The Expression Of FoxO1 Transcription Factor In Macrophages And Its Relationship With Inflammation

Objectives:1.To observe the expression of Fox O1 transcription factor before and after macrophages activation.2.To observe the effect of Fox O1 transcription factor knockdown on macrophage inflammation.3.To determine whether FoxO1 through the TGF-β to affect the inflammatory response.Methods:1.Lipopolysaccharide(LPS)activated macrophages,enzyme-linked immunosorbent assay(ELISA)to measure the optimal concentration and time of LPS-activated macrophages.Collect the LPS at various concentrations and incubate the cell supernatant.ELISA kit was used to detect the levels of IL-6 and TNF-α in the supernatant at various concentrations and time intervals,and the optimal time and concentration of LPS-activated macrophages were introduced.The expression of Foxo1,Foxo3 a,Foxo4and Foxo6 macrophages were detected by agarose gel electrophoresis before and after activation.Real-time fluorescent quantitative PCR was used to detect the mRNA level of foxo1 gene.The expression of FOXO1 protein was detected by Western blot.2.The expression of foxo1 gene was knocked down by siRNA in LPS activation group.and determined by flow cytometry,real-time quantitative PCR and Western blot.IL-6 and TNF-α levels in the supernatants of the two groups were measured by ELISA kit.The changes of IL-6 and TNF-α contents in the activated macrophages before andafter transfection were compared.3.Macrophages knock down foxo1 expression in two groups.One group did not deal with another group of LPS-activated cells.Western blot was used to compare the expression of TGF-β protein in four groups of macrophages.Result:1.Lipopolysaccharide activated macrophages,the supernatant of IL-6 and TNF-αcontent gradually increased.The levels of inflammatory factors,IL-6 and TNF-α in the supernatant of LPS-stimulated LPS were higher than those of LPS at 50 ng/ml and 36 hours after incubation,indicating that macrophage activation The best condition.The expression of FoxO1 transcription factor in the activated group was lower than that in the untreated group,and the difference was statistically significant(p <0.05)by real-time fluorescence quantitative PCR and Western blot.2.The effect of Foxo1 si RNA transfection was determined by flow cytometry,real-time quantitative PCR and Western blot.The results showed that the expression level of Fox O1 transcription factor at mRNA level and protein level was 0.47 times before transfection,and the transfection was successful.The results of ELISA showed that the content of IL-6 in the activated group was 151.2 ng/ml±49.1 ng/ml,the content of TNF-α was 746.2 ng/ml±68.2 ng/ml,the content of IL-6 in the transfected group increased To 247.2 ng/ml±56 ng/ml,TNF-α content increased to 1282 ng/ml±26.7 ng/ml.The levels of IL-6 and TNF-α in the transfected group were significantly increased,and the difference was statistically significant(p <0.01).3.Normal macrophages were divided into two groups: one group was not treated and the other group knocked down Foxo1.Two groups of macrophages were then divided into two groups: one was not treated and the other was activated with LPS.The relative expression of TGF-βprotein in four groups was analyzed by Western blot.The results showed that TGF-β protein was expressed in activated group,activated group and siRNA group.The relative expression of TGF-βprotein in active group was 0.37,andthe relative expression level of activation group and si RNA group was 0.54.Indicating that macrophage knockdown after Foxo1 activation will cause increased TGF-βfactor,indicating that FoxO1 transcription factor may be through the TGF-βfactor affect the inflammatory response.Conclusions:1.Fox O1 transcription factor may be involved in the inflammatory process.2.Transcription factor Fox O1 may have a negative regulation of the role of inflammatory response.3.Fox O1 transcription factor may affect the inflammatory response through TGF-βfactor.