通脉养心丸提取物对大鼠离体肠系膜动脉的舒张作用研究

Vasodilation effect and mechanism of extraction of Tongmai Yangxin Pills (TMYX) on isolated rat mesenteric artery

为研究通脉养心丸提取物(TMYX)对大鼠离体肠系膜动脉的舒张作用并探讨其作用机制,采用在大鼠离体肠系膜动脉血管上观察累积浓度TMYX对基础状态血管、苯肾上腺素(PE,1×10^-5 mol·L^-1)和氯化钾(KCl,60 mmol·L^-1)预收缩的血管环的作用,并观察去除血管内皮细胞对TMYX舒张作用的影响。进一步采用一氧化氮合酶抑制剂L-硝基-精氨酸甲酯(L-NAME)和环氧合酶抑制剂吲哚美辛预孵育观察TMYX的舒血管效果。同时在人脐静脉内皮细胞上观察TMYX对细胞NO释放和一氧化氮合酶eNOS磷酸化的影响。结果发现,TMYX在1~750 mg·L^-1对基础状态的内皮完整血管张力无明显影响,对KCl预收缩的内皮完整血管也无明显舒缩作用。但TMYX能有效舒张苯肾上腺素收缩的大鼠肠系膜动脉,最大舒张率为(64.71±10.03)%。用一氧化氮合酶抑制剂(L-NAME,1×10^-4 mol·L-1)预孵育或去除肠系膜动脉血管内皮后,TMYX的最大舒张率分别降为(35.77±8.93)%,(25.85±10.84)%,与未加L-NAME组和内皮完整组比较有显著差异(P<0.01)。但吲哚美辛对TMYX的舒张作用没有影响。此外TMYX 50 mg·L^-1可以显著增加人脐静脉内皮细胞P-eNOS的蛋白表达,促进NO释放,而L-NAME处理后则下调细胞P-eNOS蛋白表达水平,抑制NO的释放。结果说明TMYX具有内皮依赖性的血管舒张作用,其对苯肾上腺素预收缩的大鼠离体肠系膜动脉的舒张作用与一氧化氮-环磷酸鸟苷(NO-cGMP)通路有关。

The aim of the present study is to evaluate the vasodilation effects of Tongmai Yangxin Pills(TMYX) on rat mesenteric artery as well as its mechanism of action. The relaxation effects of TMYX extracts with different concentrations were determined on isolated rat mesenteric artery in normal condition as well as pretreating by phenylephrine and KCl. Vascular relaxation effects of TMTX were also determined in mesenteric artery preincubated with L-ANME and indomethacin or in endothelium denuded mesenteric artery. Moreover, effects of TMYX by 50 mg·L^-1 on NO secretion and the phosphorylation of eNOS in a cellular model of human umbilical vein endothelial cell(HUVEC) pretreated with or without L-NAME were also observed. The experimental results showed that TMYX has no obvious effect on vasodilation of arteries in normal or KCl pretreated condition, while it can dose-dependently relax the rat mesenteric artery with intact endothelium stimulated with phenylephrine at a maximal diastolic rate of(64.71±10.03)%. After preincubating with L-NAME for 15 min or removal of mesenteric artery endothelium, the maximal diastolic rate was decreased to(35.77±8.93)% and(25.85±10.84)% respectively. However, preincubating with indomethacin had no inhibitory effect on TMYX induced vascular relaxation. Meanwhile, TMYX at 50 mg·L^-1 could increase the expression of P-eNOS and the secretion of NO in HUVEC. L-NAME significantly inhibited NO release and phosphorylation of eNOS induced by TMYX. The results suggested TMYX exerted endothelium-dependent relaxation effects against PE-induced contractions of isolated rat mesenteric artery through NO-cGMP signaling pathway.