Polarization Of TAMs Based On ?-adrenergic Pathway Regulation: Xiaoyao Powder Inhibits Metastasis Of Colon Cancer

Objective: The previous animal experiment of the research group found that Xiaoyao Powder can inhibit liver metastasis of colon cancer caused by liver qi stagnation,and can reduce the levels of serum adrenaline and norepinephrine and reduce the degree of macrophage infiltration in tumor tissues.Based on the theoretical knowledge of traditional Chinese medicine,this topic focuses on the use of mouse colon cancer in situ tumor model,from the perspective of in vivo and in vitro experiments,using flow cytometry,co-immunoprecipitation,immunofluorescence co-localization,immunohistochemistry,LDH,etc.Xiaoyao San plays a role in the regulation of human colon cancer metastasis by tumor-associated macrophages(TAMs);it is clear that Xiaoyao Powder regulates the mechanism of TAMs inhibiting colon cancer metastasis through ?-adrenergic signaling pathway,and verifies that Xiaoyao Powder increases the polarization of M1 macrophages.It promotes the release of immune factors by macrophages,increases the activity of macrophages to kill tumor cells,and clarifies the mechanism by which polarized macrophages inhibit tumor metastasis.Methods: 1.The mouse tail vein metastasis model was used to record the body weight of the mice to observe the quality of life.The tumor metastasis of the tumor model mice was detected by small animal in vivo imaging technique,and the effect of Xiaoyao Powder on colon cancer metastasis was observed.The mice were detected by FACS.Expression of macrophage surface molecules CD86 and F4/80 in blood and spleen 2.PMA was used to induce human mononuclear cell line THP-1 into M0 macrophages,and then select appropriate concentrations of interferon-?(IFN-?)and lipopolysaccharide(LPS)to induce M0 macrophages into M1 macrotypes.Phagocytosis;MTT assay was used to detect the effect of sputum-treated macrophages on the activity of tumor cells;RT-PCR was used to detect inducible nitric oxide synthase(i NOS)and tumor necrosis factor-?(TNF-?)gene expression in M1 macrophages;ELISA detects secretion of cytokines such as IL-6 and TNF-?;3.The expression of ?-adrenergic receptors in THP-1,M0 and M1 was examined by PCR.The cytotoxicity assay was used to observe the regulation of the killing effect of ?-adrenergic receptor agonists and inhibitors on M1 macrophages.Detection and expression of M1 macrophage marker TNF-? by PCR and ELISA.Lentiviral vector-mediated RNA interference technology was used to silence the ADRB2 gene of THP-1 cells,and the knockout efficiency was verified by fluorescence microscopy,PCR and flow cytometry.The M1 macrophage marker Gene expression was detected by PCR.4.PCR was used to detect the expression of ADRB2 gene in the tumors of Xiaoyao San and normal saline mice,and the expression of ADRB2 gene in M1 macrophages after Xiaoyao San.The MTT cell activity test was used to observe whether Xiaoyao San can reverse ? adrenergic receptors.Inhibition of M1 macrophage killing by agonists,using flow cytometry to detect the decrease of M1 macrophage ratio after Xiaoyao San can reverse the agonist effect of ?-adrenergic receptor.Flow cytometry and ELISA were used to detect the change of the effect of Xiaoyao Powder on M1 macrophages and the secretion of labeled cytokines after ADRB2 gene silencing.PCR was used to detect whether Xiaoyao San affects the expression of ADRB2 receptor in THP-1 cells.Fluo4 staining to detect whether Xiaoyao San affects the activation of ADRB2 receptor in THP-1 cells.PCR was used to detect the expression of downstream signaling pathway gene after ADRB2 gene silencing;Xiaoyao San acted on M1 macrophages with ADRB2 gene silencing,and detected the activation of downstream signaling pathway.Results: 1.In vivo imaging showed that the colon cancer metastasis of Xiaoyao group was significantly decreased,and the fluorescence intensity of the tumor was significantly lower than that of the saline control group(P<0.05).The results of flow cytometry showed that the spleen and blood of Xiaoyao San mice were in the blood.M1 macrophages were significantly elevated(P<0.001;P<0.01).2.The human peripheral blood mononuclear cell line THP-1 was successfully induced to differentiate into macrophages,and the optimal concentration of interferon-?(IFN-?)and lipopolysaccharide(LPS)was selected to establish an M1 macrophage model.The killing rate of M1 macrophages in the Xiaoyao group was significantly higher than that in the control group(P<0.05).The cytokines TNF-? and IL-6 secreted by M1 macrophages in Xiaoyao San group increased significantly.(P<0.001;P<0.01);the expression of TNF-? and INOS genes in M1 macrophages of Xiaoyao San group was significantly increased(P<0.05).3.The expression of the ?-adrenergic receptor gene ADRB2 was gradually decreased during the induction of THP-1 into M1 macrophages(P<0.05);the supernatant of M1 macrophages acting on the tumors in the adrenaline and isoproterenol groups The cell killing rate decreased(P<0.05;P<0.01);the expression of TNF-? in M1 macrophage of propranolol group was significantly increased(P<0.05),and the killing rate of tumor cells was increased(P<0.01).The expression and secretion of TNF-?,a marker of M1 macrophage,was significantly decreased after adrenaline and isoproterenol(P<0.001;P<0.01).The THP-1 cell lines of sh-ADRB2 and sh-CTRL were successfully constructed.The expression of M1 macrophage markers TNF-? and INOS was significantly increased after gene silencing(P<0.05).4.The expression of ADRB2 in the mice of Xiaoyao San group was significantly lower than that in the saline group(P<0.05).The expression of ADRB2 gene was significantly decreased after Xiaoyao San and M1 macrophages(P<0.05).Xiaoyao San and ?-adrenergic receptors were observed.The combination of agonists can significantly reverse the killing inhibition of agonists(P<0.05),and the combined effect significantly reverses the proportion of M1-type cells reduced by agonists.When M1 macrophages induced by THP-1,which was silenced by ADRB2 gene,there was no significant change in the proportion of M1-type macrophages,and there was no significant difference in the secretion of cytokines.Xiaoyao San significantly reduced the expression of ADRB2 gene in THP-1 cells and did not affect ADRB2 receptor activation.After the ADRB2 gene was silenced,the downstream PTEN/PI3K/AKT/RICTOR signaling pathway was activated,which promoted polarization(P<0.05).When Xiaoyao San acted on the unbroken cells of ADRB2 gene,it played the same role in promoting polarization(P<0.05),when Xiaoyao San acts on the MRB-type macrophage with ADRB2 gene silencing,its role in promoting polarization disappears.Conclusion:Xiaoyao Powder can significantly inhibit the proliferation and metastasis of colon cancer in mice.The realization is that Xiaoyao Powder can strengthen the polarization of M1 macrophages by down-regulating the expression of ADRB2 gene in ?-adrenergic signaling pathway,thereby enhancing killing and tumor suppression.