Ghrelin在维生素D3和尼古丁诱导大鼠血管钙化中的作用

Role of Ghrelin on the vascular calcification induced by vitamin D3 combined with nicotine in rats

目的建立大鼠血管钙化(VC)模型并探讨Ghrelin在VC中的作用。方法将40只8周龄SD大鼠随机分为4组:钙化组、对照组、钙化+低浓度Ghrelin组、钙化+高浓度Ghrelin组。钙化组用尼古丁灌胃(溶解于花生油中)联合维生素D 3(VD 3)溶解于无水乙醇肌肉注射,对照组仅用花生油和无水乙醇做相同处理,钙化+低浓度Ghrelin组和钙化+高浓度Ghrelin组分别在VD 3和尼古丁处理基础上分别腹腔注射50 μg/kg、100 μg/kg剂量的Ghrelin每日1次,连续4周。用Von Kossa染色观察大鼠胸主动脉VC程度,ELISA法测血清钙离子(Ca^2+)、碱性磷酸酶(ALP)及胸主动脉骨形态发生蛋白-2、4(BMP-2、BMP-4)含量,免疫组化法检测胸主动脉BMP-2、BMP-4表达。Western blot法检测4组BMP-2、BMP-4和Ghrelin的表达。结果钙化组和对照组Von Kossa染色证实VC模型建立成功,且高浓度Ghrelin比低浓度Ghrelin有更明显的抑制VC作用,ELISA法测得血清Ca 2+、ALP、胸主动脉BMP-2、BMP-4含量以及免疫组化法、Western blot法测BMP-2、BMP-4蛋白表达验证Ghrelin有抑制VC的作用,高浓度Ghrelin抑制VC更加明显( P <0.05)。Western blot检测4组Ghrelin表达情况,其中钙化组和对照组相比较差异无统计学意义( P >0.05)。结论维生素D 3联合尼古丁可以成功建立大鼠VC模型,Ghrelin能够在一定程度上抑制VC的发展。

Objective To establish the rat vascular calcification(VC) model and explore the effect of Ghrelin on vascular calcification. Methods Forty sprague dawley rats aged 8 weeks were randomly divided into four groups: calcification group, control group, calcification + low concentration Ghrelin group, calcification + high concentration Ghrelin group. In the calcification group, nicotine was intragastrically administered(dissolved in peanut oil) and vitamin D 3(VD 3) dissolved in anhydrous ethanol was intramuscularly injected,calcification + low concentration of Ghrelin group and calcification + high concentration of Ghrelin group was intraperitoneally injected with 50 μg/kg and 100 μg/kg Ghrelin once daily for 4 weeks on the basis of vitamin D 3 and nicotine treatment. The degree of calcification of rat thoracic aorta was observed by Von Kossa section staining. ELISA was used to measure serum calcium (Ca^2+), alkaline phosphatase (ALP) and thoracic aortic bone morphogenetic protein-2, 4 (BMP-2, BMP- 4) content, immunohistochemistry was used to detect the expression of BMP-2 and BMP-4 in the thoracic aorta. Western blot assay was used to detect the expression of BMP-2, BMP-4 and Ghrelin. Results The calcification group and the control group confirmed that the vascular calcification model was established successfully by Von Kossa staining, and the high concentration of Ghrelin inhibited vascular calcification more than the low concentration of Ghrelin. The serum Ca 2+, ALP, thoracic aorta BMP-2, BMP-4 content were measured by ELISA method.The BMP-2, BMP-4 protein expression confirmed that Ghrelin inhibited vascular calcification by immunohistochemistry and Western blot, high concentration of Ghrelin inhibited vascular calcification more significantly ( P <0.05). Western blot results demonstrated that there was no significant difference between the calcification group and the control group ( P >0.05). Conclusion Nicotine combined with vitamin D 3 can establish rat vascular calcification model, and hrelin can inhibit the development of calcification.