The Inhibition Of Anemoside B4 On Liver Cancer Through Notch Signaling Pathway And Its Mechanism

ObjectiveIn this study,HepG2 and Huh-7 cell models were used in vitro and a nude mouse xenograft tumor model was established in vivo to investigate the inhibition of Anemoside B4?AB4?,and explore its possible mechanism from the perspective of the Notch signaling pathway.Methods1.Hepatocellular carcinoma?HCC?cells were randomly divided into control group,AB4 administration groups and positive control group?DAPT group?.MTT and colony formation assays were used to detect the inhibition of AB4 on HCC cells.Annexin V-FITC/PI staining assay was used to detect the apoptosis of HCC cells after AB4administration.Western Blot assay was used to detect the expression of apoptosis-related proteins after AB4 administration.2.Tumor-bearing nude mice were randomly grouped into control group,AB4administration groups,and positive control group?DAPT group?after establishing a tumor-bearing nude mouse xenograft model to explore the inhibitory effect of AB4 in vivo on tumor-bearing nude mice,and making tumor histopathological sections of tumor-bearing nude mice.The histopathological morphology after AB4 administration was observed by HE staining.The expression of Ki67 to explore the proliferation of HCC cells in tumor tissues by Immunohistochemistry.3.Western blot assay was used to detect the expression of Notch pathway-related proteins after administration of HCC cells.Immunohistochemistry was used to detect the expression of Notch-related proteins in tumor-bearing nude mice.Results1.In MTT assay,AB4 inhibited the proliferation of HepG2 and Huh-7 cells in a concentration-dependent manner within a certain range.The optimal dosing concentration and optimal dosing time of HepG2 cells were 50?mol/L and 24 h,respectively.The IC₅₀0 was 50.58±1.58?mol/L.In addition,the optimal dosing concentration and optimal dosing time of Huh-7 cells were 45?mol/L and 24 h,respectively.The IC₅₀0 was 45.05±1.77?mol/L.The colony formation assay showed that50 and 100?mol/L AB4 readily inhibited colony formation of HepG2 cells,45 and 100?mol/L AB4 significantly inhibited colony formation of Huh-7 cells?p<0.01?.Annexin V-FITC/PI staining assay showed that 50?mol/L AB4 can induce apoptosis of HepG2cells,and 45?mol/L AB4 can induce apoptosis of Huh-7 cells.In Western Blot assay,the expression of apoptosis-related proteins Cleaved Caspase-3,Cleaved PARP and Cytochrome C was significantly increased after administration of 50?mol/L AB4 in HepG2 cells?p<0.01?,and the expression of apoptosis-related proteins Cleaved Caspase-3,Cleaved PARP and Cytochrome C was significantly increased after administration of 45?mol/L AB4 in Huh-7 cells?p<0.01?.2.AB4 significantly inhibited tumor growth in nude mice.The tumor inhibition rates of 60 mg/kg AB4 group,120 mg/kg AB4 group and positive control group?DAPT group?were 24.05%,59.67%and 66.67%,respectively.In tumor tissues,HE staining assay showed that the cell outline was blurred,the cell boundary was unclear,the cell shape was irregular,the cell nucleus had become pyknotic and had then undergone karyolysis after administration of AB4 at 60 and 120 mg/kg dose.Immunohistochemistry showed that the expression of Ki67 was decreased after administration of 60 mg/kg and 120mg/kg AB4,and there was a significant difference compared with the control group?p<0.05 or p<0.01?.3.Western Blot assay showed that the expression of Notch-related proteins Notch 1,Jagged 1,NICD 1,Hes 1,Hey 1 was significantly decreased after administered at 50 and100?mol/L AB4 in HepG2 cells?p<0.05 or p<0.01?,and the expression of each protein was no significant difference compared with the control group after administration of 25?mol/L AB4.The combination of 50?mol/L AB4 and the Notch pathway inhibitor DAPT showed that DAPT enhanced the inhibition in HepG2 cells.In Huh-7 cells,the expression of Notch 1,Hes 1,Hey 1 proteins was significantly decreased after administration of 45 and 100?mol/L AB4?p<0.05 or p<0.01?,and there was no significant difference compared with the control group after administration of 25?mol/L AB4.In addition,the expression of Jagged 1 and NICD 1 proteins was significantly decreased after administration of 25,45,100?mol/L AB4?p<0.05 or p<0.01?.The combination of 45?mol/L AB4 and DAPT showed that the DAPT also enhanced the inhibition.Immunohistochemistry showed that the expression of Notch 1 and Hes 1proteins in tumor-bearing nude mice was decreased after AB4?60,120 mg/kg?administration?p<0.05 or p<0.01?.Conclusions1.AB4 inhibited the proliferation of HepG2 and Huh-7 cells,and increased the expression of apoptosis-related proteins Cleaved Caspase-3,Cleaved PARP and Cytochrome C.2.AB4 inhibited the growth of tumor-bearing nude mice.It inhibited the expression of Ki67 in tumor tissues,thereby inhibiting the proliferation of tumor tissue cells.3.The inhibitory effect of AB4 on HCC may be related to the inhibition of the expression of Notch 1,Jagged 1,NICD 1,Hes 1,Hey 1 in Notch signaling pathway.