Exploring The Molecular Mechanism Of Necroptosis And Immune Infiltration In Mice With Sepsis-induced Myocardial Dysfunction Based On RNA-Seq And Bioinformatics Analysis

Objective: This study is based on the technology of RNA Sequencing(RNA-Seq)and bioinformatics analysis of myocardial tissue in the mouse model of Sepsis-induced myocardial dysfunction(SIMD),intended to explore the pathogenesis and key differential genes to identify the related molecular pathways of SIMD,and to analyze the level of immune infiltration of necrotic apoptosis-related genes,to provide a new research direction for septic cardiomyopathy.Methods: In this study,male C57BL/6 mice were randomly divided into an experimental group(n=5)and a control group(n=5).The experimental group mice were generated by intraperitoneal injection of lipopolysaccharide(LPS)to establish the model of SIMD.In contrast,the control group mice were generated by intraperitoneal injection of the same volume of normal saline.Finally,the success of establishing the model of SIMD in mice was tested by echocardiography,the pathology of myocardial tissue pathology,and the detection of biochemical indicators.Subsequently,three mice were randomly selected from the control and experimental groups to be included in the formal experiment.RNA-Seq was carried out on the myocardial tissue of mice in the control group(n=3)and experimental group(n=3).The Differentially expressed genes(DEGs)were screened by the limma software package(adjusted p≤0.05 and |log2FC|≥1).Gene set enrichment analysis(GSEA)was used to study the molecular pathway of DEGs.Then,based on the Kyoto encyclopedia of genes and genomes(KEGG),we calculate the necroptosis-related differentially expressed genes(NRDEGs)combined with DEGs and the necroptosis pathway.And we use a database for annotation,visualization,and integrated discovery(DAVID)to conduct gene ontology(GO)enrichment analysis of NRDEGs.The above revealed the significantly enriched biological process(BP),cellular component(CC),molecular function(MF),and KEGG pathway analysis of these genes.Then,through the STRING database,the protein-protein interaction network(PPI Network)was constructed based on the protein corresponding to NRDEGs.The Cytoscape software was used to search for protein-intensive regions and essential node proteins with potential functions in SIMD.Finally,quantitative real-time PCR(q RT-PCR)was used to detect the m RNA expression of core genes in NRDEGs,Western blotting(WB),and immunohistochemistry(IHC)were used to detect the expression of key proteins.Results: In this study,compared with the control group,the echocardiogram of the experimental group showed that the left ventricle ejection fraction and fractional shortening were significantly decreased(P<0.0001,P<0.0001);interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),creatine kinase isozyme(CKMB)and cardiac troponin-T(CTNT)of the experimental group were significantly increased(P<0.001,P<0.001,P<0.001,P<0.001,P<0.01).HE staining results of myocardial pathological sections of mice in the experimental group showed that myocardial cells were uneven in size,disordered in arrangement,and had inflammatory cell infiltration.3654 DEGs were found in the two groups through bioinformatics analysis,including 1810 up-regulated and 1844 down-regulated genes.Including necroptosis,several pathways were significantly enriched in the experimental group.We have identified 35 NRDEGs,including MLKL and RIPK3,and bioinformatics analysis shows that NRDEGs strongly correlate with the immune microenvironment.The gene expression level in the myocardium of mice in the experimental group was positively correlated with the infiltration level of mast cells,M1 macrophages,and neutrophils and negatively correlated with the infiltration level of B cells and plasma cells.The results of q RT-PCR showed that the m RNA expression levels of TNFα、IL-1β、MLKL、RIPK3、CASPASE8、TLR3、TRADD、STAT1、NLRP3,and FAS in the experimental group were significantly higher than those of the control group(p<0.05).Compared with the control group,Western blotting analysis showed that the Relative protein expression level of TNFα、IL-1β、MLKL 、 RIPK3 、 CASPASE8,and TLR3 in the experimental group were significantly increased(p<0.05).Immunohistochemical staining showed that the expression of RIPK3 and MLKL in the experimental group’s myocardial tissue was increased,confirming that necroptosis occurred in myocardial cells.Conclusion: In conclusion,this study showed that LPS-induced necroptosis occurred in the mouse model of SIMD.The results of bioinformatics analysis show that NRDEGs are significantly enriched and have a strong correlation with the immune microenvironment,which indicates that necroptosis may affect the development of septic cardiomyopathy by regulating the immune microenvironment and also shows that NRDEGs may be a potential diagnostic biomarker and therapeutic target for patients with septic cardiomyopathy.