基于NF-κB/CaSR/PTHrP信号通路探讨扶正解毒法抑制肺腺癌骨转移的作用机制研究

Bone metastases significantly influence the prognosis of lung adenocarcinoma. Great attention has been given to the effect of tumor inflammatory microenvironment on the progression of lung adenocarcinoma. NF-κB is one of the most important transcriptional factors in response to tumor inflammatory microenvironment. The parathyroid hormone associated protein (PTHrP), which is one of the key molecules downstream of calcium-sensing receptor (CaSR), can promote tumor bone metastasis by inducing osteoclast maturation. Until now, there is still no report on whether the pathogenesis and development of bone metastasis of lung adenocarcinoma is related to NF-κB / CaSR / PTHrP pathway. It was indicated by our preliminary studies that CaSR may be involved in bone metastases in patients with lung adenocarcinoma. Besides, it was found that traditional Chinese medicine under the guidance of Fuzheng Jiedu can inhibit bone metastasis of lung adenocarcinoma and reduce the expression of CaSR in bone metastatic tissues in animal experiment. In this way, we speculated that the traditional Chinese medicine under the guidance of Fuzheng Jiedu decoction can inhibit the osteoblast differentiation and maturation by inhibiting the activation of NF-κB / CaSR / PTHrP signaling pathway. Thus preventing the occurrence and development of bone metastasis of lung adenocarcinoma. To confirm the aforementioned hypothesis, the effect of Fuzheng Jiedu traditional Chinese medicine on bone metastasis of lung adenocarcinoma will be carefully studied in vitro and in vivo, such as in vivo imaging and molecular biology, and the effect of Fuzheng Jiedu on lung adenocarcinoma Transfer basis. Finally, the resuls of our studies may provide basis for the treatment of bone metastasis of lung adenocarcinoma.

骨转移严重影响肺腺癌预后，炎性微环境与其发生发展密切相关。NF-κB是应答炎性微环境的重要因子。钙敏感受体（CaSR）下游的甲状旁腺激素相关蛋白（PTHrP）可通过诱导破骨细胞成熟而促进肿瘤骨转移。肺腺癌骨转移的发生发展是否涉及NF-κB/CaSR/PTHrP通路，未见报道。我们前期研究提示CaSR可能参与肺腺癌患者骨转移；体外模拟骨微环境，发现肺腺癌细胞CaSR的表达可能受NF-κB调控；动物实验发现扶正解毒中药可抑制肺腺癌骨转移并降低骨转移组织CaSR表达。据此，推测“扶正解毒法指导下的中药可通过抑制骨微环境对肿瘤细胞NF-κB/CaSR/PTHrP信号通路的激活，进而抑制破骨细胞分化及成熟，防止肺腺癌骨转移的发生与发展”。本课题拟通过体外试验和小鼠肺腺癌骨转移模型，采用活体成像、分子生物学等验证假设，探讨扶正解毒中药抑制肺腺癌骨转移的效应机制，为扶正解毒法治疗肺腺癌骨转移提供依据。

骨转移严重影响肺腺癌预后，炎性微环境与其发生发展密切相关。钙敏感受体（CaSR）是骨组织中重要的钙结合受体。CaSR的激活，可能与核因子κB(NF-κB)有关。当肿瘤细胞表面CaSR上调并激活后，可刺激肿瘤细胞释放甲状旁腺激素相关蛋白(PTHrP)。前期研究发现肺瘤平膏（FLP）能通过调控NF-κB抑制肺癌A549细胞的生长、侵袭和转移。由此本课题假设：肺瘤平膏可能通过干预NF-κB/CaSR/PTHrP信号通路诱导破骨细胞分化成熟，进而促进肺腺癌骨转移。为证实假设，本课题主要研究了骨微环境通过激活肺腺癌细胞NF-κB/CaSR/PTHrP信号通路促进破骨细胞分化成熟及肺腺癌骨转移；肺瘤平膏可通过抑制骨微环境对肺腺癌细胞NF-κB/CaSR/PTHrP信号通路的激活，进而抑制破骨细胞的分化成熟，最终防治肺腺癌骨转移的发生与发展。结果发现：肺癌患者肺癌组织中CaSR表达较癌旁组织及正常肺组织明显升高，骨转移患者肺癌组织中CaSR表达量高于骨未转移者的肺癌组织；CaSR过表达后，A549细胞的增殖和迁移能力显著提高，其对破骨前体细胞分化有直接的诱导作用，破骨基质降解相关酶的表达显著升高，而对成骨细胞却表现出显著的抑制，诱导成骨细胞成熟分化的相关酶也显著下调；同时，CaSR过表达后，NF-κB表达量增加，说明NF-κB与CaSR存在正向调控关系，促使下游信号分子PTHrP的表达增加，IL-11、IL-6、IL-8等破骨细胞分化因子的表达水平也显著提高，促进破骨细胞分化成熟。而当敲低CaSR的表达后，结果恰好相反。荧光染色结果及体外骨吸收实验结果显示，过表达CaSR的A549细胞能增加破骨细胞F-肌动蛋白环的形态和数量、牛骨切片上的骨吸收面积，而FLP能显著抑制这种变化。活体动物成像、Micro-CT及小鼠血液学结果表明，CaSR过表达能增加骨组织破坏程度、缩短生存时间，并增加血清中骨代谢相关指标的表达，而FLP能够减轻骨组织破坏程度并延长生存时间、显著提高BV/TV比值以及Tb.N数量，显著抑制小鼠肺腺癌骨转移的发生发展。初步证明具有高转移潜能的A549细胞中NF-κB与CaSR相互调控，诱导PTHrP表达，促使破骨细胞分化成熟，FLP能够抑制肺腺癌骨转移，这一作用可能是通过抑制CaSR/NF-κB/PTHrP通路，为中医药防治肺腺癌骨转移的临床应用提供有力的证据。