## 3 Results
### Enrichment and Functional Analysis  
There were 1386 genes identified as regulated by both miRNAs (MiRNet). Enrichment analysis (KEGG) identified the Toll-like receptor signaling pathway as being significantly regulated (the third in order of significance), with 22 hits (adjusted \(p\) value = 3.87 \(\times\) 10\({}^{-4}\)). Toxoplasmaosis was the most highly significant pathway regulated by the miRNAs of interest with 24 hits (adjusted \(p\) value = 3.77 \(\times\) 10\({}^{-5}\)), of which 10 were common to the Toll-like receptor signaling pathway (Tables S7 and S8) (Figures 2 and S2).  
Proteins of the Toll-like receptor signaling pathway regulated by the miRNAs of interest (Table S8) were involved (KEGG) as the five more significant pathways in the Toll-like receptor signaling pathway, [PERSON]'s sarcoma-associated herpesvirus infection, hepatitis B, influenza A, and TNF signaling pathway(Table S9). Using Reactome, the top  
Figure 1: miRNA serum expression in control and septic animals receiving low (LoR) or high (HiR) fluid resuscitation. Bars indicate median, percentile 25 th and percentile 75 th. The p values indicate the statistical significance between the group differences (Kruskal–Wallis test). * \(p\) < 0.05 versus control group. \(\dagger\)_p_ < 0.05 versus LoR group (Mann–Whitney test with [PERSON]’s correction).  
Figure 2: (**A**) Genes targeted by miR-146a-5p and miR-34a-5p participating in the Toll-like receptor signaling pathway (KEGG). (**B**) Protein–protein interaction (STRING) of the 22 hits (STRING, confidence 0.9, in 4 clusters). Purple line, experimental evidence. Light blue line, database evidence. Dotted lines, interactions appearing only after clustering. Proteins are involved in multiple immune regulation and cytokine signaling pathways.  
five were cytokine signaling in the immune system, interleukin-6 signaling, hemostasis, downstream signaling events of B cell receptors (BCRs), and PIP3 activating AKT signaling (Table S10). Finally, using Gene Ontology biological processes, the top five pathways related to the 22 Toll-like receptor signaling pathway proteins were positive regulation of immune system processes, the response to cytokine, the cytokine-mediated signaling pathway, regulation of immune response, and positive regulation of signal transduction (Table S11) (all with adjusted \(p<0.001\)).  
Similarly, proteins of the toxoplasmosis pathway regulated by the miRNAs of interest (Table S8) were involved, as the five most significant pathways (with adjusted \(p<0.001\)) were in toxoplasmosis, the Toll-like receptor signaling pathway, small-cell lung cancer, the TNF signaling pathway, and pathways in cancer (KEGG) Table S12). Using Reactome the only pathway regulated with adjusted \(p<0.001\) was hemostasis (Table S13). Finally, using Gene Ontology biological processes, the top five pathways related to the 24 toxoplasmosis pathway proteins (with adjusted \(p<0.001\)) were intracellular signal transduction, regulation of catalytic activity, cellular response to chemical stimulus, cellular response to organic substances, and response to organic substances (Table S14).  
We analyzed the PPIs for the 22 proteins of the Toll-like receptor signaling pathway (STRING, confidence of 0.9 in 4 clusters). The PPIs were highly significant (PPI enrichment \(p\) value: 2.22 \(\times\) 10\({}^{-16}\)) (Figure 2). The PPIs for the 24 proteins of the toxoplasmosis pathway were also analyzed (PPI enrichment \(p\) value \(<\) 1.0 \(\times\) 10\({}^{-16}\)) (Table S8 and Figure S2).