Mechanistic Study Of Small Peptides From Periplaneta Americana Resist Oxidative Stress-induced Apoptosis In Pig Granulosa Cells

ObjectiveIn the practical pig production,environmental factor such as intensive and large-scale feeding,high temperature and high altitude is the primary reason to cause oxidative stress of sows.Oxidative stress can induce apoptosis of granulosa cells and lead to abnormal follicular atresia,thereby reducing the sow’s breed function.Small peptides are prepared from the body of the Periplaneta americana.It has relatively strong in vitro and in vivo antioxidant activities.The aim of this study was to investigate the protective effect of small peptides from Periplaneta americana on the apoptosis of pig-ovary granulosa cells induced by hydrogen peroxide（H₂O₂）and its anti-apoptotic signaling pathway,which provides a theoretical basis for improving the litter size and reproductive efficiency of pigs and a new idea for the development and application of small peptides from Periplaneta americana in livestock and poultry ecological breeding.MethodsPig-ovary granulosa cells were collected and cultured.Granulosa cells were treated with different concentrations of H₂O₂for different periods of time.Cell viability was measured using the cell counting kit-8（CCK-8）assay,and cell apoptosis was detected using flow cytometry to determine the optimal concentration and time of H₂O₂for establishing a cell apoptosis model.Then the oxidatively damaged granulosa cells were treated with different concentrations of small peptides for different periods of time.Cell viability was detected using the CCK-8 assay,and the malondialdehyde（MDA）and nitric oxide（NO）contents,reactive oxygen species（ROS）levels and superoxide dismutase（SOD）activities were detected to determine the optimal repair concentration and time of small peptides.TUNEL assay,q RT-PCR,western blotting and Fox O1transfection were undertaken to analyze the protective effect of small peptides on oxidative stress-induced apoptosis in pig-ovary granulosa cells and its key regulatory factors.Finally,western blotting,immunofluorescence,coimmunoprecipitation and apoptosis detection were used to determine the signaling pathway of small peptides resist oxidative stress-induced apoptosis in pig-ovary granulosa cells.Results（1）Pig-ovary granulosa cells were treated with 100μmol/L H₂O₂for 6 hr,which significantly reduced cell viability and resulted in apoptosis and an apoptosis rate for cells of 32.95%（P<0.01）;（2）Cells were treated with 400μg/m L small peptides for 24hr to improved cell viability in H₂O₂-stimulated cells.The increased MDA levels,NO contents and ROS levels and decreased activities of SOD caused by H₂O₂stimulation were reversed by small peptides.Small peptides downregulated the m RNA levels of Caspase-3,Bax and Fas L,thus inhibiting H₂O₂-induced apoptosis in granulosa cells.Meanwhile 400μg/m L small peptides had no negative effect on granulosa cells（P<0.01）;（3）Compared with other members of Fox O family,the inhibition of small peptides on Fox O1 in H₂O₂-induced granulosa cells was the most obvious.Small peptides significantly increased the p-Fox O1/Fox O1 expression ratio,thereby decreasing Caspase-3 expression in H₂O₂-induced cells.The effect of small peptides was the same following Fox O1 overexpression（P<0.01）;（4）Small peptides significantly promoted the combination of Fox O1 with the 14-3-3 protein,increased Fox O1 translocation to the cytoplasm and decreased Fox O1 acetylation in H₂O₂-induced cells（P<0.01）;（5）Small peptides significantly reduced p-JNK/JNK expression ratio and increased the p-Fox O1/Fox O1 expression ratio,thereby decreasing Caspase-3 expression and cell apoptosis in H₂O₂-induced cells（P<0.01）.ConclusionSmall peptides from Periplaneta americana have the inhibited effect on the oxidative stress-induced apoptosis in pig-ovary granulosa cells,which effects the translocation of Fox O1 to the cytoplasm by the Fox O1 interaction with 14-3-3 and the JNK/Fox O1 signaling pathway,enabling reversal of the oxidative stress-induced apoptosis of pig-ovary granulosa cells.Acetylation of Fox O1 is also involved in the regulatory effect of small peptides.