Effect Of Toxoplasma Gondii ROP16 Protein On Decidualization Of Mouse Uterine Stromal Cells

Toxoplasma gondii is an obligate intracellular parasitic genus belonging to the genus Eucoccidiorid,Toxoplasmatidae,Toxoplasma,according to the virulence of the worm.The difference can be divided into 3 types of Toxoplasma gondii.Congenital vertical transmission of Toxoplasma gondii is one of the important teratogenic factors leading to adverse pregnancy outcomes.Toxoplasma can invade the fetus through the placenta,causing serious consequences at different stages of pregnancy.The current research on the mechanism of abnormal pregnancy caused by Toxoplasma gondii is mostly focused on immunology,while the interaction between Toxoplasma gondii and cells related to embryonic development is relatively rare.The various proteins secreted by Toxoplasma gondii to complete the life process in the host cell have the function of regulating the function of the host cell.For example,Toxoplasma gondii ROP18 and GRA15 can affect the gene expression of host cells through the host cell NFκB.Toxoplasma gondii ROP16 protein also has polymorphism between different strains.Type I and type III strains（eg,RH,CTG）ROP16I/III protein can phosphorylate tyrosine at position 705 of Stat3 by directly binding to Stat3 of the host cell.Toxoplasma protein ROP16 phosphorylates the transcription factor Stat3 of host cells and partially induces apoptosis in host cells and inhibits cell differentiation.The EMT phenomenon of embryo implantation in early pregnancy is also involved in Stat 3,accompanied by cell proliferation and differentiation.Objective:The effects of Toxoplasma gondii protein ROP16 on pregnancy in mice were studied from the aspects of apoptosis and MET.The interaction protein of ROP16 was revealed,and the molecular mechanism of ROP16-induced adverse pregnancy in mice was elucidated.Meterials:The in vitro experiments were mainly carried out by culturing mouse uterine stromal cells and transfecting them with recombinant ROP16_I/III lentivirus.Flow cytometry,Western blotting and Real-Time PCR were used to detect apoptosis rate and apoptosis.The expression levels of related proteins Bax and Bcl-2 were used to verify the effect of ROP16_I/III/III on MET during decidualization in mice:the uterine stromal cells of the fourth day of pregnancy were cultured to induce decidualization and transfection.Western blot and Real-Time PCR were used to detect the expression changes of MET-related marker molecules Vimentin,E-cadherin and transcription factors,and to detect the expression and transcription of apoptosis-related proteins.The in vivo experiment mainly involves injecting ROP16_I/III into the mouse uterus on the 4th day of pregnancy by lentiviral transfection,taking the mouse uterus on the8th day,separating the tissue at the implantation site,and extracting RNA or protein.The expression level of apoptosis-related proteins was detected by Real-Time PCR or Western blot.Immunohistochemistry was used to detect the level of apoptosis at the locus.The effect of ROP16_I/III decidualization was verified in vivo:the pregnancy rate,the number of embryo implantation,and the weight of the decidual tissue were weighed.In addition,the tissue of the implantation site is separated,RNA or protein is extracted,and MET-related marker molecules and transcription factors are detected by Real-Time PCR or Western blot.Results:（1）The purity of extracted mouse uterine stromal cells was（95±2.5）%.After induced decidualization in vitro,the expression of PRL mRNA increased significantly with time（p<0.05）.（2）ROP16 was overexpressed in decidual cells induced by decidualization,and cell flow cytometry results showed that the apoptosis rate of decidual cells was decreased compared with the control group（p<0.05）.The expression of BAX in the decidual cell apoptosis protein was significantly decreased,and the expression of BCL-2 protein was up-regulated.（3）ROP16 was overexpressed in decidual cells induced by decidualization,and themRNAs of ZEB1,E-Cadherin and Vimentin in each group were quantified by qRT-PCR.The results showed:Compared with the control group,the mRNA expression of ZEB1and Vimentin in the ROP16 transfection group was increased（p<0.05）,while themRNA expression of E-Cadherin was decreased.（4）On the 4th day of pregnancy,the uterine horn was injected with ROP16 lentivirus or empty lentivirus negative control.The mouse uterus was taken out on the 8th day,and the number of mouse embryo implantation was counted.The results showed:Compared with the control group,the uterine embryo implantation rate of the ROP16 transfection group was significantly decreased（p<0.05）.（5）The uterus model on the 8th day of the above procedure was used to detect the expression and distribution of Bax and Bcl-2 by immunohistochemistry.The results were consistent with the results of Western blot.Conclusions:During decidualization,Toxoplasma gondii ROP16 protein inhibits the apoptosis of decidual cells,which may affect the balance of decidual cell proliferation and apoptosis and affect embryo implantation.At the same time,in the process of enamelization.ROP16 protein affects MET in uterine stromal cells.The experimental results show that the implantation rate of embryos in the uterus of the pre-implantation uterus is significantly reduced after injection of ROP16 lentivirus into the uterine horn,indicating that the ROP16 protein of Toxoplasma gondii can cause adverse pregnancy by affecting the decidualization process in the uterus of pregnant mice.