The Role Of GABA-A Receptor Pathway In Atherosclerosis

Part Ⅰ GABA and GABA-A receptor agonist treatments inhibit the THP-1-derived foam cell formationObjective To investigate the roles of GABA and GABA agonist treatments in the THP-1-derived foam cell formationMethods THP-1-derived macrophages were randomly assigned to one of the following groups:group1treated with PBS, group2treated with ox-LDL, group3treated with GABA, group4,5,6treated with ox-LDL and low, medium and high concentrations of GABA respectively. Then, we had observed the number of foam cells in groups by Oil Red O staining. The expression levels of proteins in groups such as SR-A, CD36, LOX-1, ACAT1, ABCA1/Gland SR-BI were measured by Western blot. Instead of GABA with GABA-A receptor agonist, topiramate, we divided THP-1-derived macrophages into one of the following groups:group1treated with PBS, group2treated with ox-LDL, group3treated with topiramate, group4,5,6treated with ox-LDL and low, medium and high concentrations of topiramate respectively and repeated above process.Results GABA and GABA-A receptor agonist topiramate treatments inhibited the number of THP-1-derived foam cells. GABA and topiramate down-regulated the expression levels of proteins of SR-A, CD36, LOX-1, ACAT1and up-regulated the expression levels of proteins of ABCA1/G1、SR-BI.Conclusion GABA and GABA-A receptor agonist treatments inhibit the THP-1-derived foam cell formation. Part ⅡGABA-A receptor modulates the atherosclerotic plaque information in apoE-/-miceObjective To investigate the roles of GABA-A receptor agonist and antagonists in the atherosclerotic plaque formation in apoE-/-mice.Methods6weeks old apoE-/-mice were randomly assigned to one of the following treatments (n=12):(1) normal diet group, i.p. normal saline;(2) high cholesterol diet group, i.p.normal saline;(3) picrotoxin group, i.p. picrotoxin;(4) bicuculline group, i.p. bicuculline;(5) topiramate group, i.p. topiramate. Each group was fed on high cholesterol diet except for group1. After12weeks, we had detected the following indicators. Atherosclerotic lesion area of the aorta was quantified by Oil Red O staining. The collagen deposition and the number of macrophages and CD3+T cells in air-dried cryostat sections of atherosclerotic lesions were evaluated by histology and immunohistochemical analysis. The mRNA expression levels of monocyte chemoattractant protein-1(MCP-1) and IFNy was examined by RT-PCR. The expression levels of proteins linked to macrophage cholesterol metabolism, such as SR-A, LOX-1, ACAT1, ABCA1/G1, SR-BI and signal pathways molecules including phosphorylation expression of p38mitogen-activated protein kinase (p38MAPK), phospho-p38MAPK(P-p38MAPK), AKT and PAKT were measured in atherosclerotic plaque by Western blot. The activity of NF-κB was analyzed by Electrophoretic mobility shift analysis (EMSA). The population of T cell subsets including T helper type1(Th1) cells, IL-17-producing helper T (Th17) cells and regulatory T (Treg) cells in mice splenocytes was measured by flow cytometric analyses.Results The plaque area of normal diet-and topiramate-treated groups markedly reduced compared with control group. In contrast, picrotoxin-and bicuculline-treated groups increased significantly respectively. Collagen deposition in the plaque area of normal diet-and topiramate-treated groups was much less than other groups. The infiltration of macrophages into the plaque was reduced in normal diet-and topiramate-treated groups whereas picrotoxin-treated group increased compared with control group. No difference in the abundance of CD3-positive T cells was observed among groups. RNase protection assay results revealed that the mRNA expression of MCP-1in topiramate-treated group markedly decreased whilst there were no statistically significant differences in IFNy among the5groups. In topiramate-treated group, the protein expression levels of SR-A and LOX-1were profoundly decreased whereas ABCA1/G1and SR-BI were significantly increased and ACAT1modestly elevated compared with control group. In contrast, the protein levels of ABCAl/Gl were markedly reduced and ACAT1was enhanced both in picrotoxin-and bicuculline-treated groups and SR-A was dramatically increased in bicuculline-treated group compared with control group. The protein expression level of P-p38MAPK was significantly reduced in topiramate-treated group compared with control group. However the phosphorylation level of AKT did not altered among the groups. The level of PAKT in normal diet-treated group seemed lower than control group, but it didn’t reach statistical difference. Compared with control group, the activation of NF-κB was markedly higher in topiramate-treated group and lower in picrotoxin-and bicuculline-treated groups. The results revealed the numbers of T cell subsets had no statistical differences between control group and other groups except for the numbers of Th17cell in normal diet group was less than control group.Conclusion In this study, we demonstrated that GABA-A receptor agonist had anti-atherosclerotic effect on apoE-/-mice. Mechanistic studies had shown that GABA-A receptor agonist not only attenuated macrophage infiltration and proliferation due to the reduction of the mRNA expression level of MCP-1and protein expression level of p38MAPK, but also restored the normal macrophage cholesterol metabolism by increasing the protein expression level of ABCA1、ABCG1、SR-BI all contributing to net cholesterol efflux through reverse cholesterol transport, and decreasing of SR-A、LOX-1, both internalizing the modified LDL to facilitate lipoprotein uptake. All of those resulted in the inhibition of macrophage foam cell formation. In contrast, picrotoxin and bicuculline accelerated the formation of macrophage foam cell. However, we didn’t observe that GABA-A receptor agents have any effects on T cells either in atherosclerotic plaque or splenocytes.