Asthma is a chronic respiratory disorder of the conducting airways where the epithelial barrier working together with the adaptive and innate immune cells respond to a diverse range of exogenous inhaled stimuli such as allergens and air pollutants (Holgate, 2011). Asthma has long been considered as “type-2-high” asthma with hallmark features of airway hyperresponsiveness (AHR), infiltration of inflammatory cells and airway wall remodeling, which contribute to repeated periods of wheezing, chest tightness, and shortness of breath in susceptible people (Lambrecht and Hammad, 2015; Lambrecht et al., 2019). In asthmatic patients, the direct and indirect medical costs bring a heavy burden to their families and society. Inhaled corticosteroids (ICS) and long-acting β2-agonist (LABA) are known as the most common pharmacological options for management of asthma. Although these medications can attenuate airway inflammation and relieve respiratory symptoms, some asthmatic patients respond poorly to corticosteroid-based therapies, and even experience the severe adverse effects (Williams et al., 2004; Aalbers et al., 2016). Consequently, alternative therapeutic options designed to alleviate airway inflammation and remodeling (such as herbal medicines which has long been used for treatment of bronchial asthma) are urgently required and expected to have better efficacy and safety in asthma therapy.

Oxidative stress is involved in the pathogenesis of asthma (Adcock et al., 2008; Dozor, 2010). Allergens exposure are known to increase the production of oxidants like reactive oxygen species (ROS) and reactive nitrogen species (RNS),which contributes to the imbalance between the oxidants and antioxidants, termed as oxidative stress (Kirkham and Rahman, 2006; Mishra et al., 2018). The existing antioxidant system, composed of enzymatic and non-enzymatic molecules, has capability to remove ROS. The antioxidant enzymes include superoxide dismutases (SODs), catalase, glutathione peroxidase (GPx) and glutathione-S-transferase (GST) etc. In addition, heme oxygenase 1 (HO-1) has been considered as a crucial antioxidant protein, and regulated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) which translocates into the nucleus and initiatives the expression of antioxidant molecules (Uchida et al., 2017; Liu et al., 2019). Furthermore, oxidative stress plays a crucial role in orchestrating airway inflammation and airway remodeling. Findings from previous study have shown that ROS released by mitochondria promoted TGF-β-mediated collagen production (Jaffer et al., 2015). Elevated oxidative stress has been considered as a driving force behind the inflammation and AHR (McGovern et al., 2016). Hence, aside from alleviating airway inflammation and obstruction, suppression of oxidative stress should be considered in asthma therapy.

A growing number of evidences implicate that nuclear factor kappa β (NF-κB) signaling is involved in airway inflammation. In response to allergen challenge, cytokines, chemokines, and bacterial and viral infections, NF-κB acting as a proinflammatory transcription factor binds to the proinflammatory genes promoter region, and thereby upregulates the expression of many mediators and growth factors important in inflammatory cascade of asthma (Adcock et al., 2008; Papi et al., 2013). In addition, enhanced activity of c-Jun N-terminal kinase (JNK) has also been reported in allergen-induced inflammation and remodeling (van der Velden et al., 2014; Wu et al., 2015; Wu et al., 2016).

Formononetin (FMT, 7-Hydroxy-4′-methoxyisoflavone) is a bioactive isoflavone isolated from Radix Astragali (the root of Astragalus membranaceus var. mongholicus or A. membranaceus, described as Huangqi in Chinese herb), which has been common applied for treatment of airway inflammation and allergic disease (Shen et al., 2008; Jin et al., 2013; Luo et al., 2014; Luo et al., 2018), whose chemical structure is depicted in Figure 1 . Our previous studies determined FMT is one of main bioactive ingredients of Bu-Shen-Yi-Qi formula (BSYQF), a Chinese formula widely used to treat respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD) (Luo et al., 2014; Nurahmat et al., 2014). FMT has a bioavailability of 21.8% and shows different absorption in all gastrointestinal segments (Luo et al., 2018). An increasing body of evidence suggests that FMT exerts notable anti-inflammatory, anti-allergic, antioxidant, anti-hepatic steatosis, antiproliferative, and anticancer effects in vitro and animal models of many disease (Wang et al., 2012; Xu and An, 2017; Fei et al., 2018; Li et al., 2018; Ong et al., 2019; Wang X. S. et al., 2019). Some reports demonstrate that FMT has neuroprotective effect in LPS-stimulated BV2 microglia and high-fat diet-induced cognitive disorder mice (El-Bakoush and Olajide, 2018; Fu et al., 2019; Wang Y. et al., 2019). Recently, FMT has already been reported to exert a significant effect in protecting epithelial integrity via G protein-coupled estrogen receptor in an animal model of asthma (Yuan et al., 2020). However, its therapeutic effects to allergic asthma need to be further identified, and their possible mechanisms also need to be further explored. Therefore, our study aims to investigate whether FMT has a therapeutic effect on chronic allergic asthma. To further explore the unrevealed mechanisms, we assess the ability to attenuate lung inflammation and restore the oxidant-antioxidant balance in murine model of allergic asthma.

BSYQF has frequent application in clinical treatment of respiratory diseases such as asthma. Our previous research suggested that BSYQF exerted anti-inflammatory, anti-oxidant and anti-remodeling effects in asthma, and we identified sixteen main bioactive components including FMT (Luo et al., 2014; Nurahmat et al., 2014; Cui et al., 2019). As previously demonstrated, FMT has therapeutic properties in hyperlipidemia and obesity (Fu et al., 2019; Wang Y. et al., 2019), cancers (Hu et al., 2019; Tay et al., 2019) and inflammatory diseases (Li et al., 2018; Aladaileh et al., 2019; Luo et al., 2019; Wang X. S. et al., 2019). Recent reports have demonstrated that FMT alleviated atopic contact dermatitis and allergic asthma by protecting epithelial integrity (Li et al., 2018; Yuan et al., 2020). These inspiring published findings impeled us to explore how FMT could benefit asthma. In this study, we demonstrated that FMT inhibited the pathological progress of allergic asthma in a murine model with typical features of AHR, leukocytes infiltration, multiple cytokines overexpression, goblet cell metaplasia, and collagen deposition. Moreover, our findings indicated that FMT exerted a promising pharmacological effect in alleviating airway inflammation and lung oxidative injury in OVA-induced asthmatic mice, possibly through inhibiting NF-κB and JNK signaling and augmenting Nrf2 signaling.

AHR and lung inflammation are considered as the most prominent features of asthma, constantly evidenced by increased R_L, decreased lung Cydn and leukocytes infiltration in vivo experiments, which has led to the use of bronchodilators and corticosteroids as the mainstream of asthma treatments (Adcock et al., 2008; Lambrecht and Hammad, 2015). A previous study indicated that FMT 10 mg/kg administrated by intraperitoneal injection could significantly alleviate AHR and decrease Th2 cytokines (IL-4, IL-5, and IL-13) levels in the HDM-induced inflammation of allergic asthma (Yuan et al., 2020). Here, we developed an OVA-induced asthma model following by FMT or Dex administration and performed lung function, lung histopathology, and BALF analysis. Our results also confirmed that FMT could suppress AHR, alleviated leukocytes infiltration, mucus hypersecretion, and collagen deposition. However, FMT 10 mg/kg and FMT 20 mg/kg seemingly have modest effects or even have no effect in OVA-induced inflammation and AHR of allergic asthma. The optimal effective dose of FMT appeared to be 40 mg/kg, similar to the effects of Dex 2 mg/kg. That is because FMT was administrated intragastrically not intraperitoneally to mice, potentially consistent with the published data that the pharmacokinetics and bioavailability of formononetin between oral and intravenous administration are different (Luo et al., 2018). Interestingly, BALF cell analysis demonstrated that FMT seemingly exerted a modest decrease in eosinophils counts without statistical significance, potentially a limitation of cell counting in BALF. To identify whether FMT could alleviate eosinophils-rich inflammation in lung, we conducted flow cytometric analysis of lung tissues. We found that FMT reduced lung eosinophils infiltration by nearly half at a dose of 40 mg/kg. The airway recruitment of inflammatory cells is associated with the chemokines. CCL11 (Eotaxin) and CCL5 (RANTES) have been shown to be served as potent chemoattractant for inflammatory cells, especially eosinophils, and is highly expressed in the allergic airway inflammation (Berkman et al., 1996; Chihara et al., 1997; Geslewitz et al., 2018). It has also been reported that the two chemokines have effects on lung fibroblast migration correlated with airway remodeling (Puxeddu et al., 2006; Zhou et al., 2012; Isgro et al., 2013). Indeed, we confirmed the elevated expression of CCL5 and CCL11 in BALF samples from OVA-induced asthma model. FMT was able to remarkably inhibit CCL5 and CCL11 overproduction. These data indicated that FMT exhibits extraordinary anti-inflammatory effects in eosinophils-rich inflammation.

The renowned type-2-high immunity occurs in almost half of asthma patients, manifested as features of eosinophilia, AHR, the elevation of IL-4, IL-5, IL-13, and Ig E in lung and blood (Fahy, 2015; Lambrecht et al., 2019). The biomarkers of type-2-high asthma are believed to develop the pathological progress of asthma. IL-4 promotes the synthesis of Ig E from B cells, and IL-13 induces goblet cell metaplasia associated with mucus hypersecretion, and IL-5 accelerates influx of eosinophils into airways (Grunig et al., 1998; Xia et al., 2018; Busse et al., 2019; Melo et al., 2019). In addition, IL-17 can drive goblet cell metaplasia and mucus hypersecretion in primary human airway epithelia (Pezzulo et al., 2019).Our results suggested that FMT treatments suppressed OVA-induced inflammatory biomarkers expression of IL-4, IL-5, IL-13, IL-17A, and Ig E, and thereby contributed to the reduction of influx of leukocytes and goblet cell metaplasia evidenced by lung histopathology. Besides, Th17 cells have been reported to participate in the pathological process of asthma, especially airway inflammation and remodeling, and abolished Treg cells activity (Zhao et al., 2013). Some studies reported that Th2 and Th17 inflammation are mutually regulated in asthma, and suppression of Th2 cytokines promotes Th17 response, indicating that combination therapies inhibiting both inflammation patterns may exert optimal efficacy in asthma managements (Choy et al., 2015; Liu et al., 2017). Therefore, based upon flow cytometric detection of Th17 cell population from mice lung tissues and Elisa analysis of Th17-inducing cytokines, we found that FMT markedly inhibited OVA-induced Th17 responses. However, our observations showed that Th17 population accounted for a small proportion of CD4+T cells, potentially consistent with the published data about the frequency of cells expressing T-helper-cell-associated cytokines (Tibbitt et al., 2019). Treg cells play a crucial role in maintaining tolerance to allergens, and thereby contributed to controlling inflammation in asthma (Zheng et al., 2019), but sometimes Treg cells could convert to a pathogenic phenotype (Th17-like cells) that exacerbates airway inflammation (Massoud et al., 2016; Noval and Chatila, 2016). Our results suggested OVA exposure induced immune responses in mice, but this involved Th17 cells and not Treg cells, and FMT treatment in mice did not promote Treg cells activation as evidenced by flow cytometric analysis of lung cells. Thus, we inferred that FMT might regulate Th2 and Th17 dysfunctions to reduce the expression of inflammatory mediators, and thereby attenuate airway inflammation in asthma.

In the pathogenesis of asthma, oxidative stress is associated with inflammation and remodeling (Mahut et al., 2004; Kirkham and Rahman, 2006; Dozor, 2010). Growing evidence suggest that both airway inflammation and remodeling trigger increased ROS and NO production in asthma (Thomassen et al., 1999; Mahut et al., 2004; Silveira et al., 2019), and conversely, inhibition of excessive ROS and RNS level alleviates airway inflammation, goblet cells hyperplasia, collagen production, and AHR induced by OVA (Jaffer et al., 2015; Sebag et al., 2017). Interestingly, NO appears to play a dual role in asthma. Some studies indicate that NO exerted beneficial effects on allergen-induced AHR (Schuiling et al., 1998), inflammatory response (Thomassen et al., 1999), and downregulates NF-κB activity (Raychaudhuri et al., 1999). FMT has been reported to prevent methotrexate-induced acute kidney injury through suppressing oxidative injury as evidenced by decreased ROS and NO production (Aladaileh et al., 2019). Here, we detected the oxidative biomarkers in lung tissue. Consistent with the published studies, we found OVA exposure significantly increased ROS and NO level in asthmatic mice lung, however, FMT attenuated oxidative injury through diminishing in situ level of ROS rather than NO. In the lung oxidative biology, the endogenous antioxidant system can eradicate excessive oxidants to avoid oxidative stress. The three SOD proteins acts as the important antioxidant to neutralize and remove ROS, and that includes Cu/Zn superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2) and extracellular superoxide dismutase (SOD3) (Kirkham and Rahman, 2006). Meanwhile, the activity and production of total SOD are attenuated when oxidative stress occurs in lung inflammation (Shen et al., 2017; Ye et al., 2017). SOD1 was reported to effectively account for all SOD activity in BALF, as part of lung antioxidant defense (DiSilvestro et al., 1998). Previous published data suggested mice received FMT 10, 20, or 40 mg/kg exhibited a noticeable enhancement of SOD activity (Aladaileh et al., 2019). In accordance, the activity of SOD was decreased in the lung tissue from OVA-challenged mice. FMT treatment significantly boosted it and produced a stronger increase than DEX. Furthermore, we detected SOD1 expression in lung tissues through western blotting, and the data also confirmed this. Thus, our findings concluded that FMT treatment remarkably restoring antioxidant defenses in lung tissue where all doses protected lung from oxidative injury.

To gain more insight into the potential mechanisms of the anti-inflammatory and antioxidant effects of FMT on asthma, we detected inflammation and oxidative stress signaling. NF-κB is known to acts as one of major contributors to inflammatory pathways. The phosphorylation of NF-κB p65 and IκB kinase is highly activated in airway inflammation (Papi et al., 2013; Ye et al., 2017). Consistently, our study found that OVA exposure promoted NF-κB activation in mice from asthma group compared with control group. Moreover, NF-κB activation has been associated with mechanisms of ROS-mediated oxidative stress (Morgan and Liu, 2011; Mishra et al., 2018). Here, OVA exposure induced NF-κB activation and ROS overproduction in lung tissue of asthmatic mice, and that may be explained by the crosstalk between ROS and NF-κB. NF-κB promoted IL-1β–induced nitric oxide synthase expression so as to enhance free radical NO formation, and FMT could inhibit the activation of NF-κB signaling (Wang et al., 2012).Here, Interestingly, a previous study pointed out the role of NO in blocking NF-κB activation including IL-1β, TNF-α production (Raychaudhuri et al., 1999). In this study, upregulated NF-κB signaling was suppressed by FMT, whose efficacy was similar to Dex. In addition, JNK signaling is closely related to airway inflammation. TNF-α or TGF-β1 treatment induced increased phosphorylation of JNK in bronchial epithelial cells (Ma et al., 2016). Inhibition of JNK significantly reduced OVA-induced inflammatory cell infiltration, mucus hypersecretion and cytokine production (Wu et al., 2015; Wu et al., 2016). House dust mite exposure caused less lung collagen deposition in JNK1-/- mice than wild mice (van der Velden et al., 2014). Rhinovirus infection induced glucocorticoid resistance can be reversed by inhibitors of JNK and IκB kinase in asthma (Papi et al., 2013). Here, our data showed that OVA induction of JNK activation was completely suppressed by FMT. Nrf2/HO-1 signaling was known to act as an important part of antioxidant system. Nrf2 activation facilitated the expression of HO-1, the fundamental antioxidant enzyme, to regulate oxidative stress in asthma (Liu et al., 2019). The previous study indicated that HO-1 displayed anti-inflammatory effects in OVA-induced neutrophilic airway inflammation, as demonstrated by inhibition of Th17 cell responses (Zhang et al., 2013). OVA exposure downregulated the Nrf2 signaling, evidenced by the diminished Nrf2 and HO-1 expression (Ye et al., 2017). Nrf2 activator could enhance the lung levels of ROS scavengers and decrease the proinflammatory cytokines release when allergen exposure occurred (Uchida et al., 2017). FMT was reported to enhance Nrf2 signaling and consequent expression of HO-1 and SOD (Aladaileh et al., 2019). Consistent with these studies, our results suggested that FMT remarkably elevated the expression of HO-1, but unfortunately, the increment of Nrf2 was not significant. Absolutely, multiple mechanisms have been involved in the anti-inflammatory and antioxidant effects of FMT. For example, the induction of ROS-mediated oxidative stress and inflammation correlate with mitochondrial dysfunction. Mitochondrial-targeted antioxidant therapy abrogates AHR, inflammation, TGF-β-mediated collagen deposition, decreases ROS production, and downregulates NF-κB activity in allergic asthma (Jaffer et al., 2015; Sebag et al., 2017). However, we do not determine whether mitochondrial structural and functional alteration occurs in asthma and whether FMT could reverse mitochondrial dysfunction to alleviate airway inflammation and oxidative stress. That undoubtedly deserves further exploration in future studies.

Our study demonstrated the potential therapeutic effect of FMT in the asthmatic murine model, which might be mediated through its anti-inflammatory and antioxidant activities. In summary, our data suggested that FMT exerted anti-inflammatory effects as evidenced by (a) alleviating AHR, inflammatory cell infiltration, goblet cell hyperplasia, and collagen deposition in airways; (b) diminishing elevated expression of IL-4, IL-5, IL-13, IL-17A, Ig E, CCL5, CCL11; (c) reducing Th17 cells and eosinophil recruitment in lung tissue; (d) inhibiting the inflammatory signaling mediated through NF-κB and JNK. Our finding also indicated that FMT restored OVA-induced imbalance of the oxidation and antioxidation as demonstrated by (e) decreasing in situ ROS production, increasing SOD activity; (f) upregulating the antioxidative signaling pathway of Nrf2/HO-1. Thus, these results provided new information about the protective effects of FMT against asthma.

The original contributions presented in the study are included in the article further inquiries can be directed to the corresponding authors.

The animal study was reviewed and approved by the Animal Care and Use Committee of the Fudan University.

LY was responsible for the original ideas and designed the experiments. JC, WW, WT, FT, XZ, JQ, TW, and JS assisted LY to conduct experiments. LY analyzed the data and wrote the draft manuscript. YW and JD were responsible to supervise manuscript writing and revised it. All authors contributed to the article and approved the submitted version.

This work was supported by grants from the National Natural Science Foundation of China (grants 8177140514, 81774074, 81704154, and 81703829), Shanghai science and technology commission (grants 17401930300 and 18401971300), and Young Elite Scientists Sponsorship Program by China Association for Science and Technology (grant 2018QNRC001).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.