Atraric Acid Exhibits Anti-Inflammatory Effect in Lipopolysaccharide

International Journal of Molecular Sciences

Article Atraric Acid Exhibits Anti-Inﬂammatory Eﬀect in Lipopolysaccharide-Stimulated RAW264.7 Cells and Mouse Models

1, 2, 2 1 1 Seul-Ki Mun †, Kyung-Yun Kang †, Ho-Yeol Jang , Yun-Ho Hwang , Seong-Gyeol Hong , Su-Jin Kim 1, Hyun-Wook Cho 3, Dong-Jo Chang 1 , Jae-Seoun Hur 4 and Sung-Tae Yee 1,*

1 Department of Pharmacy, Sunchon National University, 255 Jungang-Ro, Suncheon 549-742, Korea; [email protected] (S.-K.M.); [email protected] (Y.-H.H.); [email protected] (S.-G.H.); [email protected] (S.-J.K.); [email protected] (D.-J.C.) 2 Suncheon Research Center for National Medicines, Suncheon 549-742, Korea; [email protected] (K.-Y.K.); [email protected] (H.-Y.J.) 3 Department of Biology, Sunchon National University, Suncheon 549-742, Korea; [email protected] 4 Department of Environmental Education, Korea Lichen Research Institute, Sunchon National University, Suncheon 549-742, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-61-750-3752; Fax: +82-61-750-3708 These authors contributed equally to this work. † Received: 26 August 2020; Accepted: 22 September 2020; Published: 25 September 2020

Abstract: Lichens, composite organisms resulting from the symbiotic association between the fungi and algae, produce a variety of secondary metabolites that exhibit pharmacological activities. This study aimed to investigate the anti-inflammatory activities of the secondary metabolite atraric acid produced by Heterodermia hypoleuca. The results confirmed that atraric acid could regulate induced pro-inflammatory cytokine, nitric oxide, prostaglandin E2, induced nitric oxide synthase and cyclooxygenase-2 enzyme expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Meanwhile, atraric acid downregulated the expression of phosphorylated IκB, extracellular signal-regulated kinases (ERK) and nuclear factor kappa B (NFκB) signaling pathway to exhibit anti-inflammatory effects in LPS-stimulated RAW264.7 cells. Based on these results, the anti-inflammatory effect of atraric acid during LPS-induced endotoxin shock in a mouse model was confirmed. In the atraric acid treated-group, cytokine production was decreased in the peritoneum and serum, and each organ damaged by LPS-stimulation was recovered. These results indicate that atraric acid has an anti-inflammatory effect, which may be the underlying molecular mechanism involved in the inactivation of the ERK/NFκB signaling pathway, demonstrating its potential therapeutic value for treating inflammatory diseases.

Keywords: anti-inﬂammation; endotoxin shock; atraric acid; lichen; Heterodermia hypoleuca

1. Introduction Inflammation is an inherent immune mechanism that occurs as a result of pathogen invasion in the body [1,2]. During an inflammatory immune response, macrophages release pro-inflammatory mediators, such as nitric oxide (NO), prostaglandin E2 (PGE2), induced nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), as well as pro-inflammatory cytokines and chemokines [3,4]. Nuclear factor-kappa B (NFκB), which regulates the transcription of inflammatory factors induced by lipopolysaccharide (LPS) stimulation, binds to IκB in the cytoplasm and activated by phosphorylation and IκB degradation. The activation of NFκB is regulated by a mitogen-activated protein kinase (MAPK) and an extracellular signal-regulated kinase (ERK) [5–7]. Abnormally high inflammatory

Int. J. Mol. Sci. 2020, 21, 7070; doi:10.3390/ijms21197070 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 7070 2 of 13 response results in severe tissue damage and endotoxin shock. Chronic inflammatory responses cause a series of diseases such as neurodegeneration, cardiovascular diseases (CVD), and cancer [3]. Endotoxin shock is a severe inflammatory reaction caused by Gram-negative bacteria infection of the bloodstream. LPS, an endotoxin on the surface of Gram-negative organisms, stimulates the release of pro-inflammatory cytokines and NO, resulting in increasing vasodilation and vascular permeability. This leads to impaired cardiovascular function, pulmonary dysfunction, acute renal impairment, septic shock and, finally, death. Many studies have demonstrated that the inhibition of NFκB and MAPK activation is an important strategy for the treatment of inflammatory diseases [8–11]. Lichens, symbiotic associations of algae and fungi, are abundantly found in the surroundings and can grow in cold and harsh environments [3,12]. In addition, lichens have been traditionally used as herbal tea and as a food ingredient in China and Japan. Their ability capacity to produce and accumulate secondary metabolites gives rise to their wide chemical diversity. Many secondary metabolites from Lichen, including atraric acid, atranorin, usnic acid and olivetoric acid, have been linked to different biological effects such as antibacterial, antioxidant and antiproliferation effects. Therefore, lichens are attracting attention as a new natural material. [13–15]. Although biological activities of numerous lichens have been studied, there is no information on hyterodermia hypoleuca (HH), a lichen native to Korea. Thus, we attempted to discover the biological activities of HH from the extract fractions using diverse solvents. The methanol (MeOH) extract exhibited a strong anti-inflammatory activity. However, there is no information for anti-inflammatory metabolites of HH, and also the mechanism underlying the anti-inflammatory activity of HH is not yet clear. Thus, we tried to discover active metabolites with anti-inflammatory activity from the MeOH extract fraction. We successfully isolated atraric acid with strong anti-inflammatory activity as a secondary metabolite with high concentration in the MeOH extract. Atraric acid is a phenolic compound, and has been reported to exhibit anti-cancer activity against prostate cancer. It serves as an antagonist of androgen receptor [16]. Atraric acid also possesses antioxidant and antibacterial [17] activities. However, it is not clear how atraric acid shows anti-inflammatory activity in vitro and in vivo. In this work, we show the in vitro and in vivo anti-inflammatory effects of atraric acid, which revealed that atratic acid has anti-inflammatory activity caused by the suppression of the MAPK-NFκB signaling pathway.

2. Results

2.1. HH Exhibited Anti-Inflammatory Effect The anti-inflammatory activity of heterodermia hypoleuca (HH) was investigated in LPS-stimulated RAW264.7 cells. At first, examining the cytotoxicity of HH extract in RAW264.7 cells using cell counting kit-8 (CCK-8), we observed that the growth of the cells was not affected by the HH extracts (Figure1a). As shown in Figure1, HH inhibited the production of NO stimulated by LPS in a concentration-dependent manner (Figure1b). In addition, the levels of pro-inflammatory, such as Tumor necrosis factor-alpha (TNF-α), interleukin l beta (IL-1β), interleukin 6 (IL-6) and granulocyte–macrophage colony-stimulating factor (GM-CSF) released in LPS-stimulated RAW 264.7 cells were significantly reduced by 20%, 30%, 58% and 45%, respectively, at 30 µg/mL of HH (Figure1c–f). Int. J. Mol. Sci. 2020, 21, 7070 3 of 13 Int.Int. J. J. Mol. Mol. Sci. Sci. 2020 2020, 21, 21, x, xFOR FOR PEER PEER REVIEW REVIEW 3 3of of 13 13

FigureFigureFigure 1.1. 1. The The screening screening ofof of anti-inflammatoryanti-inflammatory anti-inflammatory activityac activitytivity ofof of thethe the methanolmethanol methanol extractextract extract ofof of HeterodermiaHeterodermia Heterodermia hypoleucahypoleuca hypoleuca (HH).(HH).(HH). ((a a()a) The)The The cellscells cells werewere were treatedtreated treated withwith with didifferent differentfferentconcentrations concentrations concentrations ofof of HHHH HH forfor for 2424 24 h,h, h, andand and thethe the viabilityviability viability ofof of thethe the µ treatedtreatedtreated cellscells cells waswas was determineddetermined determined byby by cellcell cell countingcounting counting kit-8kit-8 kit-8 assay.assay. assay. TheThe The cellscells cells werewere were preincubatedpreincubated preincubated withwith with 11 1 μ gμg/mL/g/mLmL µ lipopolysaccharidelipopolysaccharidelipopolysaccharide (LPS) (LPS) (LPS) for for for 1 1 h 1h h andand and thenthen then treatedtreated treated withwith with 1-301-30 1-30 μ gμg/mL/g/mLmL HH HH HH for for for 24 24 24 h.h. h. ((b b()b) The)The The concentrationconcentration concentration ofofof nitricnitric nitric oxideoxide oxide inin in thethe the culturedcultured cultured mediummedium medium waswas was measuredmeasur measureded byby by usingusing using thethe the GriessGriess Griessreaction. reaction. reaction. TheThe The releaserelease release ofof of α c β d e TumorTumorTumor necrosisnecrosis necrosis factor-alphafactor-alpha factor-alpha (TNF-(TNF- (TNF-α)(α) )( c),(),c), interleukininterleukin interleukin ll betalbeta beta (IL-1(IL-1 (IL-1β)(β) )( d(),d),), interleukininterleukin interleukin 66 6 (IL-6)(IL-6) (IL-6) (( e()e) and)and and granulocyte–macrophage colony-stimulating factor (GM-CSF) (f) was determined by enzyme-linked granulocyte–macrophagegranulocyte–macrophage colony-s colony-stimulatingtimulating factor factor (GM-CSF) (GM-CSF) ( f()f )was was determined determined by by enzyme-linked enzyme-linked immunosorbent assay (ELISA). Data are presented as mean SD from three independent experiments immunosorbentimmunosorbent assay assay (ELISA). (ELISA). Data Data are are presented presented as as mean mean ± ±SD SD from from three three independent independent experiments experiments ### ± ( ###p < 0.001 versus the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 versus the LPS group). (###( pp < < 0.001 0.001 versus versus the the control control group; group; * *p p < < 0.05, 0.05, ** ** p p < <0.01, 0.01, *** *** p p < < 0.001 0.001 versus versus the the LPS LPS group). group). 2.2. Identification of Atraric Acid 2.2.2.2. Identification Identification of of Atraric Atraric Acid Acid The yield and purification of atraric acid attained by the high performance liquid chromatography The yield and purification of atraric acid attained by the high performance liquid (HPLC)The purification yield and (Figure purification S2, Table of S1)atraric of MeOH acid extractattained of HHby (Figurethe high S1) areperformance 2.05% and 96.5%liquid chromatography (HPLC) purification (Figure S2, Table S1) of MeOH extract of HH (Figure S1) are respectively.chromatography The structure(HPLC) purification of atraric acid (Figure was identified S2, Table using S1) of nuclear MeOH magnetic extract resonanceof HH (Figure (NMR) S1) and are 2.05% and 96.5% respectively. The structure of atraric acid was identified using nuclear magnetic mass2.05% spectroscopy and 96.5% respectively. (MS) and was The compared structure to of the at previouslyraric acid was reported identified data. using Atraric nuclear acid: 1Hmagnetic NMR resonance (NMR) and mass spectroscopy (MS) and was compared to the previously reported data. (methanol-d4,resonance (NMR) 400 MHz); and mass 6.25 (1H,spectroscopy s, H-6), 3.83 (MS) (3H, and s, H-10), was compared 2.31 (3H, s, to H-8), the 1.89previously (3H, s, H-9),reported13C NMR data. AtraricAtraric acid: acid: 1H 1H NMR NMR (methanol-d4, (methanol-d4, 400 400 MHz); MHz); 6.25 6.25 (1H, (1H, s, s, H-6), H-6), 3.83 3.83 (3H, (3H, s, s, H-10), H-10), 2.31 2.31 (3H, (3H, s, s, H-8), H-8), (methanol-d4, 10013 MHz); 172.5 (C-1), 162.1 (C-3), 160.1 (C-5), 140.0 (C-7), 111.1 (C-6), 108.4 (C-4), 1.89 (3H, s, H-9),13 C NMR (methanol-d4, 100 MHz); 172.5 (C-1), 162.1 (C-3), 160.1 (C-5), 140.0 (C-7), 104.41.89 (3H, (C-2), s, 52.4H-9), (C-10), C NMR 23.9 (methanol-d4, (C-8), 8.1 (C-9); 100 ElectrosprayMHz); 172.5 ionization(C-1), 162.1 (ESI)-MS (C-3), 160.1 showed (C-5), an140.0 [M +(C-7),H]+ 111.1111.1 (C-6), (C-6), 108.4 108.4 (C-4), (C-4), 104.4 104.4+ (C-2), (C-2), 52.4 52.4 (C-10), (C-10),+ 23.9 23.9 (C-8), (C-8), 8.1 8.1 (C-9) (C-9) ; ;Electrospray Electrospray13 ionization ionization (ESI)-MS (ESI)-MS ion at m/z = 197.0 [M+ +H] , 219.0 [M+Na] (The+ number of carbone+ in C NMR chemical shifts13 was showed an [M+H]+ ion at m/z = 197.0 [M+H]+ , 219.0 [M+Na]+ (The number of carbone in13 C NMR identifiedshowed an using [M+H] 2D ion NMR at datam/z = and 197.0 confirmed [M+H] , by219.0 comparing [M+Na] them(The number with previously of carbone reported in C NMR data) chemical shifts was identified using 2D NMR data and confirmed by comparing them with (Figurechemical2)[ 18shifts–20]. was identified using 2D NMR data and confirmed by comparing them with previouslypreviously reported reported data) data) (Figure (Figure 2) 2) [18–20]. [18–20].

Figure 2. Chemical structure of atraric acid. FigureFigure 2. 2. Chemical Chemical structure structure of of atraric atraric acid. acid.

Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 13 Int. J. Mol. Sci. 2020, 21, 7070 4 of 13 Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 4 of 13 2.3. Atraric Acid Inhibited the Production of NO and Pro-Inflammatory Cytokines

2.3.2.3. Atraric AtraricWe investigated Acid Acid Inhibited Inhibited the the theproduction Production Production of of of NO, NO NO andPGE2 and Pro-Inflammatory Pro-Inflammatory and pro-inflammatory Cytokines cytokines, such as IL-1β, IL-6 and GM-CSF, in LPS-stimulated RAW264.7 cells to determine the anti-inflammatory activity of We investigated the production of NO, PGE2 and pro-inflammatory cytokines, such as βIL-1β, atraricWe acid investigated isolated from the productionHH. Based ofon NO, the PGE2cell viab andility pro-inflammatory test, we performed cytokines, the in vitro such test as IL-1 of atraric, IL-6 IL-6 and GM-CSF, in LPS-stimulated RAW264.7 cells to determine the anti-inflammatory activity of acidand with GM-CSF, concentrations in LPS-stimulated of 1 to 300 RAW264.7 μM for the cells evaluation to determine of the the anti-inflammatory anti-inflammatory activity. activity As of shown atraric atraric acid isolated from HH. Based on the cell viability test, we performed the in vitro test of atraric inacid Figure isolated 3, fromwe confirmed HH. Based that on the atraric cell viability acid did test, not we affect performed the viability the in vitro of testRAW264.7 of atraric cells acid withat a acid with concentrations ofµ 1 to 300 μM for the evaluation of the anti-inflammatory activity. As shown concentrationconcentrations ranging of 1 to 300 fromM 1 for to the300 evaluation μM. The levels of the anti-inflammatoryof NO, PGE2 and activity.pro-inflammatory As shown incytokines Figure3, in Figure 3, we confirmed that atraric acid did not affect the viability of RAW264.7 cells at a werewe confirmed determined that after atraric the acid treatment did not aofff ectthe theLPS-st viabilityimulated of RAW264.7 RAW264.7 cells cells at awith concentration atraric acid. ranging LPS concentration µranging from 1 to 300 μM. The levels of NO, PGE2 and pro-inflammatory cytokines stimulationfrom 1 to 300 significantlyM. The levels increased of NO, the PGE2 production and pro-inflammatory of NO, PGE2 and cytokines all pro-inflammatory were determined cytokines after were determined after the treatment of the LPS-stimulated RAW264.7 cells with atraric acid. LPS inthe RAW264.7 treatment ofcells. the The LPS-stimulated Griess reaction RAW264.7 showed cells that with atraric atraric acid acid. significantly LPS stimulation reduced significantly the LPS- stimulation significantly increased the production of NO, PGE2 and all pro-inflammatory cytokines stimulatedincreased the NO production production of NO,in a dose-depen PGE2 and alldent pro-inflammatory manner (69% of cytokines inhibition in RAW264.7at 300 μM, cells.Figure The 4a). Griess The in RAW264.7 cells. The Griess reaction showed that atraric acid significantly reduced the LPS- pro-inflammatoryreaction showed thatcytokines atraric acidin the significantly culture media reduced were the measured LPS-stimulated using NOan productionenzyme-linked in a stimulated NO production in a dose-dependent mannerµ (69% of inhibition at 300 μM, Figure 4a). The immunosorbentdose-dependent assay manner (ELISA) (69% ofkit. inhibition Treatment at of 300 theM, LPS-stimulated Figure4a). The RAW264.7 pro-inflammatory cells with cytokines atraric acid in pro-inflammatory cytokines in the culture media were measured using an enzyme-linked resultedthe culture in a media dose-depende were measurednt decrease using in an PGE2, enzyme-linked IL-1β, IL-6 immunosorbent and GM-CSF levels assay induced (ELISA) by kit. LPS Treatment (100%, immunosorbent assay (ELISA) kit. Treatment of the LPS-stimulated RAW264.7 cells with atraric acid 36.3%,of the LPS-stimulated93.3% and 80.9% RAW264.7 of inhibition cells at with 300 atraricμM respectively). acid resulted The in aresults dose-dependent clearly revealed decrease that in atraric PGE2, resultedβ in a dose-dependent decrease in PGE2, IL-1β, IL-6 and GM-CSF levels induced by LPS (100%,µ acidIL-1 exhibits, IL-6 and anti-inflammatory GM-CSF levels induced activity byby LPSregulati (100%,ng the 36.3%, levels 93.3% of inflammatory and 80.9% of inhibitionfactors produced at 300 inM 36.3%, 93.3% and 80.9% of inhibition at 300 μM respectively). The results clearly revealed that atraric LPS-stimulatedrespectively). TheRAW264.7 results cells. clearly revealed that atraric acid exhibits anti-inflammatory activity by acidregulating exhibits the anti-inflammatory levels of inflammatory activity factors by regulati producedng the in levels LPS-stimulated of inflammatory RAW264.7 factors cells. produced in LPS-stimulated RAW264.7 cells.

Figure 3. Effect of atraric acid on the viability of RAW264.7 cells. The cells were treated with 1–300 μFigureM atraric 3. E ﬀacidect offor atraric 24 h. Cell acid viability on the viability was determin of RAW264.7ed using cells. cell The counting cells were kit-8. treated Data withare presented 1–300 µM Figure 3. Effect of atraric acid on the viability of RAW264.7 cells. The cells were treated with 1–300 asatraric mean acid ± SD for from 24 h.three Cell independent viability was experiments. determined using cell counting kit-8. Data are presented as μmeanM atraricSD acid from for three 24 h. independent Cell viability experiments. was determined using cell counting kit-8. Data are presented as mean± ± SD from three independent experiments.

FigureFigure 4. 4. EffectEﬀect of of atraric atraric acid acid on onthe the levels levels of nitr of nitricic oxide oxide (NO), (NO), prostaglan prostaglandindin E2 (PGE2) E2 (PGE2) and pro- and inflammatorypro-inﬂammatory cytokines cytokines produced produced by LPS-stimulat by LPS-stimulateded RAW2647 RAW2647 cells. cells.The cells The were cells werepretreated pretreated with Figure 4. Effect of atraric acid on the levels of nitric oxide (NO), prostaglandin E2 (PGE2) and pro- 1with μg/mL 1 µg LPS/mL LPSfor 1 for h 1and h and subsequently subsequently treated treated with with 1–300 1–300 μMµM atraric atraric acid acid for for 24 24 h. h. ( (aa)) The The inflammatory cytokines produced by LPS-stimulated RAW2647 cells. The cells were pretreated with concentrationconcentration of of nitric nitric oxide oxide in in the the cultured cultured medium medium was was measured measured as as an an indicator indicator of of NO NO production production 1 μg/mL LPS for 1 h and subsequently treated with 1–300 μM atraric acid for 24 h. (a) The concentration of nitric oxide in the cultured medium was measured as an indicator of NO production

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by using Griess reaction. The release of PGE2 (b), IL-1β (c), IL-6 (d) and GM-CSF (e) was determined byby usingELISA. Griess Data reaction.are presented The release as mean of ± PGE2 SD from (b), IL-1threeβ independent(c), IL-6 (d) and experiments GM-CSF ( e(###) was p < 0.001 determined versus bythe ELISA. control Data group;* are presentedp < 0.05, ** as p < mean 0.01, ***SD p from< 0.001 three versus independent the LPS group). experiments (### p < 0.001 versus ± the control group;* p < 0.05, ** p < 0.01, *** p < 0.001 versus the LPS group). 2.4. Atraric Acid Inhibited LPS-Induced Expression of iNOS and COX-2 2.4. Atraric Acid Inhibited LPS-Induced Expression of iNOS and COX-2 iNOS and COX-2 are pro-inflammatory proteins that are key enzymes in catalyzing the iNOS and COX-2 are pro-inflammatory proteins that are key enzymes in catalyzing the production production of NO and PEG2, respectively [21]. To investigate the effect of atraric acid on the of NO and PEG2, respectively [21]. To investigate the effect of atraric acid on the production of NO and production of NO and PGE2, the expressions of the enzymes iNOS and COX-2 were evaluated using PGE2, the expressions of the enzymes iNOS and COX-2 were evaluated using Western blot and flow Western blot and flow cytometry. Our result showed that in LPS-stimulated RAW264.7 cells, cytometry. Our result showed that in LPS-stimulated RAW264.7 cells, expression of iNOS and COX-2 expression of iNOS and COX-2 was significantly enhanced, whereas treatment with atraric acid was significantly enhanced, whereas treatment with atraric acid resulted in decreased expression of resulted in decreased expression of iNOS and COX-2 in a concentration-dependent manner (Figure iNOS and COX-2 in a concentration-dependent manner (Figure5). 5).

FigureFigure 5.5. EEffectffect ofof atraricatraric acidacid onon induced induced nitric nitric oxide oxide synthase synthase (iNOS) (iNOS) and and cyclooxygenase-2 cyclooxygenase-2 (COX-2) (COX- expression2) expression in LPS-stimulatedin LPS-stimulated RAW2647 RAW2647 cells. cells. The The cells cells were were pre-treated pre-treated with with 1 µg 1/mL μg/mL LPS LPS for 1 for h and 1 h subsequentlyand subsequently treated treated with 100,with 300 100,µM 300 atraric μM acidatraric for acid 18 h. for (a )18 The h. expression(a) The expression of iNOS wasof iNOS analyzed was byanalyzed flow cytometry by flow cytometry using specific using antibodies specific antibodies and isotype and controls. isotype (b controls.) Mean fluorescence (b) Mean fluorescence intensities (MFI.)intensities are shown(MFI.) are for shown each panel. for each (c) Westernpanel. (c) blot Western analysis blot was analysis performed was performed to investigate to investigate the effect the of atrariceffect of acid atraric on theacid expression on the expression of pro-inflammatory of pro-inflammatory proteins. proteins. The ratios The ratios of iNOS of iNOS (d) and (d) COX-2and COX- (e) to2 (βe)-actin to β-actin after after atraric atraric acid acid treatment treatment were were determined, determined, respectively. respectively. Data Data are are presented presented as as mean mean ## ### ± SD± SD from from two two independent independent experiments experiments ( (##p p< < 0.01, ### pp << 0.0010.001 versus versus the the control control group; group; * p *

2.5. Atraric Acid Suppressed LPS-Stimulated Phosphorylation of the Nfκb Signaling Pathway The NFκB signaling pathway holds significant importance in the inflammation process. NFκB exists in the inactive state bind to IκB which is an inhibitor of NFκB. However, an inflammatory

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2.5. Atraric Acid Suppressed LPS-Stimulated Phosphorylation of the Nfκb Signaling Pathway The NFκB signaling pathway holds significant importance in the inflammation process. NFκB Int.exists J. Mol. in Sci. the 2020 inactive, 21, x FOR state PEERbind REVIEW to IκB which is an inhibitor of NFκB. However, an inflammatory6 of 13 stimulation triggers the LPS-induced phosphorylation of IκB followed by its degradation. The NFκB stimulation triggers the LPS-induced phosphorylation of IκB followed by its degradation. The NFκB is then phosphorylated and converted to its active form. In the NFκB signaling pathway, MAPK is then phosphorylated and converted to its active form. In the NFκB signaling pathway, MAPK regulates the activation of NFκB; the phosphorylation of MAPK induces the activation of NFκB regulates the activation of NFκB; the phosphorylation of MAPK induces the activation of NFκB followed by subsequent expression of inflammatory mediators and pro-inflammatory cytokines [10,22]. followed by subsequent expression of inflammatory mediators and pro-inflammatory cytokines To investigate the effect of atraric acid on the MAPK/NFκB signaling pathway, LPS-stimulated [10,22]. To investigate the effect of atraric acid on the MAPK/NFκB signaling pathway, LPS- RAW264.7 cells were treated with atraric acid to analyze the expression of the inflammatory mediators stimulated RAW264.7 cells were treated with atraric acid to analyze the expression of the by Western blot. Figure6 showed that atraric acid reduced the expression of p-I κB, which resulted inflammatory mediators by Western blot. Figure 6 showed that atraric acid reduced the expression in the degradation of IκB, and p-ERK in LPS-stimulated RAW264.7 cells. The result showed that the of p-IκB, which resulted in the degradation of IκB, and p-ERK in LPS-stimulated RAW264.7 cells. The phosphorylation of NFκB was inhibited by the treatment with atraric acid (Figure6e), implying the result showed that the phosphorylation of NFκB was inhibited by the treatment with atraric acid potential value of atraric acid as an anti-inflammatory agent. (Figure 6e), implying the potential value of atraric acid as an anti-inflammatory agent.

FigureFigure 6. 6. EffectsEffects of atraricatraric acid acid on on the the activation activation of NF ofκ BNF signaling.κB signaling. RAW264.7 RAW264.7 cells were cells pre-incubated were pre- incubatedwith 1 µg with/mL LPS1 μg/mL for 30 LPS min for and 30 thenmin and treated then with treated 100, with 300 µ 100,M atraric 300 μM acid atraric for 4 acid h. (a for) The 4 h. inhibitory (a) The inhibitoryeffect of atrariceffect acidof atraric against acid the against phosphorylation the phosphorylation of IκB, extracellular of IκB, extracellular signal-regulated signal-regulated kinase (ERK), kinaseand N (ERK),κB was and detected NκB was by Westerndetected blot. by Western The ratios blot. of degradationThe ratios of ofdegradation IκB(b), p-I ofκB( IκcB), ( p-ERKb), p-Iκ (Bd )(c and), p-ERKp-NF κ(dB() ande) to p-NFβ-actinκB after(e) to atraricβ-actin acid after treatment atraric acid were treatment determined, were respectively.determined, respectively. Data are presented Data ## ### as mean SD from two independent experiments ( p < 0.01, ##p < 0.001 versus### the control group; are presented± as mean ± SD from two independent experiments ( p < 0.01, p < 0.001 versus the control* p < 0.05, group; ** p *< p 0.01,< 0.05, *** **p p< <0.001 0.01, versus*** p < 0.001 the LPS versus group). the LPS group). 2.6. Atraric Acid Exhibited Anti-Inflammatory Effects on LPS-Induced Endotoxin Shock in Mice 2.6. Atraric Acid Exhibited Anti-Inflammatory Effects on LPS-Induced Endotoxin Shock in Mice Based on in vitro study for the anti-inflammatory activity of atraric acid, in vivo efficacy of atraric Based on in vitro study for the anti-inflammatory activity of atraric acid, in vivo efficacy of acid was evaluated on LPS-induced endotoxin shock in mice. Endotoxin shock not only increases atraric acid was evaluated on LPS-induced endotoxin shock in mice. Endotoxin shock not only the level of cytokines in the blood, but also induces tissue damage, causing secondary disease [23,24]. increases the level of cytokines in the blood, but also induces tissue damage, causing secondary The inhibitory effect of atraric acid on the release of inflammatory cytokines from the serum and disease [23,24]. The inhibitory effect of atraric acid on the release of inflammatory cytokines from the peritoneal lavage fluid was first investigated. As shown in Figure7, the LPS-stimulated group of serum and peritoneal lavage fluid was first investigated. As shown in Figure 7, the LPS-stimulated mice exhibited significantly increased production of pro-inflammatory cytokines, whereas, for ataric group of mice exhibited significantly increased production of pro-inflammatory cytokines, whereas, acid-treated mice, the production of pro-inflammatory cytokines was inhibited (Figure7b-e). Next, for ataric acid-treated mice, the production of pro-inflammatory cytokines was inhibited (Figure 7b- we performed hematoxylin and eosin (H&E) staining to analyze the pathological characteristics of the e). Next, we performed hematoxylin and eosin (H&E) staining to analyze the pathological organs, including the kidney, liver, and lung. In the LPS group, vasodilation in glomerular atrophy, characteristics of the organs, including the kidney, liver, and lung. In the LPS group, vasodilation in bleeding, and recruitment of inflammatory cells were observed in the kidney, and similar pathological glomerular atrophy, bleeding, and recruitment of inflammatory cells were observed in the kidney, damage such as vasodilation and bleeding were observed in the liver. In addition, the alveolar septa and similar pathological damage such as vasodilation and bleeding were observed in the liver. In became thicker and the alveoli exuded, leading to the destruction of some alveolar structures in the addition, the alveolar septa became thicker and the alveoli exuded, leading to the destruction of some lung of the LPS-stimulated mice (Figure7f). On the contrary, the atraric acid-treated group of mice alveolar structures in the lung of the LPS-stimulated mice (Figure 7f). On the contrary, the atraric acid-treated group of mice exhibited reduced pathological damages such as vasodilation and bleeding. Taken together, it was confirmed that atraric acid suppresses the inflammatory response induced by LPS in vitro and in vivo.

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exhibited reduced pathological damages such as vasodilation and bleeding. Taken together, it was Int. J.conﬁrmed Mol. Sci. 2020 that, 21, atraric x FOR PEER acid REVIEW suppresses the inﬂammatory response induced by LPS in vitro and7 inof 13 vivo .

FigureFigure 7. Effects 7. Eff ectsof atraric of atraric acid on acid LPS-induced on LPS-induced endotoxin endotoxin shock in shock mice. in (a mice.) BALB/c (a) BALBmice (/nc = mice 5) were (n = 5) intraperitoneallywere intraperitoneally administered administered (i.p.) LPS (i.p.)(10 mg/kg, LPS (10 E. mg coli/kg, 011:B4)E. coli and011:B4) subsequently and subsequently treated with treated atraricwith acid atraric (10 acidmg/kg, (10 mg30 /mg/kg)kg, 30 mg or/ kg)control or control (PBS) (PBS)for 4 forh. ( 4b h.–e) ( bThe–e) Thelevels levels of pro-inflammatory of pro-inflammatory cytokinescytokines released released by by the the serum serum and and peritoneal lavagelavage were were detected detected by by ELISA ELISA assay. assay. (f) Tissue (f) Tissue samples sampleswere stainedwere stained with H&Ewith toH&E exhibited to exhibited histopathological histopathological changes changes (Abbreviations: (Abbreviations: RT, renal RT, tubule; renal RC, tubule;renal RC, corpuscle; renal corpuscle; D, bile duct; D, bile L, lymphaticduct; L, lympha vessels;tic vessels; PV, portal PV, vein; portal HA, vein; hepatic HA, artery;hepatic PC, artery; pulmonary PC, pulmonarycapillaries; capillaries; A, alveoli). A, alveoli). The arrows The showedarrows show inflammatoryed inflammatory infiltration. infiltration. Data are Data presented are presented as mean ### ± as meanSEM ± from SEM five from independent five independent experiments experiments ( p < (###0.001 p < 0.001 versus versus the control the control group; group; * p < *0.05, p < 0.05, ** p < **0.01 p < 0.01,versus *** the p < LPS 0.001 group) versus Scale the LPS bar: group) 50 µm. Scale bar: 50 μm. 3. Discussion 3. Discussion There is a great interest in the discovery of biologically active natural products due to their There is a great interest in the discovery of biologically active natural products due to their activities with relatively low toxicity as compared to other modalities such as synthetic chemicals [10]. activities with relatively low toxicity as compared to other modalities such as synthetic chemicals [10]. Numerous studies have shown that natural products are developed as beneficial dietary supplements for health and therapeutic agents against disease [25]. Lichen is a unique organism that produces biologically active secondary metabolites with various pharmacological effects including antioxidant, anti-cancer [17] and anti-viral effects [16]. In the LPS stimulated inflammation, toll-like receptor-4 (TLR4) and MD-2 in macrophages form heterodimers that recognize common patterns of

Int. J. Mol. Sci. 2020, 21, 7070 8 of 13

Numerous studies have shown that natural products are developed as beneficial dietary supplements for health and therapeutic agents against disease [25]. Lichen is a unique organism that produces biologically active secondary metabolites with various pharmacological effects including antioxidant, anti-cancer [17] and anti-viral effects [16]. In the LPS stimulated inflammation, toll-like receptor-4 Int. J.(TLR4) Mol. Sci. and 2020,MD-2 21, x FOR in PEER macrophages REVIEW form heterodimers that recognize common patterns of8 anof 13 LPS molecule [26]. Upstream signaling initiated from the interaction of LPS and TLR4 complex induced an LPS molecule [26]. Upstream signaling initiated from the interaction of LPS and TLR4 complex the activation of MAPKs/NFκB signaling pathway, which results in overexpression of inflammatory induced the activation of MAPKs/NFκB signaling pathway, which results in overexpression of factors [22]. The overexpression of the inflammatory factors, such as enzymes iNOS and COX-2 and inflammatory factors [22]. The overexpression of the inflammatory factors, such as enzymes iNOS pro-inflammatory cytokines including TNF-α, IL-6, and IL-1β, induces cellular inflammatory responses and COX-2 and pro-inflammatory cytokines including TNF-α, IL-6, and IL-1β, induces cellular and consequently leads to in vivo damage such as endotoxin shock (Figure8)[ 6,21]. LPS-stimulated inflammatory responses and consequently leads to in vivo damage such as endotoxin shock (Figure macrophages have been generally used as an in vitro model to evaluate the anti-inflammatory activity 8) [6,21]. LPS-stimulated macrophages have been generally used as an in vitro model to evaluate the of numerous natural products and synthetic compounds. anti-inflammatory activity of numerous natural products and synthetic compounds.

FigureFigure 8. Schematic 8. Schematic representation representation of the of anti-inflammatory the anti-inflammatory effects eff ectsof atraric of atraric acid acidin RAW264.7 in RAW264.7 cells. cells. Abbreviations:Abbreviations: LPS, LPS, lipopolysaccharide; lipopolysaccharide; TLR4, TLR4, Toll- Toll-likelike receptor receptor 4; MyD88, 4; MyD88, myeloid myeloid differentiation differentiation factorfactor 88; 88;MAPKs, MAPKs, mitogen-activated mitogen-activated protein protein kina kinase;se; ERK, extracellularextracellular regulated regulated protein protein kinase; kinase; NF κB, NFκnuclearB, nuclear factorfactor-κB;κ iNOS,B; iNOS, inducible inducible nitric nitric oxide oxide synthase; synthase; COX-2, COX-2, cyclooxygenase-2; cyclooxygenase-2; NO, NO, nitric nitric oxide. oxide. The purpose of this study was to discover an anti-inflammatory agent from a natural product. The same was successfully achieved by isolation and in vitro/in vivo evaluation of secondary The purpose of this study was to discover an anti-inflammatory agent from a natural product. metabolites obtained from a natural source; that is, lichens. Atraric acid has been previously reported The same was successfully achieved by isolation and in vitro/in vivo evaluation of secondary to have various biological activities such as antibacterial and anti-cancer activity [16,17]. However, we metabolites obtained from a natural source; that is, lichens. Atraric acid has been previously reported first isolated the atraric acid from hyterodermia hypoleuca, and reported its anti-inflammatory activity to have various biological activities such as antibacterial and anti-cancer activity [16,17]. However, in vitro and in vivo. In this study, we first screened several fractions of solvent extracts of heterodarmia we first isolated the atraric acid from hyterodermia hypoleuca, and reported its anti-inflammatory hypoleuca by estimating inhibitory activities for the production of inflammatory factors including NO, activity in vitro and in vivo. In this study, we first screened several fractions of solvent extracts of TNF-α, IL-6, IL-1β, and GM-CSF in LPS-stimulated macrophages, which revealed that the methanol heterodarmia hypoleuca by estimating inhibitory activities for the production of inflammatory factors extracts showed the most potent anti-inflammatory activity (Figure1). Next, we separated and including NO, TNF-α, IL-6, IL-1β, and GM-CSF in LPS-stimulated macrophages, which revealed that purified atraric acid from the MeoH extract using HPLC (Figure2, Figure S2, Table S1). The isolated the methanol extracts showed the most potent anti-inflammatory activity (Figure 1). Next, we atraric acid exhibited a strong inhibition for the production of NO, PGE2, IL-6, IL-1β and GM-CSF separated and purified atraric acid from the MeoH extract using HPLC (Figure 2, S2, Table S1). The in LPS-stimulated RAW264.7 cells (Figure4). Atraric acid also reduced the protein expression of isolated atraric acid exhibited a strong inhibition for the production of NO, PGE2, IL-6, IL-1β and enzymes iNOS and COX-2, involved in the production of inflammatory mediators such as NO and GM-CSF in LPS-stimulated RAW264.7 cells (Figure 4). Atraric acid also reduced the protein PGE2. In order to prove the anti-inflammatory effect of atraric acid in vivo, the effect of atraric acid was expression of enzymes iNOS and COX-2, involved in the production of inflammatory mediators such evaluated with LPS-induced endotoxin shock in mice. Endotoxin shock was induced by the treatment as NO and PGE2. In order to prove the anti-inflammatory effect of atraric acid in vivo, the effect of with LPS (10 mg/kg i.p. injection) in mice. Two different concentrations of atraric acid (10 and 30 mg/kg) atraric acid was evaluated with LPS-induced endotoxin shock in mice. Endotoxin shock was induced were injected 2 h before and 4 h after LPS administration. Excessive pro-inflammatory cytokines in by the treatment with LPS (10 mg/kg i.p. injection) in mice. Two different concentrations of atraric the serum and peritoneum lavage were observed due to the systematic inflammatory reactions in the acid (10 and 30 mg/kg) were injected 2 h before and 4 h after LPS administration. Excessive pro- LPS-stimulated groups. Furthermore, the LPS-stimulated mice also exhibited histological signs of inflammatory cytokines in the serum and peritoneum lavage were observed due to the systematic inflammatory reactions in the LPS-stimulated groups. Furthermore, the LPS-stimulated mice also exhibited histological signs of necrosis resulting from vasodilation and bleeding of the organs. However, in the atraric acid-treatment mice, the LPS-induced pro-inflammatory cytokine expression was inhibited. Histological staining in these mice showed reduced vasodilation and bleeding of the damaged organs (Figure 7b–f). The in vitro and in vivo data demonstrate that atraric acid can be a promising therapeutic agent against inflammatory diseases.

Int. J. Mol. Sci. 2020, 21, 7070 9 of 13 necrosis resulting from vasodilation and bleeding of the organs. However, in the atraric acid-treatment mice, the LPS-induced pro-inﬂammatory cytokine expression was inhibited. Histological staining in these mice showed reduced vasodilation and bleeding of the damaged organs (Figure7b–f). The in vitro and in vivo data demonstrate that atraric acid can be a promising therapeutic agent against inﬂammatory diseases.

4. Materials and Methods

4.1. Collection and Preparation of the Lichen Heterodermia hypoleuca (Kol.170037, HH) was provided by Allied Bioresource Center in the Korean Lichen Research Institute, Sunchon National University, Korea. HH was collected from the coastal rocks of the southern part of Korea. The dried Lichen thalli (60 g) were extracted with 2 L methanol (MeOH) at room temperature for 48 h using sonication. The extract was then ﬁltered and concentrated under vacuum at 40 ◦C using a rotary evaporator.

4.2. Chemicals and Reagents Roswell park memorial institute-1640 medium (RPMI 1640) and fetal bovine serum (FBS) were purchased from Hyclone Laboratories (Hyclone, South Logan, UT, USA). Cell counting kit–8 (CCK-8) was purchased from Dojindo Laboratories (Dojindo, Kumamoto, Japan). Dimethyl sulfoxide (DMSO), bovine serum albumin (BSA) and Lipopolysaccharides (LPS) were purchased from Sigma Aldrich (St. Louis, MO, USA). Puriﬁed rat anti-mouse (TNF-α, IL-6, IL-10, IL-1β, GM-CSF) and biotin rat anti-mouse (TNF-α, IL-6, IL-10, IL-1β and GM-CSF) were purchased from BD Biosciences (San Diego, CA, USA). PGE2 ELISA kit was purchased from R&D Systems (R&D, Minneapolis, MN, USA). HPLC grade acetonitrile, water and methanol were purchased from J.T Baker (USA), while ODS-A (40–60 mesh), Luna 5u (C 100A 250 110 nm), and YMC-actus (Triat C 100A 250 20 mm) were from Merck 18 × 18 × Co. (Germany), Phenomenex Inc (USA), and YMC (Japan), respectively. Atraric acid, as standard component, was obtained from ChemFaces Biochemical Co., Ltd. (Wuhan, China). The purity of atraric acid (over 96.5%) that was isolated from HH in our laboratory was determined by a high-performance liquid chromatography-evaporative light scattering detector (Figure S2, Table S1).

4.3. Isolation and Analysis HH extract was dissolved in ethyl acetate (EtOAc) and was partitioned with water (30% MeOH). EtOAc fraction dissolved in MeOH (50% DMSO) was separated by preparative liquid chromatography (Prep-LC, YMC-actus Triart C 250 20 nm column, YMC, Japan), 15 mL/min, UV detection at 18 × 254, 365 nm using gradient elution 5% solvent B to 100% solvent B as eluent to afford 63 fractions (1-63) (solvent A: Water + formic acid 0.5%, solvent B: Acetonitrile + formic acid 0.5%). Atraric acid (39.5 g, Rt = 36.9 min) was obtained by reversed-phase HPLC (Luna 5u C 100A 250 20 nm 18 × column, Phenomenex Inc.,USA), 1.0 mL/min, UV detections at 254 nm using isocratic elution, solvent B (Acetonitrile + formic acid 0.5%): 0–60 min; as eluent 50%. LC-MS/ELSD analysis was performed to analyze the purified atraric acid. The column used for the analysis was a Phenomenex C (5 µm, 4.6 250 mm) column. As analysis conditions, the initial 18 × solvent 5% solvent B hold at for 5 min. The gradient condition, Solvent B was then increased from 5% to 70% within 40 min, following proportions solvent B; 45–50 min, 70–100%, hold at B; 100% for 10 min. Solvent A and B in 0.1% Trifluoroacetic acid (TFA).

4.4. Instruments and Data Collection NMR spectra were recorded on a JNM-ECZS series FT 400 NMR (JEOL, Japan) spectrometer and Varian Inova spectrometers using DMSO-d6 as the solvent, obtained from Cambridge Isotope Laboratories (CIL), Inc. Chemical shifts were conﬁrmed to be Atraric acid compared with the reported studies. Mass spectra were analyzed on Agilent Technologies 6120 Quadrupole mass spectrometer Int. J. Mol. Sci. 2020, 21, 7070 10 of 13 coupled with an Agilent Technologies 1260 series HPLC, Evaporative light scattering detector (ELSD) G4260A and Electrospray ionization source (ESI) low resolution.

4.5. Cell Culture RAW264.7 cells (murine macrophage cell line, KCLB 40071) were purchased from Korean Cell Line Bank (Seoul, South Korea). The cells were grown in RPMI 1640 supplemented with 10% FBS, 100 units/mL of penicillin, 100 µg/mL of streptomycin (Invitrogen, Carlsbad, CA, USA), and 2-mercaptoethanol (50 µM) in a humidiﬁed atmosphere at 37 ◦C with 5% CO2

4.6. Cell Viability Assay Cell viability was determined by CCK-8 assay. RAW 264.7 cells (5 104 cell/well) were seeded × in a 96-well plate and incubated over-night before experimental interventions. Next, the cells were treated with diﬀerent concentrations of samples for 24 h. Thereafter, 10 µL of CCK-8 was added to each plated and incubated for 2 h at 37 ◦C with 5% CO2. The optical density was then read at 450 nm using a microplate reader (Versa Max, molecular devices, Sunnyvale, CA). The cell viability was evaluated by comparing the absorbance values of the sample groups with that of the control group, which was considered as 100%.

4.7. Measurement of NO and Cytokines The concentrations of NO, TNF-α, IL-6 IL-1β and GM-CSF were measured by Griess assay and ELISA assay. RAW 264.7 cells (5 105 cell/mL) were seeded in a 24-well plate and incubated over-night × before experimental interventions. The cells were treated with various concentrations of sample extracts in the presence of LPS (1 µg/mL) for 24 h at 37 ◦C with 5% CO2. Subsequently, the culture supernatant was assayed according to the manufacturer’s instructions [9,27].

4.8. Western Blot Analysis The cell was collected and washed twice with cold phosphate-buffer saline (PBS). Total protein was extracted by using radioimmunoprecipitation assay buffer (Thermo, Rockford, IL, USA) in the presence of protease and phosphatase inhibitor cocktail (Thermo, Rockford, IL, USA). Protein concentration was determined using the BCA protein assay kit (Thermo, Rockford, IL, USA). Equal amounts of protein were separated by 4–12% bis-tris plus gels (Thermo, Rockford, IL, USA) and transferred to nitrocellulose membranes (Thermo, Rockford, IL, USA). The membranes were incubated with blocking solution for 1 h at room temperature followed by overnight incubation at 4 ◦C with primary antibodies. The primary antibodies included specific antibody β-actin (1:1000, Thermo, Rockford, IL, USA), iNOS (1:500, Thermo, Rockford, IL, USA), COX-2 (1:1000, Thermo, Rockford, IL, USA), IκB (1:1000, Thermo, Rockford, IL, USA), p-IκB (1:1000, Thermo, Rockford, IL, USA), NFκB (1:1000, Thermo, Rockford, IL, USA) and p-NFκB (1:1000, Thermo, Rockford, IL, USA). The membranes were washed with TBST and incubated in horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000, Thermo, Rockford, IL, USA) for 1 h at room temperature while shaking. Next, the membranes were washed with TBST and developed with the enhanced chemiluminescence kit (Thermo, Rockford, IL, USA). The protein bands were captured and measured using a bio-imaging system (Microchemi 4.2 Chemilumineszenz-system, Neve Yamin, Israel) [28].

4.9. Animals and Experimental Design Female BALB/c mice (7 weeks old), weighing about 17-20 g, were purchased from orient-bio (Orientbio Inc., Seongnam, Korea). The animals were housed in a controlled environment [22 2 and 50 5% (relative humidity)] in ± ± polycarbonate cages and fed a standard animal diet with water. All mice were treated strictly according to the guidelines for laboratory animal care and use issued by the Sunchon National University Int. J. Mol. Sci. 2020, 21, 7070 11 of 13

Institutional Animal Care and Use Committee (SCNU IACUC). All procedures were approved by SCNU IACUC (license number: SCNU IACUC-2019-15, approval date: 12 Dec 2019), and all the experiments performed on mice were performed with extra care and concern. For the endotoxin shock model, a total of 20 mice were randomly divided into a control group, LPS (10 mg/kg, i.p.) group, and atraric acid-treated group (10 mg/kg, 30 mg/kg, i.p.) (n = 5). As shown in Figure7a, atraric acid was treatment was conducted 2 h prior to LPS administration standard, and atraric acid was intraperitoneally administered to mice for 4 h after LPS injection. Eight hours after the last administration, blood samples were taken, serum samples were prepared for pro-inﬂammatory cytokine measurement, and organs (kidney, liver, lung, spleen) were extracted for tissue staining [26,29].

4.10. Histopathological Examination Tissues were fixed in 4% formalin and embedded in paraffin. Sections of 4 µm thickness were obtained and stained with H&E and histological changes were monitored under the microscope at 400 magnification [10,30]. × 4.11. Statistical Analysis Data are presented as mean standard deviation (SD) or standard error of the mean (SEM). ± The statistical differences between groups were analyzed by one-way SPSS version 22 (SPSS, Chicago, IL, USA) followed by Student’s t-test. A p-value of 0.05 or less indicated statistical significance.

5. Conclusions Atraric acid isolated from the MeOH extract of hyterodermia hypoleuca significantly reduced the production of NO, PGE2, TNF-α and IL-6 in LPS-induced RAW 264.7 macrophages and suppressed the expression of enzymes iNOS, COX-2. In addition, the inhibition of NFκB/ERK in NFκB-MAPK pathways, which is one of the inflammatory signaling pathways, was confirmed in vitro. Based on in vitro results, the in vivo anti-inflammatory effect of atraric acid was evaluated in the LPS-induced mouse endotoxin shock model, showing the downregulation of inflammatory cytokines by LPS-induced endotoxin shock and the decrease in vasodilation and bleeding in damaged organs. Taken together, it was confirmed that atraric acid is a potential target compound for the development of a novel anti-inflammatory agent.

6. Patents A patent for an anti-inﬂammatory composition containing an extract component derived from lichen was ﬁled. [Patent application number: 10-2018-0128880].

Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/19/ 7070/s1. Figure S1: Procedure for fractionation and separation by HH MeOH extracts (Ext.); Figure S2: HPLC chromatograms profiles of atraric acid; Table S1: Integration of chromatogram. Author Contributions: S.-K.M. and S.-T.Y. conceived and designed the experiments; S.-K.M., K.-Y.K. and H.-Y.J. performed the experiments; S.-K.M., K.-Y.K. and H.-W.C. analyzed the data; Y.-H.H., S.-J.K., S.-G.H., and J.-S.H. contributed reagents/materials/analysis tools; S.-K.M., K.-Y.K. and D.-J.C. wrote the paper. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: This study was carried out with the support of R&D Program for Forest Science Technology (Project No. 2017024A00-1819-BA01) provided by Korea Forest Service (Korea Forestry Promotion Institute). Conflicts of Interest: The authors have no conflict of interest to declare. Int. J. Mol. Sci. 2020, 21, 7070 12 of 13

Abbreviations

LPS lipopolysaccharide HH Heterodermia hypoleuca NFκB nuclear factor kappa B NO nitric oxide COX-2 cyclooxygenase-2 PGE2 prostaglandin E2 TNF-α tumor necrosis factor-α IL-1β interleukin-1β IL-6 interleukin-6 GM-CSF granulocyte-macrophage colony-stimulating factor TLR-4 toll-like receptor 4 CCK-8 cell counting kit-8 i.p. intraperitoneal

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