摘要
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 investigate the effect of pioglitazone on cell apoptosis in human HepG2 cells and explore the potential mechanisms. Methods Cell apoptosis index was detected by Annexin V-FITC. Cell cycle phases and activated caspase 3 protein were analyzed by flow cytometry analysis （FCM）. PPARγ mRNA expression was measured by reverse transcription-polymerase chain reaction （RT-PCR）. The expressions of PPARγ protein, AMP-activated protein kinase α and phosphorylated AMPKα （p-AMPKα） were detected by Western blotting. NF-KB DNA binding activity was measured by electrophoretic mobility shift assay （EMSA）. Results Pioglitazone induced cell apoptosis, an increased proportion of cells in the G0/G1 phase and a decreased proportion of cells in S phase in a dose-dependent manner in HepG2 cells while the expression level of PPARγ/mRNA and protein remained unhanged. After being transfected with pGCsi-PPARγ, pioglitazone also induced apoptosis at a higher concentration （20 μmol/L）. Pioglitazone did not induce the expression of activated caspase 3 protein or influence the DNA binding activity of NF-KB, but resulted in an activation of the increased phos- phorylatsion of AMP-activated protein kinase α （AMPKct）. Conclusion Pioglitazone induces apoptosis and interferes with cell cycle in HepG2 cells, which may not be completely related to PPAR7 dependent signal pathway. AMPKα signals may participate in the above process.
出处
《军事医学》
CAS
CSCD
北大核心
2012年第9期666-672,共7页
Military Medical Sciences
基金
武汉市青年晨光计划资助课题(20065004116-49)
军事医学科学院创新启动基金(YC0216)
