Protective Effect Of Quercetin On NEFA Induced Apoptosis Of Bovine Endometrial Cells

Background: Ketosis is a common metabolic disease in high-yield dairy cows,which is characterized by hyperketemia,high non esterified fatty acidemia and hypoglycemia.The report points out that the occurrence of ketosis in perinatal cows will not only reduce the milk yield and milk quality,but also affect the reproductive performance and immune function of dairy cows.Studies have shown that high concentrations of non esterified fatty acids(NEFA)can induce oxidative stress and inflammatory response in dairy cows,which may be the cause of a series of reproductive disorders in ketosis dairy cows.Quercetin,as a natural antioxidant,has many biological functions,such as anti-inflammatory,antioxidant,immune regulation and so on.Taking bovine endometrial cells as the research object,this study explored the protective effect of quercetin on oxidative damage and apoptosis of bovine endometrial cells induced by NEFA from the perspective of oxidative damage and apoptosis,so as to provide a practical basis for exploring the relationship between bovine ketosis and postpartum reproductive disorders,and formulating reasonable prevention and control measures.Methods: Different concentrations of NEFA were applied to bovine endometrial cells(BEND)cultured in vitro for 6h,12 h,24h and 48 h respectively.The cell proliferation was detected by CCK8 method,and the appropriate concentration and time of administration were selected.The samples were divided into four groups: blank control group and 10 μmol / L quercetin group,1.2 mmol / L NEFA group,10 μmol / L quercetin + 1.2 mmol / L NEFA group.Biochemical methods were used to detect the indexes related to oxidative damage of BEND cells,TUNEL method was used to detect the apoptosis of BEND cells,and q PCR and Western blot were used to detect the m RNA and protein expression levels of mitochondrial apoptosis pathway related genes of BEND cells in different treatment groups.Results:(1)CCK8 test showed that the cell viability decreased gradually with the increase of NEFA concentration.High concentration of NEFA caused oxidative stress injury and apoptosis.The damage of cell membrane,the destruction of organelle structure and the formation of apoptotic bodies were observed by projection electron microscope.(2)The results of CCK8 showed that quercetin concentrtion below 10 μmol/L can increase cell viability and damage cells when it exceeded 10 μmol/L.The results of q PCR showed that compared with the control group,the expressions of Caspase-3,Cyt-C,Bax,Caspase-9 and Apaf-1 in the NEFA group increased,while the expression of Bcl-2 decreased;compared with the NEFA group,the expressions of Apaf-1,Bax,Cyt-C,Caspase-9 and Caspase-3 in the combined administration group of quercetin and NEFA decreased,and the expression of Bcl-2 increased.Western-blot results showed that compared with the control group,the expressions of Caspase-3,Cyt-C,Bax,and Caspase-9 in the NEFA group were increased,and the expression of Bcl-2 was decreased;compared with the NEFA group,the expressions of Bax,Cyt-C,Caspase-9 and Caspase-3 in the combined administration group of quercetin and NEFA decreased,while the expression of Bcl-2 increased.Conclusion: High concentrations of NEFA can induce oxidative damage in BEND cells,leading to apoptosis of BEND cells.Quercetin can effectively antagonize NEFA induced BEND cell apoptosis by inhibiting the mitochondrial apoptosis pathway and regulating the expression of apoptosis related genes.