| 109 | 0 | 69 |
| 下载次数 | 被引频次 | 阅读次数 |
目的 探讨小檗碱(BBR)在慢性淋巴细胞白血病(CLL)中的作用，明确其是否通过直接靶向并抑制Src家族激酶Lck/Yes新型酪氨酸激酶(Lyn),进而诱导白血病细胞凋亡发挥抗肿瘤作用。方法 采用分子对接技术预测BBR与Lyn激酶的结合潜力；通过生物素下拉(Pull-down)实验验证二者的直接相互作用。体外实验使用人慢性B细胞白血病细胞系(MEC-1),通过CCK-8、 TUNEL染色检测细胞活力与凋亡；通过Western blot法检测B细胞受体(BCR)通路关键蛋白Lyn、脾酪氨酸激酶(Syk)、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B(AKT)及凋亡指标B细胞淋巴瘤2基因(Bcl2)相关X蛋白(BAX)、 Bcl2相关细胞死亡激动剂(BAD)、切割型半胱天冬酶3(c-caspase-3)、 Bcl2表达；利用实时定量PCR检测下游基因细胞周期蛋白D1(Cyclin D1)、 Bcl2、骨髓细胞瘤病毒癌基因同源物(c-Myc)转录水平。通过构建Lyn慢病毒过表达稳转株进行功能挽救实验。体内通过尾静脉注射构建C-NKG小鼠白血病模型，采用HE染色评估脾、肝、肺的肿瘤浸润，通过生存分析评估BBR疗效。结果 体外实验显示，BBR浓度依赖性地抑制MEC-1细胞增殖并诱导凋亡，同时抑制BCR通路蛋白磷酸化及下游基因表达。分子对接与Pull-down实验证实BBR可直接结合Lyn。Lyn过表达可逆转BBR诱导的细胞凋亡及对通路的抑制。体内实验表明，BBR治疗能显著减轻白血病小鼠的器官浸润并延长其生存期，此效应可被Lyn过表达抵消。结论 BBR通过直接靶向Lyn激酶，抑制BCR-Lyn-PI3K-AKT信号通路，从而在体内外诱导CLL细胞凋亡、抑制肿瘤进展。本研究为将BBR作为靶向Lyn的天然药物治疗CLL提供了实验依据。
Abstract：Objective To investigate the role of berberine(BBR) in chronic lymphocytic leukemia(CLL) and to determine whether it exerts anti-tumor effects by directly targeting and inhibiting Lck/Yes tyrosine kinase(Lyn), a novel Src family kinase, thereby inducing leukemia cell apoptosis. Methods Molecular docking was employed to predict the binding potential between BBR and Lyn kinase, and biotin pull-down assay was conducted to validate their direct interaction. In vitro experiments utilized the human chronic B-cell leukemia cell line MEC-1, with cell viability and apoptosis assessed via CCK-8 and TUNEL staining, respectively. Key proteins in the B-cell receptor(BCR) pathway, including Lyn, spleen tyrosine kinase(Syk), phosphatidylinositol 3-kinase(PI3K), protein kinase B(AKT), and apoptosis-related markers Bcl2-associated X protein(BAX), Bcl2-associated agonist of cell death(BAD), cleaved caspase-3(c-caspase-3), and B-cell lymphoma 2(Bcl2), were analyzed by Western blot. Transcriptional levels of downstream genes, including cyclin D1(Cyclin D1), Bcl2, and myelocytomatosis viral oncogene homolog(c-Myc), were quantified using real-time quantitative PCR. Functional rescue experiments were performed using Lyn-overexpressing lentiviral stable cell lines. In vivo, a C-NKG mouse leukemia model was established via tail vein injection, with tumor infiltration in the spleen, liver, and lungs evaluated by HE staining, and therapeutic effect of BBR assessed by survival analysis. Results In vitro, BBR inhibited MEC-1 cell proliferation in a concentration-dependent manner and induced apoptosis, while suppressing the phosphorylation of BCR pathway proteins and downstream gene expression. Molecular docking and pull-down assays confirmed the direct binding between BBR and Lyn. The overexpression of Lyn reversed BBR-induced apoptosis and pathway inhibition. In vivo, BBR treatment significantly reduced organ infiltration and prolonged survival in leukemic mice, which can be reversed by Lyn overexpression. ConclusionBBR induces CLL cell apoptosis and inhibits tumor progression in vitro and in vivo by directly targeting Lyn kinase and suppressing the BCR-Lyn-PI3K-AKT signaling pathway. These findings provide experimental evidences supporting BBR as a natural Lyn-targeted therapeutic agent for CLL.
[1] 杨蕊雪，曾雪娇，曲建华.慢性淋巴细胞白血病中2型固有淋巴细胞通过IL-9对Treg及CD8+T细胞功能的影响[J].细胞与分子免疫学杂志，2025,41(8):673-679.
[2] 王向丽，田思锐，吴涛.B细胞受体及其相关信号通路在慢性淋巴细胞白血病中的研究进展[J].中国病理生理杂志，2025,41(9):1847-1854.
[3] Jain N,Wierda W G,O'Brien S.Chronic lymphocytic leukaemia[J].Lancet,2024,404(10453):694-706.
[4] Fiske B E,Wemlinger S M,Crute B W,et al.Lyn governs the establishment and maintenance of B cell anergy by suppressing PI3K signaling[J].Nat Commun,2026,17(1):3660.
[5] Muhowski E M,Ravikrishnan J,Gordon B,et al.Preclinical evaluation of combination nemtabrutinib and venetoclax in chronic lymphocytic leukemia[J].J Hematol Oncol,2022,15(1):166.
[6] Khan A,Taiyab A,Hasan G M,et al.Structural and functional insights into Lyn kinase:from immune regulation to therapeutic targeting[J].J Cell Biochem,2026,127(1):e70082.
[7] Patton J T,Woyach J A.Targeting the B cell receptor signaling pathway in chronic lymphocytic leukemia[J].Semin Hematol,2024,61(2):100-108.
[8] Abdollahi M,Mohammadlou M,Maral H,et al.Anti-tumor effect of berberine on chronic lymphocytic leukemia cells[J].Med Oncol,2022,39(12):217.
[9] Han Y,Guo S,Li Y,et al.Berberine ameliorate inflammation and apoptosis via modulating PI3K/AKT/NF-κB and MAPK pathway on dry eye[J].Phytomedicine,2023,121 :155081.
[10] 樊翠，张娅薇，杨蕊，等.LAIR-1通过阻断JAK2 V617F突变的人HEL细胞JAK/STAT和PI3K/AKT/mTOR信号通路抑制其增殖并促进其凋亡[J].细胞与分子免疫学杂志，2024,40(3):207-214.
[11] 李晓婉，张莉，封祖希，等.BTLA/HVEM轴在血液肿瘤中的表达及免疫靶点治疗前景[J].细胞与分子免疫学杂志，2025,41(1):64-70.
[12] Buchner M.Insights into PI3K/AKT signaling in B cell development and chronic lymphocytic leukemia[J].FEBS Lett,2025,599(20):2896-2910.
[13] Zygmunciak P,Robak T,Puła B.Treatment of double-refractory chronic lymphocytic leukemia-an unmet clinical need[J].Int J Mol Sci,2024,25(3):1589.
[14] Feng Y,Wei Z,Li R,et al.Effects and mechanism of berberine in ameliorating microglia-mediated hypothalamic inflammation by downregulating the MAPK and NF-κB pathway[J].J Ethnopharmacol,2026,355(Pt B):120695.
[15] Li J,Dan W,Zhang C,et al.Exploration of berberine against ulcerative colitis via TLR4/NF-κB/HIF-1α pathway by bioinformatics and experimental validation[J].Drug Des Devel Ther,2024,18:2847-2868.
[16] Liu D,Li L,Zhang J,et al.Berberine promotes apoptosis and inhibits the migration of oral squamous carcinoma cells through inhibition of the RAGE/PI3K/AKT/mTOR pathway[J].Biomed Pharmacother,2025,187:118147.
[17] Sun A,Yang H,Li T,et al.Molecular mechanisms,targets and clinical potential of berberine in regulating metabolism:a review focussing on databases and molecular docking studies[J].Front Pharmacol,2024,15:1368950.
[18]Alizad G,Hemati M,Bagheri B,et al.Investigating the impact of berberine on autophagy-mediated drug resistance in chronic lymphocytic leukemia tumor cells[J].Med Oncol,2025,42(7):240.
[19] Li J,Yang L,Shen R,et al.Self-nanoemulsifying system improves oral absorption and enhances anti-acute myeloid leukemia activity of berberine[J].J Nanobiotechnology,2018,16(1):76.
[20] Sun P,Wang Z,Ma Y,et al.Advance in identified targets of berberine[J].Front Pharmacol,2025,16:1500511.
[21]Zhou W,Asif A,Situ C,et al.Multiple target and regulatory pathways of berberine[J].Phytomedicine,2025,146:157030.
[22] Vervoordeldonk M Y L,Hengeveld P J,Levin M D,et al.B cell receptor signaling proteins as biomarkers for progression of CLL requiring first-line therapy[J].Leuk Lymphoma,2024,65(8):1031-1043.
[23] Sobczyńska-Konefał A,Monika J,Karabon L,et al.Insights into genetic aberrations and signalling pathway interactions in chronic lymphocytic leukemia:from pathogenesis to treatment strategies[J].Biomark Res,2024,12(1):162.
[24] Faust J R,Hamill D,Kolb E A,et al.Mesothelin promotes acute myeloid leukemia progression through LYN-dependent signaling[J].J Biol Chem,2026,302(5):111390.
[25] Wosniaki D K,Kusma B N,Marin A M,et al.The GAB2 network in action:unveiling GAB2,GRB2,PIK3R1,and LYN in chronic myeloid leukemia[J].Hematol Transfus Cell Ther,2025,47(S3):104462.
[26] Song Y,Zhou K,Jing H,et al.Phase 1/2 studies of DZD8586,a non-covalent BBB penetrant LYN/BTK dual inhibitor,in BTK inhibitor resistant chronic lymphocytic leukemia (CLL) and other B-cell non-Hodgkin lymphoma (B-NHL)[J].Blood,2024,144(Suppl 1):3248.
基本信息:
DOI：10.13423/j.cnki.cjcmi.010143
中图分类号:R733.7
引用信息:
[1]巩宏涛,王业生,马若巾,等.小檗碱通过靶向Lyn抑制BCR通路诱导慢性淋巴细胞白血病B细胞凋亡的机制研究[J].细胞与分子免疫学杂志,2026,42(07):577-585.DOI:10.13423/j.cnki.cjcmi.010143.
基金信息:
河南省医学科技攻关计划联合共建项目(LHGJ20250400)
2026-04-27
2026-04-27
2026-04-27