| Atherosclerosis is a chronic multifactorial disease of the arterial wall, which can lead to acute clinical events such as myocardial infarction and stroke if rupture of plaque and thrombosis occurs. Although several theories or hypothesis about atherogenesis such as lipid infiltration theory, smooth muscle cell proliferation theory, thrombogenesis theory, have been proposed during the past decades, up to now, none can prove entirely satisfactory. The modern theory of atherosclerosis is inflammation theory, which is based on "response to injury hypothesis" and developed by immune hypothesis, emphasizing endothelial dysfunction and immunological function disorder. This suggests that non-neuronal cholinergic system may play an important role in progression of atherosclerosis.The concept of a non-neuronal cholinergic system separate from the cholinergic nervous system was first proposed in 1998. Choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), non-neuronal muscarinic receptors (NNMRs), organic cation transporters (OCTs), and acetylcholinesterase constitute the non-neuronal cholinergic system, which differs markedly from the cholinergic nervous system with regard to synthesis, storage, release, elimination, receptors, and mode of action of acetylcholine. The non-neuronal cholinergic system appears extremely early in the evolutionary process and is found throughout human tissues and organs in epithelial cells, mesothelial cells, endothelial cells and immune cells. The non-neuronal cholinergic system plays an indispensable role in the body. It regulates basic cell functions like gene expression, proliferation, differentiation, cytoskeletal organization, cell-cell contact (tight and gap junctions, and desmosomes), locomotion, migration, ciliary activity, electrical activity, secretion, and absorption; it also plays a part in the control of specific and non-specific immune functions. The relationship between the non-neuronal cholinergic system and atherosclerosis has not been reported in the world.Our laboratory was the first to show that NNMRs have unique pharmacological characteristics and therapeutic effect against atheroscelrosis. The present study was aimed at exploration of the mechanisms by which activation of NNMRs exert antiflammatory effect to prevent the progression of atherosclerosis. The in vivo experiment was carried out in ApoE-/- mice fed the high cholesterol diet and in vitro experiment on ox-LDL-induced rat aortic endothelial cells.1. In vivo study on mechanisms underlying inhibition of atherosclerotic inflammation by activation of NNMRs in apolipoprotein E-deficient miceEight-week-old apolipoprotein E-knockout(ApoE-/-) mice fed on a high cholesterol diet were administered intragastrically with saline or arecoline for 7 weeks at final doses of 7 and 21mg/kg/d. The wild-type C57BL/6J mice were served as normal control, feeding on a normal chow diet and tap water. hematoxylin and eosin, immunohistochemistry, real-time quantitative PCR, western blot, flow cytometry and ELISA were used in this study. Compared with saline-treated ApoE-/-mice, arecoline at 7mg/kg/d and 21mg/kg/d significantly reduced the surface area of atherosclerosis plaque, downregulated VCAM-1, ICAM-1, MCP-1 mRNA and protein expression, decreased the nuclear protein expression and activity of NFkB, and upregulated the expression of PPARγmRNA expression in aorta of ApoE-/- mice.Immune responses participate in the whole inflammatory progress of atherosclerosis. The altered T cell immune responses in arecoline-treated ApoE-/- mice included the following aspects: 1) significantly attenuated T cell infiltration in the aortic root, decreased T cell proliferation rate and CD4+/CD8+ ratio in the spleen, 2) downregulated mRNA expression of co-stimulating factor CD28/CD86 and CD40L, and 3) the shift of pro-inflammatory Th1 to anti-inflammatory Th2 responses. Interferon[IFN]-γand interleukin[IL]-10 can be secreted by Th1 and Th2 cells respectively. We found that arecoline downregulated mRNA expression of IFN-γand upregulated mRNA expression of IL-10 in the aorta of ApoE-/- mice. Correspondingly, concanavalin A(ConA)-induced IFNγsecretion can be decreased and IL-10 production enhanced by arecoline in the cultured splenocytes from ApoE-/- mice. The above-mentioned inhibited T cell immune responses were in line with the suppressed NFκB-DNA binding activity. These findings suggested that the atherosclerosis in ApoE-/- mice can be improved by arecoline through modulation of T cell immune responses, at least partially via depressing NFκB activation.Our previous study demonstrated that arecoline interfered with the immune responses by targeting the non-neuronal muscarinic acetylcholine receptors. Thus, we postulate that the anti-inflammatory effect of arecoline at least is partially due to the action on T cell activity of aorta and spleen in ApoE-/- mice through modulation of NNMR, since and partially due to the direct and indirect action on NFkB and PPAR-γ.2. In vitro study on mechanisms underlying inhibition of atherosclerotic inflammation by activation of NNMRs in rat aortic endotheial cellsStimulated by ox-LDL, MCP-1 gene expression was ignificantly upregulated in rat aorta endothelial cells, the MCP-1 secretion increased and the adhesion of monocyte to endothelial cells promoted. Arecoline treatment at 1～100μM significantly inhibited the above reactions in a dose-dependent way. Immunocytochemical examination showed that arecoline significantly inhibited ox-LDL induced NFkB nuclear translocation. And electrophoretic mobility shift assay (EMSA) indicated that arecoline significantly reduced the NFkB-DNA binding activity. These effects of arecoline on MCP-1 and NFkB could be blocked by atropine, which indicated that the anti-inflammatory effect of arecoline was mediated through activation of endothelial NNMR.3. New mechanisms of Pivanampeta underlying inhibition of atherosclerotic inflammation by activation of NNMRs in rat aortic endotheial cellsPivanampeta(PPVP) is a new compound synthesized in our laboratory which targets NNMR. The protective effect on endothelial cells and against atherosclerosis has been demonstrated in our previous study. Rat aortic endothelial cells were isolated and cultured. Passage 3-5 of cultured cells were used. Nitric oxide(NO) production was measured via griess reaction. MCP-1 secretion was assayed by enzyme-linked immunosorbent assay(ELISA) and the adhesion of monocytes to endothelial cells was counted under the light microscrope. Besides, Peroxisome proliferator activated receptor gamma(PPARγ) mRNA expression was determined by RT-PCR. PPVP significantly enhanced ox-LDL-reduced NO production, decreased ox-LDL-stimulated MCP-1 secretion and monocyte adhesion, and increased ox-LDL-inhibited PPARγmRNA expression. The effect of PPVP on MCP-1 secretion and monocyte adhesion was markedly attenuated by nitric oxide inhibitor(L-NAME). In conclution, PPVP inhibits MCP-1 secretion and monocyte adhesion by affecting NO pathway, which may be associated with the enhanced expression of PPARγ. The new mechanisms involved NO production and enhanced mRNA expression of PPARγfurther validated the effect of activation of NNMR on atherosclerosis.In conclution:1. NNMR dysfunction can be observed at the very initial stage of atherosclerosis. Targeting NNMRs in endothelial cells prevents the progression of atherosclerosis.2.Targeting NNMR can inhibit atherosclerotic inflammation through modulation of T cell immune responses.3. Targeting NNMR reduces atherosclerotic inflammation by affecting NO, NFκB, PPVPγpathway directly or indirectly.4. NNMR is a promising drug target for treatment of atherosclerosis. |
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