钩吻生物碱对肺癌的抑制作用及其机制

Inhibitory effect and mechanism of alkaloids derived from Gelsemium elegans Benth on lung cancer

目的 探讨钩吻素子(koumine,KOU)及钩吻总生物碱(total alkaloids of Gelsemium elegans Benth,TAG)抗肺癌的作用及其机制。方法 采用活细胞动态分析系统、集落形成实验研究低、中、高浓度(100、150、200μg/mL)KOU对人肺腺癌细胞A549、SPCA1的增殖作用。构建肺癌细胞小鼠移植瘤模型,分为模型组(Model组)、环磷酰胺组(CTX组,剂量为20 mg/kg)、KOU组(剂量为2 mg/kg)和TAG组(剂量为0.5 mg/kg),CTX组和KOU、TAG组按0.1 mL/10 g隔天腹腔注射,给药10 d检测KOU和TAG对肺癌实体瘤生长的作用,采用HE、免疫组化(immunohistochemistry,IHC)及TUNEL染色观察肿瘤组织,并计算肿瘤体积和肿瘤抑制率。对TAG作用48 h的A549细胞进行RNA测序分析,筛选差异表达基因(differentially expressed genes,DEGs),并进行KEGG(Kyoto encyclopedia of genes and genomes,KEGG)和GO(Gene Ontology,GO)功能富集分析。采用实时荧光定量逆转录聚合酶链反应(real-time quantitative reverse transcription polymerase chain reaction,real-time-qPCR)进一步分析验证相关基因的表达。结果 活细胞动态分析系统拟合出A549、SPCA1细胞融合度下降,细胞数目减少,生长状态变差。集落形成实验结果证实KOU使细胞克隆数目形成减少,在高浓度200μg/mL时最为显著(P<0.01)。动物实验表明KOU组、TAG组的肿瘤抑制率依次可达24.55%、36.08%,与Model组相比,TAG组肿瘤体积显著下降(P<0.05)。RNA测序分析筛选出DEGs总数为2 793个,其中上调基因1 433个,下调基因1 360个。富集分析发现DEGs主要富集于IL-17信号通路、肿瘤坏死因子信号通路及p53信号通路等。RT-qPCR验证结果与RNA测序分析结果一致,GADD34、ZFP36、GADD45A、GADD45B、TP53INP2基因在TAG组显著表达增加(P<0.01)。结论 在体内、外水平钩吻生物碱均抑制肺癌增殖,转录组学发现其抑制肺癌作用的多个DEGs和通路。

Objective To determine the anti-lung cancer effect of koumine(KOU) and total alkaloids of Gelsemium elegans Benth(TAG) and investigate the underlying mechanism.Methods After low,medium and high concentrations(100,150,200 μg/mL) of KOU were used to treat human lung adenocarcinoma cell lines A549 and SPCA1,Live Cell Imaging and Analysis by Sartorius colony formation assay was employed to detect the cell proliferation.The transplanted tumor model of lung cancer cells in mice was constructed and divided into model group(model group),cyclophosphamide group(CTX group,20 mg/kg),KOU group(2 mg/kg) and TAG group(0.5 mg/kg).After the mice of the CTX,KOU and TAG groups were intraperitoneally injected with 0.1 mL/10 g corresponding agents every other day for 10 d,the growth of lung cancer solid tumors was observed grossly and with HE staining,immunohistochemical(IHC) assay and TUNEL staining to and calculate the tumor size and growth inhibitory rate.RNA sequencing analysis was performed on A549 cells treated with TAG for 48 h to screen the differentially expressed genes(DEGs) between the treatment group and the control group,and the obtained DEGs were further analyzed with Kyoto Encyclopedia of Genes and Genomes(KEGG) and Gene Ontology(GO) functional enrichment analyses.RT-qPCR was applied to further analyze and verify the expression of related genes.Results Live Cell Imaging and Analysis fitted that the confluence of A549 and SPCA1 cells was decreased and the number of cells in the treated groups was observed to decrease,with poor growth.The results of colony formation assay confirmed that KOU reduced the number of cell clones,especially at a dose of 200 μg/mL(P<0.01).Animal experiments showed that KOU and TAG treatment inhibited the tumor growth by 24.55% and 36.08%,respectively.TAG treatment resulted in significantly decreased tumor size when compared with the model group(P<0.05).RNA sequencing analysis revealed that there were totally 2 793 DEGs,including 1 433 up-regulated genes and 1 360 down-regulated ones.Enrichment analysis displayed that the DEGs were mainly enriched in IL-17 signaling pathway,tumor necrosis factor signaling pathway and P53 signaling pathway.The results of RT-qPCR were consistent with the results of RNA sequencing analysis.The expression levels of GADD34,ZFP36,GADD45 A,GADD45 B and TP53INP2 genes were significantly increased in the TAG group(P<0.01).Conclusion Alkaloids of Gelsemium elegans Benth inhibits the proliferation of lung cancer in vivo and in vitro.Transcriptomics find that KOU and TAG inhibit multiple DEGs and pathways of lung cancer.