Protection Of EPD Targeted For NNMR On Endothelial Function Damaged By Ox-LDL And Uric Acid

Atherosclerosis is a common and frequently-occurring disease, which seriously harm humanity health. In westernized societies, it is the underlying cause of about 50% of all death. In our country the atherosclerosis disease incidence rate also rose year by year in recent years. But so far, there isn't the anti-atherosclerosis ideal medicine, and FDA didn't authorizated any medicine as anti- atherosclerosis function. Therefore, the research developing the new function mechanism, the security, the effective anti-atherosclerosis medicine, has the great significance.Atherosclerosis is a chronic inflammation disease. It is well established that endothelial damage and dysfunction is the initiating event in atherosclerosis. Therefore, it is a good tactics that protecting the endothelial function. When the factors damaging the endothelial function are existing, vascular endothelial cell non-neuronal muscarinic receptor (NNMR) function become lower. Based on the literature investigation and the work foundation, we selected three Chinese native medicine monomers, code respectively A, B, C, to conduct the anti-atherosclerosis compound research. A medicine is the NNMR agonist, which may activate the endogenous protection mechanism. In the rabbit and quail atherosclerosis model, has the accurate prevention therapeutics function. The B medicine has the anti-calcium function, and the C medicine has the anti-oxidation function. The two medicines have the endothelium protective function and the anti-atherosclerosis function in view of the atherosclerosis external independent dangerous factors. In the endothelial cell damage model induced by ox-LDL, Cuige Shi and so on, using the even design, observed the influence of A, B, and C cooperation on the ICAM-1 gene expression in damaged endothelial cells. Through the mathematical statistic analysis, Shi discovered three medicines has the correlation, and in three medicines the A medicine was primary medicine, which could not lack in the association. The drug efficacy was best when the proportion is 1∶40∶30, named as EPD. In the endothelial cell damage model induced by hyperhomocysteinemia, Zhibian Duan and so on discovered that EPD, in 2, 6, 10 mmol/L concentration (A medicine concentration), remarkably suppressed ICAM-1, VCAM-1, the MCP-1 gene overexpression, and had the endothelium protective function.Basing the foundation of the earlier work, we first compared the effect of EPD to arecoline, the host ingredient of EPD, in endothelial cells model of metabolic disturbance induced by ox-LDL. We also observed the inhibition of atropine, a inhibitor of arecoline, to EPD, indirectly judging ingredients B and C function characteristic. Then we evaluated the effect of EPD in endothelial cells model of metabolic disturbance induced by high uric acid. Finally we observed the anti-atherosclerosis effect of EPD in ApoE-/-mice atherosclerosis model. We would provide the experiment relying for EPD developing into the anti-atherosclerosis medicine through this research.1. EPD protected against endothelial dysfunction in endothelial cells model of metabolic disturbance induced by high uric acid.1.1 Inhibited the overexpression of ICAM-1, VCAM-1 and MCP-1 mRNA in high uric acid damaged endothelial cells.EPD inhibited the overexpression of intercellular adhesive molecule-1 (ICAM-1), vascular cell adhesive molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) mRNA in endothelial cells damaged by high uric acid in a concentration-dependent manner at the concentrations of 0.01～100μM, and could be inhibited by atropine.1.2 Enhanced the NO production released by the high uric acid damaged endothelial cellsEPD produced concentration-dependent NO increases in high uric acid damaged endothelial cells at the concentrations of 0.01～100μM, and could be inhibited by atropine.Conclusion: EPD protected against endothelial dysfunction induced by uric acid and it's effect is better than arecoline.2. EPD protected against endothelial dysfunction in endothelial cells model of metabolic disturbance induced by ox-LDL.2.1 Suppressed endothelium-monocytes adhesion and migration induced by ox-LDLEPD suppressed endothelium-monocytes adhesion induced by ox-LDL in a concentration-dependent manner at the concentrations of 0.01～100μM. It's effect was better than arecoline and could be blocked by atropine and L-NAME. EPD suppressed U937 monocytic cells migration to ECV304 endothelial cells induced by ox-LDL in a concentration-dependent manner at the concentrations of 0.1～10μM.2.2 Inhibited the overexpression of ICAM-1, VCAM-1 and MCP-1 induced by ox-LDL.EPD inhibited the overexpression of intercellular adhesive molecule-1 (ICAM-1), vascular cell adhesive molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) in endothelial cells damaged by ox-LDL in a concentration -dependent manner at the concentrations of 0.1～10μM, and could be inhibited by atropine.2.3 Enhanced the NO production released by the ox-LDL damaged endothelial cellsEPD produced concentration-dependent NO increases in ox-LDL damaged endothelial cells at the concentrations of 0.1～10μM, and could be inhibited by atropine. Conclusion: EPD protected against endothelial dysfunction induced by ox-LDL and it's effect is better than arecoline.3. EPD alleviated atherosclerosis and inflammation responses in ApoE knockout mice3.1 Alleviated atherosclerosis in ApoE knockout miceApoE^-/- mice fed with a high cholesterol diet for 7 weeks developed fibrofatty plaques with lipid-rich core in aortic root. 7-week administration of EPD significantly reduced lesion sizes from 176178±20449μm² to 51524±7414μm² (P<0.01), and in parallel, reduced the fractional area of lesion dramatically by 73%(P<0.01) at the doses of 10mg/kg in aortic root. ApoE^-/- mice challenged with a high-fat, cholesterol-rich diet for 7 weeks developed severe hypercholesterolemia. The high serum cholesterol level in fat-fed apoE^-/- mice was not affected by EPD at 10 mg/kg administered intragastically. Thus, the atheroprotective effects of EPD could not be explained by its effects on the lipid metabolism.3.2 Suppressed inflammation immune responses in ApoE knockout mice fed with a high cholesterol diet3.2.1 Reduced T lymphocyte infiltration and adhesive molecule overexpression in the atherosclerotic plaquesThere was a 55.0%(P<0.01) reduction in CD3+ T lymphocyte infiltration in the atherosclerotic plaques in apoE^-/- mice treated with 10mg/kg EPD, when compared with untreated apoE^-/- mice. EPD at the dose of 10mg/kg significantly inhibited ICAM-1, VCAM-1, MCP-1, VLA-4 gene overexpression and the MCP-1 protein overexpression, didn' affect LFA-1 gene expression.3.2.2 Suppressed T lymphocyte proliferation, regulated the Thl/Th2 balanceTreatment with EPD at 10mg/kg resulted in 40.3%(P<0.01) reducing of T cell proliferation stimulated by concanavalin A in spleen, as compared with the apoE^-/- mice untreated with EPD. EPD didn't affect the percent of B lymphocyte and the CD4+/CD8+ ratio.EPD at the dose of 10mg/kg remarkably reduced the spleen and aorta IFNγgene overexpression, as well as the spleen IL-12 gene overexpression, simultaneously reduced aorta IFNγprotein overexpression. EPD did not affect the spleen and aorta IL-4 gene expression and spleen IL-10 the gene expression, but remarkably elevated aorta IL-10 gene overexpression. EPD inhibited Th1 factor IFNγ, IL-12 expression, promotes Th2 factor IL-10 expression, regulated the Th1/Th2 immunity balance.3.2.3 Inhibited coordination stimulates factor CD28/CD86 gene overexpressionEPD 10mg/kg inhibited coordination stimulates factor CD28/CD86 gene overexpression, inhibited CD40L gene overexpression, did not affect CD40 gene expression.3.2.4 Regulation signal passage essential molecular expressionEPD at the dose of 10mg/kg promoted the spleen and aorta NNMR gene expression, inhibited NF-kappa B gene and protein overexpression, promoted the PPARγgene expression.3.2.5 Inhibited spleen T lymphocyte proliferation and Th1 cell factor INFγoverexpression in vitro.EPD inhibited spleen T lymphocyte proliferation and Th1 cell factor INFγoverexpression stimulated by concanavalin A in a concentration-dependent manner at the concentrations of 1～100μM.Conclusion: EPD at the dose of 10 mg/kg alleviated atherosclerosis in ApoE knockout mice, suppressed T lymphocyte proliferation, regulated the Th1/Th2 balance, and inhibited coordination stimulates factor CD28/CD86 gene overexpression. EPD alleviated atherosclerosis in ApoE knockout mice through Suppressing inflammation immune responsesOur results indicated that EPD suppressed endothelium-monocytes adhesion and migration, inhibited adhesive molecule overexpression, and enhanced the NO production in a concentration-dependent manner in endothelial cells model of metabolic disturbance induced by ox-LDL and high uric acid. Protection of EPD on endothelial function was better than arocoline. EPD alleviated atherosclerosis and inflammation responses in ApoE knockout mice. This research provided experimental basis for EPD developing into new anti-atherosclerosis medicine.