The Protective Effect And Mechanism Of Mildronate On High Altitude Hypoxia-induced Lung Injury

Background:High altitude in plateau area is a complex environment and hypobaric hypoxia results in metabolic abnormality（glycolysis）and oxidative stress,which induces acute mountain sickness,such as acute lung injury（ALI）and high altitude brain edema（HACE）.Hypobaric hypoxia-induced lung injury influences the efficiency of workers and impairs the travel experience of tourists.Up to date,drugs to prevent high altitude-induced injury remains deficient.Previous reports demonstrated that glycolysis play an important role in process of hypobaric hypoxia-induced lung injury.Mildronate,an analogue of L-carnitine,can regulate metabolism.In this study,we based on glycolysis pathway to study the protective effect of mildronate on acute lung injury at high altitude.Objective:This study aims to investigate the effect and mechanism of mildronate on hypoxia-induced lung injury in high altitude.Methods:Experiment 1:Mildronate prevents hypoxia-induced lung injury in high altitude（1）Establishment of hypoxia-induced acute lung injury in vivo at high altitude.Kunming mice were divided into six groups:control group,hypoxia group,mildronate（50,100,and 200 mg/kg）and acetazolamide（50 mg/kg）groups.After intragastric treatment for 3 days,mice were placed into hypobaric hypoxia experiment chamber for 24 hours to establish lung injury animal model.The effect of mildronate pretreatment alleviating lung injury was evaluated by determining pathological changes of lung tissue,aquaporin protein expression,and blood gas analysis.（2）Establishment of hypoxia-induced acute lung injury in vitro at high altitude.Type Ⅱ alveolar epithelial cells（RLE-6TN）were divided into six group:control group,hypoxia group,mildronate group（10,20,and 40μM）and acetazolamide（10 μM）groups.Cells were pretreated with complete culture medium containing drugs in incubator for 24 hours and hypoxia incubator for another 24 hours to establish lung injury cell model.CCK-8 assay was used to determine the cell proliferation.Experiment 2:Mildronate regulates glycolysis and its potential target proteins（1）Measurement of glycolysis level.Lung injury model in vitro was established to investigate the effect of mildronate regulating glycolysis by determining glycolysis stress in Seahorse cell energy metabolism instrument and lactate acid content in cell supernatant.（2）Screening and validating pathways and targets of mildronate.HuProtTM 20K Human proteome microarrays analysis was used to investigate the potential pathway and target protein of mildronate,which rapid characterizing and quantificating the binding situation of biotin-labeled small molecule drug（mildronate）with～20,000 human full-length proteins.The genes transcription and the protein expression of glycolytic enzymes and pyruvate metabolic enzymes in vivo and in vitro,such as PFKP,PKM2,LDHA,PDK1 and PDH,were evaluated using RT-qPCR and Western Blot to investigate the regulation effect of mildronate under hypoxia condition.Molecular docking,immunofluorescence analysis,and pull-down assay were performed to confirm the target of mildronate.The regulation effect of mildronate to PFK enzyme activity under hypoxia condition was also evaluated.Experiment 3:Mildronate alleviates hypoxia-induced oxidative stress and mitochondrial damage at high altitudeThe regulation of mildronate in Nrf2 protein expression in vivo and in vitro was determined by western blot.MDA content and SOD activity in vivo were examined using assay kits.These experiments investigate mildronate alleviate hypoxia-induced oxidative stress.Mitochondrion stress in Seahorse cell energy metabolism instrument,the protein expression of mitochondrial fission and fusion relative proteins using western blot in vitro were evaluated to investigate mildronate ameliorates oxidative stress-induced mitochondrial damage and homeostasis imbalance.Results:Experiment 1:Mildronate prevents hypoxia-induced lung injury in high altitudeMildronate pretreatment for 3 days significantly alleviated hypoxia-induced lung injury and decreased lung injury score（P<0.001）.Pretreatment with mildronate significantly inhibited hypoxia-induced increase of AQP1 protein expression compared with the hypoxia group（P<0.01）.Mildronate significantly ameliorated metabolic acidosis in mice with lung injury（P<0.01）.Besides,the hypoxia-induced decrease of cell viability was reversed by pretreatment with mildronate（P<0.001）.In conclusion,pretreatment with mildronate has a significant protective effect on hypoxia-induced lung injury.Experiment 2:Mildronate regulates glycolysis and its potential target proteinsAfter hypoxia for 24 hours,the significantly increase of glycolysis level（P<0.01）induced by hypoxia in vitro was inhibited by mildronate pretreatment（P<0.05）.Human proteome microarrays found that the potential target proteins of mildronate were intracellular proteins with catalytic activity in metabolic pathways,which consist largely of proteins in glycolysis.The hypoxia-induced increase of mRNA and protein in PFKP,PKM2,LDHA and PDK1（P<0.05）were negatively regulated by mildronate（P<0.05）.Mildronate significantly increased the expression of PFKP,which suggested that PFKP is the main potential target protein of mildronate.Besides,by molecular docking,immunofluorescence co-localization and pull down experiments,mildronate could also interact with PFKP and regulated its activity（P<0.001）.Experiment 3:Mildronate alleviates hypoxia-induced oxidative stress and mitochondrial damage at high altitudeCompared with the hypoxia group,mildronate significantly increased Nrf2 protein expression in cytoplasm（P<0.01）and Nrf2 protein level in nucleus（P<0.05）,whcih indicated that mildronate promotes Nrf2 transfer from cytoplasm to nucleus.Mildronate significantly decreased MDA content and increased SOD activity in vivo compared with hypoxia group（P<0.05）.These results indicated that mildronate promoted the transfer of Nrf2 from cytoplasm to nucleus,alleviating oxidative stress-induced lung injury.Additionally,mildronate ameliorated mitochondrial function,such as the improvement of basal respiration,proton leak,and non-mitochondrial oxygen consumption（P<0.05）.Compared with control group,hypoxia in high altitude induced mitochondrial fission and fusion homeostasis imbalance,such as the significant increase of FIS 1 and OPA1 protein expression（P<0.05）,decrease of DRP1 and MFN2 protein expression（P<0.05）.But mildronate decreased FIS1 and OPA1 protein expression and increased DRP1 and MFN2 protein expression（P<0.05）.In conclusion,mildronate significantly alleviates mitochondrial function and homeostasis balance.Conclusion:（1）Mildronate alleviates the hypoxia-induced lung injury by targeting PFKP to regulate glycolysis.（2）Mildronate promotes Nrf2 translocation to the nucleus to mitigate hypoxia-induced oxidative stress and mitochondrial damage.（3）Mildronate is a potential preventive drug against hypoxia-induced lung injury.Innovation:（1）This study for the first time demonstrated that the preventive effect of mildronate on the hypoxia-induced lung injury at high altitude.（2）The mechanism of action is associated with the glycolysis-Nrf2 pathway mediating lung protective effects against hypoxia-induced oxidative stress and mitochondrial damage.（3）Besides,the PFKP protein is the main target of mildronate,and PFKP maybe a drug target of against hypoxia-induced lung injury at high altitude.