The Role Of PI3K/Akt Pathway In The Induction Of Macrophages RAW264.7 Apoptosis By Pseudomonas Aeruginosa Supernatant

ObjectiveTo detect the expression levels of the inositol-3-kinase(PI3K)/protein kinase B(Akt)signaling pathway in the apoptosis of mouse mononcyte-macrophag(RAW264.7)induced by supernatant of Pseudomonas aeruginosa(PA)and then to in vestigate the role and mechanism of PI3K/Akt pathway in the process of apoptosis.MethodsDetermined by MTT method is used to detect cell proliferation rate,get appropriate action time,PA that depending on the concentration of supernatant fluid was divided into control group complete medium(add same volume of complete medium),10%PA group,20%PA group,30%PA group⁽([1])for the PA concentration on clear liquid and fully develop volume+LY294002 group,control group,10%PA+LY294002 group,20%PA+LY294002,30%PA+LY294002 group,the cells in each group were treated for 6h,12h and 24h.LY294002 was a PI3K blocker.The concentration was 20 mol/L according to the instructions and the preliminary experimental results.According to MTT results,the PA supernatant treating for 12h was screened as the processing condition.After that,the experimental group was the same as the MTT experimental group.Hoechst 33342 staining was used to observe the nuclear morphological changes.Apoptosis rate was detected by flow cytometry.The expression of Akt and p-Akt protein was detected by Western blot.Finally,the data analysis was conducted by using SPSS23.0 software.Results1.The results of MTT showed that at 6h,12h and 24h,the result of the experiment results compared to control group,cell proliferation inhibition rate after adding the supernatant on PA were significantly increased(P<0.05).Compared with the groups with different concentrations of PA supernatant at the same time,the inhibition rate of cell proliferation increased with the increase of PA supernatant concentration(P<0.05).Compared with the group of 6h and the group of 12h at different concentrations of PA supernatant at 24h and the group of 12h at different concentrations of PA supernatant at 24h,the inhibition rate of cell proliferation increased with the extension of action time(P<0.05).The treatment time of 12h and 24h was slected for the subsequent experiments.After the addition of LY294002,the inhibition rate of cell proliferation in the 6h,12h and 24h control group was increased compared with that in the untreated group(P<0.05),indicating that the proliferation of RAW 264.7 cells could be inhibited by PA supernatant in a concentration-time dependent manner.2.The results of flow cytometry showed that the apoptosis rate of RAW264.7 increased after treatment with different concentrations of PA supernatant for 12h.The increase was statistically significant(P<0.05).The PA supernatant induced apoptosis in RAW264.7 cells in a concentration-dependent manner.LY29400 blocker can increase the apoptosis rate of macrophages.3.After staining with Hoechst 33342,compared with no PA infection group of cells,RAW 264.7 cells were treated with PA supernatant for 12h,the nuclei were changed,showing bright blue,pyknosis,fragmentation and apoptotic bodies were formed,.These nuclear morphological changes were all manifestation of apoptosis.With the increase of PA supernatant concentration,the changes of cell apoptosis were gradually obvious.It was suggested that the apoptosis of RAW264.7 cells was gradually increased with the increase of PA supernatant concentration.4.The results of Western blot showed that compared with the control group,the PA supernatant group could significantly reduce the expression of p-Akt in a concentration-dependent manner,and the difference was statistically significant(P<0.05).Meanwhile,there was no change in the expression level of Akt,and the difference was statistically significant(P>0.05).After the addition of blockers,p-Akt protein expression was further reduced in each group,and the difference was statistically significant(P<0.05).There was no change in the expression of Akt in each group after the addition of blocker,and the difference was statistically significant(P>0.05).Conclusions1.PA supernatant could induce the apoptosis of macrophage RAW264.7,and showed a concentration-dependent and time-dependent relationship.2.LY294002 inhibits the activity of PI3K/Akt,suggesting that the PI3K/Akt signaling pathway may be involved in the PA induced apoptosis of RAW264.7 cells.