Mycoplasma Genitalium Protein Of Adhesion Inhibit Urethral Epithelial Cells Apoptosis Via Cyclophilin A-CD147 Activating The PI3K/Akt/NF-κB Pathway

Background: Mycoplasma genitalium is a prokaryotic microbe that is mainly transmitted through sexual contact and causes urogenital infections.M.genitalium can adhere to and even invade cells by interacting with receptor proteins on the surface of the host cell membrane.M.genitalium protein of adhesion(MgPa)plays a pivotal role in the process of M.genitalium adhesion to host cells.The recognition of ligands on pathogenic microorganisms by corresponding receptors on host cells is a prerequisite for successful establishment of pathogens infection.And our previous studies demonstrated that cyclophilin A(CypA)on the surface of human urethral epithelial cells(SV-HUC-1)is the receptor of MgPa.Therefore,this study is dedicated to understanding the effect of MgPa interaction with its cell receptor CypA on the apoptosis of SV-HUC-1 cells,which may provide a experimental evidence for understanding the survival mechanism of M.genitalium in host cells.Objective:To investigate whether MgPa will affect the apoptosis of SV-HUC-1 cells through CypA-CD147 and discover the related pathways,so as to provide a possible molecular biological basis for the cellular immune escape of M.genitalium.Methods:1.To investigate whether MgPa inhibits or promotes the apoptosis of SV-HUC-1 cells.Using Staurosporine(STS)as a positive control for apoptosis and stimulating cells with 20μg/m L MgPa,Flow cytometry was used to detect the apoptotic rate of each group,and Western blot(WB)was used to detect the expression levels of Bax,Caspase-3 and Cleaved Caspase-3 in each group.2.To investigate the role of CypA and CD147 in MgPa’s inhibition of SV-HUC-1 cell apoptosis.The cells were transfected with CypA-si RNA and CD147-si RNA,and control-si RNA was used as the control,then the cells were treated with MgPa and/or STS respectively,Flow cytometry was used to detect the apoptotic rate of each group.Using WB to detect the expression levels of Bax,Caspase-3 and Cleaved Caspase-3 in each group.3.To investigate whether MgPa regulates the PI3K/Akt/NF-κB pathway through CypA-CD147 to inhibit the apoptosis of SV-HUC-1cells.After treating the cells with MgPa and/or STS respectively,WB was used to detect the expression levels of p-PI3 K,PI3K,p-Akt,Akt,p-IκB and IκB in each group.After pre-treated with PI3 K inhibitor LY294002 or DMSO control,the cells were treated with MgPa and STS,and the expression levels of p-IκB and IκB were detected by WB.After pre-treated with PI3K inhibitor LY294002 and IκB inhibitor BAY 11-7082,the cells were treated with MgPa and/or STS,respectively,WB detected the expression levels of Caspase-3 and Cleaved Caspase-3in each group.In order to verify that MgPa regulates the PI3K/Akt/NF-κB pathway through CypA and CD147,the cells were transfected with CypA-si RNA and CD147-si RNA,and then treated with MgPa and/or STS respectively.WB was used to detect the expression levels of p-PI3 K,PI3K,p-Akt,Akt,p-IκB and IκB in each group.4.CypA knockout C57 BL mice were constructed,and mouse primary urethral epithelial cells were obtained by collagenase I digestion.MgPa and/or STS were used to treat normal mouse primary urethral epithelial cells and CypA gene knockout C57 BL mouse primary urethral epithelial cells,Flow cytometry was used to detect the apoptotic rate of each group.Results:1.The results of WB suggested that the expression of Bax and cleaved caspase 3 in the MgPa treatment group was significantly down-regulated;after using STS to induce apoptosis,the expression of Bax and cleaved caspase 3 in the MgPa treatment group was lower than that of the STS induced group;The results of flow cytometry showed that after STS induced apoptosis,the apoptosis rate(8.79%)of the MgPa treatment group was lower than that of the STS inducer group(16.59%);2.WB results showed that after transfection with CypA-si RNA or CD147-si RNA,the expression of Bax and cleaved caspase 3 in the MgPa and STS co-treatment group was higher than that in the untransfected group.The results of flow cytometry showed that after transfection of CypA-si RNA or CD147-si RNA,the apoptosis rates of the MgPa and STS co-treatment groups were 42.8% and 46.4%,respectively,which were significantly higher than the 24.63% of the untransfected group;Interfering with the expression of CypA or CD147 obviously reversed the inhibitory effect of MgPa on STS-induced apoptosis,and MgPa inhibited the apoptosis of urothelial cells through CypA/CD147.3.After STS induced cell apoptosis,the expression of p-PI3 K,p-Akt and p-IκB in the MgPa treatment group were significantly up-regulated;after PI3 K inhibitor treatment,the expression level of p-IκB was down-regulated.After PI3 K and IκB inhibitor treatment,the expression level of Cleaved Caspase-3 in the MgPa and STS co-treatment group was significantly up-regulated compared with the uninhibited group;after transfection with CypA-si RNA or CD147-si RNA,the expression levels of p-PI3 K,p-Akt and p-IκB in the MgPa and STS co-treatment group were significantly down-regulated compared with the untransfected group;MgPa regulates the PI3K/Akt/NF-κB pathway through CypA/CD147 to inhibit the apoptosis of SV-HUC-1 cells.4.The results of flow cytometry demonstrated that after 36 hours of MgPa treatment of the cells,the apoptosis rate of the normal mouse urethral epithelial cell group was 24.38%,which was lower than that of the CypA knockout mouse urethral epithelial cell group of 33.28%;and after co-treatment with MgPa and STS,the apoptosis rate of normal mouse urethral epithelial cell group was 28.02%,which was lower than that of CypA knockout mouse urethral epithelial cell group of 45.58%,which further confirmed the role of CypA in MgPa’s inhibition effect of cell apoptosis.Conclusions: Mycoplasma genitalium protein of adhesion regulates PI3K/Akt/NF-κB pathway through CypA/CD147 to inhibit SV-HUC-1cell apoptosis.