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Introduction

Bronchial asthma is a complex inflammatory airway disease mainly characterized by the presence of inflammation, airway hyperresponsiveness (AHR) and airway remodeling. While these are disease-defining characteristics, asthma has recently been recognized as a widely heterogeneous disease [Global Initiative for Asthma (GINA), 2014 update]. To date, it is estimated that 300 million individuals are affected by the disease worldwide and an effective treatment has not yet been found (1). Common allergens such as house dust, animal dander, inhalants, drugs, pollens, and air pollutants contribute to the induction of asthma. Cumulative evidence has indicated that CC chemokine receptor 7 (CCR7) and its ligands, CCL19/CCL21, are involved in these inflammatory responses (2–4); they are also associated with the recruitment and the activation of multiple cell types, such as dendritic cells (DCs), eosinophils (EOS), T cells and B cells (5–8), and also interact with inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 (9,10).

Chemokines are small (8–14 kDa) chemotactic cytokines that bind to seven transmembrane domain G protein-coupled receptors (GPCR) and are secreted by a great variety of cell types. Chemokines are important mediators involved in the migration and activation of various subsets of leukocytes (11), as well as lymphoid development and lymphocyte homing into and out of secondary and tertiary lymphoid organs.

CCR7 is a member of the GPCR family and is commonly expressed in memory T cells, B cells and mature DCs (12). CCL19 and CCL21 are the only ligands for CCR7. T cells and antigen-presenting DCs establish close physical contacts within the lymph nodes, which jointly contribute to a myriad of adaptive immune functions, including secondary lymphoid organogenesis, and regulatory and memory T cell function (13).

It has been previously described that the CCR7 promoter includes potential binding sites for nuclear factor-κB (NF-κB) (14), which is involved in many inflammatory diseases. CCR7 is a direct target of NF-κB and the induction of CCR7 expression has been shown to be regulated by NF-κB (14), indicating that the modulation of inflammation may be beneficial to the control and treatment of asthma.

Baicalin, as a small molecule flavonoid compound (Fig. 1), which is the major active constituent of Scutellariae radix, has been extensively employed in traditional Chinese medicine (TCM) for the treatment of asthma. It has been demonstrated that baicalin exerts anti-inflammatory effects on experimental colitis (15). In a previous study of ours, we demonstrated the anti-airway remodeling effect of baicalin (16). Moreover, baicalin has been shown to rectify the Th1/Th2 and Treg/Th17 imbalance in asthmatic mice (17). However, the effects and related molecular mechanisms of action of baicalin on CCR7 and its ligands associated with airway inflammation have not been fully elucidated. The present study was designed to examine the effects and the underlying molecular mechanisms of action of baicalin on airway inflammation in a mouse model of ovalbumin (OVA)-induced asthma.

Figure 1 Molecular structure of baicalin.

Materials and methods

Animals and reagents

Seven-week-old female BALB/c mice were purchased from SIPPR/BK Laboratory Animal Co., Ltd., (Shanghai, China), and reared under specific pathogen-free conditions following the guidelines and approval of the Animal Care and Use Committee on the Ethics of Animal Experiments of Fudan University, Shanghai, China.

Baicalin (purity ≥98%; chemical structure shown in Fig. 1) was provided by Must Biotechnology Co., Ltd. (Chengdu, China). OVA (grade V), aluminum hydroxide and methacholine (Mch) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The OVA-specific IgE ELISA kit was obtained from Shibayagi (Gunma, Japan). The CCL19 and CCL21, and the TNF-α and IL-6 ELISA kits were purchased from RayBiotech (Norcross, GA, USA). Anti-mouse CCR7 antibody was purchased from Abcam (Cambridge, MA, USA).

Mouse model of OVA-induced asthma and treatment

Asthma was initiated by an intraperitoneal injection of 0.2 ml saline containing 20 µg OVA and 2 mg aluminum hydroxide on days 0, 7, 14 and 21; from day 25 to 31, each mouse was placed into an individual chamber and challenged with 3% OVA nebulization (30 min/day) utilizing an ultrasonic nebulizer model 402AI; Yuyue, Jiangsu, China).

The mice were divided into 6 experimental groups as follows (n=12/group): i) the normal control (NC) group; ii) the asthma (A) group; iii) asthmatic mice treated with 10 mg/kg baicalin (B10) group; iv) asthmatic mice treated with 25 mg/kg baicalin (B25) group; v) asthmatic mice treated with 50 mg/kg baicalin (B50) group; and vi) asthmatic mice treated with dexamethasone (DXM) (0.085 mg/kg). The mice in the NC group were sensitized and challenged with an equivalent volume of normal saline. Baicalin and DXM were intragastrically administered from day 24 to 31 prior to the OVA challenge. The following analyses were implemented within 24 h of the final OVA challenge:

Determination of lung function

AHR was detected by an invasive pulmonary facility for small animals (Buxco Electronics, Troy, NY, USA) after the final OVA challenge, as previously described by Pichavant et al (18). Briefly, each mouse was tracheostomized and intubated under anesthesia and the administration by nebulization to gradients of Mch (3.125, 6.25 and 12.5 mg/ml) was carried out to evaluate pulmonary resistance (RL) and pulmonary dynamic compliance (Cdyn). Data were expressed as a percentage change from the baseline value.

Inflammatory cell classification in bronchoalveolar lavage fluid (BALF) and collection of serum

Immediately following the assessment of AHR, and while the mice were still under anesthesia, blood was collected from the left eyeball and stored at 4°C for 2 h and then centrifuged at 5,000 × g, 4°C for 30 min. The serum was collected, repackaged and stored at −80°C for use in ELISA. The lung tissues of the mice were then exposed, the right lung was tied and BALF was collected by inserting a tracheal tube and lavaging the left lung 3 times with 0.3 ml aliquots of phosphate-buffered saline (PBS) and centrifuged at 800 × g, 4°C for 10 min. The supernatants were gathered and stored at −80°C for ELISA and the sediments were resuspended in 0.1 ml PBS for determining the cell count using a hemacytometer (Hemavet 950FS; Drew Scientific, Inc., Waterbury, CT, USA).

Lung histopathological assessment

Following the collection of BALF, the mice were euthanized and the lung tissue from the middle lobe of the right lung was detached and fixed in 4% paraformaldehyde, embedded in paraffin and thin slices (4-µm-thick) were cut from blocks and stained with hematoxylin and eosin (H&E). The histopathological changes were determined under an optical microscope (Eclipse 80i; Nikon Corporation, Tokyo, Japan) to assess the inflammatory changes in the lung tissues of the mice.

Histopathological evaluation was performed in a blinded manner on randomized sections. The severity of inflammatory cell infiltration into the lungs was assessed by a 5 point scoring system as previously described by Underwood et al (19).

Detection of IgE, chemokine and cytokine levels in BALF or serum by ELISA

The levels of CCL19, CCL21 and OVA-specific IgE in BALF, as well as the levels of IL-6 and TNF-α in serum were detected using respective ELISA kits, following the manufacturer's instructions. The assays employed antibodies specific for each cytokine coated on 96-well plates. All reagents, samples and standards were prepared as instructed. Subsequently, 100 µl standard or sample were added to each well followed by incubation for 2.5 h at room temperature. The solution was then discarded and the sampels were washed 3–4 times with 1X Wash Solution. This was followed by the addition of 100 µl prepared biotin antibody to each well and incubation for 1 h at room temperature, and further washing with 1X Wash Solution for 3 times. Subsequently, 100 µl prepared streptavidin solution was added followed by incubation for 45 minutes at room temperature. TMB One-Step Substrate Reagent (100 µl) was then added to each well, followed by incubation for 30 min at room temperature. Finally, 50 µl Stop Solution were added to each well and the absorbance was immediately read at 450 nm.

Reverse transcription-quantitative PCR (RT-qPCR)

The expression of CCR7 in the lung tissue was detected by RT-qPCR. Briefly, total RNA was extracted and reverse transcripted into first-strand cDNA. qPCR amplification was then performed according to the manufacturer's instructions using a PCR Amplifier (FTC-3000 qPCR system; Funglyn Biotech, Inc., Toronto, ON, Canada). The cycling conditions were as follows: 1 cycle at 95°C for 3 min, 40 cycles at 95°C for 5 sec and 60°C for 30 sec; and 1 cycle for melting at 94°C for 90 sec, 60°C for 3 min and 94°C for 10 sec. The fold change in the expression of each gene was calculated using the 2−ΔΔCt method, with the housekeeping gene, GAPDH, as an internal control. The final data were presented as the fold change compared to the expression level in the mice in the NC group. The sequences of the primers used are listed in Table I.

Table I Primers used for PCR amplification.

Table I

Primers used for PCR amplification.

Gene	Primer sequences
CCR7	F: 5′-CGCAACTTTGAGCGGAACAA-3′
(NM_001301713)	R: 5′-TTCGCAGCTGCTATTGGTGA-3′
GAPDH	F: 5′-TGGCCTTCCGTGTTCCTAC-3′

[i] CCR7, CC chemokine receptor 7; F, forward; R, reverse.

Western blot analysis

Lung tissues were collected for the examination of the protein expression of CCR7, as well as that of phosphorylated (p-)IκBα and p-p65 by western blot analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10%) was used to isolate the total protein following extraction. The lung protein samples were electrophoretically transferred onto PVDF membranes. Subsequently, the PVDF membranes were blocked with 5% BSA for 2 h at room temperature followed by incubation with primary and secondary antibodies. The immunoblots were incubated with the following antibodies: primary antibodies overnight at 4°C with CCR7 (1:2,000 dilution; Abcam), p-IκBα (1:1,000 dilution) and p-p65 (1:1,000 dilution) (both from Cell Signaling Technology, Inc., Danvers, MA, USA); and secondary antibodies at 37°C for 2 h with 1:2,000 dilution. Finally, the immunoblots were visualized and analyzed using Image Lab software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Statistical analysis

All analyses were undertaken using SPSS 19.0 statistical software. Data are expressed as the means ± SEM. For comparisons of multiple parameters, ANOVA with the LSD test or Dunnett's test were used to evaluate the statistical significance of the differences between groups. A P-value <0.05 was considered to indicate a statistically significant difference.

Results

Baicalin mitigates AHR in asthmatic mice

The efficacy of baicalin against OVA-induced AHR was examined by means of the airway responsiveness to aerosolized PBS or Mch. As shown in Fig. 2, only inconspicuous alterations in RL were observed in the normal mice, while the mice with OVA-induced asthma exhibited an obvious increase in RL and a decrease in Cdyn (Mch of 6.25 and 12.5 mg/ml) compared with the mice in the NC group (P<0.05). Treatment with all concentrations of baicalin led to an obvious increase in Cdyn and a reduction in RL in the asthmatic mice (P<0.05) and these effects occurred in a dose-dependent manner.

Figure 2 Baicalin reduces airway hyperresponsiveness (AHR) to elevating doses of methacholine (Mch) by decreasing airway resistance (RL) and increasing dynamic compliance (Cdyn) in asthmatic mice. Data are expressed as a percentage change from the baseline value. *P<0.05 vs. normal control (NC) group; #P<0.05 vs. asthmaticc (A) group. B10, B25 and B50 indicates treatment with baicalin at 10, 25 and 50 mg/kg, respectively.

Inflammatory cell recruitment in BALF is suppressed by baicalin

As shown in by our results, the OVA-sensitized and challenged mice exhibited a large quantity of inflammatory cells infiltrating the lungs compared to the mice in the NC group (P<0.01; Fig. 3). In order to ascertain the effects of baicalin on inflammatory cell counts, we detected general white blood cells (WBCs) and EOS. The WBC and EOS counts in BALF were significantly decreased in the baicalin-treated mice. Moreover, the mice treated with 50 mg/kg baicalin (B50) exhibited a marked reduction in the WBC and EOS counts in BALF (P<0.01), and this reduction in inflammatory cell count in the B50 group was greater than that observed in the DXM-treated mice.

Figure 3 Baicalin inhibits the influx of inflammatory cells into the bronchoalveolar lavage fluid (BALF) of asthmatic mice. Total (A) white blood cells (WBC) and (B) eosinophils (EOS) were counted in BALF collected from mice in each group. **P<0.01 vs. normal control (NC) group; #P<0.05 vs. asthmatic (A) group; ##P<0.01 vs. A group. B10, B25 and B50 indicates treatment with baicalin at 10, 25 and 50 mg/kg, respectively.

Histological lung inflammation caused by OVA inhalation is reduced in baicalin-treated mice

In the lung tissues from the mice exposed to OVA, a distinct infiltration of EOS and mucus secretion into the tracheobronchial mucosa and airway lumen was observed as compared with the mice in the NC group (P<0.05; Fig. 4). Conversely, the baicalin and DXM-treated mice exhibited less infiltration of EOS into the tracheobronchial mucosa and airway lumen (P<0.05).

Figure 4 Baicalin attenuates the infiltration of inflammatory cells into the lungs and inhibits mucus secretion in and around the airways of asthmatic mice (magnification, ×200). *P<0.05 vs. normal control (NC) group; #P<0.05 vs. asthmatic (A) group. B10, B25 and B50 indicates treatment with baicalin at 10, 25 and 50 mg/kg, respectively.

Effect of baicalin treatment on IgE, CCL19 and CCL21 levels in OVA-challenged mice

The results of ELISA indicated that the OVA-specific IgE level in BALF was significantly increased by the OVA challenge when compared to the mice in the NC group (P<0.05; Fig. 5A). Gavage of baicalin in each dose group showed an obvious reduction in level of OVA-specific IgE in BALF (P<0.05). Simultaneously, the mice with asthma exhibited a marked increase in the levels of CCL19 and CCL21 (P<0.01; Fig. 5B and C) in BALF. Treatment with 10 mg/kg baicalin (B10) markedly decreased the level of CCL19 and CCL21 (P<0.01), and treatment with baicalin at 25 and 50 mg/kg (B25 and B50) resulted in a marked reduction in the CCL19 level (P<0.05; Fig. 5B). However, treatment with baicalin at 25 and 50 mg/kg did not significantly inhibit the CCL21 level in BALF, whereas DXM did (Fig. 5C).

Figure 5 Baicalin reduces ovalbumin (OVA)-specific (A) IgE, (B) CCL19 and (C) CCL21 levels in bronchoalveolar lavage fluid (BALF) of asthmatic mice. OVA-specific IgE and chemokines in BALF were analyzed by ELISA. *P<0.05 vs. normal control (NC) group; **P<0.01 vs. NC group; #P<0.05 vs. asthmatic (A) group; ##P<0.01 vs. A group. B10, B25 and B50 indicates treatment with baicalin at 10, 25 and 50 mg/kg, respectively.

Effect of baicalin treatment on IL-6 and TNF-α levels in serum of mice with OVA-induced asthma

IL-6 and TNF-α have been shown to be implicated in many aspects of airway pathology and in evoking allergic inflammatory responses (20–22). In this study, to examine the effect of baicalin on these cytokines, the secretion of these cytokines in the serum of mice in each group were also evaluated by ELISA. As shown in Fig. 6, there were significant elevations in the expression levels of IL-6 and TNF-α in the mice with OVA-induced asthma compared with the mice in the NC group (P<0.01). The increased expression levels of these cytokines in serum were evidently suppressed by baicalin treatment (P<0.01). Enzyme immunoassays revealed that compared with the asthmatic mice, the mice treated with baicalin exhibited a marked decrease in the levels of IL-6 and TNF-α, and this effect was more prominent than that observed with DXM treatment.

Figure 6 Baicalin inhibits the expression of inflammatory cytokines in serum of asthmatic mice. (A) IL-6 and (B) TNF-α levels were measured in serum collected from mice in each group. **P<0.01 vs. normal control (NC) group; #P<0.05 vs. asthmatic (A) group; ##P<0.01 vs. A group; ###P<0.001 vs. A group. B10, B25 and B50 indicates treatment with baicalin at 10, 25 and 50 mg/kg, respectively.

Baicalin inhibits CCR7 mRNA and CCR7 protein, as well as p-IκBα and p-p65 protein expression in lung tissue

To determine whether the effects of baicalin on CCR7 and NF-κB were transcriptionally regulated, CCR7 mRNA expression was determined by RT-qPCR, and the protein expressio levels of CCR7 and p-IκBα and p-p65 were detected by western blot analysis. As shown in Fig. 7, CCR7 mRNA expression and CCR7, p-IκBα (P<0.05) and p-p65 (P<0.01) protein expression were significantly increased in the OVA-challenged mice. Notably, treatment with baicalin at 25 mg/kg (B25) had a marked inhibitory effect on the CCR7 mRNA and protein levels, as well as on the p-IκBα and p-p65 protein levels in the lung tissue of the OVA sensitized and challenged mice.

Figure 7 (A-E) Baicalin suppresses the protein expression of CC chemokine receptor 7 (CCR7) and nuclear factor (NF)-κB in the lung tissue of asthmatic mice. The levels of CCR7, as well as phosphorylated IκBα (p-IκBα) and phosphorylated p65 (p-p65) protein expression in lung tissue of mice in each group were assayed by western blot analysis. *P<0.05 vs. normal control (NC) group; #P<0.05 vs. asthmatic (A) group. B10, B25 and B50 indicates treatment with baicalin at 10, 25 and 50 mg/kg, respectively.

Discussion

Asthma is a highly complex airway inflammatory disorder, the incidence of which is continually increasing. Airway inflammation, AHR and mucus overproduction are the major pathological characteristics of the disease (1). Chemokine receptors with their ligands and NF-κB together with related cytokines are considered to be involved in the pathogenesis of asthma (4,23–25). In this study, the effects of baicalin on inflammation in a mouse model of OVA-induced asthmat and on the associated chemokine receptors and their ligands were investigated. Our results revealed that the OVA-exposed mice exhibited substantial airway inflammatory changes compared to the NC mice. Consistent with previous discoveries (26,27), in this study, AHR was markedly increased in the OVA-exposed asthmatic mice; however, treatment with baicalin led to a significant increase in Cdyn and a reduction in RL in the asthmatic mice and these effects occurred in a dose-dependent manner, which indicated that baicalin treatment had a protective effect on pulmonary function in the asthmatic mice. Furthermore, the increased amount of inflammatory cells, as well as the OVA-specific IgE level in BALF and the secretion of inflammatory cytokines into the blood of asthmatic mice were markedly reduced by baicalin.

The homeostatic chemokines, CCL19 and CCL21, are the sole ligands for CCR7, and they have a potent chemotactic activity for antigen-presenting mDCs (28) and have been shown to be important in the homing and traffic of naive lymphocytes within lymphoid tissues (29). Studies have demonstrated that the interaction of recruited DCs and T cells at the site of regional lymph nodes is promoted by CCL19 and CCL21 (30,31). Mice lacking both CCL21 and CCL19 gene expression exhibit a significantly reduced airway inflammatory response (4). In this study, treatment with various concentrations of baicalin exerted a marked inhibitory effect on the CCL19 level, and treatment with baicalin at 10 mg/kg significantly reduced the CCL21 level in BALF, which may support the anti-inflammatory effect of baicalin in asthmatic mice.

CCR7 is considered to be involved in the lymph node homing of naive T cells and DCs, and also plays a crucial role in regulating immune cell migration for the balance of immunity against pathogens and the maintenance of central and peripheral immunological tolerance (32,33). It has been shown that CCR7−/− mice exhibit a strongly impaired migration of lung-derived DCs to the bronchial lymph nodes (34). In addition, defective CCR7 expression has been shown to contribute to the reduced efficiency of the interaction between DCs and T cells, as well as to the decreased responsiveness to CCR7 ligands (35). Based on such discoveries, CCR7 has received considerable attention as a potential therapeutic target in allergies (36). In this study, the protein and mRNA expression level of CCR7 were remarkably elevated in asthmatic mice; however, CCR7 protein expression was significantly reduced by treatment with baicalin at 25 and 50 mg/kg and by DXM, and CCR7 mRNA expression was markedly decreased by treatment with baicalin at 25 mg/kg, suggesting that baicalin may be an effective medication for allergic inflammation.

Crucial to the pathogenesis of asthma, the inflammatory cytokines, IL-6 and TNF-α, which have been reported to be implicated in the airway allergic inflammatory responses, regulate many aspects in adaptive immune disorders. IL-6 and TNF-α, which can be expressed by several cell types (24,37), aggregate into the airways and lung tissues when allergy occurs. A previous study demonstrated that IL-6 levels were markedly elevated both in symptomatic and asymptomatic asthma patients (38). A recent study also found that IL-6 trans-signaling increased the expression of airway disease-related genes in airway smooth muscle (25). In addition, the anti-inflammatory effect exerted by targeting TNF-α is currently being widely investigated and studies have shown that the blockade of TNF-α contributes to the suppression of murine allergic airway inflammation (22,39). Various studies have demonstrated that the inhibition of the critical inflammatory cytokines, IL-6 and TNF-α, represent a prominent new approach for the treatment of IL-6- and TNF-α-associated inflammatory diseases (40–43). The findings of the present study demonstrated that compared with the mice in the NC group, the IL-6 and TNF-α levels in serum were significantly decreased by treatment with all concentrations of baicalin, and these effects were more prominent than those observed with DXM treatment. Our data may confirm the hypothesis that baicalin exerts an inhibitory effect on regulatory Th17 cells in asthmatic mice.

NF-κB is a master regulatory transcription factor that is crucial to the expression of inflammatory cytokines, chemokine receptors and the counterpart ligands in inflammation-related diseases (44). The inhibitor of IκB kinase (IKKs), p65/p50 complex and IκB are key cytokines in the NF-κB pathway; however, among the p65/p50 complex, only p65 has transcriptional activity (45). It has been demonstrated that the interaction between CCR7 and its ligand, CCL19, activate p-IκB and cause NF-κB to translocate to the nucleus, increasing the DNA-binding capacity of NF-κB (46). In addition, it has been demonstrated that the stimulation of DCs with CCL19 or CCL21 induces the activation of NF-κB (47). Therefore, CCR7-mediated NF-κB activation may play a key role in the pathophysiological process of asthma (Fig. 8), and the suppression of NF-κB activation and the interaction with CCR7 and its ligands are associated with reducing airway inflammation. Accordingly, we investigated whether baicalin treatment could interfere with CCR7 and the NF-κB pathway. The data from western blot analysis revealed that the expression of p-IκBα and p-p65 protein were markedly elevated in the lung tissue of OVA-exposed mice as compared to the NC mice and were markedly inhibited by treatment with baicalin at the dose of 25 mg/kg. Collectively, these data suggest the potential of baicalin to inhibit the functions of CCR7 and its ligands via NF-κB, thus attenuating inflammation, and the development and progression of asthma.

Figure 8 Possible role of CC chemokine receptor 7 (CCR7)-mediated NF-κB activation.

In conclusion, in the present study, we demonstrated that baicalin improves pulmonary function and attenuates airway inflammation. Our data provide novel mechanistic insight into the anti-inflammatory effects of baicalin. Importantly, the levels of IgE and CCR7, and those of its ligand, CCL19, were prominently suppressed by treatment with baicalin. Furthermore, baicalin exerts an inhibitory effect on the NF-κB pathway. These discoveries may demonstrate the potential of baicalin as an agent for the management of allergic asthma.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (nos. 81173390, 81403476 and 81573758) and the Development Project of Shanghai Peak Disciplines-Integrated Chinese and Western Medicine.

References