Research On The Change Mechanism Of UFBP1 In Dairy Cow Mastitis

Dairy cow mastitis is a common disease of dairy cow mammary gland,which is often caused by a variety of pathogens.It has caused huge losses to dairy industry and seriously threatened food safety and people’s health.Cronobacter sakazakii and Streptococcus agalactiae are common pathogens of cow mastitis,which often cause strong inflammatory reaction.UFBP1,as one of the substrates modified by UFM system,is mainly located in endoplasmic reticulum and plays an important role in endoplasmic reticulum stress-induced apoptosis.Therefore,this study explored the mechanism of UFBP1 in mastitis through the following aspects.1.Role of UFBP1 in dairy cow mastitisMilk samples of dairy cows with mastitis were collected from a farm in Jiangsu Province for isolation and detection of pathogenic bacteria.The main pathogenic bacteria were identified as Cronobacter sakazakii and Streptococcus agalactiae by LB medium and Cronobacter sakazakii chromogenic medium.The living breast tissues infected by Streptococcus agalactiae were collected for the following experiments: 1.H.E.staining to observe the morphological changes of breast;2.Oil red O staining to observe the changes of lipid droplets;3.TUNEL staining was used to detect apoptosis;4.Fluorescent quantitative PCR was used to detect UFBP1 m RNA expression;5.Western blot was used to detect UFBP1,NLRP3,AIM2,caspase-1 and ASC protein expression in breast tissue.The results showed that compared with the control group,the morphological structure of mammary gland in mastitis group had significant changes,including significant collapse of mammary acini,infiltration of inflammatory cells and hyperplasia of mammary epithelial cells;the lipid droplets in mammary epithelial cells in mastitis group disappeared,the number of mammary epithelial cells decreased,and the proportion of apoptotic cells increased significantly(P < 0.01);The m RNA and protein levels of UFBP1 in mastitis group were significantly lower than those in control group(P < 0.01).Meanwhile,NLRP3,Caspase-1,AIM2 and ASC in mastitis group were significantly higher than those in control group(P < 0.01).Conclusion: Mastitis caused by Streptococcus agalactiae can damage the mammary structure of dairy cows,infiltrate inflammatory cells,disappear lipid droplets,enhance tissue inflammatory response and increase cell apoptosis.The expressions of NRLP3,Caspase-1,AIM2 and ASC are significantly increased,and UFBP1 is significantly decreased.2.Expression and mechanism of UFBP1 in mouse mastitis induced by Cronobacter sakazakii and Streptococcus agalactiaeIn order to verify the change of UFBP1 expression in mastitis caused by two kinds of pathogens,a mouse mastitis model was established by using the bacterial samples collected from the above farms.After the establishment of the model,the mammary gland tissue was taken for the following experiments: 1.H.E.staining was used to observe the morphological changes of mammary gland;2.Fluorescent quantitative PCR was used to detect the expression of TNF-α,IL-1β,IL-6,Bax and Bcl2 and other inflammatory factors and apoptosis related m RNA;3.Western blot was used to detect the expression of TNF-α,IL-1β,IL-6,Bax and Bcl2 The expression of endoplasmic reticulum stress-related proteins,such as Grp78,Chop and Erdj4,and apoptosis related proteins,such as Bax and Bcl2,and ufm1 system modified proteins,such as UFBP1,was detected by blot.The results showed that both of the two pathogens could cause severe mastitis.H.E.staining showed that there were a lot of inflammatory cells infiltrating into the mammary tissue of mice in mastitis group,and the structure of acinar tissue was destroyed.Compared with the control group,the m RNA expression levels of TNF-α,IL-1 β,IL-6 and other inflammatory cytokines in C.sakazakii mastitis group were significantly higher than those in the control group(P <0.01).Bax was significantly up-regulated(P < 0.05)and Bcl2 was significantly down regulated(P < 0.01);the m RNA levels of Grp78,Chop and Erdj4 were significantly increased(P < 0.05),the protein levels of Grp78 and Chop were significantly increased(P< 0.01);the protein expression levels of UFBP1 was significantly down regulated(P <0.01);the m RNA expression levels of TNF-α,IL-1β and other inflammatory cytokines in non Streptococcus lactis mastitis group were significantly increased(P < 0.01)The expression levels of UFBP1 was significantly down regulated(P < 0.01).Conclusion: Both of the two pathogens can cause severe mastitis in mice,induce tissue inflammation and cell apoptosis,and the expressions of UFBP1 is significantly down regulated in mastitis.3.Study on the interaction mechanism between UFBP1 and NLRP3In order to investigate the interaction between ufbp1 and NLRP3,the expression of UFBP1 was verified by the knockout of bovine epithelial cells and mouse fibroblasts by tamoxifen in vitro,and apoptosis related genes were detected.Meanwhile,si RNA with high efficiency was constructed and screened.After transfection and silencing efficiency,ufbp1 expression was detected.The results showed that the expression level of ufbp1 in the breast epithelial cells of dairy cows was significantly decreased in RNA level(P < 0.01)and protein level(P < 0.05).After the ufbp1 was knocked out,the expression level of NLRP3 in fibroblasts decreased,while the levels of AIM2 and ASC were significantly increased(P< 0.05)and Caspase-1 was significantly increased(P < 0.01).NLRP3 expression was significantly down regulated after UFBP1 knockout(P < 0.01),and Bcl-2 was significantly decreased after UFBP1 was knocked out(P < 0.01).Conclusion: the expression level of NLRP3 and ufbp1 is the same,and the knockout of ufbp1 can inhibit the expression of Bcl2 and induce apoptosis.