基于3D肿瘤模型和网络药理学的威灵仙粗提物体外抗肺癌药效及潜在作用机制研究

In vitro anti-lung cancer efficacy and potential mechanism of crude extract of Clematidis Radix et Rhizoma based on 3D tumor model and network pharmacology

目的基于3D肿瘤细胞模型和网络药理学方法,研究威灵仙体外抗肺癌的药效及潜在作用机制。方法使用人肺癌细胞系A549建立体外3D肺癌模型,以依托泊苷作为阳性对照组,对水、30%乙醇、60%乙醇、70%乙醇、90%乙醇提取得到的威灵仙粗提物进行体外药效学研究。通过多种数据库筛选出药物和肺癌的核心靶点,并采用ELISA试剂盒检测信号通路中的关键因子。结果挖掘出威灵仙活性成分33个,核心成分3种,为Hederagenin、2-Deoxy-D-Ribono-1、beta-sitosterol,排名靠前的靶点为CASP3、PTGS2、IL6、TNF,推测为核心靶点。GO功能富集共筛选得到171个条目,KEGG通路富集共筛选出56条通路。ATP检测结果显示各2D、3D细胞模型处理组IC_(50)值分别为:依托泊苷(41.98±1.15),(118.63±3.93)μmol·L^(-1);水提取物(0.89±0.04),(6.37±0.78)mg·mL^(-1);30%醇提物(0.64±0.02),(5.40±0.78)mg·mL^(-1);60%醇提物(0.45±0.01),(4.30±1.16)mg·mL^(-1);70%醇提物(0.37±0.02),(3.24±0.85)mg·mL^(-1);90%醇提物(0.60±0.03),(5.46±1.47)mg·mL^(-1)。HE染色结果显示,肿瘤细胞核皱缩及胞质减少程度强弱顺序为70%醇提物>依托泊苷处理组>60%醇提物>90%醇提物>30%醇提物>水提物。免疫细胞结果显示,相比于对照组,各给药组均有肿瘤细胞核皱缩且P53表达量显著减少(P<0.05)。ELISA结果显示,威灵仙70%醇提物和60%醇提物均对Bax-Bcl-2-Caspase-3信号通路产生显著影响(P<0.05)。结论各组威灵仙粗提物均对人A5493D模型有一定的活性抑制作用,其中70%醇提物效果最佳,60%醇提物次之,这种抑制作用可能与其上调Bax、Caspase-3含量,下调Bcl-2含量相关,即威灵仙粗提物可能具有抑制肿瘤细胞增殖,促进肿瘤细胞凋亡的作用。

Objective To determine the anti-lung cancer targets and mechanism of Clematidis Radix et Rhizoma,based on 3D tumor cell model combined with network pharmacology.Methods An in vitro 3D lung cancer model was established with human lung cancer cell line A549.The in vitro pharmacodynamic studies were conducted based on the crude extract of Clematidis Radix et Rhizoma obtained with water,30%ethanol,60%ethanol,70%ethanol and 90%ethanol,respectively,and the etoposide was used as a positive control group.The core targets of the drug and lung cancer were screened by various databases,and the key factors in the signal pathway were detected with ELISA kit.Results Totally 33 active components of Clematidis Radix et Rhizoma were determined,with 3 core components,namely Hederagenin,2-Deoxy-D-Ribono-1 and beta-sitosterol;the top targets included CASP3,PTGS2,IL6 and TNF,which were considered to be the core targets.Totally 171 items were screened by GO function enrichment,and 56 pathways were screened by KEGG pathway enrichment.The ATP assay showed that the IC_(50) values of the 2D and 3D cell model by different crude extract were(41.98±1.15)and(118.63±3.93)μmol·L^(-1) for etoposide;(0.89±0.04)and(6.37±0.78)mg·mL^(-1) for water extract;(0.64±0.02)and(5.40±0.78)mg·mL^(-1) for 30%ethanol extract;(0.45±0.01)and(4.30±1.16)mg·mL^(-1) for 60%ethanol extract;(0.37±0.02)and(3.24±0.85)mg·mL^(-1) for 70%ethanol extract;(0.60±0.03)and(5.46±1.47)mg·mL^(-1) for 90%ethanol extract,respectively.The HE staining showed that the order of nucleus shrinkage and cytoplasmic decrease was 70%ethanol extract>etoposide treatment>60%ethanol extract>90%ethanol extract>30%ethanol extract>water extract.Immunohistochemical showed that tumor nucleus shrinkage and decreased P53 expression occurred in the experimental groups as compared with the control group(P<0.05).ELISA showed that both 70%and 60%ethanol extract of Clematidis Radix et Rhizoma greatly affected the Bax-Bcl-2-Caspase-3 signal pathway(P<0.05).Conclusion All groups of crude extract of Clematidis Radix et Rhizoma can inhibit the activity of human lung cancer cells by 3D model.The 70%ethanol extract has the best effect,followed by 60%ethanol extract.This inhibition may be related to up-regulating Bax and Caspase-3 content,and down-regulating Bcl-2 content,implying that the crude extract of Clematidis Radix et Rhizoma can inhibit tumor cell proliferation and promot tumor cell apoptosis.