摘要
目的 研究丹参酮ⅡA对人喉癌Hep-2细胞凋亡和自噬的影响,并基于磷脂酰肌醇3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路探讨其可能作用机制。方法 分别以不同浓度丹参酮ⅡA(0,2,4,8,16,32μmol/L)干预对数生长期人喉癌Hep-2细胞48 h, CCK-8法检测细胞增殖抑制率,计算半抑制浓度(IC50)作为后续实验药物浓度。取对数生长期人喉癌Hep-2细胞,设空白组、丹参酮ⅡA组、丹参酮ⅡA+IGF-1(PI3K激活剂)组、丹参酮ⅡA+LY294002(PI3K抑制剂)组,各组给予相应干预48 h后,CCK-8法、Annexin V-FITC/PI双染法、GFP-LC3荧光质粒转染法检测细胞增殖抑制率、凋亡率和自噬体形成情况,Western blot法检测B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、激活型半胱氨酸蛋白酶-3(Cleaved Caspase-3)、微管相关蛋白1轻链3(LC3)、PI3K、p-PI3K、Akt、p-Akt、mTOR、p-mTOR蛋白表达情况。结果 丹参酮ⅡA对Hep-2细胞增殖抑制作用呈现剂量依赖性,IC50值为9.6μmol/L。与空白组比较,丹参酮ⅡA组Hep-2细胞增殖抑制率、凋亡率均显著升高(P均<0.05),细胞内自噬体数量显著增多(P<0.05),Bax、Cleaved Caspase-3、LC3-Ⅰ、LC3-Ⅱ表达量及LC3-Ⅱ/LC3-Ⅰ、Bax/Bcl-2比值均显著升高(P均<0.05),Bcl-2、p-PI3K、p-Akt、p-mTOR表达量及p-PI3K/PI3K、p-Akt/Akt、p-mTOR/mTOR均显著降低(P均<0.05);IGF-1能够明显逆转丹参酮ⅡA对Hep-2细胞的上述调控作用,LY294002则能够明显加强丹参酮ⅡA的上述作用。结论 丹参酮ⅡA能够促进人喉癌Hep-2细胞凋亡和自噬,其作用机制可能与抑制PI3K/Akt/mTOR信号通路活化有关。
Objective It is to study the effect of TanshinoneⅡA on apoptosis and autophagy of human laryngeal carcinoma Hep-2 cells and explore its possible mechanism of action based on the phosphatidylinositol 3-kinase/protein kinase B/mammalian target protein of rapamycin(PI3K/Akt/mTOR)signaling pathway.Methods Different concentrations of TanshinoneⅡA(0,2,4,8,16,32μmol/L)were used to intervene the human laryngeal carcinoma Hep-2 cells in logarithmic growth phase for 48 h.The cell proliferation inhibition rate was detected by CCK-8 method,and the half-inhibitory concentration(IC50)was calculated and used as the drug concentration in subsequent experiments.The Hep-2 cells in logarithmic growth phase were divided into blank control group,TanshinoneⅡA group,Tanshinone IIA+IGF-1(PI3K activator)group and TanshinoneⅡA+LY294002(PI3K inhibitor)group.48 h after drug intervention,the cell proliferation inhibition rate,apoptosis rate,autophagosome formation were respectively detected by CCK-8 method,Annexin V-FITC/PI double staining method and GFP-LC3 fluorescent plasmid transfection method.The expression of B-cell lymphoma-2(Bcl-2),Bcl-2-related X protein(Bax),activated caspase-3(Cleaved Caspase-3),microtubule-associated protein 1 light chain 3(LC3),phosphatidylinositol 3-kinase(PI3K),p-PI3K,protein kinase B(Akt),p-Akt,mammalian target of rapamycin(mTOR),p-mTOR were detected by Western blot method.Results The inhibitory effect of TanshinoneⅡA on Hep-2 cell proliferation was dose-dependent,which IC50 was 9.6μmol/L.Compared with the blank control group,the cell proliferation inhibition rate and apoptosis rate in TanshinoneⅡA group were significantly increased(P<0.05),the number of intracellular autophagosomes was significantly increased(P<0.05),the expression of Bax,Cleaved Caspase-3,LC3-Ⅰ,LC3-Ⅱand LC3-Ⅱ/LC3-Ⅰand Bax/Bcl-2 were significantly increased(all P<0.05),and the expression of Bcl-2,p-PI3K,p-Akt,p-mTOR and p-PI3K/PI3K,p-Akt/Akt,p-mTOR/mTOR were significantly decreased(all P<0.05);IGF-1 could obviously reverse the above-mentioned regulatory effect of TtanshinoneⅡA on Hep-2 cells,LY294002 could enhance the above-mentioned effect of tanshinoneⅡA.Conclusion TanshinoneⅡA can promote the apoptosis and autophagy of human laryngeal carcinoma Hep-2 cells,which mechanism may be related to inhibiting the activation of PI3K/Akt/mTOR signaling pathway.
作者
王海叶
邢瑞敏
马欣
宋长亮
李静静
乔冰冰
吴庭艳
WANG Haiye;XING Ruimin;MA Xin;SONG Changliang;LI Jingjing;QIAO Bingbing;WU Tingyan(Handan Central Hospital,Handan 056000,Hebei,China;Handan Iron and Steel Hospital Co.Ltd.,Handan 056000,Hebei,China)
出处
《现代中西医结合杂志》
CAS
2023年第3期308-314,共7页
Modern Journal of Integrated Traditional Chinese and Western Medicine
基金
邯郸市科学技术研究与发展计划项目(21422083142)。
关键词
丹参酮ⅡA
喉癌
磷脂酰肌醇3激酶
蛋白激酶B
哺乳动物雷帕霉素靶蛋白
凋亡
自噬
TanshinoneⅡA
laryngeal carcinoma
phosphatidylinositol 3-kinase
protein kinase B
mammalian target of rapamycin
apoptosis
autophagy
