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、Wound healing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 protein 2，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 responsive 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（Cyclin B1）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（cleaved poly ADP-ribose polymerase，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，Western blotting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-mesenchymal transition，EMT）的发生，其分子机制可能与调控Skp2-p21/p53信号轴有关。槲皮素和表儿茶素可能是TFLS靶向Skp2发挥作用的效应物质。

Objective To explore the effect and underlying mechanism of total flavonoids from Litchi Semen (TFLS) against non-small 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 metastasis of NSCLC, induce apoptosis, block cell cycle in G2/M phase, and weaken the occurrence of epithelial-mesenchymal transition (EMT) of A549 cells in vitro. The molecular mechanism may be related to the regulation of Skp2-p21/p53 signaling axis. Quercetin and (−)-epicatechin may be the effective substances for TFLS targeting Skp2.

R285.5

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