胡桃醌诱导HeLa细胞凋亡的机制

Effects of juglone on the proliferation and apoptosis of He La cells

目的探讨胡桃醌对宫颈癌He La细胞促凋亡作用的机制,及其相关的信号转导通路。方法选取处于对数生长期的He La细胞将其分为空白对照组和30μmol/L胡桃醌组。采用MTT法观察胡桃醌对不同时间点He La细胞的抑制作用;Hoechst 33258试剂盒,流式细胞仪测定胡桃醌对不同时间点He La细胞凋亡的影响;Western blotting检测凋亡相关蛋白Bcl-2、Bax和Caspase-3,磷脂酰肌醇3-激酶(PI3K)/Akt通路中AKt及p Akt的表达。结果MTT实验显示,与对照组比较,各时间点He La细胞具有抑制作用,且差异显著均有统计学意义(P<0.05,P<0.01);Hoechst 33258检测表明,30μmol/L胡桃醌处理细胞4,8,12 h后可出现明显的细胞核典型凋亡细胞形态;细胞凋亡检测表明,30μmol/L胡桃醌培养不同时间后,与对照组相比早期凋亡率明显增高;Western blotting检测显示,与正常对照组相比,30μmol/L胡桃醌处理细胞12h后,He La细胞Bcl-2,p Akt蛋白的表达明显降低,而Bax、Cleave-Caspase-3,Akt蛋白表达明显升高。结论胡桃醌通过抑制PI3K/Akt通路,进而促进He La细胞凋亡。

Objective To explore the pro-apoptotic mechanism of juglone in human cervical cancer HeLa cells, and to study its associated signal transduction pathways. Methods Cultured HeLa cells were incubated with 30p.mol/L juglone for 24,48 and 72 hours. The proliferation of HeLa cells was detected by methyl thiazolyl tetrazolium （MTT） assay. The cell apoptosis was detected by Hoechst 33258 staining and flow cytometry （FCM） ; The expressions of Bcl-2,Bax and Caspase-3 ,Akt, pAkt and PTEN were detected by Western blotting. Results MTT results showed that the HeLa cell growth was greatly inhibited in a time dependent manner （ P 〈 0. 05, P 〈 0. 01 ） when compared with the control group. After treatment on HeLa cells for 4,8 and 12 hours, typical morphological changes of cell apoptosis such as karyopyknotic pyknic hyperfluoreseence bolus,nuclear fragmentation and apoptotic body were observed by Hoechst 33258 staining. The early apoptosis ratio was increased significantly when compared with the control group. Western blotting results showed that the expression of Bcl-2 and pAkt decreased obviously,whereas those of Bax, Caspase-3 and Akt increased significantly in the juglone treatment group when compared with the control group. Conclusion Juglone may inhibit PI3K/Akt pathway, thereby promoting HeLa cells apoptosis.