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International Immunopharmacology 22 (2014) 133140
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Schisantherin A protects lipopolysaccharide-induced acute respiratorydistress syndrome in mice through inhibiting NF-B 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: firstname.lastname@example.org (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 ta 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-B)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- (TNF-),interleukin-6 (IL-6) and interleukin-1 (IL-1) in the BALF. In addition, the phosphorylation of nuclear transcriptionfactor-kappaB (NF-B) p65, inhibitory kappa B alpha (IB-), 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.
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 . Com-mon causes of ARDS are sepsis, pneumonia, trauma, aspiration pneumo-nia, pancreatitis, and so on . Recent data indicate approximately190,000 cases of ARDS in the United States each year, with an associated74,500 death . 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 . 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 . LPS, an important component of the outermembrane of the Gram-negative bacteria has been widely recognizedas a clinically relevant model inducer of ARDS , 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 . 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-B andMAPKs signaling pathways play important roles in the development ofARDS [5,12,13]. Treatments aimed at inhibiting NF-B and MAPKssignaling pathways may have potential therapeutic advantages
134 E. Zhou et al. / International Immunopharmacology 22 (2014) 133140
for inflammatory diseases. Moreover,in vitro it has been reported thatSchisantherin A exerts anti-inflammatory properties via down-regulating MAPKs and NF-B signaling pathways in LPS-treated macro-phages . 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
Male BALB/c mice, 68 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 4080%. 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.
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-, IL-6 and IL-1 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-B p65, mouse mAb Phospho-IB- and rabbit mAb IB- 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 g LPS in 50 lPBS to induce lung injury . Controlmicewere given 50 l 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 . 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 WrightGiemsa staining method.
2.6. Cytokine assays
Inflammatory cytokines TNF-, IL-1 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 mic