Age-related Expressions Of Oxidative And Inflammatory Response In SAMP8 And The Intervention Of SS31

Objectives: Alzheimer's disease(AD) is an age-related neurodegenerative disease characterized clinically by a progressive cognitive decline in learning and memory in AD patients, pathologically by neuronal loss, accumulation of extracellular deposits of amyloid-beta(A?), granular vacuolar degeneration in hippocampus, and formation of neurofibrillary tangles(NFTs). The incidence of people to be affected with AD is 7%~10% beyond 65 years of age, which brings heavy burden to families and the society. However, the disease mechanism in AD is still not completely understood, which may be due to aging process, the accumulation of A? peptides, enhanced chronic neuroinflammation and oxidation. The accumulation of A?, the major component of senile plaques in human brain, has been suggested to be a critical hallmark of AD pathology and neurodegeneration. Nuclear factor kappa B(NF-?B) is a stress-sensing transcription factor, which regulates the expression of genes involved in immune and inflammatory responses. Stimulated by various stressors, pro-inflammatory genes are activated and encoded proteins are expressed to regulate cell division and apoptosis. Nuclear factor erythroid 2-related factor 2(Nrf2) is a transcription factor essential for the regulation of a series of genes encoding anti-oxidative and detoxifying enzymes to guard against oxidative stress, by activating the Nrf2/antioxidant response element(ARE) signaling pathway. Senescence accelerated mouse prone 8(SAMP8) is a strain of mice shows accelerated aging. This model exhibits many of the behavioral and pathological characteristics of AD and has been proposed as an excellent animal model to study the chronology of neurodegenerative changes in AD development and possible therapeutic strategies. SAMR1 shows normal agingand acts as control. SS31(H-D-Arg-Dmt-Lys-Phe-NH2), a novel mitochondria-targeted antioxidant peptide, easily penetrates the blood brain barrier and targets mitochondria. SS31 have shown the capability of reducing generation of mitochondrial reactive oxygen species(ROS) and protecting against oxidative damage and inflammation, protecting mitochondria and synapsis. To date, the molecular mechanisms underlying SS31 is not completely characterized and whether this compound has protective effect in AD is still unknown. Here we describe the age-related changes in A?, NF-?B and Nr2/ HO-1 in the hippocampus of 4, 8, 12 months SAMP8, with a special emphasis on the protective effects of the mitochondrial-targeted antioxidant SS31 in SAMP8 mice for 8 weeks, which may be a novel therapeutic strategy for AD.Methods: All clean SAMP8 and SAMR1 male mice were purchased from the Tianjin University of Traditional Chinese Medicine with their body weight from 25 g to 32 g. They were fed in the Clinical Research Center of Hebei General Hospital with standard food and water. Study 1: 18 SAMP8 mice were randomly separated into 3 groups(n=6) and cultivated till 4-month(4M group), 8-month(8M group) and 12-month(12M group) respectively. Study 2: 6 8-month-old saline-treated SAMR1 mice were cultivated 8 more weeks with standard food and water, as the control group. 12 8-month-old SAMP8 mice were randomly separated into 2 groups: saline-treated SAMP8 group were cultivated 8 more weeks with standard food and water, the mice from SS31-treated group were intraperitoneal injected with SS31 at a density of 1 n M as optimal for 8 weeks. Each group was injected at a dose of 5mg/kg/day. The hippocampus was dissected and RNA and protein were extracted from all mice. The protein levels of A??NF-?B p65?HO-1, as well as nuclear and cytoplasmic Nrf2 were detected by western blots. The gene expressions of NF-?B?Nrf2?HO-1 were tested by RT-q PCR.Results: 1 The A? deposition, NF-?B and Nrf2/ARE signaling pathway changed withage in the hippocampus of SAMP8 in accelerated aging process 1.1 The protein level of A?42 was upregulated in aging process The protein levels of A?42 increased gradually in 4M, 8M and 12 M. Compared with 4M, the levels of 12 M increased significantly(P<0.05), indicating the neurotoxic A?42 increased with age in the hippocampus of SAMP8(Fig.2B). 1.2 The activation of NF-?B signaling increased with age in aging process The gene expressions and protein levels of NF-?B p65 increased gradually in 4M, 8M and 12 M. Compared with 4M, the levels of 12 M increased significantly(P<0.05), demonstrating that the activation of NF-?B signaling was increased with age and was accompanied by increased inflammatory response(Fig.1A, Fig.2A). 1.3 Nrf2/ARE signaling pathway was activated during aging The gene expressions of Nrf2 increased gradually in 4M, 8M and 12M(Fig.1B). Protein amounts of N-Nrf2 increased slowly in 4M, 8M and 12M(Fig.3A), while C-Nrf2 decreased accordingly(Fig.3B). Results indicated that Nrf2 translocated gradually into nucleus with increasing age. The downstream detoxifying and antioxidant enzyme HO-1, which was regulated by Nrf2/ARE, changed as the way of Nrf2, indicating the slowly upregulated antioxidant effect(Fig.1C, Fig.2C). 2 The levels of A? deposition increased, with NF-?B and Nrf2/ARE signaling pathway activated in SAMP8 relative to SAMR1 2.1 The A? deposition was increased and NF-?B pathway was activated in SAMP8 Compared with SAMR1, the protein levels of A?42(Fig.4B) and NF-?B p65(Fig.4A) increased significantly in 10-month-old SAMP8, as well as the gene expressions of NF-?B p65(P<0.05)(Fig.1D). 2.2 The Nrf2 signaling pathway was activated in SAMP8 Compared with SAMR1, the gene expressions of Nrf2 and HO-1 increased significantly in 10-month-old SAMP8, as well as the protein levels of N-Nrf2?HO-1(P<0.05)(Fig.1D, E, Fig.5A, Fig.4C).3 SS31 ameliorated A? deposition,inhibited NF-?B signaling and activated Nrf2/ARE pathway 3.1 SS31 reduced A?42 deposition The protein level of A?42 decreased significantly after the treatment of SS31(P<0.01), indicating SS31 decreased A?42 level significantly(Fig.4B). 3.2 SS31 inhibited NF-?B signaling and decreased inflammatory response Compared with SAMP8, the gene expression and protein level of NF-?B p65 decreased significantly after the treatment of SS31 for 8 weeks(P<0.01), demonstrating SS31 inhibited NF-?B signaling and ameliorated inflammatory response(Fig.1D, Fig.4A). 3.3 SS31 activated Nrf2/ARE pathway and increased the anti-oxidative ability SS31-treated SAMP8 exerted higher gene expressions(Fig.1E, F) and protein levels(Fig.4C, Fig.5A) of Nrf2 and HO-1, indicating the further activated Nrf2/ARE pathway and stronger anti-oxidant effect.Conclusions: 1 The levels of A? deposition were increased and the NF-?B and Nrf2/ARE signaling pathway were activated gradually with age in the hippocampus of SAMP8 during aging process. 2 The antioxidant SS31 peptide may be a potential therapeutic treatment for AD with the ability of inhibiting NF-?B signaling, activating Nrf2/ARE pathway and reducing A? deposition, indicating that SS31 may be a potential therapeutic strategy for AD.