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目的观察吡格列酮干预对体外培养的HepG2细胞凋亡和细胞周期的影响,并探讨其药理作用机制。方法以不同浓度的吡格列酮干预体外培养HepG2细胞,采用流式细胞术检测细胞凋亡、细胞周期以及激活的胱天蛋白酶(Caspase)3蛋白表达,RT-PCR检测PPARγmRNA的表达,Western印迹检测PPARγ蛋白、磷酸腺苷激活的蛋白激酶(AMPKα)蛋白和磷酸化AMPKα(p-AMPKα)蛋白的表达;并将PPARγ小干扰RNA(pGCsi-PPARγ)表达质粒转染HepG2细胞,观察PPARγ基因沉默后吡格列酮对细胞凋亡作用的影响。结果不同浓度的吡格列酮干预HepG2细胞后,诱导细胞的凋亡,在此过程中G0/G1期细胞比例明显增加,S期细胞比例明显减少,并呈一定的剂量依赖关系;但在上述过程中,PPARγmRNA和蛋白的表达没有显著变化;吡格列酮在高浓度(20μmol/L)时对pGCsi-PPARγ表达质粒转染的HepG2细胞仍表现出凋亡诱导作用。不同浓度的吡格列酮干预后未见激活的caspase 3表达峰,对NF-κB的DNA结合活性也无明显影响,但其在高浓度(20μmol/L)时明显增加对照组和pGCsi-PPARγ转染组p-AMPKα的表达,而总AMPKα则无明显变化。结论吡格列酮能够干扰细胞周期、诱导其凋亡,这种作用不是完全通过PPARγ依赖途径实现的,AMPKα信号途径参与上述过程。
Objective To observe the effect of pioglitazone on the apoptosis and cell cycle of HepG2 cells cultured in vitro and to explore its pharmacological mechanism. Methods HepG2 cells were cultured in vitro with different concentrations of pioglitazone. The apoptosis, cell cycle and activation of caspase 3 protein were detected by flow cytometry. The expression of PPARγ mRNA was detected by RT-PCR. The expression of PPARγ protein (AMPKα) and phospho-AMPKα (p-AMPKα) protein were detected by RT-PCR. The expression of pGCsi-PPARγ was transfected into HepG2 cells. The effect of PPARγ gene silencing on the expression of pioglitazone Effect of apoptosis. Results Pioglitazone at different concentrations induced the apoptosis of HepG2 cells, the proportion of cells in G0 / G1 phase increased significantly and the proportion of cells in S phase decreased significantly in a dose-dependent manner. However, in the above process, PPARγmRNA and protein expression did not change significantly; pioglitazone still showed apoptosis-inducing effect on HepG2 cells transfected with pGCsi-PPARγexpression plasmid at high concentration (20μmol / L). No significant difference was observed in the DNA binding activity of NF-κB when pioglitazone was treated with different concentrations of pioglitazone. However, the effect of pioglitazone at the high concentration (20μmol / L) p-AMPKα expression, while the total AMPKα no significant change. Conclusion Pioglitazone can interfere with the cell cycle and induce its apoptosis. This effect is not completely mediated by the PPARγ-dependent pathway. The AMPKα signaling pathway participates in this process.