Effect Of Glycolytic Pathway Regulated By Oxygen Concentration On Fibrosis-like Changes In MLE-12 Cells

Objectives MLE-12 cells were used to establish a lung epithelial cell model infected with silicon dioxide（Si O₂）and bleomycin（BLM）in vitro,and the glycolysis rate was regulated by changing the oxygen concentration to explore the relationship between cytokine inflammation and fibrosis factors and glycolysis.Methods MLE-12 cells cultured in vitro were stimulated with 50μg/L pure Si O₂（1～5μm）dust and 50μg/L BLM,respectively.The three-gas incubator was used to regulate the oxygen concentration.The MLE-12 cells were subjected to hypoxia（2%O₂）and hyperoxia（30%O₂）intervention.They were divided into the control group,Si O₂ model group,Si O₂ hyperoxia group（30%O₂）,Si O₂ hypoxia group（2%O₂）,BLM model group,BLM hyperoxia group（30%O₂）and BLM hypoxia group（2%O₂）.Morphological changes of MLE-12 cells in each treatment group were observed,and the cell viability and inhibition rate were measured by CCK-8 method.The colorimetric method was used to determine the glucose consumption and lactic acid content of the cell culture medium supernatant.The content of hydroxyproline（HYP）was determined by alkaline hydrolysis The contents of interleukin-6（IL-6）,tumor necrosis factor-α（TNF-α）and transforming growth factor-β1（TGF-β1）in the supernatant were determined by ELISA.The protein expression levels of pyruvate kinase（PKM2）,phosphofructokinase（PFK）,hexokinase（HK1）,lactate dehydrogenase（LDHA）and hypoxia-inducible factor 1α（HIF-1α）in the cell lysates were determined by Western-blot.Results 1 The CCK-8 method was used to test the cell viability and cell inhibition rate.Compared with the control group,the cell viability was decreased and the inhibition rate was increased in each model group（P<0.05）.Under hyperoxia condition,the cell viability of each group was higher than the corresponding model group,and the inhibition rate was decreased（P<0.05）.The cell viability was decreased and the inhibition rate was increased under hypoxia condition compared with the corresponding model group（P<0.05）.2 The results of glucose consumption and lactic acid content showed that cell glucose consumption and lactic acid content in each model group were higher than the control group（P<0.05）.Glucose consumption and lactic acid content in each group were lower than the corresponding model group under hyperoxia condition（P<0.05）.Under hypoxic condition,glucose consumption and lactic acid content in each group were higher than the corresponding model group（P<0.05）.3 The results of glycolysis-related protein detection showed that the expression levels of glycolysis-related proteins LDHA,PKM2,PFK,HK,and HIF-1αin the Si O₂ model group were higher than the control group（P<0.05）.The expressions of glycolysis-related proteins LDHA,PKM2,HK,and HIF-1αin the BLM model group were higher than the control group（P<0.05）,and the expression of PFK was about the same as the control group（P>0.05）.The expressions of glycolysis-related proteins LDHA,PKM2,PFK,HK,and HIF-1αin the Si O₂ hyperoxia group were lower than the model group（P<0.05）.The expression levels of glycolysis-related proteins in the Si O₂ hypoxia group were further higher than the model group（P<0.05）.The expression levels of glycolysis-related proteins LDHA,PKM2 and PFK in the BLM hyperoxia group were higher than the model group（P<0.05）,while the expression levels of HK and HIF-1αwere decreased（P<0.05）.The expressions of glycolysis-related proteins LDHA,PKM2,PFK,HK,and HIF-1αin the BLM hypoxia group were higher than those in the model group（P<0.05）.4 The results of inflammation,fibrosis and transforming growth factor tests showed that the expressions of IL-6,TNF-α,TGF-β1 and HYP in each model group were higher than the control group（P<0.05）.The expressions of IL-6,TNF-α,TGF-β1 and HYP in the Si O₂ hyperoxia group and BLM hyperoxia group were lower than the corresponding model group（P<0.05）.The expressions of IL-6,TNF-α,TGF-β1 and HYP in the Si O₂ hypoxia group and BLM hypoxia group were further increased than the corresponding model group（P<0.05）.Conclusions 1 The stimulation of Si O₂ increased the glycolysis rate and the expression of glycolytic enzyme.It caused the extracellular lactic acid accumulation and led to the cellular inflammatory reaction.The secretion of fibrotic factor and transforming growth factor increased.Increasing the oxygen concentration reduced the glycolysis rate,reduced the expression of key glycolytic enzymes,reduced lactic acid accumulation,and reduced the inflammatory reaction,the secretion of fibrotic factor and transforming growth factor.Reducing the oxygen concentration caused the intracellular hypoxia,further enhanced the glycolysis rate and increased the expression of key glycolytic enzymes.It aggravated the extracellular lactic acid accumulation and the cellular inflammatory reaction.The secretion of fibrotic factor and transforming growth factor increased.2 The BLM stimulation improved the cell glycolysis rate,and resulted in the extracellular lactic acid accumulation,but the expression of key glycolytic enzymes was not significantly increased.It led to the cellular inflammatory reactions.The secretion of fibrotic factor and transforming growth factor also increased.Increasing the oxygen concentration could slow the glycolysis rate and increase the expression of key glycolytic enzymes.It also reduced lactic acid accumulation,the inflammatory reaction,and the secretion of fibrotic factor and transforming growth factor.Reducing the oxygen concentration could cause the intracellular hypoxia,significantly increase the glycolysis rate,increase the expression of key enzymes of glycolysis,and aggravate the extracellular lactic acid accumulation and the cellular inflammatory reaction.In addition,the secretion of fibrotic factor and transforming growth factor increased.3 To sum up,increasing the oxygen concentration could reduce the cell glycolysis rate and increase the aerobic respiration for both Si O₂ and BLM stimulated MLE-12 cells,but the effect was more obvious for Si O₂ stimulated cells.Figure 18;Table 7;Reference 113...