蟾毒它灵对非小细胞肺癌的抗癌活性及其肾毒性研究[D]

Investigation of the Antineoplastic Effects of Bufotalin on Non-Small Cell Lung Cancer and Its Associated Nephrotoxic Potential

关键词:  蟾毒它灵  非小细胞肺癌  EGFR  网络毒理学  肾毒性

Key words:  Bufotalin  Non-small cell lung cancer  EGFR  Network Toxicology  Nephrotoxicity

摘要:

目的 研究蟾毒它灵对非小细胞肺癌细胞及裸鼠的抗癌活性,并探讨蟾毒它灵的肾毒性及潜在机制。方法 (1)体外实验:以0、100、150、200、250、300、350、400、450 nM蟾毒它灵干预A549细胞24、48、72 h后,采用CCK-8法检测细胞增殖；100、200、300 nM蟾毒它灵干预后,通过细胞划痕实验检测A549细胞迁移能力；(2)体内实验:通过裸鼠右侧腋下注射A549细胞构建非小细胞肺癌荷瘤裸鼠模型,随机分成空白组和蟾毒它灵低、中、高剂量组,每组6只。记录裸鼠体重变化及皮下移植瘤瘤体抑制率,并进行瘤体及肾脏HE染色、检测血清中肾损伤指标,采用Western Blot法检测瘤体的EGFR、Bax、Bcl-2表达情况。(3)网络毒理学与分子对接:用CTD比较毒物遗传学数据库预测蟾毒它灵毒性作用靶点,通过GeneCards、OMIM数据库收集肾毒性相关靶点,获得两者交集靶点；对交集靶点构建PPI蛋白互作网络并进行GO和KEGG富集分析；之后对关键靶点进行分子对接和可视化分析；(4)通过qRT-PCR验证网络毒理学预测的肾毒性靶点。结果 (1)CCK-8结果表明,蟾毒它灵抑制A549细胞的效果成浓度和时间依赖性；与空白组相比,各蟾毒它灵给药组细胞划痕愈合率显著降低(P＜0.01)。(2)与空白组相比,蟾毒它灵给药组小鼠显著抑制非小细胞肺癌瘤体增长,且与剂量呈反比；高剂量组瘤体HE染色出现细胞密度降低、细胞皱缩等典型凋亡形态学增多；WB结果表明,与空白组相比,蟾毒它灵给药组EGFR及Bcl-2/Bax蛋白表达下调(P＜0.01)(3)网络毒理学共获得201个蟾毒它灵肾毒性潜在靶点；分子对接显示蟾毒它灵与TP53、TNF、AKT1、IL-6、STAT3靶点具有较好的结合活性。(4)与对照组相比,各给药组TP53、TNF和IL-6表达显著上调(P＜0.01)。结论 蟾毒它灵在可能通过抑制EGFR表达抑制非小细胞肺癌增殖、迁移及抑制非小细胞肺癌发展,且其可能通过调控TP53、TNF、AKT1、IL-6、STAT3发挥一定的肾毒性。

Abstract:

ABSTRACT Objective: This study aimed to evaluate the anticancer effects of bufalin on non-small cell lung cancer (NSCLC) cells and in a nude mouse model, as well as to investigate the nephrotoxic potential of bufalin and elucidate its underlying mechanisms. Methods: (1) In vitro assays: A549 NSCLC cells were exposed to varying concentrations of bufalin (0, 100, 150, 200, 250, 300, 350, 400, and 450 nM) for 24, 48, and 72 hours. Cell proliferation was quantified using the CCK-8 assay. Cell migration was assessed via wound healing assays following treatment with 100, 200, and 300 nM bufalin. (2) In vivo studies: A nude mouse xenograft model was established by subcutaneous injection of A549 cells into the right axillary region. Mice were randomly assigned to control, low-, medium-, and high-dose bufalin groups (n=6 per group). Body weight and tumor inhibition rates were monitored. Histopathological examination of tumor and kidney tissues was performed using hematoxylin and eosin (HE) staining. Serum biomarkers indicative of renal injury were measured. Western blot analysis was conducted to determine the expression levels of EGFR, Bax, and Bcl-2 in tumor tissues. (3) Network toxicology and molecular docking: Potential nephrotoxicity-related targets of bufalin were predicted utilizing the Comparative Toxicogenomics Database (CTD). Nephrotoxicity-associated targets were retrieved from GeneCards and OMIM databases, and overlapping targets were identified. A protein-protein interaction (PPI) network was constructed for these intersecting targets, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Key targets were subjected to molecular docking and visualization studies. (4) Quantitative real-time PCR (qRT-PCR) was employed to validate the nephrotoxicity-related targets predicted by network toxicology. Results: (1) The CCK-8 assay demonstrated that bufalin inhibited proliferation of A549 cells in a dose- and time-dependent manner. Wound healing assays revealed a significant reduction in migration rates across all bufalin-treated groups compared to controls (P < 0.01). (2) In vivo, bufalin administration resulted in a dose-dependent suppression of tumor growth in nude mice. HE staining of tumors from the high-dose group exhibited decreased cellular density and increased apoptotic morphological characteristics, including cell shrinkage. Western blot analysis showed significant downregulation of EGFR expression and a decreased Bcl-2/Bax protein ratio in bufalin-treated groups relative to controls (P < 0.01). (3) Network toxicology analysis identified 201 potential bufalin-associated nephrotoxicity targets. Molecular docking revealed strong binding affinities between bufalin and key targets such as TP53, TNF, AKT1, IL-6, and STAT3. (4) qRT-PCR results indicated significant upregulation of TP53, TNF, and IL-6 expression in all bufalin-treated groups compared to controls (P < 0.01). Conclusion: Bufalin exhibits inhibitory effects on proliferation, migration, and progression of non-small cell lung cancer, potentially through downregulation of EGFR expression. Furthermore, bufalin may induce nephrotoxicity via modulation of key molecular targets including TP53, TNF, AKT1, IL-6, and STAT3.