Study On The Mechanism Of HSP27 Regulating The Occurrence Of RFM In Dairy Cows Through Anti-oxidative Stress And Apoptosis

Retained fetal membranes?RFM?is a common reproductive disorder in cows.Some studies have shown that RFM is closely related to apoptosis and antioxidant status.In the previous studies,we found that HSP27 was significantly increased in the placental tissues of RFM cows,and HSP27 played an important role in the biological processes of antioxidation and apoptosis in the body.Therefore,this experiment intends to determine the correlation between HSP27,apoptosis-related factors and intracellular ROS and the occurrence of RFM in dairy cows through in vivo and in vitro experiments using biochemical and molecular biology techniques.Based on the establishment of a model of oxidative stress in primary epithelial cells of dairy cows,we explored whether HSP27 was involved in the occurrence of RFM through apoptosis and oxidative stress.And it elucidated the mechanism of HSP27 participating in the process of RFM,so as to provide a theoretical basis for further revealing the pathogenesis of RFM in dairy cows.The 7 RFM cows and 7 healthy cows in this experiment,with similar milk yield,age,weight,parity and no other diseases,were taken blood and maternal placental tissues at 12 h after calving.Serum concentrations of HSP27 were detected by ELISA,and expressions of HSP27?Akt?p-Akt?p-IKK?NF-?B p65?p-NF-?B p65?Bad?Bcl-xl?Bcl-2 in the HSP27-mediated signaling pathway were detected by qRT-PCR and Western blot.Then the changes of GSSG and GSH contents and the activities of GSH-Px and G6PD in the tissues were detected to determine the relationship between them and the occurrence of RFM in cows.The primary endometrial epithelial cell model of dairy cows was used to construct an oxidative stress model of primary endometrial epithelial cells of dairy cows.After screening to determine the optimal time and concentration of adding HSP27 promoter 17-AAG and inhibitor siRNA,cells of each group were collected 24 h after 17-AAG stimulation and 48 h after siRNA transfection.The expressions of HSP27?Akt?p-Akt?p-IKK?NF-?B p65?p-NF-?B p65?Bad?Bcl-xl?Bcl-2 in the HSP27-mediated signaling pathway were detected by qRT-PCR and Western blot.Then the cells with low and overexpression of HSP27 were stimulated with H₂O₂ at a concentration of 400?M for 2 h to detect the mRNA and protein expressions of HSP27,as well as the changes of intracellular ROS,GSSG and GSH content and the activity of GSH-Px and G6PD.Results:the concentration of HSP27 in serum of RFM cows was significantly higher than that of NO-RFM cows?P<0.05?.In the placental tissue of RFM cows,compared with controls,the mRNA expression levels of HSP27,Akt,IKK,Bcl-xl,Bcl-2?P<0.01?and NF-?B p65?P<0.05?were significantly increased,and the mRNA expression level of Bad was significantly decreased?P<0.05?.The protein expression of HSP27 and NF-?B p65 were significantly increased?P<0.05?,and the protein expression of Akt?p-Akt?p-IKK?p-NF-?B p65?Bcl-xl?Bcl-2 were significantly increased?P<0.01?.The protein expression of Bad was significantly decreased?P<0.05?.The content of GSH and the activities of GSH-Px and G6PD in maternal placental tissue of RFM cows was significantly lower than that of NO-RFM cows?P<0.01?,and the content of GSSG was significantly higher than that of NO-RFM cows?P<0.01?.The oxidative stress model of primary endometrial epithelial cells in cows was successfully established,and the optimal concentration of inhibitor and promoter was screened?17-AAG was24 h with 200?M and SiRNA was 48 h?.Results of qRT-PCR and Western blot showed that the mRNA and protein expressions of HSP27?Akt?p-Akt?p-IKK?NF-?B p65?p-NF-?B p65?Bcl-xl and Bcl-2 increased significantly?P<0.01?,and the mRNA and protein expression Bad was decreased?P<0.05?when 17-AAG stimulated cells.When siRNA was transfected,the mRNA and protein expressions of HSP27?Akt?p-Akt?p-IKK?NF-?B p65?p-NF-?B p65?Bcl-xl?P<0.01?and Bcl-2?P<0.05?were significantly decreased,and the expression of Bad was significantly increased?P<0.01?.When HSP27 was overexpressed,the mRNA and protein expression of HSP27 was significantly increased?P<0.01?.The content of GSH and the activity of GSH-Px and G6PD were significantly increased?P<0.01?,while the content of ROS and GSSG were significantly decreased?P<0.01?.When the expression of HSP27 was low,the mRNA and protein expression of HSP27 was significantly decreased?P<0.01?.The content of GSH?P<0.05?and the activity of GSH-Px and G6PD?P<0.01?were significantly decreased,and the content of ROS and GSSG were both significantly increased?P<0.01?.From the above results,it can be known that HSP27 can participate in the antioxidant and apoptosis process of endometrial epithelial cells by regulating the apoptosis signaling pathway mediated by NF-?B and the expression of key factors in the antioxidant pathway mediated by G6PD.High expression of HSP27 can affect the process of fetal membranes efflux by affecting cell apoptosis and intracellular ROS content.