Anti-inflammatory effects of natural volatile organic compounds from Pinus koraiensis and Larix kaempferi in mouse model

Changhwan Ahn, Jae-Woo Kim, Mi-Jin Park, Seung Ryul Kim, Sung-Suk Lee, Eui-Bae Jeung,✉

1Laboratory of Veterinary Biochemistry and Molecular Biology, Veterinary Medical Center and College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk 28644, Republic of Korea;

2Division of Wood Chemistry &Microbiology, Department of Forest Products, National Institute of Forest Science,Seoul 02455, Republic of Korea;

3Laboratory of Biochemistry, School of Medicine, Chungbuk National University, Cheongju, Chungbuk 28644,Republic of Korea.

Abstract

Keywords: Pinus koraiensis, Larix kaempferi, inflammation, cytokine, lungs

Introduction

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 asthmarelated 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 ofPinusin the Pinaceae family. MostPinusspecies 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 antiinflammatory effects of the pine bark and its essential oil[11].Larix kaempferi(L. kaempferi) belongs to the familyPinaceaein 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 fromL. kaempferihas been used in folk remedies to reduce allergic reactions[13]. Several previous studies have examined the alleviating effect ofP. koraiensison allergic dermatitis in a mouse model[12,14-15]. In addition,L.kaempferieffectively 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 ofP.koraiensisorL. kaempferireduces 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 withP. koraiensisorL.kaempferipanels 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)viathe 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 VOCtreated groups included the LPS+VOC ofP.koraiensisgroup (P. koraiensispanel, 1 026 cm3) and the LPS+VOC ofL. kaempferigroup (L. kaempferipanel, 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 000gfor 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 PGE2ELISA 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 ofP. koraiensis, and LPS+VOC ofL. 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 400gfor 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 fromP. koraiensisandL. kaempferiwere collected by trapping gas using the Shibata minipump(MP-∑300, Shibata, Saitama, Japan). The composition of VOCs was analyzed by GC-MS (Trace 1310/ISQLT, 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 GraphpadTMsoftware.Pvalue ＜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 ofP. koraiensisin the inflammatory BALB/c mouse model, each mouse was administered LPSviathe intraperitoneal route. Compared to the VE group,marked induction of serum IgE and PGE2was observed in the LPS-treated group. The elevated serum IgE levels were recovered in the DEX group(Fig. 1). Treatment with VOCs ofP. koraiensisalso resulted in a decrease in serum IgE levels. In addition,treatment with VOCs ofP. koraiensisandL.kaempferireduced the PGE2levels as compared to those in the LPS-treated group. These antiinflammatory effects on serum IgE and PGE2levels indicated that exposure to VOCs ofP. koraiensisandL. kaempferirelieved the systemic inflammatory condition, suggesting that VOCs of bothP. koraiensisandL. kaempferican be used for their continuous inflammation-relieving effect.

Fig. 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 ofP. koraiensisandL. kaempferiinhibited 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 bothP. koraiensisandL. kaempferirecovered 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 bothP. koraiensisandL.kaempferishowed anti-inflammatory properties.

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 ofP. koraiensisandL. kaempferiinhibited 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 bothP. koraiensisandL. kaempferirecovered 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 bothP.koraiensisandL. kaempferiexerted anin vivoantiinflammatory effect in the lungs.

Fig. 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 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 ofP.koraiensisandL. kaempferi(Fig. 4). Therefore, antiinflammatory effects of the VOCs ofP. koraiensisandL. kaempferion nasal LPS induced lung inflammation provided a promising potential for inflammation recovery similar to the positive control, dexamethasone.

VOC contents of P. koraiensis and L. kaempferi

For one month, the terpene contents in VOCs were analyzed every other day by entrapping the gas.Quantitative analysis of the components of VOCs ofP. koraiensisandL. kaempferiwere shown inTable 1.The concentrations and amount of the components that diffused fromL. kaempferiwere higher than that fromP. koraiensis. Both VOCs contained alphapinene, beta-pinene, carproaldehyde, limonene,terpinolene, alpha-terpineol, borneol, and camphor. In addition,P. koraiensisemited 3-carene, alphaterpinene, 1, 8-cineole, isopulegol, pinocarveol, 4-terpineol, verbenone, caryophyllene, and alphacedrene, whereasL. kaempferiemited beta-farnesene.The concentrations of VOCs also differed in a closed system;P. koraiensiscontained 564.37 ng/L VOC andL. kaempfericontained 80.91 ng/L VOC.

Discussion

Fig. 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.

Fig. 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).

Table 1 Contents in VOCs in P. koraiensis and L. kaempferi panel (ng/L)

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 antiinflammatory activities, thereby contributing to their chemopreventive potential[23]. Recently, elemol (a compound extracted fromC. 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 antiinflammatory 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 ofP. koraiensisandL. kaempferiin 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 withP. koraiensisshowed decreased tendency of IgE which meant certain alleviation of inflammatory response induced by LPS. The induced serum PGE2levels significantly decreased after exposure to the VOCs in the LPS-induced systemic inflammation mice model. Exposure to VOCs of bothP. koraiensisandL. kaempferiresulted 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 proinflammatory 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 ofP. koraiensisandL. kaempferimay contribute to the suppressed stimulation of cytotoxicand 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 PGE2as presented inFig. 1B.

Following the systemic anti-inflammatory effects of VOCs of bothP. koraiensisandL. 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 bothP. koraiensisandL. kaempferidoes 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 ofP. koraiensisandL.kaempferi, thereby indicating that the VOCs were capable of blocking the inflammatory cascades at the transcriptional level. Finally, treatment with dexamethasone and VOCs ofP. koraiensisandL.kaempferiresulted in the recovery of bronchial wall thickness from nasal LPS treatment.

The VOCs ofP. koraiensisandL. kaempferiwere collected by trapping the gases using a SHIBATA minipump. The GC-MS analysis revealed that the VOCs ofP. koraiensiscontained 41 volatile compounds, whereas the VOCs ofL. kaempfericontained 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, αterpineol, borneol, and camphor. All these compounds are reported to exert anti-inflammatory effects[36-41].

In conclusion, this study demonstrates the antiinflammatory effects of VOCs from bothP. koraiensisandL. kaempferiin a systemic and local inflammation mouse model. The VOCs decrease the expression of inflammatory cytokines by dissociating NF-κB from IκBviaIKKα expression. Our results further suggest that the VOCs could be potent therapeutic compounds for treating inflammation, by regulating the serum IgE and PGE2levels 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.