| Objective: Alzheimer’s disease(AD)is a nervous system disease,mainly manifested as cognitive dysfunction,of which late-onset AD accounts for more than 90%.Lipoprotein esterase(LPL)is a key enzyme in lipid metabolism,which is closely related to AD and is involved in the clearance of β-amyloid protein(Aβ)in the brain.LPL localized in cerebral microvascular endothelial cells was shown to be mainly derived from peripheral tissues.Our previous study found decreased LPL expression in cerebral microvascular endothelial cells in AD model mice,possibly caused by decreased LPL expression in adipose tissue.γ-Aminobutyric acid(GABA)is an important inhibitory neurotransmitter in the central nervous system and is abundant in the brain;GABA is also a food factor with many biological effects.Studies show that GABA is associated with many neurological diseases,such as AD.Aβ accumulation in the brain is an important link leading to the pathogenesis of AD.BBB is the main way for Aβ clearance to transport out of the brain,and the cerebral microvascular endothelial cells are an important part of BBB.Low density lipoprotein receptor-associated protein 1(LRP1),located in the basement membrane side of brain microvascular endothelial cells,plays an important role in Aβ across BBB transport.LPL could advance cellular absorb low density lipoprotein(LDL)and increase intracellular cholesterol levels.There is evidence that LDL regulates LRP 1 expression.Previous studies showed that GABA can antagonize the decrease of LPL expression in adipose tissue and brain microvascular endothelial cells in AD model mice,but it remains to be confirmed whether GABA can regulate LRP1 expression in brain microvascular endothelial cells.Histone deacetylases(HDACs)and histone acetyltransferases(HATs)are the key enzymes that affect histone acetylation.Previous studies found that GABA antagonizes AD by inhibiting HDACs,and miR-29 a plays an important mediating role in the regulation of LPL expression by HDACs.In our in vitro study,we also found that the expression of miR-29 a in human neuroblastoma(SH-SY5Y)cells showed a "U" change with the increase of Aβ treatment concentration,while the expression of HDACs gradually increased,which suggested that it was difficult for HDACs alone to interpret the change of miR-29 a expression,and the specific molecular mechanism of the regulation of miR-29 a expression needs to be further explored.With the increase of the methylation level of histone H3 tails,histone methyltransferases(Histone methyltransferase,HMTs)can recruit HATs complexes,suggesting that the occurrence of histone methylation modification plays an important role in promoting histone acetylation.SP1 can bind to the promoter region of miR-29 a and regulate miR-29 a expression.In the transcriptional regulation of genes,whether SP1 is an activator or a repressor is determined by the nature of its protein complex.Based on the above,we speculate that in AD,SP1 may recruit histone methyltransferase1/2(EHMT1/2),recruit P300,relieve the deacetylation of HDACs,promote histone acetylation,and finally make miR-29 a expression first decline and then rise.The increase of LDL-C,TC,and TG in plasma,and the decrease of HDL-C were associated with AD.APOE4 is considered a major genetic risk factor for late-onset AD,but its effects on blood lipid levels are inconsistent,and whether APOE4 affects AD through the blood lipid pathway needs to be further explored.LPL is a key enzyme in lipid metabolism,and the inheritance and variation of LPL genes may affect LPL activity,and then affect lipid metabolism.Therefore,the correlation of SNPs of LPL with the risk of AD is also of practical significance.Currently,SNPs of LPLrs285,rs320 and rs328 have been shown to be associated with AD in non-Chinese populations,but few studies on the Chinese Han population have reported.In conclusion,this study investigated the regulation of LPL expression by GABA in peripheral tissues and its mechanism by combining in vivo and in vitro experiments in antagonistic AD.SNPs and lipid levels between LPL and APOE.Methods: 1.5-month-old APP/PS1 double transgenic AD model mice were randomly assigned to AD and AD+GABA,and C57BL/6 wild-type mice born in littermates were randomly assigned to WT or WT+GABA.Five male and female mice in each group.Mice in AD+GABA and WT+GABA groups drank distilled water containing 1 mg/ml GABA,and mice in AD and WT groups drank only distilled water.The general condition of mice was observed during the experiment,and after 6 months,the cognitive function was evaluated by Morris maze experiment.Mice were killed after anesthesia,and brain tissue was collected to detect Aβ deposition by immunohistochemistry;the co-localization of Aβ,LRP1,LPL and cerebral microvascular endothelial cell marker CD31 was detected by immunofluorescence.To explore the possible mechanism of GABA regulation on LRP1 expression in brain microvascular endothelial cells in AD model mice.2.Animal grouping and treatment,the expression levels of adipose tissue LPL,miR-29 a,P300,EHMT1/2,and SP1 in each group of mice were determined by q RT-PCR and Western blotting.Mouse precursor fat(3T3-L1)cells were cultured in vitro,pretreated with a final concentration of10 n M,3T3-L1 cells were treated at different concentrations of Aβ(0-2μM)after 3h,and the effects of GABA on the levels of LPL,miR-29 a,SP1,P,300,and EHMT1/2,histone acetylation,and histone methylation in Aβ were analyzed by q RT-PCR and Western blotting.SP1,P300 and EHMT1/2 were silenced by si RNA technology for 24 h,and the cells were harvested.Cell expression levels of miR-29 a and LPL were determined by q RTPCR and Western blotting.P300 or EHMT1/2 of 3T3-L1 cells were silenced by si RNA technology,while a control group was set up and cultured for 24 h to detect the level of histone acetylation or methylation in the miR-29 a promoter region by Ch IP assay.To reveal the regulation and molecular mechanism of GABA on LPL expression in adipose tissue of AD model mice.3.Patients diagnosed with AD were included in Liaoning Jinqiu Hospital from July 2020 to December 2022,and age-and gender-matched control populations were included in the same period.Clinical data were collected from the study subjects.In the early morning,4ml of venous blood was collected with EDTA anticoagulant tube,and SNPs of LPLrs285,rs320,rs328 and APOE were detected by Taqman probe.The relations between LPL gene polymorphisms and lipid levels and AD were analyzed.Results: 1.Regulation of LRP1 expression in brain microvascular endothelial cells in AD model mice.The results of the water maze experiment showed that GABA improved the decline of cognitive function in the AD model mice(P<0.05).GABA reduces Aβ plaque deposition in the brain of AD model mice(P<0.01).GABA decreased Aβ levels in cerebral microvascular endothelial cells in AD model mice(P<0.05).GABA antagonizes the decrease of LPL protein expression in brain microvascular endothelial cells of AD model mice(P<0.01).GABA inhibited the decrease of LRP1 protein expression in brain microvascular endothelial cells of AD model mice(P<0.05).2.GABA regulation and mechanism of LPL expression in adipose tissue in AD model mice.Adipose tissue LPL in AD model(P<0.01);miR-29a(P<0.01);EHMT1/EHMT2,H3K9 methylation(P<0.01,P<0.05);P300,H3K9 acetylation(P<0.01);and SP1(P<0.01,P<0.05).GABA antagonizes the above alterations in AD model mice.As the concentration of Aβ treatment increased,the LPL expression level increased and then decreased,the miR-29 a level decreased and then increased,the EHMT1/EHMT2 expression level gradually increased,the H3K9 methylation level increased and then decreased,the P300 expression level gradually increased,the H3K9 acetylation level decreased and then increased,and the SP1 expression level gradually increased,significantly(P<0.05,P<0.01).Compared with the control group,Cells treated with 0.5μM Aβ had increased levels of LPL expression and decreased levels with miR-29 a,Groups treated with 2μM Aβ had decreased LPL expression levels(P<0.01)and increased miR-29 a levels(P<0.05),EHMT1/EHMT2 expression and H3K9 methylation levels were increased in cells treated with 0.5μM or2μM Aβ(P<0.05,P<0.01),Increased expression level of P300 and decreased H3K9 acetylation level(P<0.05,P<0.01),The SP1 expression level was increased(P<0.05).Compared to the Aβ(0.5μM)group,LPL expression decreased(P<0.01),miR-29 A increased in Aβ(2μM)and GABA + Aβ(0.5μM)groups(P<0.01),EHMT1/EHMT2 expression and H3K9 methylation level were increased in Aβ(2μM)group cells,EHMT1/EHMT2 expression and H3K9 methylation level were decreased in the GABA+Aβ(0.5μM)group(P<0.05,P<0.01),Aβ(2μM)cells showed increased P300 expression and decreased H3K9 acetylation,GABA+Aβ(0.5μM)cells had decreased P300 expression and increased H3K9 acetylation(P<0.05,P<0.01),SP1 in Aβ(2μM)group,The SP 1 expression level was decreased in the GABA+Aβ(0.5μM)group(P<0.05).GABA+Aβ(2μM)cells increased LPL expression(P<0.01),miR-29a(P<0.05),EHMT1/EHMT2 expression and H3K9 methylation(P<0.05,P<0.01),H3K9 acetylation(P<0.05),and SP1(P<0.05)compared to Aβ(2μM)group.Compared with control group,the expression of miR-29 a in si RNA-SP1 group was increased,while the expression of LPL was decreased,with statistical significance(P<0.01).The expression of miR-29 a decreased and LPL increased in si RNA-P300 group,with statistical significance(P<0.01).The expression of miR-29 a was increased in si RNA-EHMT1/EHMT2 group,while the expression of LPL was decreased,with statistical significance(P<0.01).Compared with Ig G group,SP1 antibody group had higher enrichment levels in the miR-29 a promoter region(P<0.01).Compared with the control group,H3K9 acetylation in the miR-29 a promoter region was significantly reduced in the P300 silencing group(P<0.01),H3K9 methylation in the miR-29 a promoter region was significantly decreased in the silencing EHMT1/2 group(P<0.01).3.Correlation studies of LPL gene polymorphisms and blood lipid levels with AD.A total of 360 subjects were included in the study,including 180 subjects each in the AD group and the control group.In terms of disease history,the prevalence of diabetes was higher in the AD group than in the control group(P<0.05).TC,TG,and LDL-C increased in AD group(P<0.01)and HDL-C decreased(P<0.01).The frequency of Apo E4 carriers was higher in AD groups than in control groups,and APOE4 carriers increased the risk of AD by 2.742 times(OR=2.742,95%CI=1.1756-4.282,P<0.01).The distribution of genotypes between the AD and control groups was statistically different(P=0.000),the risk of AD in the rs285 TT genotype was 3.344 times that of the AD and control genotypes at the LPL rs328(P=0.045),and the rs328 TT genotype was 2.651 times the risk of AD than the rs328 CC genotype.TC and LDL-C levels were higher in the APOE4 phenotype in AD than the APOE2(P<0.05);LDL-C in the APOE4 than the APOE2 phenotype(P<0.05),HDL-C than the APOE 2(P<0.05);TG than the LPL rs285 TT in both AD and controls(P<0.05)and HDL-C in the LPL rs285 TT than the rs285 CC genotype(P<0.05).Conclusions: 1.LPL can mediate the anti-AD effects of GABA by regulating LRP1 expression in brain microvascular endothelial cells and thereby promoting Aβ clearance in the brain.2.In adipose tissue of AD model mice,GABA can modulate the expression of LPL,and the mechanism of action may be that P300 and EHMT1/2 mediate the regulation of GABA on LPL expression via the SP1/miR-29 a pathway.3.APOE4 is a risk factor for AD,which may affect the pathogenesis of AD by increasing TC and LDL-C levels;SNP located at LPLrs285 is associated with lipid levels,SNPs located at LPLrs285 and rs328 are associated with AD,and LPLrs285 may be associated with AD by affecting TG levels.APOE and LPL genes interact on the risk of AD. |
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