Anti-tumor Effect Of CpG ODN Combination With Nifuroxazide In Mice Burdened HCC

Background As the most common digestive tract malignant tumors,hepatocellular carcinoma（HCC）seriously endangers human health.At present,the incidence rate and mortality rate of HCC increase year by year,and the prevention and treatment is still a global challenge.One specific therapeutic area,immuno-oncology（IO）,has received much renewed attention for its ability to take advantage of the body’s immune system to fight cancer.CpG-ODN triggers innate immune cell activation through TLR9 signaling,and in particular,activate plasmacytoid dendritic cells（p DCs）and B cells.Due to the complication of tumorigenesis,monotherapy shows poorly effect.In order to overcome these limitations,the combination of drugs have been explored.Previous experiments by our research team suggested that the broad-spectrum antimicrobial drug,nifuroxazide suppressed the expression of p-Stat3 in tumor cells.Studies also showed that CpG ODN combined with STAT3 inhibitor exerted an anti-prostate cancer effect.Aim To explore the therapeutic effect and mechanism of CpG ODN combined with nifuroxazide on hepatocellular carcinoma.Methods 1.In vitro experiments.Hep G2 cells were plated and cultured in incubator,and nifuroxazide with different concentrations was added respectively.After 24 and 48 hours,the cells were photographed under microscope,and the cell viability was measured by CCK-8 assay.Cell migration assay was used to detect whether nifuroxazide had an effect on cell migration.Western blot was used to further detect the expression of apoptosis,proliferation and migration relative proteins.Flow cytometry was used to detect cell apoptosis.2.In vivo experiments.1.Establish the tumor bearing mice.H22 cells were digested,resuspended,and calculated.The cell suspension with the concentration of 2 × 107 cells/ml was placed on ice and 100μl were injected subcutaneously into the right hindlimb of C57BL/6 mice.2.On the day 7th after inoculation,mice were randomly divided into four groups: PBS group,nifuroxazide group,CpG ODN group and nifuroxazide combined with CpG ODN group,with 5 mice in each group.Mice in the PBS group were injected intratumorally with100μl PBS,mice in the nifuroxazide group were injected intratumorally with 200μg nifuroxazide,mice in the CpG ODN group were injected with 20μg CpG ODN into inguinal lymph nodes,while mice in the nifuroxazide + CpG ODN group were injected with 200μg nifuroxazide and 20μg CpG ODN.CpG ODN was injected once every other day and nifuroxazide was injected once a day.All treatments lasted for 1 week.On the day7 th after treatment,mice were killed,the tumors were removed and weighted.The expression of caspase-3,Stat3 and other related proteins in tumor tissues were detected by Western blot.The infiltration of immune cells in mouse tumor tissues was detected by immunofluorescence assay.TUNEL was used to detect the apoptotic cells in tumor tissues.The proportion of CD4⁺ and CD8⁺ T lymphocytes in mouse spleen was detected by FACS.Results 1.Nifuroxazide has a killing effect on Hep G2 cells.2.Nifuroxazide inhibited the migration of Hep G2 cells and increased the apoptosis of Hep G2 cells.3.Nifuroxazide affected the expression of proliferation and apoptosis related proteins in Hep G2 cells;4.In the HCC bearing mice,nifuroxazide combined with CpG ODN effectively inhibited the tumor growth.5.Combined therapy significantly inhibited the phosphorylation of Stat3 in hepatocellular carcinoma cells.6.Combined therapy significantly increased apoptosis in hepatocellular carcinoma.7.The infiltration of T lymphocytes in tumor tissue were increased significantly,and the ratio of CD4+ and CD8+ T lymphocytes in spleen were increased in the combined treatment group.Conclusion CpG ODN combined with nifuroxazide significantly inhibited the tumor growth of HCC bearing mice,increased the apoptosis of tumor cells,increased the infiltration of T lymphocytes in tumor tissues and the ratio of spleen,and played a significant synergistic anti HCC effect.This study provides a new drug combination for the combined treatment of HCC,which has a certain experimental and theoretical value.