Isoleucilactucin Ameliorates Coal Fly Ash-Induced Inflammation Through

International Journal of Molecular Sciences

Article Isoleucilactucin Ameliorates Coal Fly Ash-Induced Inﬂammation through the NF-κB and MAPK Pathways in MH-S Cells

H. M. Arif Ullah 1 , Tae-Hyung Kwon 2,*, SeonJu Park 3 , Sung Dae Kim 1 and Man Hee Rhee 1,*

1 Department of Veterinary Medicine, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Korea; [email protected] (H.M.A.U.); [email protected] (S.D.K.) 2 Department of Research and Development, Chuncheon Bio-Industry Foundation (CBF), Chuncheon 24232, Korea 3 Chuncheon Center, Korea Basic Science Institute (KBSI), Chuncheon 24341, Korea; [email protected] * Correspondence: [email protected] (T.-H.K.); [email protected] (M.H.R.); Tel.: +82-33-258-6993 (T.-H.K.); +82-53-950-5967 (M.H.R.)

Abstract: We investigated whether isoleucilactucin, an active constituent of Ixeridium dentatum, reduces inflammation caused by coal fly ash (CFA) in alveolar macrophages (MH-S). The anti- inflammatory effects of isoleucilactucin were assessed by measuring the concentration of nitric oxide (NO) and the expression of pro-inflammatory mediators in MH-S cells exposed to CFA-induced inflammation. We found that isoleucilactucin reduced CFA-induced NO generation dose-dependently in MH-S cells. Moreover, isoleucilactucin suppressed CFA-activated proinflammatory mediators,

including cyclooxygenase-2 (COX2) and inducible NO synthase (iNOS), and the proinﬂammatory cytokines such as interleukin-(IL)-1β, IL-6, and tumor necrosis factor (TNF-α). The inhibiting

Citation: Ullah, H.M.A.; Kwon, properties of isoleucilactucin on the nuclear translocation of phosphorylated nuclear factor-kappa B T.-H.; Park, S.; Kim, S.D.; Rhee, M.H. (p-NF-κB) were observed. The effects of isoleucilactucin on the NF-κB and mitogen-activated protein Isoleucilactucin Ameliorates Coal Fly kinase (MAPK) pathways were also measured in CFA-stimulated MH-S cells. These results indicate Ash-Induced Inflammation through that isoleucilactucin suppressed CFA-stimulated inflammation in MH-S cells by inhibiting the NF-κB the NF-κB and MAPK Pathways in and MAPK pathways, which suggest it might exert anti-inflammatory properties in the lung. MH-S Cells. Int. J. Mol. Sci. 2021, 22, 9506. https://doi.org/10.3390/ Keywords: lung macrophages; isoleucilactucin; anti-inflammatory properties; NF-κB pathway; ijms22179506 AMPK pathway

Academic Editor: Stefania Marzocco

Received: 13 August 2021 1. Introduction Accepted: 29 August 2021 Published: 1 September 2021 Inflammation is a critical aspect of the immune response against cell damage and pathogens, which also provides a defense mechanism to remove harmful stimuli [1–3]. Publisher’s Note: MDPI stays neutral Increased movement of plasma and leukocytes from the blood into wounded regions with regard to jurisdictional claims in causes acute inflammation, which is an initial response of the organism to damaging published maps and institutional affil- stimuli. When it becomes chronic, inflammation causes a gradual shift in the cell types iations. present at the site of inflammation and can result in a destruction or regeneration of tissues, depending on the context [4]. Dysregulated inflammation is associated with various chronic diseases [2,5]. Macrophages play a crucial role in inflammatory processes, primarily by producing proinflammatory mediators that include nitric oxide (NO), inducible NO

Copyright: © 2021 by the authors. synthase (iNOS), cyclooxygenase-2 (COX-2), as well as the cytokines interleukin (IL)-1β, Licensee MDPI, Basel, Switzerland. IL-6, and tumor necrosis factor-α (TNF-α)[6,7]. Therefore, inhibiting proinflammatory This article is an open access article macrophage transformation is considered a critical strategy in the treatment of numerous distributed under the terms and inflammatory disorders. conditions of the Creative Commons In the last few decades, environmental contaminants were shown to cause immuno- Attribution (CC BY) license (https:// logic dysfunction in chronic respiratory inflammatory disorders: asthma, chronic respira- creativecommons.org/licenses/by/ tory diseases, chronic obstructive pulmonary disease (COPD), and various types of lung 4.0/).

Int. J. Mol. Sci. 2021, 22, 9506. https://doi.org/10.3390/ijms22179506 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 13

In the last few decades, environmental contaminants were shown to cause immuno- logic dysfunction in chronic respiratory inflammatory disorders: asthma, chronic respira- Int. J. Mol. Sci. 2021, 22, 9506 2 of 12 tory diseases, chronic obstructive pulmonary disease (COPD), and various types of lung cancer [1,6,8–10]. COPD is the world’s third leading cause of death, with 3 million individuals dying from the disease in 2016 [11]. In modern industrial countries, particulate mattercancer (PM) [1,6,8 represents–10]. COPD the is most the world’s dangerous third ai leadingr contaminant cause of [12]. death, The with main 3 millionsources individ- of PM includeuals dying outdoor from pollutants the disease from in 2016 gasoline [11]. In fuel modern burning industrial in automobiles countries, and particulate industries, matter as well(PM) as represents indoor pollutants the most from dangerous cigarette air smok contaminante and culinary [12]. The exhausts main sources [6,13]. ofAccording PM include to previousoutdoor research, pollutants exposure from gasoline to PM has fuel been burning linked in automobilesto worseningand of asthma industries, symptoms, as well as as wellindoor as increased pollutants hospitalization from cigarette smoke and mortality and culinary in individuals exhausts [6 with,13]. AccordingCOPD [14,15]. to previous Expo- sureresearch, to PM exposure could induce to PM alveolar has been macropha linked toges worsening to produce of asthmaexcessive symptoms, amounts asof wellproin- as flammatoryincreased hospitalization mediators, such and as mortalityiNOS, COX2, in individuals TNF-α, IL-1 withβ, and COPD IL-6, [14which,15]. determine Exposure toa widePM couldrange induceof pathological alveolar macrophagesand clinical symptoms to produce [9,16]. excessive amounts of proinflammatory α β mediators,The pharmaceutical such as iNOS, industry’s COX2, TNF- interest, IL-1 in, natural and IL-6, drugs which has determine grown, thus a wide including range of naturalpathological resources and clinicalin drug symptomsdevelopment [9,16 [3,17,]. 18]; indeed, over 100 medications derived The pharmaceutical industry’s interest in natural drugs has grown, thus including from natural products are now being tested in clinical trials [3,19]. Isoleucilactucin, one of natural resources in drug development [3,17,18]; indeed, over 100 medications derived the major active constituents of Ixeridium dentatum, has been reported to have amylase from natural products are now being tested in clinical trials [3,19]. Isoleucilactucin, one secretion activity after treatment with high glucose in human salivary gland cells [20]. of the major active constituents of Ixeridium dentatum, has been reported to have amylase Coal fly ash (CFA) is made up of a variety of liquid and solid PM, including asbestos, coal, secretion activity after treatment with high glucose in human salivary gland cells [20]. and combustion particles of various sizes and origins [1,6,21]. However, the effect of iso- Coal fly ash (CFA) is made up of a variety of liquid and solid PM, including asbestos, leucilactucin in the CFA-stimulated inflammatory cascade on alveolar macrophages (MH- coal, and combustion particles of various sizes and origins [1,6,21]. However, the effect S) remains elusive. The goal of this study is to determine whether isoleucilactucin has anti- of isoleucilactucin in the CFA-stimulated inflammatory cascade on alveolar macrophages inflammatory activities upon CFA-induced inflammatory stimulation of MH-S cells. (MH-S) remains elusive. The goal of this study is to determine whether isoleucilactucin has anti-inflammatory activities upon CFA-induced inflammatory stimulation of MH-S cells. 2. Results 2.1.2. ResultsIsoleucilactucin Protected against CFA-Induced Nitric Oxide Production and Cell Death in Alveolar2.1. Isoleucilactucin Macrophage Protected(MH-S) Cells against CFA-Induced Nitric Oxide Production and Cell Death in Alveolar Macrophage (MH-S) Cells Nitric oxide (NO) is a vital regulator in the inflammatory process, while excess NO contributesNitric oxideto the (NO)development is a vital regulatorof numerous in the inflammatory inflammatory disorders process, while[22–24]. excess In our NO study,contributes the NO to levels the development in murine MH-S of numerous cells in inflammatoryresponse to CFA disorders stimulation [22–24 were]. In ourassessed study, usingthe NO the levels Griess in reaction. murine MH-S Isoleucilactucin cells in response decreased to CFA NO stimulation induction werepotently assessed and dose-de- using the pendentlyGriess reaction. (Figure Isoleucilactucin 1A). decreased NO induction potently and dose-dependently (Figure1A).

FigureFigure 1. 1. EffectEffect of of isoleucilactucin isoleucilactucin on on coal coal fly ﬂy ash ash (CFA)-induced (CFA)-induced nitric nitric oxide oxide (NO) (NO) generation generation and and viabilityviability in in MH-S MH-S cells. cells. (A (A) )Cells Cells were were categorized categorized into into six six groups: groups: basal basal (control), (control), CFA CFA (2.5 (2.5 μµg/mL),g/mL), andand four four concentrations concentrations of of CFA CFA with with isoleucilactucin isoleucilactucin (12.5, (12.5, 25, 25, 50, 50, and and 100 100 μµM).M). Cells Cells were were treated treated withwith the the different different isoleucilactuc isoleucilactucinin concentrations forfor 3030min min before before CFA CFA treatment treatment and and incubated incubated for for18 18 h. Theh. The Griess Griess reagent reagent method method was wa useds used to determine to determine the NO the level. NO (level.B) The (B MTT) The reagent MTT reagent method methodwas used was to used perform to perform a cell viability a cell viability experiment. experi Ament. 24-well A 24-well plate was plate used was to used seed to the seed cells. the Values cells. Valuesfrom three from independent three independent experiments experi werements expressed were expressed as the mean as the± meanSEM +/ (n− =SEM 3). Compared(n = 3). Compared with the basal group, ns: not signiﬁcant, # p < 0.001; compared with the CFA group, * p < 0.05, ** p < 0.01, and *** p < 0.001.

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with the basal group, ns: not significant, # p < 0.001; compared with the CFA group, * p < 0.05, ** p < Int. J. Mol. Sci. 2021, 22, 9506 0.01, and *** p < 0.001. 3 of 12

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used to demonstrate cell viability, and the data showed that isoleucilactucin had no effect The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was on cell toxicity at the different concentrations examined in comparison to the control used to demonstrate cell viability, and the data showed that isoleucilactucin had no effect group (Figure 1B). Our findings together indicate that isoleucilactucin suppressed NO on cell toxicity at the different concentrations examined in comparison to the control group generation dose-dependently and at noncytotoxic levels. (Figure1B). Our findings together indicate that isoleucilactucin suppressed NO generation dose-dependently and at noncytotoxic levels. 2.2. Suppressive Effect of Isoleucilactucin on CFA-Induced Proinflammatory Cytokines in MH-S Cells2.2. Suppressive Effect of Isoleucilactucin on CFA-Induced Proinflammatory Cytokines in MH-STo Cells evaluate the anti-inflammatory effect of isoleucilactucin on CFA-activated inflam- mationTo in evaluate the MH-S the anti-inflammatorycells, reverse transcript effection of polymerase isoleucilactucin chain on reaction CFA-activated (RT-PCR) inflam- was assessed.mation in After the MH-S 30 min cells, of reversepretreatment transcription with isoleucilactucin, polymerase chain the reactionlevels of (RT-PCR)CFA-induced was proinflammatoryassessed. After 30 factors min of were pretreatment decreased with in MH-S isoleucilactucin, cells. mRNA thelevels levels of proinflammatory of CFA-induced mediators,proinflammatory such as factors iNOS and were COX-2, decreased as well in MH-S as proinflammatory cells. mRNA levels cytokines, of proinflammatory such as IL-1β, IL-6,mediators, and TNF- suchα as, were iNOS measured and COX-2, by asRT-PCR well as to proinflammatory explore the anti-inflammatory cytokines, such effects as IL-1 ofβ, isoleucilactucinIL-6, and TNF-α (Figure, were measured2A). At the by mRNA RT-PCR level, to explore proinflammatory the anti-inflammatory factors were effectsfound ofto beisoleucilactucin dose-dependently (Figure decreased2A). At the(Figure mRNA 2B–F). level, RT-PCR proinflammatory results showed factors that were isoleucilactu- found to cinbe dose-dependentlyprevented CFA-induced decreased inflammatory (Figure2B–F). cytokine RT-PCR production results showed and thatreduced isoleucilactucin the expres- sionprevented levels CFA-inducedof proinflammatory inflammatory factors. cytokine production and reduced the expression levels of proinflammatory factors.

Figure 2. Effect of isoleucilactucin on CFA-induced proinflammatory mediators and cytokines in MH-S cells analyzed using Figure 2. Effect of isoleucilactucin on CFA-induced proinflammatory mediators and cytokines in MH-S cells analyzed reverseusing reverse transcription transcription polymerase polyme chainrase reactionchain reaction (RT-PCR). (RT-PCR). (A) RT-PCR (A) RT-PCR analysis analysis was performed was performed to evaluate to evaluate the mRNA the levelsmRNA of levels proinflammatory of proinflammatory mediators, mediators, such as such cyclooxygenase as cyclooxyge 2 (COX2)nase 2 (COX2) and inducible and inducible nitric oxide nitric synthase oxide synthase (iNOS), (iNOS), as well as well proinflammatory as proinflammatory cytokines, cytokines, such as such tumor as necrosistumor necrosis factor-alpha factor-alpha (TNF-α (TNF-), interleukinα), interleukin (IL)-6, (IL)-6, IL-1β, IL-1 andβ GAPDH., and GAPDH. (B–F) ImageJ(B–F) ImageJ was used was to used perform to perform quantitative quantitati analysisve analysis of the relativeof the relative mRNA mRNA expression expression levels. Isoleucilactucinlevels. Isoleucilactucin dosages dosages of 12.5, 25,of 12.5, 50, and 25, 10050, andµM were100 μ usedM were to seedused the to seed cells inthe a cells 6-well in plate.a 6-well Values plate. from Values three from independent three inde experimentspendent experiments were expressed were # asexpressed the mean as± theSEM mean (n =+/ 3).− SEM Compared (n = 3). withCompared the basal with group, the basa ns: notl group, significant, ns: not# significant,p < 0.001; compared p < 0.001; with compared the CFA with group, the *CFAp < 0.05,group, ** p* p< < 0.01, 0.05, and ** p *** < 0.01,p < 0.001. and *** p < 0.001.

2.3. Isoleucilactucin Ameliorated CFA-Induced mRNA Expression of Proinflammatory Cytokines in MH-S Cells To validate the RT-PCR results in MH-S cells, proinflammatory mediators mRNA expression was next investigated. Following isoleucilactucin (12.5, 25, 50, and 100 µM) administration, the mRNA expression levels of the proinflammatory mediators were markedly and dose-dependently inhibited (Figure3A–E). The real-time PCR results demonstrated Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 13

2.3. Isoleucilactucin Ameliorated CFA-Induced mRNA Expression of Proinflammatory Cytokines in MH-S Cells To validate the RT-PCR results in MH-S cells, proinflammatory mediators mRNA expression was next investigated. Following isoleucilactucin (12.5, 25, 50, and 100 μM) Int. J. Mol. Sci. 2021, 22, 9506 4 of 12 administration, the mRNA expression levels of the proinflammatory mediators were markedly and dose-dependently inhibited (Figure 3A–E). The real-time PCR results demonstrated that isoleucilactucin significantly lowered the proinflammatory factors in a concentration-dependentthat isoleucilactucin signiﬁcantly manner lowered(Figure 3A–E). the proinﬂammatory factors in a concentration- dependent manner (Figure3A–E).

Figure 3. Effect of isoleucilactucin on CFA-induced mRNA expression in MH-S cells measured using real-time polymerase chainFigure reaction 3. Effect (PCR). of isoleucilactucin (A–E) The mRNA on CFA-induced levels of COX2, mRNA iNOS, expressi IL-1βon, IL-6, in MH-S and TNF-cells measuredα were analyzed using real-time by PCR afterpolymerase 18 h of chain reaction (PCR). (A–E) The mRNA levels of COX2, iNOS, IL-1β, IL-6, and TNF-α were analyzed by PCR after 18 h of incubation with CFA. Isoleucilactucin dosages of 12.5, 25, 50, and 100 µM were used. GAPDH was used as a housekeeping incubation with CFA. Isoleucilactucin dosages of 12.5, 25, 50, and 100 μM were used. GAPDH was used as a housekeeping gene. Values from the three independent experiments were expressed as the mean ± SEM (n = 3). Compared with the basal gene. Values from the three independent experiments were expressed as the mean +/− SEM (n = 3). Compared with the # p p p p group,basal group,< 0.001;# p < 0.001; compared compared with thewith CFA the group,CFA group, ns: not ns: significant, not significant, * < 0.05,* p < **0.05,< ** 0.01, p < and0.01,*** and <*** 0.001. p < 0.001. 2.4. Isoleucilactucin Inhibited the Translocation of Nuclear Factor-Kappa B in CFA-Treated 2.4.MH-S Isoleucilactucin Cells Inhibited the Translocation of Nuclear Factor-Kappa B in CFA-Treated MH- S Cells An immunofluorescence (IF) analysis was used to examine the translocation of acti- vatedAn nuclear immunofluorescence factor (NF)-κB from (IF) theanalysis cytoplasm was used to the to nucleus,examine to the determine translocation whether of acti- the vatedanti-inflammatory nuclear factor actions (NF)-κ ofB from isoleucilactucin the cytoplasm could to the be nucleus, mediated to by determine the NF-κ whetherB signaling the pathwayanti-inflammatory in CFA-stimulated actions of alveolarisoleucilactucin macrophages. could be In mediated activated by MH-S the NF- cells,κB treatmentsignaling withpathway CFA in increased CFA-stimulated NF-κB translocation alveolar macrophages. from the cytoplasm In activated to the nucleus,MH-S cells, but treatment with theCFA maximum increased doseNF-κ ofB translocation isoleucilactucin from (100 theµ M)cytoplasm inhibited to phosphorylatedthe nucleus, but (p)-NF-treatmentκB withnuclear the translocationmaximum dose (Figure of isoleucilactucin4). Here, we (100 used μ Bay-11M) inhibited as an phosphorylated NF- κB inhibitor. (p)-NF- Ourκ IFB nuclearexperiment translocation reveals that (Figure the anti-inflammatory 4). Here, we used effects Bay-11 of as isoleucilactucin an NF-κB inhibitor. may result Our IF from ex- partlyperiment inhibiting reveals phosphorylationthat the anti-inflammatory of the NF-κ Beffects signaling of isoleucilactucin cascade. may result from partly inhibiting phosphorylation of the NF-κB signaling cascade.

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κ FigureFigure 4.4. EffectEffect ofof isoleucilactucinisoleucilactucin onon CFA-inducedCFA-induced nuclearnuclear factor-kappafactor-kappa BB (NF-(NF-κB)B) translocationtranslocation inin MH-SMH-S cellscells examinedexamined byby immunoﬂuorescenceimmunofluorescence (IF)(IF) assay.assay. InIn aa 6-well6-well plate,plate, cellscells werewere seededseeded onon aa coatedcoated covercover slipslip andand divideddivided intointo fourfour groups:groups: basal,basal, CFA,CFA, CFACFA withwith isoleucilactucinisoleucilactucin (100(100µ μM),M), andand CFACFA withwith Bay-11Bay-11 (p-NF-κB inhibitor). Before Before treatment treatment with with CFA CFA (2.5 (2.5 μg/mL),µg/mL), cells cells were were treated treated for for30 min 30 min with with isoleucilactucin isoleucilactucin and and Bay-11 Bay-11 (10 (10 μM)µM) and and incubated incubated for for 18 18h. h.IF IFstaining staining was was used used to toexamine examine the the p-NF- p-NF-κB κnuclearB nuclear translocation. translocation. To observe To observe the nuclei, the nuclei, the samples the samples were weremounted mounted using usinga ProLong a ProLong Gold Antifade Gold Antifade Reagent Reagent containing containing DAPI (blue). DAPI (blue).Confocal Confocal microscopy microscopy at 1000× at magnifi- 1000× cation was used to examine the stained cells. magniﬁcation was used to examine the stained cells.

2.5.2.5. IsoleucilactucinIsoleucilactucin InhibitsInhibits thethe ActivationActivation ofof NuclearNuclear Factor-KappaFactor-Kappa BB andand Mitogen-ActivatedMitogen-Activated ProteinProtein KinaseKinase SignalingSignaling PathwaysPathways inin CFA-TreatedCFA-Treated MH-S MH-S Cells Cells ToTo exploreexplore the the molecular molecular pathways pathways underlying underlying the the protective protective effects effects of of isoleucilactucin isoleucilactu- incin our in ourin vitro in vitroCFA-induced CFA-induced inflammation inflammation model, model, we we performed performed Western Western blot blot analysis. analy- CFAsis. CFA activates activates the inflammatorythe inflammatory pathway, pathway, and theand NF- theκ NF-B andκB MAPKand MAPK signaling signaling pathways path- areways important are important in inflammatory in inflammatory processes processes [25–27]. [25–27]. Pretreatment Pretreatment with isoleucilactucin with isoleucilactucin greatly decreasedgreatly decreased the phosphorylation the phosphorylatio (p) ofn inhibitor (p) of inhibitor of kappa of Bkappa (IκB) B and (Iκ NF-B) andκB inNF- MH-SκB in cells,MH- whereasS cells, whereas treatment treatment with CFA with significantly CFA significantly enhanced enhanced the p-I κtheB andp-Iκ p-NF-B and κp-NF-B in alveolarκB in al- macrophagesveolar macrophages (Figure 5(FigureA–C). Furthermore, 5A–C). Furthermore, as compared as compared to treatment to withtreatment CFA alone,with CFA the MAPKalone, the pathways, MAPK includingpathways, phosphorylated including phosphor extracellularylated extracellular signal-regulated signal-regulated kinase (p-ERK), ki- phosphorylatednase (p-ERK), phosphorylated c-Jun N-terminal c-Jun kinase N-terminal (p-JNK), kinase and p-P38, (p-JNK), were and dose-dependently p-P38, were dose- re- duceddependently (Figure 5reducedD–G). Collectively, (Figure 5D–G). these Collectively, findings indicate these that findings the CFA-induced indicate that activation the CFA- κ κ ofinduced p-NF- activationB, p-I B, p-ERK,of p-NF- p-JNK,κB, p-I andκB, p-ERK, p-P38 in p-JNK, MH-S and cells p-P38 was significantlyin MH-S cells reduced was signif- by pretreatmenticantly reduced with by isoleucilactucin. pretreatment with isoleucilactucin.

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Figure 5.5. EffectEffect ofof isoleucilactucin isoleucilactucin on on CFA-induced CFA-induced NF- NF-κB andκB and mitogen-activated mitogen-activated protein protein kinase kinase (MAPK) (MAPK) pathways pathways in MH-S in cellsMH-S measured cells measured by Western by Western blotting. blotting. (A) Western (A) Western blot analysis blot wasanalysis done was to evaluate done to theevaluate protein the levels protein of the levels phosphorylated of the phos- (p)-IphorylatedκB and (p)-I p-NF-κBκ Band pathways. p-NF-κB ( Bpathways.,C) ImageJ (B was,C) usedImageJ to performwas used densitometric to perform densit analysisometric of the analysis protein of expression the protein levels. ex- (pressionD) Western levels. blotting (D) Western was used blotting to investigate was used the to MAPKinvestigate pathways, the MA whichPK pathways, includes which ERK, JNK,includ andes ERK, p38. JNK, (E–G and) ImageJ p38. was(E–G used) ImageJ to perform was used densitometric to perform densitometric analysis of the analysis protein of expression the protein levels. expression As a levels. loading As control, a loadingβ-actin control, was β-actin used. Isoleucilactucinwas used. Isoleucilactucin dosages of dosages 12.5, 25, of 50,12.5, and 25, 100 50, µandM were100 μ usedM were on used cells on seeded cells seeded in a 6-well in a plate.6-well Valuesplate. Values from thefrom three the three independent experiments were expressed as the mean +/− SEM (n = 3). Compared with the basal group, # p < 0.001; independent experiments were expressed as the mean ± SEM (n = 3). Compared with the basal group, # p < 0.001; compared compared with the CFA group, ns: not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001. with the CFA group, ns: not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001. 3. Discussion 3. Discussion The inflammatory process involves an abnormally high production of NO caused by The inflammatory process involves an abnormally high production of NO caused by iNOS synthesis [28,29]. In the pathophysiology of inflammatory disorders, iNOS plays a iNOS synthesis [28,29]. In the pathophysiology of inflammatory disorders, iNOS plays a crucial role in the release ofof NONO [[30,31].30,31]. Furthermore, inflammatoryinflammatory stimuli stimuli increase increase COX-2 COX- 2during during the the inflammatory inflammatory process process [ 32[32,33].,33]. InIn thethe currentcurrent study,study, onlyonly thethe CFA-activatedCFA-activated group produced more NO compared to the control group, whereas pretreatment with isoleucilactucin inhibited NO generation. Throughout thethe inflammation inflammation process, process, endotoxins endotoxins and cytokinesand cytokines cause rapidcause changes rapid changesin NO gene in NO expression, gene expression, resulting resulting in de novo in de recruitment novo recruitment of the iNOSof the andiNOS COX-2 and COX-2 pathwayspathways [34,35 [34,35].]. In the In current the current study, study, the CFA-treated the CFA-treated group group displayed displayed elevated elevated mRNA mRNA levels levelsof proinflammatory of proinflammatory factors, factors, iNOS, iNOS, COX2, COX2, TNF- αTNF-, IL-1αβ, IL-1, andβ, IL-6. and IL-6. In addition, In addition, treatment treat- withment isoleucilactucinwith isoleucilactucin decreased decreased the mRNA the mRNA levels oflevels the proinflammatoryof the proinflammatory mediators, mediators, includ- includinging cytokines. cytokines. These These findings findings indicate indicate that isoleucilactucin that isoleucilactucin might might have anti-inflammatory have anti-inflam- matoryproperties. properties. Our results Our supportresults support previous previo observationsus observations that the that COX-2 the andCOX-2 iNOS and mRNA iNOS mRNAexpression expression levels were levels elevated were elevated following following CFA stimulation CFA stimulation [1,6,8]. [1,6,8]. According to our IF staining data, CFA elevated p-NF- κB translocation into the nucleus, contrarycontrary toto isoleucilactucin isoleucilactucin (100 (100µM) μM) and and Bay-11 Bay-11 (10 µ(10M), μ whichM), which significantly significantly reduced re- ducedthe translocation. the translocation. In an earlier In an earlier investigation, investigation, we discovered we discovered a similar a similar outcome outcome [6]. Bay-11 [6]. wasBay-11 also was previously also previously shown shown to have to anti-inflammatory have anti-inflammatory properties properties through through inhibiting inhibit- the ingphosphorylation the phosphorylation of IκB[ 36of– I38κB]. [36–38]. The NF-κκBB andand MAPKMAPK signaling signaling cascades cascades were were identified identified as keyas key players players in the in inflam-the in- flammatorymatory process process [39–42 [39–42].]. NF-κ NF-B, anκB, important an important transcription transcription factor, factor, plays aplays critical a critical role in role the

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in the inflammatory response [43–45] and the MAPK signaling cascade is also important inflammatoryfor the inflammatory response [process43–45] and [27,46]. the MAPK As a signalingresult, we cascade investigated is also importantanti-inflammatory for the inflammatorymechanisms mediated process [27 by,46 isoleu]. Ascilactucin a result, we in investigateda macrophage anti-inflammatory cell line (MH-S). mechanismsWestern blot- mediatedting was byused isoleucilactucin to evaluate the in aleve macrophagels of protein cell expression line (MH-S). of Western p-IκB, p-NF- blottingκB, wasp-ERK, used T- κ κ toERK, evaluate p-JNK, the T-JNK, levels p-P38, of protein and T-P38. expression CFA treatment of p-I B, p-NF-increasedB, the p-ERK, activity T-ERK, of the p-JNK, NF-κB T-JNK, p-P38, and T-P38. CFA treatment increased the activity of the NF-κB and MAPK and MAPK signaling pathways in the MH-S cell line, but isoleucilactucin administration signaling pathways in the MH-S cell line, but isoleucilactucin administration dramat- dramatically inhibited the NF-κB and MAPK signaling. Based on these results, we pre- ically inhibited the NF-κB and MAPK signaling. Based on these results, we predicted dicted that isoleucilactucin would reduce the IκB and NF-κB phosphorylation, resulting that isoleucilactucin would reduce the IκB and NF-κB phosphorylation, resulting in a in a proinflammatory factor suppressive action. Our data show that isoleucilactucin re- proinflammatory factor suppressive action. Our data show that isoleucilactucin reduced duced CFA-activated inflammation by lowering the NF-κB and MAPK signaling pathway CFA-activated inflammation by lowering the NF-κB and MAPK signaling pathway activity activity levels (Figure 6). levels (Figure6).

Figure 6. Proposed mechanistic model of the role of isoleucilactucin on CFA-induced inﬂammation inFigure MH-S 6. cells. Proposed mechanistic model of the role of isoleucilactucin on CFA-induced inflammation in MH-S cells. 4. Materials and Methods 4.1.4. Materials Reagents and Methods 4.1. RoswellReagents Park Memorial Institute medium (RPMI-1640) for culturing macrophage, penicillin–streptomycin,Roswell Park Memorial fetal bovine Institute serum, medium and phosphate-buffered (RPMI-1640) for culturing saline were macrophage, acquired frompenicillin–streptomycin, Welgene (Gyeongsan-si, fetal Korea). bovine Oligo-dT serum, and primerphosphate-buffered sequences, including saline were COX-2, ac- iNOS,quired IL-1 fromβ, IL-6,Welgene and TNF-(Gyeongsan-si,α, were obtained Korea). from Oligo-dT Bioneer and (Daejeon, primer sequences, Republic of including Korea). OtherCOX-2, reagents iNOS, wereIL-1β purchased, IL-6, and fromTNF- Invitrogen,α, were obtained Sigma-Aldrich, from Bioneer and (Daejeon, Thermo Fisher. Republic of Korea). Other reagents were purchased from Invitrogen, Sigma-Aldrich, and Thermo 4.2. Plant Material Collection Fisher. The leaves of Ixeridium dentatum collected in the area of Jeonju-si, Jeonbuk, Korea, in4.2. May Plant 2017, Material and wereCollection certiﬁed by Professor Sang-Won Lee of the National Institute of Horticultural and Herbal Science, South Korea. A voucher specimen is kept in the The leaves of Ixeridium dentatum collected in the area of Jeonju-si, Jeonbuk, Korea, in Herbarium of the College of Pharmacy, Yonsei University, Incheon, Korea (ID201705). May 2017, and were certified by Professor Sang-Won Lee of the National Institute of Hor- 4.3.ticultural Plan Material and Herbal Extraction Science, and South Isolation Korea. A voucher specimen is kept in the Herbarium of the College of Pharmacy, Yonsei University, Incheon, Korea (ID201705). The plant extraction and isolation were conducted as previously described [20,47]. In brief, dried leaves of I. dentatum (5.0 kg) were extracted with MeOH (3 × 10 L, 50 ◦C) 4.3. Plan Material Extraction and Isolation and sonicated for 4 h after solvent evaporation, producing 290.0 g extract. The extract The plant extraction and isolation were conducted as previously described [20,47]. In was suspended in H2O and partitioned with n-hexane and EtOAc to obtain n-hexane brief, dried leaves of I. dentatum (5.0 kg) were extracted with MeOH (3 × 10 L, 50 °C) and (ID1, 91.0 g), EtOAc (ID2, 15.1 g), and H2O (ID3, 178.0 g) extracts after the solvents were removedsonicated in for vacuo. 4 h Inafter a Diaion solvent HP-20 evaporation, column, the producing H2O fraction 290.0 was g extract. chromatographed The extract with was increasingsuspended MeOH in H2O concentrations and partitioned (25, with 50, n-hexane and 75%) and to Et acquireOAc to three obtain sub-fractions: n-hexane (ID1, ID3A 91.0 (14.5g), EtOAc g), ID3B (ID2, (13.3 15.1 g), g), and and ID3C H2O (23.2 (ID3, g). 178.0 To obtain g) extracts isoleucilactucin, after the solvents the ID3C were fraction removed was in chromatographedvacuo. In a Diaion on HP-20 an RP-18 column, CC the and H eluted2O fraction with was MeOH:H chromatographed2O (1:2, v/v) with (5.2 mg).increasing The resultingMeOH concentrations isoleucilactucin (25, was 50, a and white 75%) amorphous to acquire powder. three sub-fractions: The HR-ESI-MS ID3A (Agilent (14.5 g), 6530) ID3B

Int. J. Mol. Sci. 2021, 22, 9506 8 of 12

+ [M–H2O] ion at m/z 389.1825 identiﬁed its molecular formula as C21H29NO7 (calculated for C21H27NO6, 389.1838).

4.4. Cell Culture and Treatment According to a previously published method [6], murine alveolar macrophage cell line (MH-S) was grown in RPMI supplemented with heat-inactivated 10% FBS, 100-unit/mL penicillin, and 100-g/mL streptomycin (1% antibiotics). Cells were then cultured and ◦ incubated in a humidiﬁed incubator at 37 C with 5% CO2.

4.5. Nitric Oxide Assay In accordance with our earlier investigation [48], Griess reagent A and B were used to assess the NO concentration. MH-S were seeded and cultivated for 18 h in a 24-well plate with or without CFA (2.5 µg/mL) for the basal group (control), only CFA-treated group, and CFA with isoleucilactucin (12.5, 25, 50, and 100 µM) groups at the indicated concentrations. In total, 100 µL of the Griess reagents were mixed with 100 µL of cell culture supernatants and incubated at room temperature for 10 min. On a microplate reader (Versamax, Molecular Devices, San Jose, CA, USA), the absorbance was measured at 540 nm.

4.6. Cell Viability Assay To investigate cytotoxicity, an experiment of cell viability was done as previously described [6,48], using a 10% MTT reagent at 900 µL/well in the culture media. The ◦ supernatants were removed after 3 h of incubation at 37 C in 5% CO2. Wells were ﬁlled with 100% of DMSO (500 µL/well) and incubated for 10 min with shaking at room temperature. Finally, the absorbance was measured at 560 nm using a microplate reader (Versamax, Molecular Devices, CA, USA).

4.7. PCR Analysis Using previously published methods, PCR analysis was performed [2,6,48]. The MH-S cells were nontreated or treated with isoleucilactucin (12.5, 25, 50, and 100 µM) for 30 min at the indicated doses and received CFA stimulation for 18 h. A TRIzol reagent was used to extract RNA from the cells. In total, 2 µg of total RNA were annealed for 10 min at 70 ◦C with oligo-dT, then cooled for 5 min on ice before reverse transcription (RT) in 20 µL of reaction mixture at 42.5 ◦C on a thermocycler for 90 min. To inactivate the reverse transcriptase, reaction was stopped at 95 ◦C for 5 min. cDNA from an RT reaction was used in a PCR premix to perform RT-PCR (Bioneer). On a 1% agarose gel stained with ethidium bromide (0.006%), the PCR products were also electrophoresed. To visualize the band, ImageQuant LAS 500 was used. The band density intensity was standardized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH). SYBR green was used in the real-time PCR. Table1 shows the primer sequences (Bioneer, Daejeon, Republic of Korea) for RT-PCR and real-time PCR.

4.8. Immunofluorescence Analysis The immunofluorescence (IF) experiment was carried out as described previously [2,6]. Cells were washed in PBS before being fixed in paraformaldehyde (4 %) for 10 min. Cells were then permeabilized with triton X-100 (0.2 %) in TBS (TBST) for 10 min before being rinsed three times with TBST for 5 min each time. Using 2% BSA, cells were blocked for 1 h at room temperature before being incubated overnight at 4◦C with the primary antibody rabbit anti-p-NF-κB (Cat. no: #3033). The cells were washed with TBST three times for 5 min each time. Samples were then incubated with secondary antibody, IgG Fab2 (Cat. no: #4413), for 1 h in the dark at room temperature, and washed three times with TBST before being mounted with a ProLong Gold Antifade Reagent with DAPI to visualize the cell nuclei, with confocal microscopy used to examine the samples (LSM700, Carl Zeiss, Jena, Germany). Int. J. Mol. Sci. 2021, 22, 9506 9 of 12

Table 1. Primers used for reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR analysis in this study.

H Primer Sequence F: 50-CACTCACGGCAAATTCAACGGCAC-30 GAPDH * R: 50-GACTCCACGACATACTCAGCAC-30 F: 50-CCCTTCCGAAGTTTCTGGCAGCAGC-30 iNOS * R: 50-GGCTGTCAGAGCCTCGTGGCTTTGG-30 F: 50-CACTACATCCTGACCCACTT-30 COX-2 * R: 50-ATGCTCCTGCTTGAGTATGT-30 F: 50-TTGACCTCAGCGCTGAGTTG-30 TNF-α * R: 50-CCTGTAGCCCACGTCGTAGC-30 F: 50-CTGTGGAGAAGCTGTGGCAG-30 IL-1β * R: 50-GGGATCCACACTCTCCAGCT-30 F: 50-GTACTCCAGAAGACCAGAGG-30 IL-6 * R: 5’-TGCTGGTGACAACCACGGCC-30 F: 50-CACTCACGGCAAATTCAACGGCAC-30 GAPDH ** R: 50-GACTCCACGACATACTCAGCAC-30 F: 50-GGCAGCCTGTGAGACCTTTG-30 iNOS ** R: 50-GCATTGGAAGTGAAGCGTTTC-30 F: 50-GGGTGTCCCTTCACTTCTTTCA-30 COX-2 ** R: 50-TGGGAGGCACTTGCATTGA-30 F: 50-TGCCTATGTCTCAGCCTCTTC-30 TNF-α ** R: 50-GAGGCCATTTGGGAACTTCT-30 F: 50-CAACCAACAAGTGATATTCTCCATG-30 IL-1β ** R: 50-GATCCACACTCTCCAGCTGCA-30 F: 50-TCCAGTTGCCTTCTTGGGAC-30 IL-6 ** R: 50-GTGTAATTAAGCCTCCGACTTG-30 * Primer sequence for RT-PCR; ** Primer sequence for Real-Time PCR.

4.9. Western Blot Analysis Western blot analysis was done as previously described [2,6,48]. Proteins from cells were isolated, concentrations were determined, and samples were heated in sodium dodecyl sulfate for 5 min. Proteins samples were separated using sodium dodecyl sulfate- polyacrylamide gel electrophoresis. Proteins were deposited on poly (vinylidene ﬂuoride) membranes and then blocked for 1 h with 5% skim milk at room temperature. Membranes were washed 3 times with TBST (washing buffer) for 10 min each time before being incubated overnight at 4 ◦C with primary antibodies (1:1000) against phosphorylated (p)-IκB (Cat. no: #2859), p-NF-κB, p-ERK (Cat. no: #9101S), total (T)-ERK (Cat. no: #9102S), p-JNK (Cat. no: #9251S), T-JNK (Cat. no: #9252), p-p38 (Cat. no: #9211), T-p38 (Cat. no: #9212), and β-actin ((Cat. no: #4967). The membranes were washed three times with TBST for 10 min each time and incubated with secondary antibody (1:3000) (Cat. no: #7074) for 1 h before being rinsed three times with TBST for 10 min each time. Protein bands were identiﬁed using enhanced chemiluminescence solutions (1:1 ratio) in an Imager ALS 500.

4.10. Statistical Analysis Data are presented as the means ± standard error of the mean (SEM) (n = 3). One-way analysis of variance was used to establish statistical signiﬁcance. The data was analyzed with GraphPad Prism 8.4.3 (GraphPad Software, LLC., San Diego, CA, USA). Probability values were considered signiﬁcant at * p < 0.05, ** p < 0.01, and *** p < 0.001 in comparison to the CFA group, and at # p < 0.001 in comparison to the control.

5. Conclusions Our study showed that isoleucilactucin has anti-inflammatory activities in CFA- induced MH-S cells. These findings add to our understanding of CFA-induced inflammatory responses and reveal that isoleucilactucin may decrease proinflammatory mediators and cytokines expression in murine alveolar macrophages, which is known as MH-S cells. Our results indicate that isoleucilactucin possesses an anti-inflammatory potential for Int. J. Mol. Sci. 2021, 22, 9506 10 of 12

inflammation management across a variety of inflammatory illnesses. Based on our results, the effects of isoleucilactucin on NF-κB and mitogen-activated protein kinase (MAPK) signaling have been linked to inflammation. More research is required to identify the exact molecular pathways in animal models that cause inflammation and determine how isoleucilactucin could be used as an herbal treatment to prevent inflammation.

Author Contributions: H.M.A.U. designed the study, performed the experiments, analyzed the data, and wrote the manuscript. S.D.K., T.-H.K. and S.P. performed experiments. M.H.R. supervised the study, edited the manuscript, and provided funding. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

CFA coal ﬂy ash MH-S alveolar macrophages NO nitric oxide PCR polymerase chain reaction COX2 cyclooxygenase-2 iNOS inducible NO synthase IL-6 interleukin-6 IL-1β interleukin-1 beta TNF-α tumor necrosis factor-alpha IκB inhibitor of kappa B p-NF-κB phosphorylated nuclear factor-kappa B MAPK mitogen-activated protein kinase JNK c-Jun N-terminal kinase ERK extracellular signal-regulated kinase COPD chronic obstructive pulmonary disease DMSO dimethyl sulfoxide RPMI roswell park memorial institute medium FBS fetal bovine serum IF immunoﬂuorescence DAPI 4, 6-diamidino-2-phenylindole

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