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Microsoft word - 11-이덕철Biomedical Science Letters 2014, 20(3): 173 179 Brief Communication eISSN : 2288-7415 Effect of Korean Red Ginseng on Artificial Sand Dust (ASD)
Induced Allergic Lung Inflammation
Jung-Ha Kim1, Tae-Jin Lee2, Im Jee-Aee3 and Duk-Chul Lee4,†
1Department of Family Medicine, Chung-Ang University Healthcare System, Seoul 156-756, Korea 2Department of Pathology, Chung-Ang University College of Medicine, Seoul 156-755, Korea 3Sports and Medicine Research Center, INTOTO Inc., Seoul 120-160, Korea 4Department of Family Medicine, Severance Hospital, Yonsei University, College of Medicine, Seoul 120-752, Korea Asian sand dust is known to promote various respiratory symptoms or disorders. For the prevention of harmful health effects by Asian sand dust, the best strategy is known to avoid or reduce exposure to the Asian sand dust. Several studies
have shown that Korean red ginseng (RG) has anti-inflammatory and anti-allergic effects. The study aimed to clarify the
effect of Korean red ginseng intake on lung inflammation responses to artificial sand dust (ASD) similar to Asian sand
dust. BALB/c mice were divided into five groups (n=12) of control (saline), ovalbumin (OVA), OVA with ASD, OVA
plus RG with ASD, and OVA plus dexamethasone (DEXA) with ASD. Histopathologic evaluation of lung was conducted.
Interleukin (IL)-5, IL-12, interferon (IFN)-γ, IL-13, monocyte chemotactic protein (MCP)-1, and eotaxin within
bronchoalveolar lavage (BAL) fluid were measured by ELISA. OVA+ASD group significantly increased concentrations
of IL-5, IL-13, MCP-1, and eotaxin (P<0.01) compared to the control. OVA+ASD+RG group showed significant
decreased levels of IL-2, IL-13, MCP-1 and eotaxin (P<0.01) compared with OVA+ASD. Between RG and DEXA
treatment groups, there was no significant difference in all cytokines and chemokines. The inflammatory cells were
significantly decreased in treatment groups with RG or DEXA compared to OVA+ASD group. This study suggests a
beneficial effect of Korean RG administration in preventing inflammation of lung resulting from Asian sand dust.
Key Words: Asian sand dust, Korean red ginseng, Anti-inflammation, Allergic mouse model
Asian sand dust is a meteorological phenomenon that respiratory diseases (Pope et al., 1995; Hong et al., 1999; affects much of Northeast Asia. The dust originats in the Kim, 2004). Moreover, water soluble ions (Park et al., deserts of China and Mongolia and is carried to the Korean 2004b), inorganic components (mineral elements) (Ichinose Peninsula by prevailing westerly winds, resulting in social et al., 2008b), or organic substances such as bacteria in the and economic damage (Kwon and Cho, 2004). It is known dust (Echigo et al., 2005; Ichinose et al., 2008a) have been that particulate matter (PM) less than 10 μm in diameter suggested to be associated with respiratory inflammation (PM10) in sand dust affect human health. Increased PM10 responses. The pulmonary function of asthma patients invading the lower trachea particularly promotes different deteriorates and nocturnal asthma symptoms increase during the dust storm season (Min et al., 2001; Song, 2001; Park Received: July 26, 2014 / Revised: September 21, 2014 Accepted: September 24, 2014 et al., 2003b). Also, daily mortality rate and hospital †Corresponding author: Duk-Chul Lee. Department of Family Medicine, admission rate were reported to increase due to respiratory Severance Hospital, Yonsei University, College of Medicine, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, Korea. illnesses during the Asian sand dust season (Kwon et al., Tel: +82-2-2228-2331, Fax: +82-2-362-2473 2002; Chen et al., 2004; Hwang et al., 2005). Furthermore, e-mail: firstname.lastname@example.org C The Korean Society for Biomedical Laboratory Sciences. All rights reserved.
recent studies have reported that the dust may enhance
allergic rhinitis and rise in Th2 cytokines in particular seem RG was greater than the effect of dexamethasone (DEXA) to be associated in this process (Kim et al., 2009). administration (Babayigit et al., 2008). The main components of red ginseng (RG, the steamed Therefore, this study aimed to clarify the effect of Korean root of Panax ginseng C.A. Meyer, family Araliaceae) are RG intake on lung inflammation responses in an allergic ginsenosides and polysaccharides that have various medicinal mice model using an artificial sand dust (ASD) similar to effects including anti-inflammatory (Lee and Lau, 2011; Asian sand dust. Paul et al., 2012), anti-allergic (Kim and Yang, 2011), and The study used a total of 60 five-week-old healthy anti-tumor effects (Ho et al., 2012). Strong anti-allergic and female BALB/c mice (Central Lab. Animal Inc., Korea) anti-inflammatory effects have been identified particularly weighing between 20 25 g. All mice were raised in the in ginsenoside Rh1 (Park et al., 2004a), Rh2 (Park et al., animal facility at 20 23℃ and the humidity of 45 70% 2003a), and Rb1, and its metabolic substance compound K from one week before the experiment. All experiments (Park et al., 2005; Yang et al., 2007). In a recent study of were performed in accordance with the National Institutes mice model of allergic asthma, histological changes such of Health guidelines for laboratory animals. The study was as increased mast cell numbers and thickened basement approved by the Institutional Animal Care and Use Com- membrane, epithelium, and subepithelial smooth muscle, mittees of the National Veterinary Research & Quarantine in lungs have been improved in the RG group compared with the placebo group. The histologic improvement of The study used the 5000 mesh yellow soil powder Fig. 1. Study protocol. For allergic mouse model, a mixture of 25 μg OVA and 1 mg Al(OH)3 gel was injected into the peritoneum
(systemic immunization) and intranasal sensitization was induced with 2% OVA for 4 days. OVA group inhaled 2% OVA every other day for two weeks. OVA+ASD group inhaled 2% OVA every other day and 1 mL ASD (10 mg/mL) every day for two. OVA+ASD+RG or OVA+ASD+DEXA group were orally administered with a 500 mg/kg/d of RG powder or a 1 mg/kg/d of DEXA, respectively, since the first day of peritoneal injection. OVA and ASD inhalation protocol was the same as the OVA+ASD group. Control group was sprayed with 1 mL of NaCl for two weeks. OVA: ovalbumin, ASD: artificial sand dust, RG: red ginseng, DEXA: dexamethasone (Yellow soil, Hongikbio, Korea). It was near identical to group was sprayed with 1 mL of NaCl for 15 minutes in sand dust collected during the dust storm season of Korea each session for two weeks using a nebulizer (Fig. 1). in terms of chemical components, the contents of heavy Among 12 mice in each group, 6 mice were used in metals and particle size among commercially manufactured histopathological examination of the lung. Lung samples yellow soils (Seo, 2009). ASD is comprised of ions and were fixed with 10% paraformaldehyde, and stained with heavy metals such as Ca2+ 6.1 cmol/kg, K+ 2.5 cmol/kg, haematoxylin and eosin (H&E) to evaluate the degree of Mg2+ 6.2 cmol/kg, Na+ 0.3 cmol/kg, Cd 7.9 mg/kg, Pb 20.4 inflammatory cell infiltration. The degrees of observed mg/kg, Zn 90.7 mg/kg, Cu 14.4 mg/kg, Cr 37.2 mg/kg, and inflammation were distinguished into three stages in lung Ni 17.6 mg/kg. ASD has pH 6.2 and electrical conductivity tissue specimen. Grade 0 was established as a normal stage of 35.5 dS/m (Seo, 2009). with almost no inflammatory cell infiltration while grade 1 BALB/c mice were divided into five groups (n=12) each was judged to be a mild inflammation stage with slight of control (saline), ovalbumin (OVA), OVA+ASD, OVA+ inflammatory cell infiltration around bronchioles. Moreover, ASD+RG, and OVA+ASD+DEXA. The generation of the grade 2 was defined to be a stage of inflammatory cell OVA-induced allergic animal model was described pre- cluster detection with more severe inflammatory cell viously (Kim et al., 2009). For systemic sensitization, 25 μg infiltration. The interpretation of lung tissue samples was OVA (Sigma, St. Louis, MO) and 1 mg Al(OH)3 gel (Pierce carried out by two pathologists who were not informed Chemical Co., Rockford, IL) were mixed with 3 mL of with the categorized group of specimens. saline and injected with 300 μL into the peritoneal cavity of Among 12 mice in each group, the rest 6 mice were used each mouse on day 1, 7 and 14. Intranasal sensitization was in obtaining bronchoalveolar lavage (BAL) fluid. The lungs implemented with 2% OVA for 30 minutes using a jet were lavaged with 1 mL of sterile saline at 37℃ by syringe. nebulizer (Omron, Japan) from day 21 to day 24 (Fig. 1). The supernatants of BAL fluid were stored at -80℃ until Subsequently, 2% OVA 5 mL inhalation was maintained in analysis of cytokines and chemokines. Interleukin (IL)-5, allergic mice in the OVA group for 30 minutes per session IL-12, interferon (IFN)-γ, IL-13, monocyte chemotactic every other day for two weeks. Along with OVA 5 mL protein (MCP)-1, and eotaxin (R&D systems, Minneapolis, inhalation, the OVA+ASD group inhaled 10 mg/mL ASD USA) within BAL fluid were assessed using an Enzyme- for 15 minutes per session. Moreover, RG and DEXA Linked Immunosorbent Assay (ELISA) kit. group was orally administered with 500 mg/kg/d of RG Data are presented as mean ± standard deviation (SD). powder (KT&G, Korea) and a 1 mg/kg/d of dexamethasone Statistical analyses on the pathologic inflammation degrees, (Dexa-S®, Ilsung, Korea), respectively, since the first day of the levels of cytokines and chemokines in BAL fluid were peritoneal injection for allergic animal model. The control conducted using ANOVA followed by Tukey's test. Dif- Table 1. Expression of inflammatory cytokines in bronchoalveolar larvage fluid
Control 2.50±0.59 2.24±0.08 OVA 3.65±0.59 2.13±0.64 OVA+ASD 4.57*±1.25 2.76±0.79 27.13*±2.33 1.93±0.61 2064.15*†±56.77 OVA+ASD+RG 2.61#±0.42 2.70±0.58 OVA+ASD+DEXA 3.36±1.07 2.84±0.51 1.29#†±0.42 2.84#±0.47 1833.61*#†±199.41 386.35*#±53.43 Each experiment consisted of six observations. All values were expressed as mean ± SE. *P<0.01 compared to control by ANOVA. #P<0.01 compared to OVA+ASD by ANOVA. †P<0.01 compared to OVA by ANOVA. OVA: ovalbumin, ASD: artificial sand dust, RG: Red ginseng, DEXA: dexamethasone
Fig. 2. Histopathologic changes of the lung. A: No pathological changes in the airway treated with saline (control). B: Moderate infiltration
of inflammatory cells in the airway treated with OVA alone. C: Marked infiltration of inflammatory cells in the airway treated with OVA+ ASD. D: Significantly decreased infiltration of inflammatory cells in the airway treated with OVA+ASD+RG compared to C. E: Significantly decreased infiltration of inflammatory cells in the airway treated with OVA+ASD+DEXA compared to C. H&E stain (×400). OVA: ovalbumin, ASD: artificial sand dust, RG: red ginseng, DEXA: dexamethasone ferences among groups were determined as statistically significant at a level of P<0.05. All calculations were performed using the SAS 9.1 statistics package (SAS Institute, Inc., Cary, NC, US). Table 1 shows the levels of cytokines and chemokines in BAL fluid. OVA+ASD group significantly increased con- centrations of IL-5, IL-13, MCP-1, and eotaxin (P<0.01) compared to the control group. OVA+ASD+RG group showed significant decreased levels of IL-5, IL-13, MCP-1 and eotaxin (P<0.01) compared with the OVA+ASD group. IL-5 and IL-13 levels of OVA+ASD+RG group were not significantly different from the control group. OVA+ASD+ Fig. 3. Inflammation in lung. Each experiment consisted of six
observations. Inflammation of OVA+ASD+RG group was signifi- DEXA group also showed decreased concentrations of cantly lower than in that of OVA+ASD group. *P<0.01 compared to control by ANOVA. IL-13, MCP-1, and eotaxin (P<0.01), and increased IFN-γ #P<0.01 compared to OVA+ASD by ANOVA. level (P<0.01) compared to the OVA+ASD group. Between OVA: ovalbumin, ASD: artificial sand dust, RG: red ginseng, DEXA: dexamethasone RG and DEXA treatment groups, there were no significant differences in all cytokines and chemokines. Fig. 2 and 3 shows the pathologic change and inflammation degree in lung according to the study groups. The inflammatory cells The purpose of this study was to verify the preventive were significantly decreased in treatment groups with RG or effect of Korean RG intake on pulmonary diseases generated DEXA group compared to the OVA+ASD group (P<0.01). by sand dust using allergic mice model. Asian sand dust induced inflammatory cell infiltration of lung and increased Some studies have suggested that ions (Park et al., 2004b), inflammatory cytokines and chemokines in BAL fluid. The such as Na+, Mg2+, Ca2+, NH4+, SO 2- 4 , NO3 , toxins including administration of RG reduced these lung inflammatory LPS (Ichinose et al., 2008a), and heavy metals (Ichinose et responses, which was effective comparable to steroid, well al., 2008b) in the dust act as a new allergic antigen in known as anti-inflammatory drug. healthy individuals or an activating factor in allergic patients. Several active components were reported to be involved Among the measured cytokines, the levels of MCP-1 in the mechanism of anti-allergic and anti-inflammatory and eotaxin of all groups increased in our study compared efficacy of RG. Ginsenoside Rh2 was reported to have cell to those in a previous study (Ichinose et al., 2008a). IL-12 membrane stabilizing effect by inhibiting the secretion of and IL-γ concentrations were generally low in our study β-hexosaminidase and anti-inflammatory efficacy by in- (Ichinose et al., 2008a; Ichinose et al., 2008b). These hibiting the formation of nitrogen oxide (NO) and pro- findings may be caused by different sand dusts. Despite the staglandin E2 (PGE2) (Park et al., 2003a). In particular, it use of ASD, the study obtained similar results consistent was found to be more powerful in cell membrane stabilizing with previous studies. The BAL fluids of ASD exposed effect compare with disodium cromolycate (Park et al., allergic mice model showed significant increases in not 2003a). Furthermore, ginsenoside Rh1 inhibited the release only IL-5, a known key mediator of eosinophilic inflam- of histamine in peritoneal mast cells of mice and inhibited mation, but also MCP-1, eotaxin, and the Th2 cytokine passive cutaneous anaphylaxis (PCA) by IgE in mice, IL-13 compared to those of control mice (Ichinose et al., exhibiting a stronger efficacy than disodium cromolycate 2008a; Ichinose et al., 2008b). Both cytokines of BAL (Park et al., 2004a). Moreover, ginsenoside Rb1 and its fluids and inflammatory cell infiltration of lung tissues were metabolic substance compound K were also reported to be significantly decreased in Korean RG-treated (OVA+ASD+ inhibit the expression of iNOS, COX-2, and NF-κB (Park RG) group compared to the ASD-exposed allergic mice et al., 2004a; Park et al., 2005). In addition, compound K (OVA+ASD) group. It is clinically significant outcome that has been suggested to influence the role of TLR4 and the anti-inflammatory effect of RG is not any different with TLR9 in inflammatory responses (Yang et al., 2007). Previous studies reported that respiratory diseases are However, RG or DEXA can also influence OVA-induced generated by eosinophil infiltration, increased eosinophil allergy as well as dust-exposed allergy. In our study, because relevant cytokine and chemokines, and allergic inflam- OVA+RG and OVA+DEXA group were not included, it is mations resulting from Th2 predominant response (Chen et difficult to clarify whether the RG or DEXA has a al., 2004; Hiyoshi et al., 2005; Ichinose et al., 2008b). beneficial effect on inflammation by dust-exposed allergy. However, the mechanism by which sand dust develops In conclusion, the current study shows that sand dust allergic responses is unclear. Although the dust may be one boosts the allergic responses in the allergic mice model. antigen directly involved in inducing allergies, further This study suggests the beneficial effects of Korean RG studies are essential to distinguish antigen substances among administration in preventing inflammations of lung resulted various components in sand dust. The sizes of Asian sand from Asian sand dust. Further studies in human subjects dust particles range between 0.1∼20 μm including fine are needed to verify the effect of RG intake on preventing particles under 2.5 μm and ultra-fine particles under 0.1 μm respiratory diseases caused by sand dust. (Choi et al., 2001; Mori et al., 2003). Titanium dioxide, a known fine particle found in sand dust, is assumed to be a direct cause as well as a deteriorating factor in pulmonary This work was supported by the 2010 grant from the diseases. It was suggested that phagocytosis of titanium Korean Society of Ginseng funded by Korea Ginseng dioxide by lung macrophages may affect the change and Corporation. The authors would like to thank Seong-Jin adjustment of respiratory cells (Donaldson et al., 2001). Park, Ph.D. at Climate Change & Agroecology Division, National Academy of Agricultural Science for providing the Effects of asian sand dust, Arizona sand dust, amorphous artificial sand dust. silica and aluminum oxide on allergic inflammation in the murine lung. Inhal Toxicol. 2008b. 20: 685-694. REFERENCES
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