Introduction: Demethylzeylasteral (T-96), a new extract of Tripterygium wilfordii Hook F, exerted immunomodulatory properties in autoimmune diseases, but its effect on airway inflammatory diseases remains unclear. Our study aims to explore the protective effect and underlying mechanism of T-96 in allergic asthma. Methods: The OVA-induced asthmatic mice were administered by gavage with T-96 (0.1 mg/10 g, 0.3 mg/10 g, or 0.6 mg/10 g) 1 h before each challenge. The airway hyperresponsiveness was assessed, pathological changes were evaluated by HE and PAS staining, and expressions of Th2 cytokines were determined by PCR and ELISA. The activation of MAPK/ERK and NF-κB pathway was assessed by western blot. Results: T-96 significantly relieved airway hyperresponsiveness in asthmatic mice, evidenced by reduced airway resistance (Raw) and increased lung compliance dynamic compliance (Cdyn). Also, enhanced inflammatory infiltration and mucus hypersecretion were ameliorated in lungs of asthmatic mice following increasing doses of T-96 treatment, accompanied by decreased eosinophils in bronchoalveolar lavage fluid (BALF), IgE and OVA-specific IgE levels in serum, and downregulated IL-5 and IL-13 expressions in BALF and lung tissues as well. Notably, phosphorylation levels of p38 MAPK, ERK, and p65 NF-κB were obviously increased in asthmatic mice compared with the control group, which were then abrogated upon T-96 treatment. Conclusion: This study first revealed that T-96 alleviated allergic airway inflammation and airway hyperresponsiveness via inhibiting MAPK/ERK and NF-κB pathway. Thus, T-96 could potentially act as a new anti-inflammatory agent in allergic asthma.

Bronchial asthma is a common chronic airway disease characterized by airway inflammation, airway hyperresponsiveness (AHR), and airway remodeling, leading to clinical presentations such as wheeze, chest tightness, shortness of breath, and cough [1]. It is estimated that more than 300 million people suffer from asthma throughout the world, including 45.7 million adult asthmatics (aged 20 years or older) in China [2, 3]. As its morbidity increases year by year, asthma is becoming a serious threat to public health worldwide. Inhaled corticosteroids and long-acting β2-adrenergic receptor agonists (ICS/LABA) are the cornerstone of asthma therapy. However, uncontrolled asthma remains a challenge in a substantial portion of patients owing to disease heterogeneity. Thus, new therapeutic strategies for better control of asthma still need to be developed.

Asthma often occurs in atopic subjects, and the best-known endotype is type 2-high asthma induced by allergens [4]. Despite its intricate immune network, the imbalance of T helper 1/T helper 2 (Th1/Th2) response is primarily responsible for type 2 inflammation [5]. Several Th2 cytokines, including interleukin 4 (IL-4), interleukin 5 (IL-5), and interleukin 13 (IL-13), are increased in bronchoalveolar lavage fluid (BALF) and lung tissues of allergic asthmatics, accompanied with elevated IgE, infiltrated eosinophils, degranulated mast cells, and recruited lymphocytes [6]. In such a context, mitogen-activated protein kinases (MAPKs) play a pivotal role in the activation of immune/inflammatory cascades [7]. MAPKs are a family of highly conserved serine/threonine kinases that include p38 MAPK, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase. The p38 MAPK and c-Jun N-terminal kinase are crucially involved in response to inflammatory and environmental insults, while ERK functions in the control of cell proliferation [8‒10]. In previous studies, the three subfamilies are involved in asthma pathobiology (including Th2 cell differentiation, inflammatory cell infiltration, and myofibroblast proliferation), contributing to airway inflammation, airway remodeling, and corticosteroid resistance [11‒14]. Based on this, the inhibition of MAPKs could be investigated as a suitable and promising target for asthma therapy.

Tripterygium wilfordii Hook F (TWHF), a traditional herb commonly known as “lei gong teng” in China, has been applied in the treatment of autoimmune and inflammatory disorders [15‒18]. Owing to the adverse effects caused by TWHF, several bioactive components such as wilforine, triptolide, triptonide, and celastrol are extracted for eliminating the main toxicity [19]. Demethylzeylasteral (T-96) is a new triterpenoid monomer isolated from the root of TWHF [20, 21]. In comparison with other monomers, T-96 has anti-tumor and immunomodulatory activities but much lower toxicity. It has been reported to exert protective effects in lupus nephritis, rheumatoid arthritis, atherosclerosis, and kidney transplantation [22‒26]. Notably, the anti-asthmatic potentials of triptolide and celastrol have been verified to abrogate airway inflammation via modulating Th1/Th2 balance, suppress eosinophil recruitment via targeting MAPK/NF-κB pathway, and inhibit airway remodeling via targeting transforming growth factor β1 (TGF-β1)-induced ASMC proliferation [27‒30]. However, the anti-asthmatic effect of T-96 has not been reported. Herein, the study aims to explore the protective effect of T-96 on airway inflammation and AHR in allergic asthma.

Reagents

Demethylzeylasteral (T-96) (molecular formula: C29H36O6, MW: 480) was isolated and provided by Professor Chunxin Yang (Department of Pharmacy, Zhongshan Hospital, Fudan University, Shanghai). The ovalbumin (OVA), aluminum hydroxide adjuvant, and methacholine were obtained from Sigma (Saint Louis, MO, USA). The reverse transcription kits and SYBR Green for PCR were purchased from Takara (Shiga, Japan). The TRIzol reagents were purchased from Sigma (Saint Louis, MO, USA). The mRNA primers were synthesized by Synbio Technologies (Suzhou, China). The primary antibodies against phospho-p38 MAPK, p38 MAPK, phospho-ERK, ERK, phospho-NF-κB p65, NF-κB p65, and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA). And other reagents for western blot were obtained from Beyotime Co. Ltd (Shanghai, China).

Animals

Female BALB/c mice (6∼8 weeks, 20∼22 g) were purchased from Shanghai SLAC Laboratory Animal Center (Shanghai, China) and maintained in the specific pathogen-free conditions in the animal center of Fudan University. All the protocols for animal experiments described in this study were approved by the Animal Ethics Committee of Zhongshan Hospital of Fudan University.

Protocols for Animal Experiments

Mice were randomly divided into five groups: (1) control group; (2) asthma group; (3) the low dose of T-96 group (OVA + T-96 0.1 mg/10 g); (4) the medium dose of T-96 group (OVA + T-96 0.3 mg/10 g); (5) the high dose of T-96 group (OVA + T-96 0.6 mg/10 g). The OVA-sensitized/challenged asthma model was established as previously described [31]. OVA sensitization was performed on days 1, 8, and 15 via intraperitoneal injection with 200 μL suspension of OVA 20 μg emulsified in aluminum hydroxide adjuvant (1:1 vol) (Fig. 1). Then the mice were placed in a self-made nebulization box (40 cm × 30 cm × 20 cm) and challenged with an aerosol of 1% OVA in phosphate-buffered saline (PBS) by ultrasonic nebulizer for 30 min on days 25–28. The above dose gradient of T-96 was defined based on the previous findings [22]. As powdered T-96 was insoluble in water, it was dissolved in anhydrous ethanol and then suspended in 1.0% sodium carboxymethyl cellulose solution to prepare the working solutions. The low-, medium-, and high-dose groups were administered by gavage with equivalent volumes (200 μL) of T-96 suspension at different working solutions (1, 3, 6 mg/mL, respectively) 1 h before each challenge for four consecutive days. The control and asthma groups were given the equivalent vehicle instead. All mice were anesthetized with avertin (25 mg/kg, Sigma-Aldrich, St Louis, MO, USA) and sacrificed for subsequent analysis 24 h after the last challenge.

Fig. 1.

Experimental schedule for OVA-induced asthmatic murine model.

Fig. 1.

Experimental schedule for OVA-induced asthmatic murine model.

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Airway Reactivity Test

To assess airway responsiveness, the airway resistance (Raw) and lung compliance dynamic compliance (Cdyn) were measured 24 h after the last challenge using the Buxco FinePoint (Buxco Electronics, Troy, NY, USA), as previously described [32]. Each mouse was placed in a plethysmographic chamber and then challenged with increasing concentrations of aerosolized methacholine (0, 6.25, 12.5, 25 mg/mL). The Raw and Cdyn were recorded every 5 min following each challenge until a plateau phase was reached. Results were expressed as the changing value from baseline (value at 0 mg/mL of methacholine) for each increasing concentration of methacholine.

Pathological Evaluation

The lung tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Each 5 μm section was prepared and stained with hematoxylin and eosin (HE) and periodic acid Schiff (PAS). The histopathological score of the above slides for airway inflammation was determined according to Underwood’s criteria [33, 34] (online suppl. Table S1; for all online suppl. material, see https://doi.org/10.1159/000537837) and the mucus secretion of the slides were assessed as previously described [35, 36] (online suppl. Table S2).

Analysis of Bronchoalveolar Lavage Fluid

As previously reported, BALF was collected by gentle lavage with 1 mL PBS into the trachea through a 22-inch intravenous catheter for three aspirations [35, 36]. The BALF was centrifuged at 300 g for 10 min at 4°C, and the supernatants were collected and stored at −80°C for further analyses. The remaining cell pellets were resuspended in 200 μL of PBS and the total numbers of BALF cells were counted using CellDrop®(DeNovix, Wilmington, DE, USA). Flow cytometry was used to classify eosinophils in BALF, which were identified as CD45+Ly6GCD11cSiglecF+. The number of eosinophils was calculated as percentage of eosinophils multiplied by the total numbers of BALF cells.

Enzyme-Linked Immunosorbent Assay

The levels of total IgE in serum, IL-5, and IL-13 in BALF were determined by Enzyme-Linked Immunosorbent Assay (ELISA) according to manufacturer’s protocols (Sizhengbai Co. Ltd, Beijing, China). The ELISA kit for detecting OVA-specific IgE in serum was obtained from BioLegend (San Diego, CA, USA).

Quantitative PCR

The total RNA was extracted from lung tissue using TRIzol reagents, and the cDNA was generated with PrimeScript RT reagents. The relative gene expressions of IL-4, IL-5, and IL-13 in lung tissues were detected by quantitative PCR. GAPDH was referred to as the internal control. The primer sequences of all genes were listed as follows: mouse GAPDH: forward 5′-GGG​TGT​GAA​CCA​CGA​GAA​AT-3′, reverse 5′-CCT​TCC​ACA​ATG​CCA​AAG​TT-3′; mouse IL-4: forward 5′-GCA​ACG​AAG​AAC​ACC​ACA​GA-3′, reverse 5′-TGC​AGC​TCC​ATG​AGA​ACA​CT-3′; mouse IL-5: forward 5′-GTG​GGG​GTA​CTG​TGG​AAA​TG-3′, reverse 5′-TAA​TCC​AGG​AAC​TGC​CTC​GT-3′; mouse IL-13: forward 5′-CAG​CAT​GGT​ATG​GAG​TGT​GG-3′, reverse 5′-AGG​CCA​TGC​AAT​ATC​CTC​TG-3′.

Western Blot

The total proteins of lung tissues were extracted using RIPA lysis buffer containing phenylmethanesulfonyl fluoride (Beyotime, China) and phosphatase inhibitors (Beyotime, China). After being quantified by a BCA Protein Assay Kit (Beyotime), equivalent proteins were loaded on a 10% Bis-Tris gradient gel (Invitrogen) and subsequently transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore). After blocking, the membranes were incubated with primary antibodies against p-p38 MAPK, p38 MAPK, p-ERK, ERK, and β-actin (1:1,000, CST, USA). Following incubation with horseradish peroxidase-conjugated secondary antibodies (1:5,000, Cell Signaling Technology, Danvers, MA, USA), the bands were detected with ECL Chemiluminescent Substrate Reagent Kit (Thermo Fisher Scientific, USA) on the Bio-Rad Laboratories system. The optical densities of the protein bands were analyzed using Image J 1.46 software (National Institutes of Health, Bethesda, MD, USA).

Statistical Analysis

Data were presented as mean ± standard error of the mean (SEM) and analyzed using SPSS 22.0 statistical software. Statistical analysis was performed using parametric Student’s t test between two groups or one-way ANOVA with Bonferroni test among multiple groups. p < 0.05 was considered statistically significant.

T-96 Treatment Attenuated Airway Hyperresponsiveness in OVA-Induced Asthma

To investigate the effect of T-96 on AHR, airway response to methacholine was measured. When exposed to increasing concentrations of aerosolized methacholine, the Raw and Cdyn of all mice changed reversely (i.e., increased Raw accompanied with reduced Cdyn). Compared with the control group, a significant increase in Raw was observed in the asthma group when challenged with methacholine at 25 mg/mL (p < 0.0001), while treatment with the medium and high dose of T-96 inhibited the increased Raw in asthmatic mice (p < 0.0001) (Fig. 2a). Conversely, the Cdyn of asthmatic mice was much lower when challenged with methacholine at the highest concentration (p < 0.01), whereas an obvious increase in Cdyn was observed in both the T-96 medium- and high-dose groups (p < 0.01) (Fig. 2b). These results indicated the protective effect of T-96 against AHR in OVA-induced asthma model.

Fig. 2.

T-96 treatment attenuated AHR in OVA-induced asthma. a Change of the airway resistance (Raw) in response to increasing concentrations of aerosolized methacholine in mice after last challenge. b Change of the lung compliance dynamic compliance (Cdyn) in response to increasing concentrations of aerosolized methacholine in mice after last challenge. Data were presented as mean ± SEM (n = 5–10 per group). **p < 0.01, ****p < 0.0001 versus control; ##p < 0.01, ###p < 0.001, ####p < 0.0001 versus asthma.

Fig. 2.

T-96 treatment attenuated AHR in OVA-induced asthma. a Change of the airway resistance (Raw) in response to increasing concentrations of aerosolized methacholine in mice after last challenge. b Change of the lung compliance dynamic compliance (Cdyn) in response to increasing concentrations of aerosolized methacholine in mice after last challenge. Data were presented as mean ± SEM (n = 5–10 per group). **p < 0.01, ****p < 0.0001 versus control; ##p < 0.01, ###p < 0.001, ####p < 0.0001 versus asthma.

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T-96 Treatment Alleviated Airway Inflammation and Mucus Hypersecretion in OVA-Induced Asthma

HE staining revealed that enhanced infiltration of inflammatory cells and obvious thickening of bronchial walls in the asthma group compared with the control group (p < 0.0001), and the pathological changes were alleviated as the T-96 dose increased (p < 0.001) (Fig. 3a, b). The PAS staining showed excessive mucus secretion in the bronchial lumen of the asthma group, in comparison with the control group (p < 0.0001). Then mucus hypersecretion was relieved following treatment with the increasing doses of T-96 (p < 0.0001) (Fig. 3c, d). These findings suggested that T-96 treatment could alleviate OVA-induced airway inflammation and mucus hypersecretion in the murine asthma model.

Fig. 3.

T-96 treatment alleviated airway inflammation and mucus hypersecretion in OVA-induced asthma. a Representative HE staining of lung sections. Original magnification, ×100 (up), ×200 (down). b Pathological scores of airway inflammation infiltration. c Representative PAS staining of lung sections. Original magnification, ×100 (up), ×200 (down). d Pathological scores of mucus secretion. Data were presented as mean ± SEM (n = 5–10 per group). ****p < 0.0001 versus control; ###p < 0.001, ####p < 0.0001 versus asthma.

Fig. 3.

T-96 treatment alleviated airway inflammation and mucus hypersecretion in OVA-induced asthma. a Representative HE staining of lung sections. Original magnification, ×100 (up), ×200 (down). b Pathological scores of airway inflammation infiltration. c Representative PAS staining of lung sections. Original magnification, ×100 (up), ×200 (down). d Pathological scores of mucus secretion. Data were presented as mean ± SEM (n = 5–10 per group). ****p < 0.0001 versus control; ###p < 0.001, ####p < 0.0001 versus asthma.

Close modal

T-96 Treatment Inhibited Eosinophil Infiltration in OVA-Induced Asthma

The number of total BALF cells and eosinophils were significantly increased in the asthma group (p < 0.01), compared with the control group (Fig. 4a, b). Further, T-96 differently inhibited the aforementioned increased inflammatory cells in asthma mice, with the medium-dose group yielding the best effect among the three pretreatment groups (p < 0.001). The findings indicated T-96 could attenuate allergic airway inflammation by inhibiting eosinophil infiltration.

Fig. 4.

T-96 treatment inhibited eosinophil infiltration in OVA-induced asthma. a Total number of BALF cells. b Number of eosinophils in BALF. Data were presented as mean ± SEM (n = 5–6 per group). **p < 0.01, ***p < 0.001 versus control; ##p < 0.01, ###p < 0.001 versus asthma.

Fig. 4.

T-96 treatment inhibited eosinophil infiltration in OVA-induced asthma. a Total number of BALF cells. b Number of eosinophils in BALF. Data were presented as mean ± SEM (n = 5–6 per group). **p < 0.01, ***p < 0.001 versus control; ##p < 0.01, ###p < 0.001 versus asthma.

Close modal

T-96 Treatment Inhibited IgE, OVA-Specific IgE Level, and Th2 Cytokine Expressions in OVA-Induced Asthma

In comparison with the control group, the asthmatic mice exhibited elevated levels of total IgE and OVA-specific IgE in serum (p < 0.001), which were reduced in the medium or high T-96-treated groups (p < 0.05) (Fig. 5a, b). Meanwhile, the levels of IL-5 and IL-13 in BALF were found increased in the asthma group compared with the control group (p < 0.01), which were further decreased in T-96-treated groups (Fig. 5c, d). Notably, the medium-dose group exhibited the strongest inhibitory effect on IL-13 level (p < 0.05) and a mild effect on IL-5 level.

Fig. 5.

T-96 treatment inhibited IgE and OVA-specific IgE levels, and Th2 cytokines in bronchoalveolar lavage fluid (BALF). a Total IgE levels in serum quantified by ELISA. b OVA-specific IgE levels in serum quantified by ELISA. c, d Levels of IL-5 (c) and IL-13 (d) in BALF quantified by ELISA. Data were presented as mean ± SEM (n = 5–10 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control; #p < 0.05, ###p < 0.001 versus asthma.

Fig. 5.

T-96 treatment inhibited IgE and OVA-specific IgE levels, and Th2 cytokines in bronchoalveolar lavage fluid (BALF). a Total IgE levels in serum quantified by ELISA. b OVA-specific IgE levels in serum quantified by ELISA. c, d Levels of IL-5 (c) and IL-13 (d) in BALF quantified by ELISA. Data were presented as mean ± SEM (n = 5–10 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control; #p < 0.05, ###p < 0.001 versus asthma.

Close modal

As shown in Figure 6, the mRNA expressions of IL-4, IL-5, and IL-13 in lung tissues were upregulated in the asthma group (p < 0.05) and then were downregulated after treatment with T-96 (p < 0.05) (Fig. 6a–c). Likewise, T-96 at the medium or high dose showed a stronger inhibitory effect on the mRNA expressions of Th2 cytokines than the low-dose group. The above results suggested that T-96 treatment could inhibit the airway inflammation by decreasing IgE and OVA-specific IgE levels, and Th2 cytokine expressions.

Fig. 6.

T-96 treatment inhibited Th2 cytokine expressions in lung tissues. a–c Levels of IL-4 (a), IL-5 (b), and IL-13 (c) in lung tissues determined by quantitative PCR. Data were presented as mean ± SEM (n = 5–10 per group). *p < 0.05, ***p < 0.001 versus control; #p < 0.05, ##p < 0.01 versus asthma.

Fig. 6.

T-96 treatment inhibited Th2 cytokine expressions in lung tissues. a–c Levels of IL-4 (a), IL-5 (b), and IL-13 (c) in lung tissues determined by quantitative PCR. Data were presented as mean ± SEM (n = 5–10 per group). *p < 0.05, ***p < 0.001 versus control; #p < 0.05, ##p < 0.01 versus asthma.

Close modal

T-96 Treatment Inhibited MAPK/ERK and NF-κB Pathways in OVA-Induced Asthma

We then investigated how T-96 affected MAPK/ERK and NF-κB pathways in OVA-induced asthma. It can be seen that the expressions of p-p38, p-ERK, and p-p65 were significantly increased in asthmatic mice compared with the control group (p < 0.001) (Fig. 7a, b), suggesting activation of MAPK/ERK and NF-κB pathways in allergic asthma. With the increasing doses of T-96 treatment, phosphorylation of p38 MAPK and ERK were progressively inhibited (p < 0.001), among which the medium- and high-dose groups showed the stronger effects than the low-dose group. However, phosphorylation of p65 NF-κB was significantly reduced only at the high dose of T-96 (p < 0.01). These results showed that T-96 treatment could attenuate OVA-induced asthma by inhibiting MAPK/ERK and NF-κB pathways.

Fig. 7.

T-96 treatment inhibited MAPK/ERK and NF-κB pathways in OVA-induced asthma. a Representative bands of p-p38, p38, p-ERK, ERK, p-p65, p65, and β-actin in lung tissues detected by western blot. b Densitometry quantification of p-p38, p-ERK, and p-p65 relative to the total cellular protein. Data were presented as mean ± SEM (n = 5–10 per group). ***p < 0.001, ****p < 0.0001 versus control; ##p < 0.01, ###p < 0.001, ####p < 0.001 versus asthma.

Fig. 7.

T-96 treatment inhibited MAPK/ERK and NF-κB pathways in OVA-induced asthma. a Representative bands of p-p38, p38, p-ERK, ERK, p-p65, p65, and β-actin in lung tissues detected by western blot. b Densitometry quantification of p-p38, p-ERK, and p-p65 relative to the total cellular protein. Data were presented as mean ± SEM (n = 5–10 per group). ***p < 0.001, ****p < 0.0001 versus control; ##p < 0.01, ###p < 0.001, ####p < 0.001 versus asthma.

Close modal

In this study, we demonstrated that OVA-induced asthma was successfully established in mice, evidenced by AHR, enhanced inflammatory infiltration, and excessive mucus secretion. Also, eosinophil infiltration in BALF, total IgE and OVA-specific IgE levels in serum, and Th2 cytokine expressions in BALF and lung tissues were increased, underlying type 2 immune responses in asthmatic mice. Further, enhanced phosphorylation of p38 MAPK, ERK, and p65 NF-κB was observed in lung tissues of the asthma group, which was consistent with the previous reports. Importantly, the administration of T-96 exerted a pronounced inhibitory effect on the above pathways, subsequently alleviating Th2 responses in allergic asthma. Our findings firstly revealed the therapeutic potential of T-96 on allergic asthma by acting on MAPK/ERK and NF-κB signaling pathways.

Allergic asthma, the most common phenotype of asthma, is characterized by exposure to allergens and subsequent Th2-dominant responses [37]. Because of substantial overlaps with other phenotypes, a diagnosis of allergic asthma should be based not only on elevated levels of total serum IgE but also higher levels of eosinophilia and Th2 cytokines in the peripheral blood and BALF of patients. Expectedly, the asthma murine model in this study exhibited these pathophysiological hallmarks. Previous evidence indicated both triptolide and celastrol have anti-asthmatic effects in vivo and vitro. Thus, we postulated the new bioactive exact T-96 has the similar effects, which was subsequently verified to alleviate OVA-induced airway inflammation and AHR as well.

MAPKs have been implicated in most aspects of asthma pathobiology and p38 is the most extensively studied [7, 8]. Activated p38 MAPK induces the polarization of Th2 cells and production of Th2 cytokines, inhibits eosinophil apoptosis, and promotes eosinophil recruitment [38, 39]. ERK contributes to Th2 cell differentiation and activation, and participates in IL-5-mediated eosinophilia and IL-13-dependent mucus hypersecretion [40‒42]. Although several MAPK inhibitors have been tested in pre-clinical models of asthma, the relevant safety concerns still need to be justified. Herein, our study demonstrated that pharmacological administration of T-96 could significantly inhibit p38 and ERK phosphorylation, thereby attenuating allergic asthma.

NF-κB is known to participate in the production of cytokines and adhesion molecules during inflammatory diseases [43]. Several studies have suggested that the inflammatory responses involving bronchial epithelial cells, eosinophils, and smooth muscle cells could be regulated by NF-κB activity [44, 45]. To be noted, T-96 exhibited protective effects by inhibiting NF-κB pathway and reducing the downstream inflammatory molecules in adjuvant-induced arthritis and lupus nephritis [22, 23]. Here we demonstrated that only the high dose of T-96 significantly inhibited the enhanced phosphorylation of p65 in asthmatic mice. In comparison with its potent effect on other MAPK subfamilies, it was implied that the anti-asthmatic effects of T-96 was partly dependent on p65 NF-κB pathway.

Our study has several limitations. First, T-96, extracted from a Chinese herb, may exert its protective effects on allergic asthma through multiple mechanisms beyond MAPK/ERK and NF-κB signaling pathways. Therefore, it is essential to extend the additional mechanism of T-96 in our future research. Second, given that the protective effects of T-96 appear to be dose-dependent, it is crucial to evaluate the probable adverse effects such as hepatotoxicity and nephrotoxicity in asthmatic mice.

In summary, this study firstly demonstrated that T-96, the new extract of TWHF, could effectively alleviate allergic airway inflammation and AHR by targeting MAPK/ERK and NF-κB signaling pathways. T-96 is expected to be a new option for asthma treatment in the future.

This study protocol was reviewed and approved by Animal Ethics Committee of Zhongshan Hospital affiliated to Fudan University (date of decision: 14 Aug 2017).

The authors have no conflicts of interest to declare.

This study was supported by National Natural Science Foundation of China (82000013, 82100028), Shanghai Sailing Program (20YF1405700), and the National Key Research and Development Program of China (2016YFC1304000, 2016YFC1304002).

J.H. performed the most experiments, analyzed the data, and wrote the main part of manuscript. H.C. performed the supplementary experiments and data analysis, prepared figures, and was responsible for drafting and revising the manuscript. X.Y. and G.Z. helped the animal experiments and formal analysis and data interpretation. L.Y. and C.Y. reviewed the manuscript and provided critical discussions. M.J. and J.W. designed and conducted the study and reviewed the manuscript. All authors have read and agreed to the final version of manuscript. J.H. and H.C. contributed equally and are listed as co-first authors.

Additional Information

Jianan Huang and Hui Cai contributed equally to this work.Edited by: H.-U. Simon, Bern.

All relevant data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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