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Liraglutide attenuates lipopolysaccharide-induced acute lung injury in mice
European Journal of Pharmacology 791 (2016) 735–740
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
crossmark
Liraglutide attenuates lipopolysaccharide-induced acute lung injury in mice a
b
a
a,⁎
a
Feng Zhou , Ying Zhang , Jing Chen , Xuemei Hu , Yancheng Xu a b
Department of Endocrinology, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei, China Department of Anesthesia, Critical Care Medicine & Emergency Medicine Center, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei, China
A R T I C L E I N F O
A BS T RAC T
Keywords: Liraglutide Lipopolysaccharide Acute lung injury Inflammation NLRP3 inflammasome
Liraglutide, an effective drug for the treatment of diabetes, has been proven to demonstrate anti-inflammatory and immunomodulatory effects. Hence, this study explored the effects and mechanism of action of liraglutide on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Male BALB/c mice were pre-conditioned with liraglutide or saline prior to intraperitoneal LPS or saline administration. Histopathological examination of lung, the wet/dry (W/D)weight ratio, protein content, inflammatory cell numbers and pro-inflammatory cytokine levels in broncho-alveolar lavage fluid (BAL fluid) were conducted. The effects of liraglutide on the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome signalling pathway were assessed by Western blot. Pre-treatment with liraglutide decreased the wet-to-dry weight ratio and protein concentrations in BAL fluid and neutrophil infiltration in the lung tissues. Liraglutide also significantly reduced the interleukin-1β and interleukin-18 levels in BAL fluid, as well as effectively inhibited the expression of NLRP3 inflammasome. These results indicated that liraglutide pre-treatment attenuated LPS-induced ALI by inhibiting the NLRP3 inflammasome pathway.
1. Introduction
an important role in the pathogenesis of ALI and sepsis (Dolinay et al., 2012; Mao et al., 2013). Therefore, an anti-NLRP3 inflammasome therapeutic approach is an effective strategy to treat LPS-induced ALI. Liraglutide, a long-acting glucagon-like peptide (GLP-1) analogue with 97% structural homology to the native hormone (Knudsen et al., 2000), inhibits glucagon secretion, increases insulin secretion and slows gastric emptying (Holst, 2007). This peptide analogue is widely used to treat type 2 diabetes mellitus. Liraglutide has been reported to demonstrate promising anti-inflammatory and immunomodulatory activities (Iwai et al., 2006; Blandino-Rosano et al., 2008). Liraglutide has also been found to inhibit IL-1β expression among macrophages in atherosclerosis (Dai et al., 2014). Viby et al. (2013) has identified that liraglutide reduces mortality and improves lung function in a model of experimental obstructive lung disease in female mice. However, the protective potential of liraglutide for ALI is yet to be reported. We hypothesise that liraglutide attenuates LPS-induced ALI by inhibiting the activation of the NLRP3 inflammasome pathway.
Acute lung injury (ALI) is a serious and common clinical disease that can be caused by various factors, such as sepsis, serious trauma and acute pancreatitis. The most common cause of ALI is sepsis. Despite the significant progress in ALI treatment, the morbidity and mortality rates remain high (Angus et al., 2001; Bosma et al., 2010). To date, no effective medicine has been discovered for ALI. Lipopolysaccharide (LPS) has been confirmed as a chief cause of ALI (Brigham and Meyrick, 1986). LPS exerts toxic effects on the lungs through direct injury to endothelial cells and indirect activation of neutrophils and macrophages, which consequently release pro-inflammatory cytokines, such as interleukin-1β(IL-1β) and interleukin-18(IL18). These potent pro-inflammatory cytokines initiate, amplify and perpetuate the inflammatory response, thereby severely influencing pulmonary gas exchange and inducing refractory hypoxemia. The maturation and secretion of IL-1β and IL-18 were mainly determined by inflammasome (Schroder and Tschopp, 2010; Franchi et al., 2012). The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is the most intensively studied inflammasome. The NLRP3 inflammasome consists of NLRP3, the adaptor protein known as apoptosis-associated speck-like protein containing caspase-1 activator domain (ASC) and Caspase-1 (Sutterwala et al., 2006; Faustin et al., 2007). The dysregulated NLRP3 inflammasome activation plays
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2. Materials and methods 2.1. Reagents LPS (from Escherichia coli 055: B5) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Liraglutide was purchased from
Corresponding author. E-mail address: [email protected] (Y. Xu).
http://dx.doi.org/10.1016/j.ejphar.2016.10.016 Received 7 September 2016; Received in revised form 7 October 2016; Accepted 14 October 2016 Available online 15 October 2016 0014-2999/ © 2016 Elsevier B.V. All rights reserved.
European Journal of Pharmacology 791 (2016) 735–740
F. Zhou et al.
and detection range for IL-1β assays is 9.37 pg/ml and 15.62– 1000.00 pg/ml (r2=0.99). The intra and inter coefficient of variation for IL-18 assay is 2.80%, 5.70%, detection limit and detection range for IL-18 assays is 4.68 pg/ml and 7.81–500.00 pg/ml (r2 =0.99). The protein concentrations in the supernatants of BAL fluid were quantified using the bicinchoninic acid (BCA) protein assay to evaluate the vascular permeability to airway.
Novo Nordisk (Bagsvaerd, Denmark). Mouse IL-1β and IL-18 enzymelinked immunosorbent assay (ELISA) kits were obtained from Wuhan Elabscience Biotechnology Co., Ltd. (Wuhan, China). The myeloperoxidase (MPO) assay kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). NLRP3 antibody was purchased from R & D systems (Minneapolis, MN, USA). ASC and Caspase-1 antibodies were purchased from Proteintech (Chicago, USA). 2.2. Animals
2.7. MPO activity assay
Male BALB/c mice (6–8 weeks old, purchased from the Centre for Disease Control and Prevention, Hubei, China) were housed in a lightand temperature-controlled room (21–23 °C, 12 h cycle). All protocols were approved by the Wuhan University of Science and Technology Animal Care and Use Committee (No. 02515065V).
MPO activity in the lung tissues was spectrophotometrically assayed using MPO assay kit. One hundred milligrams of lung tissue was homogenized and fluidized in extraction buffer to obtain 5% of the homogenate. The sample including 0.9 ml homogenate and 0.1 ml of buffer solution was heated to 37 °C in a water bath for 15 min, and then the enzymatic activity was determined by measuring the changes in absorbance at 460 nm using a 96-well plate reader and expressed as units per gram weight. A standard curve was created.
2.3. Experimental design After a certain habituation period, the mice were randomly divided into three groups: control (CON) group, LPS group and liraglutide treatment (LPS+Lir) group, with eight mice in each group. In the CON and LPS groups, an equal volume of normal saline was injected subcutaneously. In the LPS+Lir group, liraglutide (200 μg/kg) (Steven et al., 2015) diluted in normal saline was administered subcutaneously. After 6 h of liraglutide administration, the mice in the LPS and LPS+Lir groups received LPS intraperitoneally at a dose of 10 mg/kg (Tang et al., 2014). The mice in the CON group were administered an equal volume of normal saline in the same way. The animals were anaesthetized with 10% chloral hydrate after 4 h of LPS treatment, and the samples were then collected.
2.8. Immunohistochemistry Lung tissue sections were stained with mouse antibody against NLRP3 overnight at 4 °C and incubated with alkaline phosphatase (AP)-conjugated secondary antibody for 1 h at room temperature. Slices were visualized following incubation with diaminobenzidine and then counterstained with hematoxylin, and then observed under a microscope. The number of brown granules in each high-powered field (magnification: ×400) was quantified as the number of positively stained cells or nuclei. The measurements were expressed as the number of positively stained cells observed in 8–10 digital images per animal.
2.4. Lung W/D weight ratio Lung edema was estimated by determining the lung W/D weight ratios. The fresh upper part of the left lung was weighed and dried in an oven at 80 °C for 48 h and then weighed again when dry to calculate the lung W/D weight ratio.
2.9. Western blot assay The protein expression was measured by western blotting. The lung tissues were kept in −80 °C. The lung tissues were homogenized in PBS containing the protease inhibitor cocktail (Beyotime Biotechnology, China). The homogenates were centrifuged for 20 min at 15,000 rpm in 4 °C. Supernatants of lung tissues were collected, and protein concentration of each sample was measured with a BCA assay kit using bull serum albumin (BSA) as standard. An equal amount of protein from each sample (30 µg) was loaded onto 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) for electrophoresis. The proteins were then transferred to polyvinylidene difluoride membranes. These membranes were blocked with solution (5% dry non-fat dried milk in Tris-buffered saline with Tween20) for 2 h at room temperature to reduce non-specific binding. The membranes were incubated with antiNLRP3 (1:500), anti-ASC (1:500), anti-Caspase-1 (1:1000) or anti-βactin (1:1000) overnight at 4 °C, respectively. The horseradish peroxidase-labeled secondary antibodies (1:10000) were added and incubated for 1 h at room temperature. Protein bands were detected with the enhanced chemiluminescence Western blotting detection system and analysed by a densitometry system.
2.5. Histology examination The left lower lobe lung tissue was fixed with 10% buffered formalin, embedded in paraffin and then sliced. Pathological changes of the lung tissues were evaluated using an optical microscope after staining with hematoxylin and eosin (H & E). Each lung section was evaluated by two investigators in a double blind manner. The severity of lung damage was evaluated using a semi-quantitative scoring, as has been described previously (Parsey et al., 1998). The histological index of lung injury included the lung edema, infiltration of inflammatory cells, alveolar haemorrhage, hyaline membrane and atelectasis. Each item was graded on a five point scale from 0 to 4 (0=normal; 1=injured area ≤25%; 2=injured area 25–50%; 3=injured area 51–70%; 4=injured area > 70%). A total of 10 fields were randomly selected for each animal and the scores were averaged. 2.6. Total inflammatory cell counts, pro-inflammatory cytokines and protein concentrations in BAL fluid Mice were anaesthetized, and BAL fluid was collected by flushing the lung (0.5 ml ×3 times). BAL fluid samples were centrifuged at 2000 rpm for 10 min at 4 °C. Sediment cells were washed and resuspended in PBS, after which the total number of cells in BAL fluid was counted double blindly by using a haemocytometer. The levels of cytokines IL-1β and IL-18 in BAL fluid were measured by ELISA in accordance with the manufacturer's instructions. The intra and inter coefficient of variation for IL-1β assay is 3.70%, 4.90%, detection limit
2.10. Statistical analysis The results were expressed as mean ± SD and analysed by one-way ANOVA. Tukey's test was used for statistical analysis to compare the data among all the groups. P < 0.05 was regarded as statistically significant. 736
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Fig. 1. Effects of liraglutide on LPS-mediated lung histopathologic changes. Lungs tissues (n=8) from each experimental group were processed for histological evaluation at 4 h after LPS challenge. Representative histological changes of lungs obtained from mice of different groups. A: CON group, B: LPS group, C: LPS+Lir group. Representative histological lung sections were stained with H & E at 400× magnification. D: Severity scores of lung injury in different groups (n=8). Data are mean ± S.D. #P < 0.01 vs. CON group, **P < 0.01 vs. LPS group.
pulmonary microvascular barrier and increased the protein contents in BAL fluid compared with the CON group (P < 0.05). Liraglutide administration in the LPS+Lir group obviously decreased the protein content in BAL fluid compared with that in the LPS group (P < 0.01; Fig. 3).
3. Results 3.1. Effects of liraglutide on lung histopathologic changes in LPSinduced ALI mice The H & E staining sections of lung tissues from each group of mice are shown in Fig. 1. The alveolar structure was normal and had no abnormal changes in CON group (Fig. 1A). The lung tissue sections of the LPS group exhibited diffused pathological changes, including interstitial edema, obvious infiltration of inflammatory cells, haemorrhage and alveolar disarray (Fig. 1B). However, these histopathologic changes were markedly improved by liraglutide (Fig. 1C). The lung damage score was significantly higher in LPS group, compared to the CON group (P < 0.05). Liraglutide pretreatment significantly decreased the lung damage score (P < 0.01, Fig. 1D).
3.4. Effects of liraglutide on the IL-1β and IL-18 levels in BAL fluid of LPS-induced ALI mice The levels of cytokines IL-1β and IL-18 in BAL fluid were detected to determine whether liraglutide pretreatment decreased the levels of NLRP3 inflammasome regulated cytokines. The levels of IL-1β and IL18 in BAL fluid significantly increased in the LPS group (P < 0.01). Liraglutide pretreatment effectively decreased the levels of IL-1β and IL-18 in BAL fluid compared with those in the LPS group (P < 0.05; Fig. 4).
3.2. Effects of liraglutide on lung W/D weight ratio in LPS-induced ALI mice
3.5. Effects of liraglutide on MPO activity in LPS-induced ALI mice
The lung W/D weight ratio was used to examine pulmonary edema. As shown in Fig. 2, the lung W/D weight ratio of the LPS group was obviously higher than that of the CON group (P < 0.01). Additionally, the lung W/D weight ratio was significantly alleviated in the LPS+Lir group compared with that in the LPS group (P < 0.01).
MPO activity was used to determine the accumulation of neutrophil infiltrates. The MPO activity in the LPS group obviously increased compared with that in the CON group (P < 0.01, Fig. 5). By contrast, the MPO activity in the LPS+Lir group significantly decreased relative to that in the LPS group (P < 0.01).
3.3. Effects of liraglutide on inflammatory cell count and protein contents in BAL fluid of LPS-induced ALI mice
3.6. Effects of liraglutide on NLRP3 inflammasome expression in LPS-induced ALI mice
The total cell count in BAL fluid significantly increased in the LPS group, compared to the CON group (P < 0.01). However, liraglutide pretreatment markedly reduced the total cell count in BAL fluid (P < 0.01). The pulmonary microvascular permeability index was determined by the protein contents in BAL fluid. LPS severely damaged the
Lung tissue sections were immunostained with anti-NLRP3 antibody to locate NLRP3 in the lung tissues and determine whether liraglutide reduced the positively stained cells. We observed that the NLRP3 protein was mainly expressed in the inflammatory cells (i.e. macrophages and neutrophils). Few positively stained cells were found
Fig. 2. Effect of liraglutide on lung W/D weight ratio in lung tissues of LPS-induced ALI. Lung W/D weight ratio was determined at 4 h after LPS challenge. The values presented are mean ± S.D. (n=8) of three independent experiments. ＃P < 0.01 vs. CON group, **P < 0.01 vs. LPS group.
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Fig. 3. Effects of liraglutide on inflammatory cell infiltration and protein content in BAL fluid. The values presented are mean ± S.D. (n=8) of three independent experiments. ＃P < 0.01 vs. CON group, **P < 0.01 vs. LPS group.
in the lung tissues of the CON group (Fig. 6A). The positively stained
cells in LPS group were significantly increased comparing with the
Fig. 4. Effects of liraglutide on IL-1β and IL-18 production in BAL fluid. BAL fluid was collected at 4 h after LPS challenge for the analysis of inflammatory cytokines IL-1β and IL-18. The values presented are mean ± S.D. (n=8) of three independent experiments. ＃P < 0.01 vs. CON group, *P < 0.05 vs. LPS group.
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Fig. 5. Effects of liraglutide on MPO activity in lung tissues of LPS-induced ALI. MPO activity was determined at 4 h after LPS administration. The values presented are mean ± S.D. (n=8) of three independent experiments. ＃P < 0.01 vs. CON group, **P < 0.01 vs. LPS group.
Fig. 6. Effects of liraglutide on the NLRP3 inflammasome in the lung of the LPS-induced ALI mice model(n=8). NLRP3 expression was determined by immunohistochemistry (×400). Sections from LPS group were highly positively stained for NLRP3. Scattered positive cytoplasmic staining was noted in the inflammatory cells (B). In the liraglutide pretreated group, only some cells were stained for NLRP3 (C). NLRP3-positive cells were marked (arrows). D: NLRP3 positive cells in different groups (n=8). Data are mean ± S.D. ＃P < 0.01 vs. CON group, **P < 0.01 vs. LPS group.
Histological analysis further verified that liraglutide significantly attenuated lung tissue injury. Numerous inflammatory mediators are released into the alveolar space during lung injury (Ganter et al., 2008). Excessive cytokinemediated inflammation plays an important role in the pathogenesis of ALI/acute respiratory distress syndrome (ARDS) (Goodman et al., 2003). IL-1β, as a potent pro-inflammatory cytokine, is produced abundantly by alveolar macrophages in ALI/ARDS (Jacobs et al., 1989); this cytokine initiates and amplifies lung inflammation in patients (Dinarello, 1996). Excessive IL-1β levels damage vascular endothelial and alveolar epithelial permeability (Ganter et al., 2008), resulting in pulmonary edema. IL-18 levels are also associated with critical illness, including ALI and sepsis (Grobmyer et al., 2000; Hoshino et al., 2009). Increased levels of IL-18 are correlated with mortality in ALI/ARDS patients (Makabe et al., 2012). In the present study, liraglutide significantly decreased the production of IL-1β and IL-18. Therefore, we recognise that the protective effect of liraglutide on LPS-induced ALI may be ascribed to the reduction of IL-1β and IL18 levels. The NLRP3 inflammasome pathway is a primary intracellular
CON group (Fig. 6B). Liraglutide pretreatment decreased the amounts of positively stained cells in the lung tissues of ALI mice (Fig. 6C). NLRP3 positive cells count was significantly higher in LPS group, compared to CON group (P < 0.01). Liraglutide treatment significantly decreased the NLRP3 positive cells count (P < 0.01, Fig. 1D). The expression of NLRP3, ASC and Caspase-1 proteins was detected in the lung tissues. The results showed that LPS upregulated the expression of NLRP3, ASC and Caspase-1 proteins in the LPS group (P < 0.01) compared with the CON group. Liraglutide obviously decreased the expression of the above mentioned proteins relative to that in the LPS group (P < 0.05 or P < 0.01; Fig. 7).
4. Discussion ALI is characterized by interstitial edema, neutrophil accumulation, epithelial integrity disruption, and protein leakage into the alveolar space, severely influencing gas exchange (Matute-Bello et al., 2008). In this study, we observed the effects of liraglutide on LPS-induced ALI in mice. Liraglutide pretreatment decreased the lung W/D weight ratio, MPO activity, total cell counts and protein concentrations in BAL fluid.
Fig. 7. Effects of liraglutide on NLRP3, ASC, Caspase-1 protein expression in the lungs of the LPS-induced ALI mouse. NLRP3, ASC, Caspase-1 protein expression were determined by Western blot. The values presented are the mean ± S.D. (n=8) of three independent experiments. The density values of protein expression were normalized for β-actin. ＃P < 0.01 vs. CON group, *P < 0.05 and **P < 0.01 vs. LPS group.
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multiprotein inflammatory pathway of the innate immune system; this pathway responds to diverse exogenous and endogenous stimuli. The NLRP3 inflammasome forms through the activation of NLRP3 and recruitment of ASC and pro-Casp-1. Such phenomena result in Casp-1 activation and subsequent processing of pro-IL-1β and pro-IL-18 to attain their active forms (Kolliputi et al., 2012). Pulmonary histological changes were significantly improved in the LPS-induced ALI of NLRP3−/−mice (Grailer et al., 2014). The deletion of the NLRP3 gene diminished Caspase-1 activation and IL-1β production, thereby significantly improving hyperoxia-induced ALI (Fukumoto et al., 2013). ASC is a crucial adaptor protein required for recruiting Casp-1 to the NLRP3 platform of inflammasome; such process is important for Caspase-1 activation and IL-1β release (Tschopp et al., 2003). Deletion of ASC eliminated Klebsiella LPS-induced IL-1β release in macrophages. ALI in Caspase-1−/− or IL-18−/− knockout mice was attenuated in ventilator-induced acute lung injury (Dolinay et al., 2012). The present results demonstrated that liraglutide pretreatment significantly inhibited theNLRP3 inflammasome protein expression. These results imply that the protective effect of liraglutide on LPSinduced ALI may be attributed to its influence on the suppression of the NLRP3 inflammasome pathway. The MPO activity is associated with the number of infiltrating neutrophils in tissues (Tseng and Kung, 2012). In the development of ALI and ARDS, the infiltration of numerous neutrophils into alveolar spaces is another important pathogenic mechanism (Abraham et al., 2000). In the current study, the MPO activity obviously increased in the lung tissues of LPS-treated mice. Liraglutide pre-treatment effectively decreased the MPO activity in the lung tissues. This finding is consistent with the work of Viby (Viby et al., 2013) which demonstrated that liraglutide significantly inhibited alveoli neutrophil accumulation in a model of experimental obstructive lung disease in female mice. Overall, the results suggest that the inhibition of neutrophil infiltration may be another pathway through which liraglutide attenuates LPS-induced acute lung injury. In conclusion, liraglutide attenuated LPS-induced ALI by inhibiting the NLRP3 inflammasome pathway, thereby decreasing the production of IL-1β and IL-18 and inhibiting inflammatory responses. Collectively, the results suggest that liraglutide treatment is a novel strategy for the management of LPS-induced ALI. Conflict of interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (NO. 81370872). References Abraham, E., Carmody, A., Shenkar, R., Arcaroli, J., 2000. Neutrophils as early immunologic effectors in hemorrhage- or endotoxemia-induced acute lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 279, L1137–L1145. Angus, D.C., Linde-Zwirble, W.T., Lidicker, J., Clermont, G., Carcillo, J., Pinsky, M.R., 2001. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit. Care Med. 29, 1303–1310. Blandino-Rosano, M., Perez-Arana, G., Mellado-Gil, J.M., Segundo, C., AguilarDiosdado, M., 2008. Anti-proliferative effect of pro-inflammatory cytokines in cultured beta cells is associated with extracellular signal-regulated kinase 1/2 pathway inhibition: protective role of glucagon-like peptide-1. J. Mol. Endocrinol. 41, 35–44. Bosma, K.J., Taneja, R., Lewis, J.F., 2010. Pharmacotherapy for prevention and treatment of acute respiratory distress syndrome: current and experimental approaches. Drugs 70, 1255–1282. Brigham, K.L., Meyrick, B., 1986. Endotoxin and lung injury. Am. Rev. Respir. Dis. 133,
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
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Liraglutide attenuates lipopolysaccharide-induced acute lung injury in mice a
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Feng Zhou , Ying Zhang , Jing Chen , Xuemei Hu , Yancheng Xu a b
Department of Endocrinology, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei, China Department of Anesthesia, Critical Care Medicine & Emergency Medicine Center, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei, China
A R T I C L E I N F O
A BS T RAC T
Keywords: Liraglutide Lipopolysaccharide Acute lung injury Inflammation NLRP3 inflammasome
Liraglutide, an effective drug for the treatment of diabetes, has been proven to demonstrate anti-inflammatory and immunomodulatory effects. Hence, this study explored the effects and mechanism of action of liraglutide on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Male BALB/c mice were pre-conditioned with liraglutide or saline prior to intraperitoneal LPS or saline administration. Histopathological examination of lung, the wet/dry (W/D)weight ratio, protein content, inflammatory cell numbers and pro-inflammatory cytokine levels in broncho-alveolar lavage fluid (BAL fluid) were conducted. The effects of liraglutide on the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome signalling pathway were assessed by Western blot. Pre-treatment with liraglutide decreased the wet-to-dry weight ratio and protein concentrations in BAL fluid and neutrophil infiltration in the lung tissues. Liraglutide also significantly reduced the interleukin-1β and interleukin-18 levels in BAL fluid, as well as effectively inhibited the expression of NLRP3 inflammasome. These results indicated that liraglutide pre-treatment attenuated LPS-induced ALI by inhibiting the NLRP3 inflammasome pathway.
1. Introduction
an important role in the pathogenesis of ALI and sepsis (Dolinay et al., 2012; Mao et al., 2013). Therefore, an anti-NLRP3 inflammasome therapeutic approach is an effective strategy to treat LPS-induced ALI. Liraglutide, a long-acting glucagon-like peptide (GLP-1) analogue with 97% structural homology to the native hormone (Knudsen et al., 2000), inhibits glucagon secretion, increases insulin secretion and slows gastric emptying (Holst, 2007). This peptide analogue is widely used to treat type 2 diabetes mellitus. Liraglutide has been reported to demonstrate promising anti-inflammatory and immunomodulatory activities (Iwai et al., 2006; Blandino-Rosano et al., 2008). Liraglutide has also been found to inhibit IL-1β expression among macrophages in atherosclerosis (Dai et al., 2014). Viby et al. (2013) has identified that liraglutide reduces mortality and improves lung function in a model of experimental obstructive lung disease in female mice. However, the protective potential of liraglutide for ALI is yet to be reported. We hypothesise that liraglutide attenuates LPS-induced ALI by inhibiting the activation of the NLRP3 inflammasome pathway.
Acute lung injury (ALI) is a serious and common clinical disease that can be caused by various factors, such as sepsis, serious trauma and acute pancreatitis. The most common cause of ALI is sepsis. Despite the significant progress in ALI treatment, the morbidity and mortality rates remain high (Angus et al., 2001; Bosma et al., 2010). To date, no effective medicine has been discovered for ALI. Lipopolysaccharide (LPS) has been confirmed as a chief cause of ALI (Brigham and Meyrick, 1986). LPS exerts toxic effects on the lungs through direct injury to endothelial cells and indirect activation of neutrophils and macrophages, which consequently release pro-inflammatory cytokines, such as interleukin-1β(IL-1β) and interleukin-18(IL18). These potent pro-inflammatory cytokines initiate, amplify and perpetuate the inflammatory response, thereby severely influencing pulmonary gas exchange and inducing refractory hypoxemia. The maturation and secretion of IL-1β and IL-18 were mainly determined by inflammasome (Schroder and Tschopp, 2010; Franchi et al., 2012). The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is the most intensively studied inflammasome. The NLRP3 inflammasome consists of NLRP3, the adaptor protein known as apoptosis-associated speck-like protein containing caspase-1 activator domain (ASC) and Caspase-1 (Sutterwala et al., 2006; Faustin et al., 2007). The dysregulated NLRP3 inflammasome activation plays
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2. Materials and methods 2.1. Reagents LPS (from Escherichia coli 055: B5) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Liraglutide was purchased from
Corresponding author. E-mail address: [email protected] (Y. Xu).
http://dx.doi.org/10.1016/j.ejphar.2016.10.016 Received 7 September 2016; Received in revised form 7 October 2016; Accepted 14 October 2016 Available online 15 October 2016 0014-2999/ © 2016 Elsevier B.V. All rights reserved.
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and detection range for IL-1β assays is 9.37 pg/ml and 15.62– 1000.00 pg/ml (r2=0.99). The intra and inter coefficient of variation for IL-18 assay is 2.80%, 5.70%, detection limit and detection range for IL-18 assays is 4.68 pg/ml and 7.81–500.00 pg/ml (r2 =0.99). The protein concentrations in the supernatants of BAL fluid were quantified using the bicinchoninic acid (BCA) protein assay to evaluate the vascular permeability to airway.
Novo Nordisk (Bagsvaerd, Denmark). Mouse IL-1β and IL-18 enzymelinked immunosorbent assay (ELISA) kits were obtained from Wuhan Elabscience Biotechnology Co., Ltd. (Wuhan, China). The myeloperoxidase (MPO) assay kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). NLRP3 antibody was purchased from R & D systems (Minneapolis, MN, USA). ASC and Caspase-1 antibodies were purchased from Proteintech (Chicago, USA). 2.2. Animals
2.7. MPO activity assay
Male BALB/c mice (6–8 weeks old, purchased from the Centre for Disease Control and Prevention, Hubei, China) were housed in a lightand temperature-controlled room (21–23 °C, 12 h cycle). All protocols were approved by the Wuhan University of Science and Technology Animal Care and Use Committee (No. 02515065V).
MPO activity in the lung tissues was spectrophotometrically assayed using MPO assay kit. One hundred milligrams of lung tissue was homogenized and fluidized in extraction buffer to obtain 5% of the homogenate. The sample including 0.9 ml homogenate and 0.1 ml of buffer solution was heated to 37 °C in a water bath for 15 min, and then the enzymatic activity was determined by measuring the changes in absorbance at 460 nm using a 96-well plate reader and expressed as units per gram weight. A standard curve was created.
2.3. Experimental design After a certain habituation period, the mice were randomly divided into three groups: control (CON) group, LPS group and liraglutide treatment (LPS+Lir) group, with eight mice in each group. In the CON and LPS groups, an equal volume of normal saline was injected subcutaneously. In the LPS+Lir group, liraglutide (200 μg/kg) (Steven et al., 2015) diluted in normal saline was administered subcutaneously. After 6 h of liraglutide administration, the mice in the LPS and LPS+Lir groups received LPS intraperitoneally at a dose of 10 mg/kg (Tang et al., 2014). The mice in the CON group were administered an equal volume of normal saline in the same way. The animals were anaesthetized with 10% chloral hydrate after 4 h of LPS treatment, and the samples were then collected.
2.8. Immunohistochemistry Lung tissue sections were stained with mouse antibody against NLRP3 overnight at 4 °C and incubated with alkaline phosphatase (AP)-conjugated secondary antibody for 1 h at room temperature. Slices were visualized following incubation with diaminobenzidine and then counterstained with hematoxylin, and then observed under a microscope. The number of brown granules in each high-powered field (magnification: ×400) was quantified as the number of positively stained cells or nuclei. The measurements were expressed as the number of positively stained cells observed in 8–10 digital images per animal.
2.4. Lung W/D weight ratio Lung edema was estimated by determining the lung W/D weight ratios. The fresh upper part of the left lung was weighed and dried in an oven at 80 °C for 48 h and then weighed again when dry to calculate the lung W/D weight ratio.
2.9. Western blot assay The protein expression was measured by western blotting. The lung tissues were kept in −80 °C. The lung tissues were homogenized in PBS containing the protease inhibitor cocktail (Beyotime Biotechnology, China). The homogenates were centrifuged for 20 min at 15,000 rpm in 4 °C. Supernatants of lung tissues were collected, and protein concentration of each sample was measured with a BCA assay kit using bull serum albumin (BSA) as standard. An equal amount of protein from each sample (30 µg) was loaded onto 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) for electrophoresis. The proteins were then transferred to polyvinylidene difluoride membranes. These membranes were blocked with solution (5% dry non-fat dried milk in Tris-buffered saline with Tween20) for 2 h at room temperature to reduce non-specific binding. The membranes were incubated with antiNLRP3 (1:500), anti-ASC (1:500), anti-Caspase-1 (1:1000) or anti-βactin (1:1000) overnight at 4 °C, respectively. The horseradish peroxidase-labeled secondary antibodies (1:10000) were added and incubated for 1 h at room temperature. Protein bands were detected with the enhanced chemiluminescence Western blotting detection system and analysed by a densitometry system.
2.5. Histology examination The left lower lobe lung tissue was fixed with 10% buffered formalin, embedded in paraffin and then sliced. Pathological changes of the lung tissues were evaluated using an optical microscope after staining with hematoxylin and eosin (H & E). Each lung section was evaluated by two investigators in a double blind manner. The severity of lung damage was evaluated using a semi-quantitative scoring, as has been described previously (Parsey et al., 1998). The histological index of lung injury included the lung edema, infiltration of inflammatory cells, alveolar haemorrhage, hyaline membrane and atelectasis. Each item was graded on a five point scale from 0 to 4 (0=normal; 1=injured area ≤25%; 2=injured area 25–50%; 3=injured area 51–70%; 4=injured area > 70%). A total of 10 fields were randomly selected for each animal and the scores were averaged. 2.6. Total inflammatory cell counts, pro-inflammatory cytokines and protein concentrations in BAL fluid Mice were anaesthetized, and BAL fluid was collected by flushing the lung (0.5 ml ×3 times). BAL fluid samples were centrifuged at 2000 rpm for 10 min at 4 °C. Sediment cells were washed and resuspended in PBS, after which the total number of cells in BAL fluid was counted double blindly by using a haemocytometer. The levels of cytokines IL-1β and IL-18 in BAL fluid were measured by ELISA in accordance with the manufacturer's instructions. The intra and inter coefficient of variation for IL-1β assay is 3.70%, 4.90%, detection limit
2.10. Statistical analysis The results were expressed as mean ± SD and analysed by one-way ANOVA. Tukey's test was used for statistical analysis to compare the data among all the groups. P < 0.05 was regarded as statistically significant. 736
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Fig. 1. Effects of liraglutide on LPS-mediated lung histopathologic changes. Lungs tissues (n=8) from each experimental group were processed for histological evaluation at 4 h after LPS challenge. Representative histological changes of lungs obtained from mice of different groups. A: CON group, B: LPS group, C: LPS+Lir group. Representative histological lung sections were stained with H & E at 400× magnification. D: Severity scores of lung injury in different groups (n=8). Data are mean ± S.D. #P < 0.01 vs. CON group, **P < 0.01 vs. LPS group.
pulmonary microvascular barrier and increased the protein contents in BAL fluid compared with the CON group (P < 0.05). Liraglutide administration in the LPS+Lir group obviously decreased the protein content in BAL fluid compared with that in the LPS group (P < 0.01; Fig. 3).
3. Results 3.1. Effects of liraglutide on lung histopathologic changes in LPSinduced ALI mice The H & E staining sections of lung tissues from each group of mice are shown in Fig. 1. The alveolar structure was normal and had no abnormal changes in CON group (Fig. 1A). The lung tissue sections of the LPS group exhibited diffused pathological changes, including interstitial edema, obvious infiltration of inflammatory cells, haemorrhage and alveolar disarray (Fig. 1B). However, these histopathologic changes were markedly improved by liraglutide (Fig. 1C). The lung damage score was significantly higher in LPS group, compared to the CON group (P < 0.05). Liraglutide pretreatment significantly decreased the lung damage score (P < 0.01, Fig. 1D).
3.4. Effects of liraglutide on the IL-1β and IL-18 levels in BAL fluid of LPS-induced ALI mice The levels of cytokines IL-1β and IL-18 in BAL fluid were detected to determine whether liraglutide pretreatment decreased the levels of NLRP3 inflammasome regulated cytokines. The levels of IL-1β and IL18 in BAL fluid significantly increased in the LPS group (P < 0.01). Liraglutide pretreatment effectively decreased the levels of IL-1β and IL-18 in BAL fluid compared with those in the LPS group (P < 0.05; Fig. 4).
3.2. Effects of liraglutide on lung W/D weight ratio in LPS-induced ALI mice
3.5. Effects of liraglutide on MPO activity in LPS-induced ALI mice
The lung W/D weight ratio was used to examine pulmonary edema. As shown in Fig. 2, the lung W/D weight ratio of the LPS group was obviously higher than that of the CON group (P < 0.01). Additionally, the lung W/D weight ratio was significantly alleviated in the LPS+Lir group compared with that in the LPS group (P < 0.01).
MPO activity was used to determine the accumulation of neutrophil infiltrates. The MPO activity in the LPS group obviously increased compared with that in the CON group (P < 0.01, Fig. 5). By contrast, the MPO activity in the LPS+Lir group significantly decreased relative to that in the LPS group (P < 0.01).
3.3. Effects of liraglutide on inflammatory cell count and protein contents in BAL fluid of LPS-induced ALI mice
3.6. Effects of liraglutide on NLRP3 inflammasome expression in LPS-induced ALI mice
The total cell count in BAL fluid significantly increased in the LPS group, compared to the CON group (P < 0.01). However, liraglutide pretreatment markedly reduced the total cell count in BAL fluid (P < 0.01). The pulmonary microvascular permeability index was determined by the protein contents in BAL fluid. LPS severely damaged the
Lung tissue sections were immunostained with anti-NLRP3 antibody to locate NLRP3 in the lung tissues and determine whether liraglutide reduced the positively stained cells. We observed that the NLRP3 protein was mainly expressed in the inflammatory cells (i.e. macrophages and neutrophils). Few positively stained cells were found
Fig. 2. Effect of liraglutide on lung W/D weight ratio in lung tissues of LPS-induced ALI. Lung W/D weight ratio was determined at 4 h after LPS challenge. The values presented are mean ± S.D. (n=8) of three independent experiments. ＃P < 0.01 vs. CON group, **P < 0.01 vs. LPS group.
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Fig. 3. Effects of liraglutide on inflammatory cell infiltration and protein content in BAL fluid. The values presented are mean ± S.D. (n=8) of three independent experiments. ＃P < 0.01 vs. CON group, **P < 0.01 vs. LPS group.
in the lung tissues of the CON group (Fig. 6A). The positively stained
cells in LPS group were significantly increased comparing with the
Fig. 4. Effects of liraglutide on IL-1β and IL-18 production in BAL fluid. BAL fluid was collected at 4 h after LPS challenge for the analysis of inflammatory cytokines IL-1β and IL-18. The values presented are mean ± S.D. (n=8) of three independent experiments. ＃P < 0.01 vs. CON group, *P < 0.05 vs. LPS group.
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Fig. 5. Effects of liraglutide on MPO activity in lung tissues of LPS-induced ALI. MPO activity was determined at 4 h after LPS administration. The values presented are mean ± S.D. (n=8) of three independent experiments. ＃P < 0.01 vs. CON group, **P < 0.01 vs. LPS group.
Fig. 6. Effects of liraglutide on the NLRP3 inflammasome in the lung of the LPS-induced ALI mice model(n=8). NLRP3 expression was determined by immunohistochemistry (×400). Sections from LPS group were highly positively stained for NLRP3. Scattered positive cytoplasmic staining was noted in the inflammatory cells (B). In the liraglutide pretreated group, only some cells were stained for NLRP3 (C). NLRP3-positive cells were marked (arrows). D: NLRP3 positive cells in different groups (n=8). Data are mean ± S.D. ＃P < 0.01 vs. CON group, **P < 0.01 vs. LPS group.
Histological analysis further verified that liraglutide significantly attenuated lung tissue injury. Numerous inflammatory mediators are released into the alveolar space during lung injury (Ganter et al., 2008). Excessive cytokinemediated inflammation plays an important role in the pathogenesis of ALI/acute respiratory distress syndrome (ARDS) (Goodman et al., 2003). IL-1β, as a potent pro-inflammatory cytokine, is produced abundantly by alveolar macrophages in ALI/ARDS (Jacobs et al., 1989); this cytokine initiates and amplifies lung inflammation in patients (Dinarello, 1996). Excessive IL-1β levels damage vascular endothelial and alveolar epithelial permeability (Ganter et al., 2008), resulting in pulmonary edema. IL-18 levels are also associated with critical illness, including ALI and sepsis (Grobmyer et al., 2000; Hoshino et al., 2009). Increased levels of IL-18 are correlated with mortality in ALI/ARDS patients (Makabe et al., 2012). In the present study, liraglutide significantly decreased the production of IL-1β and IL-18. Therefore, we recognise that the protective effect of liraglutide on LPS-induced ALI may be ascribed to the reduction of IL-1β and IL18 levels. The NLRP3 inflammasome pathway is a primary intracellular
CON group (Fig. 6B). Liraglutide pretreatment decreased the amounts of positively stained cells in the lung tissues of ALI mice (Fig. 6C). NLRP3 positive cells count was significantly higher in LPS group, compared to CON group (P < 0.01). Liraglutide treatment significantly decreased the NLRP3 positive cells count (P < 0.01, Fig. 1D). The expression of NLRP3, ASC and Caspase-1 proteins was detected in the lung tissues. The results showed that LPS upregulated the expression of NLRP3, ASC and Caspase-1 proteins in the LPS group (P < 0.01) compared with the CON group. Liraglutide obviously decreased the expression of the above mentioned proteins relative to that in the LPS group (P < 0.05 or P < 0.01; Fig. 7).
4. Discussion ALI is characterized by interstitial edema, neutrophil accumulation, epithelial integrity disruption, and protein leakage into the alveolar space, severely influencing gas exchange (Matute-Bello et al., 2008). In this study, we observed the effects of liraglutide on LPS-induced ALI in mice. Liraglutide pretreatment decreased the lung W/D weight ratio, MPO activity, total cell counts and protein concentrations in BAL fluid.
Fig. 7. Effects of liraglutide on NLRP3, ASC, Caspase-1 protein expression in the lungs of the LPS-induced ALI mouse. NLRP3, ASC, Caspase-1 protein expression were determined by Western blot. The values presented are the mean ± S.D. (n=8) of three independent experiments. The density values of protein expression were normalized for β-actin. ＃P < 0.01 vs. CON group, *P < 0.05 and **P < 0.01 vs. LPS group.
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multiprotein inflammatory pathway of the innate immune system; this pathway responds to diverse exogenous and endogenous stimuli. The NLRP3 inflammasome forms through the activation of NLRP3 and recruitment of ASC and pro-Casp-1. Such phenomena result in Casp-1 activation and subsequent processing of pro-IL-1β and pro-IL-18 to attain their active forms (Kolliputi et al., 2012). Pulmonary histological changes were significantly improved in the LPS-induced ALI of NLRP3−/−mice (Grailer et al., 2014). The deletion of the NLRP3 gene diminished Caspase-1 activation and IL-1β production, thereby significantly improving hyperoxia-induced ALI (Fukumoto et al., 2013). ASC is a crucial adaptor protein required for recruiting Casp-1 to the NLRP3 platform of inflammasome; such process is important for Caspase-1 activation and IL-1β release (Tschopp et al., 2003). Deletion of ASC eliminated Klebsiella LPS-induced IL-1β release in macrophages. ALI in Caspase-1−/− or IL-18−/− knockout mice was attenuated in ventilator-induced acute lung injury (Dolinay et al., 2012). The present results demonstrated that liraglutide pretreatment significantly inhibited theNLRP3 inflammasome protein expression. These results imply that the protective effect of liraglutide on LPSinduced ALI may be attributed to its influence on the suppression of the NLRP3 inflammasome pathway. The MPO activity is associated with the number of infiltrating neutrophils in tissues (Tseng and Kung, 2012). In the development of ALI and ARDS, the infiltration of numerous neutrophils into alveolar spaces is another important pathogenic mechanism (Abraham et al., 2000). In the current study, the MPO activity obviously increased in the lung tissues of LPS-treated mice. Liraglutide pre-treatment effectively decreased the MPO activity in the lung tissues. This finding is consistent with the work of Viby (Viby et al., 2013) which demonstrated that liraglutide significantly inhibited alveoli neutrophil accumulation in a model of experimental obstructive lung disease in female mice. Overall, the results suggest that the inhibition of neutrophil infiltration may be another pathway through which liraglutide attenuates LPS-induced acute lung injury. In conclusion, liraglutide attenuated LPS-induced ALI by inhibiting the NLRP3 inflammasome pathway, thereby decreasing the production of IL-1β and IL-18 and inhibiting inflammatory responses. Collectively, the results suggest that liraglutide treatment is a novel strategy for the management of LPS-induced ALI. Conflict of interests The authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (NO. 81370872). References Abraham, E., Carmody, A., Shenkar, R., Arcaroli, J., 2000. Neutrophils as early immunologic effectors in hemorrhage- or endotoxemia-induced acute lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 279, L1137–L1145. Angus, D.C., Linde-Zwirble, W.T., Lidicker, J., Clermont, G., Carcillo, J., Pinsky, M.R., 2001. Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit. Care Med. 29, 1303–1310. Blandino-Rosano, M., Perez-Arana, G., Mellado-Gil, J.M., Segundo, C., AguilarDiosdado, M., 2008. Anti-proliferative effect of pro-inflammatory cytokines in cultured beta cells is associated with extracellular signal-regulated kinase 1/2 pathway inhibition: protective role of glucagon-like peptide-1. J. Mol. Endocrinol. 41, 35–44. Bosma, K.J., Taneja, R., Lewis, J.F., 2010. Pharmacotherapy for prevention and treatment of acute respiratory distress syndrome: current and experimental approaches. Drugs 70, 1255–1282. Brigham, K.L., Meyrick, B., 1986. Endotoxin and lung injury. Am. Rev. Respir. Dis. 133,
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