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GDF-15 prevents lipopolysaccharide-mediated acute lung injury via upregulating SIRT1
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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GDF-15 prevents lipopolysaccharide-mediated acute lung injury via upregulating SIRT1 Hengya Song a, 1, Qian Chen b, 1, Songping Xie a, Jie Huang a, Ganjun Kang a, * a b
Department of Thoracic Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, China Department of Oncology, Renmin Hospital of Wuhan University, Wuhan, 430060, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2020 Accepted 18 March 2020 Available online xxx
Inflammation and oxidative stress were involved in alveolar epithelial cells (AECs) damage and contributed to the progression of acute lung injury (ALI). Growth differentiation factor-15 (GDF-15) was reported to have important roles in pulmonary diseases, yet its role in AECs damage and ALI remains elusive. Herein, we found that GDF-15 was upregulated upon LPS stimulation in murine lungs and human AECs. GDF-15 treatment prevented, whereas Gdf-15 silence aggravated LPS-induced inflammation, oxidative stress and apoptosis. Moreover, we determined that GDF-15 alleviated AECs damage and ALI via upregulating SIRT1, and SIRT1 suppression completely abrogated the beneficial effects of GDF-15 in vivo and in vitro. GDF-15 protected against LPS-triggered AECs damage and ALI via upregulating SIRT1, and GDF-15 might be a valuable therapeutic candidate for treating ALI. © 2020 Elsevier Inc. All rights reserved.
Keywords: GDF-15 Lipopolysaccharide ALI SIRT1
1. Introduction Acute lung injury (ALI) is a devastating disease with high incidence and mortality, which ultimately predisposes the occurrence of acute respiratory distress syndrome (ARDS) related to severe refractory hypoxemia and multiple organ failure among patients in the intensive care unit [1]. Currently, there is no effective interventions for ALI patients besides mechanical ventilation, antibiotic therapy or other symptomatic therapeutic strategies [2]. The alveolar epithelial cells (AECs) serve as the main cell type in lung tissue and cover most of the alveolar inner surface area [3]. It is well-accepted that AECs compose the primary defense barrier against extrinsic insults and play critical roles in the pathogenesis of ALI [4]. Previous studies implicated that AECs were more vulnerable to injury in comparison with other lung cells and AECs damage could be observed in all ALI patients with different etiologies, which subsequently impaired gas exchange and fluid clearance [4,5]. Multiple factors contribute to AECs injury, including inflammation and oxidative stress [6,7]. In response to ALI, the expression and release of inflammatory cytokines, such as interleukin-1 beta
* Corresponding author. Department of Thoracic Surgery, Renmin Hospital of Wuhan University, No.238 Jiefang Road, Wuchang District, Wuhan, 430060, China. E-mail address: [email protected] (G. Kang). 1 These authors contributed equally to this work.
(IL-1b) and tumor necrosis factor alpha (TNF-a), were found to be elevated in human AECs and bronchoalveolar lavage fluid (BALF), and conversely, these cytokines act on AECs to elicit apoptotic pathways. Moreover, suppression of inflammation notably alleviates AECs injury and improves pulmonary function in the context of ALI [8]. Besides, oxidative stress is also involved in AECs injury and the development of ALI [9]. To neutralize the free radicals and prevent their detrimental effects, many endogenous antioxidants such as catalase (CAT) and superoxide dismutase (SOD) are synthesized under basal conditions, which however are rapidly overwhelmed during an acute injury [10]. Budinger et al. further found that SOD2 overexpression counteracted the activation of apoptotic pathways in primary AECs and prolonged the survival of ALI mice [5]. Sirtuin 1 (SIRT1) belongs to NAD þ related class III histone deacetylases and is implicated in regulating inflammation and oxidative stress [11,12]. Recent studies defined SIRT1 as a potential molecular node in AECs injury and ALI via regulating inflammation, oxidative stress and cell apoptosis [13,14]. Li et al. found that SIRT1 activation remarkably reduced the synthesis of inflammatory cytokines and blunted AECs apoptosis [15]. Whereas results from Gong et al. indicated that Sirt1 knockout promoted the release of inflammatory cytokines and aggravated oxidative damage in ALI [16]. Therefore, finding agents that significantly activate SIRT1 might help to develop novel therapeutic strategies against AECs injury and ALI. Growth differentiation factor-15 (GDF-15) is a divergent member of human transforming growth factor-b cytokine superfamily,
https://doi.org/10.1016/j.bbrc.2020.03.103 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: H. Song et al., GDF-15 prevents lipopolysaccharide-mediated acute lung injury via upregulating SIRT1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.103
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whose production could be triggered by tissue damage [17]. Previous studies verified that GDF-15 has important roles in pulmonary diseases. Husebø et al. reported that high plasma GDF-15 level was associated with the compromised pulmonary function and higher mortality in patients with chronic obstructive pulmonary disease [18]. Data from Zhang et al. found that GDF-15 expression was upregulated in bleomycin-challenged mice and human fibrotic lungs, which was inversely correlated with lung diffusion capacity and forced vital capacity [19]. Moreover, they identified AECs as the primary source of GDF-15 in human lung tissue via single-cell RNA sequencing method, yet its role in AECs injury remains elusive. The present study aims to investigate the role and potential molecular basis of GDF-15 on inflammation and oxidative stress during AECs injury and ALI progression. 2. Materials and methods 2.1. Animal ALI model and GDF-15 treatments All animal experimental procedures were supervised and approved by the Animal Ethics Committee of Renmin Hospital of Wuhan University, which were also in line with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Lipopolysaccharide (LPS), a main component of the outer membrane in Gram-negative bacteria, is identified as a major inducer of bacterial sepsis-induced ALI. Thus, ten-week-old male C57BL/6 mice were randomly exposed to intratracheal injection of LPS for 12 h (#L2630, Sigma) at a dosage of 5 mg/kg to generate LPSmediated ALI model in vivo, whereas mice assigned to control groups (Con) received equal volume of normal saline [20]. For survival study, mice were intratracheally treated with LPS at a lethal dosage (25 mg/kg) and the survival rate were counted every 12 h [20]. In addition, mice were subjected to an intraperitoneal infusion of vehicle or recombinant GDF-15 (1 mg/kg; #9279-GD, R&D Systems) for 12 h prior to LPS insult [21]. To clarify the involvement of SIRT1, a specific inhibitor, Ex527 (1 mg/kg; E7034, Sigma) was given for 24 h before LPS injection [22]. 2.2. Lung wet to dry ratio Lung wet to dry ration (wet/dry) was calculated to evaluate the degree of pulmonary edema [20]. Briefly, the left lung was harvested with the blood removed, which was then heated in a thermostatic oven at 80 C for 96 h to achieve constant dried weight.
2.5. Cell culture and treatment Human AECs cell line were purchased from the ScienCell Research Laboratories (San Diego, CA) and were resynchronized in serum-free medium for 16 h [28]. Then AECs were incubated with recombinant GDF-15 (20 ng/ml) in the presence or absence of LPS (1 mg/ml) for 24 h except specific annotation [7,17]. To knock down the expression of GDF-15 and SIRT1 in vitro, AECs were preinfected with small interfering RNA against Gdf-15 (siGdf-15) or Sirt1 (siSirt1) using Lipofectamine RNAiMAX (Invitrogen) for 24 h before LPS incubation [29,30]. Experiments were performed in cells with early passages from 3 to 7. 2.6. Biochemical analysis Lung homogenates and cell lysates were dissolved in extraction buffer for the analysis of malondialdehyde (MDA) and 3nitrotyrosine (3-NT) content, SOD, CAT, NADPH oxidase (NOX), SIRT1 and myeloperoxidase (MPO) activities according to the manufacturer’s instructions [20,23]. 2.7. Inflammatory cytokines and GDF-15 measurements IL-1b, TNF-a cytokines and GDF-15 protein level were measured by the ELISA method as previously described [20,23]. 2.8. Cell viability, lactate dehydrogenase (LDH) level and caspase-3 activity detection Cell viability was assessed by the cell counting kit-8 (CCK-8) assay [31,32]. The BALF and cell culture were collected for measuring LDH activity via a commercially available kit [20]. Caspase-3 activity was evaluated in lung homogenates and cell lysates via a fluorogenic peptide derived from DEVD-pNA [33]. 2.9. Statistical analysis All data were exhibited as mean ± standard deviation (SD) and the statistical analysis was performed by the SPSS software (Version 22.0). Comparisons between two groups were performed using the two tailed Student’s t-test, whereas multi-group’s comparisons were performed by one-way ANOVA analysis. A P value less than 0.05 were considered statistically significant. 3. Results
2.3. BALF acquisition and analysis After euthanasia, the mice were intratracheally injected with 1 ml cooled phosphate buffer saline (PBS) for three times and then the fluid was collected, centrifuged (1500 rpm, 4 C) for 10 min. Numbers of total cells, neutrophils and macrophages were counted with a hemocytometer and Wright-Giemsa staining [20,23]. The cell-free supernatant was collected for total protein content analysis using a Bio-Rad protein assay and inflammatory cytokine detection by the enzyme-linked immunosorbent assay (ELISA) method. 2.4. Western blot and quantitative real-time PCR analysis Total proteins were extracted using RIPA lysis (ThermoFisher) and separated by SDS-PAGE as previously described [22,24,25]. Special protein bands were scanned by the ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc.; Hercules, CA, USA). Total RNA was isolated and quantified by ABI real-time PCR system (7900HT FAST; Foster City, CA) [26,27].
3.1. GDF-15 suppressed LPS-induced inflammation, oxidative stress and apoptosis in AECs We first detected the expression of GDF-15 in lung tissues from LPS-injected mice and the data showed that GDF-15 expression was upregulated during ALI (Fig. 1A). Besides, we observed that Gdf-15 mRNA level in AECs was gradually increased in response to LPS insult, with the most expression in 24 h (Fig. 1B). Therefore, we selected this time course in our next study. As depicted in Fig. 1C, GDF-15 expression and the release to the medium was augmented with LPS treatment. Next, we treated AECs with GDF-15 to verify its role in vitro. As shown in Fig. 1D, GDF-15 (from 0 to 200 ng/ml) treatment showed no impact on cell survival at basal conditions. We thus selected the concentration 20 ng/ml for further study as previously described [17]. LPS incubation triggered the expression and release of IL-1b, TNF-a, which were notably suppressed with GDF-15 protection (Fig. 1EeF). Besides, we found GDF-15 treatment significantly decreased MDA and 3-NT levels in LPS-treated AECs (Fig. 1G). Accordingly, the anti-oxidant enzymes SOD and CAT
Please cite this article as: H. Song et al., GDF-15 prevents lipopolysaccharide-mediated acute lung injury via upregulating SIRT1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.103
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Fig. 1. GDF-15 suppressed LPS-induced inflammation, oxidative stress and apoptosis in AECs. (A) The mRNA and protein level of GDF-15 in murine lungs from ALI mice (n ¼ 6). (B) The Gdf-15 mRNA level in AECs after LPS (1 mg/ml) treatment for indicating time course (n ¼ 6). (C) GDF-15 protein level in AECs cell lysates or medium (n ¼ 6). (D) Cell viability with different concentration of GDF-15 incubation for 24 h (n ¼ 5). (E) The relative mRNA level in indicating groups (n ¼ 6). (F) The level of IL-1b and TNF-a in medium (n ¼ 6). (G) MDA and 3-NT content in AECs from indicating groups (n ¼ 6). (H) SOD, CAT and NOX activities in AECs from indicating groups (n ¼ 6). (I) Cell viability detected by CCK-8 assay (n ¼ 5). (J) LDH release to medium (n ¼ 8). (K) Representative western blot images and the statistical data (n ¼ 6). (L) Caspase-3 activity in AECs from indicating groups (n ¼ 8). Results are expressed as the mean ± standard deviation (SD). *P < 0.05 compared with the matched group. n.s., no significance.
activities were upregulated, whereas the pro-oxidant enzymes NOX activity was downregulated by GDF-15 incubation after LPS insult (Fig. 1H). Uncontrolled inflammation and oxidative stress elicited multiple apoptotic pathways and provoked cell death in response to LPS [8,9]. As expected, AECs with LPS incubation have increased cell apoptosis, which was alleviated in the presence of GDF-15, as evidenced by the improved cell viability and decreased LDH release, apoptosis-related protein expression and caspase-3 activity (Fig. 1IL). 3.2. GDF-15 silence aggravated LPS-induced inflammation, oxidative stress and apoptosis in AECs On the contrary, Gdf-15 silence exacerbated the expression of inflammatory cytokines in response to LPS insult (Fig. 2AeB). The
production of MDA and 3-NT was also elevated in Gdf-15-deficient cells (Fig. 2C). Besides, we observed that the activities of SOD and CAT were decreased, whereas NOX activity was further increased in LPS-treated AECs after Gdf-15 knockdown (Fig. 2DeE). Moreover, LPS-induced AECs injury and apoptosis were also aggravated in siGdf-15-infected cells with LPS treatment, as evidenced by the increased caspase-3 activity, LDH release and decreased cell viability (Fig. 2FeH). 3.3. GDF-15 alleviated AECs injury in response to LPS incubation via upregulating SIRT1 Recent studies defined SIRT1 as a molecular node in LPSinduced AECs injury and our data also showed that GDF-15 incubation preserved, whereas Gdf-15 silence further decreased SIRT1
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Fig. 2. GDF-15 silence aggravated LPS-induced inflammation, oxidative stress and apoptosis in AECs. (A) The mRNA and protein level of GDF-15 in AECs (n ¼ 6). (B) The level of IL-1b and TNF-a in medium (n ¼ 6). (C) MDA and 3-NT content in AECs from indicating groups (n ¼ 6). (DeE) SOD, CAT and NOX activities in AECs from indicating groups (n ¼ 6). (F) Caspase-3 activity in AECs (n ¼ 6). (G) LDH release to medium (n ¼ 8). (H) Cell viability detected by CCK-8 assay (n ¼ 5). Results are expressed as the mean ± SD. *P < 0.05 compared with the matched group.
expression and activity in response to LPS injury (Fig. 3AeD). To clarify the role of SIRT1, we silenced SIRT1 in AECs and the efficiency was confirmed in Fig. 3E. GDF-15 treatment notably decreased IL-1b and TNF-a expression in response to LPS treatment, yet failed to do so in Sirt1-deficient cells (Fig. 3F). In addition, GDF15 incubation significantly increased the activities of SOD, CAT and decreased MDA, 3-NT content together with NOX activity in AECs, but not in that pre-infected with siSirt1 (Fig. 3GeI). Notably, GDF15-mediated suppression on AECs damage was also abrogated in infected AECs, as revealed by the enhanced caspase-3 activity, LDH release and decreased cell survival (Fig. 3J). 3.4. SIRT1 inhibition abolished the beneficial effects of GDF-15 on lung tissue injury in ALI mice AECs serve as the primary defense barrier against extrinsic insults and its injury was essential for the development of ALI [6]. As shown in Fig. 4A, LPS injection triggered intrapulmonary inflammatory responses in mice, as evidenced by the increased level of IL1b and TNF-a in BALF, which was effectively reduced in mice with GDF-15 pretreatment. Besides, GDF-15 markedly inhibited the accumulation of total cells, neutrophils, macrophages and decreased total protein levels in BALF induced by LPS injury (Fig. 4BeC). Accordingly, the MPO activity in lung tissue was also suppressed in mice with GDF-15 protection (Fig. 4D). Consistent
with the in vitro data, we found that GDF-15 pretreatment significantly attenuated LPS-induced oxidative damage to the lung, as confirmed by the decreased MDA, 3-NT content, NOX activity and increased SOD, CAT activities (Fig. 4EeG). LDH level and caspase-3 activity, indices for cellular damage and apoptosis were increased in LPS-treated lung tissue, which were inhibited by GDF-15 treatment (Fig. 4H). In addition, pretreatment of GDF-15 remarkably attenuated pulmonary edema, as reflected by the decreased lung wet/dry ratio (Fig. 4I). Consistent with the protective phenotype, we observed that the induction of pro-apoptotic protein was downregulated, and conversely, the anti-apoptotic protein was upregulated in GDF-15-treated lung tissue after LPS insult (Fig. 4J). More importantly, GDF-15 administration significantly improved the survival condition in LPS-triggered ALI mice (Fig. 4K). Mice were then treated with Ex527 to further clarify the role of SIRT1 in vivo. As shown in Figs. S1AeC, GDF-15 pretreatment reduced IL-1b, TNF-a level in BALF and markedly blocked inflammatory cell infiltration to lung tissue in ALI mice, which was abrogated by SIRT1 inhibition. In addition, Ex527 abrogated the beneficial effects of GDF-15 on LPS-induced oxidative stress, as assessed by the increased MDA and 3-NT levels (Fig. S1D). The elevated LDH level and caspase-3 activity were restored by GDF-15 in control mice, but not in that treated with SIRT1 inhibitor (Figs. S1EeF). As expected, the improved pulmonary edema seen in GDF-15-treated mice were almost completely abolished by SIRT1 suppression (Fig. S1G).
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Fig. 3. GDF-15 alleviated AECs injury in response to LPS incubation via upregulating SIRT1. (AeB) The protein level and activity of SIRT1 in AECs with GDF-15 incubation (n ¼ 6). (CeD) The protein level and activity of SIRT1 in siGdf-15-infected AECs (n ¼ 6). (E) The efficiency of siGdf-15 in AECs detected by the mRNA and activity of SIRT1 (n ¼ 6). (F) The level of IL-1b and TNF-a in medium (n ¼ 6). (G) MDA and 3-NT content in AECs from indicating groups (n ¼ 6). (HeI) SOD, CAT and NOX activities in AECs from indicating groups (n ¼ 6). (J) Caspase-3 activity, LDH release to medium and cell viability (n ¼ 6). Results are expressed as the mean ± SD. *P < 0.05 compared with the matched group.
4. Discussion ALI is a common medical emergency and potentially lifethreatening condition with high mortality, which could rapidly become decompensation and progress to ARDS [2]. Despite an advancement of the mechanical ventilation and other adjunctive strategies, the mortality rate of ALI is still high and its prognosis remains poor worldwide. Therefore, the identification of novel therapeutic agents is greatly needed for treating ALI. In the present study, we found that GDF-15 treatment notably attenuated LPSinduced AECs injury in vitro and effectively prevented inflammation as well as oxidative stress during ALI in mice. Moreover, GDF15 administration could remarkably improve survival rate in LPSchallenged ALI. Our data clearly identified GDF-15 as a potential therapeutic agent against LPS-mediated ALI. Unrestrained inflammatory response and excessive oxidative stress are proven to play critical roles in the initiation and
progression of LPS-induced ALI [8,9]. In response to LPS stimulation, Toll-like receptors were recruited to lipid rafts and interacted with specific molecular adaptors, resulting in the activation of downstream signaling axis responsible for pro-inflammatory cytokines synthesis [34]. Besides, Huang et al. recently revealed that LPS injection could activate nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome, which is essential for the maturation and release of pro-inflammatory cytokines [20]. Consistently, we herein found that LPS treatment promoted the expression and release of IL-1b, TNF-a in AECs as well as murine lungs. Reactive oxygen species (ROS) overproduction is another feature of ALI and contributes to the development of ALI. Oxygen free radicals, such as superoxide anion and hydrogen peroxide, could directly trigger lipid and protein peroxidation, thereby causing cell membrane disruption and protein dysfunction. In accordance with these findings, we found that the byproducts of lipid/protein peroxidation in AECs and murine lung tissues were
Please cite this article as: H. Song et al., GDF-15 prevents lipopolysaccharide-mediated acute lung injury via upregulating SIRT1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.103
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Fig. 4. SIRT1 inhibition abolished the beneficial effects of GDF-15 on lung tissue injury in ALI mice. (A) The level of IL-1b and TNF-a in BALF from ALI mice (n ¼ 6). (B) Inflammatory cell counts in BALF from ALI mice (n ¼ 8). (C) Total protein concentrations in in BALF from ALI mice (n ¼ 6). (D) MPO activity in lung tissue (n ¼ 6). (E) MDA and 3-NT content in murine lungs from indicating groups (n ¼ 6). (FeG) SOD, CAT and NOX activities in murine lungs from indicating groups (n ¼ 6). (H) LDH level and caspase-3 activity in murine lung tissue (n ¼ 6). (I) Lung wet/dry ration (n ¼ 10). (J) Representative western blot images and the statistical data (n ¼ 6). (K) Survival rate in mice with 25 mg/ml LPS administration (n ¼ 20). Results are expressed as the mean ± SD. *P < 0.05 compared with the matched group.
increased upon LPS stimulation. To combat the oxidative damage, the organisms are equipped with effectively anti-oxidant enzymatic defenses to scavenge ROS, including SOD and CAT [10]. However, we identified the activities of SOD and CAT were markedly decreased by LPS treatment. AECs cover most of the alveolar surface area and are important for barrier defense and fluid homeostasis of the lung tissue [6]. A complex network of inflammatory cytokines and oxygen free radicals contributes to mediate, amplify and perpetuate AECs damage and the lung injury process, which subsequently destroyed AECs barrier integrity and promoted pulmonary edema as well as leukocyte extravasations during ALI. Herein, we also observed that LPS instillation elicited leukocyte
infiltration and fluid leakage in murine lungs that were prevented by GDF-15 administration. SIRT1 is implicated in various pathophysiological processes, including inflammatory response and oxidative stress. Yuan et al. previously found that SIRT1 activation protected against inflammatory response in doxorubicin-induced cardiotoxicity, whereas results from Yin et al. indicated that Sirt1 deletion accelerated inflammation and steatosis in response to ethanol challenge [22,35]. Anh et al. recently found that SIRT1 could interact with nuclear factor-kappa B (NF-kB) and blocked its transcriptional activity via deacetylation in arterial inflammation [36]. In addition, a previous study identified a specific CpG site on IL-1b proximal
Please cite this article as: H. Song et al., GDF-15 prevents lipopolysaccharide-mediated acute lung injury via upregulating SIRT1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.103
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promoter, and Sirt1 deficiency caused its hypomethylation and IL1b transactivation [37]. Furthermore, SIRT1 activation by resveratrol obviously inhibited NLRP3 inflammasome activation and thereby protected against LPS-induced lung injury [38]. NF-E2related factor 2 (NRF2) serves a redox sensitive transcription factor and plays a pivotal role in maintaining redox homeostasis against oxidative damage via modulating antioxidant response element (ARE) [39,40]. Previous studies verified that SIRT1 activation notably promoted NRF2/ARE axis and enhanced the antioxidant capacity [41]. Herein, we observed that SIRT1 was upregulated by GDF-15 treatment, which was further downregulated in Gdf-15-deficient AECs upon LPS stimulation. SIRT1 suppression markedly abolished the protective effects of GDF-15 on LPSinduced inflammation, oxidative stress and cell apoptosis. Our data identified GDF-15 as a valuable therapeutic candidate for treating ALI. Declaration of competing interest The authors state no conflict of interest. Acknowledgements This study is supported by the National Natural Science Foundation of China (No: 81801954) and the Fundamental Research Funds for the Central Universities (No. 2042019kf0057). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.03.103. References [1] A.J. Harris, A.S. Mirchandani, R.W. Lynch, et al., IL4Ralpha signaling abrogates hypoxic neutrophil survival and limits acute lung injury responses in vivo, Am. J. Respir. Crit. Care Med. 200 (2019) 235e246, https://doi.org/10.1164/ rccm.201808-1599OC. [2] P. Radermacher, S.M. Maggiore, A. Mercat, Fifty years of Research in ARDS. Gas exchange in acute respiratory distress syndrome, Am. J. Respir. Crit. Care Med. 196 (2017) 964e984, https://doi.org/10.1164/rccm.201610-2156SO. [3] M.S. Taylor, R.R. Chivukula, L.C. Myers, et al., Delayed alveolar epithelialization: a distinct pathology in diffuse acute lung injury, Am. J. Respir. Crit. Care Med. 197 (2018) 522e524, https://doi.org/10.1164/rccm.201706-1094LE. [4] Y. Guo, A. Mishra, T. Weng, et al., Wnt3a mitigates acute lung injury by reducing P2X7 receptor-mediated alveolar epithelial type I cell death, Cell Death Dis. 5 (2014), https://doi.org/10.1038/cddis.2014.254 e1286. [5] G.R. Budinger, G.M. Mutlu, D. Urich, et al., Epithelial cell death is an important contributor to oxidant-mediated acute lung injury, Am. J. Respir. Crit. Care Med. 183 (2011) 1043e1054, https://doi.org/10.1164/rccm.201002-0181OC. [6] X. Wang, M. Liu, M.J. Zhu, et al., Resveratrol protects the integrity of alveolar epithelial barrier via SIRT1/PTEN/p-Akt pathway in methamphetamineinduced chronic lung injury, Cell Prolif (2020), https://doi.org/10.1111/ cpr.12773 e12773. [7] C.Y. Chuang, T.L. Chen, Y.G. Cherng, et al., Lipopolysaccharide induces apoptotic insults to human alveolar epithelial A549 cells through reactive oxygen species-mediated activation of an intrinsic mitochondrion-dependent pathway, Arch. Toxicol. 85 (2011) 209e218, https://doi.org/10.1007/s00204010-0585-x. [8] R.B. Goodman, J. Pugin, J.S. Lee, et al., Cytokine-mediated inflammation in acute lung injury, Cytokine Growth Factor Rev. 14 (2003) 523e535, https:// doi.org/10.1016/s1359-6101(03)00059-5. [9] C.W. Chow, A.M. Herrera, T. Suzuki, et al., Oxidative stress and acute lung injury, Am. J. Respir. Cell Mol. Biol. 29 (2003) 427e431, https://doi.org/ 10.1165/rcmb.F278. [10] T. Li, Y.N. Wu, H. Wang, et al., Dapk1 improves inflammation, oxidative stress and autophagy in LPS-induced acute lung injury via p38MAPK/NF-kappaB signaling pathway, Mol. Immunol. 120 (2020) 13e22, https://doi.org/ 10.1016/j.molimm.2020.01.014. [11] I.C. Lee, X.Y. Ho, S.E. George, et al., Oxidative stress promotes SIRT1 recruitment to the GADD34/PP1alpha complex to activate its deacetylase function, Cell Death Differ. 25 (2018) 255e267, https://doi.org/10.1038/cdd.2017.152. [12] M. Magni, G. Buscemi, L. Maita, et al., TSPYL2 is a novel regulator of SIRT1 and p300 activity in response to DNA damage, Cell Death Differ. 26 (2019)
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GDF-15 prevents lipopolysaccharide-mediated acute lung injury via upregulating SIRT1 Hengya Song a, 1, Qian Chen b, 1, Songping Xie a, Jie Huang a, Ganjun Kang a, * a b
Department of Thoracic Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, China Department of Oncology, Renmin Hospital of Wuhan University, Wuhan, 430060, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2020 Accepted 18 March 2020 Available online xxx
Inflammation and oxidative stress were involved in alveolar epithelial cells (AECs) damage and contributed to the progression of acute lung injury (ALI). Growth differentiation factor-15 (GDF-15) was reported to have important roles in pulmonary diseases, yet its role in AECs damage and ALI remains elusive. Herein, we found that GDF-15 was upregulated upon LPS stimulation in murine lungs and human AECs. GDF-15 treatment prevented, whereas Gdf-15 silence aggravated LPS-induced inflammation, oxidative stress and apoptosis. Moreover, we determined that GDF-15 alleviated AECs damage and ALI via upregulating SIRT1, and SIRT1 suppression completely abrogated the beneficial effects of GDF-15 in vivo and in vitro. GDF-15 protected against LPS-triggered AECs damage and ALI via upregulating SIRT1, and GDF-15 might be a valuable therapeutic candidate for treating ALI. © 2020 Elsevier Inc. All rights reserved.
Keywords: GDF-15 Lipopolysaccharide ALI SIRT1
1. Introduction Acute lung injury (ALI) is a devastating disease with high incidence and mortality, which ultimately predisposes the occurrence of acute respiratory distress syndrome (ARDS) related to severe refractory hypoxemia and multiple organ failure among patients in the intensive care unit [1]. Currently, there is no effective interventions for ALI patients besides mechanical ventilation, antibiotic therapy or other symptomatic therapeutic strategies [2]. The alveolar epithelial cells (AECs) serve as the main cell type in lung tissue and cover most of the alveolar inner surface area [3]. It is well-accepted that AECs compose the primary defense barrier against extrinsic insults and play critical roles in the pathogenesis of ALI [4]. Previous studies implicated that AECs were more vulnerable to injury in comparison with other lung cells and AECs damage could be observed in all ALI patients with different etiologies, which subsequently impaired gas exchange and fluid clearance [4,5]. Multiple factors contribute to AECs injury, including inflammation and oxidative stress [6,7]. In response to ALI, the expression and release of inflammatory cytokines, such as interleukin-1 beta
* Corresponding author. Department of Thoracic Surgery, Renmin Hospital of Wuhan University, No.238 Jiefang Road, Wuchang District, Wuhan, 430060, China. E-mail address: [email protected] (G. Kang). 1 These authors contributed equally to this work.
(IL-1b) and tumor necrosis factor alpha (TNF-a), were found to be elevated in human AECs and bronchoalveolar lavage fluid (BALF), and conversely, these cytokines act on AECs to elicit apoptotic pathways. Moreover, suppression of inflammation notably alleviates AECs injury and improves pulmonary function in the context of ALI [8]. Besides, oxidative stress is also involved in AECs injury and the development of ALI [9]. To neutralize the free radicals and prevent their detrimental effects, many endogenous antioxidants such as catalase (CAT) and superoxide dismutase (SOD) are synthesized under basal conditions, which however are rapidly overwhelmed during an acute injury [10]. Budinger et al. further found that SOD2 overexpression counteracted the activation of apoptotic pathways in primary AECs and prolonged the survival of ALI mice [5]. Sirtuin 1 (SIRT1) belongs to NAD þ related class III histone deacetylases and is implicated in regulating inflammation and oxidative stress [11,12]. Recent studies defined SIRT1 as a potential molecular node in AECs injury and ALI via regulating inflammation, oxidative stress and cell apoptosis [13,14]. Li et al. found that SIRT1 activation remarkably reduced the synthesis of inflammatory cytokines and blunted AECs apoptosis [15]. Whereas results from Gong et al. indicated that Sirt1 knockout promoted the release of inflammatory cytokines and aggravated oxidative damage in ALI [16]. Therefore, finding agents that significantly activate SIRT1 might help to develop novel therapeutic strategies against AECs injury and ALI. Growth differentiation factor-15 (GDF-15) is a divergent member of human transforming growth factor-b cytokine superfamily,
https://doi.org/10.1016/j.bbrc.2020.03.103 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: H. Song et al., GDF-15 prevents lipopolysaccharide-mediated acute lung injury via upregulating SIRT1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.103
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whose production could be triggered by tissue damage [17]. Previous studies verified that GDF-15 has important roles in pulmonary diseases. Husebø et al. reported that high plasma GDF-15 level was associated with the compromised pulmonary function and higher mortality in patients with chronic obstructive pulmonary disease [18]. Data from Zhang et al. found that GDF-15 expression was upregulated in bleomycin-challenged mice and human fibrotic lungs, which was inversely correlated with lung diffusion capacity and forced vital capacity [19]. Moreover, they identified AECs as the primary source of GDF-15 in human lung tissue via single-cell RNA sequencing method, yet its role in AECs injury remains elusive. The present study aims to investigate the role and potential molecular basis of GDF-15 on inflammation and oxidative stress during AECs injury and ALI progression. 2. Materials and methods 2.1. Animal ALI model and GDF-15 treatments All animal experimental procedures were supervised and approved by the Animal Ethics Committee of Renmin Hospital of Wuhan University, which were also in line with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Lipopolysaccharide (LPS), a main component of the outer membrane in Gram-negative bacteria, is identified as a major inducer of bacterial sepsis-induced ALI. Thus, ten-week-old male C57BL/6 mice were randomly exposed to intratracheal injection of LPS for 12 h (#L2630, Sigma) at a dosage of 5 mg/kg to generate LPSmediated ALI model in vivo, whereas mice assigned to control groups (Con) received equal volume of normal saline [20]. For survival study, mice were intratracheally treated with LPS at a lethal dosage (25 mg/kg) and the survival rate were counted every 12 h [20]. In addition, mice were subjected to an intraperitoneal infusion of vehicle or recombinant GDF-15 (1 mg/kg; #9279-GD, R&D Systems) for 12 h prior to LPS insult [21]. To clarify the involvement of SIRT1, a specific inhibitor, Ex527 (1 mg/kg; E7034, Sigma) was given for 24 h before LPS injection [22]. 2.2. Lung wet to dry ratio Lung wet to dry ration (wet/dry) was calculated to evaluate the degree of pulmonary edema [20]. Briefly, the left lung was harvested with the blood removed, which was then heated in a thermostatic oven at 80 C for 96 h to achieve constant dried weight.
2.5. Cell culture and treatment Human AECs cell line were purchased from the ScienCell Research Laboratories (San Diego, CA) and were resynchronized in serum-free medium for 16 h [28]. Then AECs were incubated with recombinant GDF-15 (20 ng/ml) in the presence or absence of LPS (1 mg/ml) for 24 h except specific annotation [7,17]. To knock down the expression of GDF-15 and SIRT1 in vitro, AECs were preinfected with small interfering RNA against Gdf-15 (siGdf-15) or Sirt1 (siSirt1) using Lipofectamine RNAiMAX (Invitrogen) for 24 h before LPS incubation [29,30]. Experiments were performed in cells with early passages from 3 to 7. 2.6. Biochemical analysis Lung homogenates and cell lysates were dissolved in extraction buffer for the analysis of malondialdehyde (MDA) and 3nitrotyrosine (3-NT) content, SOD, CAT, NADPH oxidase (NOX), SIRT1 and myeloperoxidase (MPO) activities according to the manufacturer’s instructions [20,23]. 2.7. Inflammatory cytokines and GDF-15 measurements IL-1b, TNF-a cytokines and GDF-15 protein level were measured by the ELISA method as previously described [20,23]. 2.8. Cell viability, lactate dehydrogenase (LDH) level and caspase-3 activity detection Cell viability was assessed by the cell counting kit-8 (CCK-8) assay [31,32]. The BALF and cell culture were collected for measuring LDH activity via a commercially available kit [20]. Caspase-3 activity was evaluated in lung homogenates and cell lysates via a fluorogenic peptide derived from DEVD-pNA [33]. 2.9. Statistical analysis All data were exhibited as mean ± standard deviation (SD) and the statistical analysis was performed by the SPSS software (Version 22.0). Comparisons between two groups were performed using the two tailed Student’s t-test, whereas multi-group’s comparisons were performed by one-way ANOVA analysis. A P value less than 0.05 were considered statistically significant. 3. Results
2.3. BALF acquisition and analysis After euthanasia, the mice were intratracheally injected with 1 ml cooled phosphate buffer saline (PBS) for three times and then the fluid was collected, centrifuged (1500 rpm, 4 C) for 10 min. Numbers of total cells, neutrophils and macrophages were counted with a hemocytometer and Wright-Giemsa staining [20,23]. The cell-free supernatant was collected for total protein content analysis using a Bio-Rad protein assay and inflammatory cytokine detection by the enzyme-linked immunosorbent assay (ELISA) method. 2.4. Western blot and quantitative real-time PCR analysis Total proteins were extracted using RIPA lysis (ThermoFisher) and separated by SDS-PAGE as previously described [22,24,25]. Special protein bands were scanned by the ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc.; Hercules, CA, USA). Total RNA was isolated and quantified by ABI real-time PCR system (7900HT FAST; Foster City, CA) [26,27].
3.1. GDF-15 suppressed LPS-induced inflammation, oxidative stress and apoptosis in AECs We first detected the expression of GDF-15 in lung tissues from LPS-injected mice and the data showed that GDF-15 expression was upregulated during ALI (Fig. 1A). Besides, we observed that Gdf-15 mRNA level in AECs was gradually increased in response to LPS insult, with the most expression in 24 h (Fig. 1B). Therefore, we selected this time course in our next study. As depicted in Fig. 1C, GDF-15 expression and the release to the medium was augmented with LPS treatment. Next, we treated AECs with GDF-15 to verify its role in vitro. As shown in Fig. 1D, GDF-15 (from 0 to 200 ng/ml) treatment showed no impact on cell survival at basal conditions. We thus selected the concentration 20 ng/ml for further study as previously described [17]. LPS incubation triggered the expression and release of IL-1b, TNF-a, which were notably suppressed with GDF-15 protection (Fig. 1EeF). Besides, we found GDF-15 treatment significantly decreased MDA and 3-NT levels in LPS-treated AECs (Fig. 1G). Accordingly, the anti-oxidant enzymes SOD and CAT
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Fig. 1. GDF-15 suppressed LPS-induced inflammation, oxidative stress and apoptosis in AECs. (A) The mRNA and protein level of GDF-15 in murine lungs from ALI mice (n ¼ 6). (B) The Gdf-15 mRNA level in AECs after LPS (1 mg/ml) treatment for indicating time course (n ¼ 6). (C) GDF-15 protein level in AECs cell lysates or medium (n ¼ 6). (D) Cell viability with different concentration of GDF-15 incubation for 24 h (n ¼ 5). (E) The relative mRNA level in indicating groups (n ¼ 6). (F) The level of IL-1b and TNF-a in medium (n ¼ 6). (G) MDA and 3-NT content in AECs from indicating groups (n ¼ 6). (H) SOD, CAT and NOX activities in AECs from indicating groups (n ¼ 6). (I) Cell viability detected by CCK-8 assay (n ¼ 5). (J) LDH release to medium (n ¼ 8). (K) Representative western blot images and the statistical data (n ¼ 6). (L) Caspase-3 activity in AECs from indicating groups (n ¼ 8). Results are expressed as the mean ± standard deviation (SD). *P < 0.05 compared with the matched group. n.s., no significance.
activities were upregulated, whereas the pro-oxidant enzymes NOX activity was downregulated by GDF-15 incubation after LPS insult (Fig. 1H). Uncontrolled inflammation and oxidative stress elicited multiple apoptotic pathways and provoked cell death in response to LPS [8,9]. As expected, AECs with LPS incubation have increased cell apoptosis, which was alleviated in the presence of GDF-15, as evidenced by the improved cell viability and decreased LDH release, apoptosis-related protein expression and caspase-3 activity (Fig. 1IL). 3.2. GDF-15 silence aggravated LPS-induced inflammation, oxidative stress and apoptosis in AECs On the contrary, Gdf-15 silence exacerbated the expression of inflammatory cytokines in response to LPS insult (Fig. 2AeB). The
production of MDA and 3-NT was also elevated in Gdf-15-deficient cells (Fig. 2C). Besides, we observed that the activities of SOD and CAT were decreased, whereas NOX activity was further increased in LPS-treated AECs after Gdf-15 knockdown (Fig. 2DeE). Moreover, LPS-induced AECs injury and apoptosis were also aggravated in siGdf-15-infected cells with LPS treatment, as evidenced by the increased caspase-3 activity, LDH release and decreased cell viability (Fig. 2FeH). 3.3. GDF-15 alleviated AECs injury in response to LPS incubation via upregulating SIRT1 Recent studies defined SIRT1 as a molecular node in LPSinduced AECs injury and our data also showed that GDF-15 incubation preserved, whereas Gdf-15 silence further decreased SIRT1
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Fig. 2. GDF-15 silence aggravated LPS-induced inflammation, oxidative stress and apoptosis in AECs. (A) The mRNA and protein level of GDF-15 in AECs (n ¼ 6). (B) The level of IL-1b and TNF-a in medium (n ¼ 6). (C) MDA and 3-NT content in AECs from indicating groups (n ¼ 6). (DeE) SOD, CAT and NOX activities in AECs from indicating groups (n ¼ 6). (F) Caspase-3 activity in AECs (n ¼ 6). (G) LDH release to medium (n ¼ 8). (H) Cell viability detected by CCK-8 assay (n ¼ 5). Results are expressed as the mean ± SD. *P < 0.05 compared with the matched group.
expression and activity in response to LPS injury (Fig. 3AeD). To clarify the role of SIRT1, we silenced SIRT1 in AECs and the efficiency was confirmed in Fig. 3E. GDF-15 treatment notably decreased IL-1b and TNF-a expression in response to LPS treatment, yet failed to do so in Sirt1-deficient cells (Fig. 3F). In addition, GDF15 incubation significantly increased the activities of SOD, CAT and decreased MDA, 3-NT content together with NOX activity in AECs, but not in that pre-infected with siSirt1 (Fig. 3GeI). Notably, GDF15-mediated suppression on AECs damage was also abrogated in infected AECs, as revealed by the enhanced caspase-3 activity, LDH release and decreased cell survival (Fig. 3J). 3.4. SIRT1 inhibition abolished the beneficial effects of GDF-15 on lung tissue injury in ALI mice AECs serve as the primary defense barrier against extrinsic insults and its injury was essential for the development of ALI [6]. As shown in Fig. 4A, LPS injection triggered intrapulmonary inflammatory responses in mice, as evidenced by the increased level of IL1b and TNF-a in BALF, which was effectively reduced in mice with GDF-15 pretreatment. Besides, GDF-15 markedly inhibited the accumulation of total cells, neutrophils, macrophages and decreased total protein levels in BALF induced by LPS injury (Fig. 4BeC). Accordingly, the MPO activity in lung tissue was also suppressed in mice with GDF-15 protection (Fig. 4D). Consistent
with the in vitro data, we found that GDF-15 pretreatment significantly attenuated LPS-induced oxidative damage to the lung, as confirmed by the decreased MDA, 3-NT content, NOX activity and increased SOD, CAT activities (Fig. 4EeG). LDH level and caspase-3 activity, indices for cellular damage and apoptosis were increased in LPS-treated lung tissue, which were inhibited by GDF-15 treatment (Fig. 4H). In addition, pretreatment of GDF-15 remarkably attenuated pulmonary edema, as reflected by the decreased lung wet/dry ratio (Fig. 4I). Consistent with the protective phenotype, we observed that the induction of pro-apoptotic protein was downregulated, and conversely, the anti-apoptotic protein was upregulated in GDF-15-treated lung tissue after LPS insult (Fig. 4J). More importantly, GDF-15 administration significantly improved the survival condition in LPS-triggered ALI mice (Fig. 4K). Mice were then treated with Ex527 to further clarify the role of SIRT1 in vivo. As shown in Figs. S1AeC, GDF-15 pretreatment reduced IL-1b, TNF-a level in BALF and markedly blocked inflammatory cell infiltration to lung tissue in ALI mice, which was abrogated by SIRT1 inhibition. In addition, Ex527 abrogated the beneficial effects of GDF-15 on LPS-induced oxidative stress, as assessed by the increased MDA and 3-NT levels (Fig. S1D). The elevated LDH level and caspase-3 activity were restored by GDF-15 in control mice, but not in that treated with SIRT1 inhibitor (Figs. S1EeF). As expected, the improved pulmonary edema seen in GDF-15-treated mice were almost completely abolished by SIRT1 suppression (Fig. S1G).
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Fig. 3. GDF-15 alleviated AECs injury in response to LPS incubation via upregulating SIRT1. (AeB) The protein level and activity of SIRT1 in AECs with GDF-15 incubation (n ¼ 6). (CeD) The protein level and activity of SIRT1 in siGdf-15-infected AECs (n ¼ 6). (E) The efficiency of siGdf-15 in AECs detected by the mRNA and activity of SIRT1 (n ¼ 6). (F) The level of IL-1b and TNF-a in medium (n ¼ 6). (G) MDA and 3-NT content in AECs from indicating groups (n ¼ 6). (HeI) SOD, CAT and NOX activities in AECs from indicating groups (n ¼ 6). (J) Caspase-3 activity, LDH release to medium and cell viability (n ¼ 6). Results are expressed as the mean ± SD. *P < 0.05 compared with the matched group.
4. Discussion ALI is a common medical emergency and potentially lifethreatening condition with high mortality, which could rapidly become decompensation and progress to ARDS [2]. Despite an advancement of the mechanical ventilation and other adjunctive strategies, the mortality rate of ALI is still high and its prognosis remains poor worldwide. Therefore, the identification of novel therapeutic agents is greatly needed for treating ALI. In the present study, we found that GDF-15 treatment notably attenuated LPSinduced AECs injury in vitro and effectively prevented inflammation as well as oxidative stress during ALI in mice. Moreover, GDF15 administration could remarkably improve survival rate in LPSchallenged ALI. Our data clearly identified GDF-15 as a potential therapeutic agent against LPS-mediated ALI. Unrestrained inflammatory response and excessive oxidative stress are proven to play critical roles in the initiation and
progression of LPS-induced ALI [8,9]. In response to LPS stimulation, Toll-like receptors were recruited to lipid rafts and interacted with specific molecular adaptors, resulting in the activation of downstream signaling axis responsible for pro-inflammatory cytokines synthesis [34]. Besides, Huang et al. recently revealed that LPS injection could activate nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome, which is essential for the maturation and release of pro-inflammatory cytokines [20]. Consistently, we herein found that LPS treatment promoted the expression and release of IL-1b, TNF-a in AECs as well as murine lungs. Reactive oxygen species (ROS) overproduction is another feature of ALI and contributes to the development of ALI. Oxygen free radicals, such as superoxide anion and hydrogen peroxide, could directly trigger lipid and protein peroxidation, thereby causing cell membrane disruption and protein dysfunction. In accordance with these findings, we found that the byproducts of lipid/protein peroxidation in AECs and murine lung tissues were
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Fig. 4. SIRT1 inhibition abolished the beneficial effects of GDF-15 on lung tissue injury in ALI mice. (A) The level of IL-1b and TNF-a in BALF from ALI mice (n ¼ 6). (B) Inflammatory cell counts in BALF from ALI mice (n ¼ 8). (C) Total protein concentrations in in BALF from ALI mice (n ¼ 6). (D) MPO activity in lung tissue (n ¼ 6). (E) MDA and 3-NT content in murine lungs from indicating groups (n ¼ 6). (FeG) SOD, CAT and NOX activities in murine lungs from indicating groups (n ¼ 6). (H) LDH level and caspase-3 activity in murine lung tissue (n ¼ 6). (I) Lung wet/dry ration (n ¼ 10). (J) Representative western blot images and the statistical data (n ¼ 6). (K) Survival rate in mice with 25 mg/ml LPS administration (n ¼ 20). Results are expressed as the mean ± SD. *P < 0.05 compared with the matched group.
increased upon LPS stimulation. To combat the oxidative damage, the organisms are equipped with effectively anti-oxidant enzymatic defenses to scavenge ROS, including SOD and CAT [10]. However, we identified the activities of SOD and CAT were markedly decreased by LPS treatment. AECs cover most of the alveolar surface area and are important for barrier defense and fluid homeostasis of the lung tissue [6]. A complex network of inflammatory cytokines and oxygen free radicals contributes to mediate, amplify and perpetuate AECs damage and the lung injury process, which subsequently destroyed AECs barrier integrity and promoted pulmonary edema as well as leukocyte extravasations during ALI. Herein, we also observed that LPS instillation elicited leukocyte
infiltration and fluid leakage in murine lungs that were prevented by GDF-15 administration. SIRT1 is implicated in various pathophysiological processes, including inflammatory response and oxidative stress. Yuan et al. previously found that SIRT1 activation protected against inflammatory response in doxorubicin-induced cardiotoxicity, whereas results from Yin et al. indicated that Sirt1 deletion accelerated inflammation and steatosis in response to ethanol challenge [22,35]. Anh et al. recently found that SIRT1 could interact with nuclear factor-kappa B (NF-kB) and blocked its transcriptional activity via deacetylation in arterial inflammation [36]. In addition, a previous study identified a specific CpG site on IL-1b proximal
Please cite this article as: H. Song et al., GDF-15 prevents lipopolysaccharide-mediated acute lung injury via upregulating SIRT1, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.03.103
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promoter, and Sirt1 deficiency caused its hypomethylation and IL1b transactivation [37]. Furthermore, SIRT1 activation by resveratrol obviously inhibited NLRP3 inflammasome activation and thereby protected against LPS-induced lung injury [38]. NF-E2related factor 2 (NRF2) serves a redox sensitive transcription factor and plays a pivotal role in maintaining redox homeostasis against oxidative damage via modulating antioxidant response element (ARE) [39,40]. Previous studies verified that SIRT1 activation notably promoted NRF2/ARE axis and enhanced the antioxidant capacity [41]. Herein, we observed that SIRT1 was upregulated by GDF-15 treatment, which was further downregulated in Gdf-15-deficient AECs upon LPS stimulation. SIRT1 suppression markedly abolished the protective effects of GDF-15 on LPSinduced inflammation, oxidative stress and cell apoptosis. Our data identified GDF-15 as a valuable therapeutic candidate for treating ALI. Declaration of competing interest The authors state no conflict of interest. Acknowledgements This study is supported by the National Natural Science Foundation of China (No: 81801954) and the Fundamental Research Funds for the Central Universities (No. 2042019kf0057). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.03.103. References [1] A.J. Harris, A.S. Mirchandani, R.W. Lynch, et al., IL4Ralpha signaling abrogates hypoxic neutrophil survival and limits acute lung injury responses in vivo, Am. J. Respir. Crit. Care Med. 200 (2019) 235e246, https://doi.org/10.1164/ rccm.201808-1599OC. [2] P. Radermacher, S.M. Maggiore, A. Mercat, Fifty years of Research in ARDS. Gas exchange in acute respiratory distress syndrome, Am. J. Respir. Crit. Care Med. 196 (2017) 964e984, https://doi.org/10.1164/rccm.201610-2156SO. [3] M.S. Taylor, R.R. Chivukula, L.C. Myers, et al., Delayed alveolar epithelialization: a distinct pathology in diffuse acute lung injury, Am. J. Respir. Crit. Care Med. 197 (2018) 522e524, https://doi.org/10.1164/rccm.201706-1094LE. [4] Y. Guo, A. Mishra, T. Weng, et al., Wnt3a mitigates acute lung injury by reducing P2X7 receptor-mediated alveolar epithelial type I cell death, Cell Death Dis. 5 (2014), https://doi.org/10.1038/cddis.2014.254 e1286. [5] G.R. Budinger, G.M. Mutlu, D. Urich, et al., Epithelial cell death is an important contributor to oxidant-mediated acute lung injury, Am. J. Respir. Crit. Care Med. 183 (2011) 1043e1054, https://doi.org/10.1164/rccm.201002-0181OC. [6] X. Wang, M. Liu, M.J. Zhu, et al., Resveratrol protects the integrity of alveolar epithelial barrier via SIRT1/PTEN/p-Akt pathway in methamphetamineinduced chronic lung injury, Cell Prolif (2020), https://doi.org/10.1111/ cpr.12773 e12773. [7] C.Y. Chuang, T.L. Chen, Y.G. Cherng, et al., Lipopolysaccharide induces apoptotic insults to human alveolar epithelial A549 cells through reactive oxygen species-mediated activation of an intrinsic mitochondrion-dependent pathway, Arch. Toxicol. 85 (2011) 209e218, https://doi.org/10.1007/s00204010-0585-x. [8] R.B. Goodman, J. Pugin, J.S. Lee, et al., Cytokine-mediated inflammation in acute lung injury, Cytokine Growth Factor Rev. 14 (2003) 523e535, https:// doi.org/10.1016/s1359-6101(03)00059-5. [9] C.W. Chow, A.M. Herrera, T. Suzuki, et al., Oxidative stress and acute lung injury, Am. J. Respir. Cell Mol. Biol. 29 (2003) 427e431, https://doi.org/ 10.1165/rcmb.F278. [10] T. Li, Y.N. Wu, H. Wang, et al., Dapk1 improves inflammation, oxidative stress and autophagy in LPS-induced acute lung injury via p38MAPK/NF-kappaB signaling pathway, Mol. Immunol. 120 (2020) 13e22, https://doi.org/ 10.1016/j.molimm.2020.01.014. [11] I.C. Lee, X.Y. Ho, S.E. George, et al., Oxidative stress promotes SIRT1 recruitment to the GADD34/PP1alpha complex to activate its deacetylase function, Cell Death Differ. 25 (2018) 255e267, https://doi.org/10.1038/cdd.2017.152. [12] M. Magni, G. Buscemi, L. Maita, et al., TSPYL2 is a novel regulator of SIRT1 and p300 activity in response to DNA damage, Cell Death Differ. 26 (2019)
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