Study On The Vasorelaxant Effect Of Lsoapiole In Rat Thoracic Aorta And Its Mechanism

Objective:The Lingnan characteristic herbal medicine, Lemonfragrant Angelica Root (LAR) effects "promoting qi circulation and relieving pain", clinically which used to treat various kinds of pain caused by cariac blood stasis. Our previous studies have found isoapiole was the quantitative index of quality assessment for LAR. This study observes the impacts of each LAR extracts and isoapiole on blood vessel in isolated organ apsect, explores the effects of isoapiole on Vascular smooth muscle cell(VSMC) and Vascular endothelial cell(VEC) in cellular level, and discusses its related mechanism, aiming to further illustrate the scientific connotation of LAR, and to provide a theoretical basis for further clinical development and application.Methods:1. Experimental models of the rat thoracic aorta rings were prepared, norepinephrine(NE) and potassium chloride(KCl) were used to pre-contract blood vessels, and each LAR extracts and Isoapiole were added to observe the related effects on vascular ring tensions, meanwhile the change of vascular ring tensions were recorded after adding the blocker L-NAME into isoapiole.2. The content changes of nitric oxide(NO) before and after isoapiole intervention were detected with Griess method.3. The content changes of NO in human umbilical vein endothelial cell (HUVEC) before and after isoapiole intervention were tested with colorimetry, and western blot was used to analyze the expressing of NO related antibodies like eNOS and PSER1176in endothelial cells(EC).4. Vascular smooth muscle cells (VSMC) were isolated and cultured in vitro, and impacts of isoapiole with different concentration ranges on VSMC proliferation were observed by Cell Counting Kit-8.Results:1. Each LAR extracts had a certain vasorelaxant effect on vascular rings (P <0.01) and petroleum ether, ethyl acetate, butanol, water extract’s relaxation rate were92.38%,91.93%,59.81%,12.20%after administration of30min. Isoapiole did no obvious effects on vascular ring tensions with or without endothelium pre-contracted by KC1(60mmol/L)(P>0.05);2. Isoapiole could relax the vascular rings with endothelium pre-contracted by NE(P<0.05) and the different concentration’s relaxation rate were31.57%,24.12%,28.99%after administration of30min. But did none relaxant effect on vascular rings without endothelium(P>0.05).3. Pre-processed by L-NAME(0. lmmol/L), the vasorelaxant effect of isoapiole on vascular rings with endothelium disappeared(P>0.05).4. Isoapiole could promote the NO expression in rat thoracic aorta rings at0.5h,1h and12h after administration(P<0.05). The concentration of NO corresponding to each time point were88.34,93.69,105.21μmol/L.5. Isoapiole solution could increase the NO synthesis in VEC at0.5h,1h,12h and24h(P<0.05). The concentration of total NO2-corresponding to each time point were35.95,43.36,54.44,61.53μmol/L. Western blotting showed that Isoapiole can increase the expression of eNOS in HUVEC.6. Isoapiole concentration from2u g/ml to2×10-4μg/ml had a certain proliferative effect on VSMC(P<0.05), but less concentration-dependent; with the concentration increasing isoapiole began to inhibit proliferation and when the concentration greater than100μg/ml, it showed significantly proliferation inhibition of VSMC (P<0.05).Conclusions:1.Each LAR extracts had a certain vasorelaxant effect on vascular rings, especially ethyl acetate and petroleum ether extracts’ effect. Isoapiole can endothelium-dependent relaxed the isolated aortic rings, via promoting the synthesis and release of NO.2. Isoapiole can increase the NO synthesis and the expression of eNOS in VEC.3. Isoapiole has dual-directional regulation function on the proliferation of VSMC:in the range of low concentration (2-2×10-4μg/ml) can promote the proliferation of VSMC, the best concentration is about2×10-3μg/ml; while showed inhibition effcets in high concentration (more than0.1mg/ml).