摘要
目的研究白杨素提高肿瘤坏死因子-α(TNF-α)诱导肝癌细胞HepG2凋亡的能力,并对其分子机制进行初步探讨。方法白杨素以不同浓度(10、20、40μmol/L)单独或联合TNF-α(10 ng/mL)处理HepG2细胞后,于普通及荧光倒置显微镜下观察细胞形态变化,获得细胞死亡的定性资料;流式细胞术检测sub-G1峰,分析峰值变化规律,获得细胞死亡的定量资料;并以Western blotting方法检测凋亡标志蛋白caspase-3、caspase-8和PARP原蛋白和相应的裂解产物的变化情况及凋亡抑制蛋白Bcl-xL、cIAPs、xIAP、cFLIP的时间-效应变化规律。结果 形态学观察可发现白杨素联合TNF-α处理HepG2细胞后,与对照组比较细胞出现明显的死亡数量增加,而单独白杨素组、TNF-α组与对照组比较则未观察到明显的细胞减少(P>0.05);流式细胞术分析sub-G1的定量资料也支持这一结果,联合处理组sub-G1值随着白杨素剂量增加而增大,最高达到(27.84±0.54)%,与对照组比较有显著差异(P<0.05),Hochest 33342荧光染色在联合处理组可观察到明显的核固缩细胞增加;Western blotting检测到凋亡标志蛋白caspase-3、caspase-8和PARP原蛋白减少、相应的活化裂解片段出现;全caspase酶抑制剂z-VAD-fmk可有效抑制联合处理组HepG2细胞死亡、sub-G1峰消失比值减少,阻止凋亡标志蛋白caspase-3、caspase-8和PARP的活化降解;TNF-α引起的凋亡抑制蛋白cFLIP-1表达量增加,随联合处理时间延长而明显下调,与对照组比较有明显差异,Bcl-xL、xIAP等其他凋亡抑制蛋白没有明显改变。结论白杨素能够有效提高TNF-α诱导HepG2细胞凋亡的能力,NF-κB调节的凋亡抑制蛋白cFLIP-1表达减少是其重要的分子机制。
Objective To research whether chrysin can sensitize the cell death of hepatoma（HepG2） cell lines induced by TNF-α and explore the molecular mechanism of this sensitization.Methods HepG2 Cells were pretreated with designed dose of chrysin（10,20,and 40 μmol/L） for 2 h,then followed TNF-α（10 ng/mL） for 24 h,the morphologic changes were observed under inversed microscope and the percentage of sub-G1 was measured using flow cytometry,the proprotein and cleavage of caspase-3,caspase-8,and PARP,regarded as the protein mark of apoptosis induced by TNF-α,were determined by Western blotting;After treating with chrysin for different times,the time course of apoptosis inhibitory protein,such as BclxL,and cIAPs,xIAP,and cFLIP,were also detected using Western blotting.Results The cell death increasing was observed in the group with combination of chrysin and TNF-α,but no obvious cell death could be found in chrysin,TNF-α alone,and control groups.The data of sub-G1 supported this result that,with the enhancement of pretreatment dose of chrysin,the percentage of sub-G1 reached（27.84±0.54）%.There was significant difference between the combined group and the control one（1.52±0.13）%（P〈0.05） and obvious dose-dependent effect could be found.But the percentages of subG1 in chrysin and TNF-α alone groups were all under 5%,and there were no significant differences compared to the control group（P〉0.05）.Chromatin condensation which was the indication of apoptosis could be observed when the cells were stained by Hochest 33342;The proprotein of caspase-3、caspase-8,and PARP degraded,their cleavage appeared,and there were dose-dependent effects;The pancaspase inhibitor z-VAD-fmk could inhibit the apoptosis of HepG2 cells which were treated with the combination of chrysin and TNF-α,according to the percentage of sub-G1 and the activation of caspase-3,caspase-8,and PARP;The apoptosis inhibitory protein cFLIP-L could be observed the down-regulation compared to the control group in a time-dependent way.
出处
《中草药》
CAS
CSCD
北大核心
2010年第11期1828-1834,共7页
Chinese Traditional and Herbal Drugs
基金
广东省自然科学基金资助项目(9151030003000004)
广东省中医药强省科研立项资助项目(2009431)
