氯化镧通过低氧诱导因子1信号通路抑制高磷诱导的人血管平滑肌细胞钙化  

Lanthanum chloride inhibits high concentration phosphate-induced calcification in human vascular smooth muscle cells via HIF-1 signaling pathway

作　　者：王琪雯 赵璐璐 元晓荣 高原 王鲁豫 张春生 谷超 李刚 WANG Qi-wen;ZHAO Lu-lu;YUAN Xiao-rong;GAO Yuan;WANG Lu-yu;ZHANG Chun-sheng;GU Chao;LI Gang(Department of Pharmacology,School of Pharmacy,Inner Mongolia Medical University,Hohhot 010110,China)

机构地区：[1]内蒙古医科大学药学院药理学教研室,内蒙古呼和浩特010110

出　　处：《中国药理学与毒理学杂志》2022年第10期729-738,共10页Chinese Journal of Pharmacology and Toxicology

基　　金：内蒙古自治区科技重大专项(zdzx201805);内蒙古自治区自然科学基金(2022JQ01)。

摘　　要：目的研究氯化镧(LaCl_(3))对高磷诱导的人血管平滑肌细胞(hVSMC)钙化的抑制作用及其机制。方法采用高磷诱导hVSMC钙化模型。将对数生长期的hVSMC随机分为细胞对照组、LaCl_(3)60μmol·L^(-1)组、高磷模型组(磷酸盐缓冲液3 mmol·L-1)、模型+氯化钠(NaCl)180μmol·L^(-1)组、模型+LaCl_(3)15,30和60μmol·L^(-1)组、模型+二甲基乙二酰氨基乙酸(DMOG)100μmol·L^(-1)+LaCl_(3)60μmol·L^(-1)组、模型+焦磷酸盐(PPI)100μmol·L^(-1)组。hVSMC给予磷酸盐缓冲液3 mmol·L-1孵育6 d造模,第4天,按分组给予相应药物孵育2 d。茜素红染色法及Von Kossa染色法检测细胞钙沉积;试剂盒检测细胞内钙含量和碱性磷酸酶(ALP)活性;荧光染色法检测细胞内活性氧(ROS)含量;实时荧光定量PCR(RT-qPCR)检测细胞平滑肌蛋白22α(sm22α)、骨形态发生蛋白2(bmp-2)、成骨相关转录因子2(runx2)和血管内皮生长因子(vegf)mRNA表达水平;Western印迹法检测细胞质低氧诱导因子1α(HIF-1α)、VEGF、SM22α和BMP-2及细胞核Runx2和HIF-1α蛋白表达水平。结果茜素红染色结果显示,与细胞对照组相比,高磷模型组细胞钙沉积明显升高(P<0.01);与高磷模型组相比,模型+LaCl_(3)15,30和60μmol·L^(-1)组细胞钙沉积明显降低(P<0.05,P<0.01)。Von Kossa染色结果显示,高磷模型组细胞黑色沉积物呈现大量聚集并广泛分布;模型+LaCl_(3)15,30和60μmol·L^(-1)组黑色沉积物有所减少。与细胞对照组相比,高磷模型组细胞内钙含量和ALP活性均明显升高(P<0.01);与高磷模型组相比,模型+LaCl_(3)15,30和60μmol·L^(-1)组细胞内钙含量和ALP活性均显著降低(P<0.05,P<0.01);与模型+LaCl_(3)60μmol·L^(-1)组相比,模型+DMOG+LaCl_(3)60μmol·L^(-1)组细胞钙含量及ALP活性均显著升高(P<0.01)。ROS检测结果显示,与细胞对照组相比,高磷模型组ROS含量显著增加(P<0.01);与高磷模型组相比,模型+LaCl_(3)15,30和60μmol·L^(-1)组ROS含量显著减少(P<0.05,P<OBJECTIVE To investigate the inhibitory effect and mechanism of lanthanum chloride(LaCl_(3))on high concentration phosphate-induced calcification in human vascular smooth muscle cells(hVSMCs).METHODS An hVSMC calcification model was established via high phosphate induction.hVSMCs in the logarithmic growth phase were randomly divided into the cell control group,LaCl_(3)60μmol·L^(-1)group,high concentration phosphate(phosphate buffer 3 mmol·L-1)model group,and model+sodium chloride(NaCl)180μmol·L^(-1)group,model+LaCl_(3)15,30 and 60μmol·L^(-1)groups,model+dimethyloxalamidoacetic acid(DMOG)100μmol·L^(-1)+LaCl_(3)60μmol·L^(-1)group,and model+pyrophosphate(PPI)100μmol·L^(-1)group.The h VSMCs were incubated with phosphate buffer solution 3 mmol·L-1for 6 d to establish a model.On the 4thday,the cells were incubated with corresponding as grouped for2 d.The effect of LaCl_(3)on intracellular calcium deposition was detected by Alizarin red staining and Von Kossa staining.The content of intracellular calcium deposition and the activity of alkaline phosphatase(ALP)were detected by kits.The content of the reactive oxygen species(ROS)was detected by fluorescent staining.The smooth muscle 22α(sm22α),bone morphogenetic protein-2(bmp-2)and osteogenesis-related transcription factor 2(runx2)and vascular endothelial growth factor(vegf)mRNA expression levels were detected by real-time quantitative PCR(RT-qPCR).The expressions of hypoxiainducible factor 1α(HIF-1α),SM22α,BMP-2 and Runx2 were detected by Western blotting.RESULTS Alizarin red staining results showed that compared with the cell control group,calcium deposition of the model group was significantly increased(P<0.01),while compared with the high-phosphorus model group,calcium deposition in model+LaCl_(3)15,30 and 60μmol·L^(-1)groups was significantly decreased(P<0.05,P<0.01).The results of Von Kossa staining showed that the black deposits in the model model group were massively aggregated and widely distributed but they were reduced in the model+LaCl_(3)

关 键 词：氯化镧 高磷血症 低氧诱导因子1Α 信号通路 血管钙化 

分 类 号：R972[医药卫生—药品]