| Objective:Gamma-aminobutyric acid(GABA)is an important inhibitory neurotransmitter in the central nervous system,which is involved in the regulation of the occurrence and development of a variety of nervous system-related diseases.However,the increasing evidence shows that the various biological effects of GABA cannot be fully explained by inhibitory neurotransmitters.Histone acetylation modification plays an important role in the transformation of gene transcription.Histone acetylation mainly depends on the catalysis of histone acetyltransferases(HATs)and histone deacetylases(HDACs).A variety of histone acetylase inhibitors have been discovered,such as trichostatin A,β-hydroxybutyric acid(BHB),butyric acid and so on.GABA is highly similar to BHB and butyric acid in structure,but whether GABA can inhibit HDACs has not yet been eliminated.It is reported that GABA can upregulate the expression of brain-derived growth factor(BDNF),synaptophysin(Syp),glutamate receptor 1(GluR1),glutamate receptor 2(GluR2)and other gene.The above genes can be suppressed by HDACs.It suggests that GABA may have the effect on inhibiting HDACs.GABA is highly express in the brain.It is mainly synthesized by neurons and released to the synaptic cleft,and functions by binding to the alternative GABA receptor on the postsynaptic membrane.GABA in the gap is also transported to the glial cell by GABA transporter(GAT).GABA is a natural functional food factor.The human body can supplement GABA by ingesting food rich in GABA or by synthesizing GABA in the gut flora.The role of neurotransmitter in the nervous system has been widely confirmed.With the deepening of research,the role of GABA in the periphery has gradually attracted attention,especially GABA and fat.Adipose tissue expresses GAT,but there is no report about the expression of GABA receptor in adipose tissue.The mechanism of GABA’s inflammatory regulation of fat is still unclear.GABA is related to many diseases such as Alzheimer’s disease(AD),Parkinson’s disease,epilepsy and multiple sclerosis.In recent years,the role of GABA in the occurrence and development of AD has received highly attention.AD is a degenerative disease of the central nervous system characterized by progressive cognitive dysfunction and memory impairment.Amyloid β(Aβ)clearance plays an important role in AD.There are many ways to clear Aβ in the brain.Aβ enzymatic degradation is one of the key ways to clear Aβ.Nepriliysin(NEP)is one of the important proteases that degrades Aβ in the brain.A significant decreased expression of NEP was detected in the AD brain,when the expression of NEP increases,it can reduce Aβ and improve cognitive function impairment.The blood-brain barrier(BBB)transport to the periphery is also the way to clear Aβ.Previous studies have shown that the expression of lipoprotein esterase(LPL)was decrease in the adipose tissue of AD mice,which in turn reduces the LPL of brain microvascular endothelial cells and affects Aβ across the BBB transport.There is also evidence that HDACs are involved in the occurrence and development of AD,and HDAC2/3 can change the levels of histone H3K9/H4K12 and AD-related genes,thereby regulating the occurrence and development of AD.This research proved that HDACs can regulate the expression level of NEP in SH-SY5 Y cell and LPL in 3T3-L1 cell.However,whether GABA regulates NEP of brain and LPL of adipose by inhibiting HDACs,thereby antagonizing AD,has not yet been confirmed.In this study,we applied in vivo and in vitro experiments to prove that GABA can inhibit HDAC through non-receptor pathways,and further explored the anti-AD effects of GABA and its possible mechanism.The results of this research provide a basis for interpreting the extensive biological functions of GABA and provide a theoretical basis for exploring the effective prevention and treatment of AD.Methods:1.The study on the inhibitory effect of GABA on HDACs of SH-SY5 Y cells and mouse brain1.1 Detection of SH-SY5 Y cell viability treated with GABA: Treated SH-SY5 Y cells with GABA(final concentration 0～320 n M),cultured for 24 h,and used MTT to detect cell viability.1.2 Detection of HDAC1/2/3 expression and Ace-H3K9/H4K12 levels in SH-SY5 Y cells treated with GABA: Cells treated with 0～20 n M GABA were cultured for 24 h,and the expression of HDAC1/2/3 mRNA and protein,the expression of Ace-H3K9,Ace-H4K12 were detected by qRT-PCR and Western Blotting.1.3Detection of HDAC1/2/3,GluR2 expression and Ace-H3K9/H4K12 level in brain tissue of GABA-treated mouse: Four-month-old C57BL/6 mice were divided into control group and GABA-treated group,mice of GABA-treated group were injected intraperitoneally with 100 mg/kg.bw GABA,and mice in control group were injected with the same dose of normal saline,and mice were sacrificed after 12 h.Then,using spectrophotometricto detect GABA level in mouse brain,and using qRT-PCR and Western Blotting to detect HDAC1/2/3,GluR2 mRNA and protein expression levels and the expression of Ace-H3K9,Ace-H4K12.1.4 Detection of GluR2 expression in HDAC1/2/3 silencing cells: Silenced SH-SY5 Y cells HDAC1/2/3,cultured 24 h,qRTPCR and Western Blottingwere used to detect the expression levels of GluR2 mRNA and protein,and Ch IP was used to detect histone acetylation in the GluR2 promoter region.1.5 Detection of GluR2 expression in SH-SY5 Y cells treated with GABA:QRT-PCR and Western Blottingwere used to detect the expression of GluR2 mRNA and protein in cells treated with GABA(20 n M),and used Ch IP to detect histone acetylation levels in the GluR2 promoter region.2.The study on GABA inhibiting HDACs expression through non-receptor pathway2.1 Detection of HDAC1/2/3 expression in SH-SY5 H cells treated with GABA receptor agonists: Treated SH-SY5 Ycell with GABA-A/-C receptor agonist muscimol(100 n M)and GABA-B receptor agonist(R)-Baclofen(100 n M),after 24 h of culture,using qRT-PCR and Western Blotting to detect the expression of HDAC1/2/3 mRNA and protein.2.2 Detection of GABA receptor expression in SH-SY5 Y cells and 3T3-L1 cells: QRT-PCR,Western Blotting and immunofluorescence were used to detect the expression of GABA receptors in SH-SY5 Y cells and 3T3-L1 cell.2.3 Detection of3T3-L1 cell viability treated with GABA: Treated 3T3-L1 cells with GABA(final concentration 0～320 n M),culture for 24 h,and used MTT to detect cell viability.2.4Detection of HDAC1/2/3 expression and Ace-H3K9/H4K12 levels in 3T3-L1 cells treated with GABA: Cells treated with 2.5 n M,5 n M,and 10 n M GABA were cultured for 24 h,and the expression of HDAC1/2/3 mRNA and protein,the expression level of Ace-H3K9 and Ace-H4K12 were detected by qRT-PCR and Western Blotting.2.5Detection of HDAC1/2/3 expression and Ace-H3K9/H4K12 levels in adipose tissue of mice treated with GABA: The treatment of mice is the same as above(1.3).Using qRTPCR and Western Blotting to detect the expression levels of HDAC1/2/3 mRNA and protein,Ace-H3K9 and Ace-H4K12 in adipose tissue.3.The antagonistic effect of GABA on AD and its possible mechanismFive-month-old APPswe/PS1 d E9 double transgenic mice were divided into AD model group and AD+GABA group,meanwhile wild-type mice were divided into wild control group and wild+GABA group.The mice in the GABA group were fed water with 2% GABA,and the mice in the control group were fed ordinary distilled water,all mice were continuous treatd for 6 months.At the end of the experiment,the neurobehavioral testing of mice was performed and mice were sacrificed.Their brains were collected and divided into halves,their fat were also collected.Using immunohistochemical to detect Aβ deposition in mouse brain tissue.Using qRT-PCR and Western Blotting to detect the expression levels of mouse brain tissue NEP and adipose tissue LPL mRNA and protein,and using immunofluorescence to detect mouse brain microvascular endothelial cell LPL expression levels.Usin g qRT-PCR and Western Blotting to detect the expression levels of HDAC1/2/3 mRNA and protein,Ace-H3K9 and Ace-H4K12 in mouse brain tissue and adipose tissue.4.Statistical analysis: The data was performed by LSD and ANOVA in SPSS 20.0,and the difference was considered statistically when P <0.05.Results:1.Inhibitory effect of GABA on HDACs in SH-SY5 Y cells and mice brain1.1 The effect of GABA on the viability of SH-SY5 Y cells: Compared with the control,there was no statistical difference in the viability of SH-SY5 Y cells treated with 5 n M,10 n M,and 20 n M GABA(P>0.05);the cell viability of SH-SY5 Y cells treated with 40 n M and 80 n M GABA were gradually increased with the increase of GABA concentration;the cell viability of 160 n M and 320 n M GABA treatments gradually decreased with the increase of GABA concentration(P<0.01).1.2 The effect of GABA on the expression of HDAC1/2/3 and Ace-H3K9/H4K12: Compared with the control,the expression of HDAC1/2/3 in cells treated with 5 n M,10 n M,and 20 n M GABA were decreased,and the expression of Ace-H3K9/H4K12 were increased(P <0.05,P <0.01).1.3 The effect of GABA on the expression of HDAC1/2/3 and AceH3K9/H4K12 in the brain tissues of mice: Intraperitoneal injection of GABA increased the expression of GABA content in the brain tissues of mice(P<0.05),and the expression of HDAC1/2/3 in the brain tissues were decreased,and the expression of Ace-H3K9/H4K12 were increased(P <0.05,P <0.01).1.4 The effect of GABA and HDACs on the expression of GluR2 and the acetylation level of the promoter region of GluR2: The expressions of GluR2 increased in GABA-treated cells and intraperitoneal injection of GABA mice(P <0.01);silencing HDAC1/2 upregulated GluR2 expression(P <0.01),but there was no significant difference in the expression of GluR2 in silenced HDAC3(P >0.05);the expression of Ace-H3K9 in the GluR2 promoter region of cells treated with GABA and HDAC1/2 silenced were increased(P <0.01).2.GABA inhibits the expression of HDACs through a non-receptor pathway2.1 The effect of GABA receptor agonists on HDAC1/2/3 expression in SH-SY5 Y cells: There were no differences of expression of HDAC1/2/3 in GABA-A/-B/-C receptor agonists treated SH-SY5 Y cells(P >0.05).2.2 GABA receptor expression in SH-SY5 Y cells and 3T3-L1 cells: SH-SY5 Y cells expressed GABA receptors,and3T3-L1 cells have no GABA receptor expressed.2.3 The effect of GABA on the viability of 3T3-L1 cells: Compared with the control,there was no significant difference in the viability of 3T3-L1 cells treated with 2.5,5,and 10 n M GABA(P >0.05),when the GABA concentration exceeded 20 n M,the cell viability gradually decreased with the increase of GABA concentration(P <0.01).2.4 The effect of GABA on HDAC1/2/3 and Ace-H3K9/H4K12 in 3T3-L1 cells and adipose tissue: Compared with the control the expression of HDAC1/2/3 mRNA and preotin were decreased in GABA-treated 3T3-L1 cell and mouse adipose tissue(P <0.05,P <0.01),and the expression of Ace-H3K9/H4K12 were increased(P<0.01).3.The antagonistic effect of GABA on AD and its possible mechanism3.1 The effect of GABA on the cognitive function of AD mice: Compared with the wild control mice,the water maze platform residence time of AD model and AD+GABA group mice were significantly shorter,and the numbers of crossings were significantly reduced(P <0.05,P <0.01);compared with the ADvgroup,the platform stay time of the AD+GABA group was significantly longer,and the numbers of crossings were significantly increased(P <0.05,P <0.01).3.2.The effect of GABA on the Aβ deposition in the AD mice brain: Compared with the wild control mice,the number of Aβ deposition in the brain of the AD model group and the AD+GABA group were significantly increased(P <0.05,P <0.01),and compared with the AD group,the number of Aβ deposits in the brain of the AD+GABA group was significantly reduced(P <0.01).3.3 The effect of GABA on the expression of NEP in the AD mice:Compared with the wild control mice,the expression of NEP in brain of AD mice was decreased(P <0.01),and compared with AD mice,the expression of NEP in AD+GABA mice was increased significantly(P <0.01).3.4 The effect of GABA on the expression of LPL in adipose tissue and brain microvascular endothelial cells of AD mice: Compared with the wild control mice,the expression of LPL in adipose tissue and brain microvascular endothelial cells of AD model mice were decreased(P <0.01);compared with AD model mice,the expression of LPL in adipose tissue and brain microvascular endothelial cells of AD+GABA mice were increased significantly(P<0.01).3.5 The effect of GABA on the expression of HDAC1/2/3 in the brain tissue and adipose tissue of AD model mice: Compared with the wild control mice,the HDAC1/2 / 3 expression of brain and adipose of were increased(P <0.01);compared with AD model mice,the expression of HDAC1/2/3 in brain and adipose of AD+GABAgroup mice were decreased(P <0.01);compared with wild control mice,the expression of Ace-H3K9 and Ace-H4K12 in the brain tissue and adipose tissue of the AD model group were decreased(P <0.01);compared with AD model mice,the expression of Ace-H3K9 and Ace-H4K12 in brain and adipose of AD+GABAgroup mice were increased(P <0.01);Conclusions: GABA inhibits HDACs through non-receptor pathways,and the antiAD effect of GABA may be related to its inhibition of HDACs and regulation of NEP and LPL. |
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