Study On The Regulation Mechanism Of FAK Signaling Pathway On Col-? Hydrolysis During The Occurrence Of Retained Fetal Membrane In Dairy Cows

Domestic and foreign scholars generally believe that the main pathological basis of the bovine retained fetal membrane(RFM)is the adhesion between the maternal placenta and the fetal placenta,and the main substance causing adhesion is type ? collagen(Collagen-?,Col-?).Focal adhesion kinase(FAK)is a 125 kDa cytoplasmic non-receptor tyrosine kinase located at the intercellular adhesion site or intercellular matrix adhesion,which regulates the activity of MMP-2 and MMP-9.MMP-2 and MMP-9 are key enzymes for the hydrolysis of Col-? and participate in the adhesion process between cells.Then how does FAK play a role in the RFM process of dairy cows? The specific mechanism of action is still unclear.Therefore,this study aims to determine the relationship between FAK,Col-? and RFM in dairy cows by detecting the changes of key factors of FAK signaling pathway and the expression of COL-? in maternal placenta tissue of RFM dairy cows.On the basis of FAK signaling pathway and Col-?,molecular biology techniques were used to detect the expression changes of FAK signaling pathway and Col-? by overexpression and gene silencing FAK to elucidate the expulsion process of dairy cows.The molecular mechanism of FAK signaling pathway on the regulation of Col-? hydrolysis provides a theoretical basis for enriching the etiology of RFM in dairy cows.The maternal placental tissue was taken from 14 cows whose number of parity,body weight,age and milk production were similar,and they were no other diseases.Half of the 14 cows failed to discharge the membranes,and the others were normal.The key molecules(FAK,Src,MMP-2,MMP-9)in FAK signaling pathway and the distribution of Col-? were detected by immunohistochemistry.To detect the expression levels of FAK,Src,MMP-2,MMP-9 and Col-? in the maternal placenta of RFM cows and healthy cows,Q-PCR and Western blot were used.Isolate and culture primary endometrial epithelial cells of dairy cows.After screening to determine the optimal concentration of FAK inhabit 14 and Angiotensin II,each group of cells was collected 24 h after the addition,and the expression levels of FAK,Src,MMP-2,MMP-9and Col-? were detected by Western blot and Q-PCR.Results: Immunohistochemistry results showed that key molecules(FAK,Src,MMP-2,MMP-9)in the FAK signaling pathway and Col-? were mainly expressed of placental tissue,and compared with the RFM group,the expression of FAK,Src,MMP-2,and MMP-9 werehigher,while the expression of Col-? is lower.Q-PCR results showed that the mRNA and protein expression levels of FAK,Src,MMP-2 and MMP-9 in the maternal placenta of RFM cows were significantly down-regulated(p < 0.05)and the expression level of Col-? was significantly elevated(p < 0.05)compared with healthy cows.The primary endometrial epithelial cell model of dairy cows was successfully established.The optimal concentration of screening was obtained.When the concentrations of FAK inhabit 14 and Angiotensin II were 2 ?M and 0.1?M,respectively,the epithelial cell viability was the best and at this concentration,the effect of low expression and over expression of FAK is the best.The results of Western blot and Q-PCR showed that the relative expression levels of FAK,Src,MMP-2 and MMP-9 were significantly down-regulated(p < 0.05)after adding 2 ?M FAK inhabit 14,and the relative expression level of Col-? was significantly up-regulated(p < 0.05).After adding 0.1 ?M Angiotensin II,the relative expression levels of FAK,Src,MMP-2 and MMP-9 were significantly up-regulated(p <0.05),and the relative expression level of Col-? was significantly down-regulated(p < 0.05).The above experimental results clarify that FAK in maternal endometrial epithelial cells can regulate the hydrolysis of Col-? and promote collagen hydrolysis through the expression of key factors in the signaling pathway,which in turn affects the process of postpartum fetal membranes expulsion in dairy cows.