1-Piperidine Propionic Acid Protects from Septic Shock Through Protease Receptor 2 Inhibition

Abstract

Sepsis is a complex disorder caused by a dysregulated host response to infection, with high levels of morbidity and mortality. Treatment aimed to modulate immune response and maintain vascular function is still one of the major clinical challenges. This study was designed to test the effect of the small molecule 1-Piperidine Propionic Acid (1-PPA) as molecular targeted agent to block protease-activated receptor 2 (PAR2), one of the major modulators of inflammatory response in LPS-induced experimental endotoxemia. In the THP-1 cell line, LPS-induced cytokine expression was inhibited by 1-PPA in a dose-dependent manner. In LPS-injected mice, treatment with 1-PPA was effective in reducing mortality and sepsis-related symptoms and improved cardiac function parameters. After 6 h from LPS injection, a significant decrease in IL-6, IL-1β, and IL-10 was observed in the lung tissue of 1-PPA-treated mice, compared to controls. In these mice, a significant decrease in vasoactive molecules, especially kininogen-1, was also observed, mainly in the liver. Histopathological analysis confirmed typical features of sepsis in different organs and these findings were markedly reduced in mice treated with 1-PPA. These data demonstrate the effectiveness of 1-PPA in protecting the whole organism from sepsis-induced damage.

Keywords: 1-piperidine propionic acid; experimental endotoxemia; protease-activated receptor 2; septic shock.

Figure 1

Effect of 1-Piperidin Propionic Acid in LPS-induced PAR2 and cytokine expression in THP-1 cell line. Quantitative real-time PCR of PAR2 (A) and of inflammatory cytokine genes (IL-1β, TNF-α, IL-6, TGF-β, and CCL2) (B) in the monocytic THP-1 cell line treated for 2 h with LPS (10 ng/mL) in absence or in presence of different concentrations of 1-Piperidin Propionic Acid (1-PPA). Results are expressed as fold increase. * p < 0.05, ** p < 0.005 compared to untreated samples (Mann–Whitney test).

Figure 2

Comparison of human and mouse PAR2 shows conservation in the 1-PPA interaction site. (A) Pairwise alignment of human Protease-Activated Receptor 2 and mouse homologous. Green boxes indicate the main residues involved in the interaction between human PAR2 and 1-PPA. Lined boxes refer to residues that are fully conserved in the mouse homologous in the same position. (B) Superimposition of PAR2 transmembrane domain; human protein (PDB 5NDD) is represented in white, while murine AlphaFold model (AF-P55086-F1-v4) is represented in green. Zoom shows the residues of mouse PAR2 conserved in the binding of 1-PPA.

Figure 3

Effect of early and late administration of 1-PPA on LPS-injected mice. (A) Kaplan Mayer curves of cumulative survival of mice injected with LPS alone (n = 6), with LPS and treated with 1-PPA 190 μMol after 1 h (early administration, n = 6) or after 3 hrs (late administration, n = 6). * p < 0.005 LPS vs. LPS + 1-PPA at 1 or 3 hrs (Log-Rank Mantel Cox test). (B) Clinical conditions monitored over time by the Body Condition Score in LPS-injected mice treated with early or late 1-PPA injection. Results are expressed as median score values (±SEM) in different groups. Dunn’s multiple comparison test was used to compare the results in the different groups at each time point (* 4 hrs: LPS vs. LPS + 1-PPA 1 h and LPS vs. LPS + 1-PPA 3 hrs, p = 0.005; * 8 h: LPS vs. LPS + 1-PPA 1 h and LPS vs. LPS + 1-PPA 3 hrs, p = 0.0004; * 16 hrs: LPS vs. LPS + 1-PPA 1 h: p = ns and LPS vs. LPS + 1-PPA 3 hrs, p = 0.035).

Figure 4

Measurement of cardiac function in LPS-injected mice untreated or treated with 1-PPA. Cardiac function was assessed by ultrasound after 5 hrs in mice injected with LPS and untreated or treated after 1 h with 190 μMol 1-PPA (n = 8/group). Additional control groups of mice (n = 4/group) were not LPS-injected and not treated with 1-PPA (Control) or treated with 1-PPA without previous LPS injection (1-PPA). All measurements were carried out using the Vevo 2100 System (Visualsonics). (A) Representation of cardiac parameters in the different groups of mice. Data are expressed as median and interquartile range (IQR). * p =< 0.05, Mann–Whitney test. (B) Representative examples of cardiac function in mice from the different groups in B-Mode and in M-Mode. CONTROL: untreated control mouse. Blue bars represent ventricular diameter, 1: left ventricular end diastolic diameter, 2: left ventricular end systolic diameter.

Figure 5

Clinical and laboratory testing over time in LPS-injected mice untreated or treated with 1-PPA. (A) Mean rectal temperature profile detected by the VEVO instrument in the different groups of mice. (B) Mean of the clinical score monitored over time. (C) Percentage of the different types of blood cells detected after 6 hrs in the different groups of mice. (D–H) Biochemical parameters, including Prothrombin Time (D), Blood Urea Nitrogen, BUN (E), Total Bilirubin (F), Alanine Aminotransferase, ALT (G), and C Reactive Protein, C Reactive Protein, CRP (H) in the different groups of mice. The results are expressed as Mean values ± SEM (Mann–Whitney test).

Figure 6

Peritoneal fluid analysis after 6 hrs from LPS injection. (A) Example of macroscopic appearance of the peritoneal fluid in an LPS-injected mouse and in an LPS-injected mouse treated with 1-PPA; (B) Cytology from peritoneal fluid of the corresponding mice, note the absence of a significant leukocytic component in the sample treated with 1-PPA; (C) Cell count from the peritoneal fluid in mice treated with LPS (n = 7) and with LPS + 1-PPA (n = 7). The results are expressed as Mean ± SEM * p = 0.0379 (Mann–Whitney test); (D1) Neutrophil and monocytes count in the peritoneal fluid. Left panel: cytofluyorimetric analysis (Gate on CD45+ CD11b+ cells); right panel: graphical representation of the Mean values ± SEM (Mann–Whitney test); (D2,D3) T-lymphocyte subpopulations count in the peritoneal fluid (Gate on CD3+ cells). (D2) CD4 T-lymphocytes: Left panel: cytofluyorimetric analysis; right panel: graphical representation of the Mean values ± SEM in activated (ACT) vs. total (TOT) CD4 positive T-lymphocytes (Mann–Whitney test); (D3) CD8 T-lymphocytes: Left panel: cytofluyorimetric analysis; right panel: graphical representation of the Mean values ± SEM in activated (ACT) vs. total (TOT) CD8 positive T-lymphocytes (Mann–Whitney test).

Figure 7

Histological analysis of the organ injury. (A) injury score of different solid organs after 6 h from LPS administration in untreated mice (n = 12) and in the group of mice treated with 1-PPA 190 μMol (n = 12) * p =< 0.05. The results are expressed as Mean ± SEM (Mann–Whitney test); (B) Representative examples of histological results in the different organs, after hematoxylin-eosin staining in one mouse injected with LPS and in one mouse LPS-injected and treated with 1-PPA. The arrow indicates a large area of leukocyte infiltrate in the aortic valve. Magnification of the panels 5× and inserts 20×. Bar = 10 μm.

Figure 8

Gene expression of cytokines and vasoactive molecules in different organs in LPS-injected mice. Solid organs were harvested after 6 hrs of LPS administration and saline injection in untreated control mice (n = 6) and in the group of mice treated with 190 μMol 1-PPA (n = 6). Results are expressed as Mean ± SEM of gene expression, reported as 2^−ΔΔct relative to basal values. p values were reported only for significant differences, p < 0.05 (Mann–Whitney test). iNOS: inducible nitric oxide synthase; eNOS: endothelial nitric oxide synthase; BR1: bradykinin receptor1; BR2: bradykinin receptor2; KNG1: kininogen-1.