The Age-related Changes Of Apoptosis-related Factors In SAMP8 Mice And The Intervention Of SS31 Peptide

Alzheimer’s disease(AD)is the most common type of dementia and a progressive neurodegenerative disease with no cure up to date.As the world population aging,AD has become a major risk factor that threating the human health,but its etiology and pathogenesis is still not entirely clear.The typical histopathological changes in AD are: β-amyloid deposition(Aβ),neurofibrillary tangles,and extensive neuronal loss while apoptosis has been shown playing an extremely important role in neuronal death in AD.One of apoptosis regulatory protein family is Bcl-2 family,and anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax is particularly important in the process of apoptosis.However,mitochondrial fission also plays an important role in apoptosis.The core protein of mitochondrial fission is dynamin related protein 1(Drp1),which is a class of GTP enzymes localized in the outer mitochondrial membrane,and it is closely related to mitochondrial fission.And SS31 peptide,as a novel mitochondria-targeted antioxidant peptide,not only inhibit oxidative stress,but also reduce mitochondrial structural damage and dysfunction,thereby reducing apoptosis,playing the role of anti-AD.The establishment of the animal model of AD is the basis of in-depth study of AD,but there is no any animal model can fully simulate human AD.Late-onset AD is the most common form of AD(nearly 90%),while SAMP8 mice has also been proposed as an model of late-onset AD.It has several characteristics are similar to the clinical manifestations and pathological changes in AD patients,such as alterations in learning and memory,β-amyloid deposition,hyperphosphorylation of tau,cerebral atrophy,cortical and hippocampal neuron loss,and so on.Therefore,this study selected SAMP8 mice and control SAMR1 mice as AD model.Objective: To observe the changes of apoptosis-related proteins(Bax,Bcl-2 and Drp1)with aging in hippocampus of SAMP8 mice,and to explore the effect of SS31 peptide on these apoptosis-related proteins in the hippocampus of SAMP8 mice and its mechanism.Methods: The SAMP8 and SAMR1 mice were purchased from Tianjin University of Traditional Chinese Medicine First Affiliated Hospital Animal Center and carried out in the Clininal Research Center of Hebei General Hospital.We selected male SAMP8 mice aged 4,8,12-month-old randomly and divided into 3 groups: 4M,8M and 12M(n=10 per group).Using Western blot and RT-PCR methods to detect the effect of aging on the gene and protein expression of BAX,BCL-2 and DRP1 in the hippocampus of SAMP8 mice.Moreover,8-month-old male SAMR1(n=10)were randomly selected as normal control group,recorded as: SAMR1 group,8-month-old male SAMP8 mice(n=10)injected saline vehicle,recorded as: SAMP8 group,while 8-month-old male SAMP8 mice(n =10)injected SS31 peptide,denoted as: SS31 group.SS31 was diluted with saline,and 5 mg/kg/day by intraperitoneal injections for 8 weeks,with the vehicle group treated with saline by the same approach.Using Western blot and RT-PCR method detect the effect of SS31 on the gene and protein expression of BAX,BCL-2 and DRP1 in the hippocampus of 8-month-old SAMP8 mice.Results:1 The gene level changes of Bax,Bcl-2 in the hippocampus of SAMP8 mice with agingThe mRNA expression level of Bax gradually increased with aging in SAMP8 mice.The Bax mRNA expression levels of 4-month-old and 8-month-old mice were significantly lower than 12-month-old SAMP8 mice(P <0.01).Although the Bax mRNA expression levels of 4-month-old mice lower than the 8-month-old mice,there was no statistically significant difference.While the Bcl-2 mRNA expression in SAMP8 mice hippocampus gradually decreased with aging,the mRNA expression levels of Bcl-2 in 4-month-old SAMP8 mice were significantly higher than the 8-month-old(P <0.05)and 12-month-old mice(P <0.01).In addition,the mRNA expression Bax/Bcl-2 ratio of 12-month-old SAMP8 mice was significantly higher than 4,8-month-old mice(P <0.01).These results suggest that the gene expression of apoptosis-related factors in SAMP8 mice increased with aging.2 The protein level changes of Bax,Bcl-2 in the hippocampus of SAMP8 mice with agingThe protein levels of Bax gradually increased with aging in SAMP8 mice.The protein expression level of BAX in 4-month-old(P<0.01)and 8-month-old(P<0.05)SAMP8 mice were significantly lower than the 12-month-old mice.The Bax protein expression levels of 4-month-old less than 8-month-old mice,but there was no statistically significant difference.While the Bcl-2 protein expression in SAMP8 mice hippocampus gradually decreased with aging,the protein expression level of BCL-2 in 4-month-old SAMP8 mice were significantly higher than the 8,12-month-old mice(P <0.01).The Bcl-2 protein expression levels of 8-month-old higher than 12-month-old mice,but there was no statistically significant difference.Moreover,the protein expression Bax/Bcl-2 ratio of 8,12-month-old SAMP8 mice was significantly higher than 4-month-old mice(P<0.01).The protein expression trends of Bax,Bcl-2 consistent to the gene expression trends,suggest that the protein expression of apoptosis-related factors in SAMP8 mice increased with aging.3 The gene expression of Drp1 in SAMP8 mice hippocampus with agingThe mRNA expression levels of Drp1 gradually increased with aging in SAMP8 mice.The mRNA expression levels of Drp1 in 4-month-old(P <0.01)and 8-month-old(P <0.05)SAMP8 mice were significantly lower than 12-month-old mice.The Drp1 mRNA expression levels of 8-month-old mice higher than 4-month-old mice,but there was no statistically significant difference.Drp1 closely related mitochondrial fission,over-expression can cause excessive mitochondrial fission increased apoptosis,suggesting that SAMP8 mice mitochondrial fission increased,apoptosis increased with aging.4 The protein expression of Drp1 in SAMP8 mice hippocampus with agingThe protein expression levels of Drp1 gradually increased with aging in SAMP8 mice.The protein levels of Drp1 in 4-month-old SAMP8 mice significantly higher than 8,12-month-old mice(P <0.01).The Drp1 protein expression levels of 12-month-old mice higher than 8-month-old mice,but there was no statistically significant difference.The protein expression trend of Drp1 consistent to the gene expression trend,further suggest that SAMP8 mice mitochondrial fission increased,apoptosis increased with aging.5 The effect of SS31 peptide on the Bax,Bcl-2 gene expression levels in the hippocampus of SAMP8 mice.Compared with the peers of SAMR1 mice,the Bax mRNA expression levels in SAMP8 mice had no significant difference,but SS31 peptide significantly downregulated the Bax mRNA expression levels in SAMP8 mice(P <0.01).The mRNA expression levels of Bcl-2 in SAMP8 mice were significantly lower compared with SAMR1 mice(P <0.05),and the SS31 peptide significantly upregulated the Bcl-2 mRNA expression levels(P <0.05).For Bax/Bcl-2,although there was no significant difference between the SAMR1 group and SAMP8 group,but SS31 was significantly reduced the Bax/Bcl-2 ratio of SAMP8 mice(P <0.01).SS31 peptide downregulated gene expression level of Bax and up-regulated gene expression level of Bcl-2,indicate that SS31 can play a role against apoptosis through regulating Bax,Bcl-2 gene expression levels.6 The influence of SS31 peptide on the Bax,Bcl-2 protein expression levels in the hippocampus of SAMP8 mice.Compared with SAMR1 mice,SAMP8 mice had higher Bax protein level(P <0.01),and SS31 significantly downregulated the protein expression of Bax(P <0.01).And the Bcl-2 protein levels of SAMP8 mice were lower than SAMR1 mice(P <0.01),while SS31 peptide significantly increased the Bcl-2 protein levels in SAMP8 mice(P <0.05).SAMP8 mice had the higher Bax/Bcl-2 ratio than SAMR1 mice(P <0.01),while SS31 significantly reduced the Bax/Bcl-2 ratio of SAMP8 mice(P <0.05).The effects of SS31 on the protein expression trends of Bax,Bcl-2 similar to the gene expression trends,suggest that SS31 can play a role against apoptosis through regulating Bax,Bcl-2 protein expression levels.7 SS31 peptide can decrease the gene expression levels of Drp1 in the hippocampus of SAMP8 miceCompared with SAMR1 mice,SAMP8 mice had higher Drp1 mRNA levels,but there was no statistically significant difference(P >0.05).And SS31 peptide significantly downregulated the mRNA expression levels of Drp1(P<0.05).Drp1 as mitochondrial fission core protein,SS31 peptide significantly reduced mRNA expression level of Drp1,indicating SS31 peptide can play a role against mitochondrial fission and apoptosis by downregulating Drp1 gene expression.8 SS31 peptide can decrease the protein expression levels of Drp1 in the hippocampus of SAMP8 miceCompared with the SAMR1 mice,the DRP1 protein expression level was significantly increased in SAMP8 mice,the difference was statistically significant(P <0.05).And the SS31 peptide significantly downregulated the Drp1 protein expression levels in the hippocampus of SAMP8 mice(P <0.01).The effects of SS31 on the protein expression trend of Drp1 consistent to the gene expression trend,suggest that SS31 peptide can play a role against mitochondrial fission and apoptosis by downregulating Drp1 protein expression.Conclusion:1 The gene and protein expression of Bax and Drp1 in the hippocampus of SAMP8 mice increased with aging,and the anti-apoptotic protein Bcl-2 gene and protein expression levels decreased,affirmed that the cell apoptosis increased in SAMP8 mice with aging.2 The SS31 can down-regulate Bax and Drp1 gene and protein expression and up-regulate the gene and protein expression of Bcl-2,played an effect against hippocampal neuronal apoptosis,suggesting that SS31 may have potential therapeutic value for patients with AD.