目的 研究传统中药米仔兰Aglaia odorata中洛克米兰醇对肝癌细胞HepG2增殖的影响及抗肿瘤作用机制。方法 MTT、细胞克隆形成、EdU染色和CFDA染色方法考察洛克米兰醇对HepG2细胞的抗增殖效果；流式细胞仪检测洛克米兰醇对HepG2细胞细胞周期和细胞凋亡的变化；Western blotting检测洛克米兰醇对细胞周期调控蛋白和丝裂原活化蛋白激酶（MAPK）信号通路相关蛋白表达的影响。结果 洛克米兰醇可以时间和浓度依赖性抑制HepG2细胞增殖。同等浓度下洛克米兰醇抗HepG2细胞增殖的效果好于阿霉素，而对正常肝细胞（L02）的毒性弱于阿霉素。流式细胞技术检测发现洛克米兰醇给药48 h能够诱导HepG2细胞G₂/M期细胞周期阻滞，但不诱导细胞凋亡。Western blotting研究发现该化合物抑制调控G₂/M周期相关蛋白cdc25C、cdc2和cyclin B1的表达并且激活细胞外调节蛋白激酶（ERK）和c-Jun氨基末端激酶（JNK）。对其机制深入研究发现，ERK抑制剂（U0126）可以部分逆转洛克米兰醇对HepG2的抗增殖和G₂/M周期阻滞及其抑制蛋白cdc25C和cdc2表达的效果。结论 洛克米兰醇在抑制肝癌细胞增殖的效果和对正常肝细胞的选择性方面优于阿霉素。洛克米兰醇能够通过过度活化ERK，从而引起HepG2细胞G₂/M周期阻滞而达到抗增殖效果。

Objective To study the effect of rocaglaol from Aglaia odorata on HepG2 proliferation and to explore the potential anti-tumor mechanism. Methods The MTT, colony formation, EdU incorporation, and CFDA-SE assays were used to determine the anti-proliferative activity of rocaglaol in HepG2 cells. Apoptosis and cell cycle distribution effect induced by rocaglaol were carried out by flow cytometry. The effect of rocaglaol on protein involved in the G₂/M checkpoint and the MAPK pathway were performed by Western blotting analysis. Results Rocaglaol significantly inhibited the viability of HepG2 cells in a dose-dependent and time-dependent manner. Rocaglaol was more effective than doxorubicin in the growth inhibition of HepG2 cells. However, rocaglaol-induced cytotoxicity in normal human hepatic cell line L02 was lower than that of doxorubicin. Treatment with different concentrations of rocaglaol at 48 h caused G₂/M cell cycle progression inhibition, rather than apoptosis in HepG2 cells. Rocaglaol can significantly reduce the expression of G₂/M cell cycle-regulating proteins cdc25C, cdc2, and cyclin B1 as well as increase the expression of ERK and JNK phosphorylation levels. Further study found that U0126 can partly abrogate the anti-proliferative activity in HepG2 cells, G₂/M phase arrest and the reduction in the protein expression levels of cdc2 and cdc25C induced by rocaglaol. Conclusion Our results demonstrated that rocaglaol was superior to doxorubicin in the inhibition of HepG2 cells proliferation and the selectivity of L02 cell activity. We provided evidence that the rocaglaol had the ability to continuously over-activate the ERK signaling in HepG2 cells, leading to the inhibition of cell proliferation through G₂/M phase arrest.

R285.5

国家自然科学基金面上项目（81773886）；中国药科大学双一流学科创新团队建设项目（CPU2018GF03）