| Objective: Myocyte enhancer factor 2C(MEF2C)exists in the nervous system in large numbers and can regulate neurodevelopment,synaptic plasticity and inflammation levels,but the mechanism of action in Alzheimer disease(AD)is unknown.To this end,brain tissue specimens from AD patients,APPswe/PSEN1 d E9 double transgenic mice,and neuroblastoma cells(SH-SY5Y)treated with amyloid peptide oligomers(A?Os)were selected as research objects.Ad-sh RNA-Mef2 c adenovirus lateral ventricle injection was used to infect APP/PS1 mice,and GV248-sh RNA-Mef2 c was transfected into SH-SY5 Y cells to silence MEF2 C to research the following: the expression and distribution of MEF2 C and A? in the brain tissue of AD patients and the cortex of APP/PS1 mice;as well as to assess potential correlations between the expression levels of MEF2 C and A? in the brain tissue of AD patients;and whether MEF2 C can reduce the production and aggregation of A?,relieve the neurotoxic effects of A? and improve the learning and memory abilities of APP/PS1 mice by regulating the Nrf2-ARE signalling pathway in APP/PS1 mice and SH-SY5 Y cells exposed to A?Os.Methods:(1)Human brain tissue specimens: Immunohistochemistry was used to detect the expression and distribution of MEF2 C and A? in autopsied brains from AD patients and control groups(non-neurological diseases);the Pearson correlation test was used to analyse the correlations of MEF2C/A? in different human brain regions.(2)Animal experiments: Immunohistochemistry was used to detect the expression and distribution of MEF2 C and A? in APP/PS1 mice;Western blot was used to detect the expression of MEF2 C protein in APP/PS1 mice.An adenoviral vector,p DC316-sh RNA-Mef2c-mus-829,was constructed to silence MEF2 C,and an adenovirus containing the gene of interest was obtained by an adenoviral packaging method to produce a virus,named Ad-sh RNA-MEF2 C.Then,6-and 10-month-old APP/PS1 mice and C57 wild type mice was treated with the silencing virus and empty virus by intracerebroventricular injection.Morris water maze tests were used to determine the spatial learning and memory abilities of mice.Haematoxylin-eosin(HE)staining was used to assess the pathological changes the brain tissue of mice,and Congo red staining was used to detect the changes of senile plaques in the cortex of mice.Td T-mediated d UTP Nick-End Labelling(TUNEL)staining was used to detect apoptosis in the cortex of mice.Transmission electron microscopy was used to detect changes in mitochondrial morphology in the cortex of mice;immunofluorescence was used to detect expression of MEF2C/A?,IBA1,IL-6 in the cortex of mice.Enzyme-linked immunosorbent assays(ELISAs)were used to detect the expression of interleukin-6(IL-6)in the cortex of mice.Biochemical methods were used to determine the content of methane dicarboxylic aldehyde(MDA)and superoxide dismutase(SOD)activity in the cortex of mice.Western blot was used to detect the expression of the following MEF2 C protein and Nrf2-ARE signalling pathway-related proteins: nuclear factor erythroid 2-related factor(Nrf2),heme oxygenase 1(HO-1),NADPH dehydrogenase,quinone 1,NQO1,SOD2,apoptosis-related proteins BAX,Bcl-2,synapse-associated protein synaptophysin(SYN),postsynaptic density protein 95(PSD-95),?-amyloid precursor protein cleaving enzyme 1(BACE1),and BACE2.(3)Cell experiments: GV40-sh RNA-Mef2 c was constructed to silence MEF2C;a GV40-sh RNA empty plasmid and the GV40-sh RNA-Mef2 c plasmid were transfected into SH-SY5 Y cells for 48 hours,and then the cells were collected after exposure to A?O(1 ?mol/L)for 24 hours.Flow cytometry was used to detect apoptosis and reactive oxygen species(ROS),and the protein expression of MEF2 C,Nrf2,HO-1,NQO1,SOD2,BAX,Bcl-2,SYN,and PSD-95 was detected by Western blot.Results:(1)Human brain tissue specimens: Immunohistochemistry results showed that A? deposition was significant in the temporal lobe,frontal lobe,and hippocampus of AD patients,and there were lower levels in the normal control group.MEF2 C is mainly expressed in the temporal lobe and frontal lobe of AD patients and the normal control group,while it is not expressed in the hippocampus and is observed at significantly lower levels in the AD patient group than in the normal control group.Pearson correlation test analysis shows that MEF2C/A? expression in different brain regions is obviously negatively correlated.(2)Animal experiments:(1)Compared with age-matched C57 mice,in the cortex of APP/PS1 mice at 6 and 10 months old,immunohistochemistry results showed that the expression of MEF2 C was reduced and A? deposition was obvious;further,Western blot results showed that the expression of MEF2 C protein was reduced.(2)Compared with age-matched C57 mice injected with Ad-sh RNA,the Morris water maze results showed reduced learning and memory abilities in 6-and 10-month-old APP/PS1 mice injected with Ad-sh RNA.Compared with the age-matched APP/PS1 mice injected with Ad-sh RNA,the Morris water maze results showed reduced learning and memory abilities in 6-and 10-month-old APP-PS1 mice injected with Ad-sh RNA-Mef2 c.(3)Compared with the age-matched C57 mice injected with Ad-sh RNA,in the cortex of 6-and 10-month-old APP/PS1 mice injected with Ad-sh RNA and C57 mice injected with Ad-sh RNA-Mef2 c,HE staining results showed no obvious pathological changes,and transmission electron microscopy results showed increased mitochondrial damage.Compared with the age-matched APP/PS1 mice injected with Ad-sh RNA,in the cortex of 6-and 10-month-old APP/PS1 mice injected with Ad-sh RNA-Mef2 c,HE staining results showed no obvious pathological changes,and transmission electron microscopy results showed increased mitochondrial damage.(4)Compared with the age-matched C57 mice injected with Ad-sh RNA,in the cortex of 6-and 10-month-old APP/PS1 mice injected with Ad-sh RNA,MEF2C/A?,Iba1,and IL6 immunofluorescence staining results showed that the expression of MEF2 C was reduced,A? deposition was obvious,Iba1 expression was increased,and IL6 expression was increased;TUNEL staining results showed increased apoptosis,and Congo red staining showed that the senile plaques were significantly deposited.In the cortex of 6-month-and 10-month-old C57 mice injected with Ad-sh RNA-Mef2 c,no A? deposition was found,and other indicators were consistent with those of APP/PS1 mice injected with Ad-sh RNA.Compared with the same-month-old APP/PS1 mice injected with Ad-sh RNA,in the cortex of 6-and 10-month-old APP/PS1 mice injected with Ad-sh RNA-Mef2 c,MEF2C/A?,Iba1,and IL6,immunofluorescence staining results showed that the expression of MEF2 C was reduced,Iba1 expression was increased,IL6 expression was increased,and A? deposition was increased;Congo staining showed that the senile plaques were deposited increased,and TUNEL staining showed increased apoptosis.(5)Compared with the age-matched C57 mice injected with Ad-sh RNA,in the cortex of 6-and 10-month-old APP/PS1 mice injected with Ad-sh RNA and C57 mice injected with Ad-sh RNA-Mef2 c,Western blot results showed that the expressions of MEF2 C,NRF2,HO-1,SOD2,NQO1,SYN,PSD95,BACE2 and BCL-2 proteins were decreased,and the expressions of BACE1 and BAX was increased.ELISA results showed that the expression of IL-6 was increased.Biochemical results showed that the content of MDA was increased,and the activity of SOD was weakened.Compared with the age-matched APP/PS1 mice injected with the Ad-sh RNA,in the cortex of 6-and 10-month-old APP/PS1 mice injected with Ad-sh RNA-Mef2 c,Western blot results showed that the expressions of MEF2 C,NRF2,HO-1,SOD2,NQO1,SYN,PSD95,BACE2 and BCL-2 proteins were decreased,and the expressions of BACE1 and BAX was increased.ELISA results showed that the expression of IL-6 was increased.Biochemical results showed that the content of MDA was increased,and the activity of SOD was weakened.(3)Cell experiments: GV40-sh RNA and GV40-sh RNA-Mef2 c were transfected into SH-SY5 Y cells for 48 hours,and then the cells were exposed to A?O(1 ?mol/L)for 24 hours.Compared with the GV248-sh RNA group,in the GV248-sh RNA+A? and GV248-sh RNA-Mef2 c groups,Western blot results showed that the expression of MEF2 C,NRF2,HO-1,NQO1,SOD2,SYN,PSD95 and BCL-2 protein decreased,and the expression of BAX protein was increased.Flow cytometry showed increased apoptosis and ROS in cells.Compared with the GV248-sh RNA+A? group,in the GV248-sh RNA-Mef2c+A? group,Western blot results showed that the expression of MEF2 C,NRF2,HO-1,NQO1,SOD2,SYN,PSD95 and BCL-2 proteins was reduced,and the expression of BAX protein was increased.Flow cytometry results showed increased apoptosis and ROS in cells.Conclusions:(1)The expression of MEF2 C in the brains of AD patients is reduced,which may be one of the reasons for the large accumulation of A? in AD and the increase in neurotoxicity.(2)Downregulation of MEF2 C may be increase the level of BACE1,reduce the level of BACE2,increase the production and aggregation of A?,damage synaptic plasticity,and reduce the ability of learning and memory in the AD animal cortex by reducing the activation of Nrf2-ARE signaling pathway.(3)Downregulation of MEF2 C may be increase the level of oxidative stress,promote the release of inflammatory factors,and increase the damage to nerve cells in the AD animal cortex by reducing the activation of Nrf2-ARE signaling pathway. |
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