Mitochondrial Sirt3 serves as a biomarker for sepsis diagnosis and mortality prediction

The purpose of this study is to determine whether the levels of serum Sirt3 correlate with disease severity and perfusion indicators in septic patients, as well as to assess the clinical value of Sirt3 as a potential novel marker for sepsis diagnosis and mortality prediction. A total of 79 patients in the ICU were included in the study, of which 28 were postoperatively noninfectious and the remaining 51 patients were all diagnosed with sepsis during the study period. The levels of Sirt3 were detected and dynamically monitored by enzyme-linked adsorption method, Pearson or Spearman coefficient for correlation analysis between Sirt3 and clinical indicators, ROC curve for evaluation of diagnosis and mortality prediction, Kaplan–Meier method for the significance of Sirt3 in 28-day survival. The serum levels of Sirt3 were lower in the sepsis patients on day 1 (P < 0.0001), and the septic shock group had lower Sirt3 levels than the sepsis group (P = 0.013). Sirt3 had good negative correlations with SOFA scores both in sepsis and septic shock groups (Pearson: r2 = − 0.424, − 0.518; P = 0.011, 0.040), and Sirt3 correlated strongly with ScvO2 in the septic shock group (Pearson: r2 = − 0.679, P = 0.004) and with PCT in the sepsis group (Pearson: r2 = − 0.409, P = 0.015). Sirt3 not only performed well in identifying sepsis (AUC = 0.995, 95% CI 0.987–1, P < 0.0001) but also greatly enhanced lactate's specificity in detecting septic shock (from 91.43 to 94.29%). Patients in the low Sirt3 group had higher ScvO2, lactate, APACHE II score, SOFA score, longer ICU stays, and worse indicators of inflammation (TNF-α, IL-6) and infection (PCT) than those in the high Sirt3 group (P < 0.05). Additionally, Sirt3 can predict mortality of sepsis (AUC = 0.746, 95% CI 0.571–0.921, P = 0.022), patients with serum Sirt3 < 10.07 pg/ml have a lower 28-day survival (log-rank P = 0.008). Low serum levels of Sirt3 are significantly correlated with the disease severity. At the same time, Sirt3 increases the sensitivity of lactate to detect “cellular hypoxia” in septic shock. Sirt3 is a promising biomarker for the diagnosis of sepsis and predicting mortality risk in septic patients.

Patients. All patients in the study were older than 18 years. Patients with sepsis must meet the latest diagnostic criteria (Sepsis-3 defines sepsis as "life-threatening organ dysfunction caused by a dysregulated host response to infection; Sequential [Sepsis-related] Organ Failure Assessment Score (SOFA) is used to define organ dysfunction as an increase in the total SOFA score of 2 points or more) 10 . Patients with sepsis will be classified into the septic shock group when they meet the following criteria: persisting hypotension requiring vasopressors to maintain mean arterial pressure [MAP] > 65 mmHg and having serum lactate level > 2 mmol/L despite adequate volume resuscitation. Patients in the ICU control group need to have normal infection indicators.
The enrolled septic patients were assessed on each study day to determine if they remained in sepsis or septic shock. During the study, patients who were transferred from ICU due to abandonment of treatment or improvement of their condition, as well as those who were no longer diagnosed with sepsis in the ICU, were not studied on subsequent study days (D3, D5, or D7).
Patients who had been or were currently diagnosed with cancer, including all types of tumors, were excluded from the study. Patients who are pregnant were also excluded. Serum collection. We collected arterial blood through an arterial indwelling catheter. Septic patients were collected on days 1, 3, 5, and 7 after being admitted to the ICU, while patients in the control group were only collected on day 1. Blood collected in the anticoagulant tube was centrifuged at 3000 r/min for 10 min after standing for 10 min, then transferred to an EP tube and stored at − 80 °C.
Elisa for serum Sirt3 level. After defrosting the frozen serum, we used the human Sirt3 ELISA kit (Cusabio Biotech Co, Ltd, Wuhan, China, Catalog # CSB-EL021341HU) to detect the concentration of Sirt3 in the serum. Statistical analysis. We used SPSS 26.0 and GraphPad PRISM 8.0 for statistical analysis and plotting.
The Shapiro-Wilk test tested whether the data were normally distributed. The normally distributed data were expressed as mean ± standard, and compared by the One-way Anova or T-test. Non-normal distribution data were expressed as M (P25, P75), and compared by the Kruskal-Wallis test or Mann-Whitney U test. And a variance homogeneity test was also performed, and non-parametric tests were used when variances are uneven. The counting data were expressed as rates (%), and the comparison between groups was tested by χ 2 . A Pearson correlation coefficient analysis was for normally distributed data, a Spearman rank correlation coefficient analysis was for non-normally distributed data. ROC curves were used to analyze the diagnostic efficacy of various indicators. 28-day survival was estimated by the Kaplan-Meier method, and differences in survival were evaluated with the log-rank test. P < 0.05 means that the difference between the two groups is statistically significant. According to the follow-up of 28-day mortality, 9 septic patients died with a mortality rate of 17.65%, including 4 in the sepsis group (11.42%) and 5 in the septic shock group (37.5%). www.nature.com/scientificreports/ During the assessment of the patients on the first day, vital signs (heart rate, blood pressure), infection indicators (WBC, PCT), inflammatory indicators (TNF-α, IL-10, IL-6), CVP, Norepinephrine (NE), oxygenation index (OI), CTNI, and NT-pro BNP were collected. Except for MAP, P (V-A) CO 2 , ScvO 2 , WBC, IL-10, CTNI, and NT-pro BNP, the rest of the indicators were statistically significant ( Table 1).

Characteristics of changes in serum levels of Sirt3 in patients with ICU. The serum level of Sirt3
was lower in septic patients on day 1 (P < 0.0001), and the septic shock group had a lower level than the sepsis group (P = 0.013) (Fig. 1a).
We tracked changes in Sirt3 serum levels on days 3, 5, and 7. The Sirt3 levels in the sepsis group gradually increased over time, and there were statistical differences in growth between D3 and D1 (P = 0.001), D5 and D3 (P = 0.027), but there was no difference between D5 and D7 (P = 0.671) (Fig. 1b). A gradual increase in Sirt3 levels over time was also observed in the septic shock group, where there was a statistical difference between D3 and D1 (P = 0.004), but no statistical difference between D5 and D3, D7 and D5 (P = 0.052, 0.324) (Fig. 1c).
We re-diagnosed and re-grouped the patients on D3, D5, and D7 to confirm that the diagnosis was as accurate as feasible because the patients' conditions may interchange after therapy. On D3, 10 patients were transferred from the ICU, and 41 patients were studied, including 30 patients with sepsis (25 patients with sepsis and 5 patients with septic shock at D1), 11 patients with septic shock (2 patients with sepsis and 9 patients with septic shock at D1). On the fifth day, 11 patients left the ICU, and the remaining 30 patients included 23 patients with sepsis (18 patients with sepsis and 5 patients with septic shock at D3) and 7 patients with septic shock (2 patients with sepsis and 5 patients with septic shock at D3). On D7, 11 patients were moved from the ICU and 19 patients remained, including 17 patients with sepsis (14 with sepsis and 3 with septic shock from D5) and 2 with septic shock (from the septic shock group at D5). The difference in Sirt3 in the two groups was then compared again on days 3 and 5, but there was no statistical difference (Fig. 1d). Since there were only 2 patients in the septic shock group on D7 after being re-diagnosed, the difference between the two groups had a low confidence level and was therefore not shown. in patients diagnosed with septic shock on day 1. (d) Serum Sirt3 levels were measured in sepsis group and septic shock group after re-diagnosed on D3 and D5. P < 0.05 were considered statistically significant. *denotes P < 0.05, **denotes P < 0.01, ****denotes P < 0.0001, "ns" means no significance.

Correlation between Sirt3 and clinical indicators in ICU patients.
Our analysis found significant differences in Sirt3 levels among the three groups of patients on day 1, so we analyzed the correlation between Sirt3 levels and clinical indicators among the three groups on day 1. Our first analysis was to determine if Sirt3 correlated with organ dysfunction, and we found that Sirt3 had a negative correlation with SOFA scores both in sepsis and septic shock groups (Pearson: r 2 = − 0.424, − 0.518; P = 0.011, 0.040), but no significant correlation with the ICU controls (Pearson: r 2 = 0.112, P = 0.570) (Fig. 2a). No correlation was found between Sirt3 and APACHE II scores in any of the three groups (Pearson: r 2 = − 0.381, − 0.064, − 0.075 P = 0.143, 0.713, 0.705).

Difference of clinical indicators in different serum levels of Sirt3.
We got a cutoff value (15.33 pg/ ml) from the maximal Youden index of the ROC curve for screening septic shock. We divided the patients into low Sirt3 (< 15.33 pg/ml) and high Sirt3 (≥ 15.33 pg/ml) groups, with 25 patients in the low Sirt3 group (13 sepsis and 12 septic shock) and 26 patients in the high Sirt3 group (22 sepsis and 4 septic shock). After comparing and assessing the differences in clinical indicators between the two groups, we discovered that patients in the low Sirt3 group had higher APACHE II and SOFA scores, longer ICU stays, worse indicators of inflammation (TNFα, IL-6) and infection (PCT) than those in the high Sirt3 group (Table3).
Predictive value of Sirt3 for 28-day survival. A total of 9 patients died during the 28-day follow-up period in our research, we plotted the ROC curve for Sirt3 predicted mortality. The area under the curve for Sirt3 was 0.746 (95% CI 0.571-0.921, P = 0.022, Fig. 4a). Based on the cutoff value of 10.07 pg/ml of Sirt3 yielded sensitivity and specificity were 66.67% and 78.57%, and all the patients were divided into two groups.
Kaplan-Meier estimator analysis showed the 28-day survival rate of patients with low Sirt3 was markedly reduced than that of patients with high Sirt3 (log-rank P = 0.008, Fig. 4b). Calculate the predicted values for lactate + Sirt3 and lactate + ScvO2 by binary logistic regression and then calculate the ROC curve. SOFA Sequential Organ Failure Assessment Score, ScvO 2 Central venous oxygen saturation, PCT procalcitonin, P (V-A) CO 2 central venous-to-arterial blood carbon dioxide partial pressure. Table 2. The AUC and optimal study parameter cutoff points for different indicators of septic shock and their associated diagnostic and efficacy values. ScvO 2 Central venous oxygen saturation, P (V-A) CO 2 central venous-toarterial carbon dioxide difference. AUC the area under the ROC curve; Cut-off value the optimal cutoff points for the sample diagnosis of septic shock. P < 0.05 means statistically significant.

Discussion
In our research, Sirt3 showed good clinical significance in sepsis. Sirt3 correlated with PCT in the sepsis group and was substantially linked with Scvo2 in the septic shock group, and its levels were negatively correlated with illness severity. Sirt3 also improved the specificity of lactate in the diagnosis of septic shock. It was an acceptable diagnostic marker for identifying sepsis and predicting mortality risk in septic patients. Furthermore, patients with low levels of Sirt3 had a worse prognosis and lower 28-day survival.
Sirt3 is a member of the Sirtuins family, which is composed of seven highly conserved NAD-dependent deacetylases that could regulate cell survival 43 , metabolism 44 , and longevity 45 . Our research showed a strong correlation between Sirt3 and SOFA, which is consistent with the results of most basic experiments on sepsis-induced MODS. In the study of septic cardiomyopathy, Sirt3 can activate AMPK-related mitochondrial biogenesis 39 and acetylate important enzymes in the tricarboxylic acid cycle 29 to reduce sepsis-induced myocardial injury in mice. Sirt3-AMPK signaling is also important for suppressing vascular inflammation and endothelial dysfunction during early sepsis 36 . Lack of Sirt3 increases CLP-induced renal insufficiency, tubular cell death and apoptosis, mitochondrial dysfunction, and ROS production in a knockout mouse model of AKI 46 . These studies focused on the cellular energy metabolism pathway have helped us understand Sirt3's role in protecting organ function at the cellular level, and our research demonstrated a close correlation between the serum level of Sirt3 and MODS in sepsis patients. MODS has been suggested to be caused by mitochondrial damage and energy metabolism disorders in cells during sepsis 47 , thus producing a phenomenon similar to "hibernation" 48 , which closely joins energy metabolism to MODS. Sirt3 contains the switches that control mitochondrial energy metabolism, it may be a potential strategy for future sepsis treatments that target mitochondria.
As one of the diagnostic criteria for septic shock, lactate is used as a perfusion indicator 49 and a resuscitation therapy endpoint 50 in clinical practice, although it can also signify numerous causes of non-perfusion. No correlation between Sirt3 and lactate was found in our study, and the reasons for this result may include small sample size and a complex mechanism of lactate elevation. Lactate is a ubiquitously produced and utilized metabolite that has a core place in nearly all energy-related pathways in humans. There is no consensus on the source, clearance, and metabolic functions of lactate in sepsis, and even we are not certain if high levels of lactate provide protection in sepsis. Understanding the pathophysiology and clinical significance of lactate is difficult. Hyperoxia is no longer a complete explanation for elevated lactate in sepsis, and mitochondrial dysfunction and decreased PDC activity have also been questioned. Research indicated 51 that the early elevation in muscle lactate concentration during LPS infusion was not attributable to the mitochondrial dysfunction or PDC inhibition. Even if the mitochondrial function is normal, lactate levels appear to rise when pyruvate generation surpasses PDC to Acetyl Coenzyme A conversion. Elevated lactate is more likely to be a metabolic stress response under stress rather than just the result of cellular oxygenation problems in sepsis. Faced with the complexity of lactate, Sirt3, an indicator of regulating mitochondrial metabolism, is difficult to achieve desirable results in studies with small sample sizes. We still hope to have a large sample study in the future to investigate the correlation between Sirt3 and lactate.
Even though the cause of lactate production is not sufficiently clear, mitochondrial dysfunction remains a significant element in the formation of increased lactate during the septic shock phase. Lactate responds too slowly to be used to guide acute changes in sepsis 52 , Sirt3 may be an earlier indicator of the phenomenon of cellular hypoxia than lactate. So we tried to explore the potential of Sirt3 for identifying patients at risk of developing septic shock whose lactate cannot be distinguished from sepsis timely, but unfortunately, due to only 2 sepsis patients progressed to septic shock when we regrouped (on D3 and D5) during the study, we did not get such results. We may need to find a larger sample size to verify again. But we found that Sirt3 compensates for lactate deficiency in response to the phenomenon of "cytopathic hypoxia" in septic shock. Sirt3 allows high levels of circulating lactate as an indicator of impaired cellular oxygen metabolism. ScvO 2 is an indicator of the balance between oxygen supply and oxygen consumption, and it can be easily disturbed by several factors, such as mitochondria, hemoglobin, cardiac output, oxygen partial pressure, and patient agitation. But ScvO 2 cannot provide a clear explanation for the imbalance of systemic oxygen supply and demand, which is the limitation of ScvO 2 , especially during septic shock. Current research suggests that impaired microcirculation and mitochondria function cause both an insufficient supply and ineffective utilization of oxygen, and these two factors affect tissue oxygen utilization independently 53 . We found that Sirt3 has a strong correlation with ScvO 2 , but this correlation remains limited to the septic shock group, which reflects poorer cellular oxygen utilization during septic shock. Mitochondria are the main site for the utilization of oxygen, we discovered that there was a significant difference in ScvO 2 between the low Sirt3 and high Sirt3 groups when the factors that influence ScvO 2 did not differ statistically, but this result did not appear between the sepsis group and septic shock groups, which implied that Sirt3 had the potential to be an indicator of cellular oxygen utilization in response to septic shock.
The massive release of inflammatory factors is closely related to mitochondrial metabolism. During sepsis, immune cells switch to glycolysis, numerous metabolites (like lactate, citrate, and succinate), produced by glycolysis and fractured TCA, accumulate in the cell to facilitate signaling and epigenetic interactions which drive the inflammatory response and secrete a large number of inflammatory factors (TNF-α, IL-6) 54 . Sirt3 is the bridge between mitochondrial metabolism and immune cell activity, and downregulation of Sirt3 reprograms mitochondrial metabolism and promotes macrophage death 55 . Sirt3 can also affect immune cell survival by regulating mitochondrial homeostasis 56 . Our results showed that the lower the level of Sirt3, the worse the indicators of inflammation (TNF-α, IL-6) and infection (PCT), a result that was expected.
Mitochondria's role in diseases is no longer limited to provide energy to cells, they also play a role in signaling, gene control, cellular calcium regulation, and activation of cell death pathways. Sirt3 influences mitochondrial intracellular function through the homeostatic regulation of mitochondrial fission 57 , biosynthesis 58  www.nature.com/scientificreports/ autophagy 59 , in addition to altering their metabolic activity. The results of this study predicted that patients with lower serum Sirt3 levels have a worse prognosis, and our survival analysis backed up this prediction. The clinical significance of Sirt3 in sepsis was confirmed by our study.

Limitation
Despite the findings, there are still many limitations. In our study, we didn't find a stronger correlation between Sirt3 and the sepsis group, and we also didn't find a correlation between lactate and Sirt3, as well as we failed to use Sirt3 to identify patients at risk of developing septic shock from sepsis, which could be due to the limited sample size. This is a single-center study, so generalizability may be limited, and it's also unclear whether different treatments influenced the findings. In the future, we hope for bigger multicenter follow-up cohort studies to look into the role of Sirt3 in sepsis.

Conclusion
Sirt3 is a promising biomarker for the diagnosis of sepsis and predicting mortality risk in septic patients. Low serum levels of Sirt3 are significantly correlated with the disease severity. In septic shock, Sirt3 can bridge the gap in the specific response of lactate to the "cytopathic hypoxia". Patients with low levels of Sirt3 have a worse prognosis and lower 28-day survival.

Data availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.