Characterized by sensitive trachea and bronchial inflammation, asthma currently constitutes a frequently occurring chronic inflammatory respiratory disease [1
]. Asthma usually involves different reversible airflow obstruction, resulting in repeated wheezing, shortness of breath, and rapid coughing that occurs most often in the morning and at night [2
]. Environmental and climate changes have engendered a rise in the incidence of asthma worldwide. Incidence rates for allergic asthma are particularly high in children, leading to the condition taking on particular importance [4
Asthma symptoms are triggered by a variety of immune, genetic, and environment-related interactions [5
]. The activation of Type 2 T helper (Th2) cells is associated with inflammatory reactions, and this causes tracheal hypersensitivity owing to cytokine release, including interleukin (IL)-13, -5, and -4 [7
]. Additionally, B cells are spurred by Th2 cells to activate mast cells, which release more inflammatory and allergic mediators, leading to Immunoglobulin E (IgE) production [9
]. Corticosteroids are the most commonly used drugs against asthma clinically [10
]. However, these drugs are unable to prevent asthma symptoms in some patients. Furthermore, these drugs can produce unacceptable side effects, which limit their application. Therefore, new drugs must be developed that directly alleviate the symptoms of asthma without excessive side effects.
Previous studies indicated that administration of Dermatophagoides pteronyssinus
(Der p) significantly increased pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in mice lung tissues [11
]. IL-6 play a crucial role in the production of Th2 cytokines and inflammatory cell recruitment in the asthmatic airways of mice [12
]. Increased inflammatory cytokines in the airway epithelium, such as IL-1β, IL-4, and TNF-α, were also observed in asthma [13
]. Phosphoinositide 3-kinases (PI3K)/Akt and nuclear factor-κB (NF-κB) pathway participate in the regulation of inflammatory mediators [14
]. NF-κB is a key transcription factor in the pathogenesis of asthma, and phosphorylation of NF-κB regulates the physiology of many cells, including inflammation, immune response, apoptosis, and cell death [15
Garlic (Allium sativum
L.) of the family Liliaceae is a perennial herb with a well-known and pungent odor. Garlic has been widely used as a food ingredient and spice since ancient times. Garlic also has favorable physiological activity, and it has been used to treat various conditions such as cardiovascular disease [17
] and hyperlipidemia [18
]. The major active components of garlic are sulfur compounds containing alliin, allicin, diallylsuifide, diallyl disulfide, diallyltrisulfide, and S-allylcysteine (SAC) [19
]. A previous study found that water extracts of garlic can increase T cell cytotoxicity and lymphoproliferative capacity [20
]. In Taiwan, garlic is used as a traditional folk medicine to prevent cold, cough, asthma, and bronchitis. Some people also place garlic in boiling water to obtain garlic water and drink it to prevent asthma attacks. In our previous studies, we have confirmed the protective effect of purified garlic extract on dust Der p-induced allergic airway inflammation in mice [21
]; however, the mechanisms and the active components are unclear. To investigate, we used different polar solvents to obtain stratified extracts of garlic and estimated their effects on and relevant mechanisms behind mouse allergic airway inflammation engendered by Der p.
Asthma is marked by high concentrations of Th2 cytokines and increased immunoglobulin IgE and IgG1 [23
]. A Der p–induced animal model is considered a suitable platform for studying allergic asthma because it shares many symptoms with human asthma [25
]. This model is characterized by a thickening of the bronchial wall, airway mucosal edema, excessive mucus secretion, and increased inflammatory cell infiltration into the lung [26
]. By using mice with Der p–induced allergic asthma, we executed the current study to evaluate the effects exerted by different fractions of garlic extracts. In our previous study, garlic extracts inhibited Der p-induced asthmatic airway inflammation through regulation of IgE and IgG1a levels and restoring the balance of Th1 and Th2 cytokines [21
]. In the current study, we evaluated the effects and the mechanisms exerted by different fraction extracts of garlic using Der p–induced allergic asthma in mice.
Our results reveal that the WA fraction significantly reduced the observed serum levels of IgE and IgG1, inhibited the creation of chemokines, increased the levels of serum IgG2a, and balanced Th1 and Th2 cytokines in mice with Der p–induced allergic asthma. This is the first time that a WA fraction of garlic extract has been shown to grant protection against allergic asthma engendered by Der p in a murine model. The primary virulence factor of asthma is the airway, and it is related to an imbalance of Th1 and Th2 cell hormones alongside increases in various immune cells and inflammatory mediators [27
The extent of inflammatory cell infiltration in the lung affects asthma severity. Accumulation of eosinophils and lymphocytes around perivascular and per bronchial spaces causes bronchial epithelial hyperplasia and goblet cell modification in the bronchus, in addition to causing macrophage aggregation in the terminal bronchioles and alveolar spaces [28
]. In the present study, Der p–induced asthmatic mice exhibited an elevation in the total cell count in the BALF. Histopathological analysis of their lung tissue revealed an increase in inflammatory cell infiltration in the lungs. However, treatment with the WA effectively inhibited the total cells, reduced inflammatory cells, and decreased inflammatory cell infiltration. Together, these findings point toward treatment with the WA having an anti-inflammatory effect on Der p–induced allergic asthma.
The primary role of chemokines is the migration of chemotactic cells, which migrate to the source of the chemokines according to the signal given by increased chemokine concentration [29
]. Some chemokines control immune cell chemotaxis. Through releasing cytokines, T lymphocytes execute an integral function in immune and inflammatory response regulation processes [30
]. In respiratory diseases, the literature confirms imbalance and dysfunction of Th1 and Th2 cells in the subpopulation of helper T lymphocytes as a key mechanism leading to allergic asthma [31
]. Respiratory tract inflammation in asthma can be caused by Th2 cytokines [32
]. In the immune system, Th2 cytokines, namely IL-5, -13, and -4, regulate the creation of IgE [34
]. IL-13, -4, and -5 are expressed abnormally in the allergic asthma mouse model [8
]. In the present study, Th2 as well as Th1 cytokines were examined in mouse BALF. The results reveal the Der p–induced mouse Th2 cytokines IL-13, -4, and -5 to be significantly increased and IFN-γ and IL-12 (both constituting Th1 cytokines) to be significantly reduced. The WA fraction of garlic extract was noted to decrease the IL-13, -4, and -5 levels but to further increase IFN-γ levels. These findings are consistent with those from cell number analysis conducted on mouse BALF and with those from pathology analysis of mouse lung tissue. Overall, these results indicate that a WA of garlic lessens airway inflammation by regulating the Th1 and Th2 cytokine balance (i.e., Th2 cytokines are reduced and Th1 cytokines are increased).
The serum levels of Der p-specific immunoglobulins IgE and IgG1a were significantly reduced after treatment with the WA fraction. Previous studies have found that IL-13 and -4 stimulate B cells to synthesize IgE, a process associated with mast cell degranulation by cross-linking with IgE receptors [30
]. Transcription factor NF-κB activation is affiliated with the Th2 cytokine creation as well as airway inflammation during allergic reactions [35
]. Conversely, NF-κB signaling pathway inhibition was found to reduce Th2 cytokine creation and airway inflammation in asthmatic mice [36
]. In this study, Der p–specific IgE inhibition engendered by WA fraction treatment might have inhibited the phosphorylation of NF-κB and mediated IL-13 and IL-4 switching to IgE.
NF-κB is a downstream component of the IL-6/PI3K/Akt pathway and is activated by phosphorylation of IKK by the PI3K/Akt pathway, resulting in IκB degradation. Thus, the Il-6/PI3K/Akt/NF-κB pathway causes enhanced inflammatory expression in Der p-induced allergic asthma. This study indicated that the PI3K/Akt/NF-κB pathway is an effective target for the WA fraction against inflammation response induced by Der p in A549 cells. The WA fraction expressed anti-inflammatory activity by inhibiting the IL-6/PI3K/Akt/NF-κB pathway.
Allergic asthma is a chronic respiratory disease. Reactive oxygen species (ROS) in the environment can cause oxidative damage to cells, and it is very likely to participate in the process of dust mites promoting sensitization. During the inflammation of the lungs, the imbalance of oxidants and antioxidants in the body is an important factor in causing cell damage. ROS is involved in maintaining the “redox homeostasis” of cells to protect cells from oxidative stress. However, ROS is produced in excess, the most common cause of which is the promotion of inflammatory cytokines such as NF-κB. Oxidative stress is a harmful process that can cause damage to the airways and lungs. A previous study demonstrated that garlic essential oil and three of the individual compounds (diallyl trisulfide, ajoene, and allicin) inhibited spontaneous ROS production by neutrophils [37
]. Moreover, PI3K plays an important role in the regulation of ROS production by human neutrophils. In this study, we demonstrated that WA fraction expressed anti-inflammatory activity by inhibiting the IL-6/PI3K/Akt/NF-κB pathway. Also, the pathological changes of airway were reversed after treating with WA fraction. Thus, the inhibition of ROS production may be one of the possible mechanisms in this work.
Mechanisms of allergic inflammation can contribute to the development of atherosclerosis and the pathogenesis of its clinical manifestations. Also, allergy-related cells such as basophils and mast cells may play a role in the regulation of lipid metabolism. Furthermore, it had been reported that allergic asthma and serum total IgE are associated with formation of arterial thrombosis [38
]. In this study, we demonstrated that WA fraction will lower the IgE expression. Thus, the WA fraction of garlic extract may have potential in decreasing the allergy-related cardiovascular problems.
Recently, studies have indicated that obesity may have an effect on the induction of asthma as underlying the enhancement of asthma-associated inflammation due to increased (unbalanced) production of proinflammatory mediators in overgrown, inflamed, and dysregulated fat tissue [39
]. In this study, we reported that WA fraction of garlic extract is effective on the restoration of Th1/Th2-related cytokine expression. Thus, we proposed that WA fraction of garlic extract may have positive effects on the treatment of obesity-induced allergic asthma.
The results of this study reveal that the WA fraction expressed the greatest anti-inflammatory effect in mice with Der p–induced allergic asthma. These findings are of some interest and indicate that the water-soluble compounds of garlic are likely to contain major active components. A previous study indicated that sulfide-derived compounds play major roles in the pharmacological activities of garlic [40
]. SAC is an important hydrophilic component of garlic, and it was observed in the HPLC profile in this study. Previous research has found that SAC has anti-inflammatory activity [41
]. It is therefore worth investigating further whether SAC or other water-soluble components of garlic act against Der p–engendered allergic asthma.
4. Materials and Methods
4.1. Chemicals and Drugs
Dichloromethane, ethylacetate, n-butanol, and n-hexane were purchased from Sigma-Aldrich (Sigma-Aldrich Co., St. Louis, MO, USA). Akt, p-Akt, IL-6, NF-κB, p-NF-κB and PI3-K antibodies were purchased from Abcam, Cambridge, USA. Ikk antibody was purchased from Cell Signaling Technology Danvers, USA. 1 g of Der p (Thermo Fisher Scientific, Waltham, MA, USA) was extracted using diethyl ether and then the filtrate was lyophilised to obtain Der p) extract. The extract was dissolved in sterile saline, filtered through a 0.22 μm filter, and stored at −80 °C before use.
4.2. Preparation of Extracts
Garlic was collected in Taichung City, Taiwan. The peeled and non-chopped garlic (10 kg) was weighed and extracted using various polar solvents. First, the garlic was extracted using n-hexane (20L) a minimum of three times. Filtrates were subsequently collected and concentrated to obtain HE. The residues were then extracted in sequence with dichloromethane, ethyl acetate, n-butanol, and water as in the aforementioned steps, resulting in five fractional extracts of garlic: HE (21.7 g, 0.22%), DI (25.7 g, 0.26%), EA (18.1 g, 0.18%), BU (191.0 g, 1.91%), and WA (514.0 g, 5.14%), as seen in Figure 9
4.3. Experimental Animals
Male BALB/c mice that were determined to be aged six to eight weeks were procured from BioLASCO Taiwan (Taipei, Taiwan) and housed and cared for according to the recommendations set forth by the NIH Guide for the Care and Use of Laboratory Animals. The entirety of the animals were raised in a specific-pathogen-free lab involving a fixed temperature, namely 22 ± 1 °C, and relative humidity, namely 55% ± 5%, in addition to a 12-h light–dark cycle. In addition, water and regulation laboratory chow were available to the acquired animals ad libitum. The Institutional Animal Care and Use Committee of Da-Yeh University (Permit Number: 105024, 26 October 2016) ratified the procedures of the executed experiment.
4.4. Establishing an Allergic Asthma Model
Through random division, the animals were placed into seven groups (n
= 10). Group 1 (control) and Group 2 (Der p) were orally given 0.9% saline water. Groups 3–7 were orally administered with 80 mg/kg of one of the extracts (HE, DI, EA, BU, and WA, respectively). The doses were chosen according to previous studies [21
]. The investigator was blinded to the treatment groups.
As shown in Figure 10
, on days 1 and 7, Groups 2–7 were subcutaneously injected with emulsion (50 μL) containing Der p (50 μg) in incomplete Freund’s adjuvant (Difco, Detroit, MI, USA); the injections were delivered at the base of their tails; on these days, the acquired animals in Group 1 were injected with 50 μL of saline. On day 14, a shot of tiletamine/zolazepam (Zoletil®
50, Virbac Corporation, Carros, France), given intraperitoneally at 20 mg/kg body weight, was administered to anesthetize the mice. With the exception of Group 1, the acquired mice were subjected to treatment using an intratracheal instillation of 50 μL of Der p (1.0 mg/mL), followed by maintaining them upright for a period of 1 min until the restoration of normal breathing [43
]. After 72 h since the last Der p challenge, mice were sacrificed. Lung and trachea tissues were removed for histological analysis. Serum and BALF were collected for analysis.
4.5. Preparing BALF and Counting Inflammatory Cells
In the executed study, total cells and BALF were collected according to He et al. [44
]. Briefly, after 72 h since the last Der p induction, the mice were euthanized under anesthesia. Subsequently, the lungs were lavaged using a syringe containing 1 mL of ice sterile saline solution. This process was repeated three times, and then the lavage fluid was collected. The collected lavage fluid was subjected to a 10-min centrifugation process executed at 4 °C and 1200 rpm, followed by gathering and keeping the supernatant at −80 °C. PBS constituted the medium for resuspending each tube’s cell pellet; a hemocytometer was used to measure the total cell count.
4.6. Histological Lung Assessment
Each lung was placed in an embedding cassette after being fixed in 10% neutral formalin. The lung tissue was first dehydrated, and, after standing overnight, the abovementioned tissue was inserted into paraffin and chopped into approximately 4–5-μm sections by using a microtome. The experiment was performed after the paraffin was dried and fixed. After the execution of staining performed by employing hematoxylin and eosin (H&E), the specific conditions and histopathological changes of each group of lung cells were examined under a light microscope, during which the degree of injury was compared and photographed. The severity of lesions in the lungs was graded according to the methods described by Shackelford et al. [22
]. The degree of lesions was graded from one to five depending on severity: 1 = minimal (<1%); 2: slight (1–25%); 3 = moderate (26–50%); 4 = moderately severe (51–75%); 5 = severe/high (76–100%). Mean histopathological scores were calculated by dividing the sum of the score per grade of affected mice by the total number of examined mice.
4.7. Measurement of Type 1 T helper (Th1) and Th2 Cytokine Concentrations in BALF
Following previous studies [45
], IFN-γ and IL-12, -13, -4, and -5 levels in the lavage fluids of the lungs from the control, Der p, and garlic extract–fed mice ventilated ex vivo with either the protective or the injurious strategy were determined in the laboratory by using mice-specific ELISA kits (BioSource, Camarillo, CA, USA). For subsequent analyses, a Bio-Plex assay was executed to determine the IFN-γ and IL-12, -13, -4, and -5 levels, which were then read on a Luminex-100 system (Luminex Co., Austin, TX, USA). The analysis of the results was executed via Bio-Plex Manager software (Bio-Rad Laboratories, Hercules, CA, USA).
4.8. Der p–Specific Serum IgE/ IgG1/ IgG2a Concentrations
In this study, serum IgE/IgG1/IgG2a concentrations were quantified via appropriate ELISA kits per instructions provided by the relevant manufacturer (Promega, Madison, WI, USA).
4.9. Measurement of Pro-Inflammatory Cytokine Concentrations in Lung Tissues
In this study, the inflammatory cytokine IL-1β (ab100704) / IL-6 (ab100712) / TNF-α (ab100785) concentrations were quantified by appropriate ELISA Kits according to the instructions provided by the relevant manufacturer (Abcam, Cambridge, UK).
4.10. Cell Culture
A549 cells were purchased from the Culture Collection and Research Center of the Food Industry Institute (Hsin-Chiu City, Taiwan). The cells were cultured in F-12K medium containing 10% fetal bovine serum (FBS) and 1% Penicillin-streptomycin-amphotercin solution (PSA), and placed in a 37 °C, 5% CO2 incubator for growth. When the cells reached approximately 80% confluence, subculture was carried out; the cells were suspended in a liquid by adding 0.05% Trypsin-EDTA and cultured in a plate at a number of 1 × 106 cells. After 24 hours of plating, Der p (5 μg/mL) and WA extract (100, 200, 500, or 1000 μg/mL) was added and reacted for 24 h The cells were harvested via treatment with cell lysis buffer for further studies.
4.11. Western Blotting Analysis
Total protein lysate from the cells was extracted in lysis buffer containing a mixture of protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentration was determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Protein lysate (50 μg) was separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked in Tris buffered saline containing 3% bovine serum albumin in Tween at 25 °C for 1 hour. After washing, the membrane was incubated overnight at 4 °C in the following primary anti-IL-6 (1:1000), PI3-K (1:1000), IκB kinase (Ikk) (1:500), p-Akt (1:500), Akt (1:500), p-NF-κB (1:500), NF-κB (1:1000) antibodies. After washing the membrane with TBS-T, the blot was incubated in a 1/5000 dilution of horseradish peroxidase-conjugated secondary antibody at 25 °C for 1 hour. Protein bands were visualized using an enhanced chemiluminescence kit (PerkinElmer, Boston, MA, USA). Actin (Millipore) was used as an internal control. The optical density of the bands were determined by software.
4.12. HPLC Analysis of the WA
For the WA and standard (S-allylcysteine, SAC), a high-performance liquid chromatography (HPLC) profile was established. The instruments comprised a Shimadzu LC-10Avp HPLC system and SPD-M10A Diode array detector. Analyses were executed in the experiment by employing a COSMOSIL 5C18-AR II column (4.6 × 250 mm2, 5 µm), with the mobile phase being observed to contain acetonitrile and 10 mM KH2PO4 with an isocratic elution (2:98). All standards and samples in the experiment were passed through a 0.45-µm Minipore filter prior to injection (10 µL) into the column. A 220-nm detection wavelength was employed, and the determined flow rate was 1.0 mL/min. Each analysis required 20 min.
4.13. Statistical Analysis
Herein, the derived results are presented as the mean ± SEM according to each group’s sample number (n). With regard to the differences among multiple groups, the corresponding significance was evaluated using analysis of variance (ANOVA). On establishing significance between groups, Duncan’s multiple range test was employed to compare two specific groups’ mean values via SPSS for Windows, version 17 (IBM, Chicago, IL, USA). In addition, statistical significance was set in this study to be p < 0.05.