Protective Effects Of Procyanidins On Oxidative Damage Of Bovine Endometrial Cells Induced By Palmitic Acid

[Objective]The content of free fatty acid(NEFA)in blood of ketosis cows was significantly increased,while palmitic acid(PA)was the main component of NEFA.The aim of this study is to develop cow endometrial epithelial cells(BEND)by in vitro detection,shows that PA indicators of cow endometrial epithelial cells oxidative damage and explore the proanthocyanidins(PC)as its antioxidant effects on Nrf2/ARE signal pathway and help cow endometrial epithelial cells(palmitic acid resistance PA)mechanism of oxidative damage induced by it.It lays a foundation for further study of ketosis and the effects of PA and PC on the reproductive performance of dairy cattle.[Methods]BEND cells were cultured separately and co-cultured at different time points 6h,12 h,24h and 48 h with palmitic acid(PA)and proanthocyanidin(PC),respectively.The relative survival rate of BEND cells was detected by CCK-8 assay.The oxidation index of Catalase(CAT),Glutathione(GSH),Glutathione Peroxidase(GSH-PX),Malondialdehyde(MDA),Superoxide Dismutase(SOD)and Total Antioxidant Capacity(T-AOC)were detected by oxidation kit.Finally,fluorescent PCR and Western-blot was used to detect the expression of mRNA and its proteins,such as nuclear factor E2 related factor(Nrf2)and its downstream target genes,such as Heme Oxygenase1(HO-1),Quinoneoxidoreductase(NQO1),Glutamylcysteine ligase catalyticsubunit(GCLC),Superoxide Dismutase(SOD),and so on.To investigate the protective effect of procyanidin on oxidative damage of BEND cells induced by(PA).[Results]1.PA has a role in oxidative damage to BEND cells was detected by CCK-8 assay.And in a certain range,the PC can antagonize the oxidative damage caused by the palmitic acid PA to play a protective effect on BEND cells.The results showed that when the concentration of PA was100-300?mol/L,the relative survival rate of native cells was not significantly changed(P>0.05),when the concentration of PA was 400 ?mol/L,the relative survival rate of the cell was significantly decreased(P<0.05),and when the concentration of PA was greater than 600?mol/L,the relative survival rate of the cell was decreased significantly(P<0.01).And when the concentration of PC was less than 10?mol/L,it could protect BEND cells.Once the concentration of PC was higher than 10?mol/L,the BEND cells would be inhibited,and the effect was dose-dependent.When PA and PC were co-treated with BEND cells,PC could resist the oxidative damage induced by PA,and the best effect was found at different time points,such as 6h,12 h,24h,48 h,and so on.2.The oxidation index of CAT GSH GSH-PX MDA SOD and T-AOC were detected.Theresults showed that compared with the blank group,the CAT activity of BEND cells in PA group decreased significantly(P<0.05).The activity of GSH GSH-PX SOD T-AOC decreased significantly(P<0.01).The content of MDA increased significantly(P< 0.01).The activity of CAT SOD T-AOC in BEND cells of PC group increased significantly(P<0.05).GSH and GSH-PX increased significantly(P<0.01),and MDA content decreased significantly(P<0.05).Compared with the corresponding PA group,the activity of CAT in PA+PC group showed an increasing trend.The activity of T-AOC increased(P<0.05)and the activity of GSH GSH-PX and SOD increased significantly(P<0.01).The content of MDA decreased significantly(P<0.05),indicating that PC could antagonize the oxidative damage induced by PA on BEND cells.3.The mRNA of BEND cells detected by fluorescence quantitative PCR showed that compared with the blank group,the mRNA expression of Nrf2 and GCLC genes in the PA group increased,but the effect was not significant.The mRNA expression of NQO1 gene increased significantly(P<0.05)and the mRNA expression of HO-1 gene increased significantly(P <0.01),while the mRNA expression of SOD gene decreased slightly.The mRNA expression of Nrf2 and SOD genes in PC group was significantly increased(P<0.05)and the mRNA expression of GCLC?NQO1 and HO-1 gene was significantly higher increased(P<0.01).Compared with PA group,the mRNA expression of Nrf2?GCLC gene in PA+PC group was not significantly higher than that in PA group.The mRNA expression of NQO1?HO-1 and SOD gene in PA+PC group was significantly higher than that in PA group(P<0.05).4.The protein expression of BEND cells was detected by Western blot.It was found that the protein expression in the PC group of NQO1 was significantly higher than that in the blank group(P<0.05),but the increase and decrease of the protein expression in the other groups only showed a trend,but the effect was not significant.Compared with the control group,the protein expression of SOD gene in PA group showed a downward trend,and the protein expression of the remaining Nrf2?GCLC?HO-1?NQO1 gene in PA group?PC group and PA+PC group showed an increasing trend compared with the control group.[Conclusion]Palmitic acid can induce oxidative damage in BEND cells,and procyanidins can antagonize the oxidative damage induced by palmitic acid.The antagonism of palmitic acid can be achieved through the Nrf2/ARE signaling pathway,thus protecting the cells.