Studies On ATP Energy Sensing Detection And Metabolic Mechanism Of E.coli Lipopolysaccharide On Hela Cells

Most of the foodborne pathogens are Gram-negative bacteria.Lipopolysaccharide(LPS),as the main virulence factor of the cell wall of Gram-negative bacteria,can cause the body to produce strong inflammation.The occurrence of inflammation can cause abnormal energy metabolism.Adenosine triphosphate(ATP),as the universal energy currency in the body,its change in concentration can reflect change in energy metabolism.In this study,Au nanocrosses(AuNCs)-graphene quantum dots(GQDs)nanoassembly(AuNCs-GQDs)was constructed for determining the effect of Escherichia coli(E.coli)LPS on intracellular ATP concentration of HeLa cells.Furthermore,the effects of E.coli LPS on energy metabolism related physiological parameters in HeLa cells were studied.And cell metabonomics were applied to explore the effects of E.coli LPS on energy metabolism related pathways and to study the mechanism of E.coli LPS on energy metabolism in HeLa cells.AuNCs-GQDs nanoassembly was constructed for visually studying the effect of E.coli LPS on intracellular ATP concentration of HeLa cells.AuNCs were synthesized by seed growth method and surface modified.GQDs modified with ATP aptamer and AuNCs modified with complementary DNA were assembled by base complementary pairing.The fluorescence of GQDs was quenched by AuNCs due to the nano-surface energy transfer effect of AuNCs.In the presence of ATP,the ATP aptamer on the surface of GQDs bound to ATP to separate from AuNCs and restored the fluorescence of GQDs.With the increase of ATP concentration,the fluorescence intensity increased,the linear range was 0.3 mmol/L~2.0mmol/L,the standard curve was y=176.67x+335.51(R~2=0.9916),and the detection limit was0.27 mmol/L(3d,n=11).In addition,the sensor showed excellent specificity,low cytotoxicity and good response to intracellular ATP.After stimulating HeLa cells with E.coli LPS,the intracellular ATP concentration was increased,but the increased ATP came from cytoplasm instead of mitochondria according to AuNCs-GQDs.In order to study the effects of E.coli LPS stimulation on energy metabolism related physiological indicators of HeLa cells,tested related inflammatory factors,including tumor necrosis factorα(TNF-α)and interleukin 6(IL-6),verified that E.coli LPS could indeed induce inflammation in HeLa cells.In addition,found that E.coli LPS could increase ATP content,NO synthase activity and NO production in HeLa cells.Meanwhile,E.coli LPS increased reactive oxygen species and changed mitochondrial membrane potential,thus altered mitochondrial function.Furthermore,the activities of enzymes involved in glycolysis and TCA cycle in HeLa cells were measured.The activities of glycolysis-related enzymes(pyruvate kinase,hexokinase and lactate dehydrogenase)increased after E.coli LPS stimulation,while that of TCA cycle-related enzymes(succinate dehydrogenase,malic dehydrogenase and isocitrate dehydrogenase)decreased slightly.Therefore,it was presumed that E.coli LPS could increase ATP level by enhancing the glycolysis pathway of HeLa cells.The underlying mechanism of ATP changes in HeLa cells induced by E.coli LPS was studied by cell metabonomics.Through multivariate statistical analysis(PCA,PLSDA)and cluster thermogram analysis,found that there was a big difference in energy metabolism between E.coli LPS stimulation group and control group.Further analysis of their metabolites revealed that after E.coli stimulation,the metabolites involved in glycolysis and pentose phosphate pathway,including glucose-6-phosphate,fructose-6-phosphate,pyruvic acid,lactic acid,succinic acid,cis-aconitic acid,ribulose 5-phosphate,ribose 5-phosphate and erythritose4-phosphate,changed correspondingly after E.coli LPS stimulation.These changes suggested that glycolysis pathway was enhanced while TCA cycle was slightly decreased.In addition,some amino acids which provide carbon skeleton for glycolysis and TCA cycle,including serine,methionine,leucine,aspartic acid and asparagine also changed significantly,indicating changes in energy metabolism.Hence,E.coli LPS influenced the energy metabolism mechanism of HeLa cells by enhancing their"Warburg"effect.