1521-0103/361/2/268–279$25.00 https://doi.org/10.1124/jpet.116.239137THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 361:268–279, May 2017Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics

Mogroside IIIE, a Novel Anti-Fibrotic Compound, ReducesPulmonary Fibrosis through Toll-Like Receptor 4 Pathways

Lijun Tao, Jinyu Yang, Fengyan Cao, Haifeng Xie, Mian Zhang, Yanqing Gong,and Chaofeng Zhang(L.T., J.Y., F.C., M.Z., C.Z.,) Research Department of Pharmacognosy, China Pharmaceutical University, Nanjing, People’sRepublic of China; (Y.G.) Division of Translational Medicine and Human Genetics, Department of Medicine, Perelman School ofMedicine, University of Pennsylvania, Philadelphia, Pennsylvania; and (H.X.) Chengdu Biopurity Phytochemicals Ltd. Chengdu,People’s Republic of China

Received November 30, 2016; accepted March 7, 2017

ABSTRACTIdiopathic pulmonary fibrosis is a progressive fibrotic lungdisease, and eventually most patients develop respiratory failurewith amedian survival rate of 2 to 3 years after diagnosis due to thelack of clinically effective therapies. Mogroside IIIE (MGIIIE), acucurbitane-type compound, was isolated from Siraitia grosve-norii. MGIIIE has shown the strongest inhibition of nitric oxiderelease, a crucial inflammatory factor, from lipopolysaccharide(LPS)-treated RAW264.7 cells compared with other mogrosides.In the pulmonary fibrosis mouse model induced by bleomycin,MGIIIE treatment attenuated pulmonary fibrosis, indicated as areduction in myeloperoxidase activity, collagen deposition, andpathologic score. MGIIIE also significantly suppressed expressionof several important fibrotic markers, e.g., a-smooth muscleactin, collagen I, transforming growth factor-b (TGF-b) signal,and metalloproteinases-9/tissue inhibitor of metalloproteinase-1.

Furthermore, MGIIIE blocked tansdifferentiation of lung residentfibroblasts into myofibroblast-like cells induced by TGF-b or LPSand subsequently inhibited collagen production in lung fibro-blasts. These data indicate that MGIIIE is a potent inhibitor forpulmonary fibrosis. In vitro and in vivo mechanistic studies haveshown that MGIIIE significantly decreased expression of toll-likereceptor 4 (TLR4) and its downstream signals of myeloid differ-entiation factor 88 (MyD88)/mitogen-activated protein kinase(MAPK), an inflammatory signal essential for extracellular matrix(ECM) deposition in pulmonary fibroblasts. Taken together, theseresults demonstrate that MGIIIE significantly prevents pulmonaryfibrosis by inhibiting pulmonary inflammation and ECMdepositionthrough regulating TLR4/MyD88-MAPK signaling. Our studysuggests that MGIIIE may have therapeutic potential for treatingpulmonary fibrosis in clinical settings.

IntroductionIdiopathic pulmonary fibrosis (IPF) is a severe lung

disease, which is characterized by progressive and irrevers-ible destruction of lung architecture caused by excessiveextracellular matrix (ECM) deposition that ultimately leadsto respiratory failure. The median survival time of patientswith IPF is about 2 or 3 years due to limited clinical therapies(Gürbüzel et al., 2016). Extensive evidence indicates thatdysfunctions of the resident fibroblasts by lung inflammationgreatly contribute to the development of pulmonary fibrosis.Once fibroblasts become activated, they transform into a-smoothmuscle actin (a-SMA)–expressing myofibroblasts that secrete

ECM components leading to fibrotic lesions in the lung(Derynck and Zhang, 2003; Chen et al., 2012). A variety ofcytokines and proteinases have been shown to be involved inthe fibrotic process including transforming growth factor-b(TGF-b), myeloperoxidase (MPO), and metalloproteinases(MMPs) (Yoshimura et al., 2006; Bhattacharyya et al., 2013;Shi et al., 2014), which are considered as essential makers forpulmonary fibrosis. Recently, a crucial role of toll-like receptor4 (TLR4) in fibroblasts during fibrogenesis has been high-lighted (Poltorak et al., 1998; Jiang et al., 2005; Huebenerand Schwabe, 2013). Pulmonary fibrosis induced by bleomycin,lipopolysaccharide (LPS), or radiation was significantly at-tenuated in TLR4 knockout mice (He et al., 2009, 2012;Bhattacharyya et al., 2013; Rhieu et al., 2014). Moreover, ithas been reported that TLR4 is required for fibroblast activationand collagen production as indicated by the fact that TGF-bfailed to induce collagen production inTLR4knockout fibroblasts(Bhattacharyya et al., 2013). Upregulated TLR4 in fibroblastsaugmented TGF-b sensitivity and converted self-limited tissue

This work was financially supported by the National Natural ScienceFoundation of China [81573553] to C.F., the National New Drug InnovationMajor Project of China [2011ZX09307-002-02], the Priority Academic ProgramDevelopment of Jiangsu Higher Education Institutions, and National Foundfor Fostering Talents of Basic Science [J1310032] in China; and the AmericanHeart Association [12SDG9050018] to Y.G. in the United States.

https://doi.org/10.1124/jpet.116.239137.

ABBREVIATIONS: a-SMA, a-smooth muscle actin; Col I, collagen I; DMSO, dimethylsulfoxide; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; IL, interleukin; IPF, idiopathic pulmonary fibrosis; JNK, c-JUN N-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MGIIIE, mogroside IIIE; MGV, mogroside V; MMP, metalloproteinase; MPO, myeloperoxidase; MyD88, myeloiddifferentiation factor 88; NO, nitric oxide; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PLF, primary lung fibroblast; TGF- b,transforming growth factor-b; TIMP, tissue inhibitor of metalloproteinase; TLR4, toll-like receptor 4.

268

at ASPE

T Journals on A

ugust 21, 2021jpet.aspetjournals.org

Dow

nloaded from

repair into intractable fibrosis in bleomycin-induced pulmonaryfibrosis (Bhattacharyya et al., 2013). In addition, downstreamsignals of TLR4, mitogen-activated protein kinases (MAPKs),including extracellular signal-regulated kinase (ERK), c-JUNN-terminal kinase (JNK), and p38, were significantly activatedin lung tissue from IPF patients (Yoshida et al., 2002) andblockade of ERK, JNK and p38 reduced fibrosis inmousemodels(Matsuoka et al., 2002; Madala et al., 2012; Vittal et al., 2013;van der Velden et al., 2016). Therefore, disruption of TLR4overexpression in lung inflammation may represent a poten-tial target for IPF prevention.In China, the fruits of Siraitia grosvenorii (Cucurbitaceae),

a famous traditional Chinese medicine, are used to treat drycough, sore throat, extreme thirst, and constipation (Committeeof National Pharmacopoeia, 2010; Chen et al., 2011). The activecomponents of Cucurbitaceae are a group of cucurbitane-typetriterpene glycosides named mogrosides. It has been reportedthat mogrosides have various bioactivities, including anti-inflammatory, anti-obesity, anti-diabetic, and anti-allergiceffects as well as hepatoprotective effects (Chen et al., 2011;Di et al., 2011; Jin and Lee, 2012; Sun et al., 2012; Xiao et al.,2012; Xiao and Wang, 2013; Song et al., 2014). Recentresearch has shown that mogroside V (MGV) attenuatedLPS-induced acute lung injury and inflammation (Suzukiet al., 2005; Shi et al., 2014; Xu et al., 2015); however, theanti-fibrotic effects of mogrosides on respiratory diseaseshave not been reported.Mogroside IIIE (MGIIIE) is a main component of the

fruits of S. grosvenorii and has shown the strongest anti-inflammatory effects in LPS-induced macrophage activa-tion in our screening experiment. Therefore, MGIIIE wasselected to evaluate the anti-fibrotic effects in a mouse exper-imental model of pulmonary fibrosis. MGIIIE exhibited asignificant anti-fibrotic activity in both in vitro and in vivomodels. Mechanistic studies identified that the MGIIIEinhibition fibrosis may be dependent on TLR4-mediated MAPKsignaling pathways.

Materials and MethodsChemicals and Reagents. MGIIIE (purity above 99%), mogro-

side IVE (purity above 99%), siamenoside I (purity above 99%), andMGV (purity above 99%) were all purchased from Chengdu BiopurifyPhytochemical Ltd. (Chengdu, China). The purity of these mogrosideshas been tested by high-performance liquid chromatography. Bleo-mycin was obtained from Nippon Kayaku (Tokyo). Prednisone wasobtained from Zhejiang Xianju Pharmaceutical Co., Ltd. (Xianju,China). Recombinant TGF-b1 was purchased from PeproTech Inc.(Rocky Hill, NJ). TAK-242 (a TLR4 inhibitor) was purchased fromMerckMillipore Inc. (Billerica, MA). 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidewas obtained fromBiosharp TechnologyInc. (Hefei, China).

The MPO test kit was purchased from Nanjing Jiancheng Bio-engineering Institute (Nanjing, China). Antibodies against phospho-Smad2/3, p38/phospho-p38, ERK/phospho-ERK, and JNK/phospho-JNKwere all obtained from Cell Signal Technology Inc. (Danvers, MA).Antibodies against b-actin, a-SMA, MMP-9, tissue inhibitor ofmetalloproteinase (TIMP)-1, myeloid differentiation factor 88 (MyD88),TGF-b1, Smad2/3, and horseradish peroxidase–conjugated secondaryantibody were all purchased from Bioworld Technology Inc. (Dublin,OH). Antibody against collagen I (Col I) was purchased from WanleibioTechnology Inc. (Shenyang, China). Antibody against TLR4 and second-ary antibody Donkey anti-rabbit IgG H&L (Alexa Fluor 488) wereobtained from Abcam Technology Inc. (Cambridge, United Kingdom).

49, 6-diamidino-2-phenylindole was obtained from Nanjing KeyGENBiotech. Co., Ltd. (Nanjing, China).

Animals. Adult male C57BL/6 mice weighing between 20 and 2 g(6–8 weeks old) were purchased from the Comparative MedicineCenter at Yangzhou University (Yangzhou, China). Care of theanimals followed the recommended protocols of the General Recom-mendation and Provisions of the Chinese Experimental AnimalsAdministration Legislation (Permit number SYXK2012-0035). Theanimal protocol was approved by the Institutional Ethical Committeeof China Pharmaceutical University. Mice were housed in a climate-controlled room at 22°C 6 2°C and 50% 6 10% humidity with a12-hour light/dark cycle, and given conventional feed and free accessto drinking water.

RAW 264.7 Culture and Nitric Oxide (NO) Measurement.The RAW 264.7 cell line was purchased from the Cell Bank of ChineseAcademic of Sciences (Shanghai, China). RAW264.7 cells were main-tained in GIBCO Dulbecco’s modified Eagle’s medium (Grand Island,NY) supplemented with Hyclone 10% fetal bovine serum (ThermoScientific, Waltham, Massachusetts), penicillin (100 U/ml), andstreptomycin (100 mg/ml) at 37°C, with 95% humidity and 5% carbondioxide. For treatment, RAW264.7 cells were detached and seededin 96-well plates at 4� 104 cells per well. When reaching a confluenceof 70%–80%, cells were incubated with LPS (5 mg/ml), chemicals, orcontrols [phosphate-buffered saline (PBS) or dimethylsulfoxide(DMSO)] for 24 hours, and 50 ml cell medium was used for NOmeasurement according to a previous published procedure (Fan et al.,2013). Briefly, 50ml of Griess reagent (Beyotime, Shanghai)wasmixedwith 50 ml culture medium. After standing for 5 minutes, the opticaldensity of the mixture was measured by a microplate reader at a540 nm wavelength. The concentration of nitrite was determined by astandard curve using sodium nitrite.

Experimental Model of Bleomycin-Induced PulmonaryFibrosis in Mice. The method of bleomycin-induced pulmonaryfibrosis has been previously described (You et al., 2015). Briefly, after1 week of acclimation, animals were divided randomly into six groupsand anesthetized by intraperitoneal injection of chloral hydratesolution (4%, 10 ml/kg), followed by intratracheal instillation ofbleomycin (5 mg/kg) or saline as the control. Seven days afterbleomycin administration, MGIIIE-H (20 mg/kg), MGIIIE-M (10 mg/kg),and MGIIIE-L (1 mg/kg) were orally administered to mice once a dayfor 14 consecutive days. Prednisone (6.5 mg/kg) was used as thepositive drug. The control groups were given the same volume of 0.9%sterilized saline. On day 21, mice were euthanized by excessiveintraperitoneal injection of chloral hydrate. Lung tissue was excisedfor pulmonary index measurement (lung weight/body weight; mg/g).The left lower lobes were fixed in 10% formalin for histopathologicalexamination, and the rest of the lung tissue was stored at 280°C.

Histologic Analysis. The lung tissue samples were fixed with10% formalin and embedded in paraffin, sectioned, and stained withH&E or Masson’s trichrome. Pathologic evaluation was conducted byexperienced pathologists in a single-blind procedure. The results werescored in accordance with a previously reportedmethod (Szapiel et al.,1979), and score numbers 0–3 corresponded to grades of 2, 1, 11,and 111, respectively.

MPO and Interleukin (IL)-1b Assays. MPOwas an indicator ofpolymorphonuclear leukocyte accumulation. The method of MPOassay has been previously described (Shi et al., 2014). Briefly, 21 daysafter bleomycin treatment, right lungs were excised and homogenizedin extraction buffer. After centrifuging, the homogenate and reactionbuffer were mixed and heated to 37°C for 15 minutes. The enzymaticactivity wasmeasured by a 96-well plate reader at a 460 nmwavelength.The IL-1b levels in the lung tissue homogenate were measured with anenzyme-linked immunosorbent assay kit according to the instructionsrecommended by themanufacturer. The optical density of themicroplatewas read at 450 nm by a 96-well plate reader.

Isolation and Culture of Mouse Primary Lung Fibroblasts(PLFs). The mouse PLFs were isolated as described previously, withsome modifications (Bruce and Honaker, 1998). Briefly, lung tissue

MGIIIE Prevents Bleomycin-Induced Pulmonary Fibrosis 269

at ASPE

T Journals on A

ugust 21, 2021jpet.aspetjournals.org

Dow

nloaded from

collected from C57/BL6 mice were minced into 1- to 2-mm3 pieces anddigested with trypsin for 15 minutes at 37°C. The digested cellsuspensions were collected and cultured with Dulbecco’s modifiedEagle’s complete medium and incubated at 37°C in an atmosphere of5% CO2 in air over night. After cells attached, the medium waschanged the next day. The percentage of fibroblast was identified as95% by morphology under a microscope.

Cell Treatment. For TGF-b1-treated mouse PLFs, cells weredetached with 0.25% trypsinization and seeded in 96-well plates at4 � 104 cells per well or 6-well plates at 2 � 105 cells per well. Cellswere treated with TGF-b1 (10 ng/ml), MGIIIE (10 mM), or controls(PBS or DMSO) for 48 hours. For LPS-treated mouse PLFs, cells weretreated with LPS (5 mg/ml), MGIIIE (10 mM), or controls (PBS orDMSO) for 48 hours. In some experiments, cells were pretreated withor without the TLR4 inhibitor, TAK-242 (10 nM), for 1.5 hours andsubsequently incubated with or without LPS (5 mg/ml), MGIIIE(10 mM), or controls (PBS or DMSO) for 48 hours.

Cell Viability Assay. Cell viability was assayed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Briefly,cells were seeded in 96-well plates at 4�104 cells per well andincubated in Dulbecco’s modified Eagle’s medium containing 10%fetal bovine serum for 24 hours. The cells were treated as describedpreviously, and then 5 mg/ml 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added to the wells and incubatedfor an additional 4 hours at 37°C in an atmosphere of 5% CO2 in air.After mixing well, the optical density was measured at 490 nm.

Real-Time Quantitative Polymerase Chain Reaction (PCR)Analysis. Total RNA was extracted from the lung tissue using Trizolreagent (Invitrogen Life Technologies Carlsbad, CA), reverse-transcribedto cDNA using the TransScript first-Strand cDNA Synthesis kit(TOYOBO, Osaka) and stored at 280°C until reverse transcription.The relative gene expression was quantified by quantitative PCRusing SYBR Premix Ex Taq (TaKaRa, Dalian, China) in StepOneReal-Time PCR (Life Technologies, Carlsbad, CA.). In each reaction,0.5 mg of total RNAwas reverse transcribed before the following PCRconditions: 94°C for 2 minutes followed by 40 cycles at 94°C for15 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, with finalextension at 72°C for 10 minutes. The sequences (59-39) for the primersare given inTable 1 and the datawere quantified using the comparativeCt (DCt) method and are presented as mean ratio to b-actin.

Immunohistochemistry and Immunofluorescence Stainings.For immunohistochemistry staining, the paraffin sections (5 mm)were incubated with 3% H2O2 to eliminate endogenous peroxidase.After antigen retrieval by heating, nonspecific binding sites wereblocked by 5% skim milk in PBS for 1 hour. The sections wereincubated with primary antibody and then incubated with second-ary antibodies conjugated with horseradish peroxidase. Color wasvisualized by incubated section with diaminobenzidine. Imageswere acquired under an Olympus BX53 microscope (Olympus,Tokyo, Japan).

For immunofluorescence staining, cell were fixed with 4%paraformaldehyde/PBS for 15 minutes, followed by incubation with0.3% Triton X-100/PBS for 10 minutes and 2% bovine serum albumin/PBS block solution for 2 hours, and then probed with primary antibodyovernight at 4°C. Alexa Fluor 488-conjugated secondary antibodieswere used to amplify the signal. 49, 6-diamidino-2-phenylindole was

used to stain nuclei. Images were obtained by fluorescent microscopy(Olympus IX53) or confocal laser scanning microscopy (Carl Zeiss,Oberkochen, Germany).

Western Blot Analysis. Total proteins extracted from lung homog-enate or cell lysate were lysed in ice-cold radioimmunoprecipitationassay lysis buffer containing 1:100 dilution of phenylmethanesulfonylfluoride (Beyotime, China). Protein concentrations were determinedusing BCA Protein Assay Kit (Beyotime). After boiling for 10 minutes,equal amounts of the protein (50mg/lane) were separated by SDS-PAGEgel and transferred to polyvinylidene fluoride membranes (MerckMillipore) that were probed with primary antibodies overnight at4°C and horseradish peroxidase–labeled secondary antibodies at25°C for 2 hours and the bands were visualized using superenhancedchemiluminescence detection reagent (Beyotime).

Statistical Analysis. All data are presented as mean6 S.D. fromat least three independent experiments. One-way analysis of varianceand two-tailed Student’s t test were used to analyze differences amongdifferent groups (GraphPad Prism software version 5.0 GraphPadSoftware Inc., San Diego. CA). Values of P , 0.05 are regarded asstatistically significant.

ResultsAnti-Inflammatory Activity Screening of Mogrosides

on LPS-Activated RAW264.7 Cells. MGV, siamenoside I,mogroside IVE, and MGIIIE are major mogrosides from thefruits of Cucurbitaceae (Fig. 1A). It has been reported thatMGV has anti-inflammatory activity in lung inflammation(Shi et al., 2014). Using the LPS-stimulated RAW264.7 mousemacrophages activationmodel, we screened the anti-inflammatoryeffects of these fourmogrosides. No cell toxicity was observed inall four mogroside-treated groups (Fig. 1B). After cells weretreated with LPS (5 mg/ml) for 24 hours, NO production wasincreased approximately 3-fold. All four mogrosides signifi-cantly inhibited NO production induced by LPS in a dose-dependentmanner.MGIIIEhad the lowest IC50 value (10.22mM)compared with the other mogrosides (Fig. 1C), indicatingthe strongest anti-inflammatory effect of MGIIIE. Therefore,MGIIIE was selected for further in vivo studies.MGIIIE Protects against Bleomycin-Induced Pulmo-

nary Fibrosis in Mice. Bleomycin is a commonly used agentto induce an experimental IPF model in mice, which is charac-terized by activated myofibroblasts that contribute to theexuberant fibrosis (Bhattacharyya et al., 2013). In thismodel, 7–9 days is the switching point from lung inflamma-tion to the fibrotic phase. In the present study, experimentalpulmonary fibrosis was induced by a single intratrachealinstillation of bleomycin (5 mg/kg). Three doses of MGIIIE,MGIIIE-L (1 mg/kg/d), MGIIIE-M (10 mg/kg/d), andMGIIIE-H(20 mg/kg/d) were orally administered for 14 days, starting7 days after bleomycin administration. As a positive control,prednisone, a synthetic corticosteroid drug for IPF treatment in

TABLE 1Sequences of primers used for real-time quantitative PCR

Gene Forward Primer (59-39) Reverse Primer (59-39) Product Size (Base Pair)

M-a-SMA CCA CGA AAC CAC CTA TAA CAG C GGA AGG TAG ACA GCG AAG CC 236M-Col I CTG ACT GGA AGA GCG GAG AG CGG CTG AGT AGG GAA CAC AC 116M-TGF-b1 AGA GCC CTG GAT ACC AAC TAT TG TGC GAC CCA CGT AGT AGA CG 286M-TLR4 ACA CTT TAT TCA GAG CCG TTG GT CAG GTC CAA GTT GCC GTT TC 297M-MyD88 CAT GGT GGT GGT TGT TTC TGA C GAC TTG GTG CAA GGG TTG GTA T 195M-actin CTG AGA GGG AAA TCG TGC GT CCA CAG GAT TCC ATA CCC AAG A 208

270 Tao et al.

at ASPE

T Journals on A

ugust 21, 2021jpet.aspetjournals.org

Dow

nloaded from

the clinical settingwas administrated intomice after bleomycininjection. After bleomycin treatment, MGIIIE-M and MGIIIE-Hsignificantly protected mice from weight loss and death evenbetter than prednisone treatment (Fig. 2, A and B). Histologicchanges in the lung including edema, alveolar wall thickening,andneutrophil infiltration in the lungparenchymawereobservedin mice after bleomycin treatment. MGIIIE markedly inhibitedthese lung damages as shown in Fig. 2, C–F. Furthermore, wecompared several inflammatory factor levels in mice in thesegroups. Both MPO activity and cytokine IL-1b were elevated inthe bleomycin-treated group; however, theywere greatly reducedin theMGIIIE-treated group (Fig. 2, G andH), indicating an anti-inflammatory effect of MGIIIE in bleomycin-induced lung in-flammation. These data demonstrated thatMGIIIE significantlyattenuated lung inflammation and fibrosis in bleomycin-inducedpulmonary fibrosis.Fibrotic Markers Are Altered by MGIIIE during

Bleomycin-Induced Pulmonary Fibrosis. Hydroxyproline,a-SMA, Col I, and MMP-9/TIMP-1 ratios are major fibrotic

markers duringpulmonary fibrosis (Yoshimura et al., 2006;Toddet al., 2012; Li et al., 2015). Hydroxyproline and Col I indicatecollagen production in tissue (Li et al., 2015). a-SMA expressionindicates a myofibroblast transformation after fibroblast activa-tion. The ratio of protease MMP-9/TIMP-1 is essential for ECMremodeling (You et al., 2015). The TGF-b/Smad2/3 signal is amajor profibrotic factor that is responsible for switching theinflammatory phase to the fibrotic phase in lung fibrosis (Yanget al., 2012; Bhattacharyya et al., 2013). To investigate whetherMGIIIE has an anti-fibrotic effect in bleomycin-induced pulmo-nary fibrosis, we measured these markers in fibrotic lung tissuein the MGIIIE-M- and MGIIIE-H-treated groups. As shown inFig. 3, bleomycin significantly increased hydroxyproline level,a-SMA expression, and Col I content, and decreased the MMP-9/TIMP-1 ratio; however, both MGIIIE-M and MGIIIE-Htreatment reduced the magnitude of all of these changes atboth the protein and mRNA level. In addition, 21 days afterbleomycin injection, TGF-b and phosphorylation of its down-stream signals in Smad2/3 were markedly increased in mice.

Fig. 1. Mogrosides inhibitedNO production in LPS-activated RAW264.7 cells. RAW264.7 cells were incubatedwith or without LPS (5mg/ml) in the absence orpresence of four mogrosides [MGV, siamenoside I (SMI), mogroside IVE (MGIVE), andMGIIIE] at different concentrations. (A) The chemical structures of fourmogrosides (MGV, SMI, MGIVE, and MGIIIE). (B) Cell viability. (C) NO levels. Data are expressed as mean 6 S.D. (n = 5). **P , 0.01, ***P , 0.001.

MGIIIE Prevents Bleomycin-Induced Pulmonary Fibrosis 271

at ASPE

T Journals on A

ugust 21, 2021jpet.aspetjournals.org

Dow

nloaded from

Fig. 2. MGIIIE treatment attenuated bleomycin-induced pulmonary fibrosis in mice. Mice were given an intragastric administration of MGIIIE(1, 10, or 20 mg/kg, respectively) and prednisone (6.5 mg/kg) daily 7 days after an intratracheal instillation of bleomycin, and mice were killed21 days after bleomycin injection. The body weight (A), survival rate (B), and pulmonary index (F) were determined, and the representativeimages of H&E staining and Masson’s trichrome staining (C) of lung sections in mice as well as comparison of the inflammation score (D) andfibrosis score (E) among the experimental groups are shown. Data are expressed as mean 6 S.D. (n = 10). The MPO activity and IL-1b level weredetected in the lung tissue (G and H). Data are expressed as mean 6 S.D. (n = 5). ###P , 0.001 versus control group. **P , 0.01, ***P , 0.001versus model group.

272 Tao et al.

at ASPE

T Journals on A

ugust 21, 2021jpet.aspetjournals.org

Dow

nloaded from

MGIIIE significantly inhibited TGF-b/Smad2/3 pathwayactivation as shown in Fig. 4. These data suggest thatMGIIIE protects against lung fibrosis through regulatingmultiple fibrotic factors.MGIIIE downregulates TLR4/MyD88-MAPK Signals

during Pulmonary Fibrosis. It is well documented thatLPS binding to TLR4 induces MyD88-dependent pathwaysand that TLR4 is responsible for the initiation of pulmonaryinflammation and fibrosis after acute and chronic lung injury,which is integrally involved in the TGF-b1 pathway in a feed-forward loop and results in increased matrix production(Willis et al., 2005; Yang et al., 2012). To investigate whetherMGIIIE regulates pulmonary fibrosis through the TLR4receptor, we measured TLR4 and its downstream signalingin the MGIIIE-M- and MGIIIE-H-treated groups. The results

from both immunohistochemistry staining and western blot-ting showed that TLR4 expression was upregulated in lungtissue after bleomycin treatment; however, both MGIIIEmiddle and high doses (10 and 20 mg/kg) significantlyinhibited TLR4 expression as shown by the lighter browncolor in lung tissue compared with the model group (see Fig. 5,A and B). Consistently, TLR4 and MyD88 mRNA and proteinlevels were significantly downregulated after treatment withMGIIIE compared with the bleomycin group (Fig. 5, C–G),indicating that MGIIIEmay regulate TLR4 expression duringlung inflammation and fibrosis. In addition, we measuredthe activity of the MAPK family, JNK, ERK, and p38, thedownstream signals of TLR4/MyD88 activation. It has beenreported that JNK, ERK, and p38 play critical roles in thedevelopment of lung fibrosis (Matsuoka et al., 2002; Yoshida

Fig. 3. MGIIIE treatment decreased the hydroxyproline (HYP) and collagen content and other fibrotic markers in bleomycin-damaged lung tissues inmice. Mice were given an intragastric administration of MGIIIE (10 and 20 mg/kg, respectively) and prednisone (6.5 mg/kg) daily 7 days after anintratracheal instillation of bleomycin, andmice were killed 21 days after bleomycin injection. (A) The HYP content in the lung tissues was detected. ThemRNA expressions of a-SMA (B) and Col I (C) in the lung tissues were detected by real-time PCR analysis. (D–I) The protein expressions of a-SMA, Col I,MMP-9, and TIMP-1, and the ratio of MMP-9/TIMP-1 in the lung tissues were detected by western blot analysis. Data are expressed as mean 6 S.D.(n = 3–5). ###P , 0.001 versus control group. *P , 0.05, **P , 0.01, ***P , 0.001 versus model group.

MGIIIE Prevents Bleomycin-Induced Pulmonary Fibrosis 273

at ASPE

T Journals on A

ugust 21, 2021jpet.aspetjournals.org

Dow

nloaded from

et al., 2002; Madala et al., 2012; Vittal et al., 2013; van derVelden et al., 2016). In our results, phosphorylation of JNK,ERK, and p38 were significantly enhanced after bleomycininjection; however, their activation was inhibited byMGIIIE-HandMGIIIE-M, and to a similar extent to by prednisone (Fig. 5,H and I). These data indicated that MGIIIE suppressed bothTLR4/MyD88 expression and its downstream MAPK signalactivation, which may contribute to its anti-fibrotic effects inpulmonary fibrosis.MGIIIE Suppressed ECM Deposition and the TLR4/

MyD88 MAPK Signaling Pathway in Activated LungFibroblasts Induced by TGF-b. Pulmonary fibroblaststransform into myofibroblast-like cells and are one of thekey steps in initiation of pulmonary fibrosis. Myofibroblastsare the major source of ECM production in the fibrotic lung(Hay et al., 1991). To further investigate the mechanismunderlying the MGIIIE inhibitory role on fibrosis in lung,PLFs were isolated from mice and subjected to TGF-bchallenge to induce fibrosis in the in vitro model, which ischaracterized as myofibroblast transformation and exces-sive collagen production in fibroblasts. First, our datashowed that cell viability of fibroblasts was not affected byMGIIIE (0.1–10 mM) (Fig. 6A). Second, our data showed thata-SMA and Col I expressions were greatly increased in TGF-b-treated fibroblasts; however, after MGIIIE incubation,

a-SMA and Col I expressions were significantly reduced(almost to an extent similar to the control group), confirm-ing the anti-fibrotic role of MGIIIE in vitro (Fig. 6, B–D).Moreover, both western blotting and inmmunoflurescencestaining show that TLR4/MyD88 and its downstreamMAPK cascades upregulated by TGF-b are significantlyinhibited by MGIIIE (Fig. 6, B and E–I). Consistent withthe data obtained in vivo, these data confirmed thatMGIIIE inhibits fibroblast activation and ECM depositionand its anti-fibrotic effect may act through regulatingTLR4/MyD88 signaling.MGIIIE Inhibited Collagen Production Mainly

through TLR4/MyD88 MAPK Signaling in ActivatedFibroblasts Induced by LPS. LPS-induced fibrosis in lungfibroblasts is another well established in vitro model for pulmo-nary fibrosis. We investigated the anti-fibrotic role of MGIIIE inthe LPS model as well. Similarly, fibroblasts were incubatedwith LPS in the presence or absence of MGIIIE and a-SMA andCol I contents were measured by western blotting. Consistently,MGIIIE exhibited an anti-fibrotic effect as shown by themuch lower a-SMA and Col I contents compared with thecontrol group (Fig. 7, C, E, and F). To test whether or notMGIIIE acts mainly through TLR4 pathway, mouse PLFs insome groups were cotreated with MGIIIE and TAK-242, aspecific antagonist of TLR4. Single addition of TAK-242 or

Fig. 4. MGIIIE treatment inhibited TGF-b/Smad pathway in bleomycin-induced pulmonary fibrosis. Mice were given an intragastricadministration of MGIIIE (10 and 20 mg/kg, respectively) and prednisone (6.5 mg/kg) daily 7 days after an intratracheal instillation ofbleomycin, and mice were killed 21 days after bleomycin injection. (A–C) The protein expression of TGF-b1 and its relative mRNA expression weredetected by western blot and real-time PCR analyses. (B–D) Effects on the expression of Smad2/3 and its phosphorylation, phospho-Smad2/3(p-Smad2/3), were detected by western blot analysis. Data are expressed as mean 6 S.D. (n = 3). ###P , 0.001 versus control group. **P , 0.01,***P , 0.001 versus model group.

274 Tao et al.

at ASPE

T Journals on A

ugust 21, 2021jpet.aspetjournals.org

Dow

nloaded from

the combination with MGIIIE showed no cell toxicity in mousePLFs (Fig. 7, A and B). LPS induced a robust expression ofTLR4 in fibroblasts; however, TAK-242 significantly blockedLPS-induced fibroblast activation and collagen production,indicating LPS-induced fibrosis is TLR4 dependent (Fig. 7,C–F). When cotreated with TAK-242 in fibroblasts, MGIIIEfailed to further reduce a-SMA and Col I contents comparedwith the MGIIIE-only group (Fig. 7, C–F), indicating MGIIIEmay inhibit fibrosis mainly through TLR4 signals. Takentogether, these data demonstrate that MGIIIE also inhibitedfibroblast activation and collagen production induced by LPS

and its inhibitory effect may be through downregulating theTLR4/MyD88-MAPKs signaling pathways.

DiscussionThe fruit of S. grosvenorii Swingle (Cucurbitaceae) is a famous

traditional Chinese herb used in the treatment of dry cough, sorethroat, extreme thirst, and constipation (Committee of NationalPharmacopoeia, 2010). A previous study has shown that MGVhas protective effects on LPS-induced acute lung injury (Shiet al., 2014). MGIIIE was one of cucurbitane-type triterpene

Fig. 5. MGIIIE downregulated TLR4/MyD88-MAPK signals in bleomycin-induced pulmonary fibrosis in mice. Mice were given an intragastricadministration of MGIIIE (10 and 20 mg/kg, respectively) and prednisone (6.5 mg/kg) daily 7 days after an intratracheal instillation of bleomycin, andmice were killed 21 days after bleomycin injection. Representative images of immunohistochemical analysis of TLR4 expression (A and B) in the lungsection (brown). The relativemRNA levels (C and D) and protein expressions (E–G) of TLR4 andMyD88were investigated by real-time PCR and westernblot analyses. (H and I) Phosphorlations of JNK, ERK, and p38 in lung tissues. Data are expressed as mean 6 S.D. (n = 3). ###P , 0.001 versus controlgroup. *P , 0.05, **P , 0.01, ***P , 0.001 versus model group.

MGIIIE Prevents Bleomycin-Induced Pulmonary Fibrosis 275

at ASPE

T Journals on A

ugust 21, 2021jpet.aspetjournals.org

Dow

nloaded from

glycosides (mogrosides) isolated from the fruits of S. grosvenorii(Xu et al., 2015). MGIIIE showed stronger inhibitory effects onmaltase than MGV (Suzuki et al., 2005); however, no studieshave investigated the effect of MGIIIE in fibrosis. In this study,

using both in vitro and in vivo models, we demonstrated forthe first time that MGIIIE significantly reduced fibrosis inthe bleomycin-induced pulmonary fibrosis mouse model andits anti-fibrotic effect may be due to its inhibition of fibroblast

Fig. 6. MGIIIE reduces ECM deposition and TLR4/MyD88-MAPK signaling pathways in mouse PLFs induced by TGF-b1. Mouse PLFs were treatedwith or without TGF-b1 (10 ng/ml) in the absence or presence of MGIIIE (10 mM) for 48 hours. (A) Cytotoxicity of MGIIIE onmouse PLFs was detected by3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (B–H) Protein expression of a-SMA, Col I, TLR4, MyD88, and MAPK in mousePLFs; (I) Representative image of protein expression of TLR4 and a-SMA by immunofluorescence staining. Data are expressed as mean6 S.D. (n = 3–5).*P , 0.05, **P , 0.01, ***P , 0.001.

276 Tao et al.

at ASPE

T Journals on A

ugust 21, 2021jpet.aspetjournals.org

Dow

nloaded from

activation and ECM deposition through downregulating theTLR4 signal in fibroblasts.IPF is a chronic, progressive, and irreversible lung injury.

There are no clinically effective therapies (Hashimoto et al.,2004). Bleomycin-induced pulmonary fibrosis in mouse is afrequently used mouse model to study the pathogenesis ofpulmonary fibrosis. After bleomycin administration, the expres-sion of inflammatory cytokines elevates quickly and returns tobackground levels 9 days after injection, while profibrotic geneexpression peaks between 7 and 14 days and remains at a highlevel up to 21 days, suggesting a switch between inflammationand fibrosis in this interval (Chaudhary et al., 2006). Therefore,the time point for MGIIIE administration to investigate its

anti-fibrotic effect was chosen to be 7 days. Our data haveshown that MGIIIE is a potent inhibitor for pulmonaryfibrosis in the bleomycin model.In IPF, the predominant cell types are the fibroblasts and

myofibroblasts. Inflammatory damage to the effected epithe-lium results in the secretion of growth factors, which stimulatethe underlying fibroblast transformation to myofibroblasts.Myofibroblasts secrete growth factors and ECM componentsand contribute to increased collagen deposition, which even-tually leads to fibrosis (Bocchino et al., 2010). Many inflam-matory cytokines and profibrotic factors are involved in thefibrosis process, among which IL-1b, MPO, TGF-b, MMP-9/TIMP, a-SMA, andCol I are themost important players. IL-1b

Fig. 7. MGIIIE inhibited fibrosis induced by LPS mainly through regulating TLR4 expression. The mouse lung fibroblasts were pretreated with orwithout TAK-242 (10 nM) for 1.5 hours and subsequently in the absence or presence of MGIIIE (10 mM) and LPS (5 mg/ml) for 48 hours. (A and B) Effectsof TAK-242 and MGIIIE on mouse lung fibroblast proliferation were measured by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays.(C–F) Expressions of TLR4, a-SMA, and Col I in mouse lung fibroblasts by western blot analysis. Data are expressed as mean6 S.D. (n = 3–5). *P, 0.05,**P , 0.01, ***P , 0.001. Nonsignificant (NS).

MGIIIE Prevents Bleomycin-Induced Pulmonary Fibrosis 277

at ASPE

T Journals on A

ugust 21, 2021jpet.aspetjournals.org

Dow

nloaded from

and MPO are the proinflammatory factors mediating lunginflammation after bleomycin injection. TGF-b is a majorinducer of fibroblast activation to myofibroblast (Wynn, 2007).MMP-9 causes collagen degradation, while TIMPs play apivotal role in control of MMP catalytic activity in fibrogenesis(Chen et al., 2012). The ratio of MMP-9/TIMP inverselycontrols ECM deposition in lung fibrogenesis (You et al.,2015). a-SMA is a marker for myofibroblasts and Col I is thehallmark for fibrosis (Li et al., 2015). In our study, MGIIIEsignificantly inhibited expression of TGF-b, a-SMA, and Col Iand increased the MMP-9/TIMP ratio, indicating MGIIIEinhibits fibrosis through regulating fibrotic factors. In addi-tion, our data have shown that MGIIIE inhibited MPO andIL-1b levels induced by bleomycin, indicating that MGIIIEmay have anti-inflammatory effects, which may contribute tolung fibrosis attenuation. In the future, the anti-inflammatoryeffects of MGIIIE including inflammatory cytokine expressionand neutrophil or macrophage infiltration will be evaluated atearlier time points (1–6 days after bleomycin injection) duringthe inflammation phase.It is well documented that LPS or TGF-b can induce

fibroblast activation and collagen deposition in cultured lungfibroblasts (Bhattacharyya et al., 2013). In the cell model oftransdifferentiation of quiescent fibroblasts into myofibro-blasts with TGF-b1 induction, mouse PLFs acquired a mes-enchymal phenotype and higher a-SMA expression (Sun et al.,2015). LPS induces fibrosis by increasing the rapid release ofproinflammatory cytokines, which activates fibroblasts aswell (Synenki et al., 2007; Krebs et al., 2015). Using thesetwo in vitro fibrosis models, our data showed that MGIIIEsignificantly inhibited fibroblast activation and collagenproduction in mouse PLFs induced by LPS or TGF-b andconfirmed the anti-fibrotic effects of MGIIIE in lung fibrosis.TLR4 has been shown to be essential in the protective

immunity against infection during the pathogenesis of fibro-sis (Jiang et al., 2005). MyD88 serves as a key TLR4 adaptorprotein, linking the receptors to downstream kinases, anddeficiency of MyD88 protects mice from inflammation andfibrosis after bleomycin treatment, indicating that TLR4/MyD88 may be a specific target in inflammatory responses(Gasse et al., 2007; Salaun et al., 2007). In addition, extensiveevidence has shown that downstream signals of TLR4, JNK,ERK, and p38 were robustly increased in IPF patients andpharmacological inhibition of these three MAPKs signifi-cantly reduced fibrosis in mice and patients (Matsuokaet al., 2002; Yoshida et al., 2002; Moran, 2011; Madala et al.,2012; Vittal et al., 2013; van der Velden et al., 2016). InhibitedTGF-b1 and pulmonary fibrosis was observed in JNK knock-out mice, indicating JNK is required for fibrosis development(Alcorn et al., 2009). PD98059, a highly selective inhibitor ofERK activation ameliorated lung injury and pulmonaryfibrosis (Galuppo et al., 2011), and a p38 inhibitor has beenapproved for IPF treatment in Europe (Moran, 2011). In thebleomycin-induced pulmonary fibrosis mouse model, our datashowed that MGIIIE significantly decreased expressions ofTLR4/MyD88, as well as phosphorylations of MAPKs(including JNK, ERK, and p38). In addition, the TLR4-specific inhibitor TAK-242 blocked LPS-induced fibroblastactivation, confirming the essential role of TLR4 in fibro-blast activation. Consistently, MGIIIE markedly decreasedTLR4/MyD88 and collagen synthesis induced by LPS orTGF-b, suggesting that MGIIIE may inhibit fibrosis through

regulating the TLR4 signaling. Moreover, MGIIIE failed toinduce further reduction of collagen production after TAK-242pretreatment on fibroblasts, implicating that MGIIIE mayinhibit fibrosis mainly through TLR4 expression; however,more direct evidence is needed before drawing this conclu-sion. Currently, our laboratory is investigating whether TLR4is required for anti-fibrotic effects of MGIIIE by performingrescue experiments using a lentival-TLR4 overexpressingsystem in mouse PLFs.In summary, the present study demonstrated that MGIIIE

protected against bleomycin-induced pulmonary fibrosis bothin vitro and in vivo and its anti-fibrotic effect may be due toinhibiting fibroblast activation and collagen deposition regu-lated by TLR4 signals. These findings provide new insightsinto understanding the bioactivity of MGIIIE. MGIIIE mayhave therapeutic potential for the treatment of pulmonaryfibrosis in clinical applications.

Acknowledgments

The authors gratefully acknowledge Chengdu Biopurify Phyto-chemical Ltd. (Chengdu, China) for the gifts of MGIIIE (purity above99%), mogroside IVE (purity above 99%), siamenoside I (purity above99%), and MGV (purity above 99%).

Authorship Contributions

Participated in research design: Tao, Xie, Gong, C. Zhang.Conducted experiments: Tao, Gong, C. Zhang.Performed data analysis: Tao, Yang, Cao.Wrote or contributed to the writing of the manuscript: Tao, M.

Zhang, Gong, C. Zhang.

References

Alcorn JF, van der Velden J, Brown AL, McElhinney B, Irvin CG, and Janssen-Heininger YM (2009) c-Jun N-terminal kinase 1 is required for the development ofpulmonary fibrosis. Am J Respir Cell Mol Biol 40:422–432.

Bhattacharyya S, Kelley K, Melichian DS, Tamaki Z, Fang F, Su Y, Feng G, PopeRM, Budinger GR, Mutlu GM, et al. (2013) Toll-like receptor 4 signaling augmentstransforming growth factor-b responses: a novel mechanism for maintaining andamplifying fibrosis in scleroderma. Am J Pathol 182:192–205.

Bocchino M, Agnese S, Fagone E, Svegliati S, Grieco D, Vancheri C, Gabrielli A,Sanduzzi A, and Avvedimento EV (2010) Reactive oxygen species are required formaintenance and differentiation of primary lung fibroblasts in idiopathic pulmo-nary fibrosis. PLoS One 5:e14003.

Bruce MC and Honaker CE (1998) Transcriptional regulation of tropoelastin ex-pression in rat lung fibroblasts: changes with age and hyperoxia. Am J Physiol 274:L940–L950.

Chaudhary NI, Schnapp A, and Park JE (2006) Pharmacologic differentiation ofinflammation and fibrosis in the rat bleomycin model. Am J Respir Crit Care Med173:769–776.

Chen M, Cheung FW, Chan MH, Hui PK, Ip SP, Ling YH, Che CT, and Liu WK(2012) Protective roles of Cordyceps on lung fibrosis in cellular and rat models. JEthnopharmacol 143:448–454.

Chen XB, Zhuang JJ, Liu JH, Lei M, Ma L, Chen J, Shen X, and Hu LH (2011)Potential AMPK activators of cucurbitane triterpenoids from Siraitia grosvenoriiSwingle. Bioorg Med Chem 19:5776–5781.

Committee of National Pharmacopoeia (2010) Pharmacopoeia of P.R. China, Chem-ical Industry Press, Beijing, China.

Derynck R and Zhang YE (2003) Smad-dependent and Smad-independent pathwaysin TGF-b family signalling. Nature 425:577–584.

Di R, Huang MT, and Ho CT (2011) Anti-inflammatory activities of mogrosides fromMomordica grosvenori in murine macrophages and a murine ear edema model. JAgric Food Chem 59:7474–7481.

Fan H, Qi D, Yang M, Fang H, Liu K, and Zhao F (2013) In vitro and in vivo anti-inflammatory effects of 4-methoxy-5-hydroxycanthin-6-one, a natural alkaloid fromPicrasma quassioides. Phytomedicine 20:319–323.

Galuppo M, Esposito E, Mazzon E, Di Paola R, Paterniti I, Impellizzeri D,and Cuzzocrea S (2011) MEK inhibition suppresses the development of lung fi-brosis in the bleomycin model. Naunyn Schmiedebergs Arch Pharmacol 384:21–37.

Gasse P, Mary C, Guenon I, Noulin N, Charron S, Schnyder-Candrian S, Schnyder B,Akira S, Quesniaux VF, Lagente V, et al. (2007) IL-1R1/MyD88 signaling and theinflammasome are essential in pulmonary inflammation and fibrosis in mice. JClin Invest 117:3786–3799.

Gürbüzel M, Sayar I, Cankaya M, Gürbüzel A, Demirtas L, Bakirci EM, and CapogluI (2016) The preventive role of levosimendan against bleomycin-induced pulmo-nary fibrosis in rats. Pharmacol Rep 68:378–382.

Hashimoto N, Jin H, Liu T, Chensue SW, and Phan SH (2004) Bone marrow-derivedprogenitor cells in pulmonary fibrosis. J Clin Invest 113:243–252.

278 Tao et al.

at ASPE

T Journals on A

ugust 21, 2021jpet.aspetjournals.org

Dow

nloaded from

Hay J, Shahzeidi S, and Laurent G (1991) Mechanisms of bleomycin-induced lungdamage. Arch Toxicol 65:81–94.

He Z, Gao Y, Deng Y, Li W, Chen Y, Xing S, Zhao X, Ding J, and Wang X (2012)Lipopolysaccharide induces lung fibroblast proliferation through Toll-like receptor4 signaling and the phosphoinositide3-kinase-Akt pathway. PLoS One 7:e35926.

He Z, Zhu Y, and Jiang H (2009) Inhibiting toll-like receptor 4 signaling amelioratespulmonary fibrosis during acute lung injury induced by lipopolysaccharide: anexperimental study. Respir Res 10:126.

Huebener P and Schwabe RF (2013) Regulation of wound healing and organ fibrosisby toll-like receptors. Biochim Biophys Acta 1832:1005–1017.

Jiang D, Liang J, Fan J, Yu S, Chen S, Luo Y, Prestwich GD, Mascarenhas MM, GargHG, Quinn DA, et al. (2005) Regulation of lung injury and repair by Toll-likereceptors and hyaluronan. Nat Med 11:1173–1179.

Jin JS and Lee JH (2012) Phytochemical and pharmacological aspects of Siraitiagrosvenorii, luo han kuo. Orient Pharm Exp Med 12:233–239.

Krebs J, Kolz A, Tsagogiorgas C, Pelosi P, Rocco PR, and Luecke T (2015) Effects oflipopolysaccharide-induced inflammation on initial lung fibrosis during open-lungmechanical ventilation in rats. Respir Physiol Neurobiol 212–214:25–32.

Li L, Li D, Xu L, Zhao P, Deng Z, Mo X, Li P, Qi L, Li J, and Gao J (2015) Total extractof Yupingfeng attenuates bleomycin-induced pulmonary fibrosis in rats. Phytome-dicine 22:111–119.

Madala SK, Schmidt S, Davidson C, Ikegami M, Wert S, and Hardie WD (2012)MEK-ERK pathway modulation ameliorates pulmonary fibrosis associated withepidermal growth factor receptor activation. Am J Respir Cell Mol Biol 46:380–388.

Matsuoka H, Arai T, Mori M, Goya S, Kida H, Morishita H, Fujiwara H, Tachibana I,Osaki T, and Hayashi S (2002) A p38 MAPK inhibitor, FR-167653, amelioratesmurine bleomycin-induced pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol283:L103–L112.

Moran N (2011) p38 Kinase inhibitor approved for idiopathic pulmonary fibrosis. NatBiotechnol 29:301.

Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E,Silva M, Galanos C, et al. (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085–2088.

Rhieu BH, Epperly MW, Cao S, Goff J, Shields D, Franicola D, Wang H,and Greenberger JS (2014) Improved longevity of hematopoiesis in long-term bonemarrow cultures and reduced irradiation-induced pulmonary fibrosis in Toll-likereceptor-4 deletion recombinant-negative mice. In Vivo 28:441–448.

Salaun B, Romero P, and Lebecque S (2007) Toll-like receptors’ two-edged sword:when immunity meets apoptosis. Eur J Immunol 37:3311–3318.

Shi D, Zheng M, Wang Y, Liu C, and Chen S (2014) Protective effects and mechanismsof mogroside V on LPS-induced acute lung injury in mice. Pharm Biol 52:729–734.

Song KJ, Liao ZX, Liu YP, Chen XY, and Huang CH (2014) Effect of activation andapoptosis of mogroside on hepatic stellate cell. Zhongchengyao 36:481–484.

Sun BS, Chen YP, Wang YB, Tang SW, Pan FY, Li Z, and Sung CK (2012) Anti-obesity effects of mogrosides extracted from the fruits of Siraitia grosvenorii(Cucurbitaceae). Afr J Pharm Pharmacol 6:1492–1501.

Sun X, Chen E, Dong R, Chen W, and Hu Y (2015) Nuclear factor (NF)-kB p65regulates differentiation of human and mouse lung fibroblasts mediated by TGF-b.Life Sci 122:8–14.

Suzuki YA, Murata Y, Inui H, Sugiura M, and Nakano Y (2005) Triterpene glycosidesof Siraitia grosvenori inhibit rat intestinal maltase and suppress the rise in bloodglucose level after a single oral administration of maltose in rats. J Agric FoodChem 53:2941–2946.

Synenki L, Chandel NS, Budinger GR, Donnelly HK, Topin J, Eisenbart J, JovanovicB, and Jain M (2007) Bronchoalveolar lavage fluid from patients with acute lunginjury/acute respiratory distress syndrome induces myofibroblast differentiation.Crit Care Med 35:842–848.

Szapiel SV, Elson NA, Fulmer JD, Hunninghake GW, and Crystal RG (1979)Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse. AmRev Respir Dis 120:893–899.

Todd NW, Luzina IG, and Atamas SP (2012) Molecular and cellular mechanisms ofpulmonary fibrosis. Fibrogenesis Tissue Repair 5:11.

van der Velden JL, Ye Y, Nolin JD, Hoffman SM, Chapman DG, Lahue KG, AbdallaS, Chen P, Liu Y, Bennett B, et al. (2016) JNK inhibition reduces lung remodelingand pulmonary fibrotic systemic markers. Clin Transl Med 5:36.

Vittal R, Fisher A, Gu H, Mickler EA, Panitch A, Lander C, Cummings OW,Sandusky GE, and Wilkes DS (2013) Peptide-mediated inhibition of mitogen-activated protein kinase-activated protein kinase-2 ameliorates bleomycin-inducedpulmonary fibrosis. Am J Respir Cell Mol Biol 49:47–57.

Willis BC, Liebler JM, Luby-Phelps K, Nicholson AG, Crandall ED, du Bois RM,and Borok Z (2005) Induction of epithelial-mesenchymal transition in alveolarepithelial cells by transforming growth factor-b1: potential role in idiopathic pul-monary fibrosis. Am J Pathol 166:1321–1332.

Wynn TA (2007) Common and unique mechanisms regulate fibrosis in variousfibroproliferative diseases. J Clin Invest 117:524–529.

Xiao G, Chen Z, Li WN, Wang Q, and Liao CX (2012) Protective effect of mogroside oncarbon tetrachloride-induced hepatic fibrosis rats. Shandong Med 52:19–24.

Xiao G and Wang Q (2013) Experiment study on the hepatoprotective effect ofmogrosides. Chin J Exp Traditional Med Formulae 19:196–200.

Xu F, Li DP, Huang ZC, Lu FL, Wang L, Huang YL, Wang RF, Liu GX, Shang MY,and Cai SQ (2015) Exploring in vitro, in vivo metabolism of mogroside V anddistribution of its metabolites in rats by HPLC-ESI-IT-TOF-MSn. J Pharm BiomedAnal 115:418–430.

Yang HZ, Wang JP, Mi S, Liu HZ, Cui B, Yan HM, Yan J, Li Z, Liu H, Hua F, et al.(2012) TLR4 activity is required in the resolution of pulmonary inflammation andfibrosis after acute and chronic lung injury. Am J Pathol 180:275–292.

Yoshida K, Kuwano K, Hagimoto N, Watanabe K, Matsuba T, Fujita M, Inoshima I,and Hara N (2002) MAP kinase activation and apoptosis in lung tissues frompatients with idiopathic pulmonary fibrosis. J Pathol 198:388–396.

Yoshimura S, Nishimura Y, Nishiuma T, Yamashita T, Kobayashi K, and YokoyamaM (2006) Overexpression of nitric oxide synthase by the endothelium attenuatesbleomycin-induced lung fibrosis and impairs MMP-9/TIMP-1 balance. Respirology11:546–556.

You XY, Xue Q, Fang Y, Liu Q, Zhang CF, Zhao C, Zhang M, and Xu XH (2015)Preventive effects of Ecliptae Herba extract and its component, ecliptasaponin A,on bleomycin-induced pulmonary fibrosis in mice. J Ethnopharmacol 175:172–180.

Address correspondence to: Dr. Yanqing Gong, Division of TranslationalMedicine and Human Genetics, Department of Medicine, Perelman School ofMedicine, University of Pennsylvania, Smilow Center for TranslationalResearch, Room 11-142, Philadelphia, PA, 19104. E-mail: gongy@mail.med.upenn.edu or Dr. Chaofeng Zhang, Research Department of Pharmacognosy,China Pharmaceutical University, 639 Longmian Road, Nanjing 210098 P.R.China. E-mail: zhangchaofeng@cpu.edu.cn

MGIIIE Prevents Bleomycin-Induced Pulmonary Fibrosis 279

at ASPE

T Journals on A

ugust 21, 2021jpet.aspetjournals.org

Dow

nloaded from