Anti-Inflammatory Effects of a Cordyceps sinensis Mycelium Culture Extract (Cs-4) on Rodent Models of Allergic Rhinitis and Asthma

Allergic rhinitis and asthma are common chronic allergic diseases of the respiratory tract, which are accompanied by immunoglobulin E (IgE)-mediated inflammation and the involvement of type 2 T helper cells, mast cells, and eosinophils. Cordyceps sinensis (Berk.) Sacc is a fungal parasite on the larva of Lepidoptera. It has been considered to be a health-promoting food and, also, one of the best-known herbal remedies for the treatment of airway diseases, such as asthma and lung inflammation. In the present study, we demonstrated the antiallergic rhinitis effect of Cs-4, a water extract prepared from the mycelium culture of Cordyceps sinensis (Berk) Sacc, on ovalbumin (OVA)-induced allergic rhinitis in mice and the anti-asthmatic effect of Cs-4 in a rat model of asthma. Treatment with Cs-4 suppressed the nasal symptoms induced in OVA-sensitized and challenged mice. The inhibition was associated with a reduction in IgE/OVA-IgE and interleukin (IL)-4/IL-13 levels in the nasal fluid. Cs-4 treatment also decreased airway responsiveness and ameliorated the scratching behavior in capsaicin-challenged rats. It also reduced plasma IgE levels, as well as IgE and eosinophil peroxidase levels, in the bronchoalveolar fluid. Cs-4 treatment completely suppressed the increases in IL-4, IL-5, and IL-13 levels in rat lung tissue. In conclusion, our results suggest that Cs-4 has the potential to alleviate immune hypersensitivity reactions in allergic rhinitis and asthma.


Introduction
Allergic rhinitis (AR) and asthma are common chronic allergic diseases of the respiratory tract. The prevalence of AR and asthma has been estimated to range from 3% to 19% and 4%, respectively, globally [1]. Patients with AR present with one or more of the following symptoms: nasal congestion, rhinorrhea, sneezing, and itching [2]. Asthma is characterized by respiratory airway symptoms, including wheezing, coughing, shortness of breath, and a feeling of tightness in the chest [3]. As the upper respiratory mucosa is continuous with the mucosa of lower airways, up to 40% of asthmatic patients show symptoms of AR [4]. By the same token, patients with AR are likely to develop bronchial asthma three times more frequently than non-rhinitis patients [4].
AR and asthma involve complex pathological processes, which are characterized by immunoglobulin E (IgE)-mediated inflammation and the involvement of type 2 T-helper cells (Th2),

Cs-4 Treatment Significantly Decreased both IL-4 and IL-13 Levels in the NLF of OVA-Sensitized and Challenged Mice
As shown in Figure 3, the OVA challenge caused increases in the IL-4 (47%) and IL-13 (51%) levels in the NFL of OVA-sensitized mice, when compared with the unsensitized and challenged controls. The DEX treatment (1 mg/kg, i.g.) completely abrogated the OVA-induced elevation of IL-4 and IL-13 levels in the NLF of OVA-sensitized mice. The Cs-4 treatment (250 and 750 mg/kg) significantly decreased both IL-4 (31 and 65%) and IL-13 (45 and 70%) levels in the NLF of OVAsensitized and challenged mice, when compared with the untreated, OVA-sensitized, and challenged controls. Figure 4 shows the effects of Cs-4 on C48/80-activated β-hexosaminidase releases in rat peritoneal mast cells (RPMC) in vitro. While preincubation with increasing concentrations (30-1000 µ g/mL) of Cs-4 did not affect β-hexosaminidase release in unactivated RPMC, C48/80 activation

Cs-4 Treatment Significantly Decreased both IL-4 and IL-13 Levels in the NLF of OVA-Sensitized and Challenged Mice
As shown in Figure 3, the OVA challenge caused increases in the IL-4 (47%) and IL-13 (51%) levels in the NFL of OVA-sensitized mice, when compared with the unsensitized and challenged controls. The DEX treatment (1 mg/kg, i.g.) completely abrogated the OVA-induced elevation of IL-4 and IL-13 levels in the NLF of OVA-sensitized mice. The Cs-4 treatment (250 and 750 mg/kg) significantly decreased both IL-4 (31 and 65%) and IL-13 (45 and 70%) levels in the NLF of OVAsensitized and challenged mice, when compared with the untreated, OVA-sensitized, and challenged controls. Figure 4 shows the effects of Cs-4 on C48/80-activated β-hexosaminidase releases in rat peritoneal mast cells (RPMC) in vitro. While preincubation with increasing concentrations (30-1000 µ g/mL) of Cs-4 did not affect β-hexosaminidase release in unactivated RPMC, C48/80 activation

Cs-4 Treatment Significantly Decreased both IL-4 and IL-13 Levels in the NLF of OVA-Sensitized and Challenged Mice
As shown in Figure 3, the OVA challenge caused increases in the IL-4 (47%) and IL-13 (51%) levels in the NFL of OVA-sensitized mice, when compared with the unsensitized and challenged controls. The DEX treatment (1 mg/kg, i.g.) completely abrogated the OVA-induced elevation of IL-4 and IL-13 levels in the NLF of OVA-sensitized mice. The Cs-4 treatment (250 and 750 mg/kg) significantly decreased both IL-4 (31 and 65%) and IL-13 (45 and 70%) levels in the NLF of OVA-sensitized and challenged mice, when compared with the untreated, OVA-sensitized, and challenged controls. significantly increased β-hexosaminidase release by 67%, when compared with the unstimulated controls. Preincubation with Cs-4 significantly suppressed C48/80-activated β-hexosaminidase release at concentrations of 300 and 1000 µ g/mL by 43 and 58%, respectively, when compared with activated and non-Cs-4 preincubated controls.
(a) (b)  The effects of Cs-4 on the C48/80-activated β-hexosaminidase release in rat peritoneal mast cells (RPMC) in vitro. Each bar represents the mean ± SD, with n = 6. * Significantly different from the control group. # Significantly different from the C48/80 non-Cs-4-incubated control group. Figure 5 shows the effects of Cs-4 on the C48/80-activated histamine release in RPMC in vitro. While preincubation with increasing concentrations (30-1000 µ g/mL) of Cs-4 did not affect the histamine release in unactivated RPMC, C48/80 activation significantly increased the histamine release by 103%, when compared with the unstimulated controls. Preincubation with Cs-4 significantly suppressed the C48/80-activated histamine release by 59 and 73%, respectively, at concentrations of 300 and 1000 µ g/mL, when compared with the activated and non-Cs-4 preincubated controls.  Figure 4 shows the effects of Cs-4 on C48/80-activated β-hexosaminidase releases in rat peritoneal mast cells (RPMC) in vitro. While preincubation with increasing concentrations (30-1000 µg/mL) of Cs-4 did not affect β-hexosaminidase release in unactivated RPMC, C48/80 activation significantly increased β-hexosaminidase release by 67%, when compared with the unstimulated controls. Preincubation with Cs-4 significantly suppressed C48/80-activated β-hexosaminidase release at concentrations of 300 and 1000 µg/mL by 43 and 58%, respectively, when compared with activated and non-Cs-4 preincubated controls.
(a) (b)   Figure 5 shows the effects of Cs-4 on the C48/80-activated histamine release in RPMC in vitro. While preincubation with increasing concentrations (30-1000 µ g/mL) of Cs-4 did not affect the histamine release in unactivated RPMC, C48/80 activation significantly increased the histamine release by 103%, when compared with the unstimulated controls. Preincubation with Cs-4 significantly suppressed the C48/80-activated histamine release by 59 and 73%, respectively, at concentrations of 300 and 1000 µ g/mL, when compared with the activated and non-Cs-4 preincubated controls.   when compared with the unstimulated controls. Preincubation with Cs-4 significantly suppressed the C48/80-activated histamine release by 59 and 73%, respectively, at concentrations of 300 and 1000 µg/mL, when compared with the activated and non-Cs-4 preincubated controls.

Preincubation with Cs-4 Suppressed C48/80-Activated Histamine Release
Molecules 2020, 25, x FOR PEER REVIEW 5 of 18 Figure 5. The effects of Cs-4 on the C48/80-activated histamine release in rat RPMC in vitro. Each bar represents the mean ± SD, with n = 6. * Significantly different from the control group. # Significantly different from the C48/80 non-Cs-4-incubated control group. Table 1 shows the effects of Cs-4 on the body weight and splenic index of capsaicin-challenged rats. Whereas the neonatal capsaicin challenge did not affect the body weights of rats 46 days after birth, the splenic index was significantly increased by 20%, when compared with the nonchallenged controls. The DEX treatment (1 mg/kg) beginning after weaning significantly reduced the body weight (61%) and splenic index (34%) of capsaicin-challenged rats, when compared with the untreated and challenged controls. Although the Cs-4 treatments (123 and 375 mg/kg) did not produce any detectable changes in the body weights of capsaicin-challenged rats, the splenic index was significantly reduced by 78% at a dose of 0.375 g/kg, when compared with the challenged and untreated controls.

Cs-4 Treatment Lowered the Cutaneous Lesion Induced by the Capsaicin Challenge on Rats
The capsaicin challenge significantly increased the cutaneous lesion score by 19.7-fold in rats. The DEX treatment (1 mg/kg) slightly but insignificantly decreased cutaneous lesion scores in capsaicin-challenged rats. The Cs-4 treatment at a dose of 375 mg/kg significantly lowered the score by 73% (Table 2).  Table 1 shows the effects of Cs-4 on the body weight and splenic index of capsaicin-challenged rats. Whereas the neonatal capsaicin challenge did not affect the body weights of rats 46 days after birth, the splenic index was significantly increased by 20%, when compared with the nonchallenged controls. The DEX treatment (1 mg/kg) beginning after weaning significantly reduced the body weight (61%) and splenic index (34%) of capsaicin-challenged rats, when compared with the untreated and challenged controls. Although the Cs-4 treatments (123 and 375 mg/kg) did not produce any detectable changes in the body weights of capsaicin-challenged rats, the splenic index was significantly reduced by 78% at a dose of 0.375 g/kg, when compared with the challenged and untreated controls.

Cs-4 Treatment Lowered the Cutaneous Lesion Induced by the Capsaicin Challenge on Rats
The capsaicin challenge significantly increased the cutaneous lesion score by 19.7-fold in rats. The DEX treatment (1 mg/kg) slightly but insignificantly decreased cutaneous lesion scores in capsaicin-challenged rats. The Cs-4 treatment at a dose of 375 mg/kg significantly lowered the score by 73% (Table 2).  Table 3 and Figure 6 show the effect of Cs-4 on airway (trachea and bronchus) responsiveness in capsaicin-challenged rats. The capsaicin challenge increased the responsiveness of both tracheal and bronchial rings to Mch-induced contractions, as evidenced by significant increases in the effective concentration (EC 50 ) by 99 and 96%, respectively, when compared with the unchallenged controls. The DEX treatment (1 mg/kg) did not produce any detectable effects on the airway responsiveness in capsaicin-challenged rats. The Cs-4 treatment (0.123 and 0.375 g/kg) significantly decreased the EC 50 of the tracheal rings (47 and 88%) and bronchial rings (158 and 228%), when compared with the challenged and untreated controls.
Molecules 2020, 25, x FOR PEER REVIEW 6 of 18  Table 3 and Figure 6 show the effect of Cs-4 on airway (trachea and bronchus) responsiveness in capsaicin-challenged rats. The capsaicin challenge increased the responsiveness of both tracheal and bronchial rings to Mch-induced contractions, as evidenced by significant increases in the effective concentration (EC50) by 99 and 96%, respectively, when compared with the unchallenged controls. The DEX treatment (1 mg/kg) did not produce any detectable effects on the airway responsiveness in capsaicin-challenged rats. The Cs-4 treatment (0.123 and 0.375 g/kg) significantly decreased the EC50 of the tracheal rings (47 and 88%) and bronchial rings (158 and 228%), when compared with the challenged and untreated controls.

Cs-4 Treatment Reduced the Number of Scratching Events
As shown in Figure 7, the capsaicin challenge triggered scratching behavior in rats, as evidenced by a significant increase (156%) in scratching events, when compared with the unchallenged controls. The DEX treatment (1 mg/kg) completely suppressed the capsaicin-induced stimulation of scratching behavior. The Cs-4 treatment (123 and 375 mg/kg) significantly reduced the number of scratching events by 31 and 85%, respectively, when compared with the challenged and untreated controls.

Cs-4 Treatment Reduced the Number of Scratching Events
As shown in Figure 7, the capsaicin challenge triggered scratching behavior in rats, as evidenced by a significant increase (156%) in scratching events, when compared with the unchallenged controls. The DEX treatment (1 mg/kg) completely suppressed the capsaicin-induced stimulation of scratching behavior. The Cs-4 treatment (123 and 375 mg/kg) significantly reduced the number of scratching events by 31 and 85%, respectively, when compared with the challenged and untreated controls.

Cs-4 Treatment Decreased the IgE and Eosinophil Peroxidase (EPO) Levels in the Bronchoalveolar (BAL) Fluid of Capsaicin-Challenged Rats
As shown in Figure 9, the capsaicin challenge significantly increased the levels of IgE (91%) and EPO (77%) in the BAL fluid in rats, when compared with the unchallenged controls. The DEX treatment (1 mg/kg) completely abolished the capsaicin-induced increases in the IgE and EPO levels in the BAL fluid. The Cs-4 treatment (123 and 375 mg/kg) significantly decreased the IgE (36-66%) and EPO (37-64%) levels in the BAL fluid of capsaicin-challenged rats, when compared with the challenged and untreated controls.

Cs-4 Treatment Decreased the IgE and Eosinophil Peroxidase (EPO) Levels in the Bronchoalveolar (BAL) Fluid of Capsaicin-Challenged Rats
As shown in Figure 9, the capsaicin challenge significantly increased the levels of IgE (91%) and EPO (77%) in the BAL fluid in rats, when compared with the unchallenged controls. The DEX treatment (1 mg/kg) completely abolished the capsaicin-induced increases in the IgE and EPO levels in the BAL fluid. The Cs-4 treatment (123 and 375 mg/kg) significantly decreased the IgE (36-66%) and EPO (37-64%) levels in the BAL fluid of capsaicin-challenged rats, when compared with the challenged and untreated controls.

Cs-4 Treatment Decreased the IgE and Eosinophil Peroxidase (EPO) Levels in the Bronchoalveolar (BAL) Fluid of Capsaicin-Challenged Rats
As shown in Figure 9, the capsaicin challenge significantly increased the levels of IgE (91%) and EPO (77%) in the BAL fluid in rats, when compared with the unchallenged controls. The DEX treatment (1 mg/kg) completely abolished the capsaicin-induced increases in the IgE and EPO levels in the BAL fluid. The Cs-4 treatment (123 and 375 mg/kg) significantly decreased the IgE (36-66%) and EPO (37-64%) levels in the BAL fluid of capsaicin-challenged rats, when compared with the challenged and untreated controls.

Cs-4 Treatment Suppressed the Capsaicin-Induced Increases in the IL-4, IL-5, and IL-13 Levels in Rat
Lung Tissue Figure 10 shows the effects of Cs-4 on various cytokine levels in the lung tissue of capsaicin-challenged rats. The capsaicin challenge caused increases in IL-4 (51%), IL-5 (55%), and IL-13 (68%) in rat lung tissues when compared with the unchallenged controls. No detectable changes in the tumor necrosis factor (TNF)-α and interferon (IFN)-γ were observed. The DEX treatment (1 mg/kg) completely suppressed capsaicin-induced increases in IL-4, IL-5, and IL-13 levels in rat lung tissues and significantly increased the levels of TNF-α (64%) and IFN-γ (34%), when compared with both the unchallenged control and the challenged/untreated controls. The Cs-4 treatment almost completely suppressed the capsaicin-induced increases in IL-4, IL-5, and IL-13 levels in rat lung tissues at a dose of 375 mg/kg.  Figure 10 shows the effects of Cs-4 on various cytokine levels in the lung tissue of capsaicinchallenged rats. The capsaicin challenge caused increases in IL-4 (51%), IL-5 (55%), and IL-13 (68%) in rat lung tissues when compared with the unchallenged controls. No detectable changes in the tumor necrosis factor (TNF)-α and interferon (IFN)-γ were observed. The DEX treatment (1 mg/kg) completely suppressed capsaicin-induced increases in IL-4, IL-5, and IL-13 levels in rat lung tissues and significantly increased the levels of TNF-α (64%) and IFN-γ (34%), when compared with both the unchallenged control and the challenged/untreated controls. The Cs-4 treatment almost completely suppressed the capsaicin-induced increases in IL-4, IL-5, and IL-13 levels in rat lung tissues at a dose of 375 mg/kg.

Discussion
In this study, animal models of allergic rhinitis and asthma were adopted to investigate the effects of Cs-4 treatments on nasal and airway allergies. Firstly, a mouse model of allergic rhinitis was produced by OVA sensitization followed by an OVA challenge, which resulted in nasal symptoms (nose rubbing and sneezing) and increases in IgE/OVA-IgE, as well as IL-4/IL-13 levels in the nasal fluid. The treatment with DEX (1 mg/kg × 10 days, before challenge) largely abrogated the

Discussion
In this study, animal models of allergic rhinitis and asthma were adopted to investigate the effects of Cs-4 treatments on nasal and airway allergies. Firstly, a mouse model of allergic rhinitis was produced by OVA sensitization followed by an OVA challenge, which resulted in nasal symptoms (nose rubbing and sneezing) and increases in IgE/OVA-IgE, as well as IL-4/IL-13 levels in the nasal fluid. The treatment with DEX (1 mg/kg × 10 days, before challenge) largely abrogated the nasal symptoms and completely suppressed the increases in IgE/OVA-IgE and IL-4/IL-13 levels in the nasal fluid in challenged mice. Daily oral treatments with Cs-4 (250 and 750 mg/kg × 10 days) also suppressed the nasal symptoms induced in OVA-sensitized and challenged mice. This inhibition was associated with reductions in IgE/OVA-IgE and IL-4/IL-13 levels in the nasal fluid. Additionally, Cs-4, as observed in the present study, suppressed the C48/80-induced histamine release from rat RPMC in vitro. Cytokines, such as IL-4 and IL-13, produced by Th2 play an important role in the induction and the sustaining of allergic responses [14]. IL-4 and IL-13 drive the immunoglobulin class switch and antigen-specific IgE-induced production of plasma cells [15]. These IgE antibodies bind with high affinity to Fc receptors on mast cells [16,17]. Allergens can induce mast cell activation and granule/histamine release from these cells. An immediate allergic response, which can lead to nasal symptoms, occurs within minutes after antigen exposure [18][19][20][21]. The ameliorative effect of DEX and Cs-4 treatments on AR is likely mediated by modulating the proinflammatory response triggered by an allergen.
Secondly, a rat model of asthma was produced by a neonatal capsaicin challenge, which was characterized by increases in the splenic index, cutaneous lesion score, airway responsiveness (i.e., tracheal and bronchial rings to Mch-induced contractions), and scratching behavior. Capsaicin-challenged rats showed increases in plasma IgE levels, as well as IgE and EPO levels, in the BAL fluid. Th2 cytokine (IL-4, IL-5, and IL-13) levels in lung tissues were also increased. The DEX treatment (1 mg/kg × 20 days following weaning) reduced the splenic index in capsaicin-challenged rats, but it also caused a reduction in body weight. Even though the DEX treatment did not produce any detectable effects on cutaneous skin lesions or airway responsiveness, it completely suppressed the stimulation of scratching behavior in capsaicin-challenged rats. Besides, the DEX treatment inhibited the capsaicin-induced increases in proinflammatory/inflammatory marker levels in the plasma and BAL fluid, as well as in rat lung tissues. While the capsaicin challenge did not change the levels of TNF-α and IFN-γ (Type 1 T helper cell (Th1) cytokines) in lung tissues, the DEX treatment caused significant increases. While the Cs-4 treatments (123 and 375 mg/kg × 20 days) did not change the body weights of the capsaicin-challenged rats, it did reduce the splenic index and lowered the cutaneous lesion scores. Both the decrease in airway responsiveness and amelioration of the scratching behavior afforded by the Cs-4 treatments were associated with reductions in plasma and BAL fluid proinflammatory/inflammatory marker levels in capsaicin-challenged rats. However, while the Cs-4 treatments completely suppressed increases in the cytokine (IL-4, IL-5, and IL-13) levels in rat lung tissues, it did not affect the levels of TNF-α or IFN-γ. The current literature considers that a skewed ratio of Th2 cytokines to Th1 cytokines indicates allergic inflammation. Th2 cell cytokines contribute to an allergic reaction, while Th1 cytokines contribute to nonallergic inflammation [22]. Th2 cytokines have been shown to be involved in the pathogenesis of asthma [22,23]. As such, the beneficial effect of DEX and Cs-4 treatments on asthma may be mediated by immunosuppressive actions on the production of Th2 cytokines. The differential effects of DEX and Cs-4 on skin lesions and airway responsiveness in capsaicin-challenged rats may be related to their distinct modes of action in immunomodulation. It has been shown that a reduced production of IFN-γ by the T cells of asthmatic patients correlates with disease severity [24,25] and that TNF-α is associated with the pathogenesis of asthma [23]. In this regard, the DEX-induced increase in IFN-γ in rat lung tissues, as observed in the present study, may be related to its preventive effect on asthma. DEX is a synthetic glucocorticoid that has anti-inflammatory and immunosuppressant effects and is used in several conditions, such as asthma and severe allergy [26]. Clinical studies have shown that patients receiving DEX, either at a high dose or for a long time, might develop several side effects, such as hyperglycemia, weight change, or osteoporosis, due to its in vivo nonselective actions [27]. The observation in our study is consistent with the weight reduction effect of the long-term DEX treatment in rats [28].
Consistent with the anti-AR and anti-asthmatic effects of Cs-4, it has been shown that a formulation of Cordyceps militaris produces an ameliorative effect in mouse model OVA-induced AR [29]. Besides, the Cordyceps militaris mycelium extract was found to modulate airway inflammation induced by OVA sensitization in mice [30]. In particular, a Cordyceps polysaccharide was shown to reduce the extent of airway inflammation in an OVA-induced mouse model of asthma [31]. An ultra-performance liquid chromatography coupled with ultraviolet detection (UPLC-UV) quantitative analysis indicated that Cs-4 contains adenine, adenosine, and cordycepin as the major constituents. In this regard, adenine has been demonstrated to inhibit IgE/antigen-induced degranulation and TNF-α release in mast cells [32]. The mechanism underlying the antiallergic effect of adenine involves the inhibition of Syk-mediated signal transduction and IκB kinase-mediated degranulation [32]. Adenosine has been reported to reduce inflammation by inhibiting leukocyte recruitment; adenosine also prevents stimulated neutrophils from adhering to vascular endothelium, as well as preventing neutrophil-mediated injury to the endothelium [33]. Cordycepin has been shown to exert anti-asthmatic activity in mice, which involves the inhibition of Th2-type responses through the suppression of the p38-MAPK and NF-κB signaling pathways [34]. Therefore, the anti-AR and anti-asthmatic effects of Cs-4 may be contributed to by the above-mentioned constituents.
In conclusion, our results suggest that Cs-4 (an extract of Cordyceps mycelium cultures) has the potential to alleviate immune hypersensitivity reactions in AR and asthma. When compared in human-equivalent doses, the long-term treatments with Cs-4, unlike that of DEX, which caused a body weight reduction in rats, produced undetectable side effects. The isolation of active components from Cs-4 for further investigation is warranted to elucidate the molecular mechanisms underlying its ameliorative effects on AR and asthma.

Materials and Methods
All animal experimental protocols were approved by the Animal Ethics Committee of the Hong Kong University of Science & Technology (Protocol number: 2018015).

Herbal Drug Preparation and UPLC-UV Analysis
Cs-4 extract, which is a commercial health product, was manufactured and supplied by Royal Medic Group Limited (Hong Kong SAR, China), with the contents of adenine, adenosine, and cordycepin being determined by UPLC-UV quantitative analysis. Briefly, Cs-4 powder was extracted by 25 mL of a mixture of water and ethanol (1:1, v/v). The mixture was sonicated for 30 min and then centrifuged at 3000× g at room temperature. The resultant supernatant was concentrated to 5 mL by rotary evaporation at 60 • C. The concentrated extract was reconstituted to a volume of 25 mL and gave a final concentration of 8 mg/mL. The Cs-4 extract (2 µL) was separated using a Waters ACQUITY UPLC system coupled with a photodiode array detector (Waters Corporation, Milford, MA, USA). A mobile phase consisting of water (solvent A) and acetonitrile (solvent B) was applied for programmed gradient elution with varying concentrations of A and B with time (0 min: 100% A and 0% B, 10 min: 97% A and 3% B, 18 min: 80% A and 20% B, and 21-24 min: 100% A) on a Waters ACQUITY UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 mm × 100 mm) (Waters Corporation, Milford, MA, USA) at a flow rate of 0.25 mL/min and a temperature of 40 • C. The detection wavelength was set at 261 nm. The contents of adenine, adenosine, and cordycepin in the Cs-4 extract fraction were determined using a calibration curve.

Measurement of Nasal Symptoms
Nasal symptoms (sneezing and nose rubbing) were observed 1 min after the last OVA challenge. The number of sneezing and nose rubbings was counted for 20 min.

Evaluation of IgE and Cytokines in NLF
Two hours after the last OVA challenge, mice were sacrificed. A ligation was made between the larynx and trachea using a thread. NLF was collected from the nostrils by perfusing 1-mL PBS-A into the larynx. The NLF collected was centrifuged at 10,000× g for 10 min at 4 • C to separate NLF from the cellular material. The supernatant was collected for cytokine measurement [39]. Levels of total IgE and OVA-specific IgE, as well as the level of cytokines (IL-4 and IL-13) released, were measured using ELISA kits (Cusabio, Houston, TX, USA).

RPMC Histamine Release
The effect of Cs-4 incubation on the mast cell histamine release was assessed using RPMC.

RPMC Preparation
Sprague-Dawley rats (~2-6 months old) were anesthetized and intraperitoneally injected with 25 mL of Hank's balanced salt solution containing 0.25-mM phenol red. The abdomen was gently massaged for about 60 s, and the fluid in the peritoneal cavity was aspirated. The retrieved cell suspension was centrifuged at 400× g for 5 min at 4 • C and washed with Gey's solution to lyse red blood cells. The cell pellet was obtained after red blood cell lysis followed by centrifugation. The pellet was resuspended in 70% isotonic Percoll solution, which was then overlaid on a 4-(2-hydroxethyl)-1-piperazineethanesulfonic acid (HEPES)-tyrode buffer. The mixture was then centrifuged at 580× g for 15 min at 4 • C. The resultant mast cell pellet was resuspended in HEPES-tyrode buffer. The viability and purity of RPMC were assessed using Trypan blue and Toluidine blue staining, respectively [40,41].

RPMC Degranulation
Purified RPMC were incubated with Cs-4 solution or HEPES-tyrode buffer for 15 min at 37 • C, then compound 48/80 (C48/80) at a final concentration at 1 mg/mL was added, and the preparation was incubated for 15 min. The reaction was stopped by chilling on ice for 10 min. The supernatant was collected by centrifugation at 20,000× g for 5 min at 4 • C. The cell pellet was lysed with 0.1% Triton X. The degree of degranulation was determined by measuring the release of β-hexosaminidase, a reliable indicator of mast cell degranulation [20]. The percentage of β-hexosaminidase release = absorbance of supernatant/(absorbance of supernatant + absorbance of lysate) × 100.

RPMC Histamine Release
Purified RPMC were incubated with Cs-4 extract or HEPES-tyrode buffer for 15 min at 37 • C; then, C48/80 at a final concentration of 1 mg/mL was added, and the mixture incubated for 15 min. The reaction was stopped by chilling on ice for 10 min. The supernatant was collected by centrifugation at 20,000× g for 5 min at 4 • C. The pellet was lysed with water, and the solution was heated at 100 • C for 10 min. Histamine levels in the supernatant and lysate samples were measured using Elisa kits (Abcam, Cambridge, MA, USA) [42][43][44][45]. The histamine release was estimated as a percentage of the total histamine (both intracellular and extracellular) release.

The Rat Model of Asthma
A recent study has demonstrated that treating neonatal Sprague Dawley rats with capsaicin can result in an asthma-like phenotype, as evidenced by changes in the IgE levels; an increased number of eosinophils in the BAL fluid; and the increased mRNA expression of Th2 cytokines (such as IL-4, IL-5, and IL-13) in the lung tissue, as well as decreased airway resistance and compliance [46].

Experimental Design
Sprague Dawley rats (~8 weeks of age; 15-20 g) were randomly assigned to 5 groups, with 10 animals in each group. Distilled water, Cs-4 (daily dose of 123 or 375 mg/kg; the lower one was a human-equivalent dose), or DEX (1 mg/kg; in the range of a human-equivalent dose) was administered intragastrically 5 days per week for 20 doses following weaning.

Neonatal Capsaicin Treatment
Pregnant Sprague Dawley rats were obtained approximately 5 days before parturition. Within 24 h after birth, pups were randomly selected and injected subcutaneously with capsaicin (75 mg/kg; 50-mg capsaicin dissolved in 10 mL of a solution containing Tween 20:ethanol:saline in a ratio of 1:1:8 (v/v), while some pups received a vehicle and served as controls. All animals were kept in a room with a 12-h light-dark cycle at 22-25 • C, with free access to food and water. The pups were weaned at postnatal week 3, and only the male pups were used in the experiment [47].

Scratching Behavior
Two hours after the last dose of treatment, rats were observed for behavioral changes. Briefly, rats were placed in separate cages for 10 min of adaptation. The behavior of the rats was then observed and recorded using a digital camera (TG-870, Olympus, Tokyo, Japan) for 29 min. The video files were played back on a computer, and the number of scratches counted by the experimenter. Nose rubbing and the motor behavior in which the hind paw moved up to the body and touched the skin a least once was taken as one count.

Evaluation of Cutaneous Lesions
Cutaneous lesions were observed and assessed by scoring, as shown in Table 4. The skin conditions in the three regions (face, ears, and body) were subjected to scoring. The sum of the scores in the three regions was used as the dermatitis score for each rat. Twenty-four hours after the last treatment, rats were anesthetized by an intraperitoneal injection of ketamine (80 mg/kg)/xylazine (5 mg/kg) dissolved in saline. The body weight was determined, and the spleen was dissected and weighed. The splenic index was estimated by the ratio of spleen weight (g)/body weight (g).

Evaluation of AHR in Vitro
AHR following the methacholine challenge was measured in isolated tracheal and bronchial rings in vitro. Briefly, rats were anesthetized by intraperitoneal injection with a ketamine (80 mg/kg)/xylazine (5 mg/kg) mixture in saline. The thorax was opened, and 10-mL prewarmed Dulbecco's Modified Eagle's Medium (DMEM) with 1% low-melt agarose was injected into the lungs through the trachea. Lungs, trachea, and bronchi were filled with the medium, and a thread was used to tie the upper part of the trachea. Trachea and lungs were removed en bloc. The isolated lungs and the trachea were placed at 4 • C for 30 min. One or two rings of trachea and bronchi were dissected from the lung and put into prewarmed DMEM. Samples were incubated at 37 • C in a humidified atmosphere of 5% CO 2 and 95% air overnight to dissolve the agarose. After the incubation, samples were washed with prewarmed DMEM, and the airway response of the tracheal and bronchial rings to increasing concentrations of Mch (Santa Cruz Biotechnology, Dallas, TX, USA) (from 10-9 to 10-0.5 M, respectively) was evaluated. Rings were incubated with a given concentration of Mch (starting with a low concentration) for 10 min at 37 • C, followed by observation under light microscopy and the image captured. The rings were sequentially challenged with six different concentrations of Mch, from low to high. The area of the lumen of each ring was measured in terms of the number of pixels using software ImageJ (LOCI). The initial percentage of the lumen size of each ring was calculated, and a graph plotting the percentage of the initial lumen size against a log molar concentration of Mch was drawn. The concentration of Mch at the one-half maximal decrease in the initial lumen size of the ring was estimated using the software GraphPad Prism6 (GraphPad Software, San Diego, CA, USA). Data were expressed as the mean ± standard error of the mean (S.E.M.). Data were analyzed by one-way ANOVA using SPSS statistical software version17 (IBM SPSS). Significant differences between the two groups were determined by the least significant difference at p < 0.05 [15,48,49].

Sampling of Lung Tissues and Bronchoalveolar Lavage
Rats were anesthetized with a ketamine (80 mg/kg)/xylazine (5 mg/kg) mixture in saline by intraperitoneal injection 24 h after the last treatment. Blood was collected via cardiac puncture.
The airways were cannulated with a syringe needle and rinsed with PBS-A without Ca 2+ or Mg 2+ (PBS-A). The fluid collected from the lavage was defined as the BAL fluid. Both lobes of the lung were acquired; then, a 20% lung homogenate was prepared with lysis buffer containing 0.5% Triton X-100, 150-mM NaCl, 15-mM Tris, 1mM-CaCl 2 , and 1mM-MgCl 2 , pH 7.4. The lung homogenates were then centrifuged at 10,000× g for 15 min at 4 • C. Supernatants were collected and kept in −80 • C before the assay [50].

IgE Levels
IgE levels in the plasma and in the supernatant produced from the BAL fluid were measured using a rat IgE ELISA kit (Cusabio, Houston, TX, USA) [46].

Eosinophil Infiltration in the BAL Fluid
The extent of eosinophil infiltration was indirectly assessed by measuring the EPO level in the BAL fluid. The BAL fluid was centrifuged 10,000× g for 10 min at 4 • C, and the supernatant was collected for the measurement of EPO using ELISA kits (Cusabio, Houston, TX, USA).

Statistical Analysis
Unless otherwise specified, data were analyzed by one-way ANOVA using SPSS statistical software version17 (IBM SPSS). Significant differences between groups were determined by Tukey's test with p < 0.05.

Conflicts of Interest:
The authors declare no conflict of interest.  Th1  Type I T helper cell  Th2  Type II T helper cell  TNF Tumor necrosis factor UPLC-UV Ultra-Performance Liquid Chromatography coupled with Ultraviolet detection