大黄酸通过MAPK信号转导通路诱导HK-2细胞凋亡  

MAPK Signal Transduction Pathway involves in Rhein-induced Apoptosis in HK-2 Cells

作　　者：杨加培 孙浩[1] 王丹丹[1] 毛勇[1] 于锋[1] 

机构地区：[1]中国药科大学临床药学教研室药物质量与安全预警教育部重点实验室,南京211198

出　　处：《中国实验方剂学杂志》2015年第15期147-151,共5页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金面上项目(30572193)

摘　　要：目的:研究大黄酸诱导人肾小管上皮细胞系HK-2细胞凋亡作用及其凋亡机制。方法:将密度为4×104个/m L的HK-2细胞悬液接种于96孔板,培养24 h后分别加入不同浓度大黄酸(0,25,50,100μmol·L-1)作用12,24,48 h,MTT法检测大黄酸对HK-2细胞活性的抑制作用;利用Hoechst 33258染色法和流式细胞术检测大黄酸诱导HK-2细胞凋亡情况;实时荧光定量PCR(RT-q PCR)检测细胞原癌基因c-jun,活化转录调控因子2(ATF-2),半胱天冬氨酸酶3(Caspase-3)基因表达水平;Western blotting检测细胞磷酸化c-jun氨基末端激酶(p-JNK),c-jun氨基末端激酶(JNK),磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK),p38丝裂原活化蛋白激酶(p38 MAPK)和活化型半胱天冬氨酸酶3(Cleaved Caspase-3)蛋白的变化,并利用JNK抑制剂SP600125,p38抑制剂SB203580进一步验证JNK和p38所介导的丝裂原活化蛋白激酶(MAPK)信号转导通路在HK-2细胞凋亡中的作用。结果:大黄酸能够呈剂量依赖性和时间依赖性抑制HK-2细胞活性。大黄酸作用下HK-2细胞凋亡率明显增加。大黄酸作用于HK-2细胞c-jun,ATF-2及Caspase-3基因的mRNA均明显上调;p-p38,p-JNK及Cleaved Caspase-3蛋白表达显著性增加,JNK和p38总蛋白表达无明显变化。JNK特异性抑制剂SP600125与p38特异性抑制剂SB203580均能显著降低大黄酸诱导的HK-2细胞的凋亡率。结论:大黄酸能够诱导HK-2细胞凋亡,其作用机制可能是通过影响MAPK信号转导通路实现的。Objective： To investigate the apoptotic effect of rhein on human proximal tubular epithelial cell line（ HK-2） and the underlying mechanisms. Method： HK-2 cells were seeded in 96-well plates at an initial density of 4 × 104 cells / m L for 24 h,and then the four different concentration of rhein（ 0,25,50,100 μmol·L-1）were applied to test cells for 12,24,48 h respectively. MTT assay was used to detect the viability of HK-2 cells.Rhein-induced apoptosis in HK-2 cells was evaluated by Hoechst 33258 staining and Flow cytometry. The expression of proto-oncogene c-jun（ c-jun）,activating transcription factor 2（ ATF-2） and cysteine proteinases with specificity for aspartic acid residues 3（ Caspase-3） mRNA was detected by real-time quantitative polymerase chain reaction（ RT-q PCR）. The changes of phosphorylated c-jun N-terminal kinase（ p-JNK）,c-jun N-terminal kinase（ JNK）,phosphorylated p38 mitogen-activated protein kinase（ p-p38 MAPK）,p38 mitogen-activated protein kinase（ p38 MAPK） and Cleaved Caspase-3 protein were detected by Western blotting. The specific inhibitors of JNK and p38 MAPK were applied for identifying the roles of the corresponding signal pathways in rhein-induced apoptosis of HK-2 cells. Result： Rhein inhibited the viability of HK-2 cells in a dose-and timedependent manners. The apoptosis rate of HK-2 cells in rhein groups was apparently increased. The RT-q PCR found that the expression of c-jun,ATF-2,Caspase-3 mRNA was significantly increased in rhein-treated groups.The results of Western blotting showed that there was no significant change in p38 and JNK protein,but an increasing trend in p-p38,p-JNK and Cleaved Caspase-3 was observed. After treatment with SP600125（ an inhibitor of JNK） and SB203580（ an inhibitor of p38） respectively,the apoptosis rates of rhein on HK-2 cells were significantly weak. Conclusion： Rhein induces apoptosis involving MAPK signal transduction pathway in HK-2cells.

关 键 词：大黄酸 HK-2细胞 细胞凋亡 丝裂原活化蛋白激酶信号转导通路 C-JUN氨基末端激酶 P38丝裂原活化蛋白激酶 

分 类 号：R285.5[医药卫生—中药学]