银杏内酯B通过调控海马线粒体H4K5乙酰化改善AD小鼠认知功能OA

AIM:This study aims to investigate the therapeutic effects of Ginkgolide B(GB)on memory defi-cits and elucidate its underlying molecular mechanisms in APP/PS1 double-transgenic mouse models of Alzheimer's dis-ease.METHODS:Thirty-two 8-month-old male APP/PS1 transgenic mice were randomly assigned to either the model group or the GB treatment group(n=16 per group).Sixteen age matched wild type(WT)mice with the same genetic back-ground served as the control group.The GB treatment group received GB by intragastric administration at 40 mg/kg twice daily for 2 months.The model group and control group received equal volumes of saline.Spatial learning and memory were evaluated by the Morris water maze test and contextual fear conditioning test.Amyloid-β(Aβ)deposition and related pro-tein expression in the hippocampus were examined by thioflavin S staining and Western blot.Dendritic spine morphology and density were assessed by Golgi staining.Mitochondrial ultrastructure and synaptic morphology in the hippocampus were observed by transmission electron microscopy.Superoxide dismutase(SOD)activity and the levels of malondialde-hyde(MDA),reactive oxygen species(ROS),and adenosine triphosphate(ATP)were measured by biochemical assays.The mRNA and protein expression of factors related to mitochondrial biogenesis were detected by real time quantitative polymerase chain reaction(RT-qPCR)and Western blot.Chromatin immunoprecipitation(ChIP)was used to analyze the enrichment of histone H4 lysine 5 acetylation(H4K5ac)at specific gene promoters.Primary hippocampal neurons treated with Aβ1-42 oligomers were used for histone deacetylase 6(HDAC6)knockdown by small interfering RNA(siRNA)to veri-fy its role.RESULTS:In the Morris water maze test,mice in the GB group showed a shorter escape latency and more crossings over the original platform location than mice in the model group(P<0.05).In the contextual fear conditioning test,freezing time was significantly prolonged in the GB group(P<0.01).Thioflavin S staining showed that the number of Aβ plaques in the hippocampus was significantly reduced in the GB group(P<0.01).Western blot showed that soluble Aβ levels and amyloid precursor protein phosphorylation at Thr668(p-APP)were significantly decreased in the hippocam-pus of the GB group(P<0.01).Golgi staining showed that dendritic spine density in the CA1 and dentate gyrus(DG)re-gions was markedly reduced in the model group(P<0.01),and this loss was partly reversed by GB treatment(P<0.01).Transmission electron microscopy showed that GB increased postsynaptic density thickness,increased the length of the synaptic active zone,and reduced synaptic cleft width.The mRNA and protein expression of postsynaptic density protein 95(PSD95),glutamate ionotropic receptor NMDA type subunit 1(GluN1),glutamate ionotropic receptor NMDA type subunit 2A(GluN2A),and synapsin1(SYN1)were significantly increased in the hippocampus of the GB group(P<0.01).GB also improved mitochondrial swelling,vacuolization,and blurred cristae.In the hippocampus of the GB group,ATP levels and SOD activity increased,whereas MDA and ROS levels decreased(P<0.01).The mRNA levels of nuclear factor erythroid 2 related factor 2(NRF2),mitochondrial transcription factor A(TFAM),and peroxisome prolifer-ator activated receptor gamma coactivator 1 alpha(PGC-1α),as well as mitochondrial DNA(mtDNA)copy number,were restored by GB treatment(P<0.05).Western blot confirmed that PGC-1α protein expression increased in the GB group(P<0.05).RT-qPCR showed that HDAC6 and histone deacetylase 7(HDAC7)mRNA levels were significantly elevated in the hippocampus of APP/PS1 mice(P<0.01).GB selectively reduced HDAC6 mRNA level and protein expression(P<0.01).In primary hippocampal neurons,Aβ treatment increased HDAC6 protein expression(P<0.01)and caused marked dendritic shortening.GB treatment or HDAC6 siRNA transfection significantly reduced HDAC6 expression(P<0.01)and attenuated dendritic shortening.In the hippocampus of APP/PS1 mice,H4K5ac,histone H4 lysine 12 acetyla-tion(H4K12ac),and histone H3 lysine 14 acetylation(H3K14ac)levels were all reduced,and the decrease in H4K5ac was the most pronounced(P<0.01).GB significantly increased H4K5ac expression(P<0.01).ChIP-qPCR showed that GB significantly increased H4K5ac enrichment at the promoter regions of GluN2A and PGC-1α(P<0.01).CONCLU-SION:GB improves synaptic plasticity,mitochondrial biogenesis,and cognitive impairment in APP/PS1 mice.This ef-fect may depend on inhibition of HDAC6,increased H4K5ac levels,and enhanced expression of GluN2A and PGC-1α.