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Blockage of glycolysis by targeting PFKFB3 alleviates sepsis-related acute lung injury via suppressing inflammation and apoptosis of alveolar epithelial cells
Accepted Manuscript Blockage of glycolysis by targeting PFKFB3 alleviates sepsis-related acute lung injury via suppressing inflammation and apoptosis of alveolar epithelial cells Yuanqi Gong, Haibing Lan, Zhihong Yu, Meng Wang, Shu Wang, Yu Chen, Haiwei Rao, Jingying Li, Zhiyong Sheng, Jianghua Shao PII:
S0006-291X(17)31082-3
DOI:
10.1016/j.bbrc.2017.05.173
Reference:
YBBRC 37894
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 19 May 2017 Accepted Date: 29 May 2017
Please cite this article as: Y. Gong, H. Lan, Z. Yu, M. Wang, S. Wang, Y. Chen, H. Rao, J. Li, Z. Sheng, J. Shao, Blockage of glycolysis by targeting PFKFB3 alleviates sepsis-related acute lung injury via suppressing inflammation and apoptosis of alveolar epithelial cells, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.05.173. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Blockage of glycolysis by targeting PFKFB3 alleviates sepsis-related acute lung injury via suppressing inflammation and apoptosis of alveolar epithelial cells
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Yuanqi Gonga1 ; Haibing Lana1 ; Zhihong Yua ; Meng Wanga ; Shu Wanga ; Yu Chena ; Haiwei Raoa ; Jingying Lia ; Zhiyong Shenga ; Jianghua Shaob* a Department of Comprehensive Intensive Care Unit, Second Affiliated Hospital of Nanchang University, Nanchang 330006, China; b Department of Hepatobiliary Surgery, Second Affiliated Hospital of Nanchang University, Nanchang 330006, China; 1 These authors made equal contributions to this paper. *Correspondence: Dr Jianghua Shao, E-mail:[email protected] Abstract Sepsis-related acute lung injury (ALI) is characterized by excessive lung inflammation and apoptosis of alveolar epithelial cells resulting in acute hypoxemic respiratory failure. Recent studies indicated that anaerobic glycolysis play an important role in sepsis. However, whether inhibition of aerobic glycolysis exhibits benefcial effect on sepsis-induced ALI is not known. In vivo, a cecal ligation and puncture (CLP)-induced ALI mouse model was set up and mice treated with glycolytic inhibitor 3PO after CLP. The mice treated with the 3PO ameliorated the survival rate, histopathological changes, lung inflammation, lactate increased and lung apoptosis of mice with CLP-induced sepsis. In vitro, the exposure of human alveolar epithelial A549 cells to lipopolysaccharide (LPS) resulted in cell apoptosis, inflammatory cytokine production, enhanced glycolytic flux and reactive oxygen species (ROS) increased. While these changes were attenuated by 3PO treatment. Sequentially, treatment of A549 cells with lactate caused cell apoptosis and enhancement of ROS. Pretreatment with N-acetylcysteine (NAC) significantly lowered LPS and lactate-induced the generation of ROS and cell apoptosis in A549 cells. Therefore, these results indicate that anaerobic glycolysis may be an important contributor in cell apoptosis of sepsis-related ALI. Moreover, LPS specifically induces apoptotic insults to A549 cell through lactate-mediated enhancement of ROS. Key words: Sepsis-related acute lung injury, anaerobic glycolysis, 3PO, human alveolar epithelial A549 cells, apoptosis, ROS. 1.Introduction Sepsis is a complex systemic inflammatory response syndrome (SIRS) resulting in high mortality in critically ill patients and multi-organ dysfunction syndrome (MODS) including cardiovascular system, liver, kidney and lung[1]. The respiratory dysfunction is one of the most frequent complication of sepsis because of pulmonary susceptibility. So acute lung injury (ALI) and its most severe manifestation acute respiratory distress syndrome (ARDS) are the most severe outcomes of sepsis and responsible for sudden deaths of patients in the intensive care unit (ICU) [2,3]. ALI is characterized by acute hypoxemic respiratory failure, pulmonary hypertension, pulmonary alveoli edema, decreased pulmonary compliance and deteriorated gas exchange [4]. The pathophysiological process of ALI is generally believed to be excessive and uncontrolled activation of lung inflammatory response which implicates many types of inflammatory cells infiltration, alveolar-capillary destruction, excessive oxidative stress characterized by the aberrant release of reactive oxygen species (ROS) and alveolar epithelial cell apoptosis [5,6]. Extensive apoptosis of pulmonary alveolar type II epithelial cells has been shown to be responsible for the impairment of the epithelial barrier function and the remodeling of certain mesenchymal cells in
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ALI [7]. However, the precise molecular mechanisms leading to lung inflammation and apoptosis of alveolar epithelial cells in sepsis-induced ALI remains incompletely understood, limiting the development for effective drugs and therapies of curing ALI caused by sepsis. Numerous studies have demonstrated that a set of different pathophysiological changes such as hyperlactatemia are generally noticed during sepsis. Traditionally, during sepsis, and especially ALI, hyperlactatemia has been interpreted as the existence of anaerobic glycolysis due to an inadequate oxygen supply inducing cellular distress [8,9]. Aerobic glycolysis is the crucial metabolic pathway that be controlled by various glycolytic enzymes in sepsis [9]. Of these glycolytic enzymes, the PFKFB3 isozyme largely rely on glycolysis for ATP generation , regulate glycolytic flux and product fructose-2,6-bisphosphate(Fru-2,6-BP) which is the potent allosteric regulator of the key glycolytic enzyme phosphofructokinase 1 (PFK-1) [10]. A small molecule antagonist of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one(3PO), has been found to causes a rapid reduction in Fru-2,6-BP, suppress glycolytic flux to lactate, decrease glucose uptake, induces growth inhibition in several cell lines and inhibit tumor growth [11]. Thus, aerobic glycolysis may be an important contributor in the context of sepsis-related ALI. However, whether blockage of aerobic glycolysis by targeting PFKFB3 shows a protective effect on sepsis-induced ALI is not known. In the present study, we focused on the effects of the PFKFB3 inhibitor 3PO on lung inflammatory and alveolar epithelial cell apoptosis in sepsis-related ALI and the possible mechanism. 2.Materials and methods 2.1 Materials and reagents LPS was purchased from Sigma-Aldrich. N-acetylcysteine (NAC a superoxide inhibitor ) were purchased from Sigma-Aldrich. Lactate was purchased from Sigma-Aldrich. 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO, Merck Millipore, Darmstadt, Germany). Mouse TNF-α、IL-6 and IL-1β ELISA kits were purchased from eBioscience (San Diego, US). Human TNF-α、IL-6 and IL-1β ELISA kits were purchased from eBioscience (San Diego, US). Lactate Colorimetric/Fluorometric Assay Kit (K607-100, Biovision). Glucose Colorimetric/Fluorometric Assay Kit (K606-100, Biovision). LDH enzymatic assay (Roche cat.).The reactive oxygen species assay kit (DCF-DA) was purchased from Applygen. 2.2 Animal Specific-pathogen-free (SPF) C57BL/6 wide-type mice (male, 6-8 weeks old) were purchased from Nanchang University (Nanchang, China). The mice were housed in a SPF environment (room temperature 24˚C, humidity range 40-70%, 12 h light/dark cycle). Sterilized water and food were provided ad libitum. All experiments were conducted under the guidelines outlined by the committee of Nanchang University on the use and care of animals. Standard guidelines for laboratory animal care followed the Guide for the Care and Use of Laboratory Animals. 2.3 Animal experimental protocol. A classic sepsis-induced ALI model were established by cecal ligation and puncture (CLP) as previously described with mildly modification [12]. C57BL/6 mice were randomly divided into three groups (10 rats in each group ):sham-operated (control), CLP , and 3PO+CLP groups. Briefly, healthy mice underwent 24-hour deprivation of food but not water. Mice were anesthetized using sodium pentobarbital (intraperitoneally, 40 mg/kg). A midline abdominal incision was made after the ventral abdomen was shaved and cleaned with 10% povidone-iodine
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wash and the cecum was exposed. The cecum was ligated just distal to the ileocecal valve to avoid intestinal obstruction, then through-and-through punctured with a 21-gauge needle. The ligated cecum was then returned to the peritoneal cavity, and the abdominal wall was closed in two layers. As sham operated animals, the cecum underwent the same procedure but neither ligated nor punctured, and then returned to the abdominal cavity. Mice receive intragastric administration of 0.07 mg/g 3PO in 50 uL DMSO and only vehicle DMSO 0 and 6 h after CLP. The mortality of mice was recorded every 12 h for 5 days after CLP in each group. The 3PO dose was determined as described previously [10].Then the mice were euthanized for collection of blood, bronchoalveolar lavage fluid (BALF) and lungs. 2.4 Lung wet/dry(W/D) weight ratio. The ratio of lung W/D weight was calculated to assess the lung edema. The fresh upper part of the right lung was cleansed and weighed to obtain the wet weight and was then dried in an oven at -80 ˚C for at least 24 h for the measurement of the dry weight. 2.5 Analysis of serum and BALF. Blood samples were collected, stored for 2 h at room temperature, and then centrifuged (3000 rpm, 20 min). The supernatants were harvested and stored at -80˚C. TNF-α, IL-6 and IL-1β was measured by ELISA according to the manufacturer's instructions. Next, BALF samples were collected. Total cells in BALF were counted, and a cytospin sample was prepared and stained with hematoxylin and eosin (H&E) for blinded assessment of differential cell percentages in BALF. Then BALF samples were centrifuged (1000 rpm, 10 min). The supernatants were harvested and stored at -80˚C. The levels of TNF-α, IL-6 and IL-1β in the BALF samples were measured using ELISA according to the manufacturer’s instructions. Lactate in BALF was measured with Lactate Colorimetric/Fluorometric Assay Kit according to the manufacturer’s instructions. 2.6 Pathological staining and assessment of lung apoptosis. The left lungs of mice were removed and fixed in 4 % paraformaldehyde, then embedded in paraffin. Lung tissues were stained with hematoxylin and eosin (H&E) for blinded histopathologic assessment. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining, fluorescence staining was performed using a commercially available in situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions. Results are expressed as the average number of TUNEL-positive staining cells per 200X magnifcation feld. 2.7 Cell culture Human pulmonary epithelial A549 cells (Shanghai Fuxiang Biological Technology Co. Ltd., ATCC, USA) were grown in a cell culture flask in RPMI 1640 medium (Gibco, Life Technology) supplemented with 10% fetal bovine serum (Gibco, Life Technology). The cells were then incubated in a humidified incubator at 37˚C with an atmosphere of 5% CO2. 2.8 Cell apoptosis analysis A549 cells were seeded into 6-well plates. When cells reached 80% confluence, the cells were treated according to the experimental plan. Harvested cells were washed with PBS and stained with Annexin V and Propidium Iodide (Beyotime, China). Fluorescence was measured using a FACSCalibur (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo v10.1 (Oregon, USA). Annexin V+/PI+ (late apoptotic) and Annexin V+/PI- (early apoptotic) cells were quantified by the frequency of fluorescently labeled cells. 2.9 Biochemical assays and analysis of the culture medium.
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Glucose concentration of the culture medium was measured by using Glucose Colorimetric/Fluorometric Assay Kit according to the manufacturer’s instructions. Lactate dehydrogenase (LDH) activity were measured using LDH enzymatic assay according to the manufacturer’s instructions [13]. Lactate concentration of the culture medium was measured by using Lactate Colorimetric/Fluorometric Assay Kit according to the manufacturer’s instructions. The levels of TNF-α, IL-6 and IL-1β in the the culture medium were measured using ELISA according to the manufacturer’s instructions. 2.10 Detection of ROS levels Intracellular ROS production was quantified using the oxidation-sensitive probe 2,7-dichlorofluorescein diacetate (DCF-DA) (Applygen). Briefly, 10 mM DCF-DA stock solution (in methanol) was diluted 1000-fold in cell culture medium without serum to yield a 10-um working solution. After 24 h of stimulation, the cells in 24-well plates were washed twice with phosphate-buffered saline (PBS) and incubated in 500 ul working solution of DCF-DA at 37˚C in dark for 1 h. The cells were then washed twice with cold PBS and resuspended in the PBS for an analysis of intracellular ROS using a multiwell fluorescence scanner (SpectraMax M5/M5e; Molecular Devices) and microscope (Olympus). DCF-DA fluorescence was detected at an excitation wavelength of 480 nm and an emission wavelength of 525 nm [14]. 2.10 Statistical analysis. Statistical analysis was performing using the SPSS (version 19.0) software package. All data are presented as the mean ± standard deviation. One-way ANOVA were utilized to compare differences among groups. Values of P <0.05 were considered statistically significant. 3.Results 3.1 Effects of glycolytic inhibitor 3PO on Sepsis-Induced ALI in mice. To determine the effect of 3PO on the acute injury of the lungs induced by sepsis in mice, survival was observed for 120 h after CLP , a histological assessment of lung tissue was performed using H&E staining and lung edema was evaluated using the ratio of lung W/D weight. As shown in Fig.1A, sepsis resulted in a raised mortality rate within 120 h. However, mice treated with 3PO showed a significantly improved survival rate during the study period. Histological analysis of lung tissue revealed that CLP mice displayed lung exudative changes，hemorrhage, edema, fibrin deposition and heavy infltration of inflammatory cell. While they were attenuated by 3PO treatment (Fig. 1B). Pulmonary wet-to-dry weight ratios in CLP group were significantly higher lung edema compared to that of the control group which was attenuated by 3PO treatment (Fig. 1C). Then we found a increased number of total cells, neutrophils and lactate in BALF of the CLP group. These effects were partially rescued by treatment with the 3PO (Fig. 1D,E,F). 3.2 3PO inhibits the lung inflammation and lung apoptosis of Sepsis-Induced ALI in mice. ALI was successfully induced by CLP surgery. To further assess the effect of 3PO on lung inflammation and lung apoptosis in the CLP model, the TNF-α, IL-6 and IL-1β in BALF and serum were measured by ELISA and apoptotic cells in lungs were detected by labeling with an in situ TUNEL assay. There were notable increases in TNF-α, IL-6 and IL-1β in BALF and serum of sepsis-induced ALI which were reduced by 3PO (Fig. 2A-F). Lungs from mice after CLP exhibited significant ladder formation, a characteristic of apoptosis which was attenuated by 3PO treatment (Fig. 2G,H). These findings clearly indicate that CLP surgery results in a robust lung inflammation and lung apoptosis in mice, and the protective effect of the 3PO. 3.3 3PO attenuates LPS-stimulated glycolysis and inflammatory cytokines release in A549
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cell. Then in vitro we assess the effect of 3PO on LPS-induced ALI. The human pulmonary epithelial A549 cell stimulated with LPS (100 ug/ml) for the indicated periods of time (0, 2, 4 h). We then evaluated glucose and lactate concentration of the culture medium, LDH activity in cell lysates and the expression levels of TNF-α, IL-6 and IL-1β in the culture medium. In mice, we have found the increase lactate in BALF of the CLP group. In A549 cell we also found LPS caused a significant increase in extracellular glucose consumption (Fig. 3A) and lactate level (Fig. 3C) and intracellular lactate dehydrogenase (LDH) activity (Fig. 3B), indicative of an enhanced glycolytic flux. Meanwhile, the expression levels of inflammatory cytokines TNF-α, IL-6 and IL-1β remarkably increased after LPS challenge (Fig. 3D,E,F). Treatment of cells with glycolytic blockade with 3PO(20µM) prior to LPS stimulation inhibited the increased glycolytic flux and inflammatory cytokines release induced by LPS in A549 cell. 3.4 3PO attenuates LPS-stimulated apoptosis by inhibiting ROS generation in A549 cell. Then we found that the exposure of A549 cell to LPS(100 ug/ml) and lactate (5 mM) resulted in cell apoptosis. The result is similar with the effect of HCl on cell apoptosis (Fig. 4A,B). Meanwhile, we also found that LPS and lactate can cause an marked increase in ROS (Fig. 4C,D). In order to further identify the role of ROS on cell apoptosis induced by the LPS and lactate. The A549 cells were respectively pretreatment with the ROS scavenger NAC. The results showed that the antioxidant NAC (10 mM) significantly suppress the increases in ROS and cell apoptosis induced by the LPS and lactate (Fig. 4C,D). Moreover, pre-treatment with 3PO could inhibit the LPS-induced the ROS increased and cell apoptosis (Fig. 4A-D). Taken together, these results indicate that 3PO ameliorates LPS-induced apoptosis by inhibiting ROS generation in A549 cell. Discussion In this study, we indicated that a crucial role of enhanced aerobic glycolysis in lung apoptosis of mice with surgery CLP and in LPS-induced A549 cell apoptosis. For the first time, we demonstrated that a specific glycolytic inhibitors 3PO can diminish acute injury of the lungs induced by sepsis in mice and LPS-induced inflammation and apoptosis of A549 cell. Moreover, in A549 cell we found that LPS can cause a increase in lactate level, indicative of an enhanced aerobic glycolysis. Then lactate in turn induce cell apoptosis of A549 cell by elevating the ROS. Sepsis is defined as the systemic inflammatory response to severe infection and surgery which may induce multiple complications such as brain injury, kidney injury, lung injury [15,16]. More importantly, sepsis is the major cause of ALI development with high morbidity and mortality [17]. The sepsis model of CLP is a widely accepted animal model for the research of sepsis-associated mechanism and we successfully established a CLP induced sepsis model to evaluate the effects of 3PO on sepsis-induced ALI in mice. The CLP-exposed ALI mice developed a raised mortality rate, lung exudative changes，hemorrhage, edema, fibrin deposition, heavy infltration of inflammatory cell, a increased of total cells, neutrophils, lactate ,TNF-α, IL-6, IL-1β and lung apoptosis. It is known that acute lung injury is characterized by alveolar epithelial and endothelial cell injury , inflammatory cell and protein rich edema fluid influx into the alveolus, alveolar and bronchial epithelial cell apoptosis, lung edema [2,18]. The apoptosis of lung epithelial cells with lung inflammation may contribute to the development and progression of ALI by promoting inflammatory exudation, destruction of lung structure and fibrosing of lung [19,20]. LPS was reported to be one of the major causes of inflammatory cells leakage, septic shock and acute lung injury [21]. A549 is an alveolar epithelial cell line with type II cell characteristics and
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has been widely used as a ALI model to study the effects of LPS in vitro [20]. So we selected the alveolar epithelial cell line A549 to assess the effects of LPS on the apoptosis and inflammation of lung epithelial cells in vitro. Our results showed LPS can induce cell apoptosis and inflammatory cytokines release in A549 cell. These data support the conclusion that the apoptosis and inflammation of lung epithelial cells may be a key factor in the development of sepsis-associated ALI. Extensive studies have reported sepsis is concerned with a number of metabolic abnormalities, including increased glucose uptake, aerobic glycolysis and hyperlactatemia [22,23]. Accelerated aerobic glycolysis contributes to the maturation of dendritic cells in response to toll-like receptor ligands (such as TLR4, TLR2 and TLR9) [24], the differentiation of both anti-inflammatory Treg cells and pro-inflammatory Th17 cells [25] and LPS-induced release of late mediators [26]. In our study, we showed that lactate expression in BALF was significantly increased in the mouse model of CLP-induced sepsis mice, suggesting the possible involvement of aerobic glycolysis in the development of sepsis-associated ALI. Then we used A549 cells to assess the effects of LPS on aerobic glycolysis in vitro. In this study, we found that LPS exposure can induce a increase in extracellular glucose consumption and lactate level and intracellular LDH activity ,indicating aerobic glycolysis play an important role in LPS-induced ALI in A549 cells. To further address the roles of aerobic glycolysis in the CLP-induced ALI mouse model and LPS-induced ALI cell model, we investigated the effect of aerobic glycolysis inhibition with the PFKFB3 inhibitor 3PO. In the previous study, the PFKFB3 represents the most abundant PFKFB isoenzyme in endothelial cells, which largely rely on glycolysis for ATP generation and it may represents a novel pharmacological strategy for targeting angiogenesis and tumor cell growth/migration [27]. Moreover, PFKFB3 inhibitors 3PO was shown to suppress glucose uptake, which in turn causes an increase in autophagy and may prove useful as rational combinations for the treatment of cancer [10], PFKFB3 blockade by 3PO also can reduce glycolysis to decrease pathological neovascularization in ocular and inflammatory models [28] and targeting glycolysis by 3PO reduces key features of Rheumatoid Arthritis pathogenesis in vitro and can mediate TLR2-induced inflammation [29]. Consistent with these data, the blockade of PFKFB3 with 3PO ameliorated CLP-induced changes in our mouse sepsis model. The lung apoptosis and lung inflammation influence each other, which then contributes to the pathogenesis of ALI. In our experiment, pretreatment with 3PO partly reduced the heavy infltration of inflammatory cell, the elevated expression of TNF-α, IL-6 and IL-1β in BALF and serum and lung apoptosis by inhibiting the expression of lactate in mice with CLP-induced ALI. These data indicate that aerobic glycolysis participates in promoting the release of inflammatory cytokines and lung apoptosis in an animal model of CLP-induced ALI and that the 3PO counteracts the effects of glycolysis and alleviates CLP-induced changes. Meanwhile, in this report we also describe that pre-treatment with glycolytic inhibitor 3PO reduced LPS-stimulated cell apoptosis and the increased expression of inflammatory cytokines TNF-α, IL-6 and IL-1β by suppressing glycolytic flux in A549 cells. These observations provide direct support for the conclusion that treatment with 3PO alleviates lung apoptosis and lung inflammation in the CLP-induced ALI mouse model and LPS-induced ALI cell model. Nevertheless,the mechanisms by which 3PO mitigates cell apoptosis and inflammatory cytokines release in LPS-induced ALI cell model of A549 cells remain to be elucidated. Recently, ROS have been verifed to be capable of regulating many intracellular signal
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transduction pathways and abnormal ROS signal may cause oxidative stress and subsequent cell apoptosis [30]. Much research has focused on the effects of ROS on the pathogenesis of ALI [31,32] and the exposure to LPS increased the levels of cellular nitric oxide and ROS and eventually led to cell apoptosis and ROS participate in LPS-induced apoptosis of A549 cells [21]. The similar results were also obtained by us, LPS significantly induced the expression of ROS. To further elucidate the relationship between the LPS-induced cell apoptosis, inflammatory cytokines release and the ROS production, we treated A549 cells with LPS in the presence of the antioxidant NAC by scavenging of ROS and found that NAC reversed the LPS-mediated cell apoptosis and inflammatory cytokines release. These data suggest that LPS can induce cell apoptosis and inflammatory cytokines release via the ROS generation. Subsequently, we used lactate stimulate to A549 cells to assess the directly effects of lactate in vitro. These results showed lactate can cause an marked increase in ROS and cell apoptosis in A549 cells, the NAC partly restored the increased expression of ROS and cell apoptosis. These data suggest that there are interaction relationship between the upregulation of aerobic glycolysis and activation of the ROS in regulation of LPS-induced cell apoptosis in A549 cells. Meanwhile we confirmed that pre-treatment with glycolysis inhibitor 3PO could suppress the LPS-induced the ROS increased and cell apoptosis. So our results indicated that LPS can induce an enhanced aerobic glycolysis which then cause cell apoptosis of A549 cell by elevating the ROS. In conclusion, our study demonstrated that LPS exposure promote a metabolic switch to aerobic glycolysis and A549 cell apoptosis by the ROS pathway. The glycolysis inhibitor 3PO may provide favorable effects to reduce inflammation reactivity of cells and cell apoptosis of lung in CLP-induced ALI mice model and LPS-induced ALI cell model. Thus, novel therapeutic strategies targeting aerobic glycolysis can serve as a potential therapeutic target in the treatment of ALI. Conflicts of interest All authors declare no conflicts of interest associated with this manuscript. Acknowledgments This study was funded by Key Research and Development Project of Jiangxi Province [S2016SFYBG0458].
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Figure 1. Effects of glycolytic inhibitor 3PO on Sepsis-Induced ALI in mice. (A) No deaths occurred in mice that underwent sham procedure (n = 10). 3PO was administered 0 and 6 h after CLP. Mortality was observed over 120 h. (B) These representative histological changes of the lung were obtained from mice of different groups (original magnification ×200). Lung tissue sections stained with H&E. (C) Wet/dry ratio of lungs from mice of different groups. (D) Total cell number in the BALF of mice from different groups. (E) Neutrophils in the BALF of mice from different groups. (F) Lactate in the BALF of mice from different groups. Data are presented as the mean ± SD. n=8-10/group. *P<0.05 vs. the control group. #P <0.05, vs. the CLP group
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Figure 2. 3PO inhibits the lung inflammation and lung apoptosis of Sepsis-Induced ALI in mice. (A) TNF-α,(B) IL-6 and (C) IL-1β was measured by ELISA in BALF. (D) TNF-α,(E) IL-6 and (F) IL-1β was measured by ELISA in Serum. (G) Arrowheads indicate TUNEL-positive apoptotic cells (original magnification ×200). (H) Quantification of TUNEL-positive cells. Data are presented as the mean ± SD. n=8-10/group. *P<0.05 vs. the control group. #P <0.05, vs. the CLP group
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Figure 3. 3PO attenuates LPS-stimulated glycolysis and inflammatory cytokines release in A549 cell. (A) Glucose consumption in A549 cells from different groups were determined. (B) Intracellular LDH activity from different groups were measured by colorimetric assay. (C) The concentrations of lactate in the culture medium from different groups were determined. (D) TNF-α,(E) IL-6 and (F) IL-1β was measured by ELISA in the culture medium from different groups. Data are presented as the mean ± SD. n=3-5/group. *P<0.05 vs. the control group. #P <0.05, vs. the LPS group
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Figure 4. 3PO attenuates LPS-stimulated apoptosis by inhibiting ROS generation in A549 cell. (A) Flow cytometry analysis showing Annexin V and PI staining. Apoptotic cells from different groups are indicated as Annexin V+ and PI- . (B) Apoptotic cells from different groups were quantified using a flow cytometer. (C) Intracellular ROS from different groups were determined with the aid of a flow cytometer. (D) Intracellular ROS from different groups were assessed using immunofluorescence. Data are presented as the mean ± SD. n=3-5/group. *P<0.05 vs. the control ＆ group. #P <0.05, vs. the LPS group， P <0.05, vs. the lactate group
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Research highlights 1. A specific glycolytic inhibitors 3PO can diminish acute injury of the lungs induced by sepsis in mice and LPS-induced inflammation and apoptosis of A549 cell. 2. LPS exposure promote a metabolic switch to aerobic glycolysis and A549 cell apoptosis by the ROS pathway.
S0006-291X(17)31082-3
DOI:
10.1016/j.bbrc.2017.05.173
Reference:
YBBRC 37894
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 19 May 2017 Accepted Date: 29 May 2017
Please cite this article as: Y. Gong, H. Lan, Z. Yu, M. Wang, S. Wang, Y. Chen, H. Rao, J. Li, Z. Sheng, J. Shao, Blockage of glycolysis by targeting PFKFB3 alleviates sepsis-related acute lung injury via suppressing inflammation and apoptosis of alveolar epithelial cells, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.05.173. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Blockage of glycolysis by targeting PFKFB3 alleviates sepsis-related acute lung injury via suppressing inflammation and apoptosis of alveolar epithelial cells
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Yuanqi Gonga1 ; Haibing Lana1 ; Zhihong Yua ; Meng Wanga ; Shu Wanga ; Yu Chena ; Haiwei Raoa ; Jingying Lia ; Zhiyong Shenga ; Jianghua Shaob* a Department of Comprehensive Intensive Care Unit, Second Affiliated Hospital of Nanchang University, Nanchang 330006, China; b Department of Hepatobiliary Surgery, Second Affiliated Hospital of Nanchang University, Nanchang 330006, China; 1 These authors made equal contributions to this paper. *Correspondence: Dr Jianghua Shao, E-mail:[email protected] Abstract Sepsis-related acute lung injury (ALI) is characterized by excessive lung inflammation and apoptosis of alveolar epithelial cells resulting in acute hypoxemic respiratory failure. Recent studies indicated that anaerobic glycolysis play an important role in sepsis. However, whether inhibition of aerobic glycolysis exhibits benefcial effect on sepsis-induced ALI is not known. In vivo, a cecal ligation and puncture (CLP)-induced ALI mouse model was set up and mice treated with glycolytic inhibitor 3PO after CLP. The mice treated with the 3PO ameliorated the survival rate, histopathological changes, lung inflammation, lactate increased and lung apoptosis of mice with CLP-induced sepsis. In vitro, the exposure of human alveolar epithelial A549 cells to lipopolysaccharide (LPS) resulted in cell apoptosis, inflammatory cytokine production, enhanced glycolytic flux and reactive oxygen species (ROS) increased. While these changes were attenuated by 3PO treatment. Sequentially, treatment of A549 cells with lactate caused cell apoptosis and enhancement of ROS. Pretreatment with N-acetylcysteine (NAC) significantly lowered LPS and lactate-induced the generation of ROS and cell apoptosis in A549 cells. Therefore, these results indicate that anaerobic glycolysis may be an important contributor in cell apoptosis of sepsis-related ALI. Moreover, LPS specifically induces apoptotic insults to A549 cell through lactate-mediated enhancement of ROS. Key words: Sepsis-related acute lung injury, anaerobic glycolysis, 3PO, human alveolar epithelial A549 cells, apoptosis, ROS. 1.Introduction Sepsis is a complex systemic inflammatory response syndrome (SIRS) resulting in high mortality in critically ill patients and multi-organ dysfunction syndrome (MODS) including cardiovascular system, liver, kidney and lung[1]. The respiratory dysfunction is one of the most frequent complication of sepsis because of pulmonary susceptibility. So acute lung injury (ALI) and its most severe manifestation acute respiratory distress syndrome (ARDS) are the most severe outcomes of sepsis and responsible for sudden deaths of patients in the intensive care unit (ICU) [2,3]. ALI is characterized by acute hypoxemic respiratory failure, pulmonary hypertension, pulmonary alveoli edema, decreased pulmonary compliance and deteriorated gas exchange [4]. The pathophysiological process of ALI is generally believed to be excessive and uncontrolled activation of lung inflammatory response which implicates many types of inflammatory cells infiltration, alveolar-capillary destruction, excessive oxidative stress characterized by the aberrant release of reactive oxygen species (ROS) and alveolar epithelial cell apoptosis [5,6]. Extensive apoptosis of pulmonary alveolar type II epithelial cells has been shown to be responsible for the impairment of the epithelial barrier function and the remodeling of certain mesenchymal cells in
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ALI [7]. However, the precise molecular mechanisms leading to lung inflammation and apoptosis of alveolar epithelial cells in sepsis-induced ALI remains incompletely understood, limiting the development for effective drugs and therapies of curing ALI caused by sepsis. Numerous studies have demonstrated that a set of different pathophysiological changes such as hyperlactatemia are generally noticed during sepsis. Traditionally, during sepsis, and especially ALI, hyperlactatemia has been interpreted as the existence of anaerobic glycolysis due to an inadequate oxygen supply inducing cellular distress [8,9]. Aerobic glycolysis is the crucial metabolic pathway that be controlled by various glycolytic enzymes in sepsis [9]. Of these glycolytic enzymes, the PFKFB3 isozyme largely rely on glycolysis for ATP generation , regulate glycolytic flux and product fructose-2,6-bisphosphate(Fru-2,6-BP) which is the potent allosteric regulator of the key glycolytic enzyme phosphofructokinase 1 (PFK-1) [10]. A small molecule antagonist of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one(3PO), has been found to causes a rapid reduction in Fru-2,6-BP, suppress glycolytic flux to lactate, decrease glucose uptake, induces growth inhibition in several cell lines and inhibit tumor growth [11]. Thus, aerobic glycolysis may be an important contributor in the context of sepsis-related ALI. However, whether blockage of aerobic glycolysis by targeting PFKFB3 shows a protective effect on sepsis-induced ALI is not known. In the present study, we focused on the effects of the PFKFB3 inhibitor 3PO on lung inflammatory and alveolar epithelial cell apoptosis in sepsis-related ALI and the possible mechanism. 2.Materials and methods 2.1 Materials and reagents LPS was purchased from Sigma-Aldrich. N-acetylcysteine (NAC a superoxide inhibitor ) were purchased from Sigma-Aldrich. Lactate was purchased from Sigma-Aldrich. 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO, Merck Millipore, Darmstadt, Germany). Mouse TNF-α、IL-6 and IL-1β ELISA kits were purchased from eBioscience (San Diego, US). Human TNF-α、IL-6 and IL-1β ELISA kits were purchased from eBioscience (San Diego, US). Lactate Colorimetric/Fluorometric Assay Kit (K607-100, Biovision). Glucose Colorimetric/Fluorometric Assay Kit (K606-100, Biovision). LDH enzymatic assay (Roche cat.).The reactive oxygen species assay kit (DCF-DA) was purchased from Applygen. 2.2 Animal Specific-pathogen-free (SPF) C57BL/6 wide-type mice (male, 6-8 weeks old) were purchased from Nanchang University (Nanchang, China). The mice were housed in a SPF environment (room temperature 24˚C, humidity range 40-70%, 12 h light/dark cycle). Sterilized water and food were provided ad libitum. All experiments were conducted under the guidelines outlined by the committee of Nanchang University on the use and care of animals. Standard guidelines for laboratory animal care followed the Guide for the Care and Use of Laboratory Animals. 2.3 Animal experimental protocol. A classic sepsis-induced ALI model were established by cecal ligation and puncture (CLP) as previously described with mildly modification [12]. C57BL/6 mice were randomly divided into three groups (10 rats in each group ):sham-operated (control), CLP , and 3PO+CLP groups. Briefly, healthy mice underwent 24-hour deprivation of food but not water. Mice were anesthetized using sodium pentobarbital (intraperitoneally, 40 mg/kg). A midline abdominal incision was made after the ventral abdomen was shaved and cleaned with 10% povidone-iodine
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wash and the cecum was exposed. The cecum was ligated just distal to the ileocecal valve to avoid intestinal obstruction, then through-and-through punctured with a 21-gauge needle. The ligated cecum was then returned to the peritoneal cavity, and the abdominal wall was closed in two layers. As sham operated animals, the cecum underwent the same procedure but neither ligated nor punctured, and then returned to the abdominal cavity. Mice receive intragastric administration of 0.07 mg/g 3PO in 50 uL DMSO and only vehicle DMSO 0 and 6 h after CLP. The mortality of mice was recorded every 12 h for 5 days after CLP in each group. The 3PO dose was determined as described previously [10].Then the mice were euthanized for collection of blood, bronchoalveolar lavage fluid (BALF) and lungs. 2.4 Lung wet/dry(W/D) weight ratio. The ratio of lung W/D weight was calculated to assess the lung edema. The fresh upper part of the right lung was cleansed and weighed to obtain the wet weight and was then dried in an oven at -80 ˚C for at least 24 h for the measurement of the dry weight. 2.5 Analysis of serum and BALF. Blood samples were collected, stored for 2 h at room temperature, and then centrifuged (3000 rpm, 20 min). The supernatants were harvested and stored at -80˚C. TNF-α, IL-6 and IL-1β was measured by ELISA according to the manufacturer's instructions. Next, BALF samples were collected. Total cells in BALF were counted, and a cytospin sample was prepared and stained with hematoxylin and eosin (H&E) for blinded assessment of differential cell percentages in BALF. Then BALF samples were centrifuged (1000 rpm, 10 min). The supernatants were harvested and stored at -80˚C. The levels of TNF-α, IL-6 and IL-1β in the BALF samples were measured using ELISA according to the manufacturer’s instructions. Lactate in BALF was measured with Lactate Colorimetric/Fluorometric Assay Kit according to the manufacturer’s instructions. 2.6 Pathological staining and assessment of lung apoptosis. The left lungs of mice were removed and fixed in 4 % paraformaldehyde, then embedded in paraffin. Lung tissues were stained with hematoxylin and eosin (H&E) for blinded histopathologic assessment. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining, fluorescence staining was performed using a commercially available in situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions. Results are expressed as the average number of TUNEL-positive staining cells per 200X magnifcation feld. 2.7 Cell culture Human pulmonary epithelial A549 cells (Shanghai Fuxiang Biological Technology Co. Ltd., ATCC, USA) were grown in a cell culture flask in RPMI 1640 medium (Gibco, Life Technology) supplemented with 10% fetal bovine serum (Gibco, Life Technology). The cells were then incubated in a humidified incubator at 37˚C with an atmosphere of 5% CO2. 2.8 Cell apoptosis analysis A549 cells were seeded into 6-well plates. When cells reached 80% confluence, the cells were treated according to the experimental plan. Harvested cells were washed with PBS and stained with Annexin V and Propidium Iodide (Beyotime, China). Fluorescence was measured using a FACSCalibur (BD Biosciences, San Jose, CA, USA) and analyzed using FlowJo v10.1 (Oregon, USA). Annexin V+/PI+ (late apoptotic) and Annexin V+/PI- (early apoptotic) cells were quantified by the frequency of fluorescently labeled cells. 2.9 Biochemical assays and analysis of the culture medium.
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Glucose concentration of the culture medium was measured by using Glucose Colorimetric/Fluorometric Assay Kit according to the manufacturer’s instructions. Lactate dehydrogenase (LDH) activity were measured using LDH enzymatic assay according to the manufacturer’s instructions [13]. Lactate concentration of the culture medium was measured by using Lactate Colorimetric/Fluorometric Assay Kit according to the manufacturer’s instructions. The levels of TNF-α, IL-6 and IL-1β in the the culture medium were measured using ELISA according to the manufacturer’s instructions. 2.10 Detection of ROS levels Intracellular ROS production was quantified using the oxidation-sensitive probe 2,7-dichlorofluorescein diacetate (DCF-DA) (Applygen). Briefly, 10 mM DCF-DA stock solution (in methanol) was diluted 1000-fold in cell culture medium without serum to yield a 10-um working solution. After 24 h of stimulation, the cells in 24-well plates were washed twice with phosphate-buffered saline (PBS) and incubated in 500 ul working solution of DCF-DA at 37˚C in dark for 1 h. The cells were then washed twice with cold PBS and resuspended in the PBS for an analysis of intracellular ROS using a multiwell fluorescence scanner (SpectraMax M5/M5e; Molecular Devices) and microscope (Olympus). DCF-DA fluorescence was detected at an excitation wavelength of 480 nm and an emission wavelength of 525 nm [14]. 2.10 Statistical analysis. Statistical analysis was performing using the SPSS (version 19.0) software package. All data are presented as the mean ± standard deviation. One-way ANOVA were utilized to compare differences among groups. Values of P <0.05 were considered statistically significant. 3.Results 3.1 Effects of glycolytic inhibitor 3PO on Sepsis-Induced ALI in mice. To determine the effect of 3PO on the acute injury of the lungs induced by sepsis in mice, survival was observed for 120 h after CLP , a histological assessment of lung tissue was performed using H&E staining and lung edema was evaluated using the ratio of lung W/D weight. As shown in Fig.1A, sepsis resulted in a raised mortality rate within 120 h. However, mice treated with 3PO showed a significantly improved survival rate during the study period. Histological analysis of lung tissue revealed that CLP mice displayed lung exudative changes，hemorrhage, edema, fibrin deposition and heavy infltration of inflammatory cell. While they were attenuated by 3PO treatment (Fig. 1B). Pulmonary wet-to-dry weight ratios in CLP group were significantly higher lung edema compared to that of the control group which was attenuated by 3PO treatment (Fig. 1C). Then we found a increased number of total cells, neutrophils and lactate in BALF of the CLP group. These effects were partially rescued by treatment with the 3PO (Fig. 1D,E,F). 3.2 3PO inhibits the lung inflammation and lung apoptosis of Sepsis-Induced ALI in mice. ALI was successfully induced by CLP surgery. To further assess the effect of 3PO on lung inflammation and lung apoptosis in the CLP model, the TNF-α, IL-6 and IL-1β in BALF and serum were measured by ELISA and apoptotic cells in lungs were detected by labeling with an in situ TUNEL assay. There were notable increases in TNF-α, IL-6 and IL-1β in BALF and serum of sepsis-induced ALI which were reduced by 3PO (Fig. 2A-F). Lungs from mice after CLP exhibited significant ladder formation, a characteristic of apoptosis which was attenuated by 3PO treatment (Fig. 2G,H). These findings clearly indicate that CLP surgery results in a robust lung inflammation and lung apoptosis in mice, and the protective effect of the 3PO. 3.3 3PO attenuates LPS-stimulated glycolysis and inflammatory cytokines release in A549
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cell. Then in vitro we assess the effect of 3PO on LPS-induced ALI. The human pulmonary epithelial A549 cell stimulated with LPS (100 ug/ml) for the indicated periods of time (0, 2, 4 h). We then evaluated glucose and lactate concentration of the culture medium, LDH activity in cell lysates and the expression levels of TNF-α, IL-6 and IL-1β in the culture medium. In mice, we have found the increase lactate in BALF of the CLP group. In A549 cell we also found LPS caused a significant increase in extracellular glucose consumption (Fig. 3A) and lactate level (Fig. 3C) and intracellular lactate dehydrogenase (LDH) activity (Fig. 3B), indicative of an enhanced glycolytic flux. Meanwhile, the expression levels of inflammatory cytokines TNF-α, IL-6 and IL-1β remarkably increased after LPS challenge (Fig. 3D,E,F). Treatment of cells with glycolytic blockade with 3PO(20µM) prior to LPS stimulation inhibited the increased glycolytic flux and inflammatory cytokines release induced by LPS in A549 cell. 3.4 3PO attenuates LPS-stimulated apoptosis by inhibiting ROS generation in A549 cell. Then we found that the exposure of A549 cell to LPS(100 ug/ml) and lactate (5 mM) resulted in cell apoptosis. The result is similar with the effect of HCl on cell apoptosis (Fig. 4A,B). Meanwhile, we also found that LPS and lactate can cause an marked increase in ROS (Fig. 4C,D). In order to further identify the role of ROS on cell apoptosis induced by the LPS and lactate. The A549 cells were respectively pretreatment with the ROS scavenger NAC. The results showed that the antioxidant NAC (10 mM) significantly suppress the increases in ROS and cell apoptosis induced by the LPS and lactate (Fig. 4C,D). Moreover, pre-treatment with 3PO could inhibit the LPS-induced the ROS increased and cell apoptosis (Fig. 4A-D). Taken together, these results indicate that 3PO ameliorates LPS-induced apoptosis by inhibiting ROS generation in A549 cell. Discussion In this study, we indicated that a crucial role of enhanced aerobic glycolysis in lung apoptosis of mice with surgery CLP and in LPS-induced A549 cell apoptosis. For the first time, we demonstrated that a specific glycolytic inhibitors 3PO can diminish acute injury of the lungs induced by sepsis in mice and LPS-induced inflammation and apoptosis of A549 cell. Moreover, in A549 cell we found that LPS can cause a increase in lactate level, indicative of an enhanced aerobic glycolysis. Then lactate in turn induce cell apoptosis of A549 cell by elevating the ROS. Sepsis is defined as the systemic inflammatory response to severe infection and surgery which may induce multiple complications such as brain injury, kidney injury, lung injury [15,16]. More importantly, sepsis is the major cause of ALI development with high morbidity and mortality [17]. The sepsis model of CLP is a widely accepted animal model for the research of sepsis-associated mechanism and we successfully established a CLP induced sepsis model to evaluate the effects of 3PO on sepsis-induced ALI in mice. The CLP-exposed ALI mice developed a raised mortality rate, lung exudative changes，hemorrhage, edema, fibrin deposition, heavy infltration of inflammatory cell, a increased of total cells, neutrophils, lactate ,TNF-α, IL-6, IL-1β and lung apoptosis. It is known that acute lung injury is characterized by alveolar epithelial and endothelial cell injury , inflammatory cell and protein rich edema fluid influx into the alveolus, alveolar and bronchial epithelial cell apoptosis, lung edema [2,18]. The apoptosis of lung epithelial cells with lung inflammation may contribute to the development and progression of ALI by promoting inflammatory exudation, destruction of lung structure and fibrosing of lung [19,20]. LPS was reported to be one of the major causes of inflammatory cells leakage, septic shock and acute lung injury [21]. A549 is an alveolar epithelial cell line with type II cell characteristics and
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has been widely used as a ALI model to study the effects of LPS in vitro [20]. So we selected the alveolar epithelial cell line A549 to assess the effects of LPS on the apoptosis and inflammation of lung epithelial cells in vitro. Our results showed LPS can induce cell apoptosis and inflammatory cytokines release in A549 cell. These data support the conclusion that the apoptosis and inflammation of lung epithelial cells may be a key factor in the development of sepsis-associated ALI. Extensive studies have reported sepsis is concerned with a number of metabolic abnormalities, including increased glucose uptake, aerobic glycolysis and hyperlactatemia [22,23]. Accelerated aerobic glycolysis contributes to the maturation of dendritic cells in response to toll-like receptor ligands (such as TLR4, TLR2 and TLR9) [24], the differentiation of both anti-inflammatory Treg cells and pro-inflammatory Th17 cells [25] and LPS-induced release of late mediators [26]. In our study, we showed that lactate expression in BALF was significantly increased in the mouse model of CLP-induced sepsis mice, suggesting the possible involvement of aerobic glycolysis in the development of sepsis-associated ALI. Then we used A549 cells to assess the effects of LPS on aerobic glycolysis in vitro. In this study, we found that LPS exposure can induce a increase in extracellular glucose consumption and lactate level and intracellular LDH activity ,indicating aerobic glycolysis play an important role in LPS-induced ALI in A549 cells. To further address the roles of aerobic glycolysis in the CLP-induced ALI mouse model and LPS-induced ALI cell model, we investigated the effect of aerobic glycolysis inhibition with the PFKFB3 inhibitor 3PO. In the previous study, the PFKFB3 represents the most abundant PFKFB isoenzyme in endothelial cells, which largely rely on glycolysis for ATP generation and it may represents a novel pharmacological strategy for targeting angiogenesis and tumor cell growth/migration [27]. Moreover, PFKFB3 inhibitors 3PO was shown to suppress glucose uptake, which in turn causes an increase in autophagy and may prove useful as rational combinations for the treatment of cancer [10], PFKFB3 blockade by 3PO also can reduce glycolysis to decrease pathological neovascularization in ocular and inflammatory models [28] and targeting glycolysis by 3PO reduces key features of Rheumatoid Arthritis pathogenesis in vitro and can mediate TLR2-induced inflammation [29]. Consistent with these data, the blockade of PFKFB3 with 3PO ameliorated CLP-induced changes in our mouse sepsis model. The lung apoptosis and lung inflammation influence each other, which then contributes to the pathogenesis of ALI. In our experiment, pretreatment with 3PO partly reduced the heavy infltration of inflammatory cell, the elevated expression of TNF-α, IL-6 and IL-1β in BALF and serum and lung apoptosis by inhibiting the expression of lactate in mice with CLP-induced ALI. These data indicate that aerobic glycolysis participates in promoting the release of inflammatory cytokines and lung apoptosis in an animal model of CLP-induced ALI and that the 3PO counteracts the effects of glycolysis and alleviates CLP-induced changes. Meanwhile, in this report we also describe that pre-treatment with glycolytic inhibitor 3PO reduced LPS-stimulated cell apoptosis and the increased expression of inflammatory cytokines TNF-α, IL-6 and IL-1β by suppressing glycolytic flux in A549 cells. These observations provide direct support for the conclusion that treatment with 3PO alleviates lung apoptosis and lung inflammation in the CLP-induced ALI mouse model and LPS-induced ALI cell model. Nevertheless,the mechanisms by which 3PO mitigates cell apoptosis and inflammatory cytokines release in LPS-induced ALI cell model of A549 cells remain to be elucidated. Recently, ROS have been verifed to be capable of regulating many intracellular signal
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transduction pathways and abnormal ROS signal may cause oxidative stress and subsequent cell apoptosis [30]. Much research has focused on the effects of ROS on the pathogenesis of ALI [31,32] and the exposure to LPS increased the levels of cellular nitric oxide and ROS and eventually led to cell apoptosis and ROS participate in LPS-induced apoptosis of A549 cells [21]. The similar results were also obtained by us, LPS significantly induced the expression of ROS. To further elucidate the relationship between the LPS-induced cell apoptosis, inflammatory cytokines release and the ROS production, we treated A549 cells with LPS in the presence of the antioxidant NAC by scavenging of ROS and found that NAC reversed the LPS-mediated cell apoptosis and inflammatory cytokines release. These data suggest that LPS can induce cell apoptosis and inflammatory cytokines release via the ROS generation. Subsequently, we used lactate stimulate to A549 cells to assess the directly effects of lactate in vitro. These results showed lactate can cause an marked increase in ROS and cell apoptosis in A549 cells, the NAC partly restored the increased expression of ROS and cell apoptosis. These data suggest that there are interaction relationship between the upregulation of aerobic glycolysis and activation of the ROS in regulation of LPS-induced cell apoptosis in A549 cells. Meanwhile we confirmed that pre-treatment with glycolysis inhibitor 3PO could suppress the LPS-induced the ROS increased and cell apoptosis. So our results indicated that LPS can induce an enhanced aerobic glycolysis which then cause cell apoptosis of A549 cell by elevating the ROS. In conclusion, our study demonstrated that LPS exposure promote a metabolic switch to aerobic glycolysis and A549 cell apoptosis by the ROS pathway. The glycolysis inhibitor 3PO may provide favorable effects to reduce inflammation reactivity of cells and cell apoptosis of lung in CLP-induced ALI mice model and LPS-induced ALI cell model. Thus, novel therapeutic strategies targeting aerobic glycolysis can serve as a potential therapeutic target in the treatment of ALI. Conflicts of interest All authors declare no conflicts of interest associated with this manuscript. Acknowledgments This study was funded by Key Research and Development Project of Jiangxi Province [S2016SFYBG0458].
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Figure 1. Effects of glycolytic inhibitor 3PO on Sepsis-Induced ALI in mice. (A) No deaths occurred in mice that underwent sham procedure (n = 10). 3PO was administered 0 and 6 h after CLP. Mortality was observed over 120 h. (B) These representative histological changes of the lung were obtained from mice of different groups (original magnification ×200). Lung tissue sections stained with H&E. (C) Wet/dry ratio of lungs from mice of different groups. (D) Total cell number in the BALF of mice from different groups. (E) Neutrophils in the BALF of mice from different groups. (F) Lactate in the BALF of mice from different groups. Data are presented as the mean ± SD. n=8-10/group. *P<0.05 vs. the control group. #P <0.05, vs. the CLP group
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Figure 2. 3PO inhibits the lung inflammation and lung apoptosis of Sepsis-Induced ALI in mice. (A) TNF-α,(B) IL-6 and (C) IL-1β was measured by ELISA in BALF. (D) TNF-α,(E) IL-6 and (F) IL-1β was measured by ELISA in Serum. (G) Arrowheads indicate TUNEL-positive apoptotic cells (original magnification ×200). (H) Quantification of TUNEL-positive cells. Data are presented as the mean ± SD. n=8-10/group. *P<0.05 vs. the control group. #P <0.05, vs. the CLP group
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Figure 3. 3PO attenuates LPS-stimulated glycolysis and inflammatory cytokines release in A549 cell. (A) Glucose consumption in A549 cells from different groups were determined. (B) Intracellular LDH activity from different groups were measured by colorimetric assay. (C) The concentrations of lactate in the culture medium from different groups were determined. (D) TNF-α,(E) IL-6 and (F) IL-1β was measured by ELISA in the culture medium from different groups. Data are presented as the mean ± SD. n=3-5/group. *P<0.05 vs. the control group. #P <0.05, vs. the LPS group
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Figure 4. 3PO attenuates LPS-stimulated apoptosis by inhibiting ROS generation in A549 cell. (A) Flow cytometry analysis showing Annexin V and PI staining. Apoptotic cells from different groups are indicated as Annexin V+ and PI- . (B) Apoptotic cells from different groups were quantified using a flow cytometer. (C) Intracellular ROS from different groups were determined with the aid of a flow cytometer. (D) Intracellular ROS from different groups were assessed using immunofluorescence. Data are presented as the mean ± SD. n=3-5/group. *P<0.05 vs. the control ＆ group. #P <0.05, vs. the LPS group， P <0.05, vs. the lactate group
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Research highlights 1. A specific glycolytic inhibitors 3PO can diminish acute injury of the lungs induced by sepsis in mice and LPS-induced inflammation and apoptosis of A549 cell. 2. LPS exposure promote a metabolic switch to aerobic glycolysis and A549 cell apoptosis by the ROS pathway.