International Immunopharmacology 19 (2014) 103–109
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Zingerone attenuates lipopolysaccharide-induced acute lung injury in mice Xianxing Xie a,1, Shicheng Sun b,1, Weiting Zhong a, Lanan Wassy Soromou a,c, Xuan Zhou a, Miaomiao Wei a, Yanling Ren a, Yu Ding d,⁎ a
College of Veterinary Medicine, Jilin University, Changchun 130062, PR China School of Biosciences and Biotechnology, University of Camerino, Camerino, MC 62032, Italy Département de Médecine Vétérinaire, Institut Supérieur des Sciences et de Médicine Vétérinaire (ISSMV) de Dalaba, Guinea d College of Animal Science, Jilin University, Changchun 130062, PR China b c
a r t i c l e
i n f o
Article history: Received 26 April 2013 Received in revised form 17 December 2013 Accepted 21 December 2013 Available online 9 January 2014 Keywords: Zingerone Acute lung injury Lipopolysaccharide Cytokines
a b s t r a c t Zingerone, one of the active components of ginger, is a phenolic alkanone with antioxidant and antiinflammatory properties. In the present study, we analyzed the role of zingerone against RAW 264.7 cells and acute lung injury induced by lipopolysaccharide (LPS) in mice. RAW cells or BALB/c mice were pretreated with zingerone one hour before stimulated with LPS. We found that zingerone significantly inhibited the production of LPS-induced proinflammatory cytokines in vitro and in vivo. When pretreated with zingerone, pulmonary histopathologic changes, as well as alveolar hemorrhage and neutrophil infiltration were substantially suppressed in lung tissues, with evidence of reduced myeloperoxidase (MPO) activity in murine acute lung injury model. The lung wet-to-dry weight (W/D) ratios, as the index of pulmonary edema, were markedly decreased by zingerone pretreatment. Furthermore, we demonstrated that zingerone attenuates the mitogen-activated protein kinases (MAPK) and nuclear factor-kappaB (NF-κB) signaling pathways through blocking the phosphorylation of ERK, p38/MAPK and IκBα, NF-κB/P65. These results suggest that zingerone may provide protective effects against LPS-induced ALI. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are syndromes of acute respiratory failure characterized by an intense pulmonary inflammatory response, involving neutrophil recruitment, interstitial edema, a disruption of epithelial integrity, and lung parenchymal injury [1]. ALI plays a pivotal role in the death of patients with multiple transfusions, shock, sepsis, and ischemia-reperfusion; however, there are still no effective measures or specific medicines to treat this syndrome [2,3]. Lipopolysaccharide (LPS) is a cell-wall component of the gram-negative bacteria that can induce a disturbance in immune and inflammatory responses [4]. Intranasal instillation of LPS has been shown to injure epithelial cell layers, induce epithelial cell apoptosis, and lead to the release of reactive oxygen species, proinflammatory cytokines, and chemotactic factors, which cause the aggregation of neutrophilic leukocytes and ultimately lung tissue injury [5,6]. The development of an ALI model using LPS instillation has become a basic investigation and therapeutic approach [7,8]. Despite significant advances in intense care researches and diverse therapeutic trials made in the past few decades, ALI remains a severe
⁎ Corresponding author. Tel.: +86 43187836161; fax: +86 43187836160. E-mail address: [email protected] (Y. Ding). 1 These authors contributed equally to this work. 1567-5769/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2013.12.028
disease and still presents a high mortality rate of approximately 40% [9,10]. Therefore, it may be useful to seek therapeutic drugs and therapies to reduce morbidity and mortality of ALI. Zingerone (Fig. 1) is one of the pungent components of ginger, and represents the 3% of essential oil with gingerol and shogaol [11]. Previous studies showed that zingerone played important roles in several functional responses of mammals, such as enhancing antiinflammatory and antioxidant effects [12], inhibiting radiation-induced declines in endogenous antioxidant levels, scavenging radiationinduced free radicals [13], and protecting against radiation-induced cytotoxicity [14]. Although these useful effects have been demonstrated, the molecular mechanism of zingerone on LPS-induced ALI was poorly understood. Therefore, the aim of this study is to clarify the molecular mechanism of zingerone on LPS-induced ALI. 2. Materials and methods 2.1. Animals Specific pathogen-free male BALB/c mice, weighing approximately 18 to 20 g, were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). The mice were housed in micro-isolator cages and given ad libitum access to food and water. The laboratory temperature was 24 ± 1 °C, and relative humidity was 40 – 80%. Mice were housed for 2–3 days to adapt to the
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Fig. 1. The chemical structure of zingerone.
environment before experimentation. All animal experiments were performed in accordance with the guide of Jilin University Animal Care and Use Committee and the Care and Use of Laboratory Animals published by the US National Institutes of Health. 2.2. Reagents Zingerone (purity ≥ 98%) and Dexamethasone (DEX, purity N 99.6%) were purchased from Changle Pharmaceutical Co., Ltd. LPS was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mouse TNF-α, IL-6 and IL-1β ELISA kits were purchased from Biolegend (San Diego, CA, USA). The myeloperoxidase (MPO) determination kit was provided by the Jiancheng Bioengineering Institute of Nanjing (Jiangsu, China). Rabbit mAb, ERK, NF-κB/P65 and p38, and mouse mAb IκB were purchased from Cell Signaling Technology Inc (MA, USA). 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. In vitro study 2.3.1. Cell culture and treatment The RAW 264.7 mouse macrophage cell line was purchased from the China Cell Line Bank (Beijing, China) and was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 3 mM Glutamine, antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) and 10% heat-inactivated fetal bovine serum, at 37 °C under a humidified atmosphere of 5% CO2. In all experiments, macrophages were incubated in the presence or absence of various concentrations of zingerone which were always added 1 h prior to LPS (1 μg/ml) stimulation. 2.3.2. Cell viability assay Cell viability was evaluated by MTT assay. Briefly, cells were plated at a density of 4 × 105 cells/ml onto 96-well plates in a 37 °C, 5% CO2 incubator for 1 h. Then the cells were treated with different concentrations of zingerone (0–75 μg/ml). After 18 h, 20 μl of MTT (5 mg/ml) was added to each well, and incubation was continued for 4 h. The supernatant was removed and the formation of formazan was resolved with 150 μl/well of DMSO. The optical density was measured at a
Fig. 2. Effect of zingerone on the viability of RAW 264.7 cells. Cells were cultured with zingerone (0–75 μg/ml) for 24 h. Cell viability was evaluated by MTT reduction assays. Data are presented as mean ± SEM of three independent experiments. *P b 0.05, **P b 0.01 vs control group.
Fig. 3. Effect of the different concentrations of zingerone on the secretion of TNF-α and IL-6 in LPS-stimulated RAW 264.7 cells. The cells were pretreated with different concentrations (6.25, 12.5, or 25 μg/ml) of zingerone for 1 h prior to stimulation with 1 μg/ml of LPS for 6 h. Control values were obtained in the absence of LPS or zingerone. The values are means ± SEM of three independent experiments. *P b 0.05, **P b 0.01 vs LPS group; ##P b 0.01 vs control group.
wavelength of 570 nm using a microplate reader (TECAN, Austria). Concentrations were determined for three wells of each sample, and each experiment was done in triplicate. 2.3.3. Cytokine assay (TNF-α, IL-6) Zingerone solubilized in DMSO (20 mg of zingerone was solubilized into 0.1 ml of DMSO) was diluted in DMEM prior to treatment. RAW 264.7 cells were plated onto 24-well plates (4 × 105 cells/well), and incubated in the presence of either LPS alone 1 μg/ml, or LPS plus zingerone (6.25 μg/ml, 12.5 μg/ml and 25 μg/ml) for 6 h. Cell-free supernatants were collected and cytokines were assayed by enzymelinked imunosorbent assay (ELISA) kit (R&DS systems), according to the manufacturer's instructions (Bio Legend, Inc, Camino Santa Fe, Suite E, San Diego, CA, USA).
Fig. 4. Effects of zingerone on pulmonary edema. Mice were given an intragastric administration of zingerone (10, 20, and 40 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. The lung wet/dry weight ratio was determined at 24 h after LPS challenge. The values presented are the means ± S.E.M. (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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2.4. In vivo study 2.4.1. Animal model of ALI The mice were randomly divided into six groups: control group; LPS (20 mg/kg) group; LPS + zingerone (10, 20, or 40 mg/kg) groups; and LPS plus dexamethasone (DEX, 5 mg/kg) group. The doses and administration form of zingerone and dexamethasone were prepared on the basis of our preliminary experiments. The control and LPS groups were pretreated with an equal volume of vehicle. One hour later, mice were anesthetized with diethyl ether and LPS (20 mg/kg) was administrated intranasally to induce lung injury. Control mice were given an equal volume of vehicle instead. Twenty-four hours after LPS
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administration, animals were euthanized. Thereafter, bronchoalveolar lavage fluid (BALF) and lung tissue samples were harvested. 2.4.2. Lung wet-to-dry weight (W/D) ratio After lungs were excised, the left lungs were blotted dry, weighed to obtain the ‘wet’ weight. Then the lungs were placed in an oven at 80 °C for 48 h to obtain the ‘dry’ weight. Lung wet-to-dry weight ratio was used as an index of pulmonary edema formation. 2.4.3. Collection of BALF and cytokine assays with ELISA After animals were killed, the airways were lavaged three times through a tracheal cannula with 0.5 ml of autoclaved PBS. BALF collected was immediately centrifuged (4 °C, 3000 rpm, 10 min) to pellet the cells. The levels of the inflammatory cytokines TNF-α and IL-1β in the BALF were measured by enzyme-linked immunosorbent assay (ELISA) kits. All procedures were done in accordance with the manufacturer's instructions. 2.4.4. Cell counting The bronchoalveolar lavage fluid samples were centrifuged (4 °C, 800 × g, 10 min) to pellet the cells. The cell pellets were resuspended in PBS for the total cell counts using a hemacytometer, and cytospins were prepared for differential cell counts by staining with the Wright– Giemsa staining method. 2.4.5. Myeloperoxidase activity in the lung tissues As MPO activity can reflect the parenchymal infiltration of neutrophils and macrophages, we measured MPO activity in our study. After lungs were excised, the right lung tissue samples were homogenized in hydroxyethyl piperazine ethanesulfonic acid (HEPES) (pH 8.0) containing 0.5% cetyltrimethyl ammonium bromide (CTAB) and subjected to three freeze–thaw cycles. The homogenate was centrifuged (4 °C, 13,000 × g, 30 min) and the cell-free extracts were stored at − 20 °C for further use. The MPO activity was assayed using a mouse MPO kit. Samples were diluted in phosphate citrate buffer (pH 5.0). 2.4.6. Pulmonary histopathology Mice under diethyl ether anesthesia were killed 24 h after LPS administration. Part of the pulmonary tissue samples were fixed in normal 4% buffered formalin for 48 h, followed by dehydration in graded alcohol and embedding in paraffin wax, and then stained with hematoxylin and eosin (H&E). Thereafter, light microscopy was performed to evaluate pathological changes in the lung tissues.
Fig. 5. Effects of zingerone on the number of total cells, neutrophils, and macrophages in the bronchoalveolar lavage fluid in LPS-induced acute lung injury mice. Mice were given an intragastric administration of zingerone (10, 20, and 40 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. Bronchoalveolar lavage fluid was collected at 24 h after LPS administration to measure the number of total cells (A), neutrophils (B), and macrophage (C). The values presented are the means ± S.E.M. (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.
Fig. 6. Effects of zingerone on MPO activity in lung tissues of LPS-challenged mice. Mice were given an intragastric administration of zingerone (10, 20, and 40 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. MPO activity in lung tissues was determined at 24 h after LPS administration. The values presented are the means ± S.E.M. (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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2.4.7. Western blot analysis The right lung tissue samples were harvested and frozen in liquid nitrogen immediately until homogenization. The homogenate was centrifuged at 14,000 × g for 10 min at 4 °C, and protein concentration determination was performed using a BCA protein assay kit (Beyotime, China) according to the manufacturer's protocol. Equal amounts of total proteins were loaded per well on 10% sodium dodecyl sulfate (SDS)polyacrylamide gels for fractionation. Subsequently, proteins were transferred onto polyvinylidene difluoride (PVDF) membrane and incubated overnight with 5% (w/v) nonfat dried milk to reduce non-specific binding, and then probed overnight at 4 °C with primary antibodies including phosphorylated and non-phosphorylated forms of NF-κB/P65, IκBα, ERK, or p38. The membrane was then washed three times for 5 min each with TBST and incubated with a 1:7000 (v/v) dilution of horseradish peroxidase-labeled IgG at room temperature for 1 h. Immunoreactive bands were visualized with enhanced chemiluminescence (ECL) western blot kit in accordance with the manufacturer's instructions. The β-actin western blot was performed as an internal control of protein loading.
2.5. Statistical analysis Values are means ± SD. Data were entered into a database and analyzed using SPSS software (SPSS for Windows version 13.0, Chicago, USA). Differences in measured variables between experimental and control groups were assessed using one-way ANOVA with Student's t-test. Statistical significance was accepted at p b 0.05 or p b 0.01.
3. Results 3.1. Effects of zingerone on cell viability After incubating cells for 18 h, the potential cytotoxicity of zingerone was evaluated by the MTT assay. At concentrations ranging from 0 to 75 μg/ml, zingerone did not display any cellular toxicity against RAW 264.7 cells from 0 to 37.5 μg/ml (Fig. 2). But at the dose of 75 μg/ml, it had cellular toxicity.
Fig. 7. Effects of zingerone on histopathological changes in lung tissues in LPS-induced ALI mice. Mice were given an intragastric administration of zingerone (10, 20, and 40 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. Then mice were anesthetized and lung tissue samples were collected at 24 h after LPS challenge for histological evaluation. These representative histological changes of the lung were obtained from mice of different groups (hematoxylin and eosin staining, original magnification 200×, Scale bar: 50 μm).
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3.2. Effects of zingerone on the expression of proinflammatory cytokines in LPS-stimulated RAW 264.7 cells The production of TNF-α and IL-6 in the culture supernatants was measured using ELISA kits. In response to LPS stimulation, the expression of cytokines was significantly upregulated compared to the control group (Fig. 3). However, the treatment with zingerone considerably inhibited the LPS induction of TNF-α and IL-6 (Fig.3A, B). 3.3. Effects of zingerone on lung W/D ratio in LPS-stimulated ALI model Pulmonary edema is the typical feature of lung injury. Twenty-four hours after LPS was given, lungs were evaluated for edema formation by determining the lung water content. Compared to the control group, the lung W/D ratio was significantly increased after LPS challenge (Fig. 4). In contrast, zingerone and DEX effectively decreased the lung W/D ratio (Fig. 4) in LPS-induced ALI model. 3.4. Effects of zingerone on inflammatory cell count in BALF of LPS-induced ALI mice Total cells, neutrophils and macrophages in BALF were analyzed in our study. Compared with the control group, total cells, neutrophils and macrophages were significantly increased in mice treated with LPS alone. However, zingerone and DEX induced a significant decrease of total cells (Fig. 5A), neutrophils (Fig. 5B) and macrophages (Fig. 5C) in BALF of mice in a dose-dependent manner. 3.5. Effects of zingerone on MPO activity in LPS-induced ALI model
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4. Discussion Previous studies indicated that LPS stimulated macrophages to release a large number of immunoregulatory molecules including proinflammatory factors such as TNF-α, IL-1β and IL-6 [15,16]. These mediators are known to play critical roles in inflammatory diseases. In the present study, MTT assay showed that zingerone did not display any cellular toxicity against RAW 264.7 cells over 18 h at concentrations ranging from 0 to 75 μg/ml. These results lead us to conclude that the effects of zingerone on RAW 264.7 cells were not due to cell death. To study the effect of zingerone on inflammatory cytokine secretion in vitro, we measured TNF-α, IL-1β and IL-6 levels in LPS-stimulated RAW cells and zingerone was found to significantly inhibit the production of TNF-α and IL-6 in a dose-dependent manner. These results suggest that zingerone has in vitro anti-inflammatory effect. To further understand the anti-inflammatory properties of zingerone, we evaluated how zingerone protects mice from LPS-induced acute lung injury. It is well known that LPS inhalation elicits pulmonary inflammation such as acute injury, which occurs after 2 – 4 h, and maximizes at 24 – 48 h [17]. At present, glucocorticoids are the most frequently used anti-inflammatory drugs in the clinical treatment of ALI/ARDS [18]. Therefore, in this study, we used DEX as a positive control to evaluate the anti-inflammatory efficiency of zingerone in LPS-induced ALI. We found that zingerone reduced the LPS-induced pulmonary edema, proinflammatory cytokine production in the BALF, MPO activity and the phosphorylation of NF-κB/P65,IκBα, ERK and p38. All the results of this investigation demonstrated that zingerone may have an important anti-inflammatory activity during ALI. Edema is a typical symptom of inflammation not only in systemic inflammation, but also in local inflammation [19]. In order to quantify
The MPO activity in lung tissues is known as a reliable marker of neutrophil infiltration. After LPS administration, the MPO activity in lung tissues was significantly increased compared with the control group (Fig. 6). When pretreated with zingerone or DEX, MPO activity was significantly decreased in the lung tissues (Fig. 6). 3.6. Effects of zingerone on histological changes in lung tissues As shown in Fig. 7, lung sections obtained from mice in LPS group showed significant pathologic changes, such as alveolar wall thickening, alveolar hemorrhage, interstitial edema, inflammatory cell infiltration and lung tissues destruction. In contrast, these histopathological changes were obviously attenuated by zingerone or DEX treatment. 3.7. Effects of zingerone on concentrations of TNF-α and IL-1β in BALF To evaluate the levels of cytokines in BALF, BALF was collected at 24 h after LPS administration. The levels of TNF-α and IL-1β in BALF were elevated in LPS-treated mice compared to those in the control group (Fig. 8). In the zingerone + LPS group or the DEX + LPS group, the concentrations of TNF-α (Fig. 8A) and IL-1β (Fig. 8B) were significantly downregulated. 3.8. Effects of zingerone on NF-κB and MAPKs signaling pathways To fully understand the protective effects of zingerone on ALI of mice, we investigated the effects of zingerone on LPS induction of NF-κB and MAPKs signaling pathways. As shown in Fig. 9 and Supplementary Fig. 1, in LPS group, the phosphorylation levels of NF-κB and MAPKs were increased significantly and the non-phosphorylation level of IκBα in cytoplasm was notably decreased. However, the increased phosphorylations of NF-κB/P65, IκBα, ERK and p38 were dramatically blocked by zingerone or DEX pretreatment (Fig. 9).
Fig. 8. Effects of zingerone on concentrations of TNF-α and IL-1β in BALF of LPS-induced acute lung injury mice. Mice were given an intragastric administration of zingerone (10, 20, and 40 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. Bronchoalveolar lavage fluid was collected at 24 h following LPS challenge to analyze the inflammatory cytokines TNF-α (A) and IL-1β (B). The values presented are the means ± S.E.M. (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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Fig. 9. Effects of zingerone on activity of ERK, p38, and IκBa in ALI model. Mice were given an intragastric administration of zingerone (10, 20, and 40 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. Proteins, extracted from the lungs using Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology; China) were analysed by western blot. Quantification of protein expression was normalized to β-actin using a densitometer (Imaging System). Similar results were obtained in three independent experiments and one of three representative experiments is shown. ##P b 0.01 vs. control group, ⁎P b 0.05 and ⁎⁎P b 0.01 vs. LPS group.
the magnitude of pulmonary edema, we first evaluated the W/D ratio of the lung and zingerone was found to significantly decrease the lung W/D ratio which was increased by LPS administration. The result reflects that zingerone performs its protective effects on LPS-induced acute lung injury partly based on suppression of pulmonary edema. The infiltration of inflammatory cells into lung tissues is a typical characteristic of inflammatory diseases. During ALI, the predominant inflammatory cells are neutrophils and these cells are known to play an important role in the development of most cases of ALI [20,21]. Activated alveolar macrophages and neutrophils cause excessive production of NO via the expression of inducible nitric oxide synthase (iNOS) resulting in impairment of lung tissues. In the present study, zingerone markedly lessened the number of macrophages and neutrophils elevated by LPS
challenge in murine model. Neutrophils express MPO, a heme protein generally associated with the killing of bacteria and oxidative tissue injury. MPO activity is a marker of neutrophil infiltration, and directly proportional to the number of neutrophils in the tissue [22]. Upon determining MPO activity, we found that LPS exposure could induce the production of MPO, while pretreatment with zingerone dramatically reduced MPO activity. These findings suggest that zingerone may abate LPS-stimulated pulmonary inflammation and injury via reducing inflammatory cell accumulation and MPO activity. Lung histopathologic analysis also showed that zingerone had a significant anti-inflammatory activity during LPS-induced ALI. Previous study has shown that LPS induced a large number of macrophages and neutrophils in the lung tissues, increased endothelial permeability and tissue damage, and thickened intra-alveolar septa with excessive
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production of inflammatory cytokines and edema in the lungs [23]. In our present study, the histopathological changes such as infiltration of proinflammatory cells, hemorrhage, formation of hyaline membranes, alveolar edema, and airway epithelial necrosis were found to be common and prominent in the LPS group but rare in the zingerone groups. These findings confirm that zingerone can alleviate severity of histopathology changes in LPS-challenged mice. It is reported that neutrophil sequestration into the pulmonary is critical for host defense and also contributes to the development of ALI [24]. As secretory cells, monocytes and macrophages are known to bring a complex network of cytokines, including TNF-α, IL-1β, and IL-6 and other proinflammatory mediators, which initiate, amplify, and perpetuate the inflammatory response in ALI/ARDS [25]. Increased levels of TNF-α and IL-1β in the BALF have been noted in ARDS patients, and the persistent elevation of proinflammatory cytokines in humans with ALI or sepsis has been associated with more severe outcomes [26]. TNF-α can elicit the inflammatory cascade and cause damage to the vascular endothelial cells. TNF-α binds with a TNF-α receptor in lung tissue, and leads to enzyme leakage [27]. On the other hand IL-1β, an early proinflammatory cytokine playing a key role in the process of ALI can stimulate the production of other cytokine. It has earned a position of prominence at the head of the inflammatory cytokine cascade [25]. Some evidences have shown that inhibition of TNF-α and IL-1β efficiently alleviated pulmonary injury in LPS-induced murine model of ALI [28,29]. In our study, the amounts of TNF-α and IL-1β in BALF were dramatically attenuated by zingerone. These results suggest that the suppression of inflammation by zingerone in lung is associated with its inhibition on proinflammatory cytokine release. It has been known that NF-κB and MAPK pathways can modulate the expressions of proinflammatory mediators. Therefore, we looked into whether zingerone regulates TNF-α and IL-1β production via interfering with the activation of NF-κB and MAPK. NF-κB is sequestered in the cytoplasm, bound by members of the IκB family. The various stimuli cause phosphorylation of IκB, which is followed by its ubiquitination and subsequent degradation and activation of NF-κB [19]. The free NFκB will translocate into the nucleus and promotes the expression of cytokines, COX-2 and proinflammatory proteins [30]. In our study, LPS-induced IκBα and NF-κB/P65 phosphorylation was markedly inhibited by pretreatment with zingerone in mice challenged with LPS. Mitogen-activated protein kinases mediate a number of physiological and pathological changes in cell function, because of its importance in signal transduction pathways. ERK, p38 and JNK/SAPK are the three major subgroups of the MAPK family. P38 signaling pathways are reported to be activated by proinflammatory cytokines such as TNF-α [31]. Our data showed that MAPK was activated in LPS-induced lung injury. However, zingerone treatment significantly blocked LPS-induced phosphorylation of ERK and p38. Taken together, it is possible that zingerone suppressed LPS-induced proinflammatory cytokine production by inhibiting the activation of IκBα/NF-κB, NF-κB/P65, ERK/MAPK and p38/MAPK. In summary, we have now provided the first in vivo information that pretreatment of zingerone could significantly attenuate pulmonary histopathologic changes, diminish inflammatory cytokines and reduce the neutrophil infiltration in lungs in LPS-induced ALI in mice. Furthermore, zingerone was shown to inhibit not only cytokine production dose-dependently, but also MAPK and NF-κB activation. These findings suggest that zingerone has a protective effect on LPS-induced ALI in mice. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2013.12.028.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Zingerone attenuates lipopolysaccharide-induced acute lung injury in mice Xianxing Xie a,1, Shicheng Sun b,1, Weiting Zhong a, Lanan Wassy Soromou a,c, Xuan Zhou a, Miaomiao Wei a, Yanling Ren a, Yu Ding d,⁎ a
College of Veterinary Medicine, Jilin University, Changchun 130062, PR China School of Biosciences and Biotechnology, University of Camerino, Camerino, MC 62032, Italy Département de Médecine Vétérinaire, Institut Supérieur des Sciences et de Médicine Vétérinaire (ISSMV) de Dalaba, Guinea d College of Animal Science, Jilin University, Changchun 130062, PR China b c
a r t i c l e
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Article history: Received 26 April 2013 Received in revised form 17 December 2013 Accepted 21 December 2013 Available online 9 January 2014 Keywords: Zingerone Acute lung injury Lipopolysaccharide Cytokines
a b s t r a c t Zingerone, one of the active components of ginger, is a phenolic alkanone with antioxidant and antiinflammatory properties. In the present study, we analyzed the role of zingerone against RAW 264.7 cells and acute lung injury induced by lipopolysaccharide (LPS) in mice. RAW cells or BALB/c mice were pretreated with zingerone one hour before stimulated with LPS. We found that zingerone significantly inhibited the production of LPS-induced proinflammatory cytokines in vitro and in vivo. When pretreated with zingerone, pulmonary histopathologic changes, as well as alveolar hemorrhage and neutrophil infiltration were substantially suppressed in lung tissues, with evidence of reduced myeloperoxidase (MPO) activity in murine acute lung injury model. The lung wet-to-dry weight (W/D) ratios, as the index of pulmonary edema, were markedly decreased by zingerone pretreatment. Furthermore, we demonstrated that zingerone attenuates the mitogen-activated protein kinases (MAPK) and nuclear factor-kappaB (NF-κB) signaling pathways through blocking the phosphorylation of ERK, p38/MAPK and IκBα, NF-κB/P65. These results suggest that zingerone may provide protective effects against LPS-induced ALI. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), are syndromes of acute respiratory failure characterized by an intense pulmonary inflammatory response, involving neutrophil recruitment, interstitial edema, a disruption of epithelial integrity, and lung parenchymal injury [1]. ALI plays a pivotal role in the death of patients with multiple transfusions, shock, sepsis, and ischemia-reperfusion; however, there are still no effective measures or specific medicines to treat this syndrome [2,3]. Lipopolysaccharide (LPS) is a cell-wall component of the gram-negative bacteria that can induce a disturbance in immune and inflammatory responses [4]. Intranasal instillation of LPS has been shown to injure epithelial cell layers, induce epithelial cell apoptosis, and lead to the release of reactive oxygen species, proinflammatory cytokines, and chemotactic factors, which cause the aggregation of neutrophilic leukocytes and ultimately lung tissue injury [5,6]. The development of an ALI model using LPS instillation has become a basic investigation and therapeutic approach [7,8]. Despite significant advances in intense care researches and diverse therapeutic trials made in the past few decades, ALI remains a severe
⁎ Corresponding author. Tel.: +86 43187836161; fax: +86 43187836160. E-mail address: [email protected] (Y. Ding). 1 These authors contributed equally to this work. 1567-5769/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.intimp.2013.12.028
disease and still presents a high mortality rate of approximately 40% [9,10]. Therefore, it may be useful to seek therapeutic drugs and therapies to reduce morbidity and mortality of ALI. Zingerone (Fig. 1) is one of the pungent components of ginger, and represents the 3% of essential oil with gingerol and shogaol [11]. Previous studies showed that zingerone played important roles in several functional responses of mammals, such as enhancing antiinflammatory and antioxidant effects [12], inhibiting radiation-induced declines in endogenous antioxidant levels, scavenging radiationinduced free radicals [13], and protecting against radiation-induced cytotoxicity [14]. Although these useful effects have been demonstrated, the molecular mechanism of zingerone on LPS-induced ALI was poorly understood. Therefore, the aim of this study is to clarify the molecular mechanism of zingerone on LPS-induced ALI. 2. Materials and methods 2.1. Animals Specific pathogen-free male BALB/c mice, weighing approximately 18 to 20 g, were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). The mice were housed in micro-isolator cages and given ad libitum access to food and water. The laboratory temperature was 24 ± 1 °C, and relative humidity was 40 – 80%. Mice were housed for 2–3 days to adapt to the
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Fig. 1. The chemical structure of zingerone.
environment before experimentation. All animal experiments were performed in accordance with the guide of Jilin University Animal Care and Use Committee and the Care and Use of Laboratory Animals published by the US National Institutes of Health. 2.2. Reagents Zingerone (purity ≥ 98%) and Dexamethasone (DEX, purity N 99.6%) were purchased from Changle Pharmaceutical Co., Ltd. LPS was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mouse TNF-α, IL-6 and IL-1β ELISA kits were purchased from Biolegend (San Diego, CA, USA). The myeloperoxidase (MPO) determination kit was provided by the Jiancheng Bioengineering Institute of Nanjing (Jiangsu, China). Rabbit mAb, ERK, NF-κB/P65 and p38, and mouse mAb IκB were purchased from Cell Signaling Technology Inc (MA, USA). 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. In vitro study 2.3.1. Cell culture and treatment The RAW 264.7 mouse macrophage cell line was purchased from the China Cell Line Bank (Beijing, China) and was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 3 mM Glutamine, antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) and 10% heat-inactivated fetal bovine serum, at 37 °C under a humidified atmosphere of 5% CO2. In all experiments, macrophages were incubated in the presence or absence of various concentrations of zingerone which were always added 1 h prior to LPS (1 μg/ml) stimulation. 2.3.2. Cell viability assay Cell viability was evaluated by MTT assay. Briefly, cells were plated at a density of 4 × 105 cells/ml onto 96-well plates in a 37 °C, 5% CO2 incubator for 1 h. Then the cells were treated with different concentrations of zingerone (0–75 μg/ml). After 18 h, 20 μl of MTT (5 mg/ml) was added to each well, and incubation was continued for 4 h. The supernatant was removed and the formation of formazan was resolved with 150 μl/well of DMSO. The optical density was measured at a
Fig. 2. Effect of zingerone on the viability of RAW 264.7 cells. Cells were cultured with zingerone (0–75 μg/ml) for 24 h. Cell viability was evaluated by MTT reduction assays. Data are presented as mean ± SEM of three independent experiments. *P b 0.05, **P b 0.01 vs control group.
Fig. 3. Effect of the different concentrations of zingerone on the secretion of TNF-α and IL-6 in LPS-stimulated RAW 264.7 cells. The cells were pretreated with different concentrations (6.25, 12.5, or 25 μg/ml) of zingerone for 1 h prior to stimulation with 1 μg/ml of LPS for 6 h. Control values were obtained in the absence of LPS or zingerone. The values are means ± SEM of three independent experiments. *P b 0.05, **P b 0.01 vs LPS group; ##P b 0.01 vs control group.
wavelength of 570 nm using a microplate reader (TECAN, Austria). Concentrations were determined for three wells of each sample, and each experiment was done in triplicate. 2.3.3. Cytokine assay (TNF-α, IL-6) Zingerone solubilized in DMSO (20 mg of zingerone was solubilized into 0.1 ml of DMSO) was diluted in DMEM prior to treatment. RAW 264.7 cells were plated onto 24-well plates (4 × 105 cells/well), and incubated in the presence of either LPS alone 1 μg/ml, or LPS plus zingerone (6.25 μg/ml, 12.5 μg/ml and 25 μg/ml) for 6 h. Cell-free supernatants were collected and cytokines were assayed by enzymelinked imunosorbent assay (ELISA) kit (R&DS systems), according to the manufacturer's instructions (Bio Legend, Inc, Camino Santa Fe, Suite E, San Diego, CA, USA).
Fig. 4. Effects of zingerone on pulmonary edema. Mice were given an intragastric administration of zingerone (10, 20, and 40 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. The lung wet/dry weight ratio was determined at 24 h after LPS challenge. The values presented are the means ± S.E.M. (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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2.4. In vivo study 2.4.1. Animal model of ALI The mice were randomly divided into six groups: control group; LPS (20 mg/kg) group; LPS + zingerone (10, 20, or 40 mg/kg) groups; and LPS plus dexamethasone (DEX, 5 mg/kg) group. The doses and administration form of zingerone and dexamethasone were prepared on the basis of our preliminary experiments. The control and LPS groups were pretreated with an equal volume of vehicle. One hour later, mice were anesthetized with diethyl ether and LPS (20 mg/kg) was administrated intranasally to induce lung injury. Control mice were given an equal volume of vehicle instead. Twenty-four hours after LPS
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administration, animals were euthanized. Thereafter, bronchoalveolar lavage fluid (BALF) and lung tissue samples were harvested. 2.4.2. Lung wet-to-dry weight (W/D) ratio After lungs were excised, the left lungs were blotted dry, weighed to obtain the ‘wet’ weight. Then the lungs were placed in an oven at 80 °C for 48 h to obtain the ‘dry’ weight. Lung wet-to-dry weight ratio was used as an index of pulmonary edema formation. 2.4.3. Collection of BALF and cytokine assays with ELISA After animals were killed, the airways were lavaged three times through a tracheal cannula with 0.5 ml of autoclaved PBS. BALF collected was immediately centrifuged (4 °C, 3000 rpm, 10 min) to pellet the cells. The levels of the inflammatory cytokines TNF-α and IL-1β in the BALF were measured by enzyme-linked immunosorbent assay (ELISA) kits. All procedures were done in accordance with the manufacturer's instructions. 2.4.4. Cell counting The bronchoalveolar lavage fluid samples were centrifuged (4 °C, 800 × g, 10 min) to pellet the cells. The cell pellets were resuspended in PBS for the total cell counts using a hemacytometer, and cytospins were prepared for differential cell counts by staining with the Wright– Giemsa staining method. 2.4.5. Myeloperoxidase activity in the lung tissues As MPO activity can reflect the parenchymal infiltration of neutrophils and macrophages, we measured MPO activity in our study. After lungs were excised, the right lung tissue samples were homogenized in hydroxyethyl piperazine ethanesulfonic acid (HEPES) (pH 8.0) containing 0.5% cetyltrimethyl ammonium bromide (CTAB) and subjected to three freeze–thaw cycles. The homogenate was centrifuged (4 °C, 13,000 × g, 30 min) and the cell-free extracts were stored at − 20 °C for further use. The MPO activity was assayed using a mouse MPO kit. Samples were diluted in phosphate citrate buffer (pH 5.0). 2.4.6. Pulmonary histopathology Mice under diethyl ether anesthesia were killed 24 h after LPS administration. Part of the pulmonary tissue samples were fixed in normal 4% buffered formalin for 48 h, followed by dehydration in graded alcohol and embedding in paraffin wax, and then stained with hematoxylin and eosin (H&E). Thereafter, light microscopy was performed to evaluate pathological changes in the lung tissues.
Fig. 5. Effects of zingerone on the number of total cells, neutrophils, and macrophages in the bronchoalveolar lavage fluid in LPS-induced acute lung injury mice. Mice were given an intragastric administration of zingerone (10, 20, and 40 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. Bronchoalveolar lavage fluid was collected at 24 h after LPS administration to measure the number of total cells (A), neutrophils (B), and macrophage (C). The values presented are the means ± S.E.M. (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.
Fig. 6. Effects of zingerone on MPO activity in lung tissues of LPS-challenged mice. Mice were given an intragastric administration of zingerone (10, 20, and 40 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. MPO activity in lung tissues was determined at 24 h after LPS administration. The values presented are the means ± S.E.M. (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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2.4.7. Western blot analysis The right lung tissue samples were harvested and frozen in liquid nitrogen immediately until homogenization. The homogenate was centrifuged at 14,000 × g for 10 min at 4 °C, and protein concentration determination was performed using a BCA protein assay kit (Beyotime, China) according to the manufacturer's protocol. Equal amounts of total proteins were loaded per well on 10% sodium dodecyl sulfate (SDS)polyacrylamide gels for fractionation. Subsequently, proteins were transferred onto polyvinylidene difluoride (PVDF) membrane and incubated overnight with 5% (w/v) nonfat dried milk to reduce non-specific binding, and then probed overnight at 4 °C with primary antibodies including phosphorylated and non-phosphorylated forms of NF-κB/P65, IκBα, ERK, or p38. The membrane was then washed three times for 5 min each with TBST and incubated with a 1:7000 (v/v) dilution of horseradish peroxidase-labeled IgG at room temperature for 1 h. Immunoreactive bands were visualized with enhanced chemiluminescence (ECL) western blot kit in accordance with the manufacturer's instructions. The β-actin western blot was performed as an internal control of protein loading.
2.5. Statistical analysis Values are means ± SD. Data were entered into a database and analyzed using SPSS software (SPSS for Windows version 13.0, Chicago, USA). Differences in measured variables between experimental and control groups were assessed using one-way ANOVA with Student's t-test. Statistical significance was accepted at p b 0.05 or p b 0.01.
3. Results 3.1. Effects of zingerone on cell viability After incubating cells for 18 h, the potential cytotoxicity of zingerone was evaluated by the MTT assay. At concentrations ranging from 0 to 75 μg/ml, zingerone did not display any cellular toxicity against RAW 264.7 cells from 0 to 37.5 μg/ml (Fig. 2). But at the dose of 75 μg/ml, it had cellular toxicity.
Fig. 7. Effects of zingerone on histopathological changes in lung tissues in LPS-induced ALI mice. Mice were given an intragastric administration of zingerone (10, 20, and 40 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. Then mice were anesthetized and lung tissue samples were collected at 24 h after LPS challenge for histological evaluation. These representative histological changes of the lung were obtained from mice of different groups (hematoxylin and eosin staining, original magnification 200×, Scale bar: 50 μm).
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3.2. Effects of zingerone on the expression of proinflammatory cytokines in LPS-stimulated RAW 264.7 cells The production of TNF-α and IL-6 in the culture supernatants was measured using ELISA kits. In response to LPS stimulation, the expression of cytokines was significantly upregulated compared to the control group (Fig. 3). However, the treatment with zingerone considerably inhibited the LPS induction of TNF-α and IL-6 (Fig.3A, B). 3.3. Effects of zingerone on lung W/D ratio in LPS-stimulated ALI model Pulmonary edema is the typical feature of lung injury. Twenty-four hours after LPS was given, lungs were evaluated for edema formation by determining the lung water content. Compared to the control group, the lung W/D ratio was significantly increased after LPS challenge (Fig. 4). In contrast, zingerone and DEX effectively decreased the lung W/D ratio (Fig. 4) in LPS-induced ALI model. 3.4. Effects of zingerone on inflammatory cell count in BALF of LPS-induced ALI mice Total cells, neutrophils and macrophages in BALF were analyzed in our study. Compared with the control group, total cells, neutrophils and macrophages were significantly increased in mice treated with LPS alone. However, zingerone and DEX induced a significant decrease of total cells (Fig. 5A), neutrophils (Fig. 5B) and macrophages (Fig. 5C) in BALF of mice in a dose-dependent manner. 3.5. Effects of zingerone on MPO activity in LPS-induced ALI model
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4. Discussion Previous studies indicated that LPS stimulated macrophages to release a large number of immunoregulatory molecules including proinflammatory factors such as TNF-α, IL-1β and IL-6 [15,16]. These mediators are known to play critical roles in inflammatory diseases. In the present study, MTT assay showed that zingerone did not display any cellular toxicity against RAW 264.7 cells over 18 h at concentrations ranging from 0 to 75 μg/ml. These results lead us to conclude that the effects of zingerone on RAW 264.7 cells were not due to cell death. To study the effect of zingerone on inflammatory cytokine secretion in vitro, we measured TNF-α, IL-1β and IL-6 levels in LPS-stimulated RAW cells and zingerone was found to significantly inhibit the production of TNF-α and IL-6 in a dose-dependent manner. These results suggest that zingerone has in vitro anti-inflammatory effect. To further understand the anti-inflammatory properties of zingerone, we evaluated how zingerone protects mice from LPS-induced acute lung injury. It is well known that LPS inhalation elicits pulmonary inflammation such as acute injury, which occurs after 2 – 4 h, and maximizes at 24 – 48 h [17]. At present, glucocorticoids are the most frequently used anti-inflammatory drugs in the clinical treatment of ALI/ARDS [18]. Therefore, in this study, we used DEX as a positive control to evaluate the anti-inflammatory efficiency of zingerone in LPS-induced ALI. We found that zingerone reduced the LPS-induced pulmonary edema, proinflammatory cytokine production in the BALF, MPO activity and the phosphorylation of NF-κB/P65,IκBα, ERK and p38. All the results of this investigation demonstrated that zingerone may have an important anti-inflammatory activity during ALI. Edema is a typical symptom of inflammation not only in systemic inflammation, but also in local inflammation [19]. In order to quantify
The MPO activity in lung tissues is known as a reliable marker of neutrophil infiltration. After LPS administration, the MPO activity in lung tissues was significantly increased compared with the control group (Fig. 6). When pretreated with zingerone or DEX, MPO activity was significantly decreased in the lung tissues (Fig. 6). 3.6. Effects of zingerone on histological changes in lung tissues As shown in Fig. 7, lung sections obtained from mice in LPS group showed significant pathologic changes, such as alveolar wall thickening, alveolar hemorrhage, interstitial edema, inflammatory cell infiltration and lung tissues destruction. In contrast, these histopathological changes were obviously attenuated by zingerone or DEX treatment. 3.7. Effects of zingerone on concentrations of TNF-α and IL-1β in BALF To evaluate the levels of cytokines in BALF, BALF was collected at 24 h after LPS administration. The levels of TNF-α and IL-1β in BALF were elevated in LPS-treated mice compared to those in the control group (Fig. 8). In the zingerone + LPS group or the DEX + LPS group, the concentrations of TNF-α (Fig. 8A) and IL-1β (Fig. 8B) were significantly downregulated. 3.8. Effects of zingerone on NF-κB and MAPKs signaling pathways To fully understand the protective effects of zingerone on ALI of mice, we investigated the effects of zingerone on LPS induction of NF-κB and MAPKs signaling pathways. As shown in Fig. 9 and Supplementary Fig. 1, in LPS group, the phosphorylation levels of NF-κB and MAPKs were increased significantly and the non-phosphorylation level of IκBα in cytoplasm was notably decreased. However, the increased phosphorylations of NF-κB/P65, IκBα, ERK and p38 were dramatically blocked by zingerone or DEX pretreatment (Fig. 9).
Fig. 8. Effects of zingerone on concentrations of TNF-α and IL-1β in BALF of LPS-induced acute lung injury mice. Mice were given an intragastric administration of zingerone (10, 20, and 40 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. Bronchoalveolar lavage fluid was collected at 24 h following LPS challenge to analyze the inflammatory cytokines TNF-α (A) and IL-1β (B). The values presented are the means ± S.E.M. (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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Fig. 9. Effects of zingerone on activity of ERK, p38, and IκBa in ALI model. Mice were given an intragastric administration of zingerone (10, 20, and 40 mg/kg) or Dex (5 mg/kg) 1 h prior to an intranasal administration of LPS. Proteins, extracted from the lungs using Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology; China) were analysed by western blot. Quantification of protein expression was normalized to β-actin using a densitometer (Imaging System). Similar results were obtained in three independent experiments and one of three representative experiments is shown. ##P b 0.01 vs. control group, ⁎P b 0.05 and ⁎⁎P b 0.01 vs. LPS group.
the magnitude of pulmonary edema, we first evaluated the W/D ratio of the lung and zingerone was found to significantly decrease the lung W/D ratio which was increased by LPS administration. The result reflects that zingerone performs its protective effects on LPS-induced acute lung injury partly based on suppression of pulmonary edema. The infiltration of inflammatory cells into lung tissues is a typical characteristic of inflammatory diseases. During ALI, the predominant inflammatory cells are neutrophils and these cells are known to play an important role in the development of most cases of ALI [20,21]. Activated alveolar macrophages and neutrophils cause excessive production of NO via the expression of inducible nitric oxide synthase (iNOS) resulting in impairment of lung tissues. In the present study, zingerone markedly lessened the number of macrophages and neutrophils elevated by LPS
challenge in murine model. Neutrophils express MPO, a heme protein generally associated with the killing of bacteria and oxidative tissue injury. MPO activity is a marker of neutrophil infiltration, and directly proportional to the number of neutrophils in the tissue [22]. Upon determining MPO activity, we found that LPS exposure could induce the production of MPO, while pretreatment with zingerone dramatically reduced MPO activity. These findings suggest that zingerone may abate LPS-stimulated pulmonary inflammation and injury via reducing inflammatory cell accumulation and MPO activity. Lung histopathologic analysis also showed that zingerone had a significant anti-inflammatory activity during LPS-induced ALI. Previous study has shown that LPS induced a large number of macrophages and neutrophils in the lung tissues, increased endothelial permeability and tissue damage, and thickened intra-alveolar septa with excessive
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production of inflammatory cytokines and edema in the lungs [23]. In our present study, the histopathological changes such as infiltration of proinflammatory cells, hemorrhage, formation of hyaline membranes, alveolar edema, and airway epithelial necrosis were found to be common and prominent in the LPS group but rare in the zingerone groups. These findings confirm that zingerone can alleviate severity of histopathology changes in LPS-challenged mice. It is reported that neutrophil sequestration into the pulmonary is critical for host defense and also contributes to the development of ALI [24]. As secretory cells, monocytes and macrophages are known to bring a complex network of cytokines, including TNF-α, IL-1β, and IL-6 and other proinflammatory mediators, which initiate, amplify, and perpetuate the inflammatory response in ALI/ARDS [25]. Increased levels of TNF-α and IL-1β in the BALF have been noted in ARDS patients, and the persistent elevation of proinflammatory cytokines in humans with ALI or sepsis has been associated with more severe outcomes [26]. TNF-α can elicit the inflammatory cascade and cause damage to the vascular endothelial cells. TNF-α binds with a TNF-α receptor in lung tissue, and leads to enzyme leakage [27]. On the other hand IL-1β, an early proinflammatory cytokine playing a key role in the process of ALI can stimulate the production of other cytokine. It has earned a position of prominence at the head of the inflammatory cytokine cascade [25]. Some evidences have shown that inhibition of TNF-α and IL-1β efficiently alleviated pulmonary injury in LPS-induced murine model of ALI [28,29]. In our study, the amounts of TNF-α and IL-1β in BALF were dramatically attenuated by zingerone. These results suggest that the suppression of inflammation by zingerone in lung is associated with its inhibition on proinflammatory cytokine release. It has been known that NF-κB and MAPK pathways can modulate the expressions of proinflammatory mediators. Therefore, we looked into whether zingerone regulates TNF-α and IL-1β production via interfering with the activation of NF-κB and MAPK. NF-κB is sequestered in the cytoplasm, bound by members of the IκB family. The various stimuli cause phosphorylation of IκB, which is followed by its ubiquitination and subsequent degradation and activation of NF-κB [19]. The free NFκB will translocate into the nucleus and promotes the expression of cytokines, COX-2 and proinflammatory proteins [30]. In our study, LPS-induced IκBα and NF-κB/P65 phosphorylation was markedly inhibited by pretreatment with zingerone in mice challenged with LPS. Mitogen-activated protein kinases mediate a number of physiological and pathological changes in cell function, because of its importance in signal transduction pathways. ERK, p38 and JNK/SAPK are the three major subgroups of the MAPK family. P38 signaling pathways are reported to be activated by proinflammatory cytokines such as TNF-α [31]. Our data showed that MAPK was activated in LPS-induced lung injury. However, zingerone treatment significantly blocked LPS-induced phosphorylation of ERK and p38. Taken together, it is possible that zingerone suppressed LPS-induced proinflammatory cytokine production by inhibiting the activation of IκBα/NF-κB, NF-κB/P65, ERK/MAPK and p38/MAPK. In summary, we have now provided the first in vivo information that pretreatment of zingerone could significantly attenuate pulmonary histopathologic changes, diminish inflammatory cytokines and reduce the neutrophil infiltration in lungs in LPS-induced ALI in mice. Furthermore, zingerone was shown to inhibit not only cytokine production dose-dependently, but also MAPK and NF-κB activation. These findings suggest that zingerone has a protective effect on LPS-induced ALI in mice. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2013.12.028.
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