荔枝核总黄酮调控Skp2-p21/p53信号轴抑制非小细胞肺癌的增殖和转移  

Total flavonoids from Litchi Semen inhibit proliferation and metastasis of nonsmall cell lung cancer by regulating Skp2-p21/p53 signaling axis

作　　者：任红 李小兰[2,3] 蒋琳[1,2] 韩瑶瑶 廖韵诺 付杰军[2] 郭宏伟 REN Hong;LI Xiaolan;JIANG Lin;HAN Yaoyao;LIAO Yunnuo;FU Jiejun;GUO Hongwei(The First Clinical Medical School,Guangxi Medical University,Nanning 530021,China;Key Laboratory of Longevity and Aging-related Diseases of Chinese Ministry of Education&Guangxi Key Laboratory of Component-efficacy Relationship of Traditional Chinese Medicine,Guangxi Medical University,Nanning 530021,China;Guangxi Key Laboratory of Bioactive Molecules Research and Evaluation,Pharmaceutical College,Guangxi Medical University,Nanning 530021,China)

机构地区：[1]广西医科大学第一临床医学院,广西南宁530021 [2]长寿与老年相关疾病教育部重点实验室&广西中药组效学重点研究室,广西南宁530021 [3]广西生物活性分子研究与评价重点实验室,广西医科大学药学院,广西南宁530021

出　　处：《中草药》2024年第20期6940-6952,共13页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金项目(82074347);中央引导地方科技发展资金专项(防科ZY20221502);2022年青年岐黄学者培养项目;广西医科大学高水平创新团队及杏湖学者计划

摘　　要：目的 探讨荔枝核Litchi Semen总黄酮(total flavonoids from Litchi Semen,TFLS)抑制非小细胞肺癌(non-small cell lung cancer,NSCLC)的作用及其潜在的分子机制。方法 TFLS作用于NSCLC细胞后,采用CCK-8法、平板克隆形成实验、Woundhealing和Transwell实验分别考察细胞的增殖、迁移及侵袭能力。网络药理学方法预测TFLS中可能发挥作用的活性成分、作用靶点及相关作用通路;流式细胞术检测TFLS对A549细胞周期和凋亡的影响;Western blotting检测转移、周期以及凋亡相关蛋白的表达情况。分子对接技术预测TFLS靶向细胞周期S期激酶相关蛋白2(S-phase kinase associated protein2,Skp2)治疗NSCLC的潜在活性成分。药物亲和反应的靶点稳定性(drug affinity re sponsive target stability,DARTS)实验进一步检测槲皮素、表儿茶素与Skp2的结合作用。结果 TFLS可显著抑制NSCLC细胞H1437、H358、A549和H1299的体外增殖及A549细胞的克隆形成能力(P<0.05);TFLS可抑制A549细胞的迁移(P<0.05、0.01)和侵袭(P<0.01),上调上皮钙黏蛋白(epithelial cadherin,E-cadherin)的表达(P<0.05),下调波形蛋白(Vimentin)的表达(P<0.01)。TFLS可使A549细胞阻滞在G2/M期并诱导其凋亡(P<0.05),下调细胞周期蛋白B1 (CyclinB1)表达(P<0.05),上调细胞周期依赖性蛋白激酶抑制因子1A (cyclin-dependent kinase inhibitor 1A,p21)和裂解的多聚ADP核糖聚合酶(cleavedpoly ADPribo sepolymerase,cleaved PARP)的表达(P<0.05、0.01)。网络药理学预测得到的核心靶点为Skp2、p53,Westernblotting结果表明TFLS可以下调Skp2蛋白的表达(P<0.01)以及上调p53蛋白的表达(P<0.05)。分子对接和DARTS结果表明,TFLS中的槲皮素、表儿茶素与Skp2有较好的结合能力。结论 TFLS在体外可以抑制NSCLC的生长和转移,诱导A549细胞凋亡,阻滞细胞在G2/M期,减弱细胞上皮-间充质转化(epithelial-me senchymaltransition,EMT)的发生,其分子机制可能与调控Skp2-p21/p53信号轴有关。槲皮素和�Objective To explore the effect and underlying mechanism of total flavonoids from Litchi Semen(TFLS)against nonsmall cell lung cancer(NSCLC).Methods After treating NSCLC cells with TFLS,the proliferation,migration,and invasion ability of cell were evaluated by CCK-8 assay,cell plate clone formation assay,Wound healing test and Transwell assay,respectively.The possible active ingredients,target and related pathways of the therapeutic efficacy of TFLS were predicted by network pharmacology.The effects of TFLS on A549 cell cycle and cell apoptosis were examined by flow cytometry.Western blotting was conducted to detect the expression levels of proteins related to metastasis,cell cycle,and cell apoptosis.Molecular docking technology was used to predict potential active components of TFLS targeting S-phase kinase associated protein 2(Skp2)in the treatment of NSCLC.Drug affinity responsive target stability(DARTS)assay was used to further detect the binding effects of quercetin and(−)-epicatechin to Skp2.Results TFLS significantly inhibited the in vitro proliferation of H1437,H358,A549,and H1299 cells and the clone formation ability of A549 cells(P<0.05).TFLS inhibited migration(P<0.05,0.01)and invasion(P<0.01)ability of A549 cells by up-regulated the expression of Epithelial cadherin(E-cadherin,P<0.05)and down-regulated the expression of Vimentin(P<0.01).Flow cytometry results showed that TFLS could block A549 cells in G2/M phase and induce its apoptosis(P<0.05)by down-regulated the expression of Cyclin B1(P<0.05)and up-regulating the expression of Cyclin-dependent kinase inhibitor 1A(p21)and Cleaved poly ADP-ribose polymerase(Cleaved PARP,P<0.05,0.01).The core targets Skp2 and p53 predicted by network pharmacology.Western blotting results showed that TFLS could down-regulating the expression of Skp2(P<0.01)and up-regulate the expression of p53(P<0.05).Molecular docking and DARTS results showed that quercetin and(−)-epicatechin in TFLS could bind well with Skp2.Conclusion TFLS could inhibit the growth and metastasi

关 键 词：荔枝核总黄酮 非小细胞肺癌 细胞周期 凋亡 转移 槲皮素 表儿茶素 

分 类 号：R285.5[医药卫生—中药学]