Saururus chinensis-controlled allergic pulmonary disease through NF-κB/COX- 2 and PGE2 pathways MiKyung Song1,*, Soon-Young Lee2,*, Minhee Kim3, Sangwoug Park1, Juyeon Park1, Yongbum Kwon1 and Dae-Hun Park2 1 Bio Technology R&D Center, WiLab Co., Ltd., Seoul, South Korea 2 Department of Nursing, Dongshin University, Naju, South Korea 3 Department of Forestry, Chonnam National University, Gwangji, South Korea * These authors contributed equally to this work. ABSTRACT Saururus chinensis is a perennial herb found in the northeastern regions of Asia, including Korea, China, and Japan, and is used in traditional medicine. Studies have identified the four major constituents in Saururus chinensis water extract (LHF618 R ) as miquelianin (11.75 ± 0.092 mg/g), rutin (1.20 ± 0.008 mg/g), quercitrin (2.38 ± 0.389 mg/g), and quercetin (0.068 ± 0.017 mg/g). Saururus chinensis can improve the symp- toms of ovalbumin- or fine dust-induced allergic pulmonary disease by suppressing the effects of WBCs and neutrophils in BALF and IgE in the serum. Saururus chinensis dose-dependently recovered morphological changes such as mucous hyper secretion (from 2.7 ± 0.46 to 0.6 ± 0.65), pulmonary epithelial cell hyperplasia (from 2.4 ± 0.55 to 0.7 ± 0.67), and inflammatory cell infiltration (from 2.3 ± 0.45 to 0.6 ± 0.43), and effectively controlled cDNA levels and protein levels of IL-13. It inhibited NF-κB translocation and COX-2 protein synthesis and suppressed the expression of PGE2. Our results show that Saururus chinensis controlled allergic pulmonary disease via the anti-inflammatory pathways, NF-κB/COX-2 and PGE2. Saururus chinensis may be a promising drug candidate against fine dust-induced allergic pulmonary disease. Submitted 17 April 2020 Accepted 4 September 2020 Published 24 September 2020 Subjects Biochemistry, Allergy and Clinical Immunology, Drugs and Devices, Respiratory Corresponding authors Medicine Yongbum Kwon, Keywords Saururus chinensis, Allergic pulmonary disease, NF-kB/COX-2, PGE2, HPLC [email protected] Dae-Hun Park, [email protected] Academic editor INTRODUCTION Matteo Lambrughi Allergic rhinitis and asthma are respiratory system diseases classified as allergic pulmonary Additional Information and diseases. Allergic rhinitis is an allergic pulmonary disease associated with the upper airway Declarations can be found on page 13 and its typical symptoms include nasal itching and discharge, upper airway obstruction, and watery eyes caused by allergens including pollens, mold, and dander (Wallace et al., 2008). DOI 10.7717/peerj.10043 Asthma is an allergic respiratory disease affecting the bronchioaleveolar region of the lower Copyright respiratory system and symptoms include mucous hypersecretion, pulmonary epithelial 2020 Song et al. cell hyperplasia, inflammatory cell infiltration, and airway remodeling induced by allergens Distributed under including pollen, mold, dander, house mite dust, pollutants, and temperature changes Creative Commons CC-BY 4.0 (Fehrenbach, Wagner & Wegmann, 2017; Hirota & Martin, 2013). Although asthma and OPEN ACCESS allergic rhinitis can be classified as different diseases, they are highly similar and the term How to cite this article Song MK, Lee S-Y, Kim M, Park S, Park J, Kwon Y, Park D-H. 2020. Saururus chinensis-controlled allergic pul- monary disease through NF-κB/COX-2 and PGE2 pathways. PeerJ 8:e10043 http://doi.org/10.7717/peerj.10043 ``allergy rhinobronchitis'' has been suggested to link the diseases. The co-morbidity of the nose and bronchus is incredibly high with 75% of asthma patients affected by rhinitis and 20% to 40% of rhinitis patients affected by asthma (Palma-Carlos, Branco-Ferreira & Palma-Carlos, 2001). Allergic rhinitis, asthma, and eczema are frequently diagnosed allergic disorders that may occur when subjects are repeatedly exposed to an allergen. These disorders may lead to allergic inflammation (Galli, Tsai & Piliponsky, 2008; Ferreira, 2004). It is important to control inflammation in allergy patients and the NK-κB/COX-2 and prostaglandin E2 (PGE2) axes are the primary pathways of inflammation (Mishra, Banga & Silveyra, 2018a; Mishra, Banga & Silveyra, 2018b). Many cytokines are closely associated with inflammation (Feghali & Wright, 1997), including interleukin-13 (IL-13), which causes chronic inflammation by stimulating B lymphocytes (Punnonen et al., 1993) and inducing inflammation, mucous hypersecretion, and fibrosis (Zhu et al., 1999). Allergic pulmonary disease is incurable and combination therapies of corticosteroid hormones, leukotriene antagonists, and/or β2 agonists (Mishra, Yao & Levine, 2013) have been used to suppress its symptoms. However, this therapeutic method has adverse effects including growth suppression, hypertension, osteoporosis, gastric ulcers, and neurotoxicity (Wise, 2014; Ciriaco et al., 2013). Saururus chinensis (S. chinensis, SC), a perennial herb found in the northeastern regions of Asia including Korea, China, and Japan, has been used as a traditional medicine for diseases such as edema, jaundice, gonorrhea, and other inflammatory diseases (Chung & Shin, 1990). Recent studies have reported the therapeutic effects of SC to include anti- inflammation (Kim et al., 2003; Cho, Cho & Song, 2005), antihypertension, vasodilation (Ryu et al., 2008), and anti-angiogenesis effects (Yoo et al., 2008), as well as the suppression of atopic dermatitis (Choi et al., 2008). However, there are few mechanistic studies on the active and therapeutic constituents of this botanical. We investigated the co-morbidity of the nose and bronchus and the effects of SC on allergic rhinitis and asthma. We determined the biological constituents of SC and extracted the compounds that suppressed the symptoms of allergic pulmonary disease. MATERIALS & METHODS Plant Materials R Dried SC leaves (LHF618 ) were purchased from Donggwang Pharmaceutical Company (Seoul, Korea), and visually identified by the Korea Pharmaceutical Trading Association (Seoul, Korea). A voucher SC specimen was deposited at the WiLab Co. Ltd. (Seoul, Korea). SC samples were prepared as follows: samples were soaked in water at 95 ± 2 ◦C for 4 h (1 kg/15 L); the resulting water was filtered, concentrated in a water bath under a vacuum using a rotary evaporator, and then spray-dried (SD) with 25% dextrin. Chemicals and reagents All reagents were of analytical grade. Methanol and acetonitrile (ACN) were purchased from JT Baker (Phillipsburg, NJ, USA). Miquelianin, rutin, and quercetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Quercitrin was purchased from InterPharm Song et al. (2020), PeerJ, DOI 10.7717/peerj.10043 2/17 (Koyang-si, Korea). Formic acid was purchased from Daejung Chemicals & Metals Co. Ltd. (Kyunggi, Korea) and fine dust (FD, Phyllite powder, PH-DG2000) was bought from Hongik Bio Tech Co. Ltd. (Jeonnam, Korea). HPLC analysis of SC We used a Waters HPLC system with a Waters alliance e2695 and a Waters 2998 photodiode array detector. The Fortis C18 analytical column (150 × 4.6 mm, 5 mm pore size) was tested and filled with the same stationary phase. Samples A (0.01% Formic acid) and B (ACN) were used as the mobile phase under gradient conditions to analyze the samples. The mobile phase was filtered by vacuum through a 0.45 mm membrane filter. Chromatography was conducted on a gradient using a flow rate of 1.0 ml/min at 30 ◦C and was detected at various UV wavelengths for individual standards and SC samples. The SC extract powder was dissolved in 50% MeOH at 10 mg/ml. The chromatograms were processed using Empower 2 software, build 1154 (Waters, Milford, MA, USA). Animal schedule We followed the protocols of our previous study for animal testing (Lee et al., 2019). Animal euthanasia guidelines were established prior to the start of the experiment and the experimenters checked the physical condition of all animals at least twice daily. Any abnormal behaviors from an animal during the experiment warranted the euthanasia of the animal and a necropsy was performed. Euthanasia was administered by an overdose of Zoletil delivered via intraperitoneal injection (Virbac, Carros, France) to preserve the BALF or respiratory system (lung) of the animal and to evaluate the suppression effects against allergic pulmonary diseases of SC. 84 BALB/c mice were purchased from Samtako Korea (Osan, Korea) and were acclimated for seven days prior to the start of our study. The mice were supplied with food and water ad libitum at 23 ± 3 ◦C, 55 ± 15% relative humidity, and 12 hr light per day, with observations of their physical conditions both prior to and during the study period. The animals were divided into 6 groups: tap water treatment group (CON), ovalbumin (OVA, Sigma-Aldrich, St. Louis, MO, USA) and FD treatment group (OVA + FD), OVA + FM and dexamethasone treatment group (OVA + FD + DEX), and after OVA and FD treated SC administrated groups (50 mg/kg, 150 mg/kg, and 300 mg/kg). Animal experiments were conducted twice using the same methods. All groups were treated twice at a one-week interval with intraperitoneal OVA, which consisted of 20 mg ovalbumin and 1 mg aluminum hydroxide hydrate (Sigma-Aldrich) in 500 mL saline, with the exception of the CON group. All mice were also exposed to a nebulizer mist (NE-U17, OMRON, Japan) of ovalbumin and fine dust for 30 min per day for 5 days, seven days after the second injection, except for the CON group. All groups were given 3 doses of an oral administration of tap water (CON or OVA + FD) or dexamethasone (OVA + FD + DEX) on the same exposure day but prior to the inhalation of ovalbumin and fine dust mist (Fig. 1). Song et al. (2020), PeerJ, DOI 10.7717/peerj.10043 3/17 Figure 1 Experimental design for animal study. Full-size DOI: 10.7717/peerj.10043/fig-1 Ethics statement Approval for the animal study was obtained from the Institutional Animal Care and Use Committee (Animal Study Approval No. CNU IACUC_YB-2019-48). Bronchoalveolar Fluid (BALF) and Serum Analysis We applied the methods of our previous study (Lee et al., 2019) and used BALF and serum analysis to analyze the WBC, differential cell counts, and IgE levels.