Study On The Repair Of Oxidative Damage Of BAD-MSCs In Bovine Endometrial Epithelial Cells

Oxidative stress is involved in a variety of pathological processes of reproductive disorders in dairy cows,reactive oxygen species（ROS）can be involved in the occurrence and development of endometritis in cows as inflammatory molecules and inflammatory mediators.Therefore,antioxidant stress plays an important role in the prevention and treatment of endometritis in dairy cows,but research has not found an ideal alternative to antibiotic therapy so far.Mesenchymal Stem Cells（MSCs）have been widely studied and applied in many disease models and animal clinics due to their powerful biological functions.Bovine endometrial epithelial cells（BEECs）are an important part of the innate immunity to the uterus of dairy cows,hence,H₂O₂ was used to construct BEECs oxidative stress model in this study to investigate the antioxidant effect of bovine adipose-derived mesenchymal stem cells（b AD-MSCs）,the objective of this study was to provide new ideas for the prevention and treatment of endometritis in dairy cows.Experiment 1:BEECs were treated with 0,5,25,50,100 and 200μmol/L H₂O₂ for 2,4,6,8 and 12 h,MTT assay was used to detect cell activity and flow cytometry was used to detect intracellular ROS level to screen the best optimum condition of oxidative stress model of bovine endometrial epithelial cells,transwell with pore size of 0.4μm was used to construct co-culture system after cell scratch test to analyze cell migration ability and Erk and p Erk protein expression level.The results showed that the cell survival rate was between 50%and 80%when H₂O₂ concentration was 50μmol/L,and the ROS levels were significantly increased when 50μmol/L H₂O₂ was used for 2 h,and H₂O₂ treatment could significantly reduced the migration ability and the expression of p Erk of bovine endometrial epithelial cells（P<0.05）,however,the cell migration ability and the expression of p Erk of bovine endometrial epithelial cells in co-culture group with b AD-MSCs were significantly increase（P<0.05）.Experiment 2:Co-culture system was constructed with transwell with pore diameter of 0.4μm after BEECs were treated with H₂O₂,and the ROS level were determined by flow cytometry;the m RNA expression of Cu/Zn-SOD,Mn-SOD,CAT and GSH-Px were measured by q PCR;the protein expression of Nrf2,HO-1 and NQO1 in cells were determined by western blot to evaluate the antioxidant capacity of b AD-MSCs.The results showed that co-culture with b AD-MSCs significantly down-regulated the ROS level in BEECs oxidative stress model,and up-regulated the m RNA expression levels of Cu/Zn-SOD,Mn-SOD,CAT and GSH-Px,and the expression of Nrf2,HO-1 and NQO1 were also significantly increased（P<0.05）.Experiment 3:Co-culture system was constructed with transwell with pore diameter of 0.4μm after BEECs were treated with H₂O₂,and the cell apoptosis was determined by flow cytometry;western blot was used to measure Bax,Bcl2 and Bcl-xl and apoptosis-related pathway proteins such as NF-κB p65 and p38 to evaluate the anti-apoptotic ability of b AD-MSCs.（P<0.05）.The results showed that co-culture with b AD-MSCs could significantly inhibited H₂O₂ induced apoptosis（P<0.05）,and significantly down-regulated the protein expressions of Bax,NF-κB p65,p-NF-κB p65 and pp38 were（P<0.05）,and the protein expressions of Bcl2 and Bcl-xl were significantly up-regulated（P<0.05）.Taken together,these results suggested that 50μmol/L H₂O₂ using for 2 h is the best condition for constructing the oxidative stress model of BEECs,and it demonstrated that b AD-MSCs promote the migration of BEECs under oxidative stress by up-regulating the protein expression of p Erk;and b AD-MSCs inhibited the production of ROS in BEECs oxidative stress model by up-regulating the m RNA expressions of Cu/Zn-SOD,Mn-SOD,CAT and GSH-Px and the protein expressions of Nrf2,HO-1 and NQO1.b AD-MSCs down-regulated the expression of NF-κB p65,p-NF-κB p65 and p-p38 to promote the protein expression of Bcl2 and Bcl-xl and inhibit the protein expression of Bax,thereby inhibiting apoptosis.It has laid a theoretical foundation for the antibiotic replacement therapy of endometritis in dairy cows.