摘要
目的:从自噬的角度探讨预知子籽提取物(AFSE)抑制HepG2肝癌细胞增殖的分子机制。方法:(1)体外培养人肝癌HepG2细胞,分别给予不同浓度AFSE干预(0、0.6、0.7、0.8、0.9、1.0 mg/ml),细胞培养24、48 h后,MTT法检测细胞增殖情况。(2)将HepG2细胞分为空白对照组和AFSE组(0.625 mg/ml)。药物干预24、48 h后,PCR检测自噬相关分子Beclin-1、LC3和P62的mRNA表达;Western blot检测Beclin-1、LC3B-Ⅰ、LC3B-Ⅱ、P62的蛋白表达,并计算LC3B-Ⅱ/LC3B-Ⅰ的比值;透射电镜观察细胞自噬体、自噬溶酶体等结构。结果:(1)AFSE能够有效抑制HepG2细胞的增殖,≥0.8 mg/ml AFSE作用48 h后,细胞存活率显著降低(P<0.05)。(2)0.625 mg/ml AFSE作用24 h后,P62 mRNA表达显著升高(P<0.01),且LC3B-Ⅰ、LC3B-Ⅱ、P62蛋白表达水平显著升高(P<0.05)。AFSE作用48 h后,P62和LC3的mRNA表达显著升高(P<0.05,P<0.01),LC3B-Ⅱ、P62蛋白表达水平显著升高(P<0.05),LC3B-Ⅱ/LC3B-Ⅰ蛋白表达比值亦升高。电镜观察显示AFSE干预24、48 h后,细胞内自噬溶酶体明显增多,48 h亦观察到未与溶酶体融合的自噬体。结论:AFSE能够抑制HepG2细胞增殖,其作用可能与诱导细胞自噬有关。
Objective:To explore the molecular mechanism of Akebiae Fructus seed extract(AFSE)on inhibiting the proliferation of hepatocellular carcinoma HepG2 cells from the perspective of autophagy.Methods:(1)Human hepatoma HepG2 cells were cultured in vitro,and were treated with AFSE at different concentrations(0,0.6,0.7,0.8,0.9,1.0 mg/ml).After intervention for 24 and 48 hours,the cell proliferation was detected by MTT assay.(2)HepG2 cells were divided into the blank control group and AFSE group(0.625 mg/ml).After drug intervention for 24 and 48 hours,the mRNA expressions of autophagy-related molecules Beclin-1,LC3 and P62 were detected by PCR,the protein expressions of Beclin-1,LC3 B-Ⅰ,LC3 B-Ⅱand P62 were measured by Western blot,and the ratio of LC3 B-Ⅱ/LC3 B-Ⅰwas calculated.The autophagosome and autophagolysosome were observed by transmission electron microscopy.Results:(1)AFSE could effectively inhibit the proliferation of HepG2 cells.After treated with AFSE(≥0.8 mg/ml)for 48 hours,the cell survival rate was decreased significantly(P<0.05).(2)After treated with 0.625 mg/ml AFSE for 24 hours,the expression of P62 mRNA was significantly increased(P<0.01),and the protein expression levels of LC3 B-Ⅰ,LC3 B-Ⅱand P62 were significantly increased(P<0.05).After treated with AFSE for 48 hours,the mRNA expressions of P62 and LC3 were increased significantly(P<0.05,P<0.01),the protein expression levels of LC3 B-Ⅱand P62 were increased significantly(P<0.05),and the protein expression ratio of LC3 B-Ⅱ/LC3 B-Ⅰwas also increased.Electron microscopy observation showed that the autophagolysosomes were increased obviously after AFSE treatment for 24 and 48 hours,and some autophagosomes without fusion with lysosomes were also observed after treatment for 48 hours.Conclusion:AFSE can inhibit the proliferation of HepG2 cells,which may be related to the induction of autophagy.
作者
范晓燕
李铭哲
刘小美
FAN Xiaoyan;LI Mingzhe;LIU Xiaomei(School of Basic Medical Sciences,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)
出处
《上海中医药大学学报》
CAS
2021年第3期52-57,共6页
Academic Journal of Shanghai University of Traditional Chinese Medicine
基金
上海市进一步加快中医药事业发展三年行动计划项目[ZY(2018-2020)-CCCX-2001-01]
上海市教委预算内项目(2019LK065)
上海中医药大学中医基础理论学科能力提升项目(2018-2020)。
关键词
预知子
肝癌
自噬
细胞
Akebiae Fructus
hepatocellular carcinoma
autophagy
cell
