Anti-tumor Effect Of SiRNA-PD-L1 Delivered By Attenuated Salmonella Combination With Nifuroxazide In HCC-burdened Mice

Background Hepatocellular carcinoma(HCC)is one of the common malignant tumors with high morbidity and mortality,thereby becoming a serious threat to human health.Therefore,how to prevent and treat HCC effectively is very important.It has been demonstrated that nifuroxazide,a broad-spectrum antibacterial drug,has some anti-cancer effects.However,tumor progression is affected by many complicated factors,and the efficacy of monotherapy is not satisfactory.Thus,more and more combination schemes have been used for tumor treatment in clinic.Especially with the development of immunology,the combination of chemotherapy and immunotherapy has become a growing trend.It has been shown that Programmed Cell Death Ligand 1(PD-L1)is expressed excessively on the surface of HCC cells and it will inhibit the anti-tumor effect of T cells through binding with PD-1 on the surface of T cells.Consequently,blocking PD-1/PD-L1 pathway can exert anti-tumor effects.We have constructed the plasmid of the small interfering ribonucleic acid aiming at Programmed Cell Death Ligand 1(siRNA-PD-L1),which significantly inhibited the expression of PD-L1 on the surface of tumor cells.Objective To detect the anti-tumor effect and explore the mechanism of siRNA-PD-L1 delivered by attenuated Salmonella in combination with nifuroxazide in HCC bearing mice.Methods 1.The construction of HCC-bearing mice model The animal studies were approved by the management of laboratory animals and the ethics committee of Xinxiang Medical University.H22 cells were counted with trypan blue staining,and the experimenters in this study adjusted the cell concentration to 1.5×107 cells/ml.The cell suspension was placed on ice for use.C57BL/6 mice were used to establish the model.Specifically,the right hind limb of the mouse was injected subcutaneously with cell suspension(the volume is 100 μl),with the number of 1.5 × 106 cells.2.The effect of the combination therapy including siRNA-PD-L1 and nifuroxazide in HCC bearing mice Seven days after the model establishment,the mice were divided into five groups: the PBS group,the scramble group,the nifuroxazide group,the siRNA-PD-L1 group and the nifuroxazide combined with siRNA-PD-L1 group,with 6 mice in each group.The mice in the PBS group were administered with 100 μl PBS;the mice in the scramble group were administered with 100 μl(5×105 cfu)attenuated Salmonella carrying the random-sequence-si RNA plasmid;the mice in the nifuroxazide group were injected with nifuroxazide(200μg/mouse);the mice in the siRNA-PD-L1 group were administered with 100 μl(5×105 cfu)of attenuated Salmonella carrying the siRNA-PD-L1 plasmid;the mice in the combination treatment group were administered with both nifuroxazide and attenuated Salmonella carrying the siRNA-PD-L1 plasmid.Besides,nifuroxazide was administered once a day for 7 times,while attenuated Salmonella was administered twice a week.On the 7th day after the end of treatment,the mice were sacrificed,and the tumor tissues were separated for detection.(1)The tumors were photographed and weighed,and the inhibitory effect of the combination treatment on tumor growth was detected.(2)The expression of the proteins including PD-L1 and Cyclin D1 were detected by Western blot.Apoptosis was detected by TUNEL assay.Expression of Ki-67 protein in tumor tissue was detected by immunohistochemistry.The infiltration of T lymphocytes and M1 macrophages were detected by immunofluorescence.The number of T lymphocytes and NK cells in the spleen was detected by flow cytometry.Results 1.Through the detection of tumor weight in tumor-bearing mice,it was found that the tumor weight of mice in the PBS group was 3.2±0.78g;the tumor weight of mice in the scramble group was 2.95±0.36g;the tumor weight of mice in the nifuroxazide group was 1.09±0.03g(P < 0.05,compared with the control group);the tumor weight of mice in the siRNA-PD-L1 group was 1.15±0.25g(P < 0.05,compared with the control group);the tumor weight of mice in the combination group was 0.63±0.3g(P < 0.05,compared with any other group).2.The results of TUNEL apoptosis showed that compared with the PBS or the scramble group,the apoptosis of tumor cells in the nifuroxazide or the siRNA-PD-L1 group was significantly more obvious,and the combination group induced the most obvious apoptosis among all the groups.The same tendency was found in the detection of proliferation-related protein Ki-67 by immunohistochemistry.3.The results of Western blot showed that the nifuroxazide or the siRNA-PD-L1 group significantly inhibited the expressions of PD-L1 and Cyclin D1 and the phosphorylation of p-Stat3 in tumor tissues compared with the PBS or the scramble group.The expression of apoptosis-associated protein cleaved PARP increased the most in the combination treatment group..4.The results of immunofluorescence showed that the nifuroxazide or the siRNA-PD-L1 group significantly increased the infiltration of T lymphocytes and M1 macrophages in the mouse tumor tissues,and the level of cell infiltration in the tumor tissues was the highest in the combination treatment group.5.The results of flow cytometry showed that the nifuroxazide or the siRNA-PD-L1 group significantly increased the number of T lymphocytes and NK cells in the spleen of mice.Compared with any other group,the number of T lymphocytes and NK cells in the spleen of mice was the highest in the combination treatment group,P < 0.05.Conclusion The attenuated Salmonella carrying the siRNA-PD-L1 plasmid combined with nifuroxazide effectively achieved a synergistic anti-tumor effect in HCC-bearing mice,increased the apoptosis,inhibited tumor proliferation and enhanced the anti-tumor immunity of tumor bearing mice.This study provides experimental basis for the application of bacteria as a carrier for the siRNA-PD-L1 plasmid on HCC as well as a pre-clinical platform for the combination therapy including siRNA-PD-L1 nifuroxazide against HCC.