Yishen Qutong Granule Inhibits Bone Metastasis Caused By Lung Cancer Through CaN/NFAT/CXCL12 Signaling Pathway

Research 1:A discussion on the rationality of the use of Yishen Qutong Granules and a dissection of the therapeutic theory and methodObjective 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 pathwayObjective 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 downregulating 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 protein expression levels during differentiation of lung cancer cells and osteoclasts 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 cancerObjective 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 metaanalysis 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.