Effect of intranasal rosiglitazone on airway inflammation and remodeling in a murine model of chronic asthma

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1 ORIGINAL ARTICLE Koren J Intern Med 2016;31:89-97 Effect of intrnsl rosiglitzone on irwy inflmmtion nd remodeling in murine model of chronic sthm Hw Young Lee *, Chin Kook Rhee *, Ji Young Kng, Chn Kwon Prk, Sook Young Lee, Soon Suk Kwon, Young Kyoon Kim, nd Hyoung Kyu Yoon Division of Pulmonry nd Criticl Cre Medicine, Deprtment of Internl Medicine, College of Medicine, The Ctholic University of Kore, Seoul, Kore Received : August 13, 2014 Revised : October 20, 2014 Accepted : October 24, 2014 Correspondence to Hyoung Kyu Yoon, M.D. Division of Pulmonry nd Criticl Cre Medicine, Deprtment of Internl Medicine, College of Medicine, Yeouido St. Mry s Hospitl, The Ctholic University of Kore, ro, Yeongdeungpo-gu, Seoul 07345, Kore Tel: Fx: E-mil: cmcyhg@ctholic.c.kr *These uthors contributed eqully to this work. Bckground/Aims: Asthm is chrcterized by irwy hyperresponsiveness, inf lmmtion, nd remodeling. Peroxisome prolifertor-ctivted receptors hve been reported to regulte inflmmtory responses in mny cells. In this study, we exmined the effects of intrnsl rosiglitzone on irwy remodeling in chronic sthm model. Methods: We developed mouse model of irwy remodeling, including smooth muscle thickening, in which ovlbumin (OVA)-sensitized mice were repetedly exposed to intrnsl OVA dministrtion twice per week for 3 months. Mice were treted intrnslly with rosiglitzone with or without n ntgonist during OVA chllenge. We determined irwy inflmmtion nd the degree of irwy remodeling by smooth muscle ctin re nd collgen deposition. Results: Mice chroniclly exposed to OVA developed sustined eosinophilic irwy inflmmtion, compred with control mice. Additionlly, the mice developed fetures of irwy remodeling, including thickening of the peribronchil smooth muscle lyer. Administrtion of rosiglitzone intrnslly inhibited the eosinophilic inflmmtion significntly, nd, importntly, irwy smooth muscle remodeling in mice chroniclly exposed to OVA. Expression of Toll-like receptor (TLR)-4 nd nucler fctor kpp-light-chin-enhncer of ctivted B cells (NFκB) ws incresed in the OVA group nd decresed in the rosiglitzone group. Co-tretment with GW9660 ( rosiglitzone ntgonist) nd rosiglitzone incresed the expression of TLR-4 nd NF-κB. Conclusions: These results suggest tht intrnsl dministrtion of rosiglitzone cn prevent not only irwy inf lmmtion but lso irwy remodeling ssocited with chronic llergen chllenge. This beneficil effect is medited by inhibition of TLR-4 nd NF-κB pthwys. Keywords: Asthm; Remodeling; Rosiglitzone; Smooth muscle INTRODUCTION Asthm is defined in the Globl Inititive for Asthm guidelines s chronic inflmmtory disese of the irwys, chrcterized by reversible irflow obstruction [1]. Altertions in the structure of the irwy wll, referred to s irwy remodeling, hve lso been recognized s regulr fetures of sthm. The structurl chnges not- Copyright 2016 The Koren Assocition of Internl Medicine This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution Non-Commercil License ( by-nc/3.0/) which permits unrestricted noncommercil use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. pissn eissn

2 The Koren Journl of Internl Medicine Vol. 31, No. 1, Jnury 2016 ed in the sthmtic irwy include subepithelil fibrosis, incresed smooth muscle mss, ngiogenesis, nd n incresed number of mucous glnds. These chnges mke it difficult to tret chronic sthm ptients [2]. Trditionlly, irwy smooth muscles (ASM) cells hve been considered to be the min effector cells of irwy nrrowing. However, ASMs hve lso recently been shown to ply roles in irwy remodeling nd inflmmtion in sthm [3]. ASMs cn secrete immunomodultory cytokines nd chemokines [4-6] nd express surfce receptors tht re importnt for cell dhesion nd leukocyte ctivtion [7-9]. Toll-like receptors (TLRs) re clss of pthogen-recognition receptors tht ctivte innte immune responses, cusing disese excerbtions in vrious irwy inflmmtory diseses, such s sthm nd chronic obstructive pulmonry disese [9]. Beyond these post-infectious immune responses, recent studies hve implicted TLRs in the pthobiology of sthm, not only inducing T helper lymphocyte type 2 (Th2) cytokine responses, but lso the process of irwy remodeling in ASMs [10,11]. Among the subtypes of TLRs, ASMs constitutively express mr- NAs for TLR4, which elicits prosthmtic ASM tissue constriction or relxtion, medited by nucler fctor kpp-light-chin-enhncer of ctivted B cells (NF-κB) signling [12]. It lso seems tht pleiotropic proinflmmtory effects of TLR4 in ASMs re ssocited with the pthophysiology of the irwy remodeling process in sthm, lthough the exct mechnisms remin uncler. Peroxisome prolifertor-ctivted receptor (PPAR)-γ lignds re known to inhibit the relese of pro-inflmmtory cytokines from irwy epithelil cells, s well s mcrophges, vsculr smooth muscle cells, nd endothelil cells [13-15]. Our group demonstrted tht the PPAR-γ gonist ciglitzone inhibited the development of irwy hyperresponsiveness significntly [16] in murine chronic sthm model. Inhibitory effects on irwy mucus hypersecretion [17] nd inflmmtion in ASMs hve lso been reported [18]. Although potentilly preventtive effects of PPAR-γ on irwy remodeling hve emerged [19], the moleculr mechnisms remin to be investigted. Recent studies hve shown tht the PPAR-γ gonist rosiglitzone exerted nti-inflmmtory effects by inhibiting the TLR4 dependent IP 10/PKC/NF-κB signling pthwys in vsculr smooth muscle cells [20]. In the present study, we sought to determine whether intrnsl rosiglitzone suppressed irwy remodeling nd whether its effect ws ssocited with TLR-4 nd IP- 10/PKC/NF-κB pthwys. METHODS Animls nd experimentl design Femle BALB/c mice (De-Hn Experimentl Animl Center, Dejon, Kore) t 7 weeks of ge were used. Mice were ssigned rndomly to one of four groups: (1) control, (2) ovlbumin (OVA) chllenge, (3) OVA chllenge plus the PPAR-γ gonist rosiglitzone, or (4) OVA chllenge plus rosiglitzone nd the PPAR-γ ntgonist GW9660. Sensitiztion nd ntigen chllenge protocol Mice were immunized by subcutneous injection with 25 μg of OVA (Grde V; Sigm-Aldrich, St. Louis, MO, USA) dsorbed to 1 mg of luminum hydroxide (Aldrich, Milwukee, WI, USA) in 200 μl of phosphte-buffered sline (PBS). Subcutneous injections were performed on dys 0, 7, 14, nd 21 nd intrnsl OVA chllenge (20 μg/50 μl in PBS) ws dministered on dys 27, 29, nd 31 under isoflurne (Vedco, St. Joseph, MO, USA) nesthesi. Intrnsl OVA chllenges were then repeted twice per week for 3 months. Age- nd gender-mtched control mice were treted in the sme wy with PBS but without OVA. Mice were scrificed 24 hours fter the finl OVA chllenge, nd broncholveolr lvge (BAL) fluid nd lung tissues were obtined. All procedures for niml reserch were performed in ccordnce with the Lbortory Animls Welfre Act, the Guide for the Cre nd Use of Lbortory Animls, nd the Guidelines nd Policies for Rodent Experiments provided by the Institutionl Animl Cre nd Use Committee t the School of Medicine, The Ctholic University of Kore. Administrtion of rosiglitzone nd GW9660 Micronized dry rosiglitzone powder (Alexis Biochemicls; Enzo Life Sciences, Phildelphi, PA, USA) ws dissolved in sterile norml sline (20 μg/25 μl), nd given dily by intrnsl dministrtion once per dy strting on dy 38 for 3 months. The PPAR-γ ntgonist GW9660 (0.5 mg/kg, dissolved in norml sline) ws lso dministrted intrnslly once per dy on dys 35, 38, nd 90

3 Lee HY et l. Intrnsl rosiglitzone in chronic sthm once per week, continuing for 3 months, during the ovlbumin chllenge. The control mice were treted in the sme wy with norml sline. Broncholveolr lvge Mice were scrificed by CO 2 sphyxition fter mesuring irwy responsiveness. The trche ws exposed nd cnnulted with silicone tubing ttched to 23-guge needle on n 800 μl tuberculin syringe. BAL fluid ws withdrwn fter instilltion of 1 ml of sterile PBS through the trche into the lung. The totl number of cells in BAL fluid ws counted using hemocytometer. The BAL fluid ws cytospun (2,000 rpm, 7 minutes) onto microscope slides nd stined with Diff-Quick (Sysmx, Kobe, Jpn). The percentges of mcrophges, eosinophils, lymphocytes, nd neutrophils in the BAL fluid were obtined by counting 400 leukocytes on rndomly selected res of the slide using light microscopy. Superntnts were stored t 70 C. Enzyme-linked immunosorbent ssy The concentrtion of prostglndin E2 (PGE 2 ) ws mesured in the BAL fluid with n enzyme-linked immunosorbent ssy kit (R&D Systems, Minnepolis, MN, USA). The ssy ws performed ccording to the mnufcturer s protocol. Western blotting Seprted lung tissues, frozen in liquid nitrogen, were disrupted using Polytron homogenizer (Tissue-Teror, Biospec Products Inc., Brtlesville, OK, USA) nd centrifuged. The proteins were purified from the superntnt, nd the concentrtion ws ssessed using the Brdford method [21]. The protein smples were seprted by 8% sodium dodecyl sulphte-polycrylmide gel electrophoresis nd trnsferred to polyvinylidene difluoride membrne (Amershm Phrmci Biotech, Little Chlfont, UK). After blocking with 10% skimmed milk (BD Difco, Frnklin Lkes, NJ, USA), the membrne ws incubted with ntibodies to TLR4, IP-10, NF-κB (1:1,000; Snt Cruz Biotech, Snt Cruz, CA, USA), nd p-akt (1:1,000; BD Bioscience, Sn Diego, CA, USA) overnight. The membrne ws wshed three times with PBS nd incubted with secondry ntibody for 2 hours. The trget protein ws detected using the ECL Western Blotting Anlysis system (Amershm Phrmci Biotech, Pisctwy, NJ, USA) nd X-ry film. Hydroxyproline nlysis Lung tissue (60 mg) from ech mouse ws used for the hydroxyproline ssy. A smple of lung homogente ws subsequently dded to 250 μl of 12 N HCl for 16 hours t 110 C. After centrifugtion, 25 μl of ech superntnt ws ssyed. To 25-μL smple of the digested lung, 25 μl of citrte/cette buffer (5% citric cid, 7.2% sodium cette, 3.4% sodium hydroxide, nd 1.2% glcil cetic cid) nd 500 μl of chlormine T solution (1.41 g of chlormine T, 26 ml of n-propnol, 20.7 ml of distilled wter, nd 53.3 ml of citrte/cette buffer) were dded. The resulting smples were then incubted t room temperture for 20 minutes before 500 μl of Ehrlich s solution (4.5 g of p-dimethylminobenzldehyde, 18.6 ml of n-propnol, nd 7.8 ml of 70% perchloric cid) were dded. These smples were incubted for 15 minutes t 65 C, nd cooled smples were red t 550 nm in spectrophotometer. Hydroxyproline concentrtions were clculted from stndrd curve of hydroxyproline. Mesurement of smooth muscle ctin Immunohistochemicl detection of α-smooth muscle ctin (Sigm-Aldrich) ws performed s described previously [22]. The immunostined re of α-smooth muscle ctin in ech prffin wx-embedded lung ws outlined nd qulified using light microscope ttched to n imge nlysis system (BX50, Olympus, Tokyo, Jpn). Results re expressed s the immunostined re of bsement membrne of bronchioles (internl dimeter 650 to 750 μm). At lest 10 bronchioles were counted on ech slide. Dt nlysis Results from ech group were compred by nlysis of vrince with the nonprmetric Kruskl-Wllis test, followed by post hoc testing with Dunn s multiple comprison of mens. All sttisticl nlyses were performed using SPSS version 17.0 (SPSS Inc., Chicgo, IL, USA). A p < 0.05 ws considered to indicte sttisticl significnce. All results re given s men ± stndrd error of the men. 91

4 The Koren Journl of Internl Medicine Vol. 31, No. 1, Jnury 2016 RESULTS Effects of rosiglitzone on irwy inflmmtion Numbers of totl cells nd different inflmmtory cells in BAL fluid were compred between the tretment groups. After OVA chllenge, totl cells, eosinophils, nd lymphocytes were incresed in BAL fluid, which were reduced significntly (totl cells, p < 0.01; eosinophils, p < 0.05; lymphocytes, p < 0.05) in the rosiglitzone-treted group (Fig. 1). The PPAR-γ ntgonist GW9660 induced n increse in the number of totl cells (p < 0.05), eosinophils (p < 0.05), nd neutrophils (p < 0.01) (Fig. 1). Effects of rosiglitzone on lung collgen levels Totl lung collgen levels, representing pulmonry fibrosis, were mesured by hydroxyproline nlysis. Chronic OVA chllenge for 3 months induced n increse in the level of hydroxyproline (OVA vs. control, 1,932 ± 200 μg/ lung vs. 578 ± 200 μg/lung). Rosiglitzone tretment decresed the level of hydroxyproline significntly compred with the OVA group (rosiglitzone vs. OVA, 1,293 ± 180 μg/lung vs. 1,932 ± 200 μg/lung, p < 0.05). Combined tretment with rosiglitzone nd GW9660 incresed the hydroxyproline level, similr to tht of the OVA group (rosiglitzone vs. rosiglitzone + GW9660, 1,293 ± 180 μg/ lung vs. 1,900 ± 150 μg/lung, p < 0.05) (Fig. 2). Effects of rosiglitzone on ASM re Peribronchil α-smooth muscle ctin immunostining ws quntified by imging (Fig. 3). Rosiglitzone decresed the re of ASM compred with the OVA group (rosiglitzone vs. OVA, 850 ± μm 2 vs. 1,339 ± μm 2, p < 0.01). Tretment with rosiglitzone nd GW9660 incresed the re of ASM compred with the rosiglitzone-lone tretment group (rosiglitzone vs. rosiglitzone + GW9660, 850 ± μm 2 vs. 1,052 ± μm 2, p < 0.01) (Fig. 4). Effects of rosiglitzone on broncholveolr lvge fluid PGE 2 levels The concentrtion of PGE 2 in broncholveolr lvge fluid incresed in the OVA chllenge group. The level of PGE 2 decresed with rosiglitzone tretment (rosiglitzone vs. OVA, 208,893 ± 181,428 vs. 538,030 ± 10,040, p < 0.05); however, GW9660 reversed the lowering effect of rosiglitzone (rosiglitzone vs. rosiglitzone + GW9660, 208,893 ± 181,428 vs. 412,048 ± 71,126, p < 0.05) (Fig. 5). Effects of rosiglitzone on TLR4 signling pthwy proteins To investigte the effects of rosiglitzone in the TLR4 Cells in BAL fluid (X 10 4 /ml) Totl cells Mcrophges Eosinophils Lymphocytes Neutrophils b c c d Hydroxyproline (µg/lung) 2,500 2,000 1,500 1, b 0 Control OVA Rosiglitzone Rosiglitzone + GW9660 Figure 1. Effect of intrnsl rosiglitzone on totl nd differentil cell counts in broncholveolr lvge f luid. Mice were scrificed 24 hours fter the finl ovlbumin (OVA) chllenge, nd broncholveolr lvge (BAL) cells were isolted. Vlues re expressed s men ± SE (n = 6 to 9 / group). p < 0.05, b p < 0.01 compred with the OVA group, c p < 0.05, d p < 0.01 compred with the rosiglitzone group. 0 Control OVA Rosiglitzone Rosiglitzone + GW9660 Figure 2. Effects of intrnsl rosiglitzone on totl lung collgen levels. Lung tissue (60 mg) ws collected from ech mouse for the hydroxyproline ssy. Vlues re presented s men ± SE (n = 8 to 15/group). OVA, ovlbumin. p < 0.05, compred with the OVA group, b p < 0.05, compred with the rosiglitzone group. 92

5 Lee HY et l. Intrnsl rosiglitzone in chronic sthm A B C D Control OVA Rosiglitzone Rosiglitzone + GW 9660 Figure 3. Peribronchil α-smooth muscle ctin immunostin ws done. Ovlbumin (OVA) chllenged mice (B) exhibited incresed peribronchil stining (red) compred to the control mice (A) ( 200). Tretment of rosiglitzone (C) decresed the immunostined re which ws inhibited by combintion tretment of GW9660 (D). 1,600 1, ,000 Are of smooth muscle (µm 2 ) 1,200 1, b PGE 2 (pg/ml) 500, , , ,000 b ,000 0 Control OVA Rosiglitzone Rosiglitzone + GW9660 Figure 4. Effects of intrnsl rosiglitzone on irwy smooth muscle cells re. The smooth muscle re is expressed s the immunostined re of bsement membrne of the bronchioles using n imge nlyzer. Vlues re presented s men ± SE (n = 11 to 16/group). OVA, ovlbumin. p < 0.01, compred with the OVA group, b p < 0.01, compred with the rosiglitzone group. 0 Crontol OVA Rosiglitzone Rosglitzone + GW9660 Figure 5. Effects of intrnsl rosiglitzone on prostglndin E2 (PGE2) levels in broncholveolr lvge fluid. Vlues re expressed s men ± SE (n = 5/group). OVA, ovlbumin. p < 0.05, compred with the OVA group, b p < 0.05, compred with the rosiglitzone group. signling pthwy, expression of TLR4 nd downstrem signling proteins were determined in homogenized lung tissues. After the OVA chllenge, protein expression of TLR4 nd the downstrem p-akt, IP-10, nd NFκB incresed significntly, compred with the control group. Rosiglitzone effectively ttenuted the levels of protein expression, which were gin reversed with co tretment with GW9660 (Fig. 6). DISCUSSION PPAR-γ is highly expressed in dipose tissue, nd ws first identified for role in lipid nd glucose metbolism. Bsed on previous in vitro nd in vivo studies, PPAR-γ hs recently been ccepted to ply role in irwy diseses, negtively regulting the expression of proinflmmtory genes. This effect is medited through inhibiting cytokines, chemottrctnts, nd cell survivl fctors. Ptel et l. [19] reveled tht the effect of PPAR-γ on cell 93

6 The Koren Journl of Internl Medicine Vol. 31, No. 1, Jnury 2016 Control OVA Rosiglitzone Rosiglitzone + GW9660 TLR-4 p-akt IP-10 NF-κB (nucleus) Actin Figure 6. Effect of intrnsl rosiglitzone on expression of Toll-like receptor 4 (TLR4) nd downstrem proteins in lung tissue. Repetitive ovlbumin (OVA) chllenge for 3 months induced TLR4, p-akt, Interferon-gmm-induced protein 10 (IP-10) nd nucler fctor-kpp B (NF-κB) expression which were inhibited by rosiglitzone therpy. Cotretment of rosiglitzone with GW9660 re-induced the expression of the proteins. growth nd G-CSF ws greter thn tht produced by glucocorticoid, the most widely used nti-inflmmtory gent. Inoue et l. [23] reported tht PPAR-γ ctivtion suppressed cycloxygenese-2 expression by inhibiting the NF-κB signling pthwy. Also, severl groups hve used inhled or orl PPAR-γ gonists in niml models of llergic sthm; ciglitzone nd rosiglitzone showed inhibitory effects on inflmmtory cell influx nd irwy hyperresponsiveness [24-26]. These nti-inflmmtory effects demonstrte tht PPAR-γ gonists my provide new therpeutic modlities for the tretment of llergic sthm. In our study, dily dministrtion of intrnsl rosiglitzone during the 3-month OVA chllenge reduced the influx of inflmmtory cells in BAL fluid significntly (Fig. 1). Combined tretment with GW9660 reversed the effect; these results suggest tht the PPAR γ gonist hd protective effect ginst chronic irwy inflmmtion. Also, the incresed level of PGE2 in BAL fluid fter the OVA chllenge decresed with rosiglitzone tretment (Fig. 5) nd gin incresed with combined tretment with GW9660. This result is comptible with the previous study of Sun et l. [27], which showed tht rosiglitzone tretment decresed urinry PGE2 levels by 57%. 94 PGE2 is generlly recognized s meditor of inflmmtion nd Th2 immunity, key elements of sthm pthophysiology [28,29]. These results reflect the suppressive effects of rosiglitzone on the Th2-medited immune response, irwy inflmmtion, nd development of sthmtic irwys. Our study is originl in using chronic ovlbumin-chllenged sthm model insted of n cute model. Among murine llergic sthm models, the chronic model demonstrtes more similr pthologicl fetures to humn sthm ptients thn the cute model. However, until now, studies investigting the role of intrnsl PPAR-γ on the chronic irwy remodeling process hve been lcking. Recently, Fogli et l. [30] demonstrted tht the PPAR-γ lignd rosiglitzone nd β2 gonist slbutmol hd synergistic interctions on ASM prolifertion. Stephen et l. [31] reported tht the PPAR-γ lignd ciglitzone decresed humn ASM migrtion nd extrcellulr mtrix (ECM) synthesis. The irwy remodeling process in llergic sthm is chrcterized by subepithelil thickening from the deposition of ECM, incresed ASM mss, nd ngiogenesis [32]. In this study, collgen deposition, demonstrted by hydroxyproline nlysis, decresed with intrnsl rosiglitzone tretment (Fig. 2). The re of ASM lso decresed in the rosiglitzone-treted group. The effect ws reversed with GW9660 (Figs. 3 nd 4) suggesting tht PPAR-γ signling is involved in collgen deposition nd ASM hyperplsi, importnt mechnisms of the irwy remodeling process. To investigte the moleculr mechnism of PPAR-γ ction on irwy remodeling, we hypothesized tht PPAR-γ negtively regulted the TLR4 receptor pthwy in ASMs. TLR4 ctivtion with lipopolyscchride (LPS) hs been reported to induce Th2 cytokine responses in murine sthm model [11]. Severl recent studies reported tht PPAR-γ gonists my inhibit TLR4 ctivtion, when it is induced by LPS, oxyhemoglobin, or ngiotensin [20,33,34]. Also, Yin et l. [35] described tht the PPAR-γ gonist rosiglitzone regulted the incresed expression of TLR4 nd NF-κB fter chronic exposure to cigrette smoke in lveolr mcrophges. The effects of PPAR-γ on TLR4 ctivtion in n OVA-chllenged murine sthm model hve not been reported before. This is the first report of inhibitory regultion of TLR4 signling, induced by OVA, through PPAR-γ gonist.

7 Lee HY et l. Intrnsl rosiglitzone in chronic sthm In our study, the ffected signling proteins downstrem of TLR4 were AKT, IP-10, nd NF-κB in homogenized lung tissues (Fig. 6). Activtion of TLR4 stimulted both myeloid differentition fctor 88 (Myd88)-dependent nd Myd88-independent pthwy. The downstrem Myd88-dependent pthwy proteins re NF-κB, phosphtidylinositol 3-kinse/Akt, nd mitogen-ctivted protein kinses, which regulte cell vibility nd inflmmtion [36]. Also, the Myd88-independent pthwy induced expression of IFN inducible genes, such s IP-10 nd glucocorticoid-ttenuted response gene 16. Through the recruitment of dptor molecules, the Myd88-independent pthwy resulted in lte-phse ctivtion of NF-κB [36]. Thus, the suppressed expression of NF-κB, Akt, nd IP-10 in lung tissues of the rosiglitzone-treted group suggests tht rosiglitzone could ct s n inhibitor of both Myd88-dependent nd independent signling pthwys. Incresed expression of NF κb, Akt, nd IP-10 in the combined tretment group of rosiglitzone with ntgonist GW9660 reinforces the downregulting effect of TLR4 signling by rosiglitzone. Since the pprovl of rosiglitzone s n nti-dibetic gent, concerns bout incresed crdiovsculr risks due to the gent hve been suggested [37]. Although the results of rndomized study did not suggest ny incresed risk of mjor dverse crdiovsculr events recently [38], questions bout the systemic effects of rosiglitzone, such s hypoglycemi, fluid retention, nd incresed levels of low density lipoprotein cholesterol must be considered in long term usge to gin preventtive effect ginst irwy remodeling. As intrnsl rosiglitzone hd sufficient effects in mice in our study, we consider tht n inhltion method my help to reduce systemic side effects of the gent. Further clinicl studies bout drug dosing nd sfety re needed. In conclusion, we confirmed tht intrnsl dministrtion of rosiglitzone effectively reduced the chrcteristics of sthmtic irwy, such s irwy inflmmtion, fibrosis, nd smooth muscle cell hyperplsi. Moreover, decresed TLR4 receptors nd downstrem signling proteins in lung tissue suggested tht these effects were medited by regultion of TLR4 signling by rosiglitzone. The ssocition between PPAR-γ gonist nd TLR4 signling in ASMs should be demonstrted by in vitro experiments seprtely. KEY MESSAGE 1. Peroxisome prolifertor-ctivted receptor γ (PPAR-γ) gonists re known to hve nti-inflmmtory effects in chronic irwy disese. 2. This study demonstrted tht rosiglitzone inhltion hs protective effect on developing sthmtic irwys through inhibiting Toll-like receptor 4 signling pthwys in n ovlbumin-chllenged chronic sthm model. 3. Intrnsl dministrtion of the PPAR-γ gonist rosiglitzone my hve therpeutic potentil in sthm ptients with few systemic side effects. Conflict of interest No potentil conflict of interest relevnt to this rticle ws reported. REFERENCES 1. Btemn ED, Hurd SS, Brnes PJ, et l. Globl strtegy for sthm mngement nd prevention: GINA executive summry. Eur Respir J 2008;31: Wiggs BR, Bosken C, Pre PD, Jmes A, Hogg JC. A model of irwy nrrowing in sthm nd in chronic obstructive pulmonry disese. Am Rev Respir Dis 1992;145: Zuyderduyn S, Sukkr MB, Fust A, Dhliwl S, Burgess JK. Treting sthm mens treting irwy smooth muscle cells. Eur Respir J 2008;32: Chn V, Burgess JK, Rtoff JC, et l. Extrcellulr mtrix regultes enhnced eotxin expression in sthmtic irwy smooth muscle cells. Am J Respir Crit Cre Med 2006;174: Brightling CE, Ammit AJ, Kur D, et l. The CXCL10/ CXCR3 xis medites humn lung mst cell migrtion to sthmtic irwy smooth muscle. Am J Respir Crit Cre Med 2005;171: El-Shzly A, Berger P, Girodet PO, et l. Frktlkine produced by irwy smooth muscle cells contributes to mst cell recruitment in sthm. J Immunol 2006;176: Lzr AL, Albeld SM, Pilewski JM, Brennn B, Pure E, Pnettieri RA Jr. T lymphocytes dhere to irwy smooth muscle cells vi integrins nd CD44 nd induce smooth 95

8 The Koren Journl of Internl Medicine Vol. 31, No. 1, Jnury 2016 muscle cell DNA synthesis. J Exp Med 1994;180: Hkonrson H, Kim C, Wheln R, Cmpbell D, Grunstein MM. Bi-directionl ctivtion between humn irwy smooth muscle cells nd T lymphocytes: role in induction of ltered irwy responsiveness. J Immunol 2001;166: Sukkr MB, Xie S, Khorsni NM, et l. Toll-like receptor 2, 3, nd 4 expression nd function in humn irwy smooth muscle. J Allergy Clin Immunol 2006;118: Agrwl S, Agrwl A, Doughty B, et l. Cutting edge: different Toll-like receptor gonists instruct dendritic cells to induce distinct Th responses vi differentil modultion of extrcellulr signl-regulted kinse-mitogen-ctivted protein kinse nd c-fos. J Immunol 2003;171: Eisenbrth SC, Piggott DA, Hulett JW, Visintin I, Herrick CA, Bottomly K. Lipopolyscchride-enhnced, toll-like receptor 4-dependent T helper cell type 2 responses to inhled ntigen. J Exp Med 2002;196: Shn X, Hu A, Veler H, et l. Regultion of Toll-like receptor 4-induced prosthmtic chnges in irwy smooth muscle function by opposing ctions of ERK1/2 nd p38 MAPK signling. Am J Physiol Lung Cell Mol Physiol 2006;291:L324-L Belvisi MG, Hele DJ, Birrell MA. Peroxisome prolifertor-ctivted receptor gmm gonists s therpy for chronic irwy inflmmtion. Eur J Phrmcol 2006;533: Jing C, Ting AT, Seed B. PPAR-gmm gonists inhibit production of monocyte inflmmtory cytokines. Nture 1998;391: Bishop-Biley D, Hl T. Endothelil cell poptosis induced by the peroxisome prolifertor-ctivted receptor (PPAR) lignd 15-deoxy-Delt12, 14-prostglndin J2. J Biol Chem 1999;274: Rhee CK, Lee SY, Kng JY, et l. Effect of peroxisome prolifertor-ctivted receptor-gmm on irwy smooth muscle thickening in murine model of chronic sthm. Int Arch Allergy Immunol 2009;148: Shen Y, Chen L, Wng T, Wen F. PPARγ s potentil trget to tret irwy mucus hypersecretion in chronic irwy inflmmtory diseses. PPAR Res 2012;2012: Zhu M, Flynt L, Ghosh S, et l. Anti-inflmmtory effects of thizolidinediones in humn irwy smooth muscle cells. Am J Respir Cell Mol Biol 2011;45: Ptel HJ, Belvisi MG, Bishop-Biley D, Ycoub MH, Mitchell JA. Activtion of peroxisome prolifertor-ctivted receptors in humn irwy smooth muscle cells hs superior nti-inflmmtory profile to corticosteroids: relevnce for chronic obstructive pulmonry disese therpy. J Immunol 2003;170: Ji Y, Liu J, Wng Z, Liu N, Gou W. PPARgmm gonist, rosiglitzone, regultes ngiotensin II-induced vsculr inflmmtion through the TLR4-dependent signling pthwy. Lb Invest 2009;89: Kruger NJ. The Brdford method for protein quntittion. Methods Mol Biol 1994;32: Rhee CK, Kim JW, Prk CK, et l. Effect of imtinib on irwy smooth muscle thickening in murine model of chronic sthm. Int Arch Allergy Immunol 2011;155: Inoue H, Tnbe T, Umesono K. Feedbck control of cyclooxygense-2 expression through PPARgmm. J Biol Chem 2000;275: Trifilieff A, Bench A, Hnley M, Byley D, Cmpbell E, Whittker P. PPAR-lph nd -gmm but not -delt gonists inhibit irwy inflmmtion in murine model of sthm: in vitro evidence for n NF-kppB-independent effect. Br J Phrmcol 2003;139: Hond K, Mrquillies P, Cpron M, Dombrowicz D. Peroxisome prolifertor-ctivted receptor gmm is expressed in irwys nd inhibits fetures of irwy remodeling in mouse sthm model. J Allergy Clin Immunol 2004;113: Lee KS, Prk SJ, Hwng PH, et l. PPAR-gmm modultes llergic inflmmtion through up-regultion of PTEN. FASEB J 2005;19: Sun Y, Ji Z, Liu G, et l. PPARγ gonist rosiglitzone suppresses renl mpges-1/pge2 pthwy in db/db mice. PPAR Res 2013;2013: Klinski P. Regultion of immune responses by prostglndin E2. J Immunol 2012;188: Scher JU, Pillinger MH. The nti-inflmmtory effects of prostglndins. J Investig Med 2009;57: Fogli S, Stefnelli F, Picchinti L, et l. Synergistic interction between PPAR lignds nd slbutmol on humn bronchil smooth muscle cell prolifertion. Br J Phrmcol 2013;168: Stephen J, Delvecchio C, Spitle N, et l. PPAR lignds decrese humn irwy smooth muscle cell migrtion nd extrcellulr mtrix synthesis. Eur Respir J 2013;41:

9 Lee HY et l. Intrnsl rosiglitzone in chronic sthm 32. Mnuykorn W, Howrth PH, Holgte ST. Airwy remodelling in sthm nd novel therpy. Asin Pc J Allergy Immunol 2013;31: Zhng LL, Go CY, Fng CQ, et l. PPARγ ttenutes intiml hyperplsi by inhibiting TLR4-medited inflmmtion in vsculr smooth muscle cells. Crdiovsc Res 2011;92: Wu Y, Zho XD, Zhung Z, et l. Peroxisome prolifertor-ctivted receptor gmm gonist rosiglitzone ttenutes oxyhemoglobin-induced Toll-like receptor 4 expression in vsculr smooth muscle cells. Brin Res 2010;1322: Yin Y, Hou G, Li E, Wng Q, Kng J. PPARγ gonists regulte tobcco smoke-induced Toll like receptor 4 expression in lveolr mcrophges. Respir Res 2014;15: Duphinee SM, Krsn A. Lipopolyscchride signling in endothelil cells. Lb Invest 2006;86: Hitt WR, Kul S, Smith RJ. The crdiovsculr sfety of dibetes drugs: insights from the rosiglitzone experience. N Engl J Med 2013;369: Home PD, Pocock SJ, Beck-Nielsen H, et l. Rosiglitzone evluted for crdiovsculr outcomes in orl gent combintion therpy for type 2 dibetes (RECORD): multicentre, rndomised, open-lbel tril. Lncet 2009;373: