| Background:Acute lung injury(ALI)/Acute Respiratory Distress Syndrome(ARDS)caused by infection or other injury factors is a common clinical emergency,and uncontrolled inflammatory reaction is the key mechanism of its occurrence and development.Inflammation is essentially composed of a series of complex and active molecular reactions such as signal transduction,gene expression and synthesis of inflammatory mediators.Recent studies have found that inflammatory molecular response is a biological process of high energy-consuming(energy intensive),which requires high-intensity energy support.The energy metabolism of inflammatory cells is a controllable factor that significantly affects the outcome of inflammation,and the regulation of energy metabolism is expected to become a new way to limit inflammatory injury.Calorie restriction(CR),which reduces energy intake by controlling diet,is a simple and direct way to limit energy metabolism.A large number of studies have proved that CR has anti-aging,anti-inflammatory,anti-tumor effects,but strong hunger and other adverse effects limit the application and promotion of CR.CR mimetics(CRM)can simulate the beneficial effects of CR without reducing food intake.Because glucose is one of the most important energy sources for cells,glycolysis inhibitor 2-deoxyglucose(2-DG)was first used as CRM.Studies have shown that 2-DG can play an anti-inflammatory and protective effect in vivo and in vitro.So,can 2-DG be used to restrict energy metabolism and reduce inflammatory damage in ALI/ARDS?Pyruvate is a key metabolite connecting glycolysis and tricarboxylic acid cycle,which is catalyzed by pyruvate kinase(PK).There are four subtypes of pyruvate kinase,among which M2 pyruvate kinase(PKM2)can quickly regulate its activity by translocation from cytoplasm to nucleus,which is the key switch molecule to regulate inflammation-related metabolic transition.PKM2 can also play the role of protein kinase and regulate inflammatory gene expression through transcription factors such as phosphorylated signal transducer and transcriptional activator 3(STAT3).Therefore,PKM2 nuclear translocation is a key molecular event associated with energy metabolism and inflammation.So,is there any relationship between the limited energy metabolism of 2-DG and the subcellular distribution of PKM2? Objective:To explore whether CR mimic 2-DG can exert anti-inflammatory and protective effect in lipopolysaccharide(LPS)-induced ALI mouse model,and reveal the related molecular signal mechanism from PKM2-STAT3 pathway.The purpose of this study is to explain the relationship between the state of energy metabolism and the occurrence and development of ALI/ARDS,and to find a new and effective way for the prevention and treatment of ALI/ARDS.Methods:Male BALB/c mice were used to replicate ALI model by intraperitoneal injection of LPS(20 mg/kg).64 mice were randomly divided into four groups: Control group,2-DG group,LPS group and 2-DG+LPS group.When the drug was dissolved in each group,the ratio of solvent volume to body weight of mice was 0.01 ml/g.Control group: intraperitoneal injection of the same volume of normal saline.2-DG group: intraperitoneal injection of 2-DG solution of 500 mg/kg.LPS group: intraperitoneal injection of LPS solution of 20 mg/kg.2-DG+LPS group: 2-DG(500 mg/kg)was injected intraperitoneally 0.5 h before LPS(20 mg/kg)exposure.The mice in each group were killed 18 h after LPS exposure.In order to explore the possible role of CR mimetics 2-DG in ALI,the plasma and lung tissue samples of mice in each group were collected.The pathomorphological changes of lung tissue were observed by HE staining,the activity of MPO in lung tissue homogenate was detected by myeloperoxidase(MPO)detection kit(CaymanChemical,USA),and the expression levels of inflammatory factors interleukin-6(IL-6)and tumor necrosis factor-?(TNF-?)in plasma and lung tissue were detected by enzyme-linked immunosorbent assay(ELISA).In addition,8 mice in each group were injected with Evans blue staining solution(80 mg/kg)into the tail vein 1 hour before the end of the experiment.After the experiment,the intact lung tissue was removed and perfused with PBS,and then homogenized to evaluate the pulmonary capillary permeability by measuring the leakage of evans blue staining solution in the lung tissue.In order to explore the effect of 2-DG on the level of PKM2 in the nucleus of lung tissue of mice exposed to LPS,the lung tissue samples were collected and the content of PKM2 in the whole lung tissue and nucleus were detected by western blotting.In order to investigate the effect of 2-DG on the expression and activity of STAT3,the expression of STAT3 and P-STAT3 in the whole lung tissue and nucleus were detected by immunoblotting.In order to explore the effect of 2-DG on the survival rate of mice,we randomly divided 126 male BALB/c mice into two parts.In the first part,36 mice were divided into two groups(2-DG pretreatment): LPS group,2-DG(0.5 h)+ LPS group,LPS group: intraperitoneal injection of LPS solution,2-DG(0.5 h)+ LPS group: intraperitoneal injection of 2-DG,followed by intraperitoneal injection of LPS solution 0.5 h later,the dose of the drugs were the same as before;In the second part,90 mice were divided into five groups(2-DG post-treatment): LPS group,LPS+2-DG(0.5 h)group,LPS+2-DG(4 h)group,LPS+2-DG(12 h)groups,LPS+2-DG(24 h)groups,LPS group: intraperitoneal injection of LPS(20 mg/kg)solution,LPS+2-DG(0.5 h)group: intraperitoneal injection of LPS solution,followed by intraperitoneal injection of 2-DG 0.5 h later.LPS+2-DG(4 h): intraperitoneal injection of LPS solution,followed by intraperitoneal injection of 2-DG 4 h later,LPS+2-DG(12 h)group: intraperitoneal injection of LPS solution,followed by intraperitoneal injection of 2-DG 12 h later,LPS+2-DG(24 hours): intraperitoneal injection of LPS solution,followed by intraperitoneal injection of 2-DG 24 h later,the dose of the drugs were the same as before;After intraperitoneal injection of LPS,the survival number of mice in each group was recorded every 6 h,and the survival of mice in different treatment groups was analyzed by Kaplan-Meier curve and logarithmic rank test.In order to explore the effect of nuclear PKM2 expression on LPS-induced ALI,A small molecule drug ML-265,which can inhibt the translocation of PKM2 into the nucleus,was used in the experiment.24 BALB/c male mice were randomly divided into three groups: Control group,LPS group and ML-265+LPS group.ML-265,a small molecule drug that can inhibit the accumulation of PKM2 in nucleus,was used in the experiment.When the drugs were dissolved in each group,the ratio of solvent volume to body weight of mice was 0.01 ml/g.Control group: intraperitoneal injection of the same amount of normal saline.LPS group: intraperitoneal injection of LPS solution.ML-265+LPS group: ML-265(50 mg/kg)was injected intraperitoneally 0.5 h before LPS(20 mg/kg)exposure,the dose of LPS was the same as before.After the mice were exposed to LPS for18 h,the mice were killed,and the plasma and lung tissue samples of each group were collected.Subsequently,the content of PKM2 in the nucleus of lung tissue was detected by immunoblotting,the levels of plasma inflammatory factors TNF-? and IL-6 were measured by ELISA,and the pathomorphological changes of lung tissue were observed by hematoxylin-eosin(HE)staining.In order to explore the effect of nuclear PKM2 on STAT3,The content of STAT3 and P-STAT3 in the nucleus of lung tissue were detected by western blotting.In order to explore whether STAT3 was involved in ALI induced by LPS.Stattic,a small molecular inhibitor of STAT3,which can specifically inhibit the activity of STAT3,was used in our experiment.24 BALB/c male mice were randomly divided into three groups: Control group,LPS group and stattic+LPS group.Control group: intraperitoneal injection of the same amount of normal saline.LPS group: intraperitoneal injection of LPS solution.stattic+LPS group: stattic(5 mg/kg)was injected intraperitoneally 0.5 h before intraperitoneal injection of LPS,the dose of LPS was the same as before.the plasma and lung tissue samples were collected after the mice exposure to LPS 18 h later.In order to investigate whether STAT3 was involved in LPS-induced ALI,The levels of plasma inflammatory cytokines TNF-? and IL-6 were measured by enzyme linked immunosorbent assay(ELISA),and the pathomorphological changes of lung tissue were observed by HE staining.Results:1.2-DG alleviated LPS-induced ALI: Compared with LPS-induced ALI mice,2-DG pretreatment significantly decreased MPO activity in lung tissue,decreased Evans blue leakage,improved histomorphological abnormalities,which characterized by intact pulmonary lobule structure,local atelectasis,no alveolar bleeding,reduction of pulmonary interstitial edema and decrease of pulmonary interstitial inflammatory cell infiltration.2-DG also significantly decreased the levels of inflammatory cytokines IL-6 and TNF-? in lung plasma and lung tissue induced by LPS.In addition,2-DG pretreatment could significantly improve the survival rate of ALI animals induced by LPS.2-DG post-treatment(0.5 h or 4 h after intraperitoneal injection of LPS)could also significantly improve the early survival rate of ALI mice induced by LPS.However,2-DG post-treatment(12 h or 24 h after intraperitoneal injection of LPS)could not improve the late survival rate of ALI mice.2.2-DG inhibited the increase of nuclear PKM2 level in lung tissue induced by LPS: Compared with the normal group,the total PKM2 content in lung tissue of LPS group had no significant change,and 2-DG pretreatment did not change the protein level of total PKM2 in lung tissue;However,compared with the normal control group,The level of nuclear PKM2 in the lung tissue of LPS group was significantly up-regulated,and 2-DG pretreatment significantly inhibited the increase of nuclear PKM2 in the lung tissue induced by LPS.3.Inhibition of PKM2 nuclear translocation attenuated LPS-induced pulmonary inflammatory injury: ML-265 preconditioning significantly inhibited the upregulation of PKM2 in the nucleus induced by LPS,and ML-265 preconditioning also significantly down-regulated the levels of plasma inflammatory cytokines TNF-? and IL-6 induced by LPS.Pathomorphological analysis of lung tissue also showed that ML-265 pretreatment significantly improved the abnormal morphology of lung tissue induced by LPS.4.STAT3 inhibitor attenuated lung inflammatory injury: Compared with the normal control group,there was no significant change in the overall level of STAT3 in the lung tissue of mice in the LPS group,and 2-DG pretreatment did not change the overall level of STAT3 in the lung tissue of the LPS group;However,compared with the normal control group,the level of nuclear STAT3 and P-STAT3 in the lung tissue of the LPS group was increased,and 2-DG pretreatment significantly decreased the level of STAT3 and P-STAT3 in the nucleus induced by LPS.ML-265 pretreatment inhibited the nuclear accumulation of PKM2,and ML-265 pretreatment also reduced the content of nuclear STAT3 and P-STAT3 induced by LPS.Pretreatment with STAT3 activity inhibitor stattic not only significantly down-regulated the levels of plasma inflammatory cytokines TNF-? and IL-6 induced by LPS,but also improved the morphological abnormalities of lung tissue induced by LPS stimulation.Conclusion:1.The preventive intervention of CRM 2-DG can effectively inhibit the LPS induced inflammatory response and lung tissue damage,significantly improve the survival rate of experimental animals;the therapeutic intervention of CRM 2-DG can still play the above-mentioned anti-inflammatory protective effect.These data suggest that CRM 2-DG can effectively prevent and treat ALI / ARDS,and it has potential applications prospects.2.The anti-inflammatory protective effect of CR mimic 2-DG may be related to its inhibition of nuclear translocation of PKM2,which can inhibit PKM2-mediated correlation between metabolism and inflammation.3.CR mimic 2-DG can inhibit the phosphorylation of STAT3 in the nucleus by regulating the nuclear translocation of PKM2,which may be the key mechanism of 2-DG down-regulating the expression of inflammation-related genes. |
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