Abstract:

Objective To analyze the sensitization mechanisms and molecular targets of Echinococcus granulosus using bioinformatics approaches. Methods Eighteen BALB/c mice were randomly grouped as infection- unsensitized (CE), infection-sensitized (ANPC), and uninfected controls (CTRL). Mice in the infected unsensitized group were injected with protoscoleces suspension (0.2 ml/mouse), followed with saline (0.2 ml/mice) after 90 days. Mice in the infected sensitized group were injected with protoscoleces suspension (0.2 ml/mice), followed with crude cyst fluid (0.2 ml/mice) 90 days after infection. Mice in the uninfected control group were injected with an equal amount of saline. Splenic tissue from three mice of each group were collected 1 h after injection for RNA extraction and transcriptome sequencing. Genes of differential expression were identified by bioinformatics analysis. Significance analysis of differential expression differences was performed using edgeR (quantified with RNA-Seq by expectation-Maximization RSEM) software. Network interaction analysis was performed using the String database and Cytoscape software with the score set as > 500. The network analysis of the genes was then performed using the Cytoscape software and use cytoHubba to obtain the degree of nodes (degree) and the nodes with degrees over 3 were filtered for display. The analysis was performed using the DAVID online enrichment analysis tool, and the results were visualized using the R language ggplot2. Results A total of 743 genes were differentially expressed in the ANPC group compared to the CTRL group, including 568 upregulated genes and 175 downregulated genes. A total of 554 genes were differentially expressed in the CE group compared to the CTRL group, including 393 upregulated genes and 161 downregulated genes. There are 458 genes specifically expressed in the ANPC group compared to the CTRL group, 269 genes specifically expressed in the CE group compared to the CTRL group and 285 genes co-expressed. GO enrichment of 458 genes, which are unique to the ANPC group, and the CTRL group showed that 147 genes are involved in the biological process, 10 genes are involved in cellular components, and 43 genes are involved in molecular functions. The GO enrichment of shared genes showed that 56 were involved in biological processes, 23 in cellular components, and 25 in molecular functions. The GO enrichment of genes that are unique to the CE group and CTRL group revealed that 51 genes are involved in biological processes, 13 genes are involved in cellular components, and 18 genes are involved in molecular functions. The functions of the ANPC group-specific genes and the CTRL group-specific genes mainly focus on inflammatory response and immune response. The functions of genes expressed in both the ANPC and CTRL groups mainly focus on the neuropeptide signaling pathway and cellular response to interleukin-1. The function of CE group-specific genes and the CTRL group-specific genes mainly focuses on the nucleosome assembly and the positive regulation of ERK1 and ERK2 cascades. The genes expressed explicitly in the ANPC group, and the CTRL group was enriched in 32 pathways, including cytokine-cytokine receptor interaction and the JAK-STAT signalling pathway. The genes commonly expressed in all groups were enriched in 9 pathways, including the phosphatidylinositol 3-kinase/protein kinase B (PI3K-Akt) signaling pathway and complement and coagulation cascades. The CE specific genes and the CTRL group-specific genes are enriched to 12 pathways, including Tyrosine metabolism and Linoleic acid metabolism, and identified key genes through the PPI network. Conclusion The inflammatory response plays an important role in metacestode sensitizing process; high expression of IL-6 promotes the occurrance of inflammation; IL-10 functions as an anti-inflammatory factor confronting the host’s inflammation response; IL-13 may be the key gene in developing immune tolerance after metacestode infection. ERK signaling pathway may play an immunomodulatory role during the growth of metacestodes. Cytokine-cytokine receptor interaction pathway and PI3K-Akt signaling pathway were significantly correlated with the sensitization induced by Einococcus infection.

Key words: Echinococcus, Bioinformatic analysis, IL-6/IL-10, Molecular target