International Immunopharmacology 22 (2014) 311–317 Contents lists available at ScienceDirect International Immunopharmacology j ourna l homepage: www.e lsev ie r .com/ locate / in t imp Protective effect of sanguinarine on LPS-induced endotoxic shock inmice and its effect on LPS-induced COX-2 expression and COX-2 associated PGE2 release from peritoneal macrophages Weifeng Li, Huani Li, Qingli Mu, Hailin Zhang, Huan Yao, Jiaoshe Li, Xiaofeng Niu ⁎ School of Pharmacy, Xi'an Jiaotong University, Xi'an 710061, PR China ⁎ Corresponding author at: School of Pharmacy, Xi'a Western Yanta Road, Xi'an City, Shaanxi Province 71 82655139; fax: +86 29 82655138. E-mail addresses:
[email protected] (W. Li), niuxf@ http://dx.doi.org/10.1016/j.intimp.2014.07.017 1567-5769/© 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 9 April 2014 Received in revised form 3 July 2014 Accepted 15 July 2014 Available online 22 July 2014 Keywords: Sanguinarine Endotoxic shock Lipopolysaccharide Cyclooxygenase-2 Prostaglandin (PG) E2 Peritoneal macrophages The quaternary ammonium salt, sanguinarine (SG) was reported to possess widespread anti-microbial and anti- inflammatory effects in experimental animals and it has been used to treat many inflammatory diseases. The aim of this study was to evaluate the anti-inflammatory effect and the possible mechanisms underlying the anti- inflammatory activity of SG. Experimentally-induced mice ES model and LPS-induced peritoneal macrophages were used to examine the anti-inflammatory function of SG. In this study, SG pretreatment significantly in- creased the survival rate of mice from 25% to 58%, 75% and 91% respectively. The production of PGE2 in BALF, the lungMPO activity and the (W/D)weight ratios were also markedly reduced. In addition, immunohistochem- ical analysis showed that the expression of COX-2was significantly suppressed in vivo.We also evaluated the ef- fect of SG in LPS-induced peritoneal macrophages to clarify the possible mechanism. The data indicated that SG greatly inhibited theproduction of PGE2, and it also decreased COX-2 protein expression,without affecting COX-1 expression, in LPS-stimulated peritoneal macrophages. Taken all together, SG potently protected against LPS- induced ES, and our results suggest that the possible mechanism may be relevant to COX-2 regulation. n Jiaotong University, No. 76 0061, PR China. Tel.: +86 29 mail.xjtu.edu.cn (X. Niu). © 2014 Elsevier B.V. All rights reserved. 1. Introduction Endotoxic shock is a septic shock that results from infection with Gram-negative bacteria [1]. Septic shock is a serious clinical problem with highmortality [2]. According to the study, there are different phases in the process of endotoxic shock. In the early stage, pro-inflammatory mediators that release from local inflammation will reduce tissue injury. Then leukocytes, lymphocytes and platelets transfer to the infected areas. Systemicpathological changes contain endothelial damage, increasedmi- crovascular permeability, local blood flow reduction, platelet aggregation and ischemia–reperfusion injury. Excessive inflammatory response may lead to tissue injury. Finally, the cumulative inflammatory response results in multiple organ injury and it eventually causes the death [3,4]. LPS, the major constituent of the Gram-negative bacterial cell wall, can induce experimental endotoxemia, the use of which has become a valu- able and reproducible experimental model for endotoxic shock [5]. Macrophages participate in host defense, immunity and inflammatory responses. LPS exerts its profound effect on the host by activating LPS-sensitive pro-inflammatory cells, mainly activated macro- phages, to release various cytokines and other pro-inflammatory molecules including platelet activating factor, prostaglandins, enzymes, and free radicals [6–9]. In macrophages, LPS alone is one of the best-characterized stimuli to induce the transcription of genes encoding pro-inflammatory proteins, which result in cyto- kine release and synthesis of enzymes, such as cyclooxygenase-2 (COX-2) [10]. Cyclooxygenase-2 (COX-2) plays a central role in themodulation of the inflammatory process by stimulating the pro- duction of prostaglandin (PG) E2. There are two COX isoforms: one is the constitutive enzyme (COX-1), constitutively expressed in nearly all tissues and mammalian cells, and provides PGs to main- tain physiological functions, and the other is COX-2, the inducible isoform induced by pro-inflammatory stimuli, such as growth fac- tors, cytokines, and endotoxins [11]. Sanguinarine (13-methyl-[1,3]benzodioxo[5,6-c]-1,3-dioxolo[4,5,1] phen-anthrdinium, SG) is an alkaloid derived from the roots of plants Sanguinaria canadensis and other poppy fumaria species [12]. There is a positivemoiety in the aromatic ring of themolecule (Fig. 1). This alka- loid has been shown antioxidant, anti-microbial and anti-inflammatory properties and used as a naturo-pathic therapy for the treatment of infections and functions as a pain reliever, expectorant, sedative, and emetic [13,14]. SG has a broad spectrum of anti-inflammatory effects. SG can potently decrease the expression of inflammatory mediators both in vivo (acute and chronic inflammatory models) and in vitro (LPS-stimulated peritoneal macrophages). In addition, SG inhibited the activation of MAPK, which regulates inflammatory mediator http://crossmark.crossref.org/dialog/?doi=10.1016/j.intimp.2014.07.017&domain=pdf http://dx.doi.org/10.1016/j.intimp.2014.07.017 mailto:
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[email protected] http://dx.doi.org/10.1016/j.intimp.2014.07.017 http://www.sciencedirect.com/science/journal/15675769 Fig. 1. Chemical structure of SG. 312 W. Li et al. / International Immunopharmacology 22 (2014) 311–317 synthesis and release in vitro [15]. In addition, SG treatment mediates beneficial effects in a colitis model of mice induced by the acetic acid [16]. A number of studies have addressed the therapeutic potential of SG. Unfortunately, to date, the protective role of SG in ES is not well characterized. In this study, we investigated the effects of SG on LPS-induced ES in vivo. Additionally, we examined the effect of SG on LPS-stimulated peritoneal macrophages. Our findings provide experimental evidence that SG serves as a possible treatment for patients with ES. 2. Materials and methods 2.1. Reagents SG was purchased from Xi'an Honson Biotechnology Co., Ltd. (Shanxi, China) and identified by the Pharmacognosy Laboratory, School of Medicine, Xi'an Jiaotong University (Xi'an, China). LPS was purchased from Sigma (St. Louis, MO, USA). Dexamethasone (DEX), as a positive control, was purchased from Xi'an Lijun Phar- maceutical Company Limited (Shanxi, China). COX-2 kinase inhibitor (NS398) was from Beyotime Institute of Biotechnology (Jiangsu, China). RPMI-1640 was purchased from Gibco (Gibco-BRL, Gaithersburg, MD, USA). Sodium thioglycolate was obtained from Sinopharm Chemical Re- agent Co., Ltd. (Shanghai, China). The enzyme-linked immunosorbent assay (ELISA) kit for mouse PGE2 was purchased from R&D Systems (Minneapolis, MN, USA). The kit for biochemical analysis of MPO was purchased from Jiancheng Bioengineering Institute (Nanjing, China). Histostain-Plus kits, DAB (3, 3′-diaminobenzidine) staining kit and rabbit anti-cyclooxygenase-2 (COX-2) were purchased from Beijing Biosynthe- sis Biotechnology Co., Ltd. Anti-COX-1 (UniProt ID: P23219) and anti- COX-2 (UniProt ID: P35354) antibody were from Epitomics Inc. (an Abcam Company, USA). Anti-GAPDH antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Polyvinylidenefluo- ride (PVDF) membranes were from Pall Gelman Laboratory (Ann Arbor, MI, USA). Other reagentswere of commercially available analytical grade. 2.2. Animals Male (25–30 g) Kunming mice were purchased from the Experi- mental Animal Center, Xi'an Jiaotong University (Xi'an, China). They were housed in a temperature (23 ± 3 °C) and humidity (50 ± 10%) controlled room. The animals were housed under standard conditions with 12 h light and dark cycles and were acclimated to housing condi- tions for at least one week before the experiments. All experimental procedures utilizing animals were performed in accordance with the guidelines approved by the Institutional Animal Care and Ethical Com- mittee of National Institute of the Xi'an Jiaotong University. 2.3. Experimentally-induced endotoxic shock All mice were randomly divided into six groups, each consisting of 12 animals: control group, LPS group, SG (1, 5, and 10 mg/kg) treatment group, andDEX group. The SG treatment group and theDEX groupwere given SG at various doses (1, 5, and 10 mg/kg), DEX (5 mg/kg) at 12 h and 1 h prior to the injection of LPS (dissolved in saline, 17 mg/kg) respectively. Mice from the control and LPS groups received an equal volume of saline instead of SG or DEX. SG was given intragastrically, while DEX (5 mg/kg) was administrated intraperitoneally as a positive control. Six hours after induction,micewere euthanized through the ve- nous sinus. Bronchoalveolar lavage fluid (BALF) was obtained by gently washing the left lung cavities three times with a total of 3 mL of saline. BALF was centrifuged at 4000 ×g, 4 °C for 10 min and the supernatants were kept at−20 °C until use. The right lung tissues were immediately excised: the upper lobe was used for MPO assay, the middle lobe was excised for analysis of lung W/D weight ratio, and the lower lobe was used for histopathological study and immunohistochemical evaluation. Survival was monitored continuously for three days. 2.4. ELISA assay for detecting PGE2 level in BALF Concentration of PGE2 in BALFwasmeasured by using commercially available ELISA kit following the manufacturer's direction. It was mea- sured by using an antibody raised against mouse PGE2 and the absor- bance was read at 450 nm. The results were expressed as ng/mL serum. 2.5. MPO assay MPO is an enzyme, which was found primarily in neutrophil azurophilic granules, and has been used intensively as a biochemical marker for granulocyte infiltration into various tissues [17]. In the study, the upper lobe of the right lung tissue was removed at 6 h after LPS injection andMPO activity were measured. Briefly, lung tissue sam- ples were weighed and placed into buffer application solution at a ratio of 50 mg per 950 μL. Samples were homogenized and centrifuged at 3000 ×g, 4 °C for 10 min. The supernatants were analyzed for MPO ac- tivity byMPO detection kit according to the manufacturer's instruction. MPO activity was assessed with an ultraviolet spectrophotometer (756PC Shanghai Spectrum Instrument Co. Ltd., China) according to the absorbance measured at 460 nm. MPO activity was expressed as unit per gram of lung tissue. 2.6. Lung W/D weight ratio As an index of lung edema, the amount of extravascular lung water was evaluated. The middle lobe of the right lung was removed for anal- ysis of lung W/D weight ratio. The lung tissue was excised, weighed to obtain the “wet weight”, and then dried to constant weight at 80 °C for 24 h and weighed again (dry weight). The ratios of lung wet/dry weight were calculated by dividing the wet weight by the dry weight. 2.7. Histopathological study The lower lobe of the right lungs harvested fromall groups ofmice at 6 h after LPS injection were fixed in Bouin's fluid for 48 h. After fixation, the fixed organwere dehydrated in graded ethanol and transparentized in xylene, then embedded in paraffin. Five-micro-meter sections were cut by microtome and were stained with hematoxylin and eosin (HE) using a standard protocol for light microscopy examination. uniprotkb:P23219 uniprotkb:P35354 313W. Li et al. / International Immunopharmacology 22 (2014) 311–317 2.8. Immunohistochemical study For COX-2 immunohistochemistry evaluation, the paraffin sections were deparaffinized in xylene and rehydrated in graded ethanol, and immersed in 0.1M citrate buffer (pH 6.0) for antigen retrieval bymicro- wave. After they were cooled, these sections were washed thrice in PBS and incubated with 3% hydrogen peroxide to quench the endogenous peroxidase activity at 37 °C for 10 min. All the sections were sealed with goat serum at 37 °C for 20 min, and then were incubated with a primary antibody for COX-2 at a dilution of 1:200 in PBS (v/v) overnight at 4 °C. On the following day, the sections were incubated with the rab- bit anti-mouse secondary antibody at 37 °C for 30min and then stained with 3, 3′-diaminobenzidine solution in darkness at room temperature for 5 min. A peroxidase labeled secondary antibody and DAB were used for antibody detection and coloration respectively. The sections were counterstained with hematoxylin, and then they were dehydrated with an increasing concentration of ethanol. In the end, the sections weremountedwithneutral gum. The resultwas observed through ami- croscope under 400-fold magnification. 2.9. Preparation of peritoneal macrophages Female Kunmingmice were injected intraperitoneally with 1% sodi- um thioglycolate (1.5 mL/each) for seven days, then they were killed using cervical dislocation, and the peritonealmacrophageswere obtain- ed by lavaging the peritoneal cavity with 5 mL phosphate buffered saline. Lavage fluid was centrifuged at 1000 ×g for 10 min to pellet cells and the supernatant was discarded. After they were washed three times by phosphate buffered saline (PBS in mM: NaCl 137, KCl 2.7, KH2PO4 1.5, Na2HPO4·12H2O 9.8, PH 7.4), the cells were suspended in RPMI-1640 supplemented with 10% fetal bovine serum and maintained in a humidified incubator at 37 °C with 5% CO2. 2.10. ELISA for detecting PGE2 production in vitro Cells at a density of 5 × 105 cells/mL were seeded in a 24-well plate and cultured for two days. Peritoneal macrophages were pretreated with SG (1 × 10−4 to 1 μg/mL) or NS398 (5 μg/mL) (The specific COX- 2 inhibitor) for 24 h. Then the peritoneal macrophages were exposed to 10 μg/mL LPS or not for 12 h [15]. Levels of PGE2 from cell superna- tants were analyzed using a PGE2 enzyme immunometric ELISA kit fol- lowing the manufacturer's direction. 2.11. Western blotting Peritonealmacrophages (1 × 106 cells/mL)were pretreatedwith var- ious concentrations of SG (final concentrations = 1 × 10−4, 1 × 10−2, 1 μg/mL) or NS398 (5 μg/mL) for 24 h before exposure to 10 μg/mL LPS for 12 h. Cells were washed twice with PBS and total cell extracts were prepared by resuspending the cells in lysis buffer (50 mM Tris–HCl, pH 7.5; 150mMNaCl; 1mM EDTA; 20mMNaF; 0.5% NP-40; and 1% Tri- ton X-100) containing a protease inhibitor and phosphatase inhibitor cocktail. The protein concentrations were quantified using the bicinchoninic acid method (BCA protein determination kit from Pierce, distributed by KFC Chemikalien, Munchen, Germany) following the manufacturer's directions. Before electrophoresis, bromophenol blue and dithiothreitol (DTT, final concentration c = 10 mM) were added to each sample. 30 μL protein from each sample was run on a 10% sodium dodecylsulfate-polyacylamide gel and was transferred onto PVDF mem- branes. The membranes were blocked with 5% skim milk in TBST buffer (20mMTris, 500mMNaCl, pH7.5 and1%Tween20) for 2 h at room tem- perature, and then incubated with primary antibodies against GAPDH (diluted 1:1000 dilutions in 5% skim milk), COX-1 and COX-2 (diluted 1:10,000 in TBST) overnight at 4 °C. After they were washed three times with TBST, the blots were incubated with anti-rabbit horseradish peroxidase-conjugated antibody (1:40,000 dilutions in TBST), and washed again three timeswith TBST. Immunoreactive bandswere devel- oped using an enhanced chemiluminescence system (GE Healthcare, Lit- tle Chalfont, Buckinghamshire, UK). All Western blots were repeated three times. 2.12. Statistical analysis Data shown represent the mean and standard error. The difference betweenmean values of two groupswere assessed using one-way anal- ysis of variance (ANOVA) with GraphPad Prism software, followed by Dunnett' t test. Comparisons among survival rates of the groups were made using a Kaplan–Meier curve and log-rank test with SPSS software. P values less than 0.05 were considered significant. 3. Results 3.1. Effect of SG on the survival of mice of the LPS-induced ES Themicewere treatedwith SG (1, 5, and 10 mg/kg) or DEX (5 mg/kg) at 12 h and 1 h before LPS challenge and mortality was observed. As shown in Fig. 2, 75% of mice died from endotoxin shock during 72 h in the LPS group. However, pretreatment with SG before LPS ad- ministration significantly increased the survival rate of mice within 72 h in a dose-dependent manner. SG, at doses of 1, 5 or 10 mg/kg, raised the survival rate from 25% to 58% (P b 0.05), 75% (P b 0.01) and 91% (P b 0.001), respectively, and similarly, the positive con- trol (DEX) led to a high survival rate, meaning 100% (P b 0.001). 3.2. Effect of SG on PGE2 levels in BALF We assessed the level of PGE2 in BALF and the result was shown in Fig. 3. Compared with the control group (0.7 ng/mL), the PGE2 level was significantly increased after LPS injection in the LPS group (2.47 ng/mL, P b 0.001). However, pretreated with sanguinarine signifi- cantly decreased the observed increase in a dose-dependent manner compared to the group treated only with LPS (2.01 ng/mL, P b 0.05, 1.48 ng/mL, P b 0.01 and 1.08 ng/mL, P b 0.001, respectively) . The DEX group also showed a lower level of PGE2 production (0.72 ng/mL, P b 0.001) compared to the LPS group. 3.3. Effect of SG onMPO activity in lung tissues of mice with LPS-induced ES We assessed the MPO level in the lung tissue and Fig. 4 showed the results. The LPS group (0.98 U/g, P b 0.001) caused amarked increase in the MPO activity compared with the control group. However, this in- crease was apparently reduced by treating with SG 1, 5, and 10 mg/kg (0.83 U/g, P b 0.05, 0.74 U/g, P b 0.01, and 0.68 U/g, P b 0.01 respective- ly) in a dose-dependent manner. Similarly, the DEX group also signifi- cantly reduced the MPO level (0.46 U/g, P b 0.001). 3.4. Effect of SG on lung wet/dry weight ratio in mice with LPS-induced ES Six hours after LPS injection, lung tissues were evaluated for edema by measuring the lung water content. The lung wet/dry (W/D) weight ratios as an index of lung edema is shown in Fig. 5. The W/D weight ratio was significantly decreased in treatment groups receiving 5 and 10 mg/kg SG (4.75, P b 0.05 and 4.21, P b 0.01, respectively) than in the LPS group (5.92). No significant difference was found between the LPS group and the SG treatment group (1 mg/kg). The DEX group also showed a significant decrease in W/D weight ratio (3.92, P b 0.01). 3.5. Effect of SG on histological changes in lung tissues from mice with LPS-induced ES To evaluate the histological changes in the lung tissue from mice with ES following treatment with SG, lung sections obtained 6 h after Fig. 2.Effect of SGpretreatment on survival rate ofmicewith LPS-inducedES. Survival rate of allmicewasmonitoredwithin 72h (n=12 for each group). Values are expressed asmean± S.E.M., *P b 0.05, **P b 0.01 and ***P b 0.001 compared to the LPS group. 314 W. Li et al. / International Immunopharmacology 22 (2014) 311–317 injection of LPS were subjected to H&E staining and observed by a mi- croscope. Lung tissues showed normal appearancewithout obvious his- topathological changes in the control group from Fig. 6A. In contrast, in the tissues of the LPS group, obvious infiltration of inflammatory cells, widespread alveolar wall thickness, demolished structure of pulmonary alveoli, alveolus collapse and severe hemorrhage were observed (Fig. 6B). However, the destruction changes of lung tissue, especially the inflammatory cell infiltration, were significantly attenuated in SG (1, 5, and 10 mg/kg) treatment group compared with the LPS group (Fig. 6C–E). The DEX group also had comparatively better protection of lung tissue with little inflammatory reaction (Fig. 6F). 3.6. Effect of SG on the expression of COX-2 in lung tissues of mice with LPS- induced ES COX-2 protein expressionwas also evaluated by immunohistochem- istry to investigate the effect of SG. As shown in Fig. 7, in the control group, we scarcely found the COX-2 specific immunolabeling in the sur- face epithelium and mononuclear cells of lamina propria of mucosa (Fig. 7A). However, the LPS group significantly increased the expression of COX-2, in which large areas of COX-2 positive cells were observed (Fig. 7B). Treatmentwith SG significantly reduced the COX-2 expression and the DEX group showed the similar effects (Fig. 7C–F). The results in Fig. 7G were the semiquantitative analysis of COX-2 expression. Fig. 3. Effect of SG pretreatment on the concentrations of PGE2 in BALF of mice with ES. Data are presented as mean ± S.E.M. (n = 12 in each group). ###P b 0.001 compared to the normal group and *P b 0.05, **P b 0.01, ***P b 0.001 when compared with the LPS group. 3.7. Effect of SG on PGE2 production in LPS-stimulated peritoneal macrophages Peritoneal macrophages were pretreated with different concentra- tions of SG (final concentrations = 1 × 10−4, 1 × 10−2, 1 μg/mL) and then exposed to 10 μg/mL LPS or not for 12 h to examine whether or not SG regulates PGE2 production. As shown in Fig. 8, peritoneal macrophages challenged by 10 μg/mL LPS can significantly induce the expression of PGE2 (P b 0.001). It was also observed that pretreated with SG exhibited a dose-dependent decrease in PGE2 production in LPS-stimulated peritoneal macrophages. 3.8. SG inhibit COX-2 expression in LPS-stimulated peritoneal macrophages As shown in Fig. 9A, LPS can obviously increase the COX-2 protein expression level in peritoneal macrophages (P b 0.001). Pretreatment of LPS-stimulated peritoneal macrophages at 1 × 10−4 to 1 μg/mL con- centrations of SG caused a significant decrease in COX-2 protein expres- sion in a concentration-dependent manner and the specific inhibitor (NS398, 5 μg/mL) showed the similar effects, while the Western blot analysis showed that COX-1 protein expression remained unchanged under the same experiment conditions. Fig. 9B showed COX-2 protein levels (normalized toGAPDH)gradually decreasedwhen the concentra- tion of SG increased, whereas the COX-1 protein level had no statistical changes. Thus, the results indicate that SG can control the COX-2 at the protein level. Fig. 4. Effect of SG pretreatment onMPO activity in lung tissues of mice with LPS-induced ES. The values presented are themean± S.E.M. (n=12 in each group). ###P b 0.001 com- pared to the normal group; *P b 0.05, **P b 0.01, ***P b 0.001when comparedwith the LPS group. Fig. 5. Effect of SG pretreatment on the lung edema ofmice with LPS-induced ES. After LPS administration, pretreatment with SG decreased wet/dry ratios markedly. Values are expressed asmean± S.E.M. (n=12 in each group). ###P b 0.001 compared to the normal group; *P b 0.05, **P b 0.01, ***P b 0.001 vs. LPS group. 315W. Li et al. / International Immunopharmacology 22 (2014) 311–317 4. Discussion SG has been used as a naturo-pathic therapy for the treatment of in- jections and functions as a pain reliever, expectorant, sedative, and emetic [13]. It also has showed the anti-inflammatory effects in some animal model, such as acetic acid-induced colitis [16]. However, the ef- fect of SG on LPS-induced ES is poorly understood. The present study was particularly focused on studying the effect of SG on LPS induced by experimental ES, which was considered a model validated to find drugs potentially anti-inflammatory activities in this disease.We exam- ined acute intraperitoneal toxicity associated with SG and identified 10mg SG/kg body weight at the highest dose in the LPS-induced endo- toxic shock model. We demonstrated for the first time that administra- tion of SG can effectively provide protection in LPS-induced ES. In this model, lethality, lung pathological changes, lung edema, andMPO activ- ity in the lung tissue were significantly attenuated in mice treated with SG at the doses of 1, 5 or 10 mg/kg. Further, we also observed that SG was able to decrease the PGE2 level and COX-2 protein expression in the lung tissue and LPS-stimulated peritoneal macrophages. All of these findings suggest that SG is a protective in the LPS-induced ES model. Fig. 6. Effect of SG pretreatment on the pulmonary histopathological changes of mice with LPS group, alveolar wall thickness, hemorrhage, alveolus collapse and obvious inflammatory cells + SG (10 mg/kg) group, minor histopathological changes compared with LPS group. (F) LPS + LPS,which is an integral structure component on the outermembrane of Gram-negative bacteria, can be released from the bacteria during cell division, cell death [18]. LPS challenge results in pathophysiologic changes similar to sepsis shock in human and many investigators have used the model to study the pathogenesis of sepsis syndrome. Injection of 17 mg/kg LPS intomice results in 75% lethality and sepsis-like symptoms, such as piloerection, hypothermia, shivering, tachycardia, and lethargy. In this study, SG showed marked protection of mice mortality from ES in- duced by LPS in a dose-dependent manner. Moreover, pretreatment with SG also exhibited some sepsis-like symptoms, which were milder than those treatedwith LPS alone. These results suggest that SG has a po- tential to be used for sepsis therapy. Bacterial LPS in the bloodstream induces large amounts of inflam- matory mediators such as TNF-α, NO and PGE2, which are thought to contribute to the LPS-induced symptoms of septic shock and mortality [19]. In LPS-induced endotoxic shock, TNF-α is not only the first re- leased inflammatory cytokine, but also leads to rapid production of other inflammatory cytokine [20]. TNF-α is the cardinal inflammatory mediator associated with various inflammatory diseases, such as bacte- rial sepsis [21]. This cytokine is produced in the early stage of inflamma- tory stimuli and can induce the production of a variety of inflammatory mediators contributing to inflammation amplification [22]. Therefore, TNF-α plays a central role in endotoxic shock [23]. Evidence has indicat- ed that TNF-αmay play a role in the progression of multiple organ fail- ure in septic shock [24]. According to Fan et al. [15], serum TNF-α levels rose dramatically after LPS injection, peaked at 1 h, and then declined gradually. SG pretreatment significantly decreased the increase in serum TNF-α at 1 h, 3 h, 6 h in a dose-dependentmanner. These results agreed with the above study. Neutrophils are the first cells to be recruited to sites of infection when an inflammatory response is initiated. Their targets contain bacte- ria, fungi, viruses, virally infected cells, and tumor cells. Although neu- trophils have beneficial actions in eliminating microbial infections, excessive neutrophil activation, together with the release of cytokines and other pro-inflammatory mediators, leads to tissue injury and con- tributes to the development of organ injury [25].MPO is amajor constit- uent of neutrophil cytoplasmic granules. Therefore, the total activity of MPO is a direct measure of neutrophil sequestration in the tissue [26]. As expected, pretreatment of SG significantly reduced the MPO activity in the lung tissue after LPS administration. Meanwhile, histopathologic study also indicated that SG pretreatment markedly attenuated the -induced ES (original magnification ×400). (A) Control group, normal structure. (B) LPS infiltration. (C) LPS + SG (1 mg/kg) group, (D) LPS + SG (5 mg/kg) group, and (E) LPS DEX group, showing a similar result to the SG (10 mg/kg) group. Fig. 7. Effect of SG on immunohistochemical localization of COX-2. (A) Control group, therewere few specific expression of COX-2 in normal lung tissue. (B) LPS group, protein expression of COX-2 was significantly increased in alveolar macrophages and epithelial cells. (C) LPS + SG (1 mg/kg) group, (D) LPS + SG (5 mg/kg) group, and (E) LPS + SG (10 mg/kg) group, protein expression of COX-2was effectively inhibited in a dose-dependent manner compared with the LPS group. (F) LPS+DEX group, showing a significant reduction in COX-2 expres- sion when compared with the LPS group. Original magnification 400× (A, B, C, D and E). (G) Densitometry analysis of photographs of immunocytochemistry for COX-2 from lung tissues. Data are expressed as the mean ± S.E.M. ###P b 0.001 compared to the normal group; *P b 0.05, **P b 0.01, ***P b 0.001 vs. LPS group. 316 W. Li et al. / International Immunopharmacology 22 (2014) 311–317 neutrophil infiltration in lung tissues. Edema is a typical symptom of in- flammation [27]. Pulmonary edema frequently leads to acute respirato- ry failure, which is a life-threatening condition [28]. In order to quantify the magnitude of pulmonary edema, we determined the W/D weight ratio of the lung tissue. The result showed that pretreatment with SG significantly decreased the ratios in a dose-dependent manner, which Fig.8. The impact of SG on LPS-induced PGE2 production in peritoneal macrophages. Cells were pretreated with different concentrations of SG (1 × 10−4 to 1 μg/mL) or NS398 (5 μg/mL) for 24 h before exposure to 10 μg/mL LPS for 12 h. The supernatant level of PGE2 was measured using ELISA. Three independent experiments were performed and the value presents the mean ± S.E.M. *P b 0.05, **P b 0.01, ***P b 0.001 vs. LPS group. indicated that SG could inhibit the leakage of serous fluid into the lung tissue. In our study, the expression of COX-2 increased in the lung tissue after LPS administration. COX-2 is normally undetectable in most tis- sues, but the expression of COX-2 is increased by pro-inflammatoryme- diators [29]. COX-2 plays an important role in the inflammatory responses of various tissues, so inhibition of COX-2 has showed potent anti-inflammatory effects. Here, we examined the effects of SG on LPS- induced PGE2 release as an index of COX-2 activity, and COX-2 protein expression using immunohistochemical assay to confirm the result of PGE2 production. We found that pretreatment with SG significantly inhibited PGE2 release after LPS administration in a dose-dependent manner, and also significantly inhibited COX-2 protein expression at the same pattern. In vitro, studies in various cell types, such as macro- phages, endothelial, and epithelial cells, indicated an increased expres- sion of COX-2 when responding to LPS [30,31]. The present study demonstrated that SG could remarkably suppress the production of PGE2 and the expression of COX-2 in LPS-activated peritoneal macro- phages, suggesting that SG may have a same effect on the production of pro-inflammatory mediators and the expression of COX-2 protein by activating macrophages. These results indicated that SG suppressed the PGE2 secretion by inhibiting the expression of its synthetic enzyme COX-2.Meanwhile,we further proposed that theprotective effects of SG on LPS-induced ES are, to some extent, attributed to the COX-2 inhibi- tion by SG. Study has demonstrated that the transcription factor nuclear NF-κB has a important role in the regulation of COX-2 expression [32]. However, we need further study to determine whether SG regulates COX-2 expression in an NF-κB-dependant manner in vivo. Fig. 9. Effect of SG on COX protein expression in peritoneal macrophages. (A) Cells were pretreated with different concentrations of SG (1 × 10−4 to 1 μg/mL) or NS398 (5 μg/mL) for 24 h and Western blot for COX protein expression were studied. (B) Protein levels of COX-1 and COX-2 corrected to GAPDH, the loading control. *P b 0.05, **P b 0.01, and ***P b 0.001 vs. LPS group. 317W. Li et al. / International Immunopharmacology 22 (2014) 311–317 5. Conclusion Pretreatment with SG could significantly reduce lethality in LPS- treated mice and attenuate the pulmonary histological changes and lung edema, reduce the neutrophil infiltration in the lung, and inhibit the release of inflammatory cytokines. In addition, SG significantly suppresses the production of PGE2 and the expression of COX-2 in LPS-activated peritoneal macrophages. These protective effects of SG may involve the suppression of COX-2 expression. 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Introduction 2. Materials and methods 2.1. Reagents 2.2. Animals 2.3. Experimentally-induced endotoxic shock 2.4. ELISA assay for detecting PGE2 level in BALF 2.5. MPO assay 2.6. Lung W/D weight ratio 2.7. Histopathological study 2.8. Immunohistochemical study 2.9. Preparation of peritoneal macrophages 2.10. ELISA for detecting PGE2 production in vitro 2.11. Western blotting 2.12. Statistical analysis 3. Results 3.1. Effect of SG on the survival of mice of the LPS-induced ES 3.2. Effect of SG on PGE2 levels in BALF 3.3. Effect of SG on MPO activity in lung tissues of mice with LPS-induced ES 3.4. Effect of SG on lung wet/dry weight ratio in mice with LPS-induced ES 3.5. Effect of SG on histological changes in lung tissues from mice with LPS-induced ES 3.6. Effect of SG on the expression of COX-2 in lung tissues of mice with LPS-induced ES 3.7. Effect of SG on PGE2 production in LPS-stimulated peritoneal macrophages 3.8. SG inhibit COX-2 expression in LPS-stimulated peritoneal macrophages 4. Discussion 5. Conclusion Acknowledgments References