The Effect And Mechanism Of TNFα On Glycolysis In A Endothelial Cell Line

BackgroundEndothelial cells could form new blood vessels in tumor microenvironment,in order to supply more oxygen and nutrients to tumor.Tumor angiogenesis is largely regulated by angiogenic factors and angiogenesis inhibitors.The main way of tumor angiogenesis is sprouting angiogenesis.Recently,many papers have reported that endothelial cell metabolism also plays an important role in vessel sprouting and angiogenesis.During vascular sprouting,metabolic changes could drive endothelial cell into different phenotypes,and different phenotypes could perform different functions because of different metabolisms.Tip cells use glycolysis to provide energy for migration,whereas stalk cells use glycolysis to generate energy and divert glycolytic intermediates to other pathways for biomass synthesis in order to proliferate.Therefore,endothelial cell glycolysis is closely related to endothelial cell sprouting.There are many inflammatory factors in tumor microenvironment,and these inflammatory factors such as IFNγ and TNFα could regulate endothelial cell function,but whether they can regulate endothelial cell metabolism has not been reported.Tumor necrosis factor-α(TNFα),an important inflammatory factor in the tumor microenvironment,plays an important role in anti-tumor process.It has been reported that TNFα could inhibit endothelial cells proliferation and inhibit angiogenesis,then achieve a purpose of inhibiting tumors,but the mechanism is not clear.Whether tumor necrosis factor-α inhibits angiogenesis to treat tumor by downregulating endothelial cell glycolysis is not clear.So in this paper,we selected an endothelial cell line sEND.1 as our research object,and explored the effect of TNFα on endothelial cell glycolysis and its molecular mechanism for the first time.We found that TNFα downregulated the expression of Glucose transporter 1(Glut1)and hexokinase 2(HK2)by activating NFκB signal pathway,and thus inhibitting the glycolysis in sEND.1 cell.ObjectiveTo investigate the effect and mechanism of tumor necrosis factor-α(TNFα)on glycolysis in a endothelial cell line.Methods1.Different concentrations of 0,1,10,50,100ng/ml of TNFα stimulate a mouse vascular endothelial cell line(sEND.1 cell line),then detect the extracellular acid rate(ECAR)by seahorse XF96;2.Flow cytometry was used to detect the amount of 2-NBDG uptake after treated with 10ng/ml TNFα to reflect the glucose uptake;3.Microplate reader was used to measure the absorbance after treated with 10ng/ml TNFα to reflect the production of lactate;4.The mRNA expression of Glut1,HK2,PFKFB2 was detected after treated with 10ng/ml TNFα by quantitative reverse transcription polymerase chain reaction(qRT-PCR);5.The expression of Glut1,HK2,PFKFB2 was detected after treated with 10ng/ml TNFα by western blotting(WB);6.The expression of p-NFκB(p-P65),NFκB(P65),JNK2 and other proteins was detected after treated with 10ng/ml TNFα by western blotting(WB);7.After inhibiting the NFκB signaling pathway,the extracellular acid rate(ECAR)was detected by seahorse XF96.The mRNA and protein level of Glut1,HK2,PFKFB2 were detected by reverse transcription polymerase chain reaction(qRT-PCR)and western blotting(WB);8.The aortic ring sprouting assay ex vivo and tube formation assay in vitro were used to detect the angiogenesis after stimulated by TNFα with or without lactate.Results1.Seahorse XF96 energy metabolism results: After treated with different concentrations of TNFα on sEND.1 cells for 6h,the ECAR of the experimental group 1ng/ml,10ng/ml,50ng/ml,100ng/ml were downregulated.After statistical analysis,the data of each experimental group were statistically significant compared with the control group(P<0.05).2.2-NBDG uptake assay: After treated with 10 ng/ml TNFα on sEND.1 cells for 6 h,glucose uptake was downregulated.The difference between the experimental group and the control group was statistically significant(P<0.05).3.Production of lactate: After treated with 10 ng/ml TNFα on sEND.1 cells for 6 h,the absorbance was significantly decreased,indicating that the lactate production were decreased.After statistical analysis,the difference between experimental group and control group was statistically significant(P <0.05).4.QRT-PCR showed that: After 10 ng/ml TNFα was stimulated to sEND.1 cells for 6 h,the gene expressions of Glut1,HK2 and PFKFB2 were decreased.After statistical analysis,the difference between experimental group and control group was statistically significant(P <0.05).5.Western Blot showed that: After 10 ng/ml TNFα was stimulated to sEND.1 cells for 6 h,the protein expressions of Glut1,HK2 and PFKFB2 were decreased.6.Western Blot showed that: 10 ng/ml TNFα was stimulated to sEND.1 cells for different times,p-NFκB(p-P65)was activated and began from 10 minutes of stimulation.Other proteins such as JNK2 and AKT signal pathway did not change.7.After inhibition of NFκB signaling pathway on sEND.1 cells by SC75741,ECAR was reversed.PCR and WB results showed that after NFκB(P65)was inhibited,the expression of Glut1 and HK2 in both mRNA and protein level were reversed,but the expression of PFKFB2 was not.The experimental group data were showed statistical significance compared with the control group(P <0.05).8.The aortic ring sprouting assay ex vivo and tube formation assay in vitro showed that TNFα without lactate could inhibit the aortic ring sprouting and tube formation,and TNFα with lactate could return to the normal aortic ring sprouting and tube formation level.Conclusion1.TNFα downregulates the glycolysis in a vascular endothelial cell line.2.NFκB conduct the TNFα-inducecd the downregulation of glycolysis.3.Inhibition of TNFα on vessel sprouting may be due to the inhibition of glycolysis.