Gastrointestinal Adverse Effects Of PD-1 Inhibitor Combined With Platinum Containing Dual Drugs And Relevant Mechanisms In Lewis Lung Cancer Mice

Part 1.Gastrointestinal adverse effects of PD-1 inhibitor combined with platinum containing dual drugs in Lewis lung cancer model miceObjective:PD-1 inhibitor combined with platinum containing chemotherapy is the first-line clinical treatment for advanced non-small cell lung cancer without sensitive gene mutation.In this study,we established Lewis lung cancer mice model,and observed the effects of combined drugs on intestinal side effects,and compared with the single drug groups.Methods:The mice model of Lewis lung cancer was constructed and randomly divided into 4 groups with 8 mice in each group:Tumor model group(Model);Chemotherapy group(CARB/PEM);Immunotherapy group(aPD-1)and combined therapy group(CARB/PEM/aPD-1).The other 8 mice without lung cancer transplantation were control group(Control).Observe and compare the weight,water and food intake,nutrition,spleen index,diarrhea,colitis scores and other intestinal side effects of mice in each group after treatment.Results:The weight,water and food intake,serum albumin and prealbumin of mice in the combined therapy group were significantly reduced compared with the model group(p<0.05),and there was no significant difference between the combined therapy group and the chemotherapy group.The diarrhea score and colon pathological score of mice in the combined therapy group were significantly higher than those in the model group and immunotherapy group(p<0.01),and there was no significant statistical difference between the combined therapy group and the chemotherapy group.In Lewis lung cancer mouse model,the volume of transplanted tumor in the three treatment groups was significantly smaller than that in the model group(p<0.01).The volume of transplanted tumor in combined treatment group was significantly smaller than that in chemotherapy group and immunotherapy group(p<0.05).Conclusion:PD-1 inhibitor combined with platinum containing chemotherapy can inhibit the growth of tumor to the greatest extent,while its gastrointestinal side effects are not significantly aggravated due to the superposition of two kinds of drugs.Part 2.Mechanisms of immune damage in the intestinal mucosa caused by PD-1 inhibitor combined with platinum-containing dual drug therapy in Lewis mice.Objective:In previous study,we found that platinum-based dual-drug chemotherapy combined with PD-1 inhibitor also caused damage to the colon mucosa in Lewis lung cancer mice.The mechanism of gastrointestinal side effects caused by immunosuppressants is still unclear.To identify and explore the relevant mechanisms of the gastrointestinal side effects of PD-1 inhibitor combined with platinum-containing dual drug therapy.Methods:The mice model of Lewis lung cancer was constructed and randomly divided into 4 groups,including tumor model group;Chemotherapy group;immunotherapy group and combined therapy group.8 mice without lung cancer were control group.Compare the expression levels of tight junction proteins occludin and claudin-1 in mouse colon mucosa.Compare the ratios of IgA+cells and the concentration of SIgA in colon mucosa of mice in each group;Cytokine IL-17 and TGF-β content and Foxp 3 mRNA expression were also compared.Results:The mRNA level of tight junction protein occludin、claudin-1 in chemotherapy group and combined therapy group are significantly decreased(p<0.05)compared with the other experimental groups.The sIgA level、the number of IgA-secreting cells、IL-17 level and TGF-β level in colonic mucosa have no significant difference between combined therapy group and other groups(p>0.05).The mRNA level of Foxp 3 is significant decreased in the immunotherapy group and combined therapy group compared with the other groups(p<0.05).Conclusions:Compared with the chemotherapy group,the combined therapy group did not aggravate the intestinal barrier function of mice.The combined drug regimen will not aggravate the gastrointestinal side effects of the model mice in the related immune reactions.The decreased expression of Treg may be related to the application of PD-1 inhibitor itself.The decrease of Treg may be associated with gastrointestinal side effects to some extent,but its extent and mechanism need further study.Part 3.Effect of PD-1 combined with platinum containing dual drugs on intestinal flora in Lewis lung cancer model miceObjective:To explore the effects of PD-1 combined with platinum containing dual drugs on intestinal flora in Lewis lung cancer model mice.The effects of combined treatment on the intestinal flora of Lewis mice were investigated,and the effects of the altered intestinal flora on the safety and efficacy of the treatment were initially investigated.Methods:6-8 weeks old C57BL/6J male mice were used to establish the Lewis lung cancer mouse model,and randomly divided into the tumor model group,chemotherapy group,immunotherapy group,and combination therapy group,and the same batch of unmodeled mice were taken as the blank control group,8 mice in each group.The total RNA of fecal bacteria was extracted from the feces of mice in each group before treatment and after each dose,and 16S rRNA gene amplification and high-throughput sequencing were performed,and the alpha diversity analysis,beta diversity analysis,difference in composition of the flora and functional analysis were performed.Results:PD-1 inhibitor had no significant effect on the diversity of intestinal flora.Compared with the chemotherapy group,PD-1 combination chemotherapy caused an increase in the phenotypic abundance of Gram-positive bacteria and a decrease in the phenotypic abundance of Gram-negative bacteria in the intestinal flora of mice.Conclusions:PD-1 combination chemotherapy did not aggravate the imbalance of intestinal flora,reduced diversity,and reduced abundance of metabolic pathways caused by chemotherapy.The ultimate impact on the efficacy of PD-1 drugs depends on the ratio of these two groups of flora.