生长素抑制大鼠血管钙化及其可能机制

The Effects and the Possible Mechanism Involved in Vivo and Vitro of Ghrelin on Rat Vascular Calcification

为探讨生长素调节血管钙化的可能机制 ,用肌肉注射维生素D3和口服尼古丁诱导大鼠血管钙化模型和 β 甘油磷酸盐诱导血管平滑肌细胞钙化模型 ,采用原子吸收分光光度法测定血管和细胞钙含量 ,磷酸苯二钠法测定碱性磷酸酶活性 ,4 5CaCl2 摄入测定钙沉积 ,逆转录—聚合酶链反应测定生长素、骨桥蛋白和内皮素mRNA表达水平 ,放射免疫分析方法测定血浆生长素和内皮素含量。结果表明 ,维生素D3和尼古丁明显诱导大鼠血管钙化 ,β 磷酸甘油可诱导血管平滑肌细胞钙化。采用 30和 30 0nmol kg生长素治疗大鼠均可抑制血管钙化 ,且高剂量时效应强于低剂量 ,与钙化组相比较 ,血管组织钙化指标均下降并且差异有显著性 ,而骨桥蛋白mRNA的表达明显上调。 10 - 8～ 10 - 6 mol L生长素呈浓度依赖性降低血管平滑肌细胞钙化指标 ,并上调骨桥蛋白mRNA表达。此外生长素明显下调血浆内皮素浓度及血管组织的内皮素表达 ,且高剂量生长素的效应更强。结果提示 ,生长素可能部分通过拮抗血浆和血管组织局部的内皮素系统效应而产生抑制血管组织和血管平滑肌细胞钙化的作用。

Aim We employed rat vascular calcific model induced by vitamin D 3 intramuscular injection and oral nicotine, and rat calcific vascular smooth muscular cells (VSMC) induced by β-glycerophosphate to study the possible mechanism which was involved in the regulatory action of ghrelin in vascular calcification. Methods The total aorta Ca content in aorta and VSMC were measured by atom absorptive spectrophotography; the alkaline phosphatase (ALP) activity were determined by the use of phenyl diphosphate-2-sodium; aortic 45 Ca-deposition was detected with the using of 45 CaCl 2; the mRNA expression of ghrelin, osteopontin (OPN) and endothelin were analyzed by reverse-transcript PCR, and the levels of ghrelin in plasma, endothelin in plasma and aorta were determined by radioimmunoassay. Results Vitamin D 3 and nicotine induced vascular calcification in rats and β-glycerophosphate induced calcification in VSMC too. The calcification in aorta was significantly attenuated by subcutaneous administration with ghrelin 30 and 300 nmol/kg for 4 weeks, and the latter dosage was more potent than the former. After the ghrelin treatment, the total aorta Ca 2+ contents, aortic 45 Ca-deposition, ALP activity were less respectively, than those in the rats with vascular calcification, but the OPN mRNA was up-expressed. After treated with ghrelin 10 -8～10 -6 mol/L, the calcification in VSMC was also attenuated, with decrease in the total Ca 2+ content, 45 Ca-deposition and ALP activity in VSMC in a concentration-dependent manner, so did the increase in OPN mRNA expression. In addition, we observed that endothelin levels of plasma and aorta and aortic endothelin mRNA expression significantly increased in the rats with vascular calcification, and ghrelin treatment significantly decreased them, with high dosage more portent than the lower. Conclusion Our results indicated that ghrelin can significantly attenuate the calcification in aorta and VSMC partly through the antagonism with endothelin system in circulation and vascular tissues.