生脉胶囊逆转小鼠结肠癌细胞5-氟尿嘧啶耐药研究

Reversal effect of Shengmai capsule on 5-fluorouracil resistance in mouse colon cancer cells

关键词:  生脉胶囊  结肠癌  5-氟尿嘧啶  化疗耐药  DNA拓扑异构酶Ⅱα

Key words:  Shengmai capsule  colon cancer  5-fluorouracil  chemoresistance  DNA topoisomerase Ⅱα

摘要:

[目的] 考察生脉胶囊（SM）逆转小鼠结肠癌细胞5-氟尿嘧啶（5-FU）耐药的作用和机制。[方法] 利用中医药整合药理学研究平台获取生脉胶囊调节的靶基因（基因集1），在Gene Cards数据库检索到结直肠癌化疗抵抗相关基因（基因集2）和5-FU抗性相关基因（基因集3），取三者交集为研究对象，进行基因本体（GO）分析和京都基因组百科全书（KEGG）通路富集分析；筛选5-FU耐药的CT26细胞（CT26/FU），构建小鼠移植瘤模型，将小鼠分为模型组、FU组（腹腔注射5-FU）和FU+SM组（腹腔注射5-FU，同时灌胃生脉胶囊）。每天测量肿瘤大小，10 d后处死小鼠，称量移植瘤的质量，提取各组移植瘤的总RNA，通过Real time RT-PCR检测5-FU耐药相关基因胸苷酸合成酶（TYMS）、二氢嘧啶脱氢酶（DPYD）、DNA拓扑异构酶Ⅱα（TOP2A）、尼克酰胺-N-甲基转移酶（NNMT）和糖皮质激素受体（NR3C1）的表达。[结果] 网络药理学分析筛选出38个候选基因，富集到多条药物耐药通路上。实验表明，与模型组相比，FU组的肿瘤大小和质量没有差异，但FU+SM组肿瘤的大小和质量都明显降低。同时，与FU组相比，FU+SM组中TOP2A基因的表达降低。[结论] SM能够逆转CT26/FU的耐药性，其机制可能与下调TOP2A表达有关。

Abstract:

[Objective] To explore the effect and mechanism of Shengmai capsule（SM） on reversing 5-fluorouracil（5-FU） resistance in mouse colon cancer cells. [Methods] Target genes regulated by SM（gene set 1） were obtained from Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine. Chemotherapy resistance related genes（gene set 2） and 5-FU resistance related genes（gene set 3） were both retrieved from Gene Cards database. The intersection of three gene sets was used for subsequent analysis. The Gene Ontology（GO） analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG） pathway enrichment analysis were performed. 5-FU resistant CT26 cells（CT26/FU） were obtained and used to construct mouse xenograft model. The tumor-bearing mice were than divided into model group，FU group（intraperitoneal injection of 5-FU） and FU+SM group（intraperitoneal injection of 5-FU and gavage with SM）. The size of xenografts was measured every day and the mice were sacrificed 10 days later while the xenografts were weighted. Total RNA of xenografts was extracted and Realtime RT-PCR was performed to detect the expression of 5-FU resistance related genes thymidylate synthase（TYMS），dihydropyrimidine dehydrogenase（DPYD），DNA topoisomerase Ⅱ（TOP2A），nicotinamide-N-methyltransferase（NNMT） and glucocorticoid receptor（nuclear receptor subfamily 3 group C member 1，NR3C1）. [Results] The 38 candidate genes were screened with network pharmacology analysis and these genes were enriched in multiple drug resistance pathways. Animal experiments suggested that there was no difference in tumor size and weight between the FU group and the model group. However，the size and weight of xenografts were decreased in the FU+SM group. Meanwhile，compared with the FU group，the expression of TOP2A in the FU+SM group decreased. [Conclusion] SM can reverse the drug resistance of CT26/FU，and its mechanism may be related to downregulating the expression of TOP2A.