Mechanism Of IFNτ Regulating RIPOR2 To Terminate Early Pregnancy Failure Caused By Ferroptosis In Dairy Cows

The breeding rate of dairy cows greatly affects the sustainable development of dairy cows,which is directly related to the production benefit of dairy farms.Early embryo loss is one of the important factors leading to low calving rates in many intensive dairy farms.In the past few decades,although scientific research has significantly improved the reproductive rate of dairy cows,our understanding of the complicated maternal-fetal mechanism of dairy cows is still shallow due to limited technical conditions and high research costs.Endometrial receptivity is the key to embryo implantation,and implantation failure caused by adverse factors such as endometritis or endometrial immune microenvironment disorders can affect the normal progress of pregnancy.The data show that ferroptosis is a new type of cell death,which is involved in the development of many biological diseases.However,existing reports of ferroptosis have mainly focused on cancer treatment,while the research on reproductive diseases,especially early pregnancy failure,is still unclear.This study was conducted through molecular dynamics simulation,bioinformatics analysis,construction of stable transfection cell lines,and in vivo study of model animals.We systematically studied the novel interaction target proteins of IFNτ in early pregnancy by using molecular docking,co-immunoprecipitation(CO-IP),adenoassociated virus transfection,RTCA DP real-time cell analysis,immunofluorescence(IF)and pull down techniques.RIPOR2 is involved in the downstream signaling pathway of ferroptosis and the expression changes of important proteins.This study aims to clarify the specific regulatory mechanism of early pregnancy failure caused by ferroptosis termination by IFNτ,expand new insights into the role of IFNτ in pregnancy immunization,and provide a new scientific basis for improving the reproductive rate of dairy cows.The specific research results of this study were as follows:1.RIPOR2 acts as a medium of IFNτ to inhibit ferroptosisIn this study,we screened the unique interacting target RIPOR2 of IFNτ,distinguished from IFNα in early pregnancy,and established three-dimensional structural models of IFNτ and RIPOR2.Protein structure docking shows that a stretch of α-Helix structure of IFNτ is completely embedded in the surface cavity of RIPOR2,forming 16 sets of hydrogen bonds that enable stable binding of the two proteins.And the interaction between IFNτ and RIPOR2 was further verified by CO-IP experiments.In order to explore the role of IFNτ and ferroptosis,this study first showed a clear correlation between embryo implantation and ferroptosis-related genes through correlation heat map analysis,and preliminarily concluded that ferroptosis would affect normal physiological behavior in early pregnancy.Next,we found that IFNτintervention can significantly reverse the abnormal changes in the expression levels of FTH1,GPX4 and PTGS2 caused by ferroptosis,and promote the decrease of iron content and MDA and the increase of GSH in vitro tissue by Erastin induced ferroptosis model.Subsequently,further studies showed that ferroptosis would inhibit the expression of RIPOR2,and interference with RIPOR2 aggravated the damage caused by ferroptosis and inhibited the expression of LIF and ITGB3.IFNτ not only elevated the expression levels of LIF and ITGB3,but also inhibited cell membrane rupture and microvillus damage caused by ferroptosis.These results indicate that IFNτ inhibits ferroptosis in endometrium during early pregnancy by promoting RIPOR2 expression.2.IFNτ inhibits ferroptosis through the ROCK1/p38/JNK1 pathwayIn order to further investigate the role of RIPOR2 in ferroptosis,adeno-associated virus was used to construct a RIPOR2 knockdown stable endometrial epithelial cell line.The effect of IFNτ on the most differentially significant genes in the Erastin-induced ferroptosis model was analyzed by RNA sequencing.ROCK1/2 is the downstream effect factor of RIPOR2.Ferroptosis can promote the expression of ROCK1 and ROCK2.IFNτ intervention only has a significant effect on ROCK1,but less effect on ROCK2.Further experiments showed that IFNτ promotes GPX4,FTH1 and suppresses PTGS2 expression through ROCK1.KEGG analysis showed that MAPK pathway enrichment was the most significant.MAPK8(p38)and MAPK12(JNK1)of MAPK pathway members had the largest variation by data analysis.The protein expression and nuclear translocation degree of p-p38 and p-JNK1 induced by ferroptosis are significantly reduced,and this effect is synergically enhanced by IFNτ after blocking ROCK1.Furthermore,this study showed that ferroptosis disorganized the mitochondrial network and significantly increased the number of fragmented or punctate mitochondria around the nucleus.However,IFNτ maintains the integrity of the mitochondrial network by regulating ROCK1 expression and attenuates the degree of cell killing by ferroptosis.These results indicate that IFNτ alleviates oxidative damage to cells by ferroptosis by inhibiting ROCK1/p38/JNK1 pathway activation.3.IFNτ terminated the early pregnancy failure caused by ferroptosisTo further explore the functional relationship of IFNτ and RIPOR2 in ferroptosis in vivo,we first knocked down RIPOR2 expression in mice with adeno-associated virus.On this basis,the mouse early pregnancy endometrial ferroptosis model was established with PA-Erastin.IFNτ enhanced GPX4 and inhibited PTGS2 expression in endometrial epithelium.It can inhibit the excessive increase of iron content in endometrial tissue and peripheral blood serum.On the other hand,IFNτ significantly inhibited the expression of p-p38 and p-JNK1 and decreased their fluorescence density in the nucleus of endometrial epithelial cells.In order to exclude individual differences,this study explored the influence of IFNτ on early pregnancy by autologous control.The results confirmed that IFNτ prevented the adverse effects of ferroptosis on early pregnancy embryo implantation in mice and ensured normal embryo implantation.The results of RT-q PCR showed that expressions of LIF and ITGB3 increased significantly under IFNτ.The fluorescence expression level of LIF also showed similar results.The above results indicate that IFNτ maintains the normal progression of early pregnancy in mice by inhibiting ferroptosis.Conclusion:IFNτ inhibits the activation of ROCK1/p38/JNK1 pathway by promoting the expression of RIPOR2,alleviates oxidative damage and terminates early pregnancy failure caused by ferroptosis.