外源性硫化氢通过对抗氧化应激减轻血管平滑肌细胞钙化

Exogenous Hydrogen Sulfide Attenuates Vascular Smooth Muscle Cells Calcification Partly by Competing against Oxidative Stress

目的探讨外源性硫化氢减轻人脐静脉平滑肌细胞(HVSMCs)钙化的机制。方法采用组织块重复贴壁法培养原代HVSMCs,经免疫细胞化学法鉴定后,将细胞随机分为对照组、高磷组(12mmolβ-GP)、高磷+Na HS组(8.0×10-6mol Na HS)、高糖组(25mmol D-葡糖糖)、高糖+Na HS组(8.0×10-6mol Na HS)。采用茜素红S染色法定性检测平滑肌细胞钙化程度;MTT法检测细胞存活率;比色法检测细胞内钙含量、碱性磷酸酶(ALP)活性、丙二醛(MDA)含量及总超氧化物歧化酶(T-SOD)活力;DCFH-DA荧光标记染色法检测细胞活性氧(ROS)水平。结果 MTT结果显示,与正常组相比,高磷和高糖均使细胞存活率下降(P均<0.05),而加入Na HS干预的两组细胞存活率基本恢复至正常组水平。与正常组相比,高磷组和高糖组钙沉积明显增加,胞内钙含量、ALP活性显著升高(P<0.05),氧化应激指标ROS增加、MDA含量升高(P<0.05),而抗氧化应激指标T-SOD活力下降(P<0.05)。而分别加Na HS干预的两组细胞,与高磷组和高糖组相比钙沉积减轻,钙含量、ALP活性、ROS、MDA回升(P<0.05),而T-SOD活力升高(P<0.05)。结论外源性硫化氢部分通过对抗氧化应激而减轻血管平滑肌细胞钙化。

Objective To investigate the effect of exogenous hydrogen sulfide on human umbilical vein smooth muscle cells( HVSMCs) calcification. Methods Primary HVSMCs were cultured by Utilizing tissue re- adherence explant. HVSMCs were randomly divided into control group,high phosphorus group( 12 mmol β-GP),high phosphorus + Na HS group( 8. 0 × 10- 6mol Na HS),high glucose group( 25 mmol D- glucose),high glucose + Na HS group( 8. 0 × 10- 6mol Na HS). Alizarin Red S was used to test the calcification extent of HVSMCs. The survival rate of cells was detected by MTT. alkaline phosphatase( ALP) activity,malondialdehyde( MDA),and total superoxide dismutase( T- SOD) vitality were detected by Colorimetry. DCFH-DA fluorescence labelled staining of intracellular reactive oxygen species( ROS). Results MTT assay showed that,compared with the normal group,the cells survival rate of high phosphorus group and high glucose group were significantly decreased,and after add Na HS,two groups of cells survival rate recovered to the normal level( P < 0. 05). Compared with the normal group,calcium deposition,intracellular calcium content and ALP activity in the high phosphorus group and the high glucose were significantly increased( P < 0. 05),as well as the oxidative stress indicators for example ROS and MDA,and anti- oxidative stress indicator T-SOD activity was significantly decreased( P < 0. 05). The intervention groups were added Na HS,compared with the high phosphorus group and the and high glucose,calcium deposits alleviated,calcium content,ALP activity,ROS,MDA rised significantly up( P < 0. 05),while the T- SOD activity decreased significantly( P < 0. 05). Conclusion Exogenous hydrogen sulfide can attenuate vascular smooth muscle cells calcification( at least partly) by competing against oxidative stress.