摘要
目的:探讨对叶百部总生物碱对人肺癌A549细胞凋亡及磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)和c-Jun氨基末端激酶/p38丝裂原活化蛋白激酶(JNK/p38 MAPK)信号通路的影响。方法:以人肺癌A549细胞为研究对象,设置空白组和不同质量浓度(100、150、200、250、300 mg·L^(-1))对叶百部总生物碱组。利用噻唑蓝(MTT)比色法、平板克隆实验观察对叶百部总生物碱对A549细胞增殖的影响;采用Hoechst 33258染色法和流式细胞术膜联蛋白V-异硫氰酸荧光素(AnnexinV-FITC)/碘化丙锭(PI)双染法观察细胞凋亡;蛋白免疫印迹法检测凋亡相关蛋白胱天蛋白酶-3(Caspase-3)、B细胞淋巴瘤-2(Bcl-2)相关X蛋白(Bax)和Bcl-2表达及PI3K、磷酸化(p)-PI3K、Akt、p-Akt、JNK、p-JNK、p38 MAPK、p-p38 MAPK蛋白的表达。结果:与空白组比较,对叶百部总生物碱组(150、200、250、300 mg·L^(-1))细胞增殖抑制率显著增高(P<0.01);对叶百部总生物碱组(150、200、250、300 mg·L^(-1))细胞克隆抑制率显著升高,细胞克隆形成率显著降低(P<0.01)。与空白组比较,对叶百部总生物碱组细胞出现染色质凝聚,荧光反应加强等典型的细胞凋亡特征。与空白组比较,对叶百部总生物碱组细胞凋亡率显著升高(P<0.01);对叶百部总生物碱(150、200、250、300 mg·L^(-1))能够明显上调Caspase-3、Bax蛋白表达(P<0.05,P<0.01),显著下调Bcl-2蛋白表达(P<0.01);对叶百部总生物碱对PI3K、Akt、JNK、p38 MAPK总蛋白表达无明显影响,对叶百部总生物碱(150、200、250、300 mg·L^(-1))能够明显下调PI3K、Akt磷酸化水平(P<0.05,P<0.01);明显上调p38 MAPK磷酸化水平(P<0.05,P<0.01);对叶百部总生物碱(200、250、300 mg·L^(-1))能够明显上调JNK磷酸化水平(P<0.05,P<0.01)。结论:对叶百部总生物碱可抑制人肺癌A549细胞的增殖,并能诱导A549细胞凋亡,其机制可能与抑制PI3K/Akt信号通路和激活JNK/p38 MAPK信号通路有关。
Objective:To investigate the effect of Stemona tuberosa alkaloids(STA)on apoptosis and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)and c-Jun N-terminal kinase/p38 mitogen-activated protein kinase(JNK/p38 MAPK)signaling pathways in human lung cancer A549 cells.Method:A549 cells were classified into blank group and STA groups(100,150,200,250,300 mg·L-1).Thiazole blue(MTT)assay and colony formation assay were used to evaluate the proliferation of A549 cells.Apoptosis was observed based on Hoechst 33258 staining,flow cytometry,and Annexin V-FITC/PI staining.Western blot was employed to detect the expression of apoptosis-related proteins cysteine-aspartic acid protease-3(Caspase-3),B-cell lymphoma-2(Bcl-2)-associated X protein(Bax),and Bcl-2,and the expression of PI3K,phosphorylated(p)-PI3K,Akt,p-Akt,JNK,p-JNK,p38 MAPK,and p-p38 MAPK.Result:Compared with the blank group,STA groups(150,200,250,300 mg·L^(-1))demonstrated the increase in inhibition rate of cell proliferation(P<0.01)and cell clone inhibition rate,and decrease in cell clone formation rate(P<0.01).In comparison with the blank group,STA groups showed typical characteristics of apoptosis,such as chromatin condensation and enhanced fluorescence reaction.The apoptosis rate of STA groups was significantly higher than that of the blank group(P<0.01).Compared with the blank group,STA(150,200,250,300 mg·L^(-1))significantly up-regulated the protein expression of Caspase-3 and Bax(P<0.05,P<0.01)and down-regulated the expression of Bcl-2protein(P<0.01).Compared with the blank group,STA had no significant influence on the total protein expression of PI3K,Akt,JNK,and p38 MAPK.However,STA(150,200,250,300 mg·L-1)significantly decreased the levels of p-PI3K and p-Akt(P<0.05,P<0.01)and increased the level of p-p38 MAPK(P<0.05,P<0.01).Compared with the blank group,STA(200,250,300 mg·L^(-1))significantly raised the level of p-JNK(P<0.05,P<0.01).Conclusion:STA can inhibit the proliferation and induce the apoptosis of A549 cells by inhibiting PI3K/Akt signalin
作者
林思
秦慧真
李泽宇
许立拔
邓玲玉
罗静
谢凤凤
张淼
朱华
王孝勋
LIN Si;QIN Huizhen;LI Zeyu;XU Liba;DENG Lingyu;LUO Jing;XIE Fengfeng;ZHANG Miao;ZHU Hua;WANG Xiaoxun(Guangxi University of Chinese Medicine,Nanning 530200,China;Guangxi Key Laboratory of Zhuang and Yao Ethnic Medicine,Guangxi University of Chinese Medicine,Nanning 530200,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2023年第4期69-76,共8页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(82060695)
广西壮瑶药重点实验室项目(桂科基字[2014]32号)
广西壮瑶药协同创新中心项目(桂教科研[2013]20号)
广西第八批特聘专家项目(桂人才通字[2019]13号)
广西民族药资源与应用工程研究中心项目(桂发改高技函[2020]2605号)
广西中医药大学-中南大学壮瑶药研究联合实验室项目(桂科计字[2021]238号)
广西产5种壮瑶药材质量标准与产品开发研究项目(桂科计字[2021]196号)。
