丹参酮ⅡA对主动脉内皮细胞功能损伤的保护机制

Effect of tanshinone ⅡA on endothelial nitric oxide synthase gene expression and intracellular Ca^(2+) level induced by angiotensin Ⅱ in cultured porcine aortic endothelial cells

目的通过观察猪主动脉内皮细胞在血管紧张肽Ⅱ(AngⅡ)作用后,丹参酮ⅡA对血管内皮细胞分泌一氧化氮(NO)及内皮型一氧化氮合酶(eNOS)蛋白、mRNA表达和细胞内游离钙离子([Ca2+]i)浓度的变化,探讨丹参酮ⅡA对血管内皮细胞的保护作用。方法采用硝酸还原法、免疫组织化学法、逆转录聚合酶链式反应(RT-PCR)和激光共聚焦扫描显像系统,分别检测NO水平、eNOS蛋白和mRNA表达,以及细胞内游离钙离子浓度([Ca2+]i)水平的变化。结果①随着AngⅡ浓度的增加、作用时间的延长,内皮细胞NO的产生及eNOS的基因表达呈顺序下降(P<0.01),表现出剂量、时间依赖性的负性作用。②丹参酮ⅡA可抑制AngⅡ对内皮细胞分泌NO及eNOS表达的负性作用(P<0.01),但尚不能恢复到空白对照组水平(P<0.05)。在丹参酮ⅡA作用1、6h,A组的抑制效应明显强于B组(P<0.05);随着作用时间延长至24h,两组间差异无统计学意义(P>0.05)。③AngⅡ可引起主动脉内皮细胞[Ca2+]i显著升高(P<0.01),丹参酮ⅡA可部分抑制AngⅡ诱导的内皮细胞[Ca2+]i升高(P<0.05)。结论丹参酮ⅡA通过增加血管内皮细胞eNOS基因表达及细胞分泌NO水平和降低内皮细胞[Ca2+]i水平来发挥对血管内皮细胞的保护作用。

Objective To investigate the protective role of tanshinone Ⅱ A on intracellular Ca^2＋ （ [ Ca^2＋ ] i） level and endothelial nitric oxide synthase （ eNOS ） gene expression effected by different contents and time of Angiotensin Ⅱ （ AT - Ⅱ） in porcine aortic endothelial cells （ PAEC ）. Method The NO level and gene expression of eNOS was measured by nitric acid deoxidizing and Immunohistochemistry and RT - PCR methods, separately. The level of [ Ca^2＋ ] i in single PAEC was observed with laser - scanning confocal microscope. Results ①With the increasing of concentration and effect time of AT - Ⅱ , the level of NO and eNOS - mRNA in PAEC was decreased in dose - and time - dependent manner （ P 〈 0.01 ）. ②Tanshinone Ⅱ A could inhibited the effect of AT - Ⅱ on the level of NO and eNOS - mRNA （ P 〈 0.05 ）. At 1 h and 6 h, the inhibition of tanshinone Ⅱ A on AT - Ⅱ effect in A group was more than that of B group. But at 24 h, no significant difference was found between both A and B group （ P 〉 0. 05）. ③AT - Ⅱ could increase [Ca^2＋ ]i in PAEC,but it could inhibited by tanshinone Ⅱ A. Conclusion Tanshinone ⅡA have a protective role on vascular endothelial ceils by increasing NO level and eNOS gene expression and decreasing [ Ca^2＋ ] i level in PAEC effected by AT - Ⅱ.