International Immunopharmacology 23 (2014) 127–133
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Protective effects of scoparone against lipopolysaccharide-induced acute lung injury Niu Niu ⁎, Baolan Li, Ying Hu, Xuebing Li, Jie Li, Haiqing Zhang Beijing Tuberculosis and Thoracic Tumor Research Institute, Department of internal medicine, Beijing hest Hospital, CMMU, Beijing 101149, China
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Article history: Received 16 June 2014 Received in revised form 28 July 2014 Accepted 13 August 2014 Available online 22 August 2014 Keywords: Scoparone LPS Acute lung injury NF-κB TLR4
a b s t r a c t The purpose of this study was to investigate the protective effects and molecular mechanisms of scoparone on lipopolysaccharide (LPS)-induced acute lung injury in mice. Mice model of acute lung injury (ALI), induced by intranasal instillation of LPS, was used to investigate the protective effects of scoparone in vivo. The alveolar macrophages were used to investigate the molecular mechanisms of scoparone in vitro. The results showed that scoparone treatment remarkably attenuated LPS-induced pulmonary edema, histological severities, myeloperoxidase activity, and TNF-α, IL-6 and IL-1β production in vivo. We also found that scoparone inhibited LPS-induced TLR4 expression, NF-κB activation, TNF-α, IL-6 and IL-1β production in alveolar macrophages in vitro. In conclusion, our results suggest that scoparone has a protective effect on LPS-induced ALI via suppression of TLR4-mediated NF-κB signaling pathways. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acute respiratory distress syndrome (ARDS) is characterized by overwhelming lung inflammation and increased microvascular permeability [1]. ARDS often results in multi-organ failure with high mortality in critically ill patients [2]. Most causes such as sepsis, trauma and burn could induce ARDS and the most common cause of ARDS is sepsis resulting from bacterial infection [3]. Current treatments such as surfactants, glucocorticoids, and stem cells do not significantly reduce lung injury and mortality [4]. Therefore, the developments of new and effective strategies to treat ARDS are urgently needed. LPS, the outer membrane of gram-negative bacteria, has been reported to be an important risk factor of ALI [5–7]. LPS stimulates alveolar macrophages to induce TLR4 activation, which finally induces the production of inflammatory cytokines, such as TNF-α, IL-6 and IL-1β [8–10]. These cytokines amplify the inflammatory responses and lung injury [11]. The pathophysiological mechanism of ARDS is believed to be associated with the uncontrolled inflammatory response in lungs [12]. Nowadays, studies showed that treatments aimed at modulating TLR4 signaling pathway to alleviate inflammatory response may have potential therapeutic advantages for ARDS [13]. Scoparone, a major component of the shoot of Artemisia capillaris, has been reported to have anti-inflammatory and anti-tumor effects [14,15]. Several studies showed that scoparone inhibited IL-8 and MCP-1 production in U937 cells and TNF-α, IL-6 and IL-1β production in LPS-stimulated RAW264.7 cells [16,17]. Furthermore, scoparone ⁎ Corresponding author. E-mail address: [email protected] (N. Niu).
http://dx.doi.org/10.1016/j.intimp.2014.08.014 1567-5769/© 2014 Elsevier B.V. All rights reserved.
was found to have a protective effect on D-galactosamine/lipopolysaccharide-induced hepatic failure in mice [18]. However, the antiinflammatory effect of scoparone on LPS-induced ALI has not been reported. The aim of this study was to investigate the protective effects and molecular mechanisms of scoparone on lipopolysaccharide (LPS)induced acute lung injury.
2. Materials and methods 2.1. Reagents Scoparone and LPS (Escherichia coli 055:B5) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mouse TNF-α, IL-6 and IL-1β assay kits were purchased from R&D Systems (Minneapolis, MN). Anti-pNF-κB p65, anti-NF-κB p65, anti-TLR4, and anti-β-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). All other chemicals were of reagent grade.
2.2. Animals Male BALB/c mice weighing 18–22 g were obtained from the Center of Experimental Animals of Peking University (Beijing, China). The mice were maintained in a pathogen-free and light-controlled room (12 h light and 12 h dark) with free access to food and water. All animal experiments were performed in accordance with the Health's Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health.
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imbedded in paraffin and sliced. The sections stained with hematoxylin and eosin (H&E) stain. Then pathological changes of lung tissues were observed under a light microscope. 2.6. Inflammatory cell counts of BALF The BALF samples were centrifuged (4 °C, 3000 rpm, 10 min) to pellet the cells. The cell pellets were resuspended in PBS for total cell counts using a hemacytometer. We prepared cytospins for differential cell counts by staining using the Wright–Giemsa staining method. 2.7. Lung wet to dry lung weight ratio (W/D ratio) measurement
Fig. 1. Effects of scoparone on LPS-induced lung edema. The lung edema was assessed by the lung W/D ratio. Data were presented as means ± SEM (n = 12 in each group). #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
The left lungs were obtained immediately and the wet weight was determined. The lungs were placed in an oven at 80 °C for 24 h to obtain the dry weight. The ratio of the wet lung to the dry lung was calculated to assess tissue edema.
2.3. Experimental design and grouping
2.8. qRT-PCR assay for CXCL1, CXCL2 and CCL2 expressions
All mice were randomly divided into six groups: Control, LPS, LPS + scoparone (20, 40 and 80 mg/kg) and LPS + DEX group. Scoparone (20, 40 and 80 mg/kg) and DEX (5 mg/kg) were given intraperitoneally. 1 h later, mice were slightly anesthetized with an inhalation of diethyl ether, 10 μg of LPS in 50 μl PBS was instilled intranasal (i.n.) to induce lung injury.
Total RNA from mammary gland tissues was extracted using TRIzol reagent. Then the RNA was reversed transcribed following the manufacturer's instructions of first strand cDNA synthesis kit from Fermentas (Burlington, Canada). The primers were: CXCL1, sense 5′GCCTATCGCCAATGAGCT-3′and antisense 5′-TGACTTCGGTTTGGGTGC3′; CXCL2 sense 5′-ACCAACCACCAGGCTACA-3′and antisense 5′-CTTC AGGGTCAAGGCAAA-3′; CCL2, sense 5′-TGGGTCCAGACATACATT-3′ and antisense 5′- ACGGGTCAACTTCACATT-3′; β-actin, sense 5′-TAAA ACGCAGCTCAGTAACAGTCG-3′and antisense 5′-TGCAATCCTGTGGCAT CCATGAAAC-3′. The mRNA expression levels were evaluated by qRTPCR using the SYBR Green QuantiTect RT-PCR kit (TaKaRa Biotechnology Co., Ltd) and the 7500 Fast Real-Time PCR System (applied Biosystems). The relative expression of each gene was normalized to β-actin.
2.4. MPO activity assay Lung tissues were homogenized in 50 mM HEPES and subjected to three-thaw cycles. Then the homogenate was centrifuged at 13,000 g for 30 min and used for MPO assay. MPO activity was detected using test kits purchased from Nanjing Jiancheng Bioengineering Institute (China) according to the instructions.
2.9. Cell culture and treatment 2.5. Histopathologic evaluation of the lung tissue The lungs were excised and fixed in 4% paraformaldehyde in 0.1 M PBS (Ph 7.4) for 24 h. Then lung tissue was dehydrated with alcohol,
Murine alveolar macrophages were isolated as described by Liu et al. with some modifications [19]. The lungs were lavaged with fluids and then centrifuged at 1000 g for 10 min. The cells were
Fig. 2. Effects of scoparone on histopathological changes in lung tissues. Mice were administrated with scoparone 1 h before intratracheal instillation of LPS. The histopathological assays (200× magnification) in lungs were performed at 7 h after LPS instillation. A: Control group, B: LPS group, C: LPS + DEX group, D: LPS + scoparone (20 mg/kg) group, E: LPS + scoparone (40 mg/kg) group F: LPS + scoparone (80 mg/kg) group.
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resuspended and plated. 4 h later, the macrophages were washed once a day for 3 days. The nonadherent cells were removed. The attached cells were used as alveolar macrophages. The cells were cultured in RPMI 1640 with 10% FBS. Alveolar macrophages were incubated in the presence or absence of scoparone that was always added 1 h prior to LPS (1 μg/mL) treatment.
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3. Results 3.1. Effects of scoparone on LPS-induced lung wet/dry weight ratio in mice As shown in Fig. 1, LPS challenge produced a significant increase in the lung wet/dry weight ratio compared to the control group. Scoparone and DEX significantly reduced the lung wet/dry weight ratio compared to those in the LPS group (P b 0.05).
2.10. Cell viability assay 3.2. Effects of scoparone on LPS-mediated lung histopathologic changes MTT assay was used to evaluate the cytotoxicity of scoparone on alveolar macrophages. The cells were treated with 50 μl of scoparone at different concentrations (0–100 μM) for 12 h, followed by stimulation with 50 μl LPS for 12 h. MTT solution (2.0 mg/ml) was added to each well. 4 h later, formazan crystals formed in viable cells were dissolved with DMSO. The optical density was measured at 570 nm on a microplate reader (TECAN, Austria).
Lung histological changes were detected in this study. As shown in Fig. 2A, normal pulmonary histology was seen in the control group.
2.11. ELISA assay The concentrations of TNF-α, IL-1β and IL-6 in the BALF and cell-free supernatants were detected using sandwich enzyme-linked immunosorbent assay (ELISA) kits according to the protocol recommended by the manufacturer.
2.12. Western blot analysis Total proteins from cells were extracted by Total Protein Extraction Kit (BestBio). Protein concentration was determined through BCA method. Equal amounts of protein were fractionated on 12% polyacrylamideSDS gel. Then proteins were transferred to polyvinylidene difluoride membrane. Then the membrane was blocked by 5% nonfat dry milk for 2 h at room temperature followed by primary antibody (1:1000) overnight at 4 °C. Subsequently, the membrane was treated with the secondary antibody for 2 h. Blots were then developed with the ECL Plus Western Blotting Detection System (Amersham Life Science, UK).
2.13. Statistical analysis All values are expressed as means ± S.E.M. Differences between mean values of normally distributed data were analyzed using oneway ANOVA (Dunnett's t-test) and two-tailed Student's t-test. Statistical significance was accepted P b 0.05 or P b 0.01.
Fig. 3. Effects of scoparone on LPS-induced lung MPO activity. The values presented are the mean ± SEM (n = 12 in each group). #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
Fig. 4. Effects of scoparone on the number of total cells, neutrophils, and macrophages in the BALF of LPS-induced ALI mice. BALF was collected at 7 h after LPS administration to measure the number of total cells, neutrophils, and macrophages. The values presented are the mean ± SEM (n = 12 in each group). #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
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Lung tissues from LPS group were significantly damaged, including interstitial edema, hyperemia, thickening of the alveolar wall and infiltration of inflammatory cells (Fig. 2B). However, these histological changes were attenuated by scoparone and DEX treatment (Fig. 2C, D, E, F).
3.4. Effects of scoparone on inflammatory cell count in the BALF
MPO activity was determined 7 h after LPS administration. As shown in Fig. 3, LPS administration (5.68 ± 0.53 U/g) significantly increased MPO activity in comparison to control group (1.85 ± 0.10 U/g). However, scoparone treatment significantly inhibited MPO activity (4.83 ± 0.61, 3.92 ± 0.38, 2.93 ± 0.24 U/g) induced by LPS.
The number of inflammatory cells, such as neutrophils and macrophages, in BALF were detected. As shown in Fig. 4, LPS challenge significantly increased the number of total cells (13.4 ± 1.2 × 108/ml), neutrophils (8.3 ± 0.7 × 108/ml) and macrophages (4.9 ± 0.4 × 10 8 /ml) compared with the control group (P b 0.01). Meanwhile, scoparone was found to significantly decrease the number of total cells (11.6 ± 1.1 × 108/ml, 8.5 ± 0.8 × 108/ml, 6.1 ± 0.9 × 108/ml) (P b 0.01 or P b 0.05), neutrophils (7.4 ± 0.7 × 108/ml, 5.2 ± 0.6 × 108/ml, 4.0 ± 0.5 × 108/ml) (P b 0.01 or P b 0.05), and macrophages (4.1 ± 0.2 × 108/ml, 3.3 ± 0.4 × 108/ml, 2.1 ± 0.3 × 108/ml) (P b 0.01 or P b 0.05) in a dose-dependent manner (Fig. 4).
Fig. 5. Effects of scoparone on LPS-induced pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 production in the BALF. BALF was collected at 7 h following LPS challenge to analyze the inflammatory cytokines TNF-α, IL-1ß, and IL-6. The values presented are mean ± SEM (n = 12 in each group). #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
Fig. 6. Effects of scoparone on LPS-induced chemokine CXCL1, CXCL2 and CCL2 expressions in mammary tissues. The values presented are mean ± SEM (n = 12 in each group). #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
3.3. Effects of scoparone on MPO activity
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IL-6 (657.05 ± 49.28 pg/ml) and IL-1β (914.83 ± 104.20 pg/ml) increased significantly in LPS-stimulated alveolar macrophages. However, the production of TNF-α (1894.07 ± 110.92, 1174.38 ± 63.24, 612.44 ± 51.24 pg/ml), IL-6 (471.63 ± 54.12, 277.25 ± 39.02, 121.92 ± 17.03 pg/ml) and IL-1β (623.58 ± 115.24, 473.29 ± 73.02, 302.84 ± 44.09 pg/ml) were significantly inhibited in a dosedependent manner when the cells were treated with scoparone.
3.9. Effects of scoparone on LPS-induced NF-κB activation
Fig. 7. Effect of scoparone on the cell viability of alveolar macrophages. Cells were cultured with different concentrations of scoparone (0–100 μM) in the absence or presence of 1 mg/L LPS for 24 h. The cell viability was determined by MTT assay. The values presented are the means ± SEM of three independent experiments.
To test the mechanism by which scoparone inhibits LPS-induced cytokine production, the effects of scoparone on LPS-induced TLR4 expression and NF-κB activation were determined by Western blotting. As shown in Fig. 9, our results showed that scoparone significantly inhibited LPS-induced TLR4 expression, NF-κB activation and IκBα degradation.
3.5. Effects of scoparone on cytokine production in the BALF
4. Discussion
BALF was collected at 7 h after LPS administration and the cytokine levels in BALF were measured by ELISA. As shown in Fig. 5, LPS administration significantly increased TNF-α (10132.88 ± 962.34 pg/ml), IL-6 (1451.35 ± 105.25 pg/ml) and IL-1β (1523.07 ± 92.78 pg/ml) production in comparison to control group. However, scoparone inhibited TNF-α (7929.27 ± 773.25, 5880.92 ± 675.32, 3720.03 ± 323.07 pg/ml), IL-6 (1184.01 ± 107.71, 870.93 ± 93.22, 703.64 ± 132.13 pg/ml) and IL-1β (1232.21 ± 77.43, 845.83 ± 64.39, 529.82 ± 63.25 pg/ml) production induced by LPS in a dose-dependent manner (Fig. 5).
The acute respiratory distress syndrome is an important cause of acute respiratory failure that is characterized by overwhelming lung inflammation [20]. Scoparone, a major component of the shoot of Artemisia capillaris, has been reported to have anti-inflammatory effects [16,17]. In this study we found that scoparone had a protective effect on LPS-induced ALI. The anti-inflammatory mechanism of scoparone is by suppression of TLR4-mediated NF-κB signaling pathways. Excessive neutrophil activation and accumulation into the alveolar space is the characteristic of pulmonary inflammation [21]. MPO activity in the parenchyma reflects the adhesion and margination of neutrophils in the lung [22]. In the present study, scoparone treatment inhibited neutrophil and macrophage accumulation in the lungs. CXCL1, CXCL2 and CCL2 are important chemoattractants for macrophage and neutrophil during inflammation [23,24]. Previous studies showed that LPS could up-regulate the expression of CXCL1, CXCL2 and CCL2 which were responsible for macrophage and neutrophil margination in the lung [25]. Thus, the inhibition of macrophage and neutrophil accumulation in the lung may be related to the inhibition of CXCL1, CXCL2 and CCL2 expressions by scoparone. Furthermore, histological analysis showed that scoparone attenuated lung tissue injury induced by LPS. These results suggested that scoparone had a protective effect on LPS-induced ALI. Alveolar macrophages have been reported to play vital roles in lung inflammation. Stimulating alveolar macrophages with LPS could induce inflammatory cytokines such as TNF-α, IL-1β and IL-6 production [26, 27]. These cytokines are crucial mediators in a range of acute and chronic responses to inflammatory diseases. While TNF-α, IL-1β and IL-6, as the principal pro-inflammatory cytokines, are involved in the pathophysiology of endotoxin induced ALI [28]. These cytokines are important predictors of morbidity in patients with ARDS. In the present study, scoparone treatment attenuated lung inflammation by inhibition pro-inflammatory cytokines TNF-α, IL-1β and IL-6 production. These results appear to correlate well with a previous report that scoparone decreased TNF-α, IL-1β and IL-6 production in LPS-stimulated
3.6. Effects of scoparone on chemokines CXCL1, CXCL2, and CCL2 expression in mammary tissues The effects of scoparone on CXCL1, CXCL2 and CCL2 expressions were detected by qRT-PCR. As shown in Fig. 6, LPS administration significantly increased CXCL1 (13.87 ± 0.73), CXCL2 (19.07 ± 1.24) and CCL2 (9.89 ± 0.75) expressions in comparison to control group. However, scoparone inhibited CXCL1 (9.78 ± 0.61, 6.91 ± 0.69, 3.25 ± 0.43), CXCL2 (15.79 ± 0.91, 8.91 ± 1.35, 4.65 ± 0.62) and CCL2 (7.78 ± 0.82, 4.92 ± 0.68, 2.65 ± 0.71) expressions induced by LPS in a dose-dependent manner (Fig. 6). 3.7. Effects of scoparone on cell viability The potential cytotoxicity of scoparone was evaluated by MTT assay. The results showed that scoparone at concentrations from 25 to 100 μM had no cytotoxic effect on alveolar macrophages (Fig. 7). 3.8. Effects of scoparone on cytokine production in LPS-stimulated alveolar macrophages TNF-α, IL-6 and IL-1β concentrations in the culture were detected by ELISA. As shown in Fig. 8, the levels of TNF-α (2385.17 ± 139.03 pg/ml),
Fig. 8. Effects of scoparone on cytokines TNF-α, IL-1ß, and IL-6 production in LPS-stimulated alveolar macrophages. Data were presented as means ± SEM of three independent experiments. #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
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Fig. 9. Scoparone inhibits lipopolysaccharide (LPS)-induced TLR4 expression and NF-κB activation. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Students's t-test. #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
RAW264.7 cells [16]. These results suggested that scoparone may have the ability to treat LPS-induced inflammatory diseases. TLR4, a key pattern recognition receptor (PRR), has been reported to play an important role in LPS-induced inflammatory response [29]. TLR4 responds to LPS and triggers the activation of NF-κB signaling pathways [9]. Activated NF-κB signaling pathway regulates the inflammatory process by promoting the production of pro-inflammatory cytokines, such as NF-α, IL-1β and IL-6 [30-32]. In this study, we found that scoparone inhibited cytokine production by suppressing TLR4mediated NF-κB activation. Recently, TLR4 signaling has been related to many inflammatory diseases such as colitis, liver injury and neurodegenerative diseases [33-35]. Treatments aimed at modulating TLR4 signaling pathway may have potential therapeutic advantages for these inflammatory diseases. Thus, scoparone may have the ability to treat these diseases. In conclusion, our results showed that scoparone had an antiinflammatory effect against LPS-induced ALI both in vivo and in vitro. The anti-inflammatory mechanism of scoparone may be due to its ability to inhibition of TLR4-mediated NF-κB signaling pathways. Scoparone may be a potential therapeutic reagent for acute lung injury treatment. Further studies are warranted to investigate the clinical usefulness of scoparone.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Protective effects of scoparone against lipopolysaccharide-induced acute lung injury Niu Niu ⁎, Baolan Li, Ying Hu, Xuebing Li, Jie Li, Haiqing Zhang Beijing Tuberculosis and Thoracic Tumor Research Institute, Department of internal medicine, Beijing hest Hospital, CMMU, Beijing 101149, China
a r t i c l e
i n f o
Article history: Received 16 June 2014 Received in revised form 28 July 2014 Accepted 13 August 2014 Available online 22 August 2014 Keywords: Scoparone LPS Acute lung injury NF-κB TLR4
a b s t r a c t The purpose of this study was to investigate the protective effects and molecular mechanisms of scoparone on lipopolysaccharide (LPS)-induced acute lung injury in mice. Mice model of acute lung injury (ALI), induced by intranasal instillation of LPS, was used to investigate the protective effects of scoparone in vivo. The alveolar macrophages were used to investigate the molecular mechanisms of scoparone in vitro. The results showed that scoparone treatment remarkably attenuated LPS-induced pulmonary edema, histological severities, myeloperoxidase activity, and TNF-α, IL-6 and IL-1β production in vivo. We also found that scoparone inhibited LPS-induced TLR4 expression, NF-κB activation, TNF-α, IL-6 and IL-1β production in alveolar macrophages in vitro. In conclusion, our results suggest that scoparone has a protective effect on LPS-induced ALI via suppression of TLR4-mediated NF-κB signaling pathways. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Acute respiratory distress syndrome (ARDS) is characterized by overwhelming lung inflammation and increased microvascular permeability [1]. ARDS often results in multi-organ failure with high mortality in critically ill patients [2]. Most causes such as sepsis, trauma and burn could induce ARDS and the most common cause of ARDS is sepsis resulting from bacterial infection [3]. Current treatments such as surfactants, glucocorticoids, and stem cells do not significantly reduce lung injury and mortality [4]. Therefore, the developments of new and effective strategies to treat ARDS are urgently needed. LPS, the outer membrane of gram-negative bacteria, has been reported to be an important risk factor of ALI [5–7]. LPS stimulates alveolar macrophages to induce TLR4 activation, which finally induces the production of inflammatory cytokines, such as TNF-α, IL-6 and IL-1β [8–10]. These cytokines amplify the inflammatory responses and lung injury [11]. The pathophysiological mechanism of ARDS is believed to be associated with the uncontrolled inflammatory response in lungs [12]. Nowadays, studies showed that treatments aimed at modulating TLR4 signaling pathway to alleviate inflammatory response may have potential therapeutic advantages for ARDS [13]. Scoparone, a major component of the shoot of Artemisia capillaris, has been reported to have anti-inflammatory and anti-tumor effects [14,15]. Several studies showed that scoparone inhibited IL-8 and MCP-1 production in U937 cells and TNF-α, IL-6 and IL-1β production in LPS-stimulated RAW264.7 cells [16,17]. Furthermore, scoparone ⁎ Corresponding author. E-mail address: [email protected] (N. Niu).
http://dx.doi.org/10.1016/j.intimp.2014.08.014 1567-5769/© 2014 Elsevier B.V. All rights reserved.
was found to have a protective effect on D-galactosamine/lipopolysaccharide-induced hepatic failure in mice [18]. However, the antiinflammatory effect of scoparone on LPS-induced ALI has not been reported. The aim of this study was to investigate the protective effects and molecular mechanisms of scoparone on lipopolysaccharide (LPS)induced acute lung injury.
2. Materials and methods 2.1. Reagents Scoparone and LPS (Escherichia coli 055:B5) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mouse TNF-α, IL-6 and IL-1β assay kits were purchased from R&D Systems (Minneapolis, MN). Anti-pNF-κB p65, anti-NF-κB p65, anti-TLR4, and anti-β-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). All other chemicals were of reagent grade.
2.2. Animals Male BALB/c mice weighing 18–22 g were obtained from the Center of Experimental Animals of Peking University (Beijing, China). The mice were maintained in a pathogen-free and light-controlled room (12 h light and 12 h dark) with free access to food and water. All animal experiments were performed in accordance with the Health's Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health.
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imbedded in paraffin and sliced. The sections stained with hematoxylin and eosin (H&E) stain. Then pathological changes of lung tissues were observed under a light microscope. 2.6. Inflammatory cell counts of BALF The BALF samples were centrifuged (4 °C, 3000 rpm, 10 min) to pellet the cells. The cell pellets were resuspended in PBS for total cell counts using a hemacytometer. We prepared cytospins for differential cell counts by staining using the Wright–Giemsa staining method. 2.7. Lung wet to dry lung weight ratio (W/D ratio) measurement
Fig. 1. Effects of scoparone on LPS-induced lung edema. The lung edema was assessed by the lung W/D ratio. Data were presented as means ± SEM (n = 12 in each group). #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
The left lungs were obtained immediately and the wet weight was determined. The lungs were placed in an oven at 80 °C for 24 h to obtain the dry weight. The ratio of the wet lung to the dry lung was calculated to assess tissue edema.
2.3. Experimental design and grouping
2.8. qRT-PCR assay for CXCL1, CXCL2 and CCL2 expressions
All mice were randomly divided into six groups: Control, LPS, LPS + scoparone (20, 40 and 80 mg/kg) and LPS + DEX group. Scoparone (20, 40 and 80 mg/kg) and DEX (5 mg/kg) were given intraperitoneally. 1 h later, mice were slightly anesthetized with an inhalation of diethyl ether, 10 μg of LPS in 50 μl PBS was instilled intranasal (i.n.) to induce lung injury.
Total RNA from mammary gland tissues was extracted using TRIzol reagent. Then the RNA was reversed transcribed following the manufacturer's instructions of first strand cDNA synthesis kit from Fermentas (Burlington, Canada). The primers were: CXCL1, sense 5′GCCTATCGCCAATGAGCT-3′and antisense 5′-TGACTTCGGTTTGGGTGC3′; CXCL2 sense 5′-ACCAACCACCAGGCTACA-3′and antisense 5′-CTTC AGGGTCAAGGCAAA-3′; CCL2, sense 5′-TGGGTCCAGACATACATT-3′ and antisense 5′- ACGGGTCAACTTCACATT-3′; β-actin, sense 5′-TAAA ACGCAGCTCAGTAACAGTCG-3′and antisense 5′-TGCAATCCTGTGGCAT CCATGAAAC-3′. The mRNA expression levels were evaluated by qRTPCR using the SYBR Green QuantiTect RT-PCR kit (TaKaRa Biotechnology Co., Ltd) and the 7500 Fast Real-Time PCR System (applied Biosystems). The relative expression of each gene was normalized to β-actin.
2.4. MPO activity assay Lung tissues were homogenized in 50 mM HEPES and subjected to three-thaw cycles. Then the homogenate was centrifuged at 13,000 g for 30 min and used for MPO assay. MPO activity was detected using test kits purchased from Nanjing Jiancheng Bioengineering Institute (China) according to the instructions.
2.9. Cell culture and treatment 2.5. Histopathologic evaluation of the lung tissue The lungs were excised and fixed in 4% paraformaldehyde in 0.1 M PBS (Ph 7.4) for 24 h. Then lung tissue was dehydrated with alcohol,
Murine alveolar macrophages were isolated as described by Liu et al. with some modifications [19]. The lungs were lavaged with fluids and then centrifuged at 1000 g for 10 min. The cells were
Fig. 2. Effects of scoparone on histopathological changes in lung tissues. Mice were administrated with scoparone 1 h before intratracheal instillation of LPS. The histopathological assays (200× magnification) in lungs were performed at 7 h after LPS instillation. A: Control group, B: LPS group, C: LPS + DEX group, D: LPS + scoparone (20 mg/kg) group, E: LPS + scoparone (40 mg/kg) group F: LPS + scoparone (80 mg/kg) group.
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resuspended and plated. 4 h later, the macrophages were washed once a day for 3 days. The nonadherent cells were removed. The attached cells were used as alveolar macrophages. The cells were cultured in RPMI 1640 with 10% FBS. Alveolar macrophages were incubated in the presence or absence of scoparone that was always added 1 h prior to LPS (1 μg/mL) treatment.
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3. Results 3.1. Effects of scoparone on LPS-induced lung wet/dry weight ratio in mice As shown in Fig. 1, LPS challenge produced a significant increase in the lung wet/dry weight ratio compared to the control group. Scoparone and DEX significantly reduced the lung wet/dry weight ratio compared to those in the LPS group (P b 0.05).
2.10. Cell viability assay 3.2. Effects of scoparone on LPS-mediated lung histopathologic changes MTT assay was used to evaluate the cytotoxicity of scoparone on alveolar macrophages. The cells were treated with 50 μl of scoparone at different concentrations (0–100 μM) for 12 h, followed by stimulation with 50 μl LPS for 12 h. MTT solution (2.0 mg/ml) was added to each well. 4 h later, formazan crystals formed in viable cells were dissolved with DMSO. The optical density was measured at 570 nm on a microplate reader (TECAN, Austria).
Lung histological changes were detected in this study. As shown in Fig. 2A, normal pulmonary histology was seen in the control group.
2.11. ELISA assay The concentrations of TNF-α, IL-1β and IL-6 in the BALF and cell-free supernatants were detected using sandwich enzyme-linked immunosorbent assay (ELISA) kits according to the protocol recommended by the manufacturer.
2.12. Western blot analysis Total proteins from cells were extracted by Total Protein Extraction Kit (BestBio). Protein concentration was determined through BCA method. Equal amounts of protein were fractionated on 12% polyacrylamideSDS gel. Then proteins were transferred to polyvinylidene difluoride membrane. Then the membrane was blocked by 5% nonfat dry milk for 2 h at room temperature followed by primary antibody (1:1000) overnight at 4 °C. Subsequently, the membrane was treated with the secondary antibody for 2 h. Blots were then developed with the ECL Plus Western Blotting Detection System (Amersham Life Science, UK).
2.13. Statistical analysis All values are expressed as means ± S.E.M. Differences between mean values of normally distributed data were analyzed using oneway ANOVA (Dunnett's t-test) and two-tailed Student's t-test. Statistical significance was accepted P b 0.05 or P b 0.01.
Fig. 3. Effects of scoparone on LPS-induced lung MPO activity. The values presented are the mean ± SEM (n = 12 in each group). #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
Fig. 4. Effects of scoparone on the number of total cells, neutrophils, and macrophages in the BALF of LPS-induced ALI mice. BALF was collected at 7 h after LPS administration to measure the number of total cells, neutrophils, and macrophages. The values presented are the mean ± SEM (n = 12 in each group). #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
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Lung tissues from LPS group were significantly damaged, including interstitial edema, hyperemia, thickening of the alveolar wall and infiltration of inflammatory cells (Fig. 2B). However, these histological changes were attenuated by scoparone and DEX treatment (Fig. 2C, D, E, F).
3.4. Effects of scoparone on inflammatory cell count in the BALF
MPO activity was determined 7 h after LPS administration. As shown in Fig. 3, LPS administration (5.68 ± 0.53 U/g) significantly increased MPO activity in comparison to control group (1.85 ± 0.10 U/g). However, scoparone treatment significantly inhibited MPO activity (4.83 ± 0.61, 3.92 ± 0.38, 2.93 ± 0.24 U/g) induced by LPS.
The number of inflammatory cells, such as neutrophils and macrophages, in BALF were detected. As shown in Fig. 4, LPS challenge significantly increased the number of total cells (13.4 ± 1.2 × 108/ml), neutrophils (8.3 ± 0.7 × 108/ml) and macrophages (4.9 ± 0.4 × 10 8 /ml) compared with the control group (P b 0.01). Meanwhile, scoparone was found to significantly decrease the number of total cells (11.6 ± 1.1 × 108/ml, 8.5 ± 0.8 × 108/ml, 6.1 ± 0.9 × 108/ml) (P b 0.01 or P b 0.05), neutrophils (7.4 ± 0.7 × 108/ml, 5.2 ± 0.6 × 108/ml, 4.0 ± 0.5 × 108/ml) (P b 0.01 or P b 0.05), and macrophages (4.1 ± 0.2 × 108/ml, 3.3 ± 0.4 × 108/ml, 2.1 ± 0.3 × 108/ml) (P b 0.01 or P b 0.05) in a dose-dependent manner (Fig. 4).
Fig. 5. Effects of scoparone on LPS-induced pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 production in the BALF. BALF was collected at 7 h following LPS challenge to analyze the inflammatory cytokines TNF-α, IL-1ß, and IL-6. The values presented are mean ± SEM (n = 12 in each group). #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
Fig. 6. Effects of scoparone on LPS-induced chemokine CXCL1, CXCL2 and CCL2 expressions in mammary tissues. The values presented are mean ± SEM (n = 12 in each group). #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
3.3. Effects of scoparone on MPO activity
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IL-6 (657.05 ± 49.28 pg/ml) and IL-1β (914.83 ± 104.20 pg/ml) increased significantly in LPS-stimulated alveolar macrophages. However, the production of TNF-α (1894.07 ± 110.92, 1174.38 ± 63.24, 612.44 ± 51.24 pg/ml), IL-6 (471.63 ± 54.12, 277.25 ± 39.02, 121.92 ± 17.03 pg/ml) and IL-1β (623.58 ± 115.24, 473.29 ± 73.02, 302.84 ± 44.09 pg/ml) were significantly inhibited in a dosedependent manner when the cells were treated with scoparone.
3.9. Effects of scoparone on LPS-induced NF-κB activation
Fig. 7. Effect of scoparone on the cell viability of alveolar macrophages. Cells were cultured with different concentrations of scoparone (0–100 μM) in the absence or presence of 1 mg/L LPS for 24 h. The cell viability was determined by MTT assay. The values presented are the means ± SEM of three independent experiments.
To test the mechanism by which scoparone inhibits LPS-induced cytokine production, the effects of scoparone on LPS-induced TLR4 expression and NF-κB activation were determined by Western blotting. As shown in Fig. 9, our results showed that scoparone significantly inhibited LPS-induced TLR4 expression, NF-κB activation and IκBα degradation.
3.5. Effects of scoparone on cytokine production in the BALF
4. Discussion
BALF was collected at 7 h after LPS administration and the cytokine levels in BALF were measured by ELISA. As shown in Fig. 5, LPS administration significantly increased TNF-α (10132.88 ± 962.34 pg/ml), IL-6 (1451.35 ± 105.25 pg/ml) and IL-1β (1523.07 ± 92.78 pg/ml) production in comparison to control group. However, scoparone inhibited TNF-α (7929.27 ± 773.25, 5880.92 ± 675.32, 3720.03 ± 323.07 pg/ml), IL-6 (1184.01 ± 107.71, 870.93 ± 93.22, 703.64 ± 132.13 pg/ml) and IL-1β (1232.21 ± 77.43, 845.83 ± 64.39, 529.82 ± 63.25 pg/ml) production induced by LPS in a dose-dependent manner (Fig. 5).
The acute respiratory distress syndrome is an important cause of acute respiratory failure that is characterized by overwhelming lung inflammation [20]. Scoparone, a major component of the shoot of Artemisia capillaris, has been reported to have anti-inflammatory effects [16,17]. In this study we found that scoparone had a protective effect on LPS-induced ALI. The anti-inflammatory mechanism of scoparone is by suppression of TLR4-mediated NF-κB signaling pathways. Excessive neutrophil activation and accumulation into the alveolar space is the characteristic of pulmonary inflammation [21]. MPO activity in the parenchyma reflects the adhesion and margination of neutrophils in the lung [22]. In the present study, scoparone treatment inhibited neutrophil and macrophage accumulation in the lungs. CXCL1, CXCL2 and CCL2 are important chemoattractants for macrophage and neutrophil during inflammation [23,24]. Previous studies showed that LPS could up-regulate the expression of CXCL1, CXCL2 and CCL2 which were responsible for macrophage and neutrophil margination in the lung [25]. Thus, the inhibition of macrophage and neutrophil accumulation in the lung may be related to the inhibition of CXCL1, CXCL2 and CCL2 expressions by scoparone. Furthermore, histological analysis showed that scoparone attenuated lung tissue injury induced by LPS. These results suggested that scoparone had a protective effect on LPS-induced ALI. Alveolar macrophages have been reported to play vital roles in lung inflammation. Stimulating alveolar macrophages with LPS could induce inflammatory cytokines such as TNF-α, IL-1β and IL-6 production [26, 27]. These cytokines are crucial mediators in a range of acute and chronic responses to inflammatory diseases. While TNF-α, IL-1β and IL-6, as the principal pro-inflammatory cytokines, are involved in the pathophysiology of endotoxin induced ALI [28]. These cytokines are important predictors of morbidity in patients with ARDS. In the present study, scoparone treatment attenuated lung inflammation by inhibition pro-inflammatory cytokines TNF-α, IL-1β and IL-6 production. These results appear to correlate well with a previous report that scoparone decreased TNF-α, IL-1β and IL-6 production in LPS-stimulated
3.6. Effects of scoparone on chemokines CXCL1, CXCL2, and CCL2 expression in mammary tissues The effects of scoparone on CXCL1, CXCL2 and CCL2 expressions were detected by qRT-PCR. As shown in Fig. 6, LPS administration significantly increased CXCL1 (13.87 ± 0.73), CXCL2 (19.07 ± 1.24) and CCL2 (9.89 ± 0.75) expressions in comparison to control group. However, scoparone inhibited CXCL1 (9.78 ± 0.61, 6.91 ± 0.69, 3.25 ± 0.43), CXCL2 (15.79 ± 0.91, 8.91 ± 1.35, 4.65 ± 0.62) and CCL2 (7.78 ± 0.82, 4.92 ± 0.68, 2.65 ± 0.71) expressions induced by LPS in a dose-dependent manner (Fig. 6). 3.7. Effects of scoparone on cell viability The potential cytotoxicity of scoparone was evaluated by MTT assay. The results showed that scoparone at concentrations from 25 to 100 μM had no cytotoxic effect on alveolar macrophages (Fig. 7). 3.8. Effects of scoparone on cytokine production in LPS-stimulated alveolar macrophages TNF-α, IL-6 and IL-1β concentrations in the culture were detected by ELISA. As shown in Fig. 8, the levels of TNF-α (2385.17 ± 139.03 pg/ml),
Fig. 8. Effects of scoparone on cytokines TNF-α, IL-1ß, and IL-6 production in LPS-stimulated alveolar macrophages. Data were presented as means ± SEM of three independent experiments. #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
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Fig. 9. Scoparone inhibits lipopolysaccharide (LPS)-induced TLR4 expression and NF-κB activation. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Students's t-test. #p b 0.01 compared to control group, *p b 0.05 and **p b 0.01 compared to LPS group.
RAW264.7 cells [16]. These results suggested that scoparone may have the ability to treat LPS-induced inflammatory diseases. TLR4, a key pattern recognition receptor (PRR), has been reported to play an important role in LPS-induced inflammatory response [29]. TLR4 responds to LPS and triggers the activation of NF-κB signaling pathways [9]. Activated NF-κB signaling pathway regulates the inflammatory process by promoting the production of pro-inflammatory cytokines, such as NF-α, IL-1β and IL-6 [30-32]. In this study, we found that scoparone inhibited cytokine production by suppressing TLR4mediated NF-κB activation. Recently, TLR4 signaling has been related to many inflammatory diseases such as colitis, liver injury and neurodegenerative diseases [33-35]. Treatments aimed at modulating TLR4 signaling pathway may have potential therapeutic advantages for these inflammatory diseases. Thus, scoparone may have the ability to treat these diseases. In conclusion, our results showed that scoparone had an antiinflammatory effect against LPS-induced ALI both in vivo and in vitro. The anti-inflammatory mechanism of scoparone may be due to its ability to inhibition of TLR4-mediated NF-κB signaling pathways. Scoparone may be a potential therapeutic reagent for acute lung injury treatment. Further studies are warranted to investigate the clinical usefulness of scoparone.
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