肌肽对血管平滑肌细胞钙化的影响及机制研究

Study of the Effect of Carnosine on Vascular Smooth Muscle Cell Calcification and Its Mechanism

目的:探讨肌肽对β磷酸甘油诱导的大鼠血管平滑肌细胞(VSMCs)钙化形成的影响及可能的机制。方法:原代分离和培养大鼠胸主动脉VSMCs,分为对照组(正常培养液)、钙化组(10mMβ-磷酸甘油)和干预组(10mMβ-磷酸甘油和10mM肌肽),连续处理10d。茜素红染色和碱性磷酸酶(ALP)活性测定各组细胞钙化情况,Western Blot测定BMP-2、Runx2、Cleaved-caspase3、β-catenin、GSK-3β(Ser9)的蛋白表达。结果:β-磷酸甘油处理后,VSMCs出现钙化,与对照组比较,钙盐沉积显著增加,碱性磷酸酶活性明显上调,成骨标志物BMP-2、Runx2蛋白表达显著升高;与钙化组比较,肌肽能显著减少钙盐沉积、降低碱性磷酸酶活性,下调BMP-2、Runx2蛋白表达。细胞凋亡结果显示,钙化组Cleaved-caspase3蛋白表达显著上调,肌肽明显降低Cleaved-caspase3水平。信号通路蛋白检测发现,β-磷酸甘油下调GSK-3β(Ser9)蛋白表达,上调β-catenin蛋白表达,肌肽可明显反转这一作用。结论:肌肽可抑制由高磷诱导的VSMCs钙化,其机制可能与抑制β-catenin信号通路调控的成骨因子转录生成和细胞凋亡有关。

Objective:To investigate the effect and mechanism of carnosine on vascular calcification inβ-glycerophosphate(β-GP)-induced vascular smooth muscle cells(VSMCs).Methods:Primary isolated and cultured rat aortic VSMCs were divided into 3 groups,the control group(normal culture medium),calcification group(10mMβ-GP),and carnosine group(10mMβ-GP+10mM carnosine).The cells were treated consecutively for 10 days.Alkaline phosphatase(ALP)activities and alizarin red staining were used to detect the calcification of VSMCs.The expression levels of BMP-2,Runx2,Cleaved-caspase3,β-catenin and GSK-3β(Ser9)proteins in the VSMCs in various groups were measured by Western blotting method.Results:Afterβ-GP treatment,calcification occured in VSMCs.Compared with control group,calcium salt deposition and ALP activity increased significantly,and osteogenic marker BMP-2 and Runx2 proteins were significantly upregulated in calcification group.Compared with calcification group,carnosine markably reduced the calcium salt deposition,inactivated the ALP activity,and downregulated BMP-2 and Runx2 proteins.The apoptosis result showed that Cleaved-caspase3 protein was increased in calcification group and decreased in carnosine group.The signal pathway protein analysis revealed thatβ-GP downregulated GSK-3β(Ser9)and upregulatedβ-catenin,while carnosine reversed the proteins expression obviously.Conclusion:Carnosine inhibits VSMCs calcification induced by hyperphosphate and may be related to inhibition ofβ-catenin signaling pathway and its upregulating osteogenic factor transcription and apoptosis.