INTIMP-03769; No of Pages 6 International Immunopharmacology xxx (2015) xxx–xxx
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
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Suhong Bao a,b,c,1, Yun Zou c,1, Bing Wang c,1, Yinjiao Li d, Jiali Zhu c, Yan Luo d,⁎, Jinbao Li a,b,c,⁎⁎
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Article history: Received 15 February 2015 Received in revised form 31 May 2015 Accepted 16 June 2015 Available online xxxx
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Keywords: Ginsenoside Rg1 LPS ALI Inflammatory cytokines M2 macrophage
Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou 221004, Jiangsu, China Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou 221004, Jiangsu, China Department of Anesthesiology and Intensive Care, Changhai Hospital, Second Military Medical University, Shanghai 200433, China d Department of Anesthesiology, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200025, China b
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Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltration of M2 macrophages
Ginsenoside Rg1 (Rg1), the major effective component of ginseng, has been reported to have potent antiinflammatory properties. However, the effect of ginsenoside Rg1 on lipopolysaccharide (LPS) -induced acute lung injury (ALI) in mice was unknown. The present study was designed to investigate the protective role of Rg1 on LPS-induced ALI and explore the potential mechanisms. The mice were divided randomly into four groups: the sham group, the LPS group and the LPS + Rg1 (40 mg/kg or 200 mg/kg) pretreatment groups. All mice received Rg1 or an equivalent volume of phosphate buffer saline (PBS) intraperitoneally 1 h before LPS administration. Edema quantification, histology, and apoptosis were detected 6 h after LPS administration. The number of inflammatory cells, the percentage of alternative activated (M2) macrophages and the exudate quantification in bronchoalveolar lavage fluid (BALF) were evaluated. The caspase 3 expression, and the levels of phosphorylated IκB-α and p65 were tested. The results showed that the Rg1 pretreatment group markedly improved lung damage, modulated the infiltration of neutrophils and M2 macrophages, prevented the production of protein and proinflammatory cytokines in BALF, and inhibited apoptosis in lung. We also found that Rg1 suppressed NF-κB and caspase 3 activation. These data suggest that Rg1 plays a protective role against LPS-induced ALI by ameliorating inflammatory responses, regulating the infiltration of M2 macrophages, and inhibiting pulmonary cell apoptosis. © 2015 Published by Elsevier B.V.
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1. Introduction
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Acute lung injury (ALI) and the subsequent acute respiratory distress syndrome (ARDS) are the major causes of respiratory failure in the intensive care unit worldwide. Several clinical diseases or syndromes can trigger the occurrence of ALI, such as severe pneumonia, sepsis, and acute pancreatitis [1–3]. As many studies have evidenced, numerous neutrophils migrate to the lungs of patients who suffer from ALI and cause the damage to lung tissues [4–6], while reducing the infiltration of neutrophils or suppressing their activities could alleviate lung injuries [7,8]. Macrophages, the primary source of proinflammatory cytokines, display controversial roles in the progression of ALI. Previous studies demonstrated that the number of macrophages in alveoli was in accordance with the lung damage [9,10]. However, recent articles showed that a persistent decrease of alveolar macrophages was associated with the worse outcome: the animals may display
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⁎ Corresponding author. ⁎⁎ Correspondence to: J. Li, Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou 221004, Jiangsu, China. E-mail addresses: [email protected] (Y. Luo), [email protected] (J. Li). 1 Suhong Bao, Yun Zou and Bing Wang contributed equally to this work.
stronger inflammatory responses, once alveolar macrophages are cleared [11,12]. The inflammatory mediators (e.g., TNF-α and IL-1β) secreted from the inflammatory cells play a critical role in the pathogenesis of ARDS. The elevated proinflammatory cytokines in the airspaces of lungs result in the disruption of the alveolar structure and the occurrence of protein-rich pulmonary edema [13,14]. Therefore, a new therapeutic agent targeting the infiltration of immune cells and the inflammatory mediators is urgently required. Ginsenoside Rg1 (Rg1) is one of the major active ingredients purified from Panax ginseng. Previous studies demonstrated its powerful pharmacological effects against apoptosis and fibrosis in various animal models [15,16]. In the cardiomyocyte hypoxia-reoxygenation model, Rg1 is regarded as an antioxidant substance that could reduce the release of lactate dehydrogenase and intracellular ROS [17]. It can exert strong protection from ischemic reperfusion injuries in hepatocytes [18]. Furthermore, our previous experiments also found that Rg1 restrained inflammatory responses via downregulation of NF-κB activity [19–21]. These exciting effects indicate that ginsenoside Rg1 may have positive effects on ALI induced by LPS administration intratracheally. Therefore, our present study aimed to assess the influences of Rg1 in a murine ALI model and investigate its potential mechanisms.
http://dx.doi.org/10.1016/j.intimp.2015.06.022 1567-5769/© 2015 Published by Elsevier B.V.
Please cite this article as: S. Bao, et al., Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltra..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.06.022
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2. Materials and methods
2.6. Determination of protein and cytokines in bronchoalveolar lavage fluid 130
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2.1. Animals and reagents
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2.7. Quantification of apoptosis in the lung
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Male BALB/c mice, aged 6–8 weeks, were obtained from the Animal Experimentation Center of the Second Military Medical University. All mice were housed under specific pathogen-free conditions and fed a standard laboratory diet. The experiments were approved by the Institutional Animal Care and Use Committee of Changhai Hospital (Approval number CH20130723-18). Ginsenoside Rg1 (CAS 22427-39-0, purity N 98%, molecular formula C42H72O14) and LPS (Escherichia coli 055:B5) were purchased from Sigma–Aldrich Corporation (St. Louis, MO, USA) and dissolved in pathogen-free phosphate buffer saline (PBS).
The protein in the BALF was obtained through centrifugation (2000 rpm, 4 °C) for 5 min. The total protein concentration of the BALF was measured by a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The levels of TNF-α, IL-1β and IL-6 in the BALF were determined by ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.
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2.2. Experimental Design
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2.8. Western Blot Analysis
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2.3. Analysis of wet-to-dry weight ratio of lung tissues
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Pulmonary edema is one of the important characteristics of ALI. In the present study, lung tissues were removed and wet weights were recorded 6 hours after LPS administration. Lung tissues were then packed in a silver paper and placed in an incubator at 80 °C for 24 h to obtain the dry weights. The wet-to-dry ratio (W/D ratio) of lung tissues was calculated to evaluate the degree of pulmonary edema.
The lung tissues of all groups were homogenized in protein lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min. After centrifugation (12,000 rpm, 4 °C, 10 min), the protein concentration was measured by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were loaded in each well and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which subsequently was transferred onto a polyvinylidene difluoride (PVDF) membrane. After blockade of nonspecific binding sites, membranes were incubated for 2 h at room temperature with various antibodies against IκBα, phospho-IκBα, NF-κB p65, phospho-NF-κB p65 and caspase 3 (Cell Signal Technology, Danvers, MA, USA). Protein bands were demonstrated by an enhanced chemiluminescence (ECL) western blot kit (Thermo Fisher Scientific, Waltham, MA, USA). Band intensity was quantified using the Image J software. Protein expression was normalized to β-actin signals.
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All mice in the experiment were divided randomly into four groups: the sham group, the LPS group, and the LPS + ginsenoside Rg1 (40 mg/kg or 200 mg/kg) pretreatment groups (n = 6 in each group). PBS or Rg1 were given intraperitoneally according to the randomized treatment 1 h before LPS administration. Then all mice were anesthetized with an inhalation of sevoflurane, and LPS (20 μg in 50 μl PBS) was injected intratracheally to induce acute lung injury. Simultaneously, mice in the sham group received an equal volume of PBS instead of LPS. The mice were euthanized by CO2 inhalation 6 h after LPS administration. Bronchoalveolar lavage fluid (BALF) and lung tissues were harvested and preserved until use.
To assess the apoptosis in the lungs, a terminal deoxy-nucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed. We fixed the lung samples with 4% buffered paraformaldehyde for more than 24 h. Then, a FragELTM DNA Fragmentation Detection Kit (Merck Millipore, Billerica, MA, USA) was used to stain the apoptotic cells in the lung tissues. TUNEL positive cells were counted in the high-power field (HPF) under light microscopy (×400) by the IPP 6.0 software.
2.9. Statistical analysis
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All data were expressed as mean ± standard deviation (SD). The statistical analysis was performed using SPSS 16.0 Programs (SPSS Inc, USA). Graphs were plotted by Prism 5.0 (GraphPad Software, San Diego, CA). The differences among the different groups were analyzed by the one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. P b 0.05 was considered statistically significant.
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3. Results
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2.4. Histological examination
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Histopathologic examination of lungs was performed on mice that were not subjected to the BALF collection. The right upper lobes were collected for histologic examination. Tissues were fixed with 4% buffered paraformaldehyde for at least 24 h. Sections (4–5 μm) were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, and stained with hematoxylin and eosin (H&E). All sections were scored blindly by two experienced pathologists under a light microscope according to the criteria previously described [22]: 0, normal tissue; 1, minimal inflammatory change; 2, no obvious damage to the lung architecture; 3, thickening of the alveolar septae; 4, formation of nodules or areas of pneumonitis that distorted the normal architecture; and 5, total obliteration of the field.
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2.5. Cell counts of bronchoalveolar lavage fluid
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BALF was collected by washing the lungs twice with 0.5 ml of PBS via the tracheal cannula. Total cells in the BALF were calculated by a flow cytometer. Neutrophils as well as macrophages were also determined by a flow cytometer through cell-specific surface markers (Gr-1 for neutrophils and F4/80 for macrophages). To determine the percent of alternative activated (M2) macrophages in BALF, the percentage of F4/80+ CD206+ cells was detected. Data were obtained by a FACSCaliburTM flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by the Flowjo software.
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3.1. Ginsenoside Rg1 ameliorated lung damages induced by LPS challenge 170 In order to assess the severity of lung injuries, we examined the W/D ratio that reflects the formation of pulmonary edema. As shown in Fig. 1A, LPS injected intratracheally significantly increased the lung W/D ratio compared with the sham group (P b 0.01), while the Rg1 pretreatment group (40 mg/kg or 200 mg/kg) alleviated the lung W/D ratio compared with the LPS group (both P b 0.01). In parallel, LPS administration destroyed the structure of alveoli and promoted the infiltration of inflammatory cells into the lungs. In contrast, the Rg1 pretreatment group reduced the accumulation of infiltrated cells, and suppressed the thickening of alveolar walls (Fig. 1B). As shown in Fig. 1C, the scores which represent the lung injury decreased significantly after Rg1 administration.
Please cite this article as: S. Bao, et al., Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltra..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.06.022
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To explore the protective effect of Rg1 on ALI induced by LPS, we detected the counts of total cells, neutrophils and macrophages in BALF. As shown in Fig. 2A and B, the total cells and neutrophils in the BALF significantly increased after the LPS treatment. As expected, pretreatment with Rg1 reduced the number of total cells and
neutrophils in the BALF after LPS administration. On the contrary, the number of macrophages in the BALF decreased after the LPS challenge, whereas the Rg1 pretreatment group prevented the decline of macrophages after LPS administration intratracheally (Fig. 2C). We also calculated the percentage of CD206+ macrophages regarded as M2 macrophages. As Fig. 2D demonstrates the LPS challenge reduced the proportion of CD206+ macrophages, while the percentage of M2 macrophages increased after Rg1 administration. However,
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3.2. Effects of ginsenoside Rg1 on the infiltration of inflammatory cells in bronchoalveolar lavage fluid
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Fig. 1. Ginsenoside Rg1 ameliorated lung damages induced by LPS challenge. (A) Rg1 reduced lung wet/dry (W/D) weight ratio 6 h after LPS administration. (B) Representative images of H&E-stained lung sections from four experimental groups (×200). (C) The severity of lung injury was scored as described above. ⁎⁎P b 0.01 vs. sham group, ##P b 0.01 vs. LPS group.
Fig. 2. Effects of ginsenoside Rg1 on the infiltration of inflammatory cells in bronchoalveolar lavage fluid. We harvested BALF 6 h after LPS administration. (A and B) Rg1 decreased the number of total cells and neutrophils in the BALF. (C) Rg1 prevented the decline of the number of macrophages in the BALF. (D) Rg1 enhanced the percentage of M2 macrophages in BALF. ⁎⁎P b 0.01 vs. sham group, ##P b 0.01 vs. LPS group.
Please cite this article as: S. Bao, et al., Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltra..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.06.022
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Since there were no statistical differences between the two doses of Rg1 in the results mentioned above, we chose one dose of Rg1 (40 mg/kg) in the following experiments. The TUNEL assay was performed to detect apoptosis in the lungs subjected to an LPS challenge. As shown in Fig. 4, mice challenged with LPS experienced dramatic activation of apoptosis in the lungs. Under HPF examination, the apoptotic cells of the LPS group were 278.75 ± 20.74, while in the Rg1 pretreatment group, the number of apoptotic cells decreased significantly (205.5 ± 61.86). We also chose the variation of caspase 3 to support the conclusion about apoptosis. As expected, Rg1 inhibited the caspase 3 expression in the lung tissues after LPS administration.
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3.4. Ginsenoside Rg1 reduced the apoptotic cells of lungs in LPS-induced acute lung injury.
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ALI and its severe form, ARDS, are life-threatening syndromes that are characterized by uncontrolled inflammatory responses and infiltration of numerous activated cells. However, few effective treatments have been discovered to attenuate the injuries, leading to the high mortality of approximately 22% to 40% for ALI [23–25]. The data from our study revealed that LPS injected intratracheally induced pulmonary edema and destroyed the alveolar structure; however, ginsenoside Rg1 exerted protective effects on LPS-induced ALI, which was confirmed by the ameliorating W/D ratio and the degree of pathologic injuries (Fig. 1). As we demonstrated, modulating the infiltration of immune cells and inhibiting the excessive inflammatory responses were the potential mechanisms. According to an increasing number of studies, excessive neutrophils in the BALF contribute to epithelial and endothelial injury, which may lead eventually to vascular leakage and pulmonary edema [26,27]. Our results revealed that the number of neutrophils in the BALF increased significantly after an LPS challenge; nevertheless, Rg1 inhibited the influx of neutrophils induced by LPS. Besides neutrophils, alveolar macrophages (AMs) also have essential roles in ALI, including initiating and maintaining the inflammatory response and regulating the resolution of lung injury and repair [28,29]. Several studies showed that the number of macrophages in BALF were in parallel with the damage to lung tissues [9,10]. However, our results were the opposite of these previous
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4. Discussion
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As shown in Fig. 3A, compared with the sham group, the concentration of total protein in the BALF increased significantly 6 h after the LPS challenge, but the protein level decreased in the Rg1 administration groups. Furthermore, the levels of proinflammatory cytokines, including TNF-α, IL-1β and IL-6 in the BALF, were measured to evaluate the influence of Rg1 on inflammatory responses. As illustrated, the levels of the cytokines mentioned above dramatically increased in the BALF after LPS administration. Meanwhile, these conditions were effectively attenuated by the Rg1 pretreatment group. Besides, compared with the sham group, the levels of TNF-α and IL-6 in the pretreated group were higher, while the concentration of IL-1β has no statistical difference between the sham group and the pretreated group (P b 0.01).
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The activation of NF-κB can explain the inflammatory responses. We investigated the influences of Rg1 on IκB-α and p65 expression by western blot. As Fig. 5 shows, the NF-κB signaling pathway was activated by LPS administration, while the Rg1 pretreatment group inhibited the phosphorylation of IκB-α and p65 effectively in the LPS-induced ALI.
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3.5. Ginsenoside Rg1 suppressed the activation of NF-κB on LPS-induced 227 acute lung injury. 228
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Fig. 3. Ginsenoside Rg1 inhibited the concentration of protein and cytokines in the bronchoalveolar lavage fluid. The BALF was collected 6 h following the LPS challenge and the supernatant was obtained. (A) Rg1 effectively decreased the effusion of total protein. (B-D) Rg1 markedly reduced the production of proinflammatory cytokin TNF-α, IL-1β, and IL-6 in BALF. ⁎⁎P b 0.01 vs. sham group, #P b 0.05vs. LPS group, ##P b 0.01 vs. LPS group.
Please cite this article as: S. Bao, et al., Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltra..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.06.022
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calculated the change in CD206+ macrophages in the BALF. We hypothesized that Rg1 plays its positive role through reversing the proportion of M2 macrophages. Imbalanced inflammatory responses play prominent roles in the development of ALI. The increased concentration of proinflammatory cytokines (eg, TNF-α, IL-1β and IL-6) in BALF has been noted to be in accordance with the severity of ALI [13,14]. Continuous elevation of the cytokines mentioned above in patients suffering from ALI indicated a poor outcome [32]. These proinflammatory cytokines magnify the
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studies. In our study, LPS administration intratracheally induced a decline of macrophages, but Rg1 treatment inhibited the downtrend. According to our own discoveries, we hypothesized that macrophages may play a protective role during ALI. In order to explore the possible reasons, we determined the apoptosis ratio of macrophages, which may exert a beneficial role via clearing apoptotic neutrophils in the BALF; however, there was no difference between groups (data not shown). Since previous studies found that induction or depletion of CD206+ macrophages could protect or exaggerate the ALI [30,31], we
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Fig. 4. Ginsenoside Rg1 reduced the apoptotic cells of lungs in LPS-induced acute lung injury. Lung tissues were harvested 6 h after LPS administration. (A) Representative images for each group. (B) TUNEL positive cells were counted under light microscopy (×400). (C) Rg1 pretreatment inhibited the expression of caspase 3. ⁎⁎P b 0.01 vs. sham group, #P b 0.05 vs. LPS group.
Fig. 5. Ginsenoside Rg1 suppressed the activation of NF-κB on LPS-induced acute lung injury. (A) Representative images for each group. (B and C) Densitometric analysis showed that the level of phosphorylated IκB-α and p65 markedly upregulated after LPS treatment. While, Rg1 pretreatment effectively supressed the phosphorylation of IκB-α and p65. This experiment is representative of three independent experiments. ⁎⁎P b 0.01 vs. sham group, ##P b 0.01vs. LPS group.
Please cite this article as: S. Bao, et al., Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltra..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.06.022
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The authors declare that there are no conflicts of interests regarding the publication of this paper.
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This work was supported by Grants 81171788 from the National Natural Science Foundation of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Kelvin, Human immunopathogenesis of severe acute respiratory syndrome (SARS), Virus Res. 133 (2008) 13–19. [33] S. Ghosh, M.S. Hayden, New regulators of NF-kappaB in inflammation, Nat. Rev. Immunol. 8 (2008) 837–848. [34] T.R. Martin, M. Nakamura, G. Matute-Bello, The role of apoptosis in acute lung injury, Crit. Care Med. 31 (2003) S184–S188. [35] K.H. Albertine, M.F. Soulier, Z. Wang, A. Ishizaka, S. Hashimoto, G.A. Zimmerman, et al., Fas and fas ligand are up-regulated in pulmonary edema fluid and lung tissue of patients with acute lung injury and the acute respiratory distress syndrome, Am. J. Pathol. 161 (2002) 1783–1796. [36] P.M. de Souza, M.A. Lindsay, Apoptosis as a therapeutic target for the treatment of lung disease, Curr. Opin. Pharmacol. 5 (2005) 232–237.
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inflammatory cascade and attract neutrophils migrating into the alveoli. In the present study, Rg1 inhibited the production of TNF-α, IL-1β and IL-6 in BALF. These results demonstrated that the protective effects of Rg1 on ALI induced by LPS may be attributed to anti-inflammatory action. NF-κB is a critical upstream regulator for the generation of proinflammatory mediators, including TNF-α, IL-1β and IL-6 [33]. We further examined the activation of NF-κB to characterize the inhibitory effect of Rg1 on cytokine production. The results showed that IκB-α degradation and p65 activation induced by LPS were significantly inhibited by Rg1, which suggests that Rg1 suppressed excessive cytokine production by inhibiting NF-κB activation. It has been proved that the activation of the NF-κB signaling pathway coincides with the increase of pulmonary cell apoptosis [7]. Inflammatory cells and cytokines infiltrated in the airspaces of lung activate apoptosis and necrosis. Thus, pulmonary cell apoptosis is also considered to be important in the pathogenesis of ALI [34]. Activation of the apoptosis pathway could contribute to the endothelial and epithelial injury, resulting in an increase in microvascular and epithelial permeability in the lungs. Albertine et al. reported that caspase 3, one of the markers of apoptosis, was increased in lung tissue from patients who died with ALI or ARDS [35]. Evidence also suggested that apoptosis modulation might be a novel therapeutic target for the treatment of ALI [36]. In our study, we found that the Rg1 pretreatment group prevented the lung cell apoptosis in mice challenged with LPS. As our results exhibited that several parameters (eg, TNF-α, IL-6) were still increased in the treated groups than in the sham group. The principal limitation in the present study was that we do not investigate further dose gradient. Further studies are warranted to clarify whether a higher dose of Rg1 could attenuate these conditions. In a word, our results demonstrated that Rg1 regulated the inflammatory cells and inhibited the NF-κB activation and pro-inflammatory cytokine production, which subsequently leads to the alleviation of apoptosis in lungs. It suggests that Rg1 may be an agent for preventing LPS-induced ALI.
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Please cite this article as: S. Bao, et al., Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltra..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.06.022
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Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
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Suhong Bao a,b,c,1, Yun Zou c,1, Bing Wang c,1, Yinjiao Li d, Jiali Zhu c, Yan Luo d,⁎, Jinbao Li a,b,c,⁎⁎
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Article history: Received 15 February 2015 Received in revised form 31 May 2015 Accepted 16 June 2015 Available online xxxx
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Keywords: Ginsenoside Rg1 LPS ALI Inflammatory cytokines M2 macrophage
Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou 221004, Jiangsu, China Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou 221004, Jiangsu, China Department of Anesthesiology and Intensive Care, Changhai Hospital, Second Military Medical University, Shanghai 200433, China d Department of Anesthesiology, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200025, China b
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Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltration of M2 macrophages
Ginsenoside Rg1 (Rg1), the major effective component of ginseng, has been reported to have potent antiinflammatory properties. However, the effect of ginsenoside Rg1 on lipopolysaccharide (LPS) -induced acute lung injury (ALI) in mice was unknown. The present study was designed to investigate the protective role of Rg1 on LPS-induced ALI and explore the potential mechanisms. The mice were divided randomly into four groups: the sham group, the LPS group and the LPS + Rg1 (40 mg/kg or 200 mg/kg) pretreatment groups. All mice received Rg1 or an equivalent volume of phosphate buffer saline (PBS) intraperitoneally 1 h before LPS administration. Edema quantification, histology, and apoptosis were detected 6 h after LPS administration. The number of inflammatory cells, the percentage of alternative activated (M2) macrophages and the exudate quantification in bronchoalveolar lavage fluid (BALF) were evaluated. The caspase 3 expression, and the levels of phosphorylated IκB-α and p65 were tested. The results showed that the Rg1 pretreatment group markedly improved lung damage, modulated the infiltration of neutrophils and M2 macrophages, prevented the production of protein and proinflammatory cytokines in BALF, and inhibited apoptosis in lung. We also found that Rg1 suppressed NF-κB and caspase 3 activation. These data suggest that Rg1 plays a protective role against LPS-induced ALI by ameliorating inflammatory responses, regulating the infiltration of M2 macrophages, and inhibiting pulmonary cell apoptosis. © 2015 Published by Elsevier B.V.
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1. Introduction
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Acute lung injury (ALI) and the subsequent acute respiratory distress syndrome (ARDS) are the major causes of respiratory failure in the intensive care unit worldwide. Several clinical diseases or syndromes can trigger the occurrence of ALI, such as severe pneumonia, sepsis, and acute pancreatitis [1–3]. As many studies have evidenced, numerous neutrophils migrate to the lungs of patients who suffer from ALI and cause the damage to lung tissues [4–6], while reducing the infiltration of neutrophils or suppressing their activities could alleviate lung injuries [7,8]. Macrophages, the primary source of proinflammatory cytokines, display controversial roles in the progression of ALI. Previous studies demonstrated that the number of macrophages in alveoli was in accordance with the lung damage [9,10]. However, recent articles showed that a persistent decrease of alveolar macrophages was associated with the worse outcome: the animals may display
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⁎ Corresponding author. ⁎⁎ Correspondence to: J. Li, Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou 221004, Jiangsu, China. E-mail addresses: [email protected] (Y. Luo), [email protected] (J. Li). 1 Suhong Bao, Yun Zou and Bing Wang contributed equally to this work.
stronger inflammatory responses, once alveolar macrophages are cleared [11,12]. The inflammatory mediators (e.g., TNF-α and IL-1β) secreted from the inflammatory cells play a critical role in the pathogenesis of ARDS. The elevated proinflammatory cytokines in the airspaces of lungs result in the disruption of the alveolar structure and the occurrence of protein-rich pulmonary edema [13,14]. Therefore, a new therapeutic agent targeting the infiltration of immune cells and the inflammatory mediators is urgently required. Ginsenoside Rg1 (Rg1) is one of the major active ingredients purified from Panax ginseng. Previous studies demonstrated its powerful pharmacological effects against apoptosis and fibrosis in various animal models [15,16]. In the cardiomyocyte hypoxia-reoxygenation model, Rg1 is regarded as an antioxidant substance that could reduce the release of lactate dehydrogenase and intracellular ROS [17]. It can exert strong protection from ischemic reperfusion injuries in hepatocytes [18]. Furthermore, our previous experiments also found that Rg1 restrained inflammatory responses via downregulation of NF-κB activity [19–21]. These exciting effects indicate that ginsenoside Rg1 may have positive effects on ALI induced by LPS administration intratracheally. Therefore, our present study aimed to assess the influences of Rg1 in a murine ALI model and investigate its potential mechanisms.
http://dx.doi.org/10.1016/j.intimp.2015.06.022 1567-5769/© 2015 Published by Elsevier B.V.
Please cite this article as: S. Bao, et al., Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltra..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.06.022
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2. Materials and methods
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Male BALB/c mice, aged 6–8 weeks, were obtained from the Animal Experimentation Center of the Second Military Medical University. All mice were housed under specific pathogen-free conditions and fed a standard laboratory diet. The experiments were approved by the Institutional Animal Care and Use Committee of Changhai Hospital (Approval number CH20130723-18). Ginsenoside Rg1 (CAS 22427-39-0, purity N 98%, molecular formula C42H72O14) and LPS (Escherichia coli 055:B5) were purchased from Sigma–Aldrich Corporation (St. Louis, MO, USA) and dissolved in pathogen-free phosphate buffer saline (PBS).
The protein in the BALF was obtained through centrifugation (2000 rpm, 4 °C) for 5 min. The total protein concentration of the BALF was measured by a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The levels of TNF-α, IL-1β and IL-6 in the BALF were determined by ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.
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2.8. Western Blot Analysis
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2.3. Analysis of wet-to-dry weight ratio of lung tissues
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Pulmonary edema is one of the important characteristics of ALI. In the present study, lung tissues were removed and wet weights were recorded 6 hours after LPS administration. Lung tissues were then packed in a silver paper and placed in an incubator at 80 °C for 24 h to obtain the dry weights. The wet-to-dry ratio (W/D ratio) of lung tissues was calculated to evaluate the degree of pulmonary edema.
The lung tissues of all groups were homogenized in protein lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min. After centrifugation (12,000 rpm, 4 °C, 10 min), the protein concentration was measured by a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were loaded in each well and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which subsequently was transferred onto a polyvinylidene difluoride (PVDF) membrane. After blockade of nonspecific binding sites, membranes were incubated for 2 h at room temperature with various antibodies against IκBα, phospho-IκBα, NF-κB p65, phospho-NF-κB p65 and caspase 3 (Cell Signal Technology, Danvers, MA, USA). Protein bands were demonstrated by an enhanced chemiluminescence (ECL) western blot kit (Thermo Fisher Scientific, Waltham, MA, USA). Band intensity was quantified using the Image J software. Protein expression was normalized to β-actin signals.
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All mice in the experiment were divided randomly into four groups: the sham group, the LPS group, and the LPS + ginsenoside Rg1 (40 mg/kg or 200 mg/kg) pretreatment groups (n = 6 in each group). PBS or Rg1 were given intraperitoneally according to the randomized treatment 1 h before LPS administration. Then all mice were anesthetized with an inhalation of sevoflurane, and LPS (20 μg in 50 μl PBS) was injected intratracheally to induce acute lung injury. Simultaneously, mice in the sham group received an equal volume of PBS instead of LPS. The mice were euthanized by CO2 inhalation 6 h after LPS administration. Bronchoalveolar lavage fluid (BALF) and lung tissues were harvested and preserved until use.
To assess the apoptosis in the lungs, a terminal deoxy-nucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed. We fixed the lung samples with 4% buffered paraformaldehyde for more than 24 h. Then, a FragELTM DNA Fragmentation Detection Kit (Merck Millipore, Billerica, MA, USA) was used to stain the apoptotic cells in the lung tissues. TUNEL positive cells were counted in the high-power field (HPF) under light microscopy (×400) by the IPP 6.0 software.
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All data were expressed as mean ± standard deviation (SD). The statistical analysis was performed using SPSS 16.0 Programs (SPSS Inc, USA). Graphs were plotted by Prism 5.0 (GraphPad Software, San Diego, CA). The differences among the different groups were analyzed by the one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. P b 0.05 was considered statistically significant.
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Histopathologic examination of lungs was performed on mice that were not subjected to the BALF collection. The right upper lobes were collected for histologic examination. Tissues were fixed with 4% buffered paraformaldehyde for at least 24 h. Sections (4–5 μm) were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol, and stained with hematoxylin and eosin (H&E). All sections were scored blindly by two experienced pathologists under a light microscope according to the criteria previously described [22]: 0, normal tissue; 1, minimal inflammatory change; 2, no obvious damage to the lung architecture; 3, thickening of the alveolar septae; 4, formation of nodules or areas of pneumonitis that distorted the normal architecture; and 5, total obliteration of the field.
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BALF was collected by washing the lungs twice with 0.5 ml of PBS via the tracheal cannula. Total cells in the BALF were calculated by a flow cytometer. Neutrophils as well as macrophages were also determined by a flow cytometer through cell-specific surface markers (Gr-1 for neutrophils and F4/80 for macrophages). To determine the percent of alternative activated (M2) macrophages in BALF, the percentage of F4/80+ CD206+ cells was detected. Data were obtained by a FACSCaliburTM flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed by the Flowjo software.
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3.1. Ginsenoside Rg1 ameliorated lung damages induced by LPS challenge 170 In order to assess the severity of lung injuries, we examined the W/D ratio that reflects the formation of pulmonary edema. As shown in Fig. 1A, LPS injected intratracheally significantly increased the lung W/D ratio compared with the sham group (P b 0.01), while the Rg1 pretreatment group (40 mg/kg or 200 mg/kg) alleviated the lung W/D ratio compared with the LPS group (both P b 0.01). In parallel, LPS administration destroyed the structure of alveoli and promoted the infiltration of inflammatory cells into the lungs. In contrast, the Rg1 pretreatment group reduced the accumulation of infiltrated cells, and suppressed the thickening of alveolar walls (Fig. 1B). As shown in Fig. 1C, the scores which represent the lung injury decreased significantly after Rg1 administration.
Please cite this article as: S. Bao, et al., Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltra..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.06.022
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To explore the protective effect of Rg1 on ALI induced by LPS, we detected the counts of total cells, neutrophils and macrophages in BALF. As shown in Fig. 2A and B, the total cells and neutrophils in the BALF significantly increased after the LPS treatment. As expected, pretreatment with Rg1 reduced the number of total cells and
neutrophils in the BALF after LPS administration. On the contrary, the number of macrophages in the BALF decreased after the LPS challenge, whereas the Rg1 pretreatment group prevented the decline of macrophages after LPS administration intratracheally (Fig. 2C). We also calculated the percentage of CD206+ macrophages regarded as M2 macrophages. As Fig. 2D demonstrates the LPS challenge reduced the proportion of CD206+ macrophages, while the percentage of M2 macrophages increased after Rg1 administration. However,
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3.2. Effects of ginsenoside Rg1 on the infiltration of inflammatory cells in bronchoalveolar lavage fluid
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Fig. 1. Ginsenoside Rg1 ameliorated lung damages induced by LPS challenge. (A) Rg1 reduced lung wet/dry (W/D) weight ratio 6 h after LPS administration. (B) Representative images of H&E-stained lung sections from four experimental groups (×200). (C) The severity of lung injury was scored as described above. ⁎⁎P b 0.01 vs. sham group, ##P b 0.01 vs. LPS group.
Fig. 2. Effects of ginsenoside Rg1 on the infiltration of inflammatory cells in bronchoalveolar lavage fluid. We harvested BALF 6 h after LPS administration. (A and B) Rg1 decreased the number of total cells and neutrophils in the BALF. (C) Rg1 prevented the decline of the number of macrophages in the BALF. (D) Rg1 enhanced the percentage of M2 macrophages in BALF. ⁎⁎P b 0.01 vs. sham group, ##P b 0.01 vs. LPS group.
Please cite this article as: S. Bao, et al., Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltra..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.06.022
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Since there were no statistical differences between the two doses of Rg1 in the results mentioned above, we chose one dose of Rg1 (40 mg/kg) in the following experiments. The TUNEL assay was performed to detect apoptosis in the lungs subjected to an LPS challenge. As shown in Fig. 4, mice challenged with LPS experienced dramatic activation of apoptosis in the lungs. Under HPF examination, the apoptotic cells of the LPS group were 278.75 ± 20.74, while in the Rg1 pretreatment group, the number of apoptotic cells decreased significantly (205.5 ± 61.86). We also chose the variation of caspase 3 to support the conclusion about apoptosis. As expected, Rg1 inhibited the caspase 3 expression in the lung tissues after LPS administration.
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ALI and its severe form, ARDS, are life-threatening syndromes that are characterized by uncontrolled inflammatory responses and infiltration of numerous activated cells. However, few effective treatments have been discovered to attenuate the injuries, leading to the high mortality of approximately 22% to 40% for ALI [23–25]. The data from our study revealed that LPS injected intratracheally induced pulmonary edema and destroyed the alveolar structure; however, ginsenoside Rg1 exerted protective effects on LPS-induced ALI, which was confirmed by the ameliorating W/D ratio and the degree of pathologic injuries (Fig. 1). As we demonstrated, modulating the infiltration of immune cells and inhibiting the excessive inflammatory responses were the potential mechanisms. According to an increasing number of studies, excessive neutrophils in the BALF contribute to epithelial and endothelial injury, which may lead eventually to vascular leakage and pulmonary edema [26,27]. Our results revealed that the number of neutrophils in the BALF increased significantly after an LPS challenge; nevertheless, Rg1 inhibited the influx of neutrophils induced by LPS. Besides neutrophils, alveolar macrophages (AMs) also have essential roles in ALI, including initiating and maintaining the inflammatory response and regulating the resolution of lung injury and repair [28,29]. Several studies showed that the number of macrophages in BALF were in parallel with the damage to lung tissues [9,10]. However, our results were the opposite of these previous
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As shown in Fig. 3A, compared with the sham group, the concentration of total protein in the BALF increased significantly 6 h after the LPS challenge, but the protein level decreased in the Rg1 administration groups. Furthermore, the levels of proinflammatory cytokines, including TNF-α, IL-1β and IL-6 in the BALF, were measured to evaluate the influence of Rg1 on inflammatory responses. As illustrated, the levels of the cytokines mentioned above dramatically increased in the BALF after LPS administration. Meanwhile, these conditions were effectively attenuated by the Rg1 pretreatment group. Besides, compared with the sham group, the levels of TNF-α and IL-6 in the pretreated group were higher, while the concentration of IL-1β has no statistical difference between the sham group and the pretreated group (P b 0.01).
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The activation of NF-κB can explain the inflammatory responses. We investigated the influences of Rg1 on IκB-α and p65 expression by western blot. As Fig. 5 shows, the NF-κB signaling pathway was activated by LPS administration, while the Rg1 pretreatment group inhibited the phosphorylation of IκB-α and p65 effectively in the LPS-induced ALI.
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Fig. 3. Ginsenoside Rg1 inhibited the concentration of protein and cytokines in the bronchoalveolar lavage fluid. The BALF was collected 6 h following the LPS challenge and the supernatant was obtained. (A) Rg1 effectively decreased the effusion of total protein. (B-D) Rg1 markedly reduced the production of proinflammatory cytokin TNF-α, IL-1β, and IL-6 in BALF. ⁎⁎P b 0.01 vs. sham group, #P b 0.05vs. LPS group, ##P b 0.01 vs. LPS group.
Please cite this article as: S. Bao, et al., Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltra..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.06.022
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calculated the change in CD206+ macrophages in the BALF. We hypothesized that Rg1 plays its positive role through reversing the proportion of M2 macrophages. Imbalanced inflammatory responses play prominent roles in the development of ALI. The increased concentration of proinflammatory cytokines (eg, TNF-α, IL-1β and IL-6) in BALF has been noted to be in accordance with the severity of ALI [13,14]. Continuous elevation of the cytokines mentioned above in patients suffering from ALI indicated a poor outcome [32]. These proinflammatory cytokines magnify the
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studies. In our study, LPS administration intratracheally induced a decline of macrophages, but Rg1 treatment inhibited the downtrend. According to our own discoveries, we hypothesized that macrophages may play a protective role during ALI. In order to explore the possible reasons, we determined the apoptosis ratio of macrophages, which may exert a beneficial role via clearing apoptotic neutrophils in the BALF; however, there was no difference between groups (data not shown). Since previous studies found that induction or depletion of CD206+ macrophages could protect or exaggerate the ALI [30,31], we
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Fig. 4. Ginsenoside Rg1 reduced the apoptotic cells of lungs in LPS-induced acute lung injury. Lung tissues were harvested 6 h after LPS administration. (A) Representative images for each group. (B) TUNEL positive cells were counted under light microscopy (×400). (C) Rg1 pretreatment inhibited the expression of caspase 3. ⁎⁎P b 0.01 vs. sham group, #P b 0.05 vs. LPS group.
Fig. 5. Ginsenoside Rg1 suppressed the activation of NF-κB on LPS-induced acute lung injury. (A) Representative images for each group. (B and C) Densitometric analysis showed that the level of phosphorylated IκB-α and p65 markedly upregulated after LPS treatment. While, Rg1 pretreatment effectively supressed the phosphorylation of IκB-α and p65. This experiment is representative of three independent experiments. ⁎⁎P b 0.01 vs. sham group, ##P b 0.01vs. LPS group.
Please cite this article as: S. Bao, et al., Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltra..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.06.022
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The authors declare that there are no conflicts of interests regarding the publication of this paper.
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This work was supported by Grants 81171788 from the National Natural Science Foundation of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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inflammatory cascade and attract neutrophils migrating into the alveoli. In the present study, Rg1 inhibited the production of TNF-α, IL-1β and IL-6 in BALF. These results demonstrated that the protective effects of Rg1 on ALI induced by LPS may be attributed to anti-inflammatory action. NF-κB is a critical upstream regulator for the generation of proinflammatory mediators, including TNF-α, IL-1β and IL-6 [33]. We further examined the activation of NF-κB to characterize the inhibitory effect of Rg1 on cytokine production. The results showed that IκB-α degradation and p65 activation induced by LPS were significantly inhibited by Rg1, which suggests that Rg1 suppressed excessive cytokine production by inhibiting NF-κB activation. It has been proved that the activation of the NF-κB signaling pathway coincides with the increase of pulmonary cell apoptosis [7]. Inflammatory cells and cytokines infiltrated in the airspaces of lung activate apoptosis and necrosis. Thus, pulmonary cell apoptosis is also considered to be important in the pathogenesis of ALI [34]. Activation of the apoptosis pathway could contribute to the endothelial and epithelial injury, resulting in an increase in microvascular and epithelial permeability in the lungs. Albertine et al. reported that caspase 3, one of the markers of apoptosis, was increased in lung tissue from patients who died with ALI or ARDS [35]. Evidence also suggested that apoptosis modulation might be a novel therapeutic target for the treatment of ALI [36]. In our study, we found that the Rg1 pretreatment group prevented the lung cell apoptosis in mice challenged with LPS. As our results exhibited that several parameters (eg, TNF-α, IL-6) were still increased in the treated groups than in the sham group. The principal limitation in the present study was that we do not investigate further dose gradient. Further studies are warranted to clarify whether a higher dose of Rg1 could attenuate these conditions. In a word, our results demonstrated that Rg1 regulated the inflammatory cells and inhibited the NF-κB activation and pro-inflammatory cytokine production, which subsequently leads to the alleviation of apoptosis in lungs. It suggests that Rg1 may be an agent for preventing LPS-induced ALI.
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Please cite this article as: S. Bao, et al., Ginsenoside Rg1 improves lipopolysaccharide-induced acute lung injury by inhibiting inflammatory responses and modulating infiltra..., Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.06.022
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