从TLR4/NF-kB信号通路探讨中医扶正解毒抑制乳腺癌肺转移的作用机制

T Toll-like receptor 4(TLR4) is an important molecule in the NF-κB signaling, which expresses highly in diverse cancers and is associated closely with immunosuppression and neoplasm invasiveness in tumor microenvironment.The past study shows the mechanism of inhibiting the breast cancer cell invasion by "Fuzhengjiedu" therapeutic principles of traditional Chinese medicine may be related to down-regulating the gene expressions of VEGF and MMP-9.The purpose of this study is to explore the mechanisms of the effects of "Fuzhengjiedu" therapeutic principles of TCM in anti-metastasis of breast cancer .In vivo ,the female BALB/c mice will be randomly divided into normal control group,CTX-treated group, Chinese herbal medicine "Fuzhengjiedu"group and its decomposed formulas "Fuzheng" Recipe and "Jiedu" Recipe groups .The animal model will be established by injecting mice breast cancer cells 4T1 into the subcutaneous of nipples of BALB/c mice. The mice in different groups will be administered with corresponding drugs and their breast tumor, lung metastases of breast cancer and angiogenesis in tumor will be evaluated. In vitro,SD female rats will be randomly divided into "Fuzhengjiedu"group and its decomposed formulas "Fuzheng" Recipe and "Jiedu" Recipe groups,CTX-treated group and normal control group to make medicated serums. Methods of MTT, matrigel basement membrane will be employed to investigate the ability of generation and invasion of 4T1 cells after these cells will be treated with the medicated serums. Methods of real time polymerase chain reaction, immunohistochemisty and immunoblotting will be employed to investigate the relevant gene expressions of TLR4/NF-kB signaling ,such as TLR4、NF-κB、MMP-9、VEGF、CXCR4. The result of the research will reveal different target points of different therapeutic principles of TCM. This research will provide new ideas of anti- metastasis of breast cancer by TCM.

脂多糖受体4（TLR4）是核转录因子-κB（NF-κB）信号通路上游的重要分子，在多种癌组织中表达增强，且与免疫抑制、肿瘤微环境和侵袭转移密切相关。我们既往报道的临床及体内外实验研究表明扶正解毒中药可部分通过抑制VEGF、降低MMP-9发挥抗乳腺癌作用。本课题拟从TLR4/ NF-κB信号通路进一步探讨扶正解毒中药预防乳腺癌远处转移的作用机制。通过建立自发性小鼠乳腺癌肺转移移植瘤模型，研究扶正、解毒单用及合用对体内瘤体生长、转移、细胞增殖及血管生成的影响；制备扶正、解毒及其合剂药物血清，研究对体外乳腺癌细胞生长、侵袭能力的影响；应用定量PCR技术结合免疫组化、免疫印迹检测中医扶正解毒法在抑制体内乳腺癌肺转移及抑制体外培养乳腺癌细胞的过程中对TLRs/NF-κB信号通路及其下游信号分子的影响。研究结果将不仅为扶正与解毒中药治疗乳腺癌的机制提供理论依据，也为中药治疗乳腺癌转移提供新的干预靶点

课题立足于“养正积自除”治疗乳腺癌的理论，以扶正解毒代表方剂乳癌术后方及其拆方（扶正方、解毒方）作为研究对象，对乳腺癌肺转移开展了一系列的研究：首先，采用水提醇沉和血清药理学两种方法制备乳癌术后方水提醇沉剂和大鼠药物血清，以化疗药物环玲酰胺为对照组，对乳癌术后方全方、扶正方、解毒方对4T1细胞增殖的影响进行研究，并从与乳腺癌免疫抑制、肿瘤微环境和侵袭转移密切相关的TLR4/NF-κB通路着手, 寻找乳癌术后方抑制乳腺癌肺转移的分子靶点。研究结果发现：乳癌术后方全、扶正方、解毒方的水提醇沉剂能抑制乳腺癌4T1细胞的增殖，对4T1细胞增殖抑制作用随药物浓度的增大而增大，且随时间的延长而增大。相同浓度下，解毒组抑制率>全方组>扶正组。其中，乳癌术后方全方、扶正方、解毒方的药物血清的研究未得到稳定、可重复的研究结果。在体内实验方面，通过对自发性小鼠乳腺癌肺转移移植瘤裸鼠的研究，发现移植瘤的瘤体积、肺转移抑制率全方组>解毒组>扶正组。根据体内与体外实验结果的差异，我们认为乳癌术后方对乳腺癌肺转移的抑制作用可能是通过调节TLR4/NF-κB信号通路中磷酸化的NF-κB蛋白（p-NF-κB）及磷酸化的IκB蛋白（p-IκB）平衡实现的，另外，乳癌术后方还通过Th1/Th2平衡、增强NK细胞活性发挥抗肿瘤及抑制肺转移作用。