白杨素联合顺铂调控JNK磷酸化促进Hep G2细胞凋亡的实验研究  

Chrysin combined with cisplatin regulates the apoptosis of HepG2 through JNK phosphorylation

作　　者：王剑宁[1] 李欣[2] 彭宝莹 杨美玲[2] 陈美芬[2] 黄俊明[2] WANG Jian-ning;LI Xin;PENG Bao-ying;YANG Mei-ling;CHENG Mei-feng;HUANG Jun-ming(Guanghua School of Stomatology,Institute of Stomatological Research,Sun Yats-Sen University,Guangdong Provincial Key Laboratory of Stomatology,Guangzhou,510055,China;Guangdong Provincial Center for Disease Control and Prevention,Guangzhou,511430,China)

机构地区：[1]中山大学光华口腔医学院附属口腔医院口腔颌面外科口腔医学研究所广东省口腔医学重点实验室,广东广州510055 [2]广东省疾病预防控制中心卫生毒理所,广东广州511430

出　　处：《时珍国医国药》2019年第4期775-778,共4页Lishizhen Medicine and Materia Medica Research

基　　金：国家自然科学基金(81202199);广东省科技计划项目(2013B021800044;2014A020212437;2015A020210040);广东省广州市科技计划项目(2014J4100090);广东省中医药局科研项目(20181065);广东省自然科学基金项目(2018A030313704)

摘　　要：目的研究白杨素联合顺铂对肝癌细胞Hep G2凋亡的影响,并对其分子机制作初步探讨。方法以不同浓度白杨素(10,20,40μmol/L)单独或联合顺铂(5μg/ml)处理Hep G2细胞后,CCK-8法测定细胞活性;Hoechst 33342染色后于荧光倒置显微镜下观察细胞形态变化;Western blot方法检测凋亡标志蛋白caspase家族及PARP、凋亡抑制蛋白Bcl-2以及c-Jun氨基末端激酶(JNK)的变化情况;分离提取细胞质蛋白,Western blot检测凋亡促进蛋白Bax与细胞色素C的变化情况。结果 CCK-8法测定结果显示白杨素联合顺铂处理HepG2细胞后,细胞活性明显下降,联合作用组与对照组及其他单独处理组比较均有统计学意义(P<0.05);形态学观察发现,与对照组比较,白杨素联合顺铂处理Hep G2细胞后,细胞出现凋亡增加,而单独白杨素、顺铂处理组则未观察到明显细胞凋亡;Western blot检测到凋亡标志蛋白Caspase-3、Caspase-8、Caspase-9、PARP原蛋白减少;全Caspase酶抑制剂z-VAD-fmk可明显抑制联合处理引起的细胞凋亡,阻止caspase家族及PARP的活化降解;白杨素联合顺铂处理细胞后能恢复顺铂单独处理引起的凋亡抑制蛋白Bcl-2的表达上调并且激活JNK1蛋白;Western blot检测细胞质蛋白在联合处理后凋亡促进蛋白Bax与细胞色素C表达明显上调。结论白杨素和低剂量顺铂单独用药对Hep G2细胞无明显影响,但联合用药能促进Hep G2细胞凋亡,其机制为JNK1磷酸化、激活JNK通路使促凋亡分子Bax和细胞色素C的释放从而引起细胞凋亡。Objective To investigate the effects of chrysin on the apoptosis of hepatoma cells induced by cisplatin, and to discuss the molecular mechanism of the combinative effect. Methods Hep G2 cells were pretreated with different concentration of chrysin(10,20,40 μM) for 2 h, then treated with cisplatin(5 μg/ml)for 24 h. The morphologic changes were observed under inversed microscope and the cell viability was measured using CCK-8. The proteins of caspase family, PARP, Bcl-2 and JNK were determined by Western blot. Then the cells’ cytoplasm was separated and the protein was extracted. The protein of Bax and cytochrome C were determined by Western blot.Results Morphological observation showed that the number of cell death significantly increased in the group treated with combination of chrysin and cisplatin compared to the control. Chromatin condensation were obviously observed when the cells were stained by Hoechst 33342 in the combination of chrysin and cisplatin, while in the other groups these were not observed significantly. The proportions of caspase family and PARP degraded induced by the combination of chrysin and cisplatin, then pan-caspase inhibitor z-VAD-fmk could reverse the degradation. Chrysin combined with cisplatin could reduce the expresstion of Bcl-2 upregulated by cisplatin alone and activate JNK protein. The expression of Bax and cytochrome C was obviously upregulated in cytoplasm.Conclusion Chrysin could promote the apoptosis of Hep G2 cells induced by cisplatin which may be related to the activation of JNK pathway to promote the release of Bax and cytochrome C.

关 键 词：白杨素 顺铂 凋亡 HEP G2细胞 JNK蛋白 

分 类 号：R962[医药卫生—药理学]