脂多糖诱导小鼠肺成纤维细胞有氧酵解机制的生物信息学研究

A bioinformatics study on the mechanism of lipopolysaccharide -induced mouse lung fibroblast aerobic glycolysis

目的 明确脂多糖（lipopolysaccharide, LPS）对小鼠肺成纤维细胞有氧酵解的诱导作用并探讨其相关机制。 方法 将培养至4~7代的小鼠肺成纤维细胞接种于6孔培养板。采用随机数字表法将其分为4组（每组3孔）：空白对照组（Con组），0.5 mg/L LPS组（LPS0.5组），2.0 mg/L LPS组（LPS2.0组），4.0 mg/L LPS组（LPS4.0组）。除Con组外，分别加入以上终浓度的LPS，在加入LPS后24 h收集细胞培养上清液，采用ELISA法检测不同浓度LPS刺激小鼠肺成纤维细胞24 h后细胞培养上清液中有氧酵解代谢产物乳酸的表达水平；以基因芯片比较LPS诱导组与Con组小鼠肺成纤维细胞基因表达水平，筛选获得两组间差异表达基因，通过生物信息学分析方法将差异基因在KEGG数据库中进行统计分析，寻找与糖代谢相关显著富集的信号通路以明确LPS诱导小鼠肺成纤维细胞有氧酵解的相关机制。 结果 与Con组比较，不同浓度LPS刺激小鼠肺成纤维细胞均可引起细胞培养上清液中有氧酵解代谢产物乳酸含量显著增加（P〈0.05），并随LPS刺激浓度的升高而升高；基因芯片筛选出LPS诱导组与Con组差异表达的基因，KEGG通路富集分析获得4条与糖代谢密切相关的信号转导通路，分别为胰岛素信号通路、Janus激酶/信号转导子和转录激活子（janus kinase/signal transducers and activators of transcription, JAK？蛳STAT）信号通路、脂肪酸代谢通路以及鞘糖脂生物合成通路？蛳乳糖和新乳糖系列。 结论 LPS可促进肺成纤维细胞有氧酵解产物乳酸的形成，该过程与胰岛素信号通路、JAK？蛳STAT信号通路、脂肪酸代谢通路以及鞘糖脂生物合成通路？蛳乳糖和新乳糖系列活化有关，可能是脓毒症相关性肺纤维化发生的重要机制之一。

Objective 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 and were divided into 4 groups （n=3） according to random number table, PBS control group （group Con）, LPS 0.5 μg/mL （group LPS0.5）, LPS 2 μg/mL （group LPS2） and LPS 4 μg/mL （group LPS4）. PBS and the corresponding concentrations of LPS were added to the cells and then the fibroblasts were incubated for 24 h. ELISA was used to detect the level of lactic acid in the supernatant of lung fibroblasts after stimulation of LPS withTo 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 and were divided into 4 groups（n=3） according to random number table as follows： PBS control group（group Con）, LPS 0.5 mg/L（group LPS0.5）, LPS 2.0 mg/L（group LPS2.0） and LPS 4.0 mg/L（group LPS4.0）. PBS and the corresponding concentrations of LPS were added to cultured fibroblasts. After incubating for 24 h, ELISA was used to detect the level of lactic acid in the supernatant of lung fibroblasts after stimulation of LPS with different concentrations. Gene？蛳chip was used to detect differentially expressed genes of mouse lung fibroblasts between LPS group and PBS group. Also, KEGG pathway enrichment analysis was performed to explore glycometabolism related signaling pathways to clarify the mechanisms of LPS？蛳induced aerobic glycolysis of mouse lung fibroblasts. Results The expression of lactic acid in the supernatant in group LPS0.5 （P〈0.05）, group LPS2.0 （P〈0.05） and group LPS4.0（P〈0.05） was significantly higher than group Con. The tendency of increasing is corresponded to LPS concentration raising. DNA microarray data screened out differentially expressed genes between LPS group and group Con. Four enrichment pathways is closely related to glycometabolism obtained, including insulin signaling pathway, janus kinase/signal transducers and activators of transcription（JAK-STAT） signaling pathway, fatty acid metabolism, glycosphingolipid biosynthesis-lacto and neolacto series pathway. Conclusions 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 of the pathogenesis of sepsis-associated pulmonary fibrosis.