Effects Of TLR4 On The Apoptosis Of RAW264.7 Macrophages Induced By Pseudomonas Aeruginosa Supernatant

ObjectiveThe aim of this paper is to explore the effects of TLR4 on the apoptosis of RAW264.7 macrophages induced by pseudomonas aeruginosa supernatent.It provides an important basis for the study of the mechanism between PA infection and macrophages,and provides a new idea for the development of clinical treatment for PA infection and the solution to the increasingly severe problem of multiple drug resistance to PA.MethodsFirstly,inhibition of cell proliferation was determined by MTT assay.The apporopriate concentration and culture time were screened after treatment with PA supernatant.RAW264.7 macrophages were dividied into con group,MOD group,con+TAK-242 group,MOD+TAK-242 group.The con group without any treatment.Cultured by medium which were distributed byfixed propotion.The MOD group were cultured by PA supernatant with 0.10,0.20,0.30concentration and medium with fixed propotion.The con+TAK-242 group and the MOD+TAK-242 group were respectively added 20?mol·L^-1TAK-242 into cells without any treatment and cells treated with 0.10,0.20,0.30 PA supernatant.the cells of each group were cultured for 4.5h,9h,18h.The MTT assay was used to detect the inhibition rate of cellproliferation in each group.When each group were cultured for 9h,hoechst staining was used to observe the nuclear mophological changes after macrophages apoptosis,flow cytometr analysis was used to test cell apoptosis,TLR4 were detected by Western blot.Results1.The result of Hoechst staining showed that a part of apoptosis appeared on con group.We observed that the nuclear chromatin was pyknosis,marginalization,cracking into different sizes and cracking into irregular crumbs.The crumbs were dense and deep staining.Not only granular and tatter massive fluorescence can be presented,but also apoptotic bodies can be showed.Compared with con group,the MOD group showed morphological changes too.The morphological changes of apoptosis were mostobviously when treated with0.30 concentration of PA supernatant.2.MTT assay showed that when the concentration of PA supernatantand the culture time were the same,after adding the TAK-242,the cell proliferation inhibition rate decreased and the difference between them was statistically significant(P<0.05).When the concentration of PA supernatant were the same,with the culture time(4.5h,9h,18h)increased proportionally,The cell proliferation inhibition rate increased gradually in different degreesand the difference between them was statistically significant(P<0.05).When the culture time were the same,with the concentration of PA supernatant(0.10,0.20,0.30)increased proportionally,The cell proliferation inhibitionrate increased gradually in different degrees and the difference between them was statistically significant(P<0.05).3.Flow cytometry suggested that compared with con group,MOD group showed different degrees of apoptosis(P<0.05),and with the concentration of PA supernatant increased gradually,the rate of apoptosis in MOD group was gradually increased(P<0.05).After the addition of TLR4 blocker,the rate of apoptosis was decreased when compared with con group(P<0.05)and MOD group(P<0.05).4.The result of western blot showed that,compared with con group,the expression of TLR4 protein was increased in MOD group(P<0.05).and with the concentration of PA supernatant increased gradually,the expression of TLR4protein was gradually increased(P<0.05).After the addition of TLR4 blocker,the expression level of TLR4 protein was decreased when compared with con group(P<0.05)and MOD group(P<0.05).conclusions1.PA supernatant can induce the apoptosis of macrophages,and has time and concentration dependence.2.PA supernatant can induce the expression of TLR4 and promote the apoptosis of macrophages.However,the addition of TLR4 blocker couldsignificantly inhibit the expression of TLR4 and the apoptosis of macrophages,suggesting that TLR4 could promote the apoptosis of RAW264.7macrophages induced by PA supernatant.