The Study On The Activation Of PI3K AKT Bcl-2 Bax Pathway Induced By Respiratory Tract Infection Of Chlamydia Trachomatis

BackgroundCD4+T cell belongs to the T cell subset class involved in immune responses.CD4+Th1 and CD4+Th17 cell play crucial role in immunological protection against Cm respiratory tract infection.Activated Th1 and Th17 cell play roles in immune regulation by secreting IFN-?and IL-17when stimulated by antigen.It has reported that PI3K/AKT signaling pathway could be activated and regulated by key stockiness in some pathogenic microorganism-induced immune responses,which results in apoptosis of host cells.The current study was aimed to explore Chlamydia muridarum(Cm)induced apoptosis of CD4~+T cell and the variation of protein level which is related with PI3K/AKT pathway.It was benefit to clarify the potential immune mechanism of Cm infection,expecting providing theoretical foundations for treatments of clinical Cm-related disorders and developments of vaccines.Materials and Methods6-8-week-old female C57BL/6 mice were administrated with 1×10~3inclusion forming units(IFU)Cm via nasal to establish mouse model of Cm respiratory tract infection model and induced mouse chlamydia pneumonia.Routine detected mouse weight and physical activity daily.Mice were sacrificed at different infection point and lung tissues were infused with perfusion to prepare mononuclear cells.Total RNA of lung tissue was extracted by use of Trizol kit.Expression of PI3K P110?at the mRNA level was detected by RT-PCR.Total protein of mononuclear cells of mice lung tissue was extracted with RIPA buffer to confirm the change of p-AKT and the ratio of Bcl2 versus Bax in different time points,we detected the expression of p-AKT,Bcl-2 and Bax at the protein level by Western Blot.Mononuclear cells of mice lung tissuewas stained with PECY7 anti-mouse CD3,APC anti-mouseCD4for surface-molecular staining,combined with Annexin?PI flow cytometryto further investigate the changes of CD4~+T cells apoptosis in the process of Cm respiratory tract infection over time.ResultsC57BL/6 mice were administrated with Cm via nasal to induce Cm pneumonia.Firstly,we investigate the role of the key factor of PI3K catalytic subunit p110?.Cm respiratory tract infection induced highly expression of PI3K catalytic subunit p110?in lung tissues.Compared with non-infected group,the mRNA level of p110?was makedly upregulated on day 3,reached to the highest level on day 7 and and decreased on day 14.Moreover,the expression of p-AKT and the ratio of Bcl-2/Bax were tested at the protein levels.Western Blot showed that the expression of p-AKT,a downstream protein of PI3K,was evidently increased in Cm infection mice lung tissues than those in non-infected group on day 3,reached to the highest level on day7 andsimilarly,decreased on day 14.The variation of p-AKT was in accordance with the mRNA level of p110?.In addition,p-AKT protein level was largely reduced by PI3K inhibitor LY294002 at different infection time points.Besides,our study demonstrated that anti-protein Bcl-2 and pro-protein Bax,downstream proteins of PI3K/AKT,played a vital role in regulating the process of CD4~+T cell apoptosis.The expression of Bcl-2 increased at the protein level on day 3(p<0.001),reached to the highest level on day 7(p<0.001)and and decreased on day 14(p<0.01).Similarly,the expression of Bax was in consistent with the changes of Bcl-2.However,the ratio of Bcl-2/Bax was decreased with infection time,which suggested that the anti-apoptosis effect gradually decreased in Cm infection mice lung tissues.And the flow cytometry detection revealed the same results.ConclusionAt the early stage of Cm respiratory infection induced p110?high expression and activation of PI3K/AKT pathway.Simultaneously,the variation tendency of p-AKT at the protein level was in line with the expression of PI3Kp110?at the mRNA level.Our study suggested that AKT involved in Cm respiratory infection induced CD4~+T cell apoptosis PI3K-dependent or PI3K-independent manner.Anti-protein Bcl-2 and pro-protein Bax were downstream factors of PI3K/AKT,which played an essential role in regulating the process of CD4~+T cell apoptosis and survival.We further found that Cm induced the expression of Bcl-2 and Bax,and reduction of the ratio of Bcl-2/Bax over time.All data demonstrated that Cm induced the activation of PI3K/AKT,the reduction of the anti-apoptosis effects and elevation of the apoptosis,which resulted in the apoptosis of CD4~+T cell in infection area of mice lung tissues.Finally,we concluded that Cm-induced the activation of PI3K/AKT/Bcl-2/Bax,which promoted the apoptosis of CD4~+T cell.