## 4 Discussion  
The main findings of our study in a large animal model of sepsis were that (1) the serum expression of miR-146a-5p and miR-34a-5p was upregulated after a septic challenge, and (2) miR-146a-5p expression was modulated by the intensity of the fluid resuscitation. These findings are of paramount importance for the discovery of biomarkers of sepsis and resuscitation, as well as the identification of novel therapeutic targets. Our animal model features human sepsis, including systemic vasodilation and pulmonary vasoconstriction, hyperlactatemic acidosis, marked pro-inflammatory response, and substantial mortality. The concentrations of IL-1\(\beta\), TNF-\(\alpha\), and IL-6 in the serum, as well as the concentration of IL-1\(\beta\) in the renal tissue ( medulla and renal cortex), increased after the septic challenge, demonstrating an inflammatory response both systemically and at the tissue level as part of the immune response to sepsis. Fluid resuscitation attenuated some of the hemodynamic changes (e.g., the decrease in MAP and RAP), the hyperlactatemic acidosis, and the magnitude of the inflammatory response (i.e., increased TNF-\(\alpha\) and, nonsignificantly, IL-6 serum concentration and IL-1\(\beta\) serum and kidney tissue concentrations). Recent studies have investigated the role of different miRNAs as biomarkers of sepsis in small animal models and in human sepsis [12; 14]. The novelty of the present investigation is the study of the expression of different miRNAs in a large animal model of sepsis and their response to fluid resuscitation. In this context, we found that sepsis was associated with increased serum expression of miR-34a-5p and miR-146a-5p. In addition, we found that the serum expression of miR-146a-5p was significantly lower in the HiR group compared with the LoR group. The expression of miR-155-5p followed the same pattern, albeit with differences that did not reach statistical significance (\(p=0.051\)). The miRNAs measured in the present investigation were chosen based in their known role in the inflammatory response, a key element in the pathobiology of sepsis (discussed in detail in the Supplementary Materials) [15; 16; 17; 18; 19; 20; 21; 22]. Our findings of increased levels of **miR-34a-5p** in sepsis are in line with the results of other in vitro studies [16; 17] and with the results of sepsis models in mice treated with cecal ligation and puncture [23] and rats given LPS [24]. We found significant upregulation of **miR-146a-5p** in sepsis. These findings confirm and expand previous results showing increased expression of this miRNA in blood from mice undergoing cecal ligation and puncture [25; 26] and in muscle tissue from pigstreated with LPS [27]. However, those findings seem to contradict the reported changes in **miR-146a-5p** expression in patients with sepsis, in whom **miR-146a-5p** was either downregulated [28; 29] or unchanged [30] compared with patients with systemic inflammatory response (SIRS) and healthy controls. These discrepancies could be explained by differences in the sampling time, which in the experimental model occurred very early after the septic challenge. Notwithstanding these apparently discrepant results, our findings confirmed the role of miR-146a-5p as a biomarker of sepsis in a large animal model of sepsis. In addition, we provided for the first time evidence that the expression of miR-146a-5p was sensitive to the dose of fluid resuscitation. In general, despite the significant changes observed in the expression of **miR-34a-5p** and **miR-146a-5p**, we could not conclude the role of the other miRNAs measured herein as biomarkers of sepsis and resuscitation in our model, as some observed trends did not reach statistical significance.