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目的:探讨过氧化物酶体增殖物活化受体γ(PPARγ)激动剂吡格列酮(PIO)对高磷诱导大鼠血管平滑肌细胞(VSMC)钙化的作用及其相关机制。方法:利用10 mmol/Lβ甘油磷酸(β-GP)诱导大鼠VSMC钙化,建立钙化模型(即钙化组);同时以完全培养基培养大鼠VSMC为正常对照组,并分别以不同浓度(5μmol/L、10μmol/L、15μmol/L、20μmol/L)PIO干预,观察培养12d,用茜素红染色法检测细胞钙沉积并检测细胞外基质钙离子浓度来观察VSMC钙化程度。Western Blot检测大鼠VSMC的α平滑肌动蛋白(α-SMA)、Runx2、BMP2、Wnt/β-catenin通路相关蛋白(β-catenin、GSK-3β、p-GSK-3β)及核蛋白cyclin-D1的表达情况。选择合适的PIO浓度(20μmol/L)并以PPARγ拮抗剂GW9662(20μmol/L)干预,观察以上指标的变化。结果:(1)钙化组钙离子浓度较正常对照组明显增高(P<0.05),而不同浓度PIO均可减轻VSMC细胞外基质的钙离子浓度(P<0.05),同时钙化组茜素红染色较正常对照组明显,而20μmol/L PIO干预组茜素红染色较钙化组减轻最为明显;(2)钙化组大鼠VSMC表达Runx2、β-catenin、p-GSK-3β、BMP2、cyclin-D1较正常对照组升高;20μmol/L PIO可显著下调钙化大鼠VSMC表达Runx2、β-catenin、p-GSK-3β、BMP2和cyclin-D1,并上调α-SMA的表达;(3)PPARγ拮抗剂GW9662可部分阻断PIO对钙化大鼠VSMC的干预作用。结论:PPARγ激动剂PIO可减轻β-GP诱导的大鼠VSMC的钙化,其作用机制与下调Wnt/β-catenin信号通路活性有关。
AIM: To investigate the effect of peroxisome proliferator-activated receptor γ (PPARγ) agonist pioglitazone (PIO) on the calcification of vascular smooth muscle cells (VSMCs) induced by high phosphorus and its related mechanisms. Methods: Calcification was induced by 10 mmol / L β-GP in rat VSMCs, and then calcified (calcification group) was established. At the same time, VSMCs were cultured in complete medium as normal control group and treated with different concentrations of 5μmol After treated with PIO for 12 days, the calcium deposition in the cells was detected by alizarin red staining and the calcium concentration in extracellular matrix was measured to observe the degree of calcification in VSMC. Western Blot was used to detect the expressions of α-smooth muscle actin (α-SMA), Runx2, BMP2 and Wnt / β-catenin pathway-related proteins (GSK-3β and p-GSK-3β) and cyclin-D1 The expression of the situation. Select the appropriate concentration of PIO (20μmol / L) and PPARγ antagonist GW9662 (20μmol / L) intervention to observe the above changes. Results: (1) Calcium concentration in calcification group was significantly higher than that in normal control group (P <0.05), while PIO in different concentrations could reduce the calcium concentration in extracellular matrix of VSMC (P <0.05) Compared with normal control group, the expression of Runx2, β-catenin, p-GSK-3β, BMP2, cyclin-D1 in calcification group VSMC was significantly lower than that in 20μmol / L PIO intervention group 20μmol / L PIO could significantly down-regulate the expressions of Runx2, β-catenin, p-GSK-3β, BMP2 and cyclin-D1 and up-regulate the expression of α-SMA in VSMC of calcified rats; Agent GW9662 can partially block the intervention of PIO on calcified rat VSMC. CONCLUSION: PPARγ agonist PIO can attenuate the β-GP-induced calcification of rat VSMCs by down-regulating the activity of Wnt / β-catenin signaling pathway.