基于CaN/NFAT信号通路探讨扶正解毒化瘀法抑制溶骨性骨转移的机制研究

Bone metastasis is a common distant metastasis in malignant tumors affecting patient quality of life and overall survival. For this, traditional Chinese medicine therapy has a promising prospect. Our previous research proved that Yishen Gukang (YSGK), a patented self-made formula, based on the principle of Fuzheng Jiedu Huayu could significantly reduce the incidence of bone metastasis and relieve osteoclasis. Our pre-experiment indicated that YSGK Decoction could downregulate the expression of CaN, CXCL12 and CXCR4 to inhibit tumor cell chemotaxis and bone damage. On the basis of the above mentioned research, we focus on the CaN/NFAT signaling pathway and downstream molecules, and establish a tibial bone metastasis together with in vitro study. The mechanism of CaN/NFAT pathway in osteolytic bone metastasis will be verified using Western blotting, polymerase chain reaction, and pathway inhibitors. And the mechanism of osteolytic bone metastasis inhibited by YSGK Decoction will be clarified by micro computed tomography, tartrate-resistant acid phosphatase staining, and enzyme linked immunosorbent assay. The hypothesis will be confirmed from the two levels in vivo and in vitro: YSGK Decoction regulates CaN/NFAT signaling pathway by inhibiting the expression of CaN, reduces the expression of CXCL12, and inhibits the chemotaxis of tumor cells to bone matrix. Meanwhile, the formula down-regulates the expression of downstream molecules such as interleukin-6 and tumor necrosis factor-β, and suppress osteoclast differentiation. Both of them can inhibit osteolytic bone destruction. In this study, we expect to provide a new target for the inhibition of bone metastasis in traditional Chinese medicine therapy and enrich the scientific connotation of Fuzheng Jiedu Huayu theory.

骨转移是恶性肿瘤常见的远处转移,严重影响患者生活质量及生存期，中医药治疗前景广阔。前期研究证实以扶正解毒化瘀法为治则的专利方益肾骨康方可明显减少骨转移发生率及骨破坏；预实验提示益肾骨康方可下调CaN、CXCL12、CXCR4表达，抑制肿瘤细胞趋化及骨损害。在此基础上，我们以CaN/NFAT通路及下游分子为切入点，建立胫骨骨转移模型结合体外研究，利用蛋白印迹、PCR、通路抑制剂等技术验证CaN/NFAT通路在溶骨性骨转移中的作用机制；应用显微CT、TRAP及酶联反应等阐明益肾骨康方抑制溶骨性骨转移的作用机制。从体内外两个层面证实假说：益肾骨康方通过抑制CaN表达调节CaN/NFAT信号通路，减少CXCL12表达，抑制肿瘤细胞向骨基质趋化；同时下调下游分子IL-6、TNF-β表达，抑制破骨细胞分化；二者共同抑制溶骨性骨破坏，从而为中医抗骨转移治疗提供新靶点并丰富“扶正解毒化瘀”理论的科学内涵。

课题组前期研究证实，益肾骨康方（已更名益肾祛痛颗粒）能通过降低骨微环境中趋化因子CXCL12的浓度水平，一定程度上消除原发部位与骨微环境间的趋化因子浓度梯度，阻断骨微环境对CXCR4+肺癌细胞的招募和趋化，抑制骨转移形成。但是，有两个问题尚不明确：（1）益肾祛痛颗粒抑制骨转移微环境中何种细胞的CXCL12释放；（2）益肾祛痛颗粒通过何种信号通路抑制该种细胞CXCL12的释放。.本项目根据任务书计划，阐明了以上两个问题：（1）益肾祛痛颗粒能够抑制骨转移微环境中肺癌细胞及破骨细胞的CXCL12释放；（2）益肾祛痛颗粒通过调控肺癌细胞及破骨细胞中的CaN/NFAT信号通路，起到抑制CXCL12释放的作用。.本次研究首先在体内及体外层面验证了益肾祛痛颗粒在肺癌骨转移治疗中的疗效。随后采用mRNA测序、比较转录组学及验证实验，证实益肾祛痛颗粒能够干预肺癌细胞内的CaN/NFAT信号通路，抑制其CXCL12的释放。其次，对动物模型骨质的检测及体外细胞功能实验提示益肾祛痛颗粒还能影响破骨细胞的分化成熟；Western-blot等的结果证实益肾祛痛颗粒能够调控破骨细胞内的CaN/NFAT信号通路，抑制了破骨细胞CXCL12的释放。.以上结果表明，益肾祛痛颗粒通过干预骨转移微环境中肺癌细胞及破骨细胞内的CaN/NFAT信号通路，抑制这两种细胞的CXCL12释放，起到降低微环境CXCL12浓度水平，阻断CXCR4+肿瘤细胞趋化的作用。.此外，动物实验结果还显示，益肾祛痛颗粒能显著减小小鼠胫骨肿瘤的体积，提示其对肺癌病灶生长的潜在抑制作用。基于此，课题组通过克隆形成等细胞功能实验及裸鼠皮下移植瘤模型，证实益肾祛痛颗粒能通过下调HMOX1蛋白表达，显著抑制肺癌细胞增殖及病灶生长，并诱导细胞凋亡。随后，通过创新性结合预测手段及HPLC-MS等验证实验，初步识别出槲皮素及金雀异黄酮为益肾祛痛颗粒在肺癌原发灶治疗中的关键活性药物成分。.本项目形成的成果包括：SCI论文3篇；培养博士后3人，博士研究生2人；益肾祛痛颗粒相关成果荣获2022年北京市科技进步二等奖，并完成标的2000万元的科技成果转化。