| Alzheimer’s disease (AD) is a progressive neurodegenerative disease, which is clinically characterized by the presence of cognitive impairment and daily living ability deterioration. The cause of AD is hidden, the cost for treatment is high and it is not cured, which increases psychological pressure and financial burden for families and communities. Phycial activity and foods rich in resveratrol is an effective intervention for prevention and remission of AD, however, the exact molecular mechanism is unclear. Accumulating evidence from research revealed that abnormal amyloid (Ap) deposit is the most important pathogenic factor of AD, which result from more Aβ production or reduction of Aβ clearance. Further researches provide evidences that different pathological changes in the AD mice are closely associated with Aβ deposit, such as energy metabolism disorders, autophagosome accumulation, and microglial activation. Based on the above, we focused on the effect of aerobic exercise and resveratrol on amyloid deposit in hippocampus of APP/PS1 transgene mice and its molecular mechanisms.OBJECTIVEIn this study, we explore the effect of 3 months aerobic treadmill exercise and resveratrol on memory and hippocampal amyloid deposit of APP/PS1 transgenic mice. And we discussed the possible molecular mechanisms of reduction of amyloid by aerobic exercise and resveratrol from the generation of amyloid, clearance of amyloid and activation of microglia, with AMPK/Sirtl as a starting point.METHODS1. Twenty-four six-month-old Tg APP/PS1 mice and twenty-four littermates wide-type mice were divided into transgenic group (Tg, n=24) and wild type group (WT, n=24).They were subjected to Morris water maze test and new object recognition test. Twenty-four hours later, six mice from each group were randomly picked and heart perfusion was done by 0.9% Nacl and 4% paraformaldehyde in turn to pre-fixed brain. The fixed brain was used as paraffin section. Six mice from each group were anesthetized and the hippocampus was isolated. Left hippocampus were used for qRT-PCR experiment, right hippocampus were used for western blot. Another six mice from each group were killed to isolate the hippocampus. Left hippocampus were used to test cholesterol by Elisa, right hippocampus were used for HMGCR activity. The rest of six mice from each group were subjected to heart perfusion of 0.9% Nacl toisolate the hippocampus, which were used to gain single cell suspension for flow cytometry.2. Ninety-six three-month-old Tg APP/PS1 mice were divied into transgenic control group (TC, n=24), transgenic exercise group (TE, n=24), transgenic resveratrol group (TR, n=24), and transgenic exercise and resveratrol group (TRE, n=24).TE and TRE group were subjected to treadmill exercise while TC and TR group stay on the treadmill and move freely, 1h after exercise training, TR and TRE receive resveratrol lavage (resveratrol dissolved in 0.5% carboxymethylcellulose sodium, 1mg/ml, a dose of 20mg/kg body weight).TC and TE group receive 0.5% carboxymethylcellulose sodium lavage. After 12 weeks intervention, they were subjected to Morris water maze test and new object recognition test. Twenty-four hours later, six mice from each group were randomly picked and heart perfusion was done by 0.9% Nacl and 4% paraformaldehyde in turn to pre-fixed brain. The fixed brain was used as paraffin section. Six mice from each group were anesthetized and the hippocampus wereisolated. Left hippocampus were used for qRT-PCR, right hippocampus were used for western blot. Another six mice from each group were anesthetized and the hippocampus was isolated. Left hippocampus was used for cholesterol levels by Elisa, right hippocampus were used for HMGCR. The rest of six mice were subjected to heart perfusion of 0.9% Nacl toisolate the hippocampus, which were used to gain single cell suspension for flow cytometry.Detection index of 1 and 2 are as follows:1) Indicators related to behavior and Senile plaques:Morris water maze was used to examine spatial learning and memory. New object recognition test was used to to examine recognition memory, Immunohistochemistry was used to examine Aβ deposit.2) Indicators Related to APP hydrolysis and AMPK/Sirtl pathway:qRT-PCR was used to determine mRNA levels of ADAM 10, BACE1, AMPK, Sirtl, CaMMKβ, HMGCR; Western blot was used to examine protein levels of APP, ADAM 10, BACE1, PS1, Aβ, AMPK, p-AMPK-Thrl72. Sirtl. CaMMKβ\Flotillinl; Elisa was used to test cholesterol levels and HMGCR activity.3) Indicators related to autophagy flux:qRT-PCR was used to determine mRNA levels of ULK1, Beclinl, Atg5, Atg12, Atg16L, LC3, p62, LAMP1; Western blot was used to examine protein levels of ULK1, p-ULK1-Ser555, p-ULK1-Ser757, Beclinl, Atg5, Atg12, Atg16L, LC3Ⅱ,LC3 Ⅰ,p62, LAMP1.4) Indicators related to microglia:qRT-PCR was used to determine mRNA levels of IL-1β, IL-6, TNFα, IL-10, TGF-β, NF-αB p65; Western blot was used to examine protein levels of NF-αB p65; FACS sorting CD11b+/CD45+cells, CD11b+/CD86+cells, CD11b+/ CD206+cells.RESULTS1)Comparing with WT group, in the orientation navigation experiment, the escape latency and pathlength of Tg were increased significantly, however the pathlength and time percent in platform quadrant of Tg had a significant reduction; the crossing times of Tg was downregulated in space probe test; and the discrimination index was decreased significangly. At the same time, the Aβ deposit aera and numbers of Tg were upregulated.2)The protein expression of APP, BACE1, PS1, Aβ, Flotillinl were higher (p<0.05) while the mRNA and protein levels of AMPK CaMKKP and activity of HMGCR were lower (p<0.05) in Tg group than WT group.3) Compared to WT, the protein expression of Atgl2, Atg16L were downregulated (p<0.05), while the mRNA expression of p62, the protein levels of p62, LAMP1, and rate of LC3 II /LC3 I were upregulated (p<0.05) in Tg group.4) The quantity of CD11b+/CD45+cells and the mRNA expresson of TNFa, IL-Aβ, IL-6, IL-10 were more (p<0.05) while the rate of M2/M1 were less (p<0.05) in Tg group than WT group.5) In the orientation navigation experiment, compared with TC, the escape latency and pathlength were decreased significantly, however the pathlength and time percent in platform quadrant of TE, TR, TRE had a significant increased; In space probe test, the crossing times and pathlength percent in platform quadrant were upregulated in TE, TR, TRE, compared to TC; In the new object recognition test, the discrimination index of TRE more than TC; And, the Aβ deposit aera and numbers were downregulated in TE, TR, TRE than TC.6) Compared with TC, the protein levels of Aβ, Flotillinl, the activity of HMGCR, the content of cholesterol were decreased (p<0.05) while the the protein levels of ADAM 10, AMPK, Sirtl, CaMKKβ and phosphorylation of AMPK were increased (p<0.05) in TE group; the protein levels of Aβ, Flotillinl were decreased (p<0.05) while the the protein levels of AMPK, Sirtl, CaMKKβ and phosphorylation of AMPK were increased (p<0.05) in TR group; the protein levels of APP, BACE1, PS1, Aβ, Flotillinl, the activity of HMGCR, the content of cholesterol were decreased (p<0.05) while the the protein levels of ADAM 10, Sirtl, CaMKKβ and phosphorylation of AMPK were increased (p<0.05) in TRE group.7) The protein expression of ULK1 and Atg5, the phosphorylation of ULK1 Ser555, the rato of LC3Ⅱ/LC3 Ⅰ higher (p<0.05) while phosphorylation of ULK1 Ser 757, the protein levels of p62 and LAMP1 lower (p<0.05) in group TE than TC; The protein expression of Atg12, the phosphorylation of ULK1 Ser555, the rato of LC3Ⅱ/LC3 Ⅰ higher (p<0.05) while phosphorylation of ULK1 Ser 757, the protein levels of p62 lower (p<0.05) in group TR than TC; The protein expression of Beclin1, Atg5 and Atg12, the phosphorylation of ULK1 Ser555, the rato of LC3 Ⅱ/LC3 Ⅰ higher (p<0.05) while phosphorylation of ULKl Ser 757, the protein levels of p62 lower (p<0.05) in group TRE than TC.8) Compared to TC, The quantity of CDllb+/CD45+cells, the NF-κB p65 protein expression and the mRNA expresson of TNFα, IL-1β, IL-6 were downregulated (p<0.05) while the rate of M2/M1 and the mRNA expresson of IL-10, TGF-β were upregulated (p<0.05) in TE, TR, TRE.CONCLUSIONS1) In Six-month-old APP/PS1 mice, the spatial learning and memory, recognition memorywere impaired, accompanied with hippocampal amyloid deposit. However, aerobic exercise and resveratrol could inhibited amyloid deposit, enhanced spatial learning and memory, recognition memory.2) The APP hydrolysis swithched to amyloidogenic pathway, which increased amyloid levels in six-month-old APP/PS1 mice. However, on the one hand, aerobic exercise and resveratrol could increase ADAM 10 levels by AMPK/Sirtl pathway to inhibit production of amyloid. On the other hand, it could decrease BACE1 expression by AMPK/Sirtl to inhibit amyloid production. In additon, aerobic exercise and resveratrol could inhibit HMGCR activity by activating AMPK to downregulate cholesrol content as well as lipid rafts, which promoted the hydrolysis of APP thusreducing production of amyloid.3) The autophagy degradation pathway was abnormal in six-month-old APP/PS1 mice, leading to autophagosome accumulation which inhibited the clearance of amyloid. However, aerobic exercise and resveratrol could increase the autophagic flux and promote amyloid clearance.4) The quantity of microglia was upregulated while the ratio of M2/M1 was downregualted in six-month-old APP/PS1 mice, which induced inflammation. However, aerobic exercise and resveratrol could increase microglia to reduce inflammation by activating AMPK and inhibiting NF-κB. And aerobic exercise and resveratrol decreased M1 microglia to suppresse pro-inflammatory cytokines secretion, while increasing M2 microglia to promote anti-inflammatory cytokines secretion. |
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