Mechanism Of Attenuated Salmonella Carrying PD-1-siRNA Combined With Nifuroxazide For Colon Cancer Therapy

BackgroundColon cancer is a common malignant tumor of the digestive tract.Nowadays,tumor-targeted therapy has attracted people's attention,but the effect of separate targeted therapy is limited,so we are looking for a joint tumor-targeted therapy.PD-1 and its ligand PD-L1 are important immunosuppressive molecules.It is important to use PD-1 as a target for immunomodulation to fight tumors and improve survival rate.Studies have shown that inhibition of Stat3 can inhibit the expression of PD-L1,Nifuroxazid is an intestinal bacteriostatic drug.In this study,Nifuroxazide was found to inhibit the expression of Stat3.Attenuated Salmonella has been shown to be an effective vector for gene therapy.Therefore,we envisage the use of attenuated Salmonella to carry PD-1 siRNA in combination with Nifuroxazide as a novel method for the treatment of colon cancer.ObjectiveExploring the effect and mechanism of attenuated Salmonella carrying PD-1 siRNA combined with Nifuroxazide against colon cancerMethods1.Cell experimentThe best CT-26 cells were plated in 6-well plates and incubated at 37°C for 24 h.The control group was added with PBS,DMSO,and the other four wells were added with different concentrations of Nifuroxazide,and then photomicrograph was taken at 24h and48h;the activity of CT-26 cells was determined by CCK8 experiment;the effect of Nifuroxazide on cell migration was detected by cell migration assay;the expression of protein related to cell proliferation,apoptosis and migration was detected by western blot.2.Animal experimentThe cultured CT-26 cells were injected into the skin of the mice to construct a tumor-bearing mouse model.On the 8th day of modeling,the model mice were randomly divided into 5 groups:PBS group,Scrmble group,Nifuroxazide group,PD-1 siRNA group,PD-1 siRNA combined with Nifuroxazide group,PBS(100 ul),Nifuroxazide(2 mg/).Only mice were treated with attenuated Salmonella 4×10~5 CFU/ML on the first and eighth days of treatment.The survival and tumor yield of the mice were recorded and the tumor size and weight were measured.Western blot was used.The expression changes of Cleaved Caspase 3,Stat3,MMP2,CD~+4,CD~+8 and other related proteins in tumor tissues were detected.Hematoxylin-eosin staining(HE)was used to observe the changes of tumor tissue morphology in different treatment groups.The expression levels of immune cells and apoptotic cells in mouse tumor tissues were detected by immunohistochemistry and TUNEL apoptosis.The proportion of CD4~+T and CD8~+T and NK1.1 cells in spleen was detected by FACS.Results1.Nifuroxazide can inhibit the growth of CT-26 cells,and with the increase of concentration,the inhibition is enhanced.2.Nifuroxazide can affect the expression of proliferation and apoptosis-related proteins after acting on CT-26 cells.3.Animal experiments HE staining results showed that compared with the model group,the treatment group showed different degrees of cell debris,the combined group was the most obvious.4.Western results of animal experiments showed that CD4 and CD8 were significantly increased in the combined group compared with the model group.5.FACS results of animal experiments showed that the proportion of CD4T cells,CD8 T cells and NK1.1 cells in the spleen of the combined group was the highest among the groups.Conclusion1.Nifuroxazide can inhibit the proliferation and migration of CT-26 cells.2.Combination therapy can inhibit tumor growth in colon cancer-bearing mice and improve survival rate in mice.3.The combination treatment group significantly improved the anti-tumor immunity of tumor-bearing mice.