研究1：益肾祛痛颗粒用药合理性讨论及理法剖析

目的 探讨益肾祛痛颗粒中中药组成的合理性。方法 检索国家知识基础设施数据库(CNKI)、中国学术期刊数据库(CSPD)和PubMed数据库中中药治疗恶性肿瘤骨转移的临床研究或经验探析。将所用处方中的药物建立数据库，通过对药物、性味归经的频数分析、SPSS聚类分析及Clementine关联规则分析，探索用药规律。结果 共纳入研究144项，获得治疗恶性肿瘤骨转移的中药处方150首，中药198味。药物频数分析中熟地黄、补骨脂、甘草、骨碎补、黄芪等应用最多；性味频数分析结果显示，温性药、甘味、苦味及辛味药使用较多；归经以肝、肾两经最多；聚类分析得到药对2对，3味药药组1组，聚类方1首；关联规则分析结果以补骨脂，当归—骨碎补等置信度最高。益肾祛痛颗粒在以上各方面的用药规律与其他150首处方基本一致；在药物归经频数分析中，益肾祛痛颗粒用药以归于肾经的药物最多。结论 益肾祛痛颗粒组方在符合中医学对于恶性肿瘤骨转移治疗的普遍认知的前提下，又创新性基于“肾主骨生髓”理论指导药物配伍，是组方合理的有效治疗药物。

研究2：益肾祛痛颗粒通过CaN/NFAT/CXCL12信号通路抑制肺癌骨转移

目的 研究益肾祛痛颗粒及其扶正、解毒和化瘀拆方对肺癌细胞表型及破骨细胞分化成熟的影响。同时，初步验证益肾祛痛颗粒通过下调肺癌细胞、破骨细胞及其前体中的钙调神经磷酸酶/活化T细胞核因子（Calcineurin/Nuclear factor of activated T cells, CaN/NFAT）信号通路，抑制CXCL12的释放，进而阻断肺癌骨转移形成。方法 首先，采用益肾祛痛颗粒及各拆方大鼠含药血清，通过体外细胞功能实验，细化研究该处方及拆方对Lewis肺癌细胞增殖、迁移及对骨基质黏附能力等肿瘤细胞在骨转移微环境中形成转移灶所涉及的表型的影响。其次，在体外通过RAW264.7细胞诱导分化破骨细胞，并采用抗酒石酸酸性磷酸酶（TRAP）染色观察该处方及拆方对破骨细胞分化成熟的影响。最后，通过Western-blot检测各组含药血清干预后对肺癌细胞、破骨细胞及其前体中CaN、NFAT及CXCL12在蛋白表达水平的影响。结果 益肾祛痛颗粒大鼠含药血清在体外抑制了Lewis肺癌细胞的增殖；该处方及各拆方组在体外显著抑制了Lewis细胞的迁移、黏附能力及对骨基质的黏附。对Lewis细胞的Western-blot检测显示，益肾祛痛颗粒及各拆方均显著下调了肺癌细胞中CaN、NFAT及CXCL12的表达。对经RAW264.7细胞诱导分化破骨细胞的TRAP染色结果显示，益肾祛痛颗粒显著抑制了破骨细胞分化成熟。对破骨细胞分化成熟过程中的Western-blot检测显示，益肾祛痛颗粒及各拆方均显著下调了破骨细胞及其前体中CaN、NFAT及CXCL12的表达。结论 益肾祛痛颗粒通过调控肺癌细胞、破骨细胞及其前体中的CaN/NFAT信号通路，抑制细胞中CXCL12的表达及释放，从而降低骨转移微环境中趋化因子浓度，阻断CXCR4⁺肺癌细胞的远程趋化，起到治疗骨转移的目的。同时，潜在地经由此通路，干预肺癌细胞的增殖、迁移、黏附及破骨细胞的分化成熟。

研究3：益肾祛痛颗粒在肺癌原发灶治疗中的有效成分研究

目的 研究益肾祛痛颗粒对肺癌原发灶的治疗效果、尝试识别其关键活性药物成分并探讨潜在的作用机制。方法 首先通过裸鼠异种移植瘤模型及体外细胞功能实验，探讨益肾祛痛颗粒在肺癌原发灶中的疗效。随后，采用高分辨液质联用（HPLC-MS）检测该药中的推定化合物成分，并基于公共数据库对这些化合物成分进行作用靶点的预测。之后，通过对肿瘤组织的mRNA测序对预测靶点中实际发生了变化的部分进行识别，采用富集分析挖掘潜在的作用机制并筛选核心基因。在对核心基因在蛋白水平的表达进行验证后，采用分子对接方法对推定化合物成分与靶点间的直接结合进行预测。最后对候选化合物进行荟萃分析，判断候选化合物的潜在有效性。结果 实验证实益肾祛痛颗粒显著抑制了肺癌细胞的克隆形成及在体内病灶的生长，同时还诱导了肺癌细胞凋亡。HPLC-MS、mRNA测序及富集分析显示氧化应激相关信号通路是益肾祛痛颗粒在肺癌原发灶治疗中的潜在作用机制。Western-blot检测显示血红素加氧酶-1（heme oxygenase-1, HMOX1）是作用靶点，分子对接和荟萃分析提示染料木素（Genistein）和槲皮素（Quercetin）是益肾祛痛颗粒的关键活性药物成分。结论 益肾祛痛颗粒及其中的活性药物成分染料木素和槲皮素，潜在地通过氧化应激相关靶点HMOX1起到对肺癌原发灶的治疗作用。

Research 1: A discussion on the rationality of the use of Yishen Qutong Granules and a dissection of the therapeutic theory and method

Objective Exploring the rationality of the composition of the Chinese herbal medicine in Yishen Qutong Granules (YSQTG). Methods Search the Chinese database CNKI and Wanfang, English database Pubmed, Including clinical studies or empirical analysis of Chinese medicine for bone metastasis and bone cancer pain. Establish a database of prescription drugs through Excel, and use frequency analysis, SPSS cluster analysis, and Clementine association rule analysis to explore the regularity of traits, tastes, and meridians of Chinese medicine. Results A total of 144 studies were included. We obtained 150 prescriptions and 198 herbs for the treatment of bone metastasis and bone cancer pain. In the analysis of drug frequency, prepared rehmannia root, fructus psoraleae, liquorice, drynaria rhizome, astragalus membranaceus were used most, the most common property was mild ones, sweet, bitter and acrid flavour were the most common and attributive to the liver and kidney meridians. Two pairs of drugs, a group of three herbs and a clustering formula were obtained in Cluster analysis. The results of association rule analysis showed that psoralen and angelica sinensis-psoraleae had the highest confidence. The dosing pattern of YSQTG in all of the above aspects is generally consistent with the other 150 prescriptions. In the drug attribution frequency analysis, The herbs in YSTQG are most often found in drugs attributed to the kidney meridian. Conclusion Traditional Chinese medicine follows the principles of relieving phlegm, nourishing liver and kidney, relieving pain through collaterals and special drugs for specific diseases in the treatment of bone metastasis and bone cancer pain.

Research 2: Yishen Qutong Granule inhibits bone metastasis caused by lung cancer through CaN/NFAT/CXCL12 signaling pathway

Objective We investigated the effects of YSQTG and its recipe of Fu Zheng, Jie Du, and Hua Yu on the phenotype of lung cancer cells and the differentiation and maturation of osteoclasts. At the same time, we tried to preliminarily validate the effect of YSQTG on lung cancer cell phenotype and osteoclast differentiation and maturation by down-regulating the Calcineurin/Nuclear factor of activated T cells (CaN/NFAT) signaling pathway in lung cancer cells, osteoclasts, and their precursors, inhibiting the release of CXCL12 and thus blocking the formation of lung cancer bone metastasis formation. Methods First, the effects of this prescription and the recipe on the phenotype of Lewis lung cancer cells involved in the formation of metastases in the bone metastatic microenvironment, such as proliferation, migration, and adhesion to the bone matrix, were studied in detail in vitro cell function assays using the rats' drug-containing serum of YSQTG and each recipe. Secondly, differentiated osteoclasts were induced by RAW264.7 cells in vitro and the effect of this prescription and each recipe on the differentiation and maturation of osteoclasts was observed by anti-tartrate acid phosphatase (TRAP) staining. Finally, the effects of each group of drug-containing serum intervention on CaN, NFAT, and CXCL12 in lung cancer cells, osteoclasts, and their precursors at the protein expression level were examined by Western-blot. Results In vitro proliferation of Lewis lung cancer cells was inhibited by the rats' drug-containing serum of YSQTG; Meanwhile, the prescription and each recipe group significantly inhibited the migration, adhesion ability, and adhesion to the bone matrix of Lewis cells. Western blotting of Lewis cells showed that the expression of CaN, NFAT, and CXCL12 in lung cancer cells was significantly down-regulated by YSQTG and its recipe. TRAP staining of differentiated osteoclasts induced by RAW264.7 cells showed that YSQTG significantly inhibited the differentiation and maturation of osteoclasts. Western blotting of osteoclasts during differentiation showed that YSQTG and each recipe significantly down-regulated the expression of CaN, NFAT, and CXCL12 in osteoblasts and their precursors. Conclusion Through regulating the CaN/NFAT signaling pathway in lung cancer cells, osteoclasts, and their precursors, YSQTG inhibits the expression and release of CXCL12 in cells, reduces the concentration of chemokines in the microenvironment of bone metastasis, and blocks the remote chemotaxis of CXCR4⁺ lung cancer cells to treat bone metastasis. At the same time, YSQTG potentially interferes with the proliferation, migration and adhesion of lung cancer cells and the differentiation and maturation of osteoclasts via this pathway.

Research 3: Identification of the key active pharmaceutical ingredients of Yishen Qutong Granules, a Chinese medicine formula, in the treatment of primary lung cancer

Objective This study aimed to investigate the antitumor efficacy of Yishen Qutong Granules (YSQTG) in primary LC treatment, to identify its key active pharmaceutical ingredients (APIs), and to explore its possible mechanism of action. Methods The antitumor role of YSQTG was validated via cell function assays and a xenograft tumor model. Then, high-performance liquid chromatography-mass spectrometry (HPLC–MS) was performed to determine the objective precipitation components of YSQTG, followed by target prediction through reference to databases. Subsequently, the proportion of the predicted targets that underwent actual changes was identified via RNA-sequencing. Enrichment analysis was performed to explore the possible mechanisms of action. Hub genes were screened, and western blotting was used to verify their protein expression levels to identify the core target. Molecular docking between the active compounds and the verified core target was performed, combined with an evaluation of the potential efficacy of candidate compounds using meta-analysis to screen the candidate key APIs. Results Experiments confirmed that YSQTG could inhibit LC cell clone formation, induce apoptosis in vitro, and inhibit lung tumor growth in vivo. HPLC-MS, RNA-seq, and enrichment analysis showed that oxidative stress-related pathways were the possible mechanism of YSQTG in primary LC treatment. Western blot verification indicated that heme oxygenase 1 (HMOX1, HO-1) could be the core target. Molecular docking and meta-analysis suggested that genistein and quercetin were the candidate key APIs. Conclusions YSQTG and its active ingredients, genistein and quercetin, may have therapeutic effects against LC through their action on the downregulation of oxidative stress-related HMOX1 protein expression.