The critical role of pancreatic stone protein/regenerating protein in sepsis-related multiorgan failure

Abstract

Introduction: Multiple organ dysfunction syndrome (MODS) is common in patients with sepsistic admitted to an intensive care unit (ICU) and greatly increases mortality. Pancreatic stone protein/regenerating protein (PSP/Reg) is a type of C-type lectin protein that is overexpressed during sepsis. This study aimed to evaluate the potential involvement of PSP/Reg in MODS development in patients with sepsis.

Materials and methods: The relationship between circulating PSP/Reg levels, patient prognosis, and progression to MODS was analyzed in patients with sepsis admitted to the ICU of a general tertiary hospital. Furthermore, to examine the potential involvement of PSP/Reg in sepsis-induced MODS, a septic mouse model was established per the cecal ligation and puncture procedure, randomized into three groups, and subjected to a caudal vein injection of recombinant PSP/Reg at two different doses and phosphate-buffered saline. Survival analyses and disease severity scoring were performed to evaluate the survival status of the mice; enzyme-linked immunosorbent assays were performed to detect the levels of inflammatory factors and organ-damage markers in murine peripheral blood; terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to measure apoptosis levels in lung, heart, liver, and kidney tissue sections and to visualize the degree of organ damage in the mouse model; myeloperoxidase activity assay, immunofluorescence staining, and flow cytometry were performed to detect neutrophil infiltration levels in vital murine organs and the activation indexes of neutrophils.

Results and discussion: Our findings indicated that Circulating PSP/Reg levels were correlated with patient prognosis and sequential organ failure assessment scores. Furthermore, PSP/Reg administration increased disease severity scores, shortened survival time, increased the TUNEL-positive staining rate, and increased the levels of inflammatory factors, organ-damage markers, and neutrophil infiltration in the organs. Neutrophils can be activated by PSP/Reg to an inflammatory state, both in vivo and in vitro, which is characterized by the increased levels of intercellular adhesion molecule 1 and CD29.

Conclusion: Patient prognosis and progression to MODS can be visualized by monitoring PSP/Reg levels upon ICU admission. Additionally, PSP/Reg administration in animal models exacerbates the inflammatory response and severity of multiorgan damage, which may be accomplished by promoting the inflammatory state of neutrophils.

Keywords: infiltration; multiple organ dysfunction syndrome; neutrophils; pancreatic stone protein; sepsis.

Figure 1

Correlation between circulating Pancreatic stone protein/regenerating protein (PSP/Reg) levels and prognosis as well as progression to multiple organ dysfunction syndrome (MODS) in patients with sepsis admitted to an intensive care unit. (A) Circulating PSP/Reg levels were compared according to disease severity in patients. Mann–Whitney U test showed a significant increase in PSP/Reg levels in patients with septic shock compared with an increase in those with severe sepsis (p < 0.001). (B) PSP/Reg levels were evaluated in patients stratified by Sequential Organ Failure Assessment (SOFA) scores, and Kruskal–Wallis test showed a significant increase in PSP/Reg levels (median and interquartile range) with increasing SOFA scores (p < 0.001). Mann–Whitney U test indicated significant differences in PSP/Reg levels between patients with and without MODS (C) and between survivors and non-survivors of sepsis. (D) ^*p < 0.05, ^**p < 0.01. (E) According to the quartile division of PSP/Reg at admission, the Kaplan–Meier survival curve for 28 days is shown. The receiver operating characteristic curve analysis of PSP/Reg for the prediction of MODS (F) and 28d mortality (G).

Figure 2

Pancreatic stone protein/regenerating protein (PSP/Reg) exacerbates multiple organ failure in a cecal ligation and puncture (CLP) mouse model. Mice were randomly selected to undergo CLP surgery and were intravenously administered with phosphate-buffered saline (PBS), PSP/Reg (40 ng/kg), or PSP/Reg (400 ng/kg) 30 min later. (A) Murine sepsis score at 24 h after CLP was significantly increased by PSP/Reg treatment by one-way analysis of variance (ANOVA). (B) Survival curves and log-rank test showed that compared with PBS, PSP/Reg significantly shortened the survival time of the mice. Serum levels of inflammatory cytokines and organ-damage markers were evaluated at 24 h after CLP. (C) tumor necrosis factor-α levels (ng/mL), (D) interleukin (IL)-6 levels (pg/mL), (E) IL-1β levels (pg/mL), (F) lactate dehydrogenase (U/L), (G) troponin I (ng/mL), (H) creatinine (mg/dL), and (I) lung wet/dry (W/D) ratio were measured. Data are expressed as the mean ± standard error of the mean and analyzed by one-way ANOVA. ^*p < 0.01, ^**p < 0.001, compared with the PBS group.

Figure 3

Pancreatic stone protein/regenerating protein exacerbated the level of cellular apoptosis in multiple organ tissues of cecal ligation and puncture (CLP) mice. After 24 h of CLP surgery, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to assess the level of cellular apoptosis (represented by red fluorescence) in lung (A), heart (B), liver (C), and kidney (D) tissue sections and quantified using the ImageJ software with the TUNEL-positive percentage analyzed by one-way analysis of variance. ^*p < 0.01, ^**p < 0.001 compared with the phosphate-buffered saline group.

Figure 4

Pancreatic stone protein/regenerating protein (PSP/Reg) increased neutrophil infiltration in multiple organs of cecal ligation and puncture (CLP) mice. The level of myeloperoxidase (MPO) activity and neutrophil (Ly6G+) infiltration in multiple organ tissues were measured 24 h after CLP in mice treated with PSP/Reg. (A): lung tissue MPO activity, (B): kidney tissue MPO activity, (C): heart tissue MPO activity, and (D): liver tissue MPO activity. The data were analyzed by one-way analysis of variance. ^*p < 0.01, ^**p < 0.001 compared with the PBS group. Representative images of Ly6G+ (green fluorescence) areas in lung (E), kidney (F), heart (G), and liver (H) tissues. Scale bar, 20 mm.

Figure 5

Pancreatic stone protein/regenerating protein (PSP/Reg) increased the levels of neutrophil surface adhesion molecules. Cecal ligation and puncture mice were stimulated with PSP/Reg 30 min after surgery, and 24 h later, flow cytometry was performed to detect the percentage of intercellular adhesion molecule 1 (ICAM-1)-positive (A) and CD29-positive (B) cells in mouse bone marrow neutrophils. Neutrophils were isolated from the mouse bone marrow and stimulated with 40 ng/ml and 400 ng/ml of PSP/Reg overnight. (C) Representative histograms and MFI quantification analysis of ICAM-1 levels in PMN under 40 ng/ml stimulation. (D) Representative histograms and MFI quantification analysis of CD29 levels in PMN under 400 ng/ml stimulation.The data were analyzed by one-way analysis of variance. ^*p < 0.01, ^**p < 0.001 compared with the PBS group.