中性鞘磷脂酶2/神经酰胺调节高磷高钙诱导的血管平滑肌细胞成骨样分化和钙化

nSMase2/ceramide Regulates High Calcium and Phosphate-Induced Osteogenic Differentiation and Calcification of Vascular Smooth Muscle Cells

【目的】阐明nSMase2是否调控高磷高钙诱导的血管平滑肌细胞钙化。【方法】采用人血管平滑肌细胞钙化体外模型,用高磷高钙诱导血管平滑肌细胞钙化。分别用鞘磷脂酶抑制剂GW4869和siRNA干扰敲低nSMase2处理血管平滑肌细胞,检测骨相关蛋白BMP2、Runx2、Osterix的表达和细胞钙化程度。【结果】在细胞钙化过程中nSMase2的表达水平和神经酰胺的含量增高(P<0.05);GW4869药理抑制或nSMase2 siRNA敲低nSMase2均能减轻高磷高钙诱导的细胞钙化和下调Runx2、BMP2、Osterix的表达水平(P<0.05)。相反,ceramide可加速高磷高钙诱导的细胞钙化和增加ALP的活性(P<0.05)。【结论】nSMase2/ceramide促进高磷高钙诱导的血管平滑肌细胞钙化,提示nSMase2/ceramide可能参与慢性肾病血管钙化的过程。

【Objective】Vascular calcification is a gene-regulated biological process similar to bone formation. It is very common in the patients with chronic kidney disease. Neutral sphingomyelinase 2（nSMase2）is a key regulator of bone development,and is responsible for ceramide generation through sphingomyelin hydrolysis,but the role of nSMase2 in vascular calcification remains unclear. The aim of this study is to determine whether nSMase2 regulates high calcium and phosphate-induced calcification of vascular smooth muscle cells（VSMC）.【Methods】In vitro model of human VSMC calcification was used in this study and high calcium and phosphate were used to induce calcification of VSMCs. GW4869 was used to inhibit nSMase2 activity and nSMase2 was knockdowned in cultured VSMC using nSMase2 siRNA. The expression of Runx2,BMP2 and Osterix was analyzed by q RT-PCR and calcification was assessed by alizarin red staining. 【Results】We found that nSMase2 expression and ceramide levels were increased in the process of VSMC calcification（P〈0.05）. Inhibition of nSMase2 activity by GW4869 and knockdown of nSMase2 attenuated high calcium and phosphate-induced VSMC calcification and down-regulated the expression level of Runx2,BMP2 and Osterix（P〈0.05）. By contrast,ceramide accelerated rat VSMC calcification and increased ALP activity（P〈0.05）.【Conclusion】We demonstrate that nSMase2/ceramide promotes high calcium and phosphate-induced VSMC calcification,suggesting that nSMase2/ceramide could participate in the progression of vascular calcification in patients with chronic kidney disease.