摘要
目的:观察冬凌草甲素(oridonin,ORI)对人骨髓瘤H929细胞株增殖、凋亡的影响,探讨其可能的作用机制。方法:分别用ORI 0、4、8、12、16、20、24、28、32μmol/L处理H929细胞12、24和36 h,应用四甲基偶氮唑蓝(MTT)检测H929细胞增殖抑制率,TUNEL法和荧光显微镜检观察Here29细胞形态变化及凋亡,AnnexinV-FITC/PI双集流式细胞术检测细胞凋亡率,蛋白印迹技术(Western blot)检测BAX、BCL-2、caspase-3、PARP、p38、JNK、ERK以及PI3K、Akt的表达变化。结果:ORI 8-16μmol/L体外显著抑制H929细胞增殖,并与浓度及时间呈正相关(P<0.05,P<0.05;r=0.961,r=0.973)。TUNEL染色法检出细胞核固缩、核碎裂;流式细胞术显示,H929细胞凋亡率呈浓度依赖性递增(P<0.05,r=0.88);Western blot检测结果提示,pro-caspase-3、BCL-2、p-PI3K、p-Akt表达呈浓度依赖性递减(r=0.9861,r=0.9725,r=0.9413,r=0.9373),而BAX、Cleaved PARP及p-p38、p-JNK、p-ERK表达递增(r=0.9178, r=0.8877,r=0.882,r=0.9645,r=0.8623)。结论:一定浓度ORI在体外能有效抑制骨髓瘤细胞H929的增殖,并诱导细胞凋亡,其可能的分子机制与上调MAPK及下调PI3K/Akt信号通路相关蛋白有关。
Objective: To investigate the effects of oridonin(ORI) on the proliferation and apoptosis of human multiple myeloma cell line H929 and its possible mechanism. Methods: H929 cells were exposed to ORI 0、4、8、12、16、20、24、28、32 μmol/L for 12, 24 and 36 hours respectively. The prolifcration inhibitory effect of ORI on H929 cells was determined by MTT assay and then the working concentrations of ORI were determined. The morphological changes and apoptosis of H929 cells were observed by TUNEL(TdT-mediated dUTP Nick-End Labeling)and fluorescence microscopy. The apoptosis rate of H929 cells was detected by flow cytometry with Annexin V-FITC/PI staining. The protein expressions of pro-caspase-3, BCL-2,p-PI3 K, p-Akt, BAX, Cleaved PARP and p-JNK, p-ERK and p-p38 in H929 cells were detected by Western blot. Results: Compared with the control group, the proliferation of H929 cells treated with the ORI of 8-16 μmol/L was significantly inhibited and the apoptosis of H929 cells was obviously increased in dose-and time-dependent manners. As for morphological changes, the characteristics of apoptotic cells were presented in H929 cells treated with ORI for 24 hours. The protein levels of pro-caspase-3, BCL-2,p-PI3 K, p-Akt were down-regulated with increasing of ORI concentration(r=0.9861, r=0.9725, r=0.9413, r=0.9373), while the BAX,Cleaved PARP and p-JNK, p-ERK and p-p38 were up-regulated(r=0.9178, r=0.8877, r=0.882, r=0.9645, r=0.8623).Conclusion: The ORI possesses anti-myeloma effects, can inhibit the proliferation and induce the apoptosis of H929 cell line in vitro. Its potential mechanism may be related with up-regulating the MAPK and down-regulating the PI3K/Akt signal pathways.
引文
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