Ghrelin protects against lipopolysaccharide-induced acute respiratory distress syndrome through the PI3K/AKT pathway

Abstract

Pulmonary endothelial barrier dysfunction is a major pathophysiology observed in acute respiratory distress syndrome (ARDS). Ghrelin, a key regulator of metabolism, has been shown to play protective roles in the respiratory system. However, its effects on lipopolysaccharide (LPS)-induced pulmonary endothelial barrier injury are unknown. In this study, the effects of ghrelin on LPS-induced ARDS and endothelial cell injury were evaluated in vivo and in vitro. In vivo, mice treated with LPS (3 mg/kg intranasal application) were used to establish the ARDS model. Annexin V/propidium iodide apoptosis assay, scratch-wound assay, tube formation assay, transwell permeability assay, and Western blotting experiment were performed to reveal in vitro effects and underlying mechanisms of ghrelin on endothelial barrier function. Our results showed that ghrelin had protective effects on LPS-induced ARDS and endothelial barrier disruption by inhibiting apoptosis, promoting cell migration and tube formation, and activating the PI3K/AKT signaling pathway. Furthermore, ghrelin stabilized LPS-induced endothelial barrier function by decreasing endothelial permeability and increasing the expression of the intercellular junction protein vascular endothelial cadherin. LY294002, a specific inhibitor of the PI3K pathway, reversed the protective effects of ghrelin on the endothelial cell barrier. In conclusion, our findings indicated that ghrelin protected against LPS-induced ARDS by impairing the pulmonary endothelial barrier partly through activating the PI3K/AKT pathway. Thus, ghrelin may be a valuable therapeutic strategy for the prevention or treatment of ARDS. Pulmonary endothelial barrier dysfunction is a major pathophysiology observed in acute respiratory distress syndrome (ARDS). Ghrelin, a key regulator of metabolism, has been shown to play protective roles in the respiratory system. However, its effects on lipopolysaccharide (LPS)-induced pulmonary endothelial barrier injury are unknown. In this study, the effects of ghrelin on LPS-induced ARDS and endothelial cell injury were evaluated in vivo and in vitro. In vivo, mice treated with LPS (3 mg/kg intranasal application) were used to establish the ARDS model. Annexin V/propidium iodide apoptosis assay, scratch-wound assay, tube formation assay, transwell permeability assay, and Western blotting experiment were performed to reveal in vitro effects and underlying mechanisms of ghrelin on endothelial barrier function. Our results showed that ghrelin had protective effects on LPS-induced ARDS and endothelial barrier disruption by inhibiting apoptosis, promoting cell migration and tube formation, and activating the PI3K/AKT signaling pathway. Furthermore, ghrelin stabilized LPS-induced endothelial barrier function by decreasing endothelial permeability and increasing the expression of the intercellular junction protein vascular endothelial cadherin. LY294002, a specific inhibitor of the PI3K pathway, reversed the protective effects of ghrelin on the endothelial cell barrier. In conclusion, our findings indicated that ghrelin protected against LPS-induced ARDS by impairing the pulmonary endothelial barrier partly through activating the PI3K/AKT pathway. Thus, ghrelin may be a valuable therapeutic strategy for the prevention or treatment of ARDS. Acute respiratory distress syndrome (ARDS) is a rapidly progressive disease that usually occurs in the intensive critical care unit; given the limited availability of effective treatments for ARDS, more progress is needed to further reduce mortality and morbidity from ARDS. Disorders of pulmonary endothelial barrier function are pivotal early steps in the occurrence and development of ARDS. Disruption of the endothelial cell barrier leads to vascular hyperpermeability and leakage of albumin and fluid, resulting in tissue edema, which is a characteristic of ARDS (1Yuan S.Y. Rigor R.R. Regulation of endothelial barrier function. Morgan and Claypool Life Sciences, San Rafael, CA2011: 146Google Scholar, 2Narula T. Deboisblanc B.P. Ghrelin in critical illness.Am. J. Respir. Cell Mol. Biol. 2015; 53: 437-442Crossref PubMed Scopus (22) Google Scholar, 3Matthay M.A. Zemans R.L. Zimmerman G.A. Arabi Y.M. Beitler J.R. Mercat A. Herridge M. Randolph A.G. Calfee C.S. Acute respiratory distress syndrome.Nat. Rev. Dis. Primers. 2019; 5: 18Crossref PubMed Scopus (272) Google Scholar). Both cell–cell junction disruption and decreased endothelial cell numbers contribute to pulmonary endothelial barrier disruption (4Liu H. Yu X. Yu S. Kou J. Molecular mechanisms in lipopolysaccharide-induced pulmonary endothelial barrier dysfunction.Int. Immunopharmacol. 2015; 29: 937-946Crossref PubMed Scopus (53) Google Scholar). Previous studies have shown that lipopolysaccharide (LPS) has dual effects on pulmonary endothelial cell barrier function through the PI3K/AKT signaling pathway. High doses of LPS inhibit human pulmonary microvascular endothelial cell migration, reduce the expression of vascular endothelial cadherin (VE-cadherin) protein, and block AKT phosphorylation, whereas low doses have the opposite effects (5Zheng X. Zhang W. Hu X. Different concentrations of lipopolysaccharide regulate barrier function through the PI3K/AKT signalling pathway in human pulmonary microvascular endothelial cells.Sci. Rep. 2018; 8: 9963Crossref PubMed Scopus (36) Google Scholar). Similarly, several studies have suggested that changes in cell migration, intercellular junction formation, and cell differentiation, which are associated with endothelial barrier, are mediated by the PI3K/AKT pathway (6Qi D. Tang X. He J. Wang D. Zhao Y. Deng W. Deng X. Zhou G. Xia J. Zhong X. Pu S. Omentin protects against LPS-induced ARDS through suppressing pulmonary inflammation and promoting endothelial barrier via an AKT/eNOS-dependent mechanism.Cell Death Dis. 2016; 7e2360Crossref PubMed Scopus (49) Google Scholar, 7Laakkonen J.P. Lappalainen J.P. Theelen T.L. Toivanen P.I. Nieminen T. Jauhiainen S. Kaikkonen M.U. Sluimer J.C. Ylä-Herttuala S. Differential regulation of angiogenic cellular processes and claudin-5 by histamine and VEGF via PI3K-signaling, transcription factor SNAI2 and interleukin-8.Angiogenesis. 2017; 20: 109-124Crossref PubMed Scopus (21) Google Scholar). Ghrelin, a gastrointestinal hormone peptide, has attracted much attention because of its ability to affect different types of systems (2Narula T. Deboisblanc B.P. Ghrelin in critical illness.Am. J. Respir. Cell Mol. Biol. 2015; 53: 437-442Crossref PubMed Scopus (22) Google Scholar). Notably, several studies have shown that ghrelin plays protective roles in animal lung injury models (8Wu R. Dong W. Cui X. Zhou M. Simms H.H. Ravikumar T.S. Wang P. Ghrelin down-regulates proinflammatory cytokines in sepsis through activation of the vagus nerve.Ann. Surg. 2007; 245: 480-486Crossref PubMed Scopus (155) Google Scholar, 9Zhou X. Xue C. Ghrelin attenuates acute pancreatitis-induced lung injury and inhibits substance P expression.Am. J. Med. Sci. 2010; 339: 49-54Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 10Imazu Y. Yanagi S. Miyoshi K. Tsubouchi H. Yamashita S. Matsumoto N. Ashitani J. Kangawa K. Nakazato M. Ghrelin ameliorates bleomycin-induced acute lung injury by protecting alveolar epithelial cells and suppressing lung inflammation.Eur. J. Pharmacol. 2011; 672: 153-158Crossref PubMed Scopus (49) Google Scholar). Our previous studies also showed that ghrelin suppresses apoptosis in alveolar macrophages by inhibiting the c-Jun N-terminal kinase and Wnt/β-catenin signaling pathways (11Li B. Zeng M. He W. Huang X. Luo L. Zhang H. Deng D.Y. Ghrelin protects alveolar macrophages against lipopolysaccharide-induced apoptosis through growth hormone secretagogue receptor 1a-dependent c-Jun N-terminal kinase and Wnt/β-catenin signaling and suppresses lung inflammation.Endocrinology. 2015; 156: 203-217Crossref PubMed Scopus (52) Google Scholar). As an endogenous growth factor, ghrelin promotes cell migration and cell proliferation via the PI3K/AKT pathway in intestinal epithelial cells, A549 human lung cancer cells, and renal cells (12Lin T.C. Liu Y.P. Chan Y.C. Su C.Y. Lin Y.F. Hsu S.L. Yang C.S. Hsiao M. Ghrelin promotes renal cell carcinoma metastasis via Snail activation and is associated with poor prognosis.J. Pathol. 2015; 237: 50-61Crossref PubMed Scopus (31) Google Scholar, 13Waseem T. Duxbury M. Ashley S.W. Robinson M.K. Ghrelin promotes intestinal epithelial cell proliferation through PI3K/AKT pathway and EGFR trans-activation both converging to ERK 1/2 phosphorylation.Peptides. 2014; 52: 113-121Crossref PubMed Scopus (47) Google Scholar, 14Zhu J. Yao J. Huang R. Wang Y. Jia M. Huang Y. Ghrelin promotes human non-small cell lung cancer A549 cell proliferation through PI3K/AKT/mTOR/P70S6K and ERK signaling pathways.Biochem. Biophys. Res. Commun. 2018; 498: 616-620Crossref PubMed Scopus (21) Google Scholar). However, it is unclear whether ghrelin has positive effects on impairment of lung endothelial cell injury through the PI3K/AKT pathway. Accordingly, in this study, we investigated the roles and mechanism of ghrelin in lung endothelial cell injury in LPS-induced ARDS and explored the roles of the PI3K/AKT signaling pathway in mediating the effects of ghrelin. Our results provided important insights into the roles of ghrelin in protecting the endothelial cell barrier against LPS-induced barrier disruption through the PI3K/AKT pathway and suggested that ghrelin may be a potential therapeutic target in the treatment of ARDS. We used a mouse model of LPS-induced ARDS to examine whether ghrelin administration could affect the progression of LPS-induced ARDS. As shown in Figure 1, administration of LPS alone into the lungs of mice resulted in pathological changes, including significant inflammatory cell infiltration, interstitial and intra-alveolar edema, hemorrhage, interalveolar septal thickening, alveolar collapse, and destruction of lung structure, compared with that in mice receiving saline or ghrelin only. Compared with the LPS group, the ghrelin-pretreated group exhibited marked improvement in lung architecture, reduced alveolar edema and hemorrhage, decreased inflammatory cell infiltration, and lower lung injury scores (Fig. 1A). Three parameters including hemorrhage, edema, and leukocyte infiltration were used as the criteria for lung injury, and the total score of these parameters was calculated according to the above system (0 points for nonexistent, 1 point for mild, 2 points for moderate, and 3 points for severe). The results of lung damage scores implied that the efficacy of ghrelin was statistically significant (Fig. 1B). Cell viability was measured using Cell Counting Kit-8 (CCK-8) assays to identify the concentrations of LPS and ghrelin for subsequent experiments. LPS decreased cell viability in a concentration- and time-dependent manner, with a half-maximal inhibitory concentration of 150 μg/ml, for 24 h (Fig. 2A). Ghrelin increases cell viability in a concentration-dependent manner, with a significant increase observed at 100 nM (Fig. 2B). From these results, we selected 150 μg/ml LPS and 100 nM ghrelin as working concentrations for subsequent experiments. Next, we evaluated the effects of ghrelin on LPS-induced apoptosis. We randomly observed and photographed four fields of view and counted the number of each view. The number of cells in each field ranges from 150 to 200. Because LPS can cause cell shrinkage and apoptosis, the cells in the LPS group are smaller than other groups, and more cells are observed in the same field of view. However, this did not affect the results because the percentage of apoptosis in each field of view was observed. The results of TUNEL staining showed that there were fewer apoptotic cells in both the control and ghrelin groups, whereas the number of apoptotic cells in the LPS group increased significantly (Fig. 3, A–C). Compared with the LPS group, pretreatment with 100 nM ghrelin caused an obvious decrease in the number of TUNEL-positive cells (Fig. 3D). Annexin V-FITC and propidium iodide (PI) dual staining showed that the percentage of apoptotic cells was higher in the LPS group than in the untreated control group (3.7% versus 22.31%, respectively; p < 0.05). Moreover, an evidently lower percentage of apoptotic cells was observed after ghrelin pretreatment than that after LPS treatment alone (10.56% versus 22.31%, respectively; p < 0.05; Fig. 3, B and D). Furthermore, Western blotting showed that ghrelin reduced the expression of Bax protein and increased Bcl-2 expression compared with that in the LPS group, indicating that apoptosis was inhibited (Fig. 3, E and F). Both TUNEL staining and flow cytometry analysis demonstrated that ghrelin markedly suppressed endothelial cell apoptosis induced by LPS, as supported by the reduced expression of Bax protein and increased expression of Bcl-2 protein (Fig. 3, A–F). In vitro scratch-wound healing tests were conducted to evaluate the effects of ghrelin on the migration of endothelial cells. The results showed that wounds in the ghrelin + LPS group recovered more significantly than those in the LPS group at 24 h after scratching (Fig. 4A). Next, to evaluate the roles of ghrelin in angiogenesis in vitro, tube formation of endothelial cells was assessed. Compared with the control group, the LPS group showed almost no tubular structure formation. However, tube formation was observed when cells were pretreated with ghrelin before LPS treatment. Thus, these results indicated that ghrelin significantly increased tube formation and branching (Fig. 4B). We then examined whether ghrelin decreased the permeability of EA.hy 926 cells using Transwell permeability assays (Fig. 5, A and B). Our findings showed that addition of ghrelin significantly blocked the LPS-induced disruption of cell permeability compared with that in the LPS group. Importantly, high-permeability pulmonary edema is attributed to disruption of endothelial adhesion junctions, which contain the transmembrane adhesion protein VE-cadherin. In this study, our Western blotting results demonstrated that EA.hy 926 cells exposed to LPS exhibited reduced VE-cadherin protein expression, whereas administration of ghrelin reversed the harmful effects of LPS on EA.hy 926 cells, as supported by an increase in the total abundance of VE-cadherin protein (Fig. 5, C and D). Taken together, these findings showed that ghrelin reinforced the pulmonary endothelial cell barrier by promoting cell migration and tube formation and stabilizing adherens junctions. To evaluate the effects of ghrelin on AKT-related signal activation in vitro, phosphorylation of PI3K and AKT was assessed by Western blotting. We found that LPS decreased PI3K and AKT phosphorylation compared with that in the control group, whereas ghrelin pretreatment obviously increased PI3K and AKT phosphorylation. As expected, we observed that LY294002 inhibited the stimulation of PI3K and AKT phosphorylation by ghrelin (Fig. 6, A and B). These results indicated that ghrelin was a stimulatory factor for PI3K/AKT signaling pathways. To clarify whether PI3K/AKT activation was involved in mediating the protective effects of ghrelin in vitro, EA.hy 926 cells were treated with LY294002 1 h before LPS challenge. We observed that changes in the levels of Bax and Bcl-2 proteins were reversed after treatment with LY294002, indicating that the antiapoptotic effects of ghrelin were markedly reduced by addition of the PI3K inhibitor LY294002 (Fig. 6, C and D). Moreover, LY294002 also reversed the stimulatory effects of ghrelin on cell migration and differentiation after LPS treatment (Fig. 6, E–H). Therefore, these findings suggested that ghrelin may play a protective role in endothelial cells, at least partially through PI3K/AKT signaling. To confirm the relationship between ghrelin and PI3K/AKT signaling pathways in vivo, lung staining with “p-Akt antibody” was performed. We found that the expression of p-Akt was increased in the ghrelin group while decreased in the LPS group (Fig. 7, A and B). In vivo, before exposure to LPS, mice were pretreated with the PI3K inhibitor LY294002 to confirm that PI3K/AKT signaling was involved in ghrelin-mediated LPS-induced ARDS in vivo. Indeed, our findings demonstrated that pretreatment with LY294002 aggravated histological injury in ghrelin-pretreated mice after LPS challenge (Fig. 7, C and D). To further explore the effects of LPS on pulmonary endothelial cells and clarify whether ghrelin played a preventive effect in LPS-induced ARDS by affecting the endothelial cell barrier, VE-cadherin protein immunofluorescence was conducted because VE-cadherin functions to maintain vascular integrity. The immunofluorescence results showed that VE-cadherin expression in the LPS group was reduced compared with that in the control group. However, compared with the LPS group, the expression of VE-cadherin was increased in the ghrelin + LPS group (Fig. 7, E and F). In addition, LY294002 obviously decreased the expression of VE-cadherin increased by ghrelin. The results implied that ghrelin might affect VE-cadherin expression through PI3K/Akt signaling pathway. In this study, we evaluated the roles of ghrelin in ARDS in a mouse model in vivo and in cells in vitro. Our results showed that ghrelin protected against LPS-induced ARDS by improving the pulmonary vascular endothelial barrier, providing insights into the potential applications of ghrelin as a therapeutic target in ARDS. According to a study by Vila et al., (15Vila G. Maier C. Riedl M. Nowotny P. Ludvik B. Luger A. Clodi M. Bacterial endotoxin induces biphasic changes in plasma ghrelin in healthy humans.J. Clin. Endocrinol. Metab. 2007; 92: PubMed Scopus Google ghrelin is of the to a increase after LPS indicating that ghrelin levels in vivo may be to inflammatory caused by of ghrelin effects on LPS-induced ARDS A. P. M. Ghrelin protects against sepsis by inhibiting group 1 and by PubMed Scopus Google Scholar). In this study, we found that of ghrelin lung injury in mouse models of ARDS, to previous However, ghrelin is shown to protective effects on LPS-induced ARDS through including inhibiting the pathway and suppressing proinflammatory in lung macrophages R. Dong W. Zhou M. Zhang Ravikumar T.S. Wang P. Ghrelin attenuates acute lung injury and mortality in J. Respir. Med. 2007; PubMed Scopus Google Scholar, J. Liu X. S. Luo Ghrelin attenuates lipopolysaccharide-induced acute lung injury through Sci. Google Scholar). we observed that ghrelin reversed the of VE-cadherin protein caused by VE-cadherin is a of the cadherin that is in different types of vascular endothelial cells. VE-cadherin plays key roles in the of endothelial cells barrier M. M. VE-cadherin and endothelial adherens of vascular Full Text Full Text PDF PubMed Scopus Google Scholar). In to previous we found that ghrelin protected the endothelial cell barrier by increasing the expression of protective effects in LPS-induced ARDS. Moreover, ghrelin had dual effects on endothelial cells at different at concentrations of than 100 cell viability whereas at concentrations of nM or cell viability decreased (Fig. and Fig. is with the study conducted by and function of the ghrelin including a in human cancer and cell PubMed Scopus Google Scholar). ARDS is by injury to the barrier, resulting in increasing permeability M.A. Zemans R.L. The acute respiratory distress and Rev. Pathol. 2011; PubMed Scopus Google Scholar). The of the pulmonary endothelial barrier in ARDS has been demonstrated N. M. of pulmonary endothelial barrier function in acute and therapeutic Res. 2014; PubMed Scopus (53) Google Scholar). Therefore, a of the mechanisms that are involved in the of the endothelial barrier is for the development of therapeutic to ARDS. Ghrelin, as a factor that the of inflammatory of in apoptosis, cell and angiogenesis in in vivo and in vitro studies A. P. M. Ghrelin protects against sepsis by inhibiting group 1 and by PubMed Scopus Google Scholar, M. S. N. S. R. S. The role of ghrelin on apoptosis, cell proliferation and system in the of Biol. 2015; PubMed Scopus Google Scholar, R. S. H. T. Y. P. K. Kangawa K. M. Ghrelin promotes angiogenesis in a mouse model of critical through activation of 2016; PubMed Scopus Google Scholar, B. Zeng M. H. Huang C. He W. G. X. Y. R. of ghrelin on the apoptosis of human in J. Mol. Med. 2016; PubMed Scopus Google Scholar). Because the of the endothelial cell barrier on between endothelial cells through intercellular and the decreased cell number and induced by LPS could cause disruption of the endothelial cell barrier (4Liu H. Yu X. Yu S. Kou J. Molecular mechanisms in lipopolysaccharide-induced pulmonary endothelial barrier dysfunction.Int. Immunopharmacol. 2015; 29: 937-946Crossref PubMed Scopus (53) Google Scholar, C. A.G. The pulmonary in acute respiratory distress and therapeutic 2016; PubMed Scopus Google Scholar). Taken together, our findings demonstrated that these potential mechanisms may be to endothelial cell barrier dysfunction the development of ARDS. Therefore, we that antiapoptotic of cell migration, improvement of tube formation and of endothelial permeability may be for of the endothelial cell barrier. Ghrelin antiapoptotic effects by decreasing Bax and increasing Bcl-2 in cells, including cells, and human microvascular endothelial cells D. Liu Zhang H. Luo Ghrelin protects human pulmonary endothelial cells against injury via PubMed Scopus Google Scholar, L. L. H. H. Ghrelin inhibits apoptosis induced by and in through the signaling PubMed Scopus Google Scholar, R. Yang G. Wang Wang H. ghrelin protects in of by inhibiting cell Biol. Rep. PubMed Scopus Google Scholar). In our study, we found that administration of ghrelin inhibited the LPS-induced increase in apoptosis in ARDS through including TUNEL flow and Western indicating the antiapoptotic effects of ghrelin in vitro. The effects of ghrelin on cell migration and angiogenesis have on cancer The ghrelin it have an for cancer Rev. PubMed Scopus Google and is the roles of ghrelin in promoting cell migration and angiogenesis in ARDS. Our suggested that ghrelin not cell migration also increased tube formation in endothelial cells after LPS To the of cell we in the In the increased endothelial permeability can to acute lung with our in vivo results, we that the of VE-cadherin protein was markedly decreased after exposure to LPS in vitro and that ghrelin administration could this Because the ghrelin group has been in the previous to confirm the effect of ghrelin the ghrelin group has not been in the The PI3K/AKT pathway cell apoptosis, and cell studies have shown that the PI3K/AKT pathway plays key roles in lung injury signalling in lung and 2007; PubMed Scopus Google Scholar). In addition, ghrelin inhibits injury and apoptosis of pulmonary endothelial cells through activating the PI3K/AKT signaling pathway D. Liu Zhang H. Luo Ghrelin protects human pulmonary endothelial cells against injury via PubMed Scopus Google Scholar). with previous we showed that treatment of endothelial cells with ghrelin the PI3K/AKT signaling pathway, the antiapoptotic protein and the protein Bax in LPS-induced lung Furthermore, our results that the PI3K inhibitor LY294002 partially reversed the protective effects of ghrelin on LPS-induced endothelial barrier including of endothelial cell migration and Taken together, these results showed that ghrelin protected against LPS-induced ARDS and endothelial barrier dysfunction partially through the PI3K/AKT signaling pathway. To whether the effect of ghrelin in the lung is mediated by endothelial cells, an immunofluorescence experiment of VE-cadherin protein was Because the VE-cadherin is a transmembrane adhesion protein on the of vascular endothelial cells, the expression of VE-cadherin the endothelial cells. The immunofluorescence results showed that LY294002 obviously decreased the expression of VE-cadherin increased by which implied that there was a effect of ghrelin in the endothelial cells. there are in our study to be in our study used VE-cadherin staining experiment to the effect of ghrelin in the lung mediated by the endothelial cells. However, whether cells play a protective role of ghrelin in lung through PI3K pathway unknown. In addition, the PI3K/AKT signaling pathway is not the potential A study suggested that ghrelin protected human endothelial cells by activating the and target of signaling pathways J. C. J. Luo J. Su B. Huang Y. Su W. Cui T. Ghrelin protects human endothelial cells against apoptosis via signaling 2014; 52: PubMed Scopus Google Scholar). In addition, et P. Yang D. Liu D. Y. and ghrelin apoptosis and of human microvascular endothelial 2017; PubMed Scopus Google suggested that ghrelin endothelial apoptosis induced by concentrations via of the c-Jun N-terminal kinase 1/2 and signaling pathways. Therefore, studies are needed to the mechanisms involved in these our indicated that ghrelin exhibited protective effects against LPS-induced ARDS on the pulmonary vascular endothelial barrier by inhibiting apoptosis and promoting cell migration and differentiation, which could have in the treatment of with ARDS.

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