GLUT1 Regulates The Release Of VEGF-A In The Alveolar Epithelium Of Lipopolysaccharide-induced Acute Lung Injury

Background:Featuring pulmonary edema and progressive hypoxemia,acute lung injury(ALI)is an early stage of acute respiratory distress syndrome(ARDS),with high morbidity and mortality.ALI is characterized by uncontrolled inflammatory cascades,which leads to dysfunction of endothelial and epithelial barriers,increase of vascular and alveolar permeability,and finally edema of lung interstitial and alveoli.Vascular endothelial growth factor A(VEGF-A),as a highly specific growth factor for vascular endothelium,plays a role in wound healing and vascular permeability.Recent studies have shown that VEGF-A,as a pre-inflammatory factor,is involved in mediating the inflammatory reaction process of ALI in the occurrence and development of ALI,especially in the acute phase of ALI.On the other hand,Studies have shown that glucose transporter protein 1(GLUT1),a widely distributed glucose transporter in many tissues and organs,exerts pro-inflammatory effects in varieties of inflammatory diseases.It remains unclear whether GLUT1 is involved in the production of VEGF-A in ALI alveolar epithelium,and whatever mechanism is to be clarified.Objective:This study is to evaluate the possible and mechanism of GLUT1 in alveolar epithelial VEGF-A production in lipopolysaccharide(LPS)-induced ALI.Methods:BALB/c mice were randomly divided into three groups:LPS group,GLUTl inhibitor(WZB117)+LPS group and PBS group.ALI model was built by intratracheal LPS injection.Mice in WZB117+LPS group were intraperitoneally injected with GLUT1 inhibitor WZB117(30 mg/kg)at 1 h,24 h and 48 h after induction of ALI state,respectively.Mice in PBS group were injected with the same amount of PBS as LPS intratracheally without any other treatment.After 72 hours,the mice were sacrificed to collect serum,lung tissue and bronchoalveolar lavage fluid(BALF).HE staining was used to observe the pathological changes of lung tissue.The expression of GLUT1 and VEGF-A in lung tissues were detected by immunohistochemistry(IHC)and western blot.ELISA was exploited to determine the contents of TNF-α,IL-6,IL1β and VEGF-A in BALF.To further verify the results from in vivo experiments in vitro,we stimulated A549 cells with 100 ng/mL LPS to construct ALI cell model.WZB117 or BAY876 were given to inhibit GLUT1,respectively.The protein expression of GLUT1,VEGF-A,pAkt,Akt and HIF-1α were detected by western blot.The mRNA expression of VEGFA was measured by qPCR.The secretion of VEGF-A in the supernatant was detected by ELISA.Results:After LPS stimulation,the expression of GLUT1 and VEGF-A were upregulated in lung tissue.While after treatment of WZB117,the expression of GLUT1 and VEGFA,the inflammatory cells infiltration(predominantly neutrophils),the contents of inflammatory factors(IL-6,IL-1β,and TNF-α)in BALF were down-regulated.In the A549 cell model,the expression of VEGF-A,p-Akt and HIF-la were increased after LPS stimulation.The treatment of BAY876 not only significantly reduced the expression of GLUT1 and VEGF-A,but also inhibited the expression of p-Akt and HIF-1α,while WZB 117 did not exhibit a similar effect.Conclusion:GLUT1 is involved in up-regulating LPS-induced release of VEGF-A from the alveolar epithelium in acute lung injury.Mechanism studies suggest that GLUT1 upregulates VEGF-A during ALI by phosphorylating Akt(Ser473)and up-regulating HIF-1α downstream.