The Mechanism Of Zinc Glycine Protecting Oxidative Stress Chick Embryos Based On PI3K/Akt/GSK-3β Signaling Pathway

Eggs are susceptible to oxidative stress caused by the external environment due to physiological reasons in the late incubation period,thus reducing the survival rate of chicken embryos in the late incubation period and affecting the hatching rate of eggs.The Nrf2endogenous antioxidant pathway is essential for cells to resist external stress responses.Our previous studies have found that the addition of zinc glycine（Zn-Gly）to the broiler breeder diet can reduce the late mortality of chicken embryos by improving the antioxidant capacity of chicken embryos;at the same time,the establishment of chicken embryo oxidative stress model showed that maternal zinc glycine increased the expression of Nrf2,increased the antioxidant enzyme activity of chicken embryo tissue,and decreased the activity of apoptosis enzyme,thereby alleviating the oxidative stress injury of chicken embryo and reducing the mortality of chicken embryo.However,the mechanism of maternal zinc glycine in reducing the late mortality of chicken embryos remains to be studied.PI3K/Akt pathway is one of the important cell survival pathways,and it is an important signal transduction pathway for cell growth,proliferation,apoptosis and anti-oxidative stress in many different cell types.Recent studies have shown that zinc can activate the PI3K/Akt signaling pathway and play a protective role in related tissues.At the same time,PI3K/Akt signaling pathway can regulate Nrf2 through GSK-3β,so we speculate that PI3K/Akt/GSK-3βsignaling pathway mediates the protective effect of maternal zinc glycine on chicken embryos under oxidative stress by affecting Nrf2 expression.The purpose of this study was to explore the protective effect of zinc glycine on oxidative stress cells and the change rule of PI3K/Akt/GSK-3βsignaling pathway in this process by establishing an oxidative stress model of chicken liver cells,and to establish a cell inhibition model of PI3K/Akt/GSK-3βsignaling pathway by adding inhibitors to clarify its role in zinc glycine anti-oxidative stress.In this experiment,CCK-8 method and ELISA method were used to detect the survival rate and LDH enzyme activity of LMH cells,and the H₂O₂oxidative stress model of LMH cells was established to determine the dosage and treatment time of H₂O₂ and glycine zinc.LMH cells were treated with 2（2 kinds of H₂O₂ treatment concentration）×2（2 kinds of glycine zinc treatment concentration）factor completely randomized experimental design.The apoptosis rate was detected by flow cytometry.ELISA was used to detect oxidative stress-related oxidation indicators（MDA,protein carbonyl and8-OHd G）and antioxidant indicators（Cu Zn-SOD）.The m RNA and protein levels of Nrf2 in the antioxidant pathway were detected by immunofluorescence and real-time fluorescence quantitative PCR.Subsequently,q RT-PCR and Western blot were used to detect the changes of PI3K/Akt/GSK-3βsignaling pathway related genes in the process of Zn-Gly anti-oxidative stress.The changes of PI3K/Akt/GSK-3βsignaling pathway related genes and proteins in the process of Zn-Gly anti-oxidative stress were detected by q RT-PCR and Western blot.In order to further explore the anti-oxidative stress effect of Zn-Gly on PI3K/Akt/GSK-3βsignaling pathway in LMH cells,a cell inhibition model of PI3K/Akt/GSK-3βsignaling pathway was established to detect the expression of genes and proteins related to PI3K/Akt/GSK-3βsignaling pathway.The test results show that:1.The optimal treatment time and concentration of H₂O₂ oxidative stress model of LMHcells were 6 h and 800μmol/L,respectively.The optimal treatment time and concentration of Zn-Gly were 6 h and 20μmol/L,respectively.2.Compared with the blank control group,the apoptosis rate and the levels of MDA,protein carbonyl and 8-OHd G in the H₂O₂ treatment group were significantly increased（P<0.05）.The activity of Cu Zn-SOD,the m RNA expression of Nrf2 and the relative fluorescence intensity of Nrf2 protein were significantly decreased（P<0.05）.The nuclear expression of Nrf2 protein was decreased,the m RNA expression of PI3K and Akt was significantly increased（P<0.05）,and the p-Akt/Akt and p-GSK-3β/GSK-3βwere significantly increased（P<0.05）.The m RNA expression of GSK-3βwas significantly decreased（P<0.05）.After LMH cells were treated with 20μmol/L Zn-Gly+800μmol/L H₂O₂,compared with the H₂O₂ treatment group,the apoptosis rate,MDA,protein carbonyl and 8-OHd G levels in the Zn-Gly+H₂O₂ treatment group were significantly decreased（P<0.05）,the Cu Zn-SOD activity and the relative fluorescence intensity of Nrf2 protein were significantly increased（P<0.05）,the nuclear expression of Nrf2 protein was increased,and the m RNA expression of PI3K and Akt was significantly decreased（P<0.05）.The p-Akt/Akt and p-GSK-3β/GSK-3βwere significantly decreased（P<0.05）,and the m RNA expression of GSK-3βwas significantly increased（P<0.05）.3.The optimal treatment time and concentration of GSK-3βinhibitor SB216763 were 6 h and 10μmol/L,respectively.After LMH cells were treated with inhibitor+Zn-Gly+H₂O₂,compared with the Zn-Gly+H₂O₂ treatment group,the apoptosis rate and protein carbonyl activity of the inhibitor+Zn-Gly+H₂O₂ treatment group were significantly decreased（P<0.05）,the Cu Zn-SOD activity,Nrf2 m RNA expression and Nrf2 protein relative fluorescence intensity were significantly increased（P<0.05）,the nuclear expression of Nrf2protein was increased,and the expression of Akt and GSK-3βprotein was significantly decreased（P<0.05）.The p-Akt/Akt and p-GSK-3β/GSK-3βwere significantly decreased（P<0.05）.In summary,zinc glycine may protect oxidative stress cells by reducing p-Akt/Akt and p-GSK-3β/GSK-3βin PI3K/Akt/GSK-3βsignaling pathway,increasing Nrf2 protein expression and nuclear distribution in cells,and ultimately increasing antioxidant enzyme Cu Zn-SOD activity and reducing apoptosis rate.The results of this study showed that PI3K/Akt/GSK-3βsignaling pathway mediates the protective effect of zinc glycinate on oxidative stress cells,which provides a theoretical basis for zinc glycinate to improve the hatching rate of eggs in meat poultry production.