International Immunopharmacology 22 (2014) 133–140

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International Immunopharmacology

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Preliminary report

Schisantherin A protects lipopolysaccharide-induced acute respiratorydistress syndrome in mice through inhibiting NF-κB and MAPKssignaling pathways

Ershun Zhou a,1, Yimeng Li a,1, Zhengkai Wei a, Yunhe Fu a, He Lei b, Naisheng Zhang a,Zhengtao Yang a, Guanghong Xie a,⁎a Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province, People's Republic of Chinab TongLe School, Nanshan ShenZhen, Guangdong Province, People's Republic of China

⁎ Corresponding author at: Department of Clinical VVeterinary Medicine, Jilin University, 5333#, Xian Ro130062, People's Republic of China. Tel.: +86 431 879816

E-mail address: zhouershun594926@163.com (G. Xie)1 Ershun Zhou and Yimeng Li contributed equally to th

http://dx.doi.org/10.1016/j.intimp.2014.06.0041567-5769/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 March 2014Received in revised form 4 June 2014Accepted 4 June 2014Available online 27 June 2014

Keywords:Schisantherin ALipopolysaccharide (LPS)Acute lung injury (ARDS)Anti-inflammatoryNuclear transcription factor-kappaB (NF-κB)Mitogen activated protein kinases (MAPKs)

Acute respiratory distress syndrome (ARDS) is characterized by polymorphonuclear neutrophils (PMNs) adhesion,activation, sequestration and inflammatory damage to alveolar-capillary membrane. Schisantherin A, adibenzocyclooctadiene lignan isolated from the fruit of Schisandra sphenanthera, has been reported to have anti-inflammatory properties. In the present study, we aimed to investigate the protective effects of schisantherin Aon LPS-inducedmouseARDS. Thepulmonary injury severitywas evaluated 7h after LPS administration and the pro-tective effects of schisantherin A on LPS-induced mouse ARDS were assayed by enzyme-linked immunosorbentassay andWestern blot. The results revealed that the wet/dry weight ratio, myeloperoxidase activity, and the num-ber of total cells, neutrophils andmacrophages in the bronchoalveolar lavagefluid (BALF)were significantly reducedby schisantherin A in a dose-dependent manner. Meanwhile, pretreatment with schisantherin A markedly amelio-rated LPS-induced histopathologic changes and decreased the levels of tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6) and interleukin-1β (IL-1β) in the BALF. In addition, the phosphorylation of nuclear transcriptionfactor-kappaB (NF-κB) p65, inhibitory kappa B alpha (IκB-α), c-jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 induced by LPS were suppressed by schisantherin A. These findings indicated thatschisantherin A exerted potent anti-inflammatory properties in LPS-induced mouse ARDS, possibly throughblocking the activation of NF-KB and mitogen activated protein kinases (MAPKs) signaling pathways. Therefore,schisantherin A may be a potential agent for the prophylaxis of ARDS.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Acute respiratory distress syndrome (ARDS) was first described in1967, and is characterized by the abrupt onset of clinically significanthypoxaemia with presence of diffuse pulmonary infiltrates [1]. Com-mon causes of ARDS are sepsis, pneumonia, trauma, aspiration pneumo-nia, pancreatitis, and so on [2]. Recent data indicate approximately190,000 cases of ARDS in the United States each year, with an associated74,500 death [3]. The pathogenesis of ARDS is complex, hallmarkfeatures including immune and endothelial cell activation, loss of vascu-lar integrity, and accumulation of protein-rich fluid in the airspaces ofthe lung [4]. To expediently study the pathophysiologic mechanisms

eterinary Medicine, College ofad, Changchun, Jilin Province88; fax: +86 431 8798168..is work.

of ARDS, several animal models have been established, including LPS-induced mouse model [5]. LPS, an important component of the outermembrane of the Gram-negative bacteria has been widely recognizedas a clinically relevant model inducer of ARDS [6], for it can activatethe host receptor TLR4, subsequently trigger a series of inflammatoryresponses, and ultimately lead to ARDS. In the last decades, several can-didate therapy strategies have been applied to ARDS, such as fluidman-agement, surfactants and glucocorticoids [7,8]. However, the mortalityof ARDS is still high. Therefore, the development of novel therapystrategies for ARDS is urgently needed.

Schisantherin A, a dibenzocyclooctadiene lignan isolated from thefruit of Schisandra sphenanthera, has been reported to possess variedbeneficial pharmacological effects [9]. It is awidely used herbalmaterialin traditional Chinese medicine used as antitussive, tonic, and sedativeagents and a component of dietary supplement products in the USA[10,11]. Recently, studies have shown that the activation of NF-κB andMAPKs signaling pathways play important roles in the development ofARDS [5,12,13]. Treatments aimed at inhibiting NF-κB and MAPKssignaling pathways may have potential therapeutic advantages

134 E. Zhou et al. / International Immunopharmacology 22 (2014) 133–140

for inflammatory diseases. Moreover,in vitro it has been reported thatSchisantherin A exerts anti-inflammatory properties via down-regulating MAPKs and NF-κB signaling pathways in LPS-treated macro-phages [14]. However, there have been few in vivo studies regarding theanti-inflammatory effects of Schisantherin A on LPS-induced mouseARDS. In the present study, we evaluated the anti-inflammatory effectsof Schisantherin A on LPS-induced mouse ARDS through many kinds ofexperimental methods and explored its potential mechanism.

2. Materials and methods

2.1. Animals

Male BALB/c mice, 6–8 weeks old, were purchased from the Centerof Experimental Animals of Baiqiuen Medical College of Jilin University(Jilin, China). The mice were housed in microisolator cages suppliedwith food and water ad libitum. The temperature of the animal housewas 24 ± 1 °C and the humidity was 40–80%. All cages were washedcarefully and sterilized by autoclaving. This study was approved bythe Jilin University Animal Care and Use Committee and all animal ex-periments were performed in accordance with the National Institutesof Health guide for the Care and Use of Laboratory Animals.

2.2. Reagents

Schisantherin A (Fig. 1) was purchased from the National Institutefor the Control of Pharmaceutical and Biological Products (Beijing,China) and suspended in phosphate-buffered saline (PBS) with 0.1%dimethylsulfoxide (DMSO). LPS (Escherichia coli 055:B5) waspurchased from Sigma Chemical Co. (St. Louis, MO, USA). Dexametha-sone (Purity N 99.6%) was purchased from Changle PharmaceuticalCo. (Xinxiang, Henan, China). The myeloperoxidase (MPO) kit wasprovided by the Jiancheng Bioengineering Institute of Nanjing (Nanjing,Jiangsu, China). Mouse TNF-α, IL-6 and IL-1β enzyme-linked immuno-sorbent assay (ELISA) kits were purchased from Biolegend (CA, USA).Mouse monoclonal phospho-specific p38 antibody, mouse monoclonalphospho-specific ERK antibody, mouse monoclonal phospho-specificJNK antibody, mouse mAb Phospho-NF-κB p65, mouse mAb Phospho-IκB-α and rabbit mAb IκB-α were purchased from Cell Signaling Tech-nology Inc (Beverly, MA). HRP-conjugated goat anti-rabbit and goat-mouse antibodies were provided by GE Healthcare (Buckinghamshire,UK). All other chemicals were of reagent grade.

2.3. Experimental design

All mice were randomly divided into six groups: control group,schisantherin A (40 mg/kg) group, LPS group, schisantherin A (10, 20and 40 mg/kg) + LPS group, and Dexamethasone (DEX) + LPS group.DEX + LPS group was used as a positive control.

Fig. 1. Chemical structure of schisantherin A.

Schisantherin A and DEX (5 mg/kg) were conducted intraperitone-ally. Mice of control and LPS groups were given an equal volume ofPBS. One hour later, after slightly anesthetized with an inhalation ofdiethyl ether, mice were instilled intranasally (i.n.) 10 μg LPS in 50 μlPBS to induce lung injury [15]. Controlmicewere given 50 μl PBS insteadof LPS. All mice were alive after 7 h of LPS treatment.

2.4. Lung wet-to-dry weight ratio

The lungs were collected at 7 h after LPS challenge, blotted dry,weighed to obtain the wet weight, and then placed in an oven at 80 °Cfor 48 h to gain the dry weight. The ratio of wet lung to dry lung wascalculated to evaluate tissue edema.

2.5. Bronchoalveolar lavage fluid collection and inflammatory cell counting

Bronchoalveolar lavage fluid (BALF) was collected as described pre-viously with slight modification [16]. Briefly, after euthanasia, tracheawas exposed and intubated with a tracheal cannula. BALF collectionwas performed by douching the airways and lungs with 1 ml coldPBS repeatedly for three times. Subsequently, BALF was centrifuged at500 × g for 10 min at 4 °C. The cell-free supernatant was transferredinto a new centrifuge tube and stored at −20 °C for cytokines assays.The bottom cell pellets were resuspended in PBS for the total cell countsusing a hemacytometer and differential cell counts were carried outwith cytospins by the Wright–Giemsa staining method.

2.6. Cytokine assays

Inflammatory cytokines TNF-α, IL-1β and IL-6 in BALF were assayedby ELISA kits according to the instruction recommended by the manu-factures (Biolegend, Inc. Camino Santa Fe, Suite E San Diego, CA, USA).The optical density (OD) of the microplate was read at 450 nm.

2.7. Myeloperoxidase activity assay

Myeloperoxidase (MPO) activity indirectly represents the parenchy-mal infiltration of neutrophils and macrophages. The accumulation ofneutrophils in the lung tissue was assessed by MPO activity measuredusing test kits purchased from Nanjing jiancheng BioengineeringInstitue (China). Briefly, 7 h after LPS challenge, mice were killedunder diethyl ether anesthesia, and the right lungs were excised. Lungtissues were homogenized in cold normal PBS (lung tissues to PBS,1:9). Then the homogenates were performed according to themanufacturer's instruction. Finally, MPO activity was measured with aspectrophotometer at 450 nm.

2.8. Histopathologic evaluation of lung tissues

Histopathologic examination was performed on mice that were notsubjected to BALF collection. Lung tissues were fixed with 10% bufferedformalin, dehydrated through a series of graded alcohols, embedded inparaffin and sliced. After staining with hematoxylin and eosin (H & E),the pathological changes in the lung tissues were observed under alight microscope.

2.9. Western blot analysis

Lung tissues were harvested at 7 h after LPS challenge and frozen inliquid nitrogen immediately until homogenization. Proteins were ex-tracted from the lungs using T-PER Tissue Protein Extraction ReagentKit according to themanufacturer's instructions. Protein concentrationswere determined by BCAprotein assay kit and equal amounts of proteinwere loaded perwell on a 10% sodiumdodecyl sulphate polyacrylamidegel (SDS-PAGE). Subsequently, proteins were transferred ontopolyvinylidene difluoride membrane. The resulting membrane was

135E. Zhou et al. / International Immunopharmacology 22 (2014) 133–140

blocked with Tris-buffered saline containing 0.05% Tween-20 (TBST),supplemented with 5% skim milk (Sigma) at room temperature for 2 hon a rotary shaker and then washed three times for 10 min each inTBST. The specific primary antibody, diluted in TBST, was incubatedwith the membrane at 4 °C overnight. Subsequently, the membranewas washed with TBST followed by incubation with the peroxidase-conjugated secondary antibody at room temperature for 1 h. Finally,the proteins were detected by using an enhanced chemiluminescence(ECL) western blotting detection kit.

2.10. Statistical analysis

All of the data are expressed as the mean ± SEM. Differences be-tween mean values of normally distributed data were analyzedusing one-way ANOVA (Dunnett's t-test) and two-tailed Student'st-test. p b 0.05 was considered to be statistically significant.

3. Results

3.1. Schisantherin A reduced LPS-induced lungwet/dryweight ratio inmice

The lung wet/dry weight ratio (Fig. 2) was evaluated at 7 h after theintranasal instillation of LPS. The results showed that there were no dif-ferences between control group and schisantherin A (40 mg/kg) group(#p N 0.05). LPS caused a significant increase in lung wet/dry weightratio (#p b 0.01) compared with the control group. Schisantherin Adose-dependently decreased the lung wet/dry weight ratio (*p b 0.05)compared to those in the LPS group.

3.2. Schisantherin A decreased the inflammatory cell count in the BALF ofLPS-induced ARDS mice

To detect the amounts of leukocytes in BALF, the inflammatory cellcounting and classification were performed via the modified Wright–Giemsa staining method. As shown in Fig. 3, no differences wereobserved in the number of neutrophils, macrophages and total cellsbetween control group and Schisantherin A (40 mg/kg) group(#p N 0.05). LPS challenge significantly increased the number of neu-trophils, macrophages and total cells compared with the controlgroup (#p b 0.01). However, pretreatment with schisantherin A

Fig. 2. Effects of schisantherin A on the lungwet/dryweight ratio of LPS-induced acute lung injury1 h prior to an i.n. administration of LPS. The lung wet/dry weight ratio was determined atgroup). #P b 0.01 vs. control group, **P b 0.05 and **P b 0.01 vs. LPS group.

(40 mg/kg) significantly inhibited LPS-induced increases in the totalcells, especially neutrophils and macrophages (*p b 0.01).

3.3. Schisantherin A inhibited the production of cytokines in the BALF ofLPS-induced ARDS mice

The effect of schisantherin A on TNF-α, IL-1ß and IL-6 production(Fig. 4) was analyzed at 7 h after LPS challenge by ELISA. There wereno significant differences between control group and schisantherin Agroup (#p N 0.05). LPS challenge caused a significant increase in TNF-α, IL-1ß and IL-6 in the lung tissue when compared with the controlgroup (#p b 0.01). Schisantherin A in a dose-dependent manner signif-icantly reduced the levels of TNF-α, IL-1ß and IL-6 in LPS-induced ARDSmice.

3.4. Schisantherin A supressed MPO activity

The MPO activity (Fig. 5) was determined to assess the neutrophilaccumulation within pulmonary tissues. Schisantherin A (40 mg/kg)alone had no obvious effects onMPO activity comparedwith the controlgroup (#p N 0.05). LPS significantly increased the MPO activity in thepulmonary tissue (#p b 0.01). However, the increase was obviouslyabated by schisantherin A (*p b 0.01).

3.5. Schisantherin A ameliorated pulmonary histopathological changes

As shown in Fig. 6, pulmonary tissue of the control group andschisantherinA (40 mg/kg) groupdisplayedno abnormal histopatholog-ical changes (Fig. 6A). LPS challenge caused significant pathologic chang-es, such as inflammatory cell infiltration, thickening of the alveolar walland pulmonary congestion (Fig. 6B). However, those pathologic changeswere significantly improved by the pretreatment of schisantherin A(Fig. 6D-F) and DEX (Fig. 6C).

3.6. Schisantherin A supressed NF-κB and MAPK signaling pathways inLPS-induced ARDS mice

NF-κB and MAPK signaling pathways play key roles in LPS-inducedARDS pathogenesis. Western blot analysis showed that LPS challengemarkedly increased the phosphorlation of p65 and degradation of IκB-α(Fig. 7). Moreover, MAPKs phosphorylation was significantly up-

mice.Micewere given an intraperitoneal injection of schisantherinA (10, 20 and 40 mg/kg)7 h after LPS challenge. The values presented are the means ± S.E.M. (n = 4–6 in each

Fig. 3. Effects of schisantherin A on the number of total cells, neutrophils, and macrophages in the BALF of LPS-induced ARDS mice. Mice were given an intraperitoneal injection ofschisantherin A (10, 20 and 40 mg/kg) 1 h prior to an i.n. administration of LPS. BALF was collected at 7 h after LPS administration to measure the number of total cells (A), neutrophils(B), and macrophage (C). The values presented are the mean ± SEM (n = 4–6 in each group). #P b 0.01 vs. control group, **P b 0.05 and **P b 0.01 vs. LPS group.

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regulated by LPS, including ERK, p38 and JNK pathways (Fig. 8).Whereas,pretreatment with schisantherin A inhibited the phosphorlation ofp65, ERK, p38, JNK, and the degradation of IκB-α in a dose-dependentmanner.

4. Discussion

ARDS is a complicated clinical syndrome, and its progression is at-tributed to numerous etiologies [17]. Though several candidate thera-pies have been applied to ameliorate ARDS, there are still few effectivemeasures or specific medicines to treat it. Intranasal instillation of LPS

can induce an ideal experimental model of ARDS, which is well usedfor preliminary pharmacological studies of new drugs or other thera-peutic agents, without causing systemic inflammation. Recently,schisantherin A isolated from the fruit of Schisandra sphenanthera hasbeen shown to exert anti-inflammatory effects through NF-κB andMAPK signaling pathways in LPS-treated RAW 264.7 cells [14]. In thepresent study, we examined the effect of schisantherin A on LPS-induced mouse ARDS. Our results revealed that pretreatment withschisantherin A reduced thewet/dry weight ratio, decreased inflamma-tory cell count, inhibited pro-inflammatory cytokine production,ameliorated the histopathological changes of pulmonary tissue, and

Fig. 4. Effects of schisantherin A on the production of inflammatory cytokine TNF-α, IL-1ß, and IL-6 in the BALF of LPS-induced ARDSmice. Mice were given an intraperitoneal injection ofschisantherin A (10, 20 and 40 mg/kg) 1 h prior to an i.n. administration of LPS. BALFwas collected at 7 h following LPS challenge to analyze the inflammatory cytokines TNF-α (A), IL-1ß(B), and IL-6 (C). The values presented are mean ± SEM (n = 6 in each group). #P b 0.01 vs. control group, **P b 0.05 and **P b 0.01 vs. LPS group.

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blocked the activation of NF-κB and MAPKs signaling pathways. Inaddition, schisantherin A (40 mg/kg) alone had no significant negativeeffects compared to control group. All data indicated that schisantherinA exerts potent anti-inflammatory effects on mouse ARDS inducedby LPS.

Tissue edema was firstly assessed by the wet/dry weight ratios oflungs, for it is a representative symptom of ARDS. The result showedthat pretreatment with schisantherin A markedly attenuated pulmo-nary edema from the decreased wet/dry weight ratio compared withLPS group. ARDS is characterized by the infiltration of neutrophils inthe lung, displaying increased MPO activity [18]. MPO is a marker ofneutrophil function and activation that can be used as a surrogate forthe level of neutrophilic inflammation and activation. A variety of

stimuli induce PMN migration into the lung, such as LPS. Neutrophiltrafficking into the vascular compartment of the lung, also known as“margination”, and into the bronchoalveolar space has been studiedextensively in various models of acute lung injury [19,20]. Neutrophilsexcrete MPO into the extracellular medium, resulting in the generationof MPO-derived oxidants and leading to tissue damage [21]. Our datashowed that schisantherin A significantly reduced the LPS-induced in-creased MPO activity. Furthermore,the number of total cells, neutro-phils, and macrophages was markedly decreased by the pretreatmentof schisantherin A in LPS-inducedmouse ARDS. Additionally, histopath-ological changes of pulmonary tissue induced by LPS were significantlyimproved by pretreatment with schisantherin A. These results sug-gested that the protective effects of schisantherin A on LPS-induced

Fig. 5. Effects of schisantherin A on MPO activity in lung tissues of LPS-induced ARDS. Mice were given an intraperitoneal injection of schisantherin A (10, 20 and 40 mg/kg) 1 h prior toan i.n. administration of LPS. MPO activity was determined at 7 h after LPS administration. The values presented are themean± SEM (n= 4–6 in each group). #P b 0.01 vs. control group,**P b 0.05 and **P b 0.01 vs. LPS group.

138 E. Zhou et al. / International Immunopharmacology 22 (2014) 133–140

mouseARDSmaydependon its inhibition of inflammatorymediators inthe lung.

Some cytokines triggered by LPS, such as TNF-α,and IL-1β, stimulatevascular endothelial cells to induce the upregulation of vascularendothelial intercellular adhesion molecule expression, leading to en-dothelial cell and leukocyte adhesion, leukocytemigration, and granulo-cyte degranulation as well as capillary permeability increases andmigration to inflammatory sites [22]. During inflammation, neutrophilsurvival or function was prolonged by the expression of various pro-inflammatory cytokines as well as the formation of neutrophil extracel-lular traps [23]. The pro-inflammatory cytokines TNF-α, IL-6, and IL-1β

Fig. 6.Effects of schisantherinA onhistopathological changes in lung tissues in LPS-inducedARDS1 h prior to an i.n. administration of LPS. Lungs (n= 4–6) from each experimental group werechanges of lung obtained from mice of different groups. A: Control group, B: SchisantherinF: LPS + schisantherin A (20 mg/kg) group G: LPS + schisantherin A (40 mg/kg) group (Hem

play an important role in many inflammatory diseases, inluding ARDS[24]. TNF-α generated by monocytes/macrophages can trigger an in-flammatory cascade, cause damage to the vascular endothelial cellsand induce other cells to produce additional factors, such as IL-6[25]. IL-1β plays a key role in the progression multiple organ failure inLPS-induced endotoxic shock. IL-6 is also an important cytokinemedia-tor which is induced to release by other cytokines, especially TNF-α[26]. In the present study, the levels of TNF-α, IL-6 and IL-1β in BALFweremarkedly decreased by schisantherin A. Accordingly, schisantherinA may protect the lung through the inhibition of inflammatorycytokines.

mice.Micewere given a intraperitoneal injection of schisantherinA (10, 20 and40 mg/kg)processed for histological evaluation at 7 h after LPS challenge. Representative histologicalA (40 mg/kg) group, C: LPS group, D: LPS + DEX group, E: LPS + (10 mg/kg) group,atoxylin and eosin staining, magnification 200×).

Fig. 7. Schisantherin A inhibited LPS-induced activation of NF-κB with western blotting. #P b 0.01 vs. control group, *P b 0.05 and **P b 0.01 vs. LPS group.

139E. Zhou et al. / International Immunopharmacology 22 (2014) 133–140

NF-κB is implicated in the regulation of inflammatory and immuneresponses which plays an important role in the development of ARDS.MAPKs also have a critical role in the regulation of cell growth, differen-tiation, and control of cellular responses to cytokines and stress [27]. Inresting cells, NF-κB family members are sequestered in the cytoplasm,for they are bound to their inhibitors which belong to the IκB family.Upon cell activation, IκB proteins are phosphorylated and degraded by

Fig. 8. Schisantherin A inhibited LPS-induced activation of p38, ERK and JNK with we

the proteasome. Subsequently, NF-κB could enter the nucleus andbind to the DNA, resulting in promoting transcription [28]. NF-κB andMAPKs signaling pathways can be activated by LPS to regulate the re-lease of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β[29]. Our results showed that schisantherin A restrained LPS-inducedIκB-α degradation and NF-κB p65 activation in mice. Moreover, thephosphorylation of ERK, p38 and JNK were blocked by pretreatment

stern blotting. #P b 0.01 vs. control group, *P b 0.05 and **P b 0.01 vs. LPS group.

140 E. Zhou et al. / International Immunopharmacology 22 (2014) 133–140

with schisantherin A. These findings suggest that schisantherin A mayexert anti-inflammatory effects on LPS-induced mouse ARDS throughinhibiting the activation of NF-κB and MAPKs signaling pathways.

In conclusion, our results demonstrated that pretreatment withschisantherin A develops a potent protective effect on LPS-inducedmouse ARDS which may be associated with its inhibition of NF-κB andMAPKs signaling pathways activation. Accordingly, We believe thatschisantherin A is a promising agent for ARDS, whichmay be a potentialalternative in the prophylaxis and treatment of ARDS. However, furtherand more comprehensive studies are needed before schisantherin A isused for the clinical practice.

Acknowledgments

This work was supported by a grant from the National NaturalScience Foundation of China (No. 30972225, 30771596).

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