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目的:采用分子对接法筛选酪氨酸蛋白激酶受体B2（EphB2）小分子抑制剂，研究其对皮肤鳞状细胞癌（CSCC）的影响及可能机制。方法:利用Schrodinger对接工具预测EphB2蛋白的三维结构及其配体结合位点，通过分子对接进行高通量虚拟筛选EphB2抑制剂，通过体内外实验验证筛出的EphB2抑制剂山奈苷与芦荟大黄素（AE）抗CSCC的作用及机制。体外实验中，将人CSCC细胞系A431、SCL-1及人永生化表皮细胞HaCaT分别分为空白对照组、二甲基亚砜组、AE组与山奈苷组，通过MTT实验（AE浓度：20、40、80、160 μmol/L；山奈苷浓度：12.5、25、50、100 μmol/L）、划痕实验及Transwell小室实验（AE浓度：80 μmol/L，山奈苷浓度：50 μmol/L）分析EphB2抑制剂对CSCC细胞增殖、迁移、侵袭的影响。体内实验中，SPF级BALB/c雌性裸鼠皮下注射0.2 ml A431细胞悬液，待成功长出瘤体以后，随机分为4组（n n = 6），空白对照组、二甲基亚砜组、AE组（腹腔注射AE 20 mg·kgn -1·dn -1 AE）与山奈苷组（腹腔注射山奈苷25 mg·kgn -1·dn -1）；每周测量裸鼠的肿瘤大小和体重；连续给药28 d后，剥取裸鼠移植瘤进行HE染色，qRT-PCR与Western blot分析AE和山奈苷对裸鼠移植瘤中上皮钙黏着蛋白、波形蛋白、磷酸化葡萄糖合成激酶3β（p-GSK-3β）、β联蛋白及GSK-3β表达的影响。组间比较采用单因素方差分析及n t检验。n 结果:筛选出对EphB2具有较高抑制活性的两个小分子化合物AA-504/20999031（山奈苷）和AA-466/21162055（AE）。MTT实验结果表明，与HaCaT细胞相比，AE对SCL-1和A431细胞具有强烈的细胞毒性，且随AE浓度升高毒性变强（n F = 17.95，n P<0.001），作用48 h时，IC50分别为124.59 μmol/L和80.85 μmol/L；山奈苷对SCL-1和A431细胞具有强烈的细胞毒性，且随山奈苷浓度升高毒性变强（n F = 11.34，n P<0.001），作用48 h时，IC50分别为119.64 μmol/L和64.96 μmol/L。划痕实验显示，与二甲基亚砜组细胞迁移距离（88.1±1.4） μm相比，AE组和山奈苷组A431细胞迁移距离[（36.7±1.0） μm和（44.7 ± 3.5） μm]显著缩短（n F = 52.34，n P < 0.001），而HaCaT细胞迁移距离差异无统计学意义（ n F = 1.73，n P = 0.238）。Transwell小室实验表明，与二甲基亚砜组A431细胞跨膜细胞数量（195.3 ± 5.7）相比，AE组和山奈苷组A431细胞显著抑制（145.0 ± 2.5和94.7 ± 4.1，n F = 72.85，n P < 0.001），而对HaCaT细胞则无明显抑制作用（ n F = 3.91，n P = 0.055）。动物实验表明，与二甲基亚砜组裸鼠移植瘤体积（841.88 ± 84.63） mmn 3相比，AE组和山奈苷组显著下降[（407.42 ± 70.37） mmn 3与（368.77 ± 62.7） mmn 3，n F = 73.78，n P < 0.001]。HE染色证实，AE和山奈苷干预可改善其病理变化。qRT-PCR与Western印迹结果显示，AE和山奈苷明显上调瘤体组织中上皮钙黏着蛋白与p-GSK-3β mRNA和蛋白表达水平（均 n P < 0.001），下调波形蛋白、β联蛋白及GSK-3β mRNA和蛋白表达水平（均 n P < 0.001）。n 结论:分子对接筛选的小分子抑制剂与EphB2可形成稳定复合物，并通过影响Wnt/β联蛋白通路诱导的上皮间质转化现象来抑制CSCC进程。“,”Objective:To screen small-molecule inhibitors of tyrosine kinase receptor B2 (EphB2) by using a molecular docking method, and to investigate their effect on cutaneous squamous cell carcinoma (CSCC) and possible mechanisms of action.Methods:The three-dimensional structure of EphB2 protein and its ligand binding sites were predicted by using the docking tool Schrodinger, and high-throughput virtual screening of EphB2 inhibitors was carried out by molecular docking. The anti-CSCC effect and mechanism of action of the screened EphB2 inhibitors kaempferitrin and aloe-emodin (AE) were verified in n in vitro and n in vivo experiments. In the n in vitro experiments, human CSCC cell lines A431 and SCL-1, as well as the human immortalized keratinocyte HaCaT, were all divided into blank control group, dimethyl sulfoxide (DMSO) group, AE group and kaempferitrin group. Methyl thiazol tetrazolium (MTT) assay (AE at concentrations of 20, 40, 80, 160 μmol/L, kaempferitrin at concentrations of 12.5, 25, 50, 100 μmol/L), scratch and Transwell assays (AE at a fixed concentration of 80 μmol/L, kaempferitrin at a fixed concentration of 50 μmol/L) were performed to analyze the effect of EphB2 inhibitors on the proliferation, migration and invasion of CSCC cells. In the n in vivo experiments, specific pathogen-free BALB/c female nude mice were subcutaneously injected with 0.2 ml of A431 cell suspension. After tumor growth, 24 tumor-bearing mice were randomly and equally divided into 4 groups: AE group and kaempferitrin group intraperitoneally injected with 20 mg·kgn -1·dn -1 AE and 25 mg·kgn -1·dn -1 kaempferitrin respectively, blank control group and DMSO group intraperitoneally injected with the same volume of sodium chloride physiological solution and DMSO respectively; the tumor size and body weight of nude mice were measured weekly; after consecutive treatment for 28 days, transplanted tumors were resected from the nude mice for hematoxylin and eosin (HE) staining, and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were performed to analyze the effect of AE and kaempferitrin on the mRNA and protein expression of E-cadherin, vimentin, glycogen synthase kinase 3β (GSK-3β), phosphorylated GSK-3β (p-GSK-3β) and β-catenin respectively. One-way analysis of variance andn t test were used for comparisons between groups.n Results:Two small-molecule compounds AA-504/20999031 (kaempferitrin) and AA-466/21162055 (AE) with high inhibitory activity against EphB2 were screened out. MTT assay showed that both AE and kaempferitrin exhibited strong cytotoxicity to SCL-1 and A431 cells compared with HaCaT cells, and their toxicity increased with the increase of their concentration (n F = 17.95, 11.34, respectively, both n P < 0.001) ; after 48-hour treatment, the 50% inhibitory concentrations (IC50s) of AE against SCL-1 and A431 cells were 124.59 and 80.85 μmol/L respectively, and the IC50s of kaempferitrin against SCL-1 and A431 cells were 119.64 and 64.96 μmol/L respectively. Scratch assay showed that the migration distance of A431 cells was significantly shorter in the AE group and kaempferitrin group (36.7 ± 1.0 μm, 44.7 ± 3.5 μm, respectively) than in the DMSO group (88.1 ± 1.4 μm, n F = 52.34, n P < 0.001), while there was no significant difference in the migration distance of HaCaT cells among the above groups ( n F = 1.73, n P = 0.238). Transwell assay showed that the number of A431 cells crossing the Transwell membrane significantly decreased in the AE group and kaempferitrin group (145.0 ± 2.5, 94.7 ± 4.1, respectively) compared with the DMSO group (195.3 ± 5.7, n F = 72.85, n P < 0.001), while neither AE nor kaempferitrin showed significant inhibitory effects of on the number of HaCaT cells crossing the Transwell membrane ( n F = 3.91, n P = 0.055). The animal experiment revealed significantly decreased volumes of transplanted tumors in nude mice in the AE group and kaempferitrin group (407.42 ± 70.37 mmn 3, 368.77 ± 62.7 mmn 3, respectively) compared with the DMSO group (841.88 ± 84.63 mmn 3, n F = 73.78, n P < 0.001). HE staining confirmed that AE and kaempferitrin could improve pathological changes of transplanted tumors. qRT-PCR and Western blot analysis showed that AE and kaempferitrin significantly up-regulated the mRNA and protein expression of E-cadherin and p-GSK-3β in tumor tissues (all n P < 0.001), and down-regulated the mRNA and protein expression of vimentin, β-catenin and GSK-3β (all n P < 0.001) .n Conclusion:The small-molecule inhibitors screened by molecular docking can form a stable complex with EphB2, and inhibit the progression of CSCC by affecting the Wnt/β-catenin pathway-induced epithelial-mesenchymal transition.