Expression And Mechanism Of KLF6 And INOS During Apoptosis Of Macrophages Induced By Pseudomonas Aeruginosa Supernatant

ObjectiveTo detect the expression levels of krupple-like factor 6(KLF6),inducible nitric oxide synthase(iNOS)and the content of one nitric oxide(NO)in the apoptosis of mouse monocyte-macrophag(RAW264.7)induced by supernatant of Pseudomonas aeruginosa(PA)and then to in vestigate the role and mechanism of KLF6 and iNOS in the process of apoptosis.MethodsThe MTT assay was used to detect the cell proliferation rate.Considering the different volume concentrations(the volume ratio of PA supernatant to complete medium)of PA supernatant,the experiment groups were divided into control group(add same volume of complete medium),15% PA group,30% PA group,45% PA group,the cells in each group were treated for 12 h and 24 h respectively.In addition,SMT(+)group(SMT was the iNOS blocker)and SMT(-)group(without SMT)were designed for the experiment,the concentration was selected as 6?M according to the manual and preliminary results.According to MTT results,30% PA supernatant treating for 24 h was screened as the processing condition.Flow cytometry was used to detect the apoptosis rate.Hoechst 33342 staining was used to observe the nuclear morphological changes.Western blot was used to detect the expression of KLF6 and iNOS protein.The expression levels of KLF6 mRNA and iNOS mRNA were detected by real-time PCR.The NO content detection kit was used to detect the content of NO.Finally,the data analysis was conducted by using SPSS16.0 software.Results1.The results of MTT showed that the proliferation rate of RAW264.7 increased with the increase of PA supernatant concentration,and increased with the prolongation of action time.The treatment time of 12 h and 24 h was slected for the subsequent experiments.After 12 h and 24 h treatment,the cell proliferation rate by the same PA supernatant was increased with the prolongation of the action time,and the difference was statistically significant(P<0.05).When the PA supernatant at different concentrations treated the cells for the same time interval,the proliferation rate increased in a concentration dependant manner,and the difference was statistically significant(P<0.05),indicating that the PA supernatant inhibited the proliferation of RAW264.7 cells in a time-concentration-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 24 h.The increase was statistically significant(P<0.05).The PA supernatant induced apoptosis in RAW264.7 cells in a concentration-dependent manner.3.After staining with Hoechst 33342,the nucleus of RAW264.7 in the control group was blue or oval,and the chromosomes were uniform.After treatment with different concentrations of PA supernatant for 24 h,the nucleus of RAW264.7 cells was pyknosis,deep stained,dense and dense blocky particles were observed in the nucleus.These nuclear morphological changes were all manifestations of apoptosis,which was most obviously in the 45% PA supernatant treatment group,suggesting that PA supernatant induced RAW264.7 cells apoptosis was concentration-dependent manner.4.The results of Western blot showed that the expression of KLF6 and iNOS protein in RAW264.7 cells were significantly higher than those in the control group(P<0.05).After treatment with 30% PA supernatant for 12 h and 24 h,the expression levels of KLF6 and iNOS in RAW264.7 cells increased significantly,and the difference was statistically significant(P<0.05).5.The results of real-time PCR showed that the expression levels of KLF6 mRNA and iNOS mRNA in different concentrations of PA supernatant were higher than those in the control group at 24 h,and the difference was statistically significant(P<0.05).The expression of KLF6 mRNA and iNOS mRNA in the 30% PA supernatant was higher than that of the control group at 12 h and 24 h,and the difference was statistically significant(P<0.05).6.The results of NO content detection showed that the NO content in the RAW264.7 cells infected with different concentrations of PA supernatant increased in a concentration-dependent manner,the difference was statistically significant(P<0.05).After RAW264.7 cells were infected with 30% PA supernatant for 12 h and 24 h,the NO content increased in a time-dependent manner,and the difference was statistically significant(P<0.05).7.The cells in the SMT(+)group were treated with the same PA supernatant,and the expression level of iNOS protein was decreased compared with the SMT(-)group(P<0.01).The difference was statistically significant.Meanwhile,the NO content in the cell supernatant treated with PA supernatant was detected.Compared with the SMT(-)group,the NO content decreased(P<0.05),and the difference was statistically significant.Apoptosis rate after SMT intervention was detected by flow cytometry.After treatment with PA supernatant,the apoptosis rate was significantly decreased compared with SMT(-)group(P<0.01),and the difference was statistically significant.Conclusions1.PA supernatant can inhibit the proliferation of RAW264.7 cells,and its inhibitory effect is concentration-time dependent.2.The mechanism by which PA induces apoptosis of RAW264.7 cells may be caused by the production of excess NO by KLF6 and iNOS.3.The reduction of iNOS expression has a protective effect on the cytotoxicity caused by PA supernatant infectied RAW264.7 cells.