PD-1 Inhibitors Participate In The Occurrence And Development Of Myocarditis By Influencing Intestinal Flora

Part Ⅰ Establishment of a PD-1 inhibitor associatedmyocarditis modelObjective:Establishment of a PD-1 inhibitor associated myocarditis model.Methods:Scheme 1: 10 C57 BL /6 mice were subcutaneously inoculated with 0.1ml of Lewis lung cancer cells,and the tumor implantation status of the mice was observed and recorded.On the 8th day,a standard mouse tumor model was obtained.They were randomly divided into two groups,with 5 mice in each group: a blank control group(on the 10 th,13th,and 16 th days after tumor implantation,0.2 ml of 0.9% Nacl injection was injected into the abdominal cavity);Immunotherapy group(intraperitoneal injection of RMP1-14:0.2 mg/animal on the10 th,13th,and 16 th days after tumor implantation).Scheme 2:Twelve male BALB/c mice(weighing 18-20 g)at 6-8 weeks were randomly divided into 4 groups of 3 mice each: A:Normal + IgG group(n=3),B: Normal + RMP1-14 group(n=3),C: EAM + IgG group(n=3)and D: EAM + RMP1-14 group(n=3).Normal + IgG group was injected subcutaneously with 0.1 mL saline on days 0 and 7,and IgG was injected intraperitoneally on days 14,16,18 and 20;Normal + RMP1-14 group was injected subcutaneously with 0.1 mL saline on days 0 and 7,and RMP1-14 0.2 mg was injected intraperitoneally on days 14,16,18 and 20;EAM + IgG group was injected subcutaneously on days 0 and 7 with 0.1 mLMy HC-α on days 0 and 7,and IgG on days 14,16,18,and 20;EAM+RMP1-14 group was injected subcutaneously with 0.1 mLMy HC-α on days 0 and 7,and RMP1-14 0.2 mg on days 14,16,18,and 20.Mice were treated on day 21 of the experiment,and feces,blood and hearts were collected to determine serum troponin and creatine kinase isoenzyme levels by enzymelinked immunosorbent assay,to score the degree of myocardial inflammation using hematoxylin-eosin staining of heart tissues。Results:Scheme 1 Results: There was no significant difference in serum cTn-1 and CK-MB concentrations between the experimental group and the control group.The experimental group and the control group did not have myocardial fibrosis and necrosis,inflammatory cell infiltration,and fibrous tissue proliferation,and it was not possible to grade the degree of inflammation.Scheme 2 Results: Cardiac marker levels: On day 21 of the experiment,the serum concentration of CK-MB in mice in the EAM+RMP1-14 group was higher than that in the normal+RMP1-14 group(10.976±0.393 vs.6.292±0.614,P<0.01),the serum concentration of CK-MB in mice in the EAM+IgG group was higher than that in the normal+IgG group(8.011±0.875 VS 7.086±0.438,P>0.05),and the serum concentration of CK-MB in mice in the EAM+RMP1-14 group was higher than that in the EAM+IgG group(10.976±0.393 vs.8.011±0.875,P<0.01);the serum concentration of c Tn-1 in mice in the EAM+RMP1-14 group was significantly higher than that in the normal+RMP1-14group(75.238±5.938 vs.50.320±3.111,P<0.01),c Tn-1 concentration in serum of mice in EAM+IgG group was higher than that in normal+IgG group(62.979±8.766 vs.51.832±3.004,P<0.05),CK-MB concentration in serum of mice in EAM+RMP1-14 group was higher than that in EAM +IgG group(75.238±5.938 vs.62.979±8.766,P<0.05).HE staining of heart: less myocardial fibrous degeneration necrosis,inflammatory cell infiltration,and fibrous tissue hyperplasia in the EAM + IgG group,with mild(+)predominant grading and presence of a small amount of mild(+++),and more myocardial fibrous degeneration necrosis,inflammatory cell infiltration,and fibrous tissue hyperplasia in the EAM + RMP1-14 group,with increased grading and presence of more mild(+++)and a small amount of moderate(++++).Conclusion:1.Direct use of RMP1-14 to induce myocarditis was unsuccessful.2.The experimental autoimmune myocarditis model can be successfully established with My HC-α,and the application of RMP1-14 has promoted the occurrence and development of myocarditis.Part Ⅱ Effect of PD-1 inhibitor on intestinal flora in mice with autoimmune myocarditisObjective:To detect the structural composition and functional changes of intestinal microflora in mice.Methods:After extracting fecal DNA,16 Sr RNA sequencing of intestinal microorganisms was performed.Changes in the composition,structure,and function of intestinal microorganisms were comprehensively evaluated through alpha diversity,beta diversity,species diversity analysis,and KEGG pathway analysis.Results:Intestinal flora: alpha diversity showed no significant abnormalities in each group,Beta diversity showed significant differences between groups,species difference analysis showed differences in microbial composition between groups at the phylum and genus levels,functional analysis showed that genes involved in HCM pathways were upregulated in group D compared to group C.Conclusion:RMP1-14 can change the structural composition and function of intestinal microflora in mice.