Molecular Mechanism Research On Sanguinarine Targeting Wnt/β-catenin Pathway In The Regulation Of Tumor-associated Macrophages Against Angiogenesis In The Lung Cancer

Objective: Tumor-associated macrophages(TAM)-mediated neovascularization is a prerequisite for lung cancer growth and metastasis.Therefore,targeting TAM to block its mediated angiogenesis is supposed to be a breakthrough in inhibiting the growth,invasion and metastasis of lung cancer.This study aims to investigate the effects of tumor-associated macrophages in regulation by Sanguinarine and the potential mechanism of antiangiogenesis in lung cancer.Methods: In the first part,the Lewis lung carcinoma model was established to evaluate the effect of Sanguinarine on tumor growth of transplanted tumor in mice,and the angiogenesis was observed by immunofluorescence technique in tissue sections.Secondly,mice macrophage clearance of Lewis lung carcinoma model was established for the antitumor angiogenesis effect of Sanguinarine after macrophage clearance.In the second part,a mouse bone marrow-derived macrophages model was constructed,and the co-culture system of macrophages and endothelial cells was further constructed to simulate the microenvironment of angiogenesis in vivo.Tubule formation assay,scratch assay,transwell assay and Western blot assay were used to observe the effect of Sanguinarine on macrophage-mediated tubule formation,endothelial cell proliferation and migration,endothelial cell marker CD31 and pro-vascular growth factor VEGF protein.In the third part,we first used the RNA-seq technique to screen for possible signaling pathways of Sanguinarine regulating macrophage polarization.Secondly,the mRNA expression of Wnt ligands in M2 macrophages before and after Sanguinarine treatment was detected by PCR and the protein expression of β-catenin,a key nuclear transcription factor,was detected by Western blot.Finally,we treated macrophages with the activator of β-catenin,a potential target,to observe the M2 polarization of macrophages again.After the intervention of β-catenin activator,macrophages were co-cultured with endothelial cells,and the effects of tubule formation,proliferation and migration of endothelial cells were observed by tubule formation experiment,scratch experiment and transwell experiment.Results: In the first part,the results showed that Sanguinarine inhibited the growth of transplanted tumor and angiogenesis in mice,and the anti-angiogenic effect of Sanguinarine is linked to the regulation of macrophage M2 phenotype.In the second part,the results showed that Sanguinarine could inhibit the polarization of M2-type macrophages and could inhibit the M2 macrophages-mediated formation of tubules,proliferation and migration of endothelial,and the high expression of VEGF.In the third part,the results showed that Sanguinarine regulates the Wnt /β-catenin signaling pathway to affect the M2 polarization of macrophages,mainly by down-regulating Wnt1 ligand、Wnt7b ligand and β-catenin,a key nuclear transcription factor.When β-catenin activator was applied to macrophages,the expression of CD206 was increased by Western blot.Macrophages treated with β-catenin activator co-cultured with endothelial cells to promote tubule formation,proliferation and migration of endothelial cells.While Sanguinarine can antagonize β-catenin activator and inhibit tubule formation,proliferation and migration of endothelial cells.Conclusion: Sanguinarine inhibits angiogenesis of lung cancer by preventing M2 macrophage polarization via targeting the Wnt/β-catenin pathway.