Study On The Role Of PI3K/Akt/HIF-1α Glycolysis Signaling Pathway In The Fibrotic Processes In Silico

Objectives Cellular experiments were performed to investigate the effects and roles of factors involved in the PI3K/Akt/HIF-1αglycolytic signaling pathway in cellular processes stimulated by MLE-12 by silica dust.Methods Mouse alveolar epithelial MLE-12 cells were cultured to establish an in vitro silicosis model.They were divided into five groups:control（MLE-12）,silica model（SiO₂）,silica PI3K inhibitor（SiO₂+LY294002）,silica PI3K promoter（SiO₂+RH-IGF-1）,and silica HIF-1αinhibitor（SiO₂+2-methoxyestradiol）.Cells and supernatants were collected respectively after 4h,8h,16h,24h of dust staining.Cell viability was assessed by CCK8assay in different groups.The content of hydroxyproline,an indicator of fibrosis,was detected by alkaline hydrolysis method.The viability of key enzymes of glycolysis,hexokinase and pyruvate kinase,as well as the content of lactic acid,an end product of glycolytic metabolism,in different groups were determined using biochemical kits.The contents of IL-6 in different groups were detected by ELISA.The protein expressions of PI3K,p-Akt,and HIF-1αin different groups were determined by Western blot.Results 1 With the extension of dust staining time,the cell survival rate of each group gradually decreased,and the cell survival rate of the silica intervention agent group was lower than that of the control group and the silica model group.2 Changes in hydroxyproline content:Compared with the control group,the hydroxyproline content in the SiO₂ model group all increased from the 8th to the 24th hour significantly.Compared with the SiO₂model group,at hours 8 to 24,the hydroxyproline content in the silica PI3K inhibitor group all decreased.The hydroxyproline content of the silica PI3K promoter group increased from the 4th to the 24th hour.At hours 8 to 24,the hydroxyproline content in the silica HIF-1αinhibitor group all decreased significantly.With the extension of time,the hydroxyproline content of the SiO₂ model group and the silica PI3K promoter group had a gradually increasing trend,and the magnitude of the change was not large at 16h and 24h.The hydroxyproline content of the silica PI3K inhibitor group had a tendency to gradually decrease and the magnitude of the change was not large at 16h and 24h.The remaining hydroxyproline content also did not change significantly at 16 and 24 hours.3 Changes in lactic acid content:Compared with the control group,at 16h and 24h,lactic acid increased in the SiO₂ model group.Compared with the SiO₂ model group,the amount of lactic acid in the silica PI3K inhibitor group was slightly reduced at the 4th hour.At hours 8 to 24,lactic acid was reduced in the silica PI3K inhibitor group,and the differences were all statistically significant（P<0.05）.Lactic acid increased in the silica PI3K enhancer group from the 4th to the 24th hour.At hours 8 to 24,lactic acid was reduced in all silica HIF-1αinhibitor groups significantly.With the extension of time,the lactic acid content in the SiO₂ model group increased gradually and tended to be stable at the 16th and 24th hours.And the lactic acid levels of the remaining groups changed little at 16 and 24 hours.4 Hexokinase viability changes:At 4～24 hours,compared with the control group,hexokinase activity in the SiO₂model group all increased.Compared with the SiO₂ model group,the hexokinase activity decreased in the silica PI3K inhibitor group,both hexokinase activities increased in the silica PI3K promoter group,and the hexokinase activity decreased in the silica HIF-1αinhibitor group,all differences being statistically significant（P<0.05）.Hexokinase activity in the SiO₂model group and the silica PI3K promoter group increased with time,whereas hexokinase activity in the silica HIF-1αinhibitor group had a tendency to decrease with time.The change in hexokinase activity over time in the silica PI3K inhibitor group was not consistent.5Pyruvate kinase activity changes:Compared with the control group,the pyruvate kinase activities of the SiO₂ model group all increased from the 8th to the 24th hours significantly.At the 4th～24th hours,compared with the SiO₂ model group,the viability of pyruvate kinase decreased in the silica PI3K inhibitor group,the viability of pyruvate kinase increased in the silica PI3K promoter group,and the viability of pyruvate kinase decreased in the silica HIF-1αinhibitor group,all differences were statistically significant（P<0.05）.With the extension of time,the viability of pyruvate kinase between the SiO₂ model group and the silica HIF-1αinhibitor group had a gradually increasing trend.6 IL-6 content changes:Compared with the control group,the contents of IL-6 in the SiO₂ model group all increased at the 4th～24th hours.Compared with the SiO₂ model group,the content of IL-6 in the silica PI3K inhibitor group all decreased at the 4th～24th hours.At 4～16 hours,the amount of IL-6 in the silica PI3K promoter group all increased,and the differences were statistically significant（P<0.05）.At 4～24hours,the IL-6 contents of the silica HIF-1αinhibitor group all decreased.The amount of IL-6 in the silica PI3K promoter group had a trend of gradually increasing with time.7 Western blot protein expression results:At 4～24 hours,compared with the control group,the protein expression of PI3K,p-Akt and HIF-1αin the SiO₂ model group all increased.Compared with the SiO₂ model group,the protein expressions of PI3K,p-Akt and HIF-1αwere decreased in the silica PI3K inhibitor group.The protein expression levels of PI3K,p-Akt,and HIF-1αwere increased in the silica PI3K promoter group.The protein expression of PI3K and p-Akt in silica HIF-1αinhibitor group changed little,while the protein expression of HIF-1αdecreased.Conclusions 1 After stimulation of MLE-12 cells by silico dust,it could promote the development of glycolysis,aggravate the level of inflammatory response and the degree of fibrosis like changes,and up regulate the expression levels of PI3K,p-Akt,and HIF-1α.2Administration of inhibitors of the PI3K/Akt/HIF-1αsignaling pathway was able to inhibit the progression of glycolysis and reduce the level of inflammation as well as the degree of fibrotic like changes.Administration of PI3K/Akt/HIF-1αsignaling pathway promoters was able to promote glycolysis progress and aggravate the level of inflammation and the extent of fibrosis like changes.3 Administration of PI3K/Akt/HIF-1αsignaling inhibitors or promoters failed to improve cell survival over time.4 In silico dust stimulated MLE-12 cells,modulating PI3K/Akt/HIF-1αsignaling related factors were able to influence the progression of glycolysis as well as fibrosis like changes.And thereby constitutes a positive feedback loop of hypoxia,enhanced glycolysis,and fibrogenesis,which contributes to progressive lung fibrosis.Figure 10;Table11;Reference 136...