| Part Ⅰ Effect of PD-1 inhibitors on immune function in the colonic mucosa of Lewis micePurpose:Explore the effect of PD-1 inhibitor on colonic mucosal immune function in Lewis mice and discuss the meaning of its anti-tumor effect on the host based on the alteration of colonic mucosal immune function.Methods:Mouse-derived LLC lung cancer cells and C57BL/6 mice were used to establish the Lewis lung cancer transplantation tumor mouse model,divided into the model group(Model),PD-1 group(aPD-1),and another healthy mice were taken as the blank control group(Control),8 mice in each group.The mice in the PD-1 group were injected with mouse-anti-PD-1(aPD-1)200μg intraperitoneally twice a week,while the mice in the blank control group and the model group were given equal amounts of saline intraperitoneally.During the experiment,the mice were weighed and the tumor volume was measured every 2 days;at the end of the experiment,the tumors,spleen,and colon were stripped from each group of mice,and each tissue and organ was set aside.The spleen was weighed and the spleen index was calculated;the IgA+cell ratio in the intestinal mucosa of each group of mice was measured by immunohistochemistry;the sIgA content in the colonic mucosa of each group of mice was measured by immunoenzyme-linked adsorption.Results:The body weight of mice in the Model group was significantly higher than that in the Control group(p<0.01),the body weight(p<0.01)and tumor volume of mice in the aPD-1 group were significantly lower than those in the Model group(p<0.01),the thymus index of mice in each group was not markedly different,the spleen index of mice in the aPD-1 group was significantly higher than that in the Model group(p<0.01).In the colonic mucosa,the IgA+cells of mice in the Model group were significantly lower than those in the Control group(p<0.05),and the IgA+cells of mice in the aPD-1 group were significantly more than those in the Model group(p<0.05).The sIgA content in the colonic mucosa of mice in the aPD-1 group was significantly higher than that in the Model group(p<0.01).Conclusion:PD-1 inhibitor can significantly enhance the immune function of colon mucosa in Lewis mice,which contributes to the local anti-tumor effect of the PD-1 inhibitor.Part Ⅱ Effect of PD-1 inhibitor on the level of inflammatory factors in the colonic mucosa of Lewis micePurpose:Explore the effect of PD-1 inhibitors on the level of inflammatory factors in the colonic mucosa of Lewis mice,and evaluate the implications for the host antitumor effect based on the alteration of inflammatory factors.Methods:The Lewis mouse model was established as in part 1.Tissue homogenates were prepared from tumor tissues of each group of mice,and the contents of IL-10 and IFN-γtumor inflammatory factors were detected by immunoenzyme-linked sorbent assay in each group of mice.At the end of the experiment,the colon lengths of the mice in each group were compared,and the expressions of Claudin-1 and Occludin mRNA in the colon mucosa of each group were detected by real-time fluorescence quantitative PCR.Results:In the tumor site:IL-10 content was significantly lower in the aPD-1 group compared with the Model group(p<0.05);IFN-γ content was significantly lower in the Model group compared with the Control group(p<0.05),while IFN-γ content was significantly higher in the aPD-1 group compared with the Model group(p<0.05).In the colonic site:IL-10 expression,IL-10 protein expression was significantly increased in the Model group compared with the Control group(p<0.01).and the mRNA level(p<0.05)and protein expression(p<0.01)of IL-10 were significantly decreased in the aPD-1 group compared with the Model group.As for IFN-y expression,IFN-γ protein expression in the Model group was significantly lower than that in the Control group(p<0.01),and IFN-y mRNA level(p<0.05)and protein expression(p<0.01)in the aPD-1 group were significantly higher compared with the Model group.As for the assessment of intestinal side effects:the colon length of mice in the aPD-1 group was significantly shorter compared with that in the Model(p<0.05);the DAI score of mice in the Model group was significantly higher than that in the Control group at the end of the experiment(p<0.05),and the DAI score of mice in aPD-1 group was significantly higher than that in Model group(p<0.05).Claudin-1 mRNA expression in the aPD-1 group was significantly lower than that in the Model group(p<0.05),and Occludin mRNA expression in the three groups of mice was not statistically different.Conclusion:PD-1 inhibitors caused increased expression of pro-inflammatory factor IFN-γand decreased expression of anti-inflammatory factor IL-10 in the colonic mucosa of Lewis mice,and the changes of inflammatory factors in the colonic mucosa cooperated with PD-1 inhibitors to exert anti-tumor effects locally in the tumor;however,the increased expression of intestinal pro-inflammatory factors easily caused damage to the intestinal epithelial barrier and induced local inflammatory side effects,and the search for PD-1 inhibitors.The balance between optimal efficacy and the lowest incidence of adverse effects still needs to be further explored.Part Ⅲ Effect of PD-1 inhibitors on intestinal flora and metabolites of short-chain fatty acids in Lewis micePurpose:Explore the effects of PD-1 inhibitors on intestinal flora and metabolites of short-chain fatty acids in Lewis mice,and investigate the impact on host antitumor effect based on the alteration of flora and short-chain fatty acids.Methods:The Lewis mouse model was established as in Part I.On day 23,the feces of each group of mice were collected,and the intestinal flora of each group of mice was sequenced by 16 SrDNA technology,and the sequencing results were analyzed by Alpha diversity analysis,Beta diversity analysis,and dominant flora analysis;the differences of short-chain fatty acid content were quantified by liquid chromatography-mass spectrometry.Results:Alpha diversity:The species richness and uniformity of intestinal flora of mice in each group were in the order of the aPD-1 group,Control group,and Model group.The Shannon index(Model group:p<0.01)and Simpson index(Model group:p<0.01)of Model group were significantly lower than those of the Control group.There was no statistical difference between the Alpha diversity index of the aPD-1 group and the Model group.For Beta diversity:between Control group and Model group(bray:p<0.01;Weighted Unifrac:p<0.05),Model group and aPD-1 group(bray:p<0.01;Weighted Unifrac:p<0.05),Control group and aPD-1 group(bray:p<0.01;Weighted Unifrac:p<0.01),the largest difference in bacterial population structure was found between Control group and aPD-1 group.As for the dominant species:compared with the Model group,the Control group had 7 species of dominant bacteria at the genus level,and the Model group had 4 species;compared with the aPD-1 group,the Model group had 2 species of dominant bacteria at the genus level,and the aPD-1 group had 5 species.aPD-1 group had significantly more isobutyric acid as the metabolite of the flora compared with the Model group(p<0.05).Conclusion:PD-1 inhibitor increased the richness and uniformity of intestinal flora species,the abundance of beneficial flora with antitumor effect,and the content of isobutyric acid in Lewis mice.The improvement of intestinal flora and metabolites in Lewis mice after the application of PD-1 inhibitor was beneficial for PD-1 inhibitor to exert anti-tumor effects and improve host immunity. |
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