild cyanosis, loud wheezing can be heard in some rats, and there are white sticky secretions in the mouth and nose. We found model rats suffered body cold stimulation, appearing symptoms and signs of yang de ciency, for example, limbs not warm, nose, ears and tail pale, model rats suffered drink cold and fatigue stimulation appear symptoms and signs of spleen and lung qi de ciency, such as body emaciation, rough and lusterless fur or thinning, enuresis, loose stools, ecphysesis, mental fatigue, slow in reacting or agitated state, and weight loss, Symptoms and signs of model rats appear identically with the syndrome of accumulation of cold uid in the lung in chronic bronchitis asthma in clinic.
The airway of asthmatic patients may be overreactive due to airway in ammation, showing sensitive and over strong contraction of the bronchial smooth muscle, resulting in airway constriction and increased airway resistance. It causes cough, chest tightness, wheezing, and other symptoms. The important features of bronchial asthma are airway obstruction and airway hyperresponsiveness.
In experimenting, we determined that inspiratory resistance and expiratory resistance increased signi cantly, while lung ventilation compliance decreased signi cantly. It suggested modeling factors increase of pulmonary airway resistance in rats, inducing hyperresponsiveness of the airway. In the study of pathomorphological of lung tissue of model rats, there were a lot of in ammatory cell in ltration around the bronchus and epithelial cells of bronchial mucous membrane, especially lymphocytes and EOS, cilia adhered; there were in ammatory cells and increased mucus content in bronchial lumen; blood cells seeped, the alveolar walls are thickened; Pulmonary alveolus seeped much liquids, evenly ruptured and merged. The phenomenon was consistent with the pathomorphological features of clinical bronchial asthma.
According to the characteristic of bronchial asthma with the syndrome of accumulation of cold uid in the lung, we drafted WYHY. It was composed of the classical prescription Xiao Qinglong Decoction with astragalus and ginseng. It has reached the therapeutic effect of warming yang for resolving uid retention and freeing lung and relieving asthma. Ephedrine in ephedra in decoction has an obvious relaxant effect on bronchial smooth muscle, especially during bronchial spasm 18 . The volatile oil of Cassia twig has antiin ammatory effect on respiratory in ammation 19 . Licorice avonoids, licorice extract, and glycyrrhetinic acid have remarkable preventing cough, eliminating phlegm effects. meanwhile, they strengthen anti-in ammatory effects by inhibiting macrophages producing PEG2 and IL-6 20,21 . Rhizoma Zingiber asarum and Paeonia lacti ora show anti-in ammatory effects by inhibiting the production of proin ammatory mediators in macrophages and monocytes. While Asarum volatile oil may indeed relieve bronchial spasm. Alkaloids from Pinellia ternata can prevent cough, Eliminate phlegm by inhibiting cough center [22][23][24] . The effective components saponins and polysaccharides of ginseng and Astragalus have anti-in ammatory, antibacterial, and regulated smooth muscle activity effects. Meanwhile, they may protect macrophages to enhance its phagocytosis 25 . The pharmacological actions of the effective ingredients in the prescription indicate that WYHY can effectively inhibit the in ammatory reaction of the respiratory tract and reverse the pathological process of airway remodeling.
TCM could treat bronchial asthma by blocking in ammation and reversing airway remodeling. In this study, we observed that WYHY can relieve the main symptoms and signs of model rats, such as shortness of breath, wheezing, secretion of airway mucus, improve lung function, inhibit the in ltration of in ammatory cells around the epithelial cells and alveoli (mainly EOS and lymphocyte), and reduce the e EOS induced cytokine, and correct the Th1/Th2 immune imbalance.
AHR is also a major symptom of asthma. After the treatment of WYHY, we determined that inspiratory resistance and expiratory resistance decreased signi cantly, while lung ventilation compliance increased signi cantly. Airway in ammation induction was one of the main causes of hyperresponsiveness 26,27 . EOS, lymphocytes, and macrophages are important in ammatory cells in asthma.
Cytokines and chemokines induced by in ammatory cells in the airway aggravate airway hyperresponsiveness. After the treatment, the epithelial cells of the bronchial mucosa of the rat showed slight swelling and hyperplasia. A small amount of mucus and in ammatory cells were found in the bronchial tube. The alveolar wall was slightly thickened, and a small amount of EOS and lymphocyte in ltration could be seen. They pointed out that WYHY can reduce the number of EOS and lymphocyte in ammatory response and relieve airway hyperresponsiveness.
TGF-β 1 is a major regulator of airway remodeling. During asthma attacks, macrophages, epithelial cells, smooth muscle cells, and EOS in the airway secrete a large amount of TGF-β 1 . TGF-β1 stimulates proliferation and hypertrophy of airway smooth muscle cells, induces the division and proliferation of broblasts, and promotes to transform broblasts into myo broblasts 28 . The expression of TGF-β 1 was positively correlated with the thickness of airway basement membrane, the number of broblasts, and (or) the severity of asthma attacks 29 . In this study, we observed that there were high levels of TGF-β1 in blood and asthma attacks heavily. After treatment of WYHY, the level of TGF-β1was reduced, the clinical symptoms were alleviated, it is possible that the decoction could reverse the level of TGF-β1.
A large number of studies have con rmed that Th2 cells play an important role in the pathogenesis of asthma. IL-13 is one of Th2 cytokines, which may cause in ammatory cells to adhere to the vascular endothelium, and then transmembrane wandering gather around the airway 30 . Meanwhile, it can inhibit the apoptosis of EOS and B cells, so that airway in ammation persists 31 . IL-13 induces and activates brotic medium TGF-β, which stimulates myo broblast proliferation and participates in airway brosis 32 . The antagonistic effect of Th1 cells on Th2 cells is an important way to prevent asthma and allergic reactions. INF-, which Th1 secreted, has the function of phagocytosis of microorganisms in the cell that can downregulate the expression of EOS chemokine receptor and inhibit EOS in ltration 33 . IFN-can also inhibit the proliferation of broblasts and the synthesis of collagen, promote the apoptosis of broblasts, and repair the damaged alveolar epithelium, and reduce the expression of TGF-β 34 . Therefore, IFN-plays an important role in suppressing airway in ammatory cell in ltration and reversing airway remodeling. TNF-is a proin ammatory cytokine, produced by mononuclear macrophage 35 , which makes the adhesion molecules of endothelial cells highly expressed. These adhesion molecules combine with the corresponding receptors on the in ammatory cells, thereby promoting the accumulation of in ammatory cells across the endothelium and activating them to induce and aggravate asthma 36 . While TNF-can stimulate airway smooth muscle cells to secrete endothelin, which exacerbate the contraction of smooth muscle and cause airway remodeling.
Our experimental results showed that the expression of IL-13 and TNF-in the peripheral blood of model rats decreased signi cantly, while the expression of INF--increased signi cantly after the intervention of WYHY. These results suggested that the decoction that regulates the expression of IL-13, TNF-, INF-, and TGF-β1 can inhibit the accumulation and activation of in ammatory cells, alleviate airway in ammation, slow down the contraction of smooth muscle, and reverse the airway remodeling.

Conclusion
WYHY decreased the expression of IL-13 and TNF-, TGF-β1 in peripheral blood, while increased the expression of INF-. WYHY can relieve the main symptoms and signs of model rats, such as shortness of breath, wheezing, secretion of airway mucus, improve lung function, inhibit the in ltration of in ammatory cells, slow down the contraction of smooth muscle, and reverse airway remodeling.

Declarations Ethics Approval
The study was approved by the comments of the laboratory animal care committee of Shandong University of Traditional Chinese Medicine (NO. SDUTCM2015020101).

Consent to publish
The authors declare that they all agree to publish the paper.

Availability of data and materials
The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Con ict of Interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.

Fundings
This study was supported by the National Natural Science Foundation of China (NSFC, Nos. 81470189, 81774169, and 81874419).

Authors' Contributions
Zhang Qingxiang designed the experiment scheme, guided the whole experiment, and reviewed and revised the manuscript. Yan    Compared with group 1, *P 0.05, **P 0.01 Compared with group 2, # P 0.05, ## P 0.01.