对叶百部总生物碱抑制非小细胞肺癌NCI-H460细胞增殖和诱导细胞凋亡的作用机制  

Mechanism of Stemona tuberosa Alkaloids in Inhibiting Proliferation and Inducing Apoptosis of Non-small Cell Lung Cancer NCI-H460 Cells

作　　者：林思 秦慧真 许立拔 龙莉 朱华[1] 王孝勋[1] LIN Si;QIN Huizhen;XU Liba;LONG Li;ZHU Hua;WANG Xiaoxun(Guangxi Key Laboratory of Zhuang and Yao Ethnic Medicine,Guangxi University of Chinese Medicine,Nanning 530200,China;Guangxi International Zhuang Medicine Hospital,Nanning 530201,China)

机构地区：[1]广西中医药大学壮瑶药重点实验室,南宁530200 [2]广西国际壮医医院,南宁530201

出　　处：《中国实验方剂学杂志》2023年第22期71-78,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：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(壮瑶药质量标准研究,桂人才通字[2019]13号);广西重点研发项目(桂科AB21196016);广西科技基地和人才专项(2021AC16020);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)。

摘　　要：目的:观察对叶百部总生物碱对人非小细胞肺癌NCI-H460细胞增殖和凋亡的影响,并探讨其可能的作用机制。方法:以非小细胞肺癌NCI-H460细胞作为研究对象,设置空白组和对叶百部总生物碱组(50、100、150、200、250 mg·L^(-1))。通过噻唑蓝(MTT)比色法和平板克隆形成实验观察对叶百部总生物碱对人非小细胞肺癌NCI-H460细胞增殖的影响;Hoechst33258染色法和流式细胞术观察细胞凋亡;实时荧光定量聚合酶链式反应(Real-time PCR)检测对叶百部总生物碱对胱天蛋白酶-3(Caspase-3)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)和表皮生长因子受体(EGFR)mRNA的表达水平;蛋白免疫印迹法(Western blot)检测对叶百部总生物碱对Caspase-3、Bax、Bcl-2、蛋白激酶B(Akt)、磷酸化(p)-Akt、EGFR、c-Jun氨基末端激酶(JNK)、p-JNK、p38丝裂原活化蛋白激酶(p38 MAPK)及p-p38 MAPK蛋白表达的影响。结果:与空白组比较,对叶百部总生物碱组细胞增殖抑制率显著升高(P<0.01);细胞克隆形成数目和克隆形成率明显降低(P<0.05,P<0.01);细胞核固缩、细胞质凝聚及细胞凋亡率显著增加(P<0.01);对叶百部总生物碱组(100、150、200、250 mg·L^(-1))Caspase-3 mRNA表达明显升高(P<0.05);对叶百部总生物碱组Bax mRNA表达显著升高(P<0.01),Bcl-2 mRNA表达显著降低(P<0.01);对叶百部总生物碱组(100、150、200、250 mg·L^(-1))EGFR mRNA表达明显降低(P<0.05,P<0.01);对叶百部总生物碱组(100、150、200、250 mg·L^(-1))Caspase-3、p-JNK蛋白表达显著上调(P<0.01);对叶百部总生物碱组Bax、p-p38 MAPK蛋白表达显著上调(P<0.01),Bcl-2蛋白表达显著下调(P<0.01);对叶百部总生物碱组(100、150、200、250 mg·L^(-1))EGFR、p-Akt蛋白表达明显下调(P<0.05,P<0.01)。结论:对叶百部总生物碱可抑制人非小细胞肺癌NCI-H460细胞增殖,并能诱导其凋亡,其机制可能与抑制EGFR蛋白的表达和Akt蛋白的磷酸化及激活JNK/p38 MAPK信号通路�Objective:To study the effect and underlying mechanism of Stemona tuberosa alkaloids on the proliferation and apoptosis of human non-small cell lung cancer NCI-H460 cells.Method:Non-small cell lung cancer NCI-H460 cells were divided into a blank group and S.tuberosa alkaloids groups(50,100,150,200,and 250 mg·L^(-1)).The effect of S.tuberosa alkaloids on the proliferation of human NCI-H460 cells was observed by thiazolyl blue tetrazolium bromide(MTT)assay and colony formation assay.Cell apoptosis was observed by Hoechst 33258 staining and flow cytometry.Real-time fluorescence-based polymerase chain reaction(Real-time PCR)was used to detect the effect of S.tuberosa alkaloids on the mRNA expression of cysteinyl aspartate-specific protease 3(Caspase-3),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),and epidermal growth factor receptor(EGFR).The protein expression levels of Caspase-3,Bax,Bcl-2,protein kinase B(Akt),phosphorylated(p-)Akt,EGFR,c-Jun N-terminal kinase(JNK),p-JNK,p38 mitogenactivated protein kinase(p38 MAPK),and p-p38 MAPK were measured by Western blot.Result:Compared with the blank group,the S.tuberosa alkaloids groups showed increased inhibition rate on cell proliferation(P<0.01),reduced number of cell clones formed and the rate of cell clonal formation(P<0.05,P<0.01),and increased karyopyknosis,cytoplasmic aggregation,and cell apoptosis rate(P<0.01).The S.tuberosa alkaloids groups at 100,150,200,and 250 mg·L^(-1) showed increased Caspase-3 mRNA expression(P<0.05),decreased EGFR mRNA expression(P<0.05,P<0.01),up-regulated protein expression of Caspase-3 and p-JNK(P<0.01),and down-regulated protein expression of EGFR and p-Akt(P<0.05,P<0.01).Additionally,compared with the blank group,the S.tuberosa alkaloids groups showed increased expression of Bax mRNA(P<0.01),decreased expression of Bcl-2 mRNA(P<0.01),up-regulated protein expression of Bax and p-p38 MAPK(P<0.01),and down-regulated protein expression of Bcl-2(P<0.01).Conclusions:S.tuberosa alkaloids can inhibit proliferation and induce apop

关 键 词：对叶百部总生物碱 非小细胞肺癌 NCI-H460细胞 凋亡 表皮生长因子(EGFR) 蛋白激酶B(Akt) 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 

分 类 号：R2-0[医药卫生—中医学] R22R242R2-031R285.5