Introduction
Asthma is a heterogeneous disease, characterized by chronic airway inflammation and bronchial hyperresponsiveness.[1,2] [3] [4-6] [7] [8] [9] [10] [11]
Both clinical and laboratory studies have demonstrated the beneficial effects of electroacupuncture (EA) in asthma when administered alone or as an adjunct to conventional western therapy.[12] [13] [14] [15] [16]
EA has been shown to suppress inflammation through neural-immune-endocrine interactions.[17-19] [20,21]
Methods
Study design
Our research was divided into two parts. In experiment 1, mice were randomly divided into four groups: control group (Control), control group treated with EA (EA), asthma group (ovalbumin [OVA]), and asthma group treated with EA (OVA + EA). The level of GABA and the expressions of GABAAR in TLR4/MyD88/NF-κB signaling pathway were assessed using Western blot and histological staining. In experiment 2, mice were randomly divided into four groups: control group (Control), asthma group (OVA), asthma group treated with EA (OVA + EA), and asthma group treated with EA plus GABAAR antagonist (OVA + EA + antagonist). GABAAR antagonist was employed to further explore the role of GABAergic system in mediating the therapeutic effect of EA in asthma. Each group was composed of five mice.
Animals
The experimental protocol was approved by the Animal Ethical and Welfare Committee of China–Japan Friendship Hospital (No. zryhyy12-20-01-1). All experiments were performed in accordance with institutional guidelines on the use of live animals for research. Female BALB/c mice (age: 4–6 weeks; weight: 20–25 g) were purchased from the animal experimental center of China–Japan Friendship Hospital. Mice were bred in a specific pathogen-free room in a controlled environment (12 h light/dark cycle, temperature: 22–24°C, and humidity: 50–70%), and provided ad libitum access to standard rodent chow and water. The mice were acclimatized in this environment for 1 week before the study.
Establishment of the experimental animal model of asthma
OVA (Sigma Aldrich, Louis, MO, USA) and aluminum hydroxide (Al(OH)3 ; Sigma Aldrich) were prepared in concentrations of 250 and 10 mg/mL, respectively. Mice were exposed to OVA via intraperitoneal (i.p.) injection of a sensitization dose (200 μL normal saline suspension containing 50 μg OVA and 2 mg Al(OH)3 ) on days 1 and 8. Subsequently, OVA-sensitized mice were challenged by intranasal (i.n.) injection of 20 μL normal saline containing 200 μg OVA on days 21, 23, 25, and 28 under isoflurane (RWD Life Science Co. Ltd, Shenzhen, China) anesthesia.[9,22] Figure 1 A]. The criteria for the successful establishment of the asthma model were exaggerated airway response to stimuli (airway hyperresponsiveness) and eosinophil-rich airway inflammation.[23]
Figure 1: Schematic illustration of the experimental design for asthma model and intervention. (A) Experimental protocol for asthma model; (B) Schematic diagram of EA. EA: Electroacupuncture; GABAAR: γ-aminobutyric acid type A receptor; i.n.: Intranasal; i.p.: Intraperitoneal; OVA: Ovalbumin.
EA treatment
We determined the location of acupoints with reference to the atlas of experimental animals and Experimental Acupuncture Science (Sixth Edition). The acupoints used in the study were BL13, located at the depression lateral to the lower border of the spinous process of the third thoracic vertebra (approximately 0.3 cm from the midline in mice), and CV22, located at the center of the suprasternal fossa [Figure 1 B]. Disposable stainless steel acupuncture needles measuring 0.2 × 25 mm (Hwato, Suzhou, China) were inserted to a depth of 2 mm. Electrodes were fixed to these needles, and electrical stimulation (2 Hz and 2 mA) with compressional waves was administered for 30 min using an EA device (KWD-808I, Great Wall Brand, Changzhou, China). EA treatment was started 24 h after OVA sensitization, and it was administered every other day for 2 weeks.[24,25]
Measurement of lung function
Twenty-four hours after the last challenge with OVA, mice were anesthetized with 1% pentobarbital sodium (90 mg/kg, Merck, Darmstadt, Germany) and 1 mg rocuronium (MSD, Shanghai, China). Invasive measurements were performed in tracheostomized, mechanically ventilated mice using the FlexiWare 8.0 (SCIREQ, Montreal, Canada) experimental platform. Aerosolized methacholine (Mch; Sigma Aldrich) was administered in incremental concentrations (0, 3.125, 6.250, 12.500, 25.000, 50.000, and 100.000 mg/mL), which were nebulized at a volume of 20 μL.[26]
Bronchoalveolar lavage fluid (BALF) collection and analysis
Lungs were lavaged four times with 0.5 mL of 4°C phosphate buffer saline (PBS) through a tracheal cannula and a total volume of 1.5 mL BALF was placed into an eppendorf tube. The supernatant was collected and stored at −80°C until further processing for enzyme-linked immunosorbent assay (ELISA) for cytokines. The cells were resuspended in 100 μL PBS for determining the cell counts.
The differential cell counts were determined using BALF smears processed by Wright-Giemsa staining (Baso, Zhuhai, China). Cells were classified and counted for each slide using the brightfield microscope according to cell morphology.
Levels of interleukin (IL)-4, IL-5, and IL-13 in the BALF were determined by ELISA kits (4A Biotech, Beijing, China), according to the manufacturer's instructions. All samples were assessed in triplicate.
Histological assessment
The left lung tissues of mice were fixed in 4% paraformaldehyde for more than 24 h and then dehydrated, paraffin-embedded, and cut into 5-μm-thick sections. The sections were dewaxed, rehydrated, and stained by hematoxylin–eosin (H&E) or periodic acid–Schiff (PAS). The severity of peribronchial and perivascular inflammation was graded according to a semi-quantitative scoring system using a scale of 0–5 (0: normal, 1: low grade, 2: low to moderate grade, 3: moderate grade, 4: moderate to high grade, and 5: high grade).[27] [28]
Immunofluorescence staining
Lung tissues were collected, fixed in 4% paraformaldehyde overnight at 4°C, and then dehydrated in sucrose. Later, tissues were embedded in optimum cutting temperature (Tissue-Tek; Sakura, Tokyo, Japan) and serially sectioned in a cryostat into 15-μm-thick slices. The tissue sections were blocked for 1 h and incubated overnight with primary antibodies (anti-GABAA R α1-6 [sc-376282, Santa Cruz, Dallas, TX, USA, 1:50]) at 4°C in a wet box. Subsequently, the sections were incubated with the corresponding secondary antibodies for 1 h at room temperature. An inverted fluorescence microscope was used to capture the fluorescent images, and the intensity of GABAAR α1-6 staining was measured with Image J (National Institutes of Health, Bethesda, MD, USA).
Immunohistochemical staining
Paraffin sections were baked overnight at 65°C. Deparaffinization and hydration were performed in xylene and graded ethanol to distilled water. Antigen retrieval was performed in tris-ethylenediamine tetraacetic acid (EDTA) buffer in a microwave oven and cooled to room temperature. After washing, the endogenous peroxidase was removed using 0.3% H2 O2 , and the sections were subjected to blocking for 15 min. Then, the sections were incubated with primary antibody at room temperature for 2 h. The antibodies used were: anti-GABAAR α1-6 (sc-376282, Santa Cruz, California, USA 1:50); anti-GABAAR α1 (ab33299, Abcam, Cambridge, UK, 1:100); anti-GABA (ab8891, Abcam, 1:200); anti-TLR4 (sc-293072, Santa Cruz, 1:100); anti-MyD88 (ab2064, Abcam, 1:200); anti-NF-κB p65 (ab86299, Abcam, 1:200). After washing, the tissues were labeled with secondary antibodies for 1 h and developed with 3,3′-diaminobenzidine, followed by washing with tap water. Counterstaining was performed using hematoxylin, and the slides were rinsed and mounted with dibutylphthalate polystyrene xylene mounting medium. Finally, the sections were observed under a microscope and the results were measured with Image J.
Western blot
Lung tissues were washed with PBS and homogenized in radio immunoprecipitation assay lysis buffer to extract protein. Protein concentrations were measured using bicinchoninic acid (BCA) protein assays (Solarbio, Beijing, China). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the fractionated proteins were transferred to a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Waltham, MA, USA). The membranes were blocked with 5% (w/v) bovine serum albumin (Bioss, Beijing, China) and incubated with the diluted primary antibodies followed by incubation with secondary antibodies. The primary antibodies used were: anti-GABAA R α1-6 (sc-376282, Santa Cruz, 1:1000); anti-GABAA R α1 (ab33299, Abcam, 1:10,000); anti-TLR4 (14358S, Cell Signaling Technology, Danvers, MA, USA, 1:1000); anti-MyD88 (4283S, Cell Signaling Technology, 1:1000); anti-NF-κB p65 (8242T, Cell Signaling Technology, 1:1000); and anti-GAPDH (5174T, Cell Signaling Technology, 1:1000). The bands were scanned by electrochemiluminescence Western Blotting Substrate (Tanon, Shanghai, China) and analyzed by Image J.
Statistical analysis
All data were presented as mean ± standard deviation, or median (interquartile range). Comparison between groups was analyzed using Student t test or Wilcoxon rank sum test. Between-group differences were assessed using one-way analysis of variance followed by Bonferroni's post hoc test for multiple comparisons. GraphPad Prism 6.0 (GraphPad Inc., La Jolla, CA, USA) was used for statistical analysis. Excel software (Microsoft, Seattle, WA, USA) was used for processing of data on lung function. P values < 0.05 were considered indicative of statistical significance.
Results
Establishment of the asthma mouse model
When the concentration of methacholine were 25 to 100 mg/mL, significant differences were recorded in Rrs between the Control group and OVA group (25 mg/mL methacholine: 3.01 ± 1.94 cmH2 O·s·mL−1 vs. 7.03 ± 2.88 cmH2 O·s·mL−1 , P = 0.003; 50 mg/mL methacholine: 3.56 ± 2.39 cmH2 O·s·mL−1 vs. 8.84 ± 3.55 cmH2 O·s·mL−1 , P = 0.004; 100 mg/mL methacholine: 5.03 ± 3.32 cmH2 O·s·mL−1 vs. 11.40 ± 3.04 cmH2 O·s·mL−1 , P = 0.002). Compared with the OVA group, OVA + EA group showed a marked reduction in the Rrs at 25 to 100 mg/mL concentration of methacholine (25 mg/mL methacholine: 7.03 ± 2.88 cmH2 O·s·mL−1 vs . 3.57 ± 1.10 cmH2 O·s·mL−1 , P = 0.008; 50 mg/mL methacholine: 8.84 ± 3.55 cmH2 O·s·mL−1 vs . 4.65 ± 1.93 cmH2 O·s·mL−1 , P = 0.035; 100 mg/mL methacholine: 11.40 ± 3.04 cmH2 O·s·mL−1 vs. 7.02 ± 3.33 cmH2 O·s·mL−1 , P = 0.048) [Figure 2 ].
Figure 2: Respiratory resistence were assessed after incremental concentrations of methacholine (0, 3.125, 6.250, 12.500, 25.000, 50.000, and 100.000 mg/mL). EA: Electroacupuncture; OVA: Ovalbumin.
Mice in OVA group exhibited typical pathological features with extensive inflammatory cell infiltration at multiple sites compared with the Control group (inflammation score 5.00 [4.00–5.00] vs. 0 [0–1.00], P < 0.001). Inflammatory infiltration was significantly reduced in OVA + EA group compared with the OVA group (inflammation score 3.00 [3.00–3.25] vs. 5.00 [4.00–5.00], P = 0.010) [Figure 3 A, B].
Figure 3: Results of histological staining. (A) Micrographs of H&E stained lung tissue sections (100 × original magnification); (B) Micrographs of PAS-stained lung tissue sections (200 × original magnification); (C) H&E inflammation scores of lung tissue in each group; (D) PAS scores of lung tissue in each group. n = 5. ∗ P< 0.05, † P < 0.01. EA: Electroacupuncture; H&E: Hematoxylin–eosin; OVA: Ovalbumin; PAS: Periodic acid–Schiff.
The PAS score representing the goblet cells was significantly increased in the OVA group compared to the control group (PAS score 4.00 [3.00–4.00] vs. 0 [0–1.00], P < 0.001). In contrast to the OVA group, goblet cells were significantly reduced in the OVA + EA group (PAS score 4.00 [3.00–4.00] vs. 1.50 [1.00–2.00], P = 0.002) [Figure 3 C, D].
EA decreased inflammatory cell counts and cytokine levels in the BALF
The eosinophils, neutrophils, macrophages, and lymphocytes in the BALF were significantly increased in the OVA group (eosinophils 107.28 ± 48.71; neutrophils 44.94 ± 20.66; macrophages 41.67 ± 16.10; and lymphocytes 34.00 ± 15.65) compared to the Control group (eosinophils 0.70 ± 0.48; neutrophils 2.29 ± 2.14; macrophages 1.07 ± 0.26; and lymphocytes 6.38 ± 5.34) (P < 0.001). As compared with OVA group, treatment with EA significantly decreased counts of inflammatory cells in the BALF (P < 0.001), including eosinophils (15.56 ± 8.68); neutrophils (13.56 ± 6.55); macrophages (15.50 ± 8.82); and lymphocytes (11.63 ± 6.70) [Table 1 ].
Table 1 -
Inflammatory cell counts in each group.
Inflammatory cells
Control (n = 5)
OVA (n = 5)
OVA+EA (n = 5)
OVA+EA+antagonist (n = 5)
t values, P values∗
t values, P values†
t values, P values‡
Eosinophils
0.70 ± 0.48
107.28 ± 48.71
15.56 ± 8.68
26.06 ± 12.10
6.918, <0.001
5.861, <0.001
2.228, 0.039
Neutrophils
2.29 ± 2.14
44.94 ± 20.66
13.56 ± 6.55
22.50 ± 10.85
6.493, <0.001
4.578, <0.001
2.230, 0.038
Macrophages
1.07 ± 0.26
41.67 ± 16.10
15.50 ± 8.82
31.72 ± 13.53
7.972, <0.001
4.507, <0.001
3.176, 0.005
Lymphocytes
6.38 ± 5.34
34.00 ± 15.65
11.63 ± 6.70
23.50 ± 11.70
5.285, <0.001
4.158, <0.001
4.785, 0.012
Data are shown as mean ± standard deviation.
∗ OVA group versus Control group.
† OVA + EA group versus OVA group.
‡ OVA + EA + antagonist group versus OVA + EA group. EA: Electroacupuncture; OVA: Ovalbumin.
The levels of IL-4, IL-5, and IL-13 in the OVA group (IL-4 564.47 ± 21.26 pg/mL; IL-5 166.40 ± 32.37 pg/mL; and IL-13 175.46 ± 5.48 pg/mL) were significantly higher than those in the Control group (IL-4 50.39 ± 7.38 pg/mL; IL-5 12.25 ± 11.75 pg/mL; and IL-13 85.59 ± 5.51 pg/mL) (P < 0.01). Compared with the OVA group, the levels were significantly reduced in OVA + EA group (IL-4 77.77 ± 11.66 pg/mL; IL-5 24.93 ± 6.78 pg/mL; and IL-13 115.45 ± 10.22 pg/mL) (P < 0.01) [Table 2 ].
Table 2 -
Cytokine levels in the bronchoalveolar lavage fluid for each group.
Cytokine levels (pg/mL)
Control (n = 5)
OVA (n = 5)
OVA+EA (n = 5)
OVA+EA+antagonist (n = 5)
t values, P values∗
t values, P values†
t values, P values‡
IL-4
50.39 ± 7.38
564.47 ± 21.26
77.77 ± 11.66
111.24 ± 17.02
39.560, <0.001
34.760, <0.001
2.809, 0.048
IL-5
12.25 ± 11.75
166.40 ± 32.37
24.93 ± 6.78
54.79 ± 15.19
7.755, 0.002
7.410, 0.002
3.109, 0.036
IL-13
85.59 ± 5.51
175.46 ± 5.48
115.45 ± 10.22
147.90 ± 7.03
20.020, <0.001
8.962, <0.001
4.530, 0.011
Data are shown as mean ± standard deviation. IL: Interleukin.
∗ OVA group versus Control group.
† OVA + EA group versus OVA group.
‡ OVA + EA + antagonist group versus OVA + EA group. EA: Electroacupuncture; OVA: Ovalbumin.
Expression of GABAAR and level of GABA were increased in asthmatic mice treated with EA
Results of Western blot and immunohistochemical staining showed the expression of GABAAR α1 and GABAAR α1-6 [Figures 4 A, D, and F]. There were no significant differences between Control and EA groups (P > 0.05). Compared with the Control group, both GABAAR α1 and GABAAR α1-6 were up-regulated in the OVA group (P < 0.01), and both were up-regulated in OVA + EA group compared with the OVA group (P < 0.05) [Figures 4 B, C, E, and G]. The immunofluorescence results of the expressions of GABAAR α1-6 [Figure 4 H] were consistent with the above results [Figure 4 I].
Figure 4: Expressions of GABA and GABAAR subunits in lung tissue. (A) Western blots showing the expressions of GABAAR α1 and GABAAR α1-6 proteins; (B) Quantitative analysis of GABAAR α1; (C) Quantitative analysis of GABAAR α1-6; (D) Expression of GABAAR α1 by immunohistochemical staining; (E) Quantitative analysis of GABAAR α1; (F) Expression of GABAAR α1-6 by immunohistochemical staining; (G) Quantitative analysis of GABAAR α1-6; (H) Expression of GABAAR α1-6 by immunofluorescence staining; (I) Quantitative analysis of GABAAR α1-6; (J) Expression of GABA by immunohistochemical staining; (K) Quantitative analysis of GABA. Data are presented as mean ± SD, n = 5. ∗ P < 0.05, † P < 0.01. EA: Electroacupuncture; GABA: γ-aminobutyric acid; GABAAR: GABA type A receptor; OVA: Ovalbumin; SD: Standard deviation. EA: Electroacupuncture; OVA: Ovalbumin.
In addition, there were no significant differences in the expression of GABA between the Control and EA groups (P = 0.429). GABA was significantly up-regulated in the OVA group compared with the Control group (P < 0.001), and it was increased in OVA + EA group compared with the OVA group (P < 0.001) [Figures 4 J, K].
GABAAR antagonist attenuated anti-inflammation effects of EA in asthmatic mice
GABAAR antagonist weakened the curative effects, as Rrs in the OVA + EA + antagonist group were higher than those in the OVA + EA group (25 mg/mL methacholine: 5.29 ± 0.89 cmH2 O·s·mL−1 vs. 3.57 ± 1.10 cmH2 O·s·mL−1 , P = 0.045; 50 mg/mL methacholine: 7.67 ± 1.86 cmH2 O·s·mL−1 vs. 4.65 ± 1.93 cmH2 O·s·mL−1 , P = 0.049) [Figure 2 ].
After inhibition of GABAAR, the OVA + EA + antagonist group showed more severe infiltration of inflammatory cells in alveoli, congestion of blood capillaries, and thickening of alveolar septa than in the OVA + EA group (inflammation score 4.50 [4.00–5.00] vs. 3.00 [3.00–3.25], P = 0.038) [Figures 3 A, B].
The PAS scores in the OVA + EA + antagonist group were significantly greater than those in the OVA + EA group (PAS score 3.00 [2.75–3.25] vs. 1.50 [1.00–2.00], P = 0.047) [Figures 3 C, D].
GABAAR antagonist affected inflammatory cell counts and cytokine levels in the BALF
The therapeutic efficacy of EA was attenuated after the administration of GABAAR antagonist, as the inflammatory cells in OVA + EA + antagonist group were significantly higher than those in the OVA + EA group, including eosinophils (26.06 ± 12.10 vs. 15.56 ± 8.68; P = 0.039); neutrophils (22.50 ± 10.85 vs. 13.56 ± 6.55; P = 0.038); macrophages (31.72 ± 13.53 vs. 15.50 ± 8.82; P = 0.005); and lymphocytes (23.50 ± 11.70 vs. 11.63 ± 6.70; P = 0.012) [Table 1 ].
Similarly, the cytokine levels in the OVA + EA + antagonist group were significantly higher than those in the OVA + EA group (IL-4 111.24 ± 17.02 pg/mL vs. 77.77 ± 11.66 pg/mL, P = 0.048; IL-5 54.79 ± 15.19 pg/mL vs. 24.93 ± 6.78 pg/mL, P = 0.036; IL-13 147.90 ± 7.03 pg/mL vs. 115.45 ± 10.22 pg/mL, P = 0.011) [Table 2 ].
TLR4/MyD88/NF-κB signaling pathway was inhibited in asthmatic mice treated with EA
The expressions of TLR4, MyD88, and NF-κB in the OVA group were significantly higher than those in the Control and EA groups (P < 0.01). Compared with the OVA group, TLR4, MyD88, and NF-κB were significantly decreased in OVA + EA group (P < 0.05) [Figure 5 ].
Figure 5: Effects of EA on TLR4/MyD88/NF-κB signaling pathway. (A) Western blots showing the expressions of TLR4, MyD88, and NF-κB proteins; (B) Quantitative analysis of TLR4; (C) Quantitative analysis of MyD88; (D) Quantitative analysis of NF-κB. Data are presented as mean ± SD, n = 5. ∗ P < 0.05, † P < 0.01. EA: Electroacupuncture; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; MyD88: Myeloid differentiation factor 88; NF-κB: Nuclear factor-kappa B; OVA: Ovalbumin; SD: Standard deviation; TLR4: Toll-like receptor 4.
GABAAR antagonist affected the down-regulation of TLR4/MyD88/NF-κB signaling pathway in asthmatic mice treated with EA
Compared with the OVA + EA group, TLR4, MyD88, and NF-κB were up-regulated in OVA + EA + antagonist group (P < 0.01). The results of immunohistochemical staining were consistent with those of Western blot assay (P < 0.01) [Figure 6 ].
Figure 6: Effects of GABAAR antagonist on TLR4/MyD88/NF-κB signaling pathway. (A) Western blots showing the expressions of TLR4, MyD88, and NF-κB proteins; (B) Quantitative analysis of TLR4; (C) Quantitative analysis of MyD88; (D) Quantitative analysis of NF-κB; (E) Expressions of TLR4 by immunohistochemical staining; (F) Quantitative analysis of TLR4; (G) Expressions of MyD88 by immunohistochemical staining; (H) Quantitative analysis of MyD88; (I) Expressions of NF-κB by immunohistochemical staining; (J) Quantitative analysis of NF-κB. Data presented as mean ± SD, n = 5. ∗ P < 0.01. EA: Electroacupuncture; GABAAR: GABA type A receptor; MyD88: Myeloid Differentiation Factor 88; NF-κB: Nuclear factor-kappa B; OVA: Ovalbumin; SD: Standard deviation; TLR4: Toll-like receptor 4.
Discussion
In this study, we successfully established a mouse model of asthma and demonstrated that EA alleviated airway hyperresponsiveness and inflammation in asthmatic mice. The level of GABA and expression of GABAAR were elevated in OVA + EA group compared with the OVA group, which suggested an association between the GABAergic system and EA in relieving asthma. Besides, we found the protective effects of EA in asthma were weakened by GABAAR antagonist, as indicated by an increase in inflammatory infiltration, airway resistance, inflammatory cells, and cytokine levels in the OVA + EA + antagonist group. In addition, TLR4/MyD88/NF-κB signaling pathway was down-regulated in asthmatic mice treated with EA, while GABAAR antagonist inhibited the down-regulation.
We confirmed that EA could alleviate asthma symptoms, which was consistent with previous studies.[15] [14] [29] [30]
This study found an increased level of GABA and increased expressions of GABAAR α1 and GABAAR α1-6 in the lung tissue of asthmatic mice, suggesting the involvement of the GABAergic system in mediating the therapeutic effect of EA in asthma, which was consistent with previous research.[31] [32] [33] [34] [35,36]
In our study, the administration of GABAAR antagonist diminished the therapeutic effects of EA in asthma, which further confirmed that the EA-induced alleviation of airway inflammation in asthma may be associated with activation of the GABAergic system. GABAARs are expressed in ASM, airway epithelium, and inflammatory cells, and GABA is associated with goblet cells; therefore, the GABAergic system is considered as an emerging target for asthma.[37,38] [5] [39]
We found that TLR4/MyD88/NF-κB were inhibited in OVA + EA group, suggesting that EA suppresses asthma airway inflammation by inhibiting TLR4/MyD88/NF-κB. Inhibition of TLR4/MyD88/NF-κB can attenuate pathological mechanisms of asthma and plays an important role in the protection of airways against allergic response and inflammatory pathology.[40] [10,41] [42] [43] [44] [45] [46]
Some limitations of our study should be acknowledged. First, we did not use GABAAR knockdown mice, which would have provided more convincing results. Second, different kinds of EA treatments may have different effects, while we only used one intervention method in this study. Third, this mouse model represents only one phenotypic expression of asthma and may not be representative of all types or experimental models of asthma. Preclinical animal models cannot be fully valid due to the uncertainties introduced by species differences, and may not reliably and consistently predict human responses.[47]
In conclusion, the activation of GABAergic system may be involved in mediating the therapeutic effect of EA in asthma by suppressing the TLR4/MyD88/NF-κB signaling pathway. Our research may provide a novel perspective of the mechanism of the therapeutic effects of EA in asthma. Our findings may support further application and development of EA in clinical practice. Further studies are required to characterize the specific mechanism by which EA attenuates asthma.
Funding
This work was supported a grant from the Scientific Research Fund of China–Japan Friendship Hospital (No. 2017-RC-3).
Conflicts of interest
None.
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