基于LPS介导的TLR4/MyD88/NF-κB通路探讨协定Ⅰ号方治疗鼻咽癌的机制

Literature review and preliminary work shows that No.ⅠFormula attenuates lipopolysaccharide（LPS）-induced the development of nasopharyngeal carcinoma (NPC) responses by suppressing Toll-like receptor 4-nuclear factor kappa B(TLR4/NF-kB)signaling pathways. We investigate the mechanism of antitumor activity by the herbs. We establish NPC animal model to investigate the herbs sera pharmacological effect of NPC on the proliferation, cell cycle and apoptosis in NPC rat model and different NPC cell lines, by using MTT assays and flow cytometry. The expression of mRNA and protein were detected by realtime-PCR and western blot. The activity of NF-κB binding DNA was examined by dual luciferase assay. Furthermore, we investigate the possible effects and changes of mammalian target of TLR4 and its downstream signaling pathway in cells induced by herbs sera. The protein expression profiling in 5-8F cells was used by differential in-gel electrophoresis (DIGE) and MALDI-TOF-MS. The underlying mechanisms may be partly due to suppression of TLR4/NF-κB and its downstream signaling and associated protein. This study will excavate the classical theory of traditional Chinese medicine(TCM) and broadening the treatment system of modern medicine, also improve the innovation level of new high effective drugs on the basis of classical prescriptions in TCM. Integrative medicine will do obtain greater achievements in creating new medicine and pharmacology and make more tremendous contributions for the rejuvenation of TCM and NPC therapy.

前期临床及基础研究均显示：协定Ⅰ号方临床论治鼻咽癌疗效较好，且对炎症信号通路上的关键分子有明确抑制作用。故推断清热解毒法可通过抗炎而发挥抗鼻咽癌作用，其机制为抑制LPS介导的TLR4/NF-κB炎症通路。本课题拟从TLR4/MyD88/NF-κB通路探讨该方的作用途径及分子机制。1、抗肿瘤效应研究：建立鼻咽癌动物模型制备药物血清，并培养细胞株，研究组方对体内、体外肿瘤生长、增殖及凋亡的影响。2、探讨机制：应用Real-time PCR技术结合免疫组化、免疫印迹检测药物血清在体内、体外对该通路及相关因子转录活性的影响，明确组方作用途径。3、最后，应用蛋白指纹图谱检测药物血清对鼻咽癌5-8F细胞蛋白质表达谱的影响。本研究不仅为中药组方治疗鼻咽癌提供理论依据，清热解毒法治疗鼻咽癌提供实验证据, 也验证了中医药通过延缓炎症因子浸润，从而抑制“炎癌链”转化，最终发挥抗肿瘤作用的新思路。

鼻咽癌病机复杂，“正虚毒瘀”为核心，病理因素多为热毒、气滞、血瘀，通过因子分析法归纳中医证型，得出本病多为复合证型，病性多为虚实夹杂。本科室根据多年临床经验拟定协定Ⅰ号方共奏清热解毒，活血消癥之功符合中医基本理论。联合治疗在鼻咽癌中疗效肯定，前期研究结果认为中药联合治疗或可成为鼻咽癌综合治疗中极有优势的方法。申请人在前期国家青年基金的资助下，发现协定Ⅰ号方在鼻咽癌移植瘤模型上发挥与PI3K通路抑制剂相似的的治疗效果（SCI已发表）。并拓展PD1抗体联合协定Ⅰ号方的实验研究，发现联合治疗具有协同功效，显著增强抗肿瘤作用，增加分泌抗病毒分子的能力。前期基础：1.临床发现协定Ⅰ号方是治疗鼻咽癌的有效方剂；2.在鼻咽癌细胞及动物模型中该方具有明确的抗增殖促凋亡作用，并能通过磷脂酰肌醇3激酶(PI3K)通路抑制血管内皮生长因子(VEGF)表达，诱导肿瘤血管发生“正常化”改善肿瘤免疫微环境；3.协定Ⅰ号方联合程序性死亡配体-1(PDL1)抗体治疗鼻咽癌具有协同效应。综上推测协定Ⅰ号方通过多靶点多途径提高PDL1抗体在鼻咽癌治疗中的疗效，初步阐释协定Ⅰ号方联合PDL-1抗体提高鼻咽癌治疗疗效的分子机制及免疫学机制，为临床应用提供理论依据。