Inflammatory diseases are associated with cytokine and adhesion molecule expression levels[1–2]. Asthma and chronic obstructive pulmonary disease are obstructive airway diseases involving chronic inflammation of the respiratory tract, but having two distinct modes of action with different patterns of inflammatory cell and mediator involvement being observed[3]. Allergic asthma is characterized by airway hyper-responsiveness to a variety of specific and nonspecific stimuli including chronic pulmonary eosinophilia, elevated serum immunoglobulin E (IgE), and excessive airway mucus production[4]. IgE is an important mediator of allergic reactions including allergic asthma and plays a central role in asthma-related symptoms, airway inflammation, and possibly airway remodeling[5]. The pathophysiology of asthma is thought to be mediated by CD4+T lymphocytes producing a type 2 cytokine profile[6]. Binding of IgE molecules to the surface of an immune cell sensitizes the cell to the specific allergen. The sensitized immune cell immediately expresses an inflammatory response, including the release of histamine, which induces the early phase of an allergic reaction. After IgE release, the immune cells synthesize other inflammatory molecules such as interleukins (ILs)[7–8].
Immunosuppressive drugs are currently being used to control undesired immune responses, such as autoimmune diseases, allergies, and allograft rejection. FK506, cyclophosphamide (CTX) and prednisone are typical immunosuppressive drugs that are being used in the clinical treatment for many years[9].
Pinus koraiensis (P. koraiensis) also known as the Korean pine, is a species of Pinus in the Pinaceae family. Most Pinus species grow in the northern hemisphere and some have been used in folk medicine for a long time. Previous studies have reported the anti-oxidant and anti-inflammatory activities of the pine pollen[10] and the anti-nociception and anti-inflammatory effects of the pine bark and its essential oil[11]. Larix kaempferi (L. kaempferi) belongs to the family Pinaceae in the Pinopsida class. It is a medium-sized to large deciduous coniferous tree reaching 20−40 meters in height, with a trunk up to 1 meter in diameter[12]. The extract oil from L. kaempferi has been used in folk remedies to reduce allergic reactions[13]. Several previous studies have examined the alleviating effect of P. koraiensis on allergic dermatitis in a mouse model[12,14– 15]. In addition, L. kaempferi effectively suppresses the levels of serum IgE and proinflammatory cytokines such as ILs, and affects mast cell appearance[4,16– 17]. L. kaempferi, regarded as an effective medicinal plant, contains active terpene compounds with effective pharmacological molecules.
Volatile organic compounds (VOCs) are reported to be associated with asthma and immune responses[18]. However, to the best of our knowledge, there are no reports indicating that exposure to VOCs of P. koraiensis or L. kaempferi reduces inflammatory symptoms and, in particular, affects specific IgE and cytokine release. In addition, the mechanisms underlying the alleviative effects of VOCs have not been elucidated. The aim of the current study was to determine whether exposure to VOCs improves the inflammation in a mouse LPS-induced inflammatory model and is a suitable candidate for use as a pharmaceutical and functional material.
Materials and methods
Animal experiments
BALB/c mice (7 weeks old) were purchased from Koatech (Pyeongtaek, Republic of Korea) and housed in polycarbonate cages with P. koraiensis or L. kaempferi panels and corncob bedding, in an environmentally controlled room [temperature, (23±2) °C; relative humidity, (50±10)%; frequent ventilation; and a 12:12 hours light-dark cycle]. The animal experiments were approved by the Chungbuk National University Animal Care and Use Committee (Cheongju, Korea) and all procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (Bethesda, MD, USA). Lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO, USA) dissolved in phosphate buffered saline (PBS) was used for the induction of inflammation in BALB/c mice. LPS was regularly administered (100 μg/kg i.p. with 100 μL of PBS, 1 mg/kg i.n. with 50 μL of PBS) via the intraperitoneal and intranasal route for 7 days. The VOC-untreated groups included the vehicle-treated (VE) group, the LPS-treated (LPS) group, and the LPS with anti-inflammatory drug dexamethasone-treated (DEX) group. The VOC-treated groups included the LPS+VOC of P. koraiensis group (P. koraiensis panel, 1 026 cm3) and the LPS+VOC of L. kaempferi group (L. kaempferi panel, 1 026 cm3). After completion of treatments, the mice were sacrificed by ether inhalation, and the lungs and blood were collected for analysis.
Serological analysis of serum
At the end of the experiment, blood samples were collected directly from the inferior vena cava using a 1 mL syringe. Serum was obtained by centrifuging the blood at 3 000 g for 10 minutes at 4 °C and stored at −70 °C for further use. Serum IgE and prostaglandin E2 (PGE2) levels were measured using the Mouse IgE Ready-Set-Go ELISA kits (eBioscience, San Diego, CA, USA) and Mouse PGE2 ELISA Ready-Set-Go kits (eBioscience, San Diego, CA, USA) according to the manufacturer's instructions.
Isolation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of each treatment group (VE, LPS, DEX, LPS+VOC of P. koraiensis, and LPS+VOC of L. kaempferi) as previously described[17]. Briefly, peripheral blood drawn from the inferior vena cava was collected in heparin vials, immediately diluted with an equal volume of PBS without calcium and magnesium and overlaid 1 : 1 on a Percoll® solution. After centrifugation at 400 g for 45 minutes at room temperature, the cells at the interface between the plasma and Percoll solution were harvested and treated with 0.83% NH4Cl in a tris-base buffer (pH7.2) for 5 minutes to lyse the remaining erythrocytes. The resulting PBMCs were prepared for RNA isolation with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA).
Total RNA extraction and real-time polymerase chain reaction amplification
Total RNA was extracted from mouse skin using TRIzol reagent according to the manufacturer's instructions. RNA concentrations were assessed using a microplate spectrophotometer (Epoch; BioTek Instruments, Winooski, VT, USA) at 260 nm. RNA quality was evaluated by performing electrophoresis on 1% agarose gel. Total RNA (1 μg) was reverse transcribed into first-strand complementary DNA (cDNA) using the Moloney murine leukemia virus reverse transcriptase (Invitrogen Life Technologies) and random primers (9-mer; Takara Bio, Otsu, Shiga, Japan). Each cDNA sample (1 μL) was amplified with 10 μL of 2 × SYBR Premix Ex Taq (Takara Bio) and 10 pmol/L of each primer. Quantitative polymerase chain reaction (PCR)-based amplification was performed using a 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA) with the following parameters: denaturation at 95 °C for 5 minutes followed by 40 cycles of denaturation at 95 °C for 30 seconds, annealing at 60 °C for 30 seconds, and extension at 72 °C for 45 seconds. Relative expression levels in each gene (normalized to that of 1A gene) were determined using the RQ software (version 1.3; Applied Biosystems).
Histopathological analysis
The lung tissues were fixed by inflating with 10% formalin. The tissues were then embedded in paraffin, cut into sections (5 micrometers) and stained with hematoxylin and eosin (H&E). All tissue samples were examined, and photographed by light microscopy (BX51; Olympus, Tokyo, Japan). Images were captured using an Olympus DP controller and manager at × 400 magnification.
Collection of volatile organic compounds in P. koraiensis and L. kaempferi panel
VOCs from P. koraiensis and L. kaempferi were collected by trapping gas using the Shibata minipump (MP-∑300, Shibata, Saitama, Japan). The composition of VOCs was analyzed by GC-MS (Trace 1310/ISQ-LT, Thermo Scientific, USA). TR-5MS capillary column (30.00 cm × 0.25 mm × 0.25 μm, Thermo Scientific, USA) was used for entrapping the gas, and He (1 mL/minute, 25 psi) was used as the carrier gas. The compounds analyzed in the VOCs were matched with total ion chromatogram (TIC) and the NIST 11 (National Institute of Standards and Technology, USA) mass spectral library. The entrapped gas was analyzed using the Thermal Desorption (TD) gas chromatography-mass spectrometer (GC-MS; GC model Agilent 7890, MS model Agilent 5975, Agilent, Santa Clara, USA); components were detached from the Tantalum Tube at 270 °C, and concentrated in a −20 °C cold trap. Concentrated components were then analyzed with GC/MS.
Statistical analysis
The results of all experiments were presented as mean±standard deviation (SD) values. Data were analyzed with a nonparametric one-way analysis of variance (ANOVA) and Tukey's test for multiple comparisons, and then ranked accordingly. All statistical analyses were performed using the GraphpadTM software. P value <0.05 was considered statistically significant.
Results
Effects of VOCs of P. koraiensis and L. kaempferi on serum inflammatory cytokines IgE and PGE2 in LPS-treated mice
To investigate the anti-inflammatory effects of VOCs of P. koraiensis in the inflammatory BALB/c mouse model, each mouse was administered LPS via the intraperitoneal route. Compared to the VE group, marked induction of serum IgE and PGE2 was observed in the LPS-treated group. The elevated serum IgE levels were recovered in the DEX group (Fig. 1). Treatment with VOCs of P. koraiensis also resulted in a decrease in serum IgE levels. In addition, treatment with VOCs of P. koraiensis and L. kaempferi reduced the PGE2 levels as compared to those in the LPS-treated group. These anti-inflammatory effects on serum IgE and PGE2 levels indicated that exposure to VOCs of P. koraiensis and L. kaempferi relieved the systemic inflammatory condition, suggesting that VOCs of both P. koraiensis and L. kaempferi can be used for their continuous inflammation-relieving effect.
Figure
1.
Effects of VOCs on serum concentration of IgE and PGE2 in LPS-treated mice.
The serum IgE and PGE2 levels in BALB/c mice were measured at the end of the experiment using an ELISA kit. Groups: VE, vehicle; LPS, negative control; LPS + DEX (DEX), positive control; P. koraiensis (P.) and L. kaempferi (L.) with LPS treatment, experimental groups. Values are expressed as the mean±standard deviation. *P<0.05 vs. VE; #P<0.05 vs. LPS treated group.
Effects of VOCs of P. koraiensis and L. kaempferi on the expression of inflammatory cytokines in peripheral blood mononuclear cells in LPS-treated mice
We investigated whether the VOCs of P. koraiensis and L. kaempferi inhibited the expression of inflammatory cytokines in PBMCs of LPS-treated mice. Expression levels of COX-2, TNF-α, IL-1β, and IL-13 mRNA in PBMCs were examined by performing real-time PCR (Fig. 2). Exposure to VOCs of both P. koraiensis and L. kaempferi recovered the COX-2, TNF-α, IL-1β, and IL-13 mRNA expression levels as compared to the LPS-treated group. Based on the observed effects on serum cytokines (Fig. 1) and the changes in expression of inflammatory cytokines in PBMCs, the VOCs of both P. koraiensis and L. kaempferi showed anti-inflammatory properties.
Figure
2.
Effects of VOCs on expression of inflammatory cytokines (Cox-2, TNF-α, IL-1β, and IL-13) in PBMCs from mice.
Groups: VE, vehicle; LPS, negative control; LPS + DEX (DEX), positive control; P. koraiensis (P.) and L. kaempferi (L.) with LPS treatment, experimental groups. Values are expressed as the mean±standard deviation. *P<0.05 vs. VE; #P<0.05 vs. LPS treated group.
Effects of VOCs of P. koraiensis and L. kaempferi on reducing expression of inflammatory cytokines in lungs in nasal-LPS treated mice
We investigated whether the VOCs of P. koraiensis and L. kaempferi inhibited the expression of pulmonary inflammatory cytokines in PBMCs of nasal-LPS treated mice. We examined the expression levels of COX-2, TNF-α and NF-κB mRNA in lung cells by performing real-time PCR (Fig. 3). Exposure to VOCs of both P. koraiensis and L. kaempferi recovered the COX-2, TNF-α, and NF-κB mRNA expression levels compared to that in the LPS-treated group. These results indicated that VOCs of both P. koraiensis and L. kaempferi exerted an in vivo anti-inflammatory effect in the lungs.
Figure
3.
Effects of VOCs on expression of inflammatory cytokines (Cox-2, IL-1β, IL-13, NF-κB, and TNF-α) in lung tissues of mice.
Groups: VE, vehicle; LPS, negative control; LPS + DEX(DEX), positive control; P. koraiensis (P.) and L. kaempferi (L.) with LPS treatment, experimental groups. Values are expressed as the mean±standard deviation. *P<0.05 vs. VE; #P<0.05 vs. LPS treated group.
Effects of VOCs of P. koraiensis and L. kaempferi on attenuating lung damage of nasal-LPS treated mice
For evaluation of lung damage, especially bronchus thickness and mucus secretion, lung (bronchial) tissues damaged by LPS treatment were stained with H&E. LPS induced an increase in bronchial wall thickness compared to that in the vehicle-treated mice, while the increased bronchial wall thickness was recovered by dexamethasone and VOCs of P. koraiensis and L. kaempferi (Fig. 4). Therefore, anti-inflammatory effects of the VOCs of P. koraiensis and L. kaempferi on nasal LPS induced lung inflammation provided a promising potential for inflammation recovery similar to the positive control, dexamethasone.
Figure
4.
Effects of VOCs on histological lung (bronchus) region of nasal LPS-treated mice.
Histological recovery of the bronchial wall in nasal LPS-treated mice with DEX and VOC of P. koraiensis and L. kaempferi was observed in H&E staining (magnification ×400).
For one month, the terpene contents in VOCs were analyzed every other day by entrapping the gas. Quantitative analysis of the components of VOCs of P. koraiensis and L. kaempferi were shown in Table 1. The concentrations and amount of the components that diffused from L. kaempferi were higher than that from P. koraiensis. Both VOCs contained alpha-pinene, beta-pinene, carproaldehyde, limonene, terpinolene, alpha-terpineol, borneol, and camphor. In addition, P. koraiensis emited 3-carene, alpha-terpinene, 1, 8-cineole, isopulegol, pinocarveol, 4-terpineol, verbenone, caryophyllene, and alpha-cedrene, whereas L. kaempferi emited beta-farnesene. The concentrations of VOCs also differed in a closed system; P. koraiensis contained 564.37 ng/L VOC and L. kaempferi contained 80.91 ng/L VOC.
Table
1.
Contents in VOCs in P. koraiensis and L. kaempferi panel
Various medications are used to control systemic or local inflammation, such as corticosteroids, calcineurin inhibitor, and immune-suppressants[19], of which corticosteroids are the most widely used[19–20]. However, long term use of corticosteroids is associated with side effects and drug tolerance in the endocrine system[20–21]. To avoid these side effects, numerous natural products from plants are being investigated for the treatment of inflammation[22]. Lately, studies in Asia and Europe have focused on herbal therapy due to its efficacy and safety. A wide variety of phenolic substances derived from plants are reported to retain marked anti-oxidant and anti-inflammatory activities, thereby contributing to their chemopreventive potential[23]. Recently, elemol (a compound extracted from C. Obtusa) has been reported as an anti-inflammation agent, and treatment with elemol is expected to prevent the onset of allergic diseases and ameliorate allergic symptoms[14–15,24–25]. In addition, VOCs from plants are reported to exhibit anti-oxidant and anti-inflammatory activities, either supporting or directly acting as effective anti-inflammatory reagents[12,26]. Although the mechanisms of herbal remedies have not been fully elucidated, many medications may have scientific merits and clinical benefits which help in alleviating patient symptoms[12–13,24,27–29].
The current study investigated the effects of VOCs of P. koraiensis and L. kaempferi in an LPS-induced systemic and local inflammation mouse model. IgE is an immunoglobulin that plays a role in acute and chronic inflammatory allergic diseases[30]. Lowered IgE implicates for alleviation of inflammation. The treatment with P. koraiensis showed decreased tendency of IgE which meant certain alleviation of inflammatory response induced by LPS. The induced serum PGE2 levels significantly decreased after exposure to the VOCs in the LPS-induced systemic inflammation mice model. Exposure to VOCs of both P. koraiensis and L. kaempferi resulted in successful alleviation of the inflammatory cytokines in the bloodstream suggesting that they may contribute to the suppressed stimulation of T cell-mediated cytokines. Similar to the systemic concentrations of inflammatory cytokines, the mRNA expressions of inflammatory cytokines (COX-2, TNF-α, IL-1β and IL-13) from the PBMCs of systemic LPS-treated mice were also significantly inhibited. COX-2 and pro-inflammatory cytokines such as TNF-α, IL-1β and IL-13 are expressed by many cells including macrophages, NK cells, monocytes, and neutrophils. These enzyme and cytokines are involved in the proliferation, differentiation, and apoptosis of cells as well as immune cell migration toward inflammatory sites; hence, inhibition of these cells suggested that the VOCs of P. koraiensis and L. kaempferi may contribute to the suppressed stimulation of cytotoxic- and helper T cell-mediated cytokines. Since Th1 and Th2 types of reactions mutually regulate several immune signaling cascades, balancing the Th1/Th2 types of reactions may be fundamental to the treatment of inflammation[31]. Prostaglandins are lipid autacoids derived from arachidonic acid, involving the inflammatory response. They are generated from arachidonate by the action of cyclooxygenase (COX) isoenzymes[32]. The decreased expression of COX-2 resulted in the decreased serum concentration of its enzymatic product PGE2 as presented in Fig. 1B.
Following the systemic anti-inflammatory effects of VOCs of both P. koraiensis and L. kaempferi, we investigated their effect on the alleviation of local inflammation in a mouse model by inoculating LPS as an intranasal spray. In the lung tissue, the inflammatory T-cell mediated cytokine COX-2 significantly decreased after exposure to the VOCs. Also, exposure to the VOCs resulted in decreased levels of the transcription factor for inflammatory cytokine NF-κB. However, no decrease was observed in the levels of TNF-α. Release of TNF-α from human PBMC are correlated with documented inflammatory activity. But both P. koraiensis and L. kaempferi does not decrease TNF-α in both PBMCs and lung tissues[33]. The levels of NF-κB and IKKα, which initiate the transcription of inflammatory cytokines, decreased after exposure to the VOCs of P. koraiensis and L. kaempferi, thereby indicating that the VOCs were capable of blocking the inflammatory cascades at the transcriptional level. Finally, treatment with dexamethasone and VOCs of P. koraiensis and L. kaempferi resulted in the recovery of bronchial wall thickness from nasal LPS treatment.
The VOCs of P. koraiensis and L. kaempferi were collected by trapping the gases using a SHIBATA minipump. The GC-MS analysis revealed that the VOCs of P. koraiensis contained 41 volatile compounds, whereas the VOCs of L. kaempferi contained 46 volatile compounds. The main contents of both VOCs were similar[16–17] and consisted of monoterpenes and sesquiterpenes, especially abundant quantities of limonene and α-pinene. Terpenes and sesquiterpenes are well-known anti-inflammatory compounds[34–35]. Both VOCs contained alpha-pinene, beta-pinene, carproaldehyde, limonene, terpinolene, α-terpineol, borneol, and camphor. All these compounds are reported to exert anti-inflammatory effects[36–41].
In conclusion, this study demonstrates the anti-inflammatory effects of VOCs from both P. koraiensis and L. kaempferi in a systemic and local inflammation mouse model. The VOCs decrease the expression of inflammatory cytokines by dissociating NF-κB from IκB via IKKα expression. Our results further suggest that the VOCs could be potent therapeutic compounds for treating inflammation, by regulating the serum IgE and PGE2 levels and T cell-derived cytokines such as TNF-α, IL-1β, and IL-13 and isoenzyme COX-2 in inflammatory lesions of systemic and local inflammation mouse model.
Acknowledgments
This work was financially supported by Forest Science Technology Program from Korean National Institute of Forest Science.
Authors and Reviewers
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