Reserch On Jiedu Sangen Decoction Combined With PD-L1 Inhibitor Inhibiting The Invasion And Metastasis Of Colon Cancer Through Epithelial Mesenchymal Transition

Objective This study was designed to investigate the relationship between Jiedu Sangen Decoction(JSD,consisting of Polygonum Cuspidatum Root,Geum Japonicum Thumnb,Radix Actinidiae)combined with PD-L1 inhibitor and EMT(Epithelial Mesenchymal Transformation),PI3K/AKT signal pathway-associated protein in In vivo and in vitro,repectively.What’s more,we aim to explore and verify the anti-metastasis effect of combination of JSD and PD-L1 inhibitor,and the genes or protein they target.Methods(1)In vitro: the CT-26 cell was intervened by EGF for 48 hours to establish colon cancer cell line EMT model,which followed by corresponding agents intervention in five groups(control group,EGF group,JSD group,PD-L1 inhibitor group and combination group).Then the protein throughput,metastasis ablility,cytoskeleton were measured using Western Blot,Wound scratch assay and transwell assay,Immunofluorescence and laser scanning confocal microscopy,respectively.(2)In vivo: to establish mice model of metastasis of colon cancer,the CT-26 cells were inoculated into 100 BALB/c mice by splenic subcapsular injection.Then they were divided into model group,JSD group,PD-L1 inhibitor group and combination group(JSD plus PD-L1 inhibitor).After 14-days intervention of corresponding agents,the liver,spleen and lung of mice were stripped and collected for Immunohistochemistry(ICH),Western Blot analysis,in order to detect the expression levels of phosphorylated PD-L1 signal pathway,PI3K/AKT signal pathway and metastasis-associated protein,etc.Results in vitro:Transwell migration assay: The numbers of CT-26 cell passing through the membrane in combination group are less than PD-L1 inhibitor group(P < 0.01).Transwell invasion assay: The numbers of CT-26 cell passing through the membrane in JSD group and combination group are less than PD-L1 inhibitor group(P < 0.01).Western Blot assay: Snail expression in EGF group is higher than control group(P < 0.05);E-Cadherin expression is up-regulated in combination group compared with JSD group and PD-L1 inhibitor group(P < 0.05);N-Cadherin expression is down-regulated in combination group compared with JSD group and PD-L1 inhibitor group(P < 0.01).Twist expression is down-regulated in combination group compared with JSD group(P < 0.05).in vivo:Immunohistochemistry: In tissue of lung,spleen and liver,the level of CD4+T cell in PD-L1 inhibitor group is higher than model group(P < 0.05).In lung tissue,E-cadherin expression level is higher in combination group than model group(P < 0.05),while N-cadherin expression level is lower in combination group than model group(P < 0.01);In spleen tissue,compared with JSD group,PD-L1 inhibitor group and model group,E-cadherin expression level is higher in combination group(P < 0.01);In tissue of spleen and liver,N-cadherin expression is decreased in combination group compared with other three groups(P < 0.01).Western Blot assay: In spleen tissue,N-cadherin expression is down-regulated in combination group compared with PD-L1 inhibitor group(P < 0.05);Snail expression is down-regulated in combination group compared with JSD group(P <0.01);The expression of Twist and Vimentin in combination group is less than JSD group and PD-L1 inhibitor group(P < 0.01);PI3K expression in combination group is down-regulated compared with JSD group(P < 0.05);P-AKT expression in combination group is down-regulated compared with JSD group and PD-L1 inhibitor group(P < 0.05);Compared with JSD group,PD-L1 inhibitor group and model group,PD-L1 expression is down-regulated in combination group(P < 0.01).In liver tissue,E-cadherin expression is increased in combination group compared with JSD group and PD-L1 inhibitor group(P <0.05);The expression of N-cadherin,Vimentin and PI3 K in combination group is less than JSD group,PD-L1 inhibitor group and model group(P < 0.05);Slug expression in combination group is down-regulated compared with JSD group(P < 0.05);P-AKT is up-regulated in combination group,JSD group and PD-L1 inhibitor group(P < 0.01);PD-L1 is down-regulated in combination group compared with JSD group and PD-L1 inhibitor group(P < 0.05)Conclusion The PD-L1 inhibitor can enhance the cellular immunity of mice.The combination of JSD and PD-L1 inbibitor can inhibit the process of EMT and the ability of metastasis,possibly by regulating PI3K/AKT signaling pathway.