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Objective To investigate the role of bufalin(BU)in inhibiting M2-type macrophage-mediated colorec-tal cancer metastasis.Methods Human acute leukemia mononuclear cells(THP-1)were differentiated into M0 macrophages using phorbol ester induction(PMA)for 48 hours.The M0 macrophages were then treated with IL-4 and IL-13 medium.Surface markers and morphological changes were observed through ELISA,morphology,and RT-qPCR experiments.RT-PCR and ELISA experiments were conducted to detect the surface markers TGF-β and IL-10 of M2 macrophages.The secretion level of IL-6 in the supernatant of M2 macrophages and colorectal cancer cells HCT116 was compared using ELISA.Additionally,the effect of conditioned medium on colorectal cancer cell HCT116 was assessed through Transwell,Wound healing,RT-qPCR,and Western blot experiments.Subsequent-ly,bufalin was added to the conditioned medium and the changes in AKT/PI3K protein,migration,and epithelial-mesenchymal transition ability in HCT116 were observed using Western blot,Transwell,Wound healing and RT-qPCR experiments.Results THP-1 were successfully differentiated into M2 macrophages.The activation of AKT/PI3K protein in HCT116 cells was induced by the secretion of IL-6 from M2 macrophages,which in turn promoted the migration and epithelial-mesenchymal transition ability of the HCT116 cells.The migration and epithelial-mes-enchymal transition mediated by M2 macrophages in HCT116 cells were effectively inhibited by Bufalin.Conclu-sion The release of IL-6 from M2 macrophages activates the AKT/PI3K signaling pathway in colorectal cancer cells,thereby promoting their migration and epithelial-mesenchymal transition capacity.Moreover,bufalin exhibits inhibitory effects on this effect.
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ObjectiveTo investigate whether Xiayuxue decoction exerts an anti-liver fibrosis effect by inhibiting glial cell line-derived neurotrophic factor (GDNF). MethodsA total of 24 C57BL/6 mice were randomly divided into control group, model group, and Xiayuxue decoction group. The mice in the model group and the Xiayuxue decoction group were given intraperitoneal injection of 10% CCl4, and those in the Xiayuxue decoction group were given 0.4678 g/kg Xiayuxue decoction by gavage since week 4. The liver function parameters alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, and liver histopathology was observed. Immunohistochemistry was used to measure the protein expression of alpha-smooth muscle actin (α-SMA) and GDNF. GFP-Col-HSC and human primary hepatic stellate cells (HSCs) were treated with GDNF (10 ng/ml), and HSC activation was measured. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the control group, the model group had significant increases in the levels of ALT and AST, and compared with the model group, the Xiayuxue decoction group had significant reductions in the levels of ALT and AST (all P＜0.01). Liver histopathology showed that the model group had marked inflammatory cell infiltration and formation of fibrous septa by proliferated collagen fibers, and the Xiayuxue decoction group had loose fibrous septa and alleviated inflammatory cell infiltration. Immunohistochemistry showed that compared with the control group, the model group had significant increases in the expression of α-SMA and GDNF (both P＜0.01), which were observed in fibrous septa, and compared with the model group, the Xiayuxue decoction group had significant reductions in the expression of α-SMA and GDNF (both P＜0.05). Western blotting showed that the control group had relatively low expression of GDNF in liver tissue, the formation of liver fibrosis at week 6 of CCl4 modeling, and an around 10-fold increase in the expression of GDNF, and the Xiayuxue decoction group had significantly inhibited protein expression of GDNF (P＜0.01); there were significant increases in the expression of α-SMA and collagen type I α1 (Col1) in mice with liver fibrosis, with significant reductions in α-SMA and Col1 after treatment with Xiayuxue decoction (all P＜0.01). The in vitro experiment showed that GDNF induced the significant increases in the protein expression of α-SMA and Col1 in HSCs, which was significantly inhibited by Xiayuxue decoction (all P＜0.01). ConclusionThe expression of GDNF is significantly upregulated in the formation of liver fibrosis. GDNF can induce HSC activation, and Xiayuxue decoction can exert an anti-liver fibrosis effect by inhibiting GDNF.