摘要
目的 探讨紫草素调节环磷酸腺苷(cyclic adenosine monophosphate,cAMP)/蛋白激酶(protein kinase,PK)A/cAMP 反应元件结合蛋白(cAMP response element binding protein,CREB)信号通路对多发性骨髓瘤(multiple myeloma,MM)细胞的影响.方法 取对数生长的RPMI-8226细胞,分为A(空白)组、B(250 μmol/L紫草素)组、C(500 μmol/L紫草素)组、D(1 000 μmol/L 紫草素)组、E(SQ22536)组、F(1 000 μmol/L 紫草素+SQ22536)组.Hoechst 33324 染色、流式细胞术观察细胞凋亡变化;细胞计数(cell counting kit,CCK)-8法检测细胞增殖;酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测 cAMP 水平;蛋白质印迹法(western blot,WB)检测通路相关蛋白及 cyclinD1、cleavedcaspase-3表达水平.结果 与A组相比,E组RPMI-8226细胞增殖率、cyclinD1表达显著增加,凋亡率、cAMP水平、cleaved caspase-3、p-PKA/PKA、p-CREB/CREB 表达显著降低(P＜0.05),但 B、C、D 组细胞增殖率、cyclin D1 表达显著降低,凋亡率、cAMP水平、cleaved caspase-3、p-PKA/PKA、p-CREB/CREB表达显著增加,不同浓度差异具有统计学意义(P＜0.05);与E组相比,F组细胞增殖率、cyclin D1表达显著降低,凋亡率、cAMP水平、cleaved caspase-3、p-PKA/PKA、p-CREB/CREB表达显著增加(P＜0.05);与D组相比,F组细胞增殖率、cyclin D1表达显著增加,凋亡率、cAMP 水平、cleaved caspase-3、p-PKA/PKA、p-CREB/CREB 表达显著降低(P＜0.05).结论 紫草素调节 cAMP/PKA/CREB信号通路抑制MM细胞增殖,促进细胞凋亡.
Abstract
Objective To investigate the effect of shikonin on cyclic adenosine monophosphate(cAMP)/protein kinase(PK)A/cAMP response element binding protein(CREB)signaling pathway on multiple myeloma cells.Methods The logarithmically grown RPMI-8226 cells were divided into A(blank)group,B(250 μmol/L shikonin)group,C(500 μmol/L shikonin)group,D(1 000 μmol/L)shikonin group,E(SQ22536)group and F(1 000 μmol/L shikonin+SQ22536)group.The apoptosis was observed by Hoechst 33324 staining and flow cytometry.Cell counting kit(CCK)-8 was used to detect cell proliferation.The level of cAMP was detected by enzyme linked immunosorbent assay(ELISA).The expression levels of pathway-related proteins,cyclin D1 and cleaved caspase-3 were detected by western blot.Results Compared with group A,the proliferation rate and cyclin D1 expression of RPMI-8226 cells in group E were significantly increased,and the apoptosis rate,cAMP level,cleaved caspase-3,p-PKA/PKA,and p-CREB/CREB expressions were significantly decreased(P＜0.05).However,cell proliferation rate and cyclin D1 expression were significantly decreased in groups B,C and D,and apoptosis rate,cAMP level,cleaved caspase-3,p-PKA/PKA,and p-CREB/CREB expression were significantly increased,with statistical differences among different concentrations(P＜0.05).Compared with group E,cell proliferation rate and cyclin D1 expression in group F were significantly decreased,apoptosis rate,cAMP level,cleaved caspase-3,p-PKA/PKA,and p-CREB/CREB expression were significantly increased in group F(P＜0.05).Compared with group D,cell proliferation rate and cyclin D1 expression in group F were significantly increased,apoptosis rate,cAMP level,cleaved caspase-3,p-PKA/PKA,and p-CREB/CREB expression were significantly decreased(P＜0.05).Conclusion Shikonin inhibits the cell proliferation and promote apoptosis of MM cells by regulating the cAMP/PKA/CREB signaling pathway.
基金项目
福建医科大学科研发展基金项目(2021QH1041)
NETL
NSTL
万方数据