The Therapeutic Role Of Soluble Epoxide Hydrolase Inhibitor AUDA In The Atherosclerosis And Related

Background:Cardiovascular disease (CVD) is the leading cause of morbidity, disability, and mortality worldwide. And atherosclerosis, the principal cause of myocardial and cerebral infarction, accounts for the majority of these deaths. Effective prevention of atherosclerosis and reasonable inhibition of its progress will be key measures to the prevention and control of CVD. Epoxyeicosatrienoic acids (EETs) have been shown to have anti-inflammatory effects and therefore may play a role in preventing vascular inflammatory and atherosclerotic diseases. Soluble epoxide hydrolase (sEH) converts EETs into less bioactive dihydroxyeicosatrienoic acids (dHET). Thus, inhibition of s-EH can prevent degradation of EETs and prolong their effects. Several studies have shown that a variety of sEH inhibitors can reduce blood pressure in hypertensive rats and decrease inflammation in a murine sepsis model, and that sEH inhibitors possess antinociceptive properties in inflammatory pain models. Given that sEH is widely expressed in the heart and its inhibition is anti-inflammatory in a range of disease models, we tested the hypothesis that inhibition of sEH may attenuate atherosclerotic lesion formation. Since atherosclerosis is now considered to be an inflammatory disease and is frequently associated with hypertension, we investigated the use of sEH inhibitors as potential Pharmaceuticals for the treatment of this disease.Objective:The aim of present study was to assess the therapeutic effect of sEH inhibitor, AUDA in a mouse model of atherosclerosis and to explore the possible mechanisms, which could provide evidences for the application of clinical practice in view of theory and experiment. The thesis will be presented according to following parts:A. Therapeutic effect of AUDA:Establishing a mouse model of atherosclerosis to provide study model for therapeutic effect evaluation; Optimization the concentration of sEH inhibitor, AUDA and assessment of the therapeutic role of AUDA in atherosclerosis.B. Exploring the mechanisms of sEH inhibitor, AUDA in the treatment of atherosclerosis.MethodsA. Therapeutic effect of AUDA:a. Establishment the mouse model of atherosclerosis:Eight weeks old ApoE-/-mice were fed with high cholesterol diet for 2 weeks and then were performed with perivacular constrictive silica collars placement on the left common carotid artery to establish mice model. Litter-mate C57BL/6 mice were used as control.b. Biochemical analyses to assess the plasma lipid level. The plasma of atherosclerosis and control mice at different time points (8 weeks,16 weeks and 24 weeks) were collected and the plasma lipid level, including TC (total cholesterol), TG (Triglyceride), LDL (Low density lipoprotein) and HDL (high density lipoprotein) were analyzed to detect the progression of the disease.c. Histopathological analyses to assess the disease course of atherosclerosis. Left carotid arteries were isolated and prepared into paraffin or frozen sections. Sections were stained with hematoxylin and eosin (H&E) to observe the features of the mice vessels.d. Based on the atherosclerosis model established above, a series of concentration of sEH inhibitor AUDA were applied into the mouse model for treatment from Week 10. At the time of Week 18, the plasma of atherosclerosis mice was obtained for inflammatory cytokines (TNF-α, IL6 and TGF-β), adhesion molecules (ICAM-1, VCAM-land P-selectin) and extracellular matrix proteins (MMP-2 and MMP-9) were quantified by using Enzyme-linked Immunosorbent Assay. Optimized treatment concentration of AUDA was conducted by analyzing the ELISA data.e. The atherosclerosis mice were applied with optimized concentration of AUDA and the therapeutic effect was evaluated. The mice weight and blood lipid level was quantified. Moreover, the plaque size and the percentage of plaque to lumen were also calculated.B. Exploring the mechanisms of AUDA in the treatment of atherosclerosis.a. The atherosclerosis mice were applied with optimized concentration of AUDA and carotid arteries were collected to perform the histopathological analyses. The infiltration of monocyte/macrophage and the amount of the smooth muscle cells were determined.b. The atherosclerosis mice were applied with optimized concentration of AUDA and carotid arteries were collected to perform the immunoblot. The protein expression of Toll like receptor 4 and nuclear factor κb were analyzed.ResultsA. Therapeutic effect of AUDA:a. Establishment the mouse model of atherosclerosis:The blood lipid level results showed that significant increasing levels of TC, TG, HDL and LDL were found at 8,16 and 24 weeks on ApoE-/-mice than the control mice.The HE staining showed that no significant plaque formation in carotids and only slight thickened vascular intima were found on the 8 week old ApoE-/-mice. After performing with the surgery and feeding with high cholesterol diet until 16h week, the ApoE-/-mice showed significant intima thickening and thickened fibrous cap was also found due to the hyperproliferation of smooth muscle cells. When the mice were fed with high cholesterol diet until 24th week, significantly increased vessel stenosis with thin fibrous cap and necrosis core was found. No significant plaque formation was found on the litter-mate control C57BL/6 mice.b. Effects of AUDA on the levels of inflammatory cytokines, adhesion molecules and extracellular matrix proteins and determination of the optimized concentration of AUDAThe atherosclerosis mice were applied with different concentration of AUDA and we found 0.35 mg/kg and 0.45 mg/kg AUDA could significantly decrease the level of inflammatory cytokines (TNF-a, IL6 and TGF-P), adhesion molecules (ICAM-1, VCAM-land P-selectin) and extracellular matrix proteins (MMP-2 and MMP-9). By comparing those factors in wild type C57BL/6, we conducted 0.35 mg/kg AUDA was the optimized concentration of AUDA.c. Therapeutic effectsThe results of mice weighing showed that the weight of AUDA treated atherosclerosis mice was significant lighter than saline treated atherosclerosis mice. The results of blood lipid level showed that the level of TC, TG and LDL was significantly lower in AUDA treated atherosclerosis mice than saline treated atherosclerosis mice while HDL was higher in AUDA treated atherosclerosis mice.The histopathological analyses of the aorta root sections showed that plaque area was significantly lower in AUDA treated atherosclerosis mice than saline treated atherosclerosis mice. Moreover, the percentage of plaque to lumen was also significant lower in AUDA treated atherosclerosis mice than saline treated atherosclerosis mice.B. The mechanisms of AUDA in the treatment of atherosclerosisa. AUDA can significantly decrease the infiltration of monocytes/macrophage and the number of smooth muscle cells in AUDA treated atherosclerosis mice than saline treated atherosclerosis mice.b. AUDA can significantly decrease the expression of TLR4 and NF-kB in AUDA treated atherosclerosis mice than saline treated atherosclerosis mice.Conclusionsa. sEH inhibitor AUDA exerted a therapeutic effect by controlling the weight and blood lipid level and decreasing plaque area and stenosis of the vessel.b. sEH inhibitor AUDA exerted its therapeutic effect by affecting the level of inflammatory cytokines (TNF-α, IL6 and TGF-β), adhesion molecules (ICAM-1, VCAM-1 and P-selectin) and extracellular matrix proteins (MMP-2 and MMP-9); decreasing the monocyte/macrophage infiltration and smooth muscle cells number; and suppressing the expression of TLR4 and NF-Kb.c. The cardioprotective effect was exerted by AUDA and it might be applied into the clinical practice in the future.Novelty and significanceSignificance:Here, we presented a study to elucidate the therapeutic role of sEH inhibitor AUDA in the treatment of a mouse of atherosclerosis and explore the possible mechanisms, which might have an impact on the prevention of atherosclerosis and show its ability in the treatment both in clinic in the near future.Novelty:a. Optimized concentration of sEH inhibitor AUDA in the treatment of atherosclerosis was 0.35 mg/kg and the effective role of sEH inhibitor AUDA in the treatment of atherosclerosis might provide novel insight into the disease.b. The mechanisms of sEH inhibitor AUDA in the treatment of atherosclerosis was explored as following ways:1. Decreasing the level of inflammatory cytokines (TNF-α, IL6 and TGF-β), adhesion molecules (ICAM-1, VCAM-1 and P-selectin) and extracellular matrix proteins (MMP-2 and MMP-9); 2. Decreasing the monocyte/macrophage infiltration and smooth muscle cells number; 3. Down regulating the expression of TLR4 and NF-Kb. These will provide novel explanation of the mechanisms.Limitation of this studyA. The drug target of AUDA was needed to be clarified.B. The mechanism of AUDA in the treatment of atherosclerosis was needed to be further elucidated.