Qiongyu Paste Inhibits The Aging Of Zea Fish Induced By H₂O₂ Through Sirt1/p53 Signaling Pathway

Purpose:By building H₂O₂induced oxidative damage model of zebrafish embryos,use qiongyugao to intervention,,analyze ROS,SOD,gsh-px,the survival rate and the generation of ROS of zebrafish determined the influence of qiongyugao on zebrafish embryos oxidative damage;Through the analysis of the expression levels of aging-related proteins sirt1,p53,p21 and its gene mRNA expression levelsOf zebrafish embryo,compared the more normal zebrafish embryos group,H₂O₂induction of zebrafish embryos oxidative damage and aging group and different concentrations of qiongyugao intervention group of the differences in gene and protein expression,discussion about the molecular mechanism of qiongyugao against oxidative damage through the sirt1/p53signaling pathways.It provides experimental reference and data support for the possible molecular mechanism of anti-aging of qiongyugao.Method:4 hours after fertilization of zebrafish?HPF?,the zebrafish embryo is divided into A,B,C,D,E five groups,group A as the normal control group,group B was 0.5 mM H₂O₂final concentration of the fertilized egg incubation48 h,C,D,E group,respectively,200,100,50 ug/ml of qiongyugao incubation1 h in the reproduction of the preparation of water of H₂O₂-induced group,record the growth rate of seedlings?The activity of superoxide dismutase?SOD?and glutathione peroxidase?gsh-px?in zebrafish embryos was detected by spectrophotometry.Observe The expression levels of senescence related protein and its gene mRNA of H₂O₂induced oxidative damage in zebrafish embryos,Western Blot detected the protein expression level of Sirt1,p53 and p21.The expression level of mRNA of Sirt1,p53 and p21 gene was detected by real-time PCR.SPSS19.0 statistical software was used for statistical analysis.Immuno of luorescence method was used to detect the expression of sirt1 in the hypothalamus of zebrafish.Rusults:1.Compared with untreated normal embryo group?control group?,H₂O₂induction group significantly reduced cell viability.The C?D?E group?200,100 and 50 ug/ml?treated by qiongyugao increased significantly?p<0.05?.2.Qiongyugao under a dose dependent significantly inhibits the generation of reactive oxygen species,compared with H₂O₂induced group,qiongyugao group generation of reactive oxygen species significantly reduced,and the results show that qiongyugao on H₂O₂induced oxidative stress has a strong protection.3.The activity of SOD and gsh-px in zebrafish embryo of qiongyugao group was significantly increased?P<0.05?,compared with the normal group and H₂O₂group,the content of SOD and gsh-px in the qiongyugaogroup increased significantly.4.compared with H2O2 induction group,zebrafish beta galactose glucoside enzyme staining positive rate has no obvious influence with 50 ug/ml concentration of Qiongyugao,in Qiongyugao concentration to 100 ug/ml staining positive rate drop,when Qiongyugao 200 ug/ml concentration effective intervention in zebrafish aging.5.The expression level of Sirt1 protein and gene mRNA was significantly increased in the qiongyugao group,and there was significant difference between the normal group and the H₂O₂induction group?p<0.05?.6.The expression levels of p53,protein and gene mRNA were significantly reduced in the qiongyugao group,and there was a significant difference between the normal group and the H₂O₂induction group?p<0.05?.7.The expression level of p21 protein and gene mRNA was significantly reduced in the qiongyugao group,and there was significant difference between the normal group and the H₂O₂induction group?p<0.05?.8.for cell survival,the generation of reactive oxygen species and the doseofQiongyugaoexistcertaindependencies,200ug/mlqiongyugao intervention group compared with H₂O₂induced model group cell viability recovered significantly?p<0.05?,and 200 ug/ml qiongyugao intervention group compared with H₂O₂induced model group the generation of reactive oxygen species decreased significantly?p<0.05?.9.The immunofluorescence method detected a clear red fluorescent signal in sirt1 in the hypothalamus of zebrafish,indicating that sirt1 was positive in the hypothalamus of zebrafish.Conclusion:1.Normal cells?control group?and H₂O₂induced cells and qiongyugao group processing cells survive have obvious difference,that qiongyugao can increase the survival rate of zebrafish embryonic cell in dose dependent.2.The intervention of qiongyugao has a strong oxidative protective effect on the oxidative damage of zebrafish embryo,and can effectively reduce the oxidative damage of the cells in zebrafish embryo tissue.3.Sirt1,p53,p21 protein and gene mRNA expression levels in the normal control group and H₂O₂induced group and qiongyugao intervention group have significant difference,indicating p53?p21protein and gene mRNA expression level in qiongyugao showed a trend of gradually reduce the intervention.Sirt1protein and gene mRNA expression level in qiongyugao increased.4.The possible molecular mechanism of qiongyu paste anti-aging is to anti-ageing possible molecular mechanism is to reduce the expression of p53,p21,increase the expression of sirt1,which regulates the sirt1/p53 signal pathway,lower the content of active oxygen in the cells and strengthen the protection of oxidative damage.