A Study On The Mechanism Of Lipopolysaccharide-induced Mouse Lung Fibroblast Aerobic Glycolysis

Part I A bioinformatics study on the mechanism of lipopolysaccharide-induced mouse lung fibroblast aerobic glycolysisObjective:To investigate the molecular mechanism of lipopolysaccharide(LPS)-induced aerobic glycolysis of lung fibroblasts in vitro to identify the pathogenesis of sepsis associated pulmonary fibrosis.Methods:Primary cultured mouse lung fibroblasts were seeded in 6-well plates with the density of 1×104/ml.After being cultured for 6 h,the cells were found adhere to the surface of culture dish.The complementary culture medium was replaced by serum-free medium before the cells were divided into 4 groups(n=3)according to random number table,PBS control group(group Con),LPS 0.5?g/ml(group LPS0.6),LPS 2?g/ml(group LPS2)and LPS 4?g/ml(group LPS4).PBS and the corresponding concentrations of LPS were added to the cells and then the fibroblasts were incubated for 24 hours.ELISA was used to detect the level of lactic acid in the cell culture supernatant of lung fibroblasts after stimulation of LPS with different concentrations.Gene chip was used to detect differentially expressed genes of mouse lung fibroblasts between LPS group and PBS group,then KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway enrichment analysis was performed to explore glycometabolism related signaling pathways to clarify the mechanisms of LPS-induced aerobic glycolysis of mouse lung fibroblasts.Results:The expression of lactic acid in the supernatant was significantly higher in group LPS0.5(P<0.01)?group LPS2(P<0.05)and group LPS4(P<0.005)compared with group Con,and showed the tendency of increasing with LPS concentration raised?DNA microarray data screened out differentially expressed genes between LPS group and Con group,and 4 enrichment pathways closely related to glycometabolism were obtained,including insulin signaling pathway,JAK-STAT signaling pathway,fatty acid metabolism and Glycosphingolipid biosynthesis-lacto and neolacto series pathway.Conclusion:LPS could initiate the process of aerobic glycolysis and promote the production of lactic acid in lung fibroblasts,which is associated with insulin signaling pathway,JAK-STAT signaling pathway,fatty acid metabolism and Glycosphingolipid biosynthesis-lacto and neolacto series pathway.It may be one of the internal mechanisms for the pathogenesis of sepsis-associated pulmonary fibrosis.Part ?A study on the mechanism of MAPK10-mediated lipopolysaccharide-induced aerobic glycolysis in mouse lung fibroblastsObjective:To observe the expression of MAPK10 after LPS stimulation on mouse lung fibroblasts,and clarify the role of JNK3 protein encoded by MAPK10 gene in the process of aerobic glycolysis induced by LPS.Methods:Primary cultured mouse lung fibroblasts were seeded in 6-well plates with the density of 1×104/ml.After being cultured for 6 h,the cells were found adhere to the surface of culture dish.The complementary culture medium was replaced by serum-free medium before the cells were divided into 4 groups(n=3)according to random number table-PBS control group(group Con),LPS 0.25?g/ml(group LPS0.25),LPS 0.5p,g/ml(group LPS0.5)and LPS 1?g/ml(group LPS1).PBS and the corresponding concentrations of LPS were added to the cells and then the fibroblasts were incubated for 6,12,24 hours respectively,qPCR was used to detect mRNA expression level of MAPK10 gene.The JNK inhibitor SP600125 of different concentrations(1?m,5?m,lO?m)were used to pre-treat mouse lung fibroblasts,then effect of different concentrations of SP600125 on the expression levels of p-JNK?JNK protein was detected by Western blot,and the level of lactic acid in the cell culture supernatant was detected by ELISA after LPS stimulation for 24 hours.Cells were divided into 4 groups according to random number table—Negative control group,LPS Group,siRNA Group and LPS+siRNA Group,then effect of MAPK10 siRNA transfection on the expression level of p-JNK,JNK protein was detected by Western blot,and the level of lactic acid in the cell culture supernatant was detected by ELISA after LPS stimulation for 24 hours.Results:Compared with Control group,LPS stimulation on lung fibroblasts for 6 hours,12 hours and 24 hours induced a significant increase in MAPK10 mRNA expression,and the difference was statistically significant.(6 hour,group LPS0.25:P<0.01,group LPS0.5:P<0.05,group LPS1:P<0.01;12 hour,group LPS0.25:P<0.05,group LPS0.5:P<0.01,group LPS1:P<0.05;24 hour,group LPS0.25:P<0.01,group LPS0.5:P<0.01,group LPS1:P<0.01).Western Blot showed that LPS-induced JNK phosphorylation of mouse lung fibroblasts could be suppressed by different concentrations of JNK inhibitor SP600125 and MAPK10 siRNA transfection,and the p-JNK/JNK ratio decreased significantly(all P<0.05).ELISA showed that different concentrations of JNK inhibitor SP600125 and MAPK10 siRNA transfection could inhibit the level of lactic acid in cell culture supernatant induced by LPS(all P<0.05).Conclusion:LPS induced-aerobic glycolysis of lung fibroblasts could be mediated by MAPK10 upregulation in the insulin signaling pathway,and downregulating MAPK10 expression or suppressing JNK3 protein inhibits LPS-induced lactic acid production.It may be one of the internal mechanisms for the pathogenesis of sepsis-associated pulmonary fibrosis.