| Retained fetal membranes(RFM)is a common reproductive disorder in dairy cows during perinatal period,and its causative factors are complex and variable.Studies have confirmed that the nutritional metabolism of dairy cows directly affects the efflux of placenta,which affects the overall metabolic status of dairy cows with RFM.The study on the relationship between the overall metabolism of dairy cows with RFM and the occurrence of RFM is helpful to reveal the mechanism of RFM and formulate timely and effective prevention and control measures.Therefore,liquid chromatography-mass spectrometry(LC-MS)was used to identify the serum differential metabolites of RFM and analyze the metabolic pathway involved in them.Glutamine(Gln)was selected as the research object to explore its role and mechanism in the pathogenesis of RFM,to explore the protective effect of Gln supplementation on endometrial epithelial cells from oxidative damage in dairy cows,and to explore the role and mechanism of Gln in the pathogenesis of RFM.In this research,eight dairy cows with RFM of similar age,body condition and parity without other diseases and eight healthy cows were selected,and their serum and maternal placenta samples were collected respectively.The differential metabolites in RFM cows were screened by LC-MS technology combined with multivariate statistical analysis,and these differential metabolites were analyzed by multivariate statistical analysis and bioinformatics analysis.q RT-PCR and ELISA were used and other methods were used to detect the oxidation / antioxidant molecular index and Gln content.Finally,the cells were pretreated with Gln at different concentrations(0,0.25,0.5,2,4,8 mm)for 24 hours,and then stimulated with H2O2.The cell survival rate,ROS content,Nrf2 activation state and oxidation / antioxidant molecule expression were detected by CCK-8,flow cytometry,laser confocal technology and q RT-PCR.The results of serum metabonomics showed that 57 differential metabolites were identified in RFM cow serum,41 of which were up-regulated and 16 were down regulated.These differential metabolites are mainly involved in 11 biological processes such as glycerolipid metabolism,D-Glutamine and D-glutamate metabolism,glutathione metabolism,among which the glutathione synthesis substrate Gln is significantly down regulated(P < 0.05),and the lack of Gln may be closely related to the occurrence of RFM.The results of in vivo experiments showed that the m RNA expression levels of Mn SOD(P <0.01),Cu / Zn SOD(P <0.05)and GSH-Px(P <0.05)in the placenta tissue of RFM cows were significantly reduced,and the content of MDA was higher(P <0.05),the GSH content and GSH-Px activity are low(P <0.05);the content of MDA(P <0.01)and GSH(P <0.05)in the serum of RFM cows is significantly increased(P <0.01),SOD,GSH-Px activity(P <0.01)and Gln content(P <0.01)were significantly reduced.The suitable condition for the model of oxidative damage to endometrial epithelial cells in dairy cows is 400 ?M H2O2 for 2 h;compared with the higher concentration(2,4 m M)Gln treatment group,the lower concentration(0,0.25 m M)Gln treatment group uterus Endometrial epithelial cell survival rate and GSH content were lower(P <0.05);compared with the lower concentration Gln(0,0.25,0.5 m M)pretreatment group,2 m M Gln pretreatment added H2O2 stimulation,cells The internal GSH content was significantly increased(P <0.01),the cell survival rate and the m RNA levels and activities of SOD and GSH-Px were higher(P <0.05),and the ROS(P <0.05)and MDA content(P <0.01)were significantly decreased(P <0.05),the activation of Nrf2 was enhanced.The above results showed that there were significant differences between dairy cows with RFM and healthy cows.The abnormality of 57 metabolites was closely related to the occurrence of RFM.Dairy cows with RFM are in the state of oxidative stress.Gln can protect endometrial epithelial cells from oxidative stress,improve the antioxidant capacity of endometrial epithelial cells,and thus participate in the process of placenta expulsion. |
PDF Full Text Request