Gastrin-releasing peptide receptor antagonist RC-3095 inhibits Porphyromonas gingivalis lipopolysaccharide-accelerated atherosclerosis by suppressing inflammatory responses in endothelial cells and macrophages.

OBJECTIVE: Porphyromonas gingivalis (P. gingivalis), one of the major periodontopathogens, is associated with the progression and exacerbation of atherosclerosis. In this study, we aimed to investigate whether the gastrin-releasing peptide receptor antagonist, RC-3095, could attenuate P. gingivalis LPS-induced inflammatory responses in endothelial cells and macrophages, as well as atherosclerosis in an ApoE-/- mouse model treated with P. gingivalis LPS. METHODS: The effect of RC-3095 on P. gingivalis LPS-induced endothelial inflammation was examined using HUVECs and rat aortic endothelium. THP-1 cells were polarized into M1 macrophages by exposure to P. gingivalis LPS, with or without RC-3095. The effect of RC-3095 on atherosclerosis progression was assessed in high-fat-fed male ApoE-/- mice through injections of P. gingivalis LPS, RC-3095, or a combination of both. RESULTS: RC-3095 significantly reduced P. gingivalis LPS-induced leukocyte adhesion to endothelial cells and aortic endothelium by suppressing NF-κB-dependent expressions of ICAM-1 and VCAM-1. In addition, RC-3095 inhibited the P. gingivalis LPS-induced polarization of M1 macrophages by blocking the MAPK and NF-κB signaling pathways. Moreover, RC-3095 decreased the area of atherosclerotic lesions in ApoE-/- mice, which was accelerated by P. gingivalis LPS injection, and lowered the expressions of ICAM-1 and VCAM-1 in the aortic tissue of mice with atherosclerosis. CONCLUSIONS: RC-3095 can alleviate P. gingivalis LPS-induced endothelial inflammation, macrophage polarization, and atherosclerosis progression, suggesting its potential as a therapeutic approach for periodontal pathogen-associated atherosclerosis.

Protective effects of gastrin-releasing peptide receptor antagonist RC-3095 in an animal model of hepatic ischemia/reperfusion injury.

AIM: We aimed to evaluate effects of RC-3095 on mice with hepatic ischemia followed by reperfusion (I/R) injury and further explore the possible underlying mechanism. METHODS: Mice were subjected to partial hepatic ischemia for 60 min followed by different durations of reperfusion. Levels of gastrin-releasing peptide (GRP) and GRP receptor (GRPR) in the blood and liver were detected by enzyme-linked immunosorbent assay (ELISA) or western blotting (WB) after 3, 6, 12, or 24 h of reperfusion. RC-3095 or normal saline (control) was given i.p. at the time of reperfusion. Expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, and IL-10 in blood and liver samples were examined with ELISA. Neutrophil influx into the liver was assessed by flow cytometry and myeloperoxidase assay. Hematoxylin-eosin staining of the liver and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling assay were used to determine hepatic injury and hepatocellular necrosis. Activation of nuclear factor (NF)-κB and p38/extracellular regulated protein kinase (ERK) mitogen activated protein kinase (MAPK) was investigated with WB. RESULTS: The expression of GRP was upregulated within 3 h after reperfusion and remained elevated for up to 24 h in the liver, whereas GRPR was also upregulated after 3 or 6 h of reperfusion, but returned to baseline levels within 24 h. RC-3095 significantly reduced the inflammatory hepatic injury, liver neutrophil accumulation, and hepatocellular apoptosis, probably by inhibiting activation of NF-κB or p38/ERK MAPK. CONCLUSION: These findings supported that GRP-GRPR played an important role in hepatic I/R injury, and RC-3095 ameliorated liver damage by suppressing the inflammatory response and hepatocellular necrosis.

Gastrin-releasing peptide and its receptor increase arthritis fibroblast-like synoviocytes invasiveness through activating the PI3K/AKT pathway.

Rheumatoid arthritis (RA) is an autoimmune disease that leads to joint destruction. The fibroblast-like synoviocytes (FLS) has a central role on the disease pathophysiology. The present study aimed to examine the role of gastrin-releasing peptide (GRP) and its receptor (GRPR) on invasive behavior of mice fibroblast-like synoviocytes (FLS), as well as to evaluate GRP-induced signaling on PI3K/AKT pathway. The expression of GRPR in FLS was investigated by immunocytochemistry, western blot (WB) and qRT-PCR. The proliferation and invasion were assessed by SRB and matrigel-transwell assay after treatment with GRP and/or RC-3095 (GRPR antagonist), and/or Ly294002 (inhibitor of PI3K/AKT pathway). Finally, AKT phosphorylation was assessed by WB. GRPR protein was detected in FLS and the exposure to GRP increased FLS invasion by nearly two-fold, compared with untreated cells (p<0.05), while RC-3095 reversed that effect (p<0.001). GRP also increased phosphorylated AKT expression in FLS. When Ly294002 was added with GRP, it prevented the GRP-induced increased cell invasiveness (p<0.001). These data suggest that GRPR expression in FLS and that exogenous GRP are able to activate FLS invasion. This effect occurs at least in part through the AKT activation. Therefore, understanding of the GRP/GRPR pathway could be relevant in the development of FLS-targeted therapy for RA.

Long-term behavioral effects of neonatal blockade of gastrin-releasing peptide receptors in rats: similarities to autism spectrum disorders.

Gastrin releasing peptide, the mammalian counterpart of the amphibian peptide, bombesin, has been increasingly implicated in regulating normal brain function as well as in the pathogenesis of psychiatric and/or neurodevelopmental disorders. We have previously shown that the neonatal blockade of the gastrin-releasing peptide receptor (GRPr) in rats produces long-lasting consequences during central nervous system development that are commonly observed in neurodevelopmental disorders such as autism spectrum disorders. The present investigation assessed in further detail, long-term behavioral effects of neonatal GRPr blockade. During postnatal days 1-10, male Wistar rat pups (n=5-10/litter) were injected (subcutaneously) with the GRPr antagonist, RC-3095 (1 mg/kg), or a vehicle (control), twice daily. Following the drug treatment regimen, several behaviors were assessed (starting on postnatal day 14) including specific social behaviors (namely, group huddling characteristics, social interaction, and social approach), restrictive/repetitive and stereotyped behaviors (y-maze, repetitive novel object contact task, observation for stereotypies) and anxiety/fear-related responses (open field, elevated plus maze and contextual fear conditioning). Rats treated neonatally with RC-3095 showed reduced sociability, restrictive interests, motor stereotypies and enhanced learned fear response compared to the controls (vehicle-treated rats). These behavioral abnormalities are consistent with those observed in autism spectrum disorders and provide further evidence that neonatal blockade of GRPr could potentially serve as a useful model to gain a better understanding of the underlying neurodevelopmental disruptions contributing to the expression of autism-relevant phenotypes.