Abstract

   Allergic asthma is a stubborn chronic inflammatory disease, and is
   considered a co-result of various immune cells, especially mast cells,
   eosinophils and T lymphocytes. At present, the treatment methods of
   allergic asthma are limited and the side effects are obvious.
   Traditional Chinese medicine has been used to treat diseases for
   thousands of years in China. One such example is the treatment of
   allergic asthma, which take the characteristics of less adverse
   reactions and obvious curative effect. Tuo-Min-Ding-Chuan Decoction
   (TMDCD) is a traditional Chinese medicine compound for the treatment of
   allergic asthma optimized from Ma-Xing-Gan-Shi Decoction (MXGSD), which
   was put forward in Treatise on Febrile Diseases by Zhang Zhongjing in
   the Eastern Han Dynasty. The compound shows a significant clinical
   effect, but the mechanism of its influence on the immune system is
   still unclear. The purpose of this study was to observe whether TMDCD
   could alleviate the symptoms of ovalbumin (OVA) challenged allergic
   asthma mice, and to explore its immune regulatory mechanism, especially
   on mast cell (MC) degranulation. The results showed TMDCD could not
   only reduce the airway hyperresponsiveness (AHR), inflammatory cell
   infiltration and mucus secretion in the lung tissue of OVA challenged
   mice, but also decrease the levels of total IgE, OVA-specific IgE,
   histamine and LTC4 in serum. We found that TMDCD can downregulate the
   expression of Fractalkine, Tryptase ε, IL-25, CCL19, MCP-1, OX40L, Axl,
   CCL22, CD30, G-CSF, E-selectin, OPN, CCL5, P-selectin, Gas6, TSLP in
   OVA challenged mice serum by using mouse cytokines antibody array. It
   has been reported in some literatures that these differentially
   expressed proteins are related to the occurrence of allergic asthma,
   such as tryptase ε, MCP-1, CCL5, etc. can be released by MC. And the
   results of in vitro experiments showed that TMDCD inhibited the
   degranulation of RBL-2H3 cells stimulated by DNP-IgE/BSA. Taken
   together, we made the conclusion that TMDCD could reduce the
   infiltration of inflammatory cells in lung tissue and alleviate airway
   remodeling in mice with allergic asthma, showed the effects of
   anti-inflammatory and antiasthmatic. TMDCD could also reduce the levels
   of IgE, histamine, LTC4, Tryptase ε, and other MC related proteins in
   the serum of allergic asthma mice, and the in vitro experiments showed
   that TMDCD could inhibit IgE mediated degranulation and histamine
   release of RBL-2H3 cells, proved its anti allergic effect.

   Keywords: TMDCD, allergic asthma, Chinese herbal compound, mast cells,
   differential expression proteins

Introduction

   Allergic asthma is a chronic inflammatory disease, which is known as
   the result of hypersensitivity to inhaled antigens and is driven by
   aberrant innate and adaptive immune responses, the cardinal features
   include mucus hypersecretion, pulmonary eosinophilic infiltration, and
   AHR ([42]Thio et al., 2018). According to the ([43]The Global Asthma
   Report 2018, 2018), over 339 million people worldwide suffer from
   asthma (E, 2018). Currently, asthma occurs in any countries, and over
   80% of asthma cases occur in low- and lower-middle income countries.
   The increase of asthma patients is associated with increasing air
   pollution by rapid urbanization and industrialization, and adds a
   serious burden on the health system and patients’ quality of life
   ([44]Dhar et al., 2020). At present, corticosteroids were considered as
   the most effective treatment in asthma, for they may effectively
   control symptoms and prevent asthma exacerbation ([45]Ora et al.,
   2020). The 2019 Global Initiative for Asthma (GINA) guidelines
   suggested adult asthma patients be treated with corticosteroids
   inhaling daily, or as needed, and reduced asthma triggers as much as
   possible ([46]Mauer and Taliercio, 2020). However, the clinical safety
   of long-term intaking corticosteroids is still controversial. Some
   researchers have found that long-term or repeated short-term use of
   corticosteroids may lead to certain side effects on systemic health
   ([47]Price et al., 2018; [48]Matsunaga et al., 2020). Therefore, it’s
   an urgent problem for researchers to find some safe and effective drugs
   for asthma, alternatively.

   MC undertakes important roles in the development of allergic asthma.
   Clinical study has shown that the level of lung function in young
   adults with allergic asthma is related to blood circulating mast cell
   progenitors (MCPs) ([49]Salomonsson et al., 2019). MCPs migrate from
   the blood into the airway, exposed to some key factors, which could
   induce them to “mature,” then finally transferred into a pivotal
   immunomodulatory cell ([50]Penn, 2020). Different biological inducers
   (e.g. SCF, CCL5, CXCL8, and CXCL10, etc.) are responsible for
   recruiting MC to airway epithelium and airway smooth muscle (ASM)
   ([51]Elieh Ali Komi et al., 2019). The cross-linking of MC binding IgE
   by allergens leads to the release of biologically active mediators
   (e.g. histamine, PGD2, LTC4) through degranulation, which promotes ASM
   contraction, mucosal edema and mucus secretion in allergic asthma
   directly ([52]Penn, 2020). Moreover, the activated MC secrete a variety
   of cytokines, leading to anaphylactic reaction through recruitment and
   activation of eosinophils, neutrocyte, and Th2 cells, together with the
   interaction between MC and histocytes in the lesion site ([53]Mukai et
   al., 2018). Thus, MC is considered as a key driver of long-term
   pathophysiological changes and tissue remodeling, which are related to
   chronic allergic inflammation in asthma ([54]Galli and Tsai, 2012).

   Antihistamines and steroids are effective drugs in the treatment of
   allergic diseases, but serious side effects hinder their long-term use
   ([55]Lim et al., 2020). TMDCD is a Chinese herbal compound optimized
   based on the compound of MXGSD and GuoMinKang, and also is a summary of
   clinical experience on allergic asthma by professor Wang Qi from
   Beijing university of Chinese medicine. The MXGSD, recorded in Treatise
   on Febrile Diseases by Zhang Zhongjing in Han Dynasty, was used to
   dispersing lung and relieving asthma. Some researchers have found that
   MXGSD could alleviate inflammatory reaction and reduce airway
   remodeling in lung tissue of asthma animal model ([56]Yu et al., 2017;
   [57]Song et al., 2018). MXGSD was also shown the benefit in improving
   lung function and the level of fractional exhaled nitric oxide (FeNO)
   ([58]Ma and Gong, 2019; [59]Cai et al., 2020). Our previous studies
   have shown that Guo-Min-Kang could decrease the serum specific IgE,
   IL-4, IL-5 and IL-13 levels in patients with allergic diseases, and
   improve the tolerance of the human body to antigen ([60]Bao, 2017).
   TMDCD can ameliorate asthma symptoms and reduce the frequency of asthma
   attacks clinically, but the mechanism is not clear. Based on the above
   situation, this study focuses on the immunomodulatory effect of TMDCD
   on allergic asthma and mast cells (RBL-2H3) in mice with allergic
   asthma, to elucidate the mechanism of TMDCD in allergic asthma.

Materials and Methods

Reagents and Instruments

   In this study, we used the following reagents: Acetonitrile and Alum
   were purchased from Thermo Fisher (USA); while Formic acid (HPLC
   grade), albumin (OVA), Dexamethasone (for in vitro experiments),
   Methacholine (MeCh), 0.25% Trypsin-EDTA, 4-Nitrophenyl
   N-acetyl-β-D-glucosaminide, and mouse monoclonal anti-Dinitrophenyl
   antibody (DNP-IgE) from Sigma (USA); Glycogen periodic acid schiff
   (PAS) stain Kit, Penicillin-Streptomycin liquid, Triton X-100,
   Toluidine Blue O solution from Solarbio (China). Dexamethasone (for in
   vivo experiments) from Lisheng-Pharma (China); Mouse IgE ELISA Kit and
   Mouse OVA sIgE ELISA Kit from CUSABIO (China); Histamine ELISA Kit and
   Mouse LTC4 ELISA Kit from CLOUD-CLONE (China); the Mouse cytokine
   antibody array (QAM-CAA-4000) from RayBiotech (China). The FBS, MEM
   medium, and GlutaMAX were purchased from Gibco (USA), DMSO (MPBIO,
   China), PIPES Buffer solution (pH 7.2) and 0.1 M carbonate buffer from
   Coolaber (China), DNP-conjugated bovine serum albumin (DNP-BSA,
   Biosearch, China), CCK-8 Kit (Dojindo, Japan) were prepared,
   respectively.

   The following instruments were employed: Ultra performance liquid
   chromatography (UPLC, Shimadzu, Japan), Votex and CO[2] incubator
   (Thermo, USA), Mass spectrometer (AB SCIEX, USA), Electronic balance
   (Shimadzu, Japan), Atomizer (OMRON, Japan), Unconstrained whole-body
   plethysmography system (WBPs, DSI, USA), Resistance and Compliance
   Plethysmographs (RCs, DSI, USA), high-speed benchtop refrigerated
   centrifuge (Microfuge22R, Beckman Coulter, Germany), Blood cell
   analyzer (NIHON KOHDEN, Japan), Microtome (Leica, Germany),
   Enzyme-labeled instrument (BioTeK, USA), Fluorescence scanner
   (InnoScan, France).

TMDCD Preparation

   TMDCD is composed of 12 kinds of traditional Chinese medicine
   ([61]Table 1), the ingredients were purchased from Beijing Tong Ren
   Tang Technology Development Co., Ltd. (Beijing, China), and were
   certified as a qualified product. Prunus mume (Siebold) Siebold and
   Zucc (batch No.190728004), Cicadae Periostracum (batch No. 900002134),
   Reynoutria multiflora (Thunb.) Moldenke (batch No. 90706003), Ganoderma
   lucidum (Leyss. ex Fr.) Karst. (batch No.001001212A), Saposhnikovia
   divaricata (Turcz. ex Ledeb.) Schischk (batch No.90917001), Gastrodia
   elata Blume (Orchidaceae) (batch No.20041302), Ephedra sinica Stapf
   (batch No.18090402), Prunus armeniaca L. (batch No.900021975), Gypsum
   Fibrosum (batch No.190724001), Glycyrrhiza uralensis Fisch. ex DC.
   (batch No.200352), Bombyx mori L. (batch No.180642), Fagopyrum cymosum
   (Trevir.) Meisn. (batch No.901001876). The preparation method of TMDCD
   was as follow: 1) A total of 780 g of the 12 medicinal materials were
   weighed, respectively, according to the proportion of TMDCD in
   [62]Table 1, then they were soaked together in 7800 ml (10 times the
   weight of medicinal materials) deionized water for 30 min, followed a
   100 min of decoction under 100°C, and the extracting solution was
   collected. 2) Added 7800 ml deionized water again, the same decocting
   conditions, and collected the solution. 3) Repeat the second step once.
   Finally, all the collected solution was concentrated to 770 ml (the
   relative density of 1.01 g/ml, equals to 20.2 g/kg/d). More details can
   be found in references ([63]Wu et al., 2019). The voucher for raw