摘要
目的:探讨桑黄水提物对胃癌SGC-7901细胞凋亡及自噬相关通路的影响。方法:体外培养SGC-7901细胞,用桑黄水提物0.5、1、2、4、6、8、10 mg/mL作用于细胞24、48和72 h,噻唑蓝(MTT)比色法检测细胞活力;乳酸脱氢酶(LDH)试剂盒测定细胞LDH释放率;Hoechst 33342染色法检测细胞核形态;碘化丙啶(PI)染色法检测细胞周期;流式细胞术检测细胞凋亡率;细胞划痕法检测细胞迁移能力;细胞克隆形成法检测细胞增殖能力;吖啶橙(AO)法染色检测细胞内酸性细胞器生成情况;蛋白免疫印迹(Western blot)法检测线粒体凋亡途径和自噬相关Pro Caspase-9、Pro Caspase-3、Cleaved Caspase-3、PARP、BAX、BCL-2、mTOR、P-ULK1、p62、Beclin-1及LC3-II蛋白表达水平。结果:桑黄水提物能呈浓度和时间依赖性地抑制SGC-7901细胞增殖;Hoechst染色可见细胞核皱缩碎裂;与空白对照组比较,桑黄水提物4 mg/mL可明显提高细胞LDH释放率(P<0.05);流氏结果显示桑黄水提物8 mg/mL可使细胞凋亡率明显上升(P<0.05),并将细胞周期阻滞在G2/M期(P<0.01);桑黄水提物各给药组细胞迁移率、细胞克隆形成率均显著下降(P<0.01);细胞内酸性细胞器增多。Western blot实验显示桑黄水提物各给药组显著上调Beclin-1、LC3-II、P-ULK1的蛋白表达(P<0.01),下调Pro Caspase-9、PARP、p62、mTOR的蛋白表达,显著增加BAX/BCL-2及Cleaved Caspase-3/Pro Caspase-3的比值(P<0.05或P<0.01)。结论:桑黄水提物呈现浓度和时间依赖性抑制胃癌SGC-7901细胞的增殖,并诱导细胞凋亡和自噬,其机制可能是激活了Caspase依赖性细胞线粒体凋亡途径和mTOR/ULK1自噬相关信号通路。
Objective:To investigate the effects of Phellinus igniarius water extract on apoptosis and autophagy pathways of gastric cancer SGC-7901 cells.Methods:SGC-7901 cells were cultured in vitro and treated with P.igniarius water extract at different concentrations(0.5,1,2,4,6,8,and 10 mg/mL)for 24,48,and 72 h,respectively,followed by the detection of cell viability using methyl thiazolyl tetrazolium(MTT)colorimetry.The lactate dehydrogenase(LDH)release rate was evaluated using a LDH kit.The cells were stained with Hoechst 33342 for observing the nuclear morphology and then with propidium iodide(PI)for determining the cell cycle.The cell apoptosis was detected by flow cytometry,cell migration by scratch test,and cell proliferation by colony-forming test.Acridine orange(AO)staining was conducted to assess the acidic organelle formation.Western blotting was carried out to measure the expression of autophagy and apoptosis related proteins including Pro Caspase-9,Pro Caspase-3,Cleaved Caspase-3,PARP,BAX,BCL-2,mTOR,P-ULK1,p62,Beclin-1 and LC3-II.Results:P.igniarius water extract inhibited the proliferation of SGC-7901 cells in a concentration and time-dependent manner.Hoechst 33342 staining revealed nuclear shrinkage and fragmentation.Compared with the blank control group,4 mg/mL P.igniarius water extract significantly increased the LDH release rate(P<0.05).The results of flow cytometry demonstrated that 8 mg/mL P.igniarius water extract significantly increased the apoptosis rate(P<0.05),and blocked the cells in the G2/M phase(P<0.01).P.igniarius water extract at all doses resulted in a significant reduction in cell migration rate and colony-forming efficiency(P<0.01)but an increase in acidic organelles.As revealed by Western blot assay,P.igniarius water extract at each dose significantly up-regulated the protein expression levels of Beclin-1,LC3-II and P-ULK1(P<0.01),down-regulated the protein expression levels of Pro Caspase-9,PARP,p62 and mTOR(P<0.05 or P<0.01),and increasing BAX/BCL-2 and Cleaved Caspase-3/Pro Caspase-3
作者
陶裕玲
李杨蕾
陈冬铭
蒋晨磊
陆红
Tao Yuling;Li Yanglei;Chen Dongming;Jiang Chenlei;Lu Hong(College of Pharmaceutical Sciences,Zhejiang Chinese Medical University,Hangzhou 311402)
出处
《中药药理与临床》
CAS
CSCD
北大核心
2021年第3期120-127,共8页
Pharmacology and Clinics of Chinese Materia Medica
关键词
桑黄水提物
胃癌细胞
凋亡
自噬
Phellinus igniarius water extract
gastric cancer cells
apoptosis
autophagy
