Objective: To investigate the expression and significance of protease activated receptor 2 (PAR2) in ovarian epithelial carcinoma. Methods: PAR2 mRNA expression levels in 410 cases of epithelial ovarian carcinoma and 88 cases of human normal ovary were analyzed from cancer Genome Atlas (TCGA) database and tissue genotypic expression database (GTEx). Immunohistochemical (IHC) staining of PAR2 protein was performed in 149 patients with ovarian cancer who underwent primary surgical treatment at Cancer Hospital of Chinese Academy of Medical Sciences. Then the relationship between mRNA/protein expression of PAR2 and clinicopathological features and prognosis was analyzed. Gene functions and related signaling pathways involved in PAR2 were studied by enrichment analysis. Results: The mRNA expression of PAR2 in epithelial ovarian carcinoma was significantly higher than that in normal ovarian tissue (3.05±0.72 vs. 0.33±0.16, P=0.004). There were 77 cases showing positive and 19 showing strong positive of PAR2 IHC staining among the 149 patients, accounting for 64.4% in total. PAR2 mRNA/protein expression was closely correlated with tumor reduction effect and initial therapeutic effect (P<0.05). Survival analysis showed that the progression free survival time (P=0.033) and overall survival time (P=0.011) in the group with high PAR2 mRNA expression was significantly lower than that in the low PAR2 mRNA group. Multivariate analysis showed tumor reduction effect, initial therapeutic effect were independent prognostic factors on both progression-free survival and overall survival (P<0.05). The progression-free survival (P=0.016) and overall survival (P=0.038) of the PAR2 protein high expression group was significantly lower than that of the low group. Multivariate analysis showed PAR2 expression, initial treatment effect and chemotherapy resistance were independent prognostic factors on both progression-free survival and overall survival (P<0.05). Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), PAR2 target genes were mainly enriched in function related to intercellular connection, accounting for 40%. Gene enrichment analysis (GSEA) showed that the Wnt/β-catenin signaling pathway (P=0.023), the MAPK signaling pathway (P=0.029) and glycolysis related pathway (P=0.018) were enriched in ovarian cancer patients with high PAR2 mRNA expression. Conclusions: PAR2 expression is closely related to tumor reduction effect, initial treatment effect and survival of ovarian cancer patients. PAR2 may be involved in Wnt/β-catenin signaling pathway and intercellular connection promoting ovarian cancer invasion and metastasis.
Female ; Humans ; Carcinoma, Ovarian Epithelial ; Receptor, PAR-2 ; Ovarian Neoplasms/pathology* ; Prognosis ; RNA, Messenger/metabolism*

Platelet activation has a major role in hemostasis and thrombosis. Various agonists including adenosine diphosphate (ADP) and thrombin interact with G protein-coupled receptors (GPCRs) which transduce signals through various G proteins. Recent studies have elucidated the role of GPCRs and their corresponding G proteins in the regulation of events involved in platelet activation. However, agonist-induced platelet activation in companion animals has not been elucidated. This study was designed to characterize the platelet response to various agonists in dog platelets. We found that 2-methylthio-ADP-induced dog platelet aggregation was blocked in the presence of either P2Y₁ receptor antagonist MRS2179 or P2Y₁₂ receptor antagonist AR-C69931MX, suggesting that co-activation of both the P2Y₁ and P2Y₁₂ receptors is required for ADP-induced platelet aggregation. Thrombin-induced dog platelet aggregation was inhibited in the presence of either AR-C69931MX or the PKC inhibitor GF109203X, suggesting that thrombin requires secreted ADP to induce platelet aggregation in dog platelets. In addition, thrombin-mediated Akt phosphorylation was inhibited in the presence of GF109203X or AR-C69931MX, indicating that thrombin causes Gi stimulation through the P2Y₁₂ receptor by secreted ADP in dog platelets. Unlike human and murine platelets, protease-activated receptor 4 (PAR4)-activating peptide AYPGKF failed to cause dog platelet aggregation. Moreover, PAR1-activating peptide SFLLRN or co-stimulation of SFLLRN and AYPGKF failed to induce dog platelet aggregation. We conclude that ADP induces platelet aggregation through the P2Y₁ and P2Y₁₂ receptors in dogs. Unlike human and murine platelets, selective activation of the PAR4 receptor may be insufficient to cause platelet aggregation in dog platelets.
Adenosine Diphosphate ; Animals ; Blood Platelets ; Dogs ; GTP-Binding Proteins ; Hemostasis ; Humans ; Pets ; Phosphorylation ; Platelet Activation ; Platelet Aggregation ; Receptors, Proteinase-Activated ; Thrombin ; Thrombosis

PURPOSE: Protease-activated receptor 2 (PAR2) reportedly triggers the immune response in allergic asthma. We aimed to investigate the mechanism on allergic inflammation mediated by PAR2. METHODS: Human lung epithelial cells (A549 cells) were used for in vitro, and the German cockroach extract (GCE)-induced mouse model was developed for in vivo studies. RESULTS: In A549 cells, the levels of reactive oxygen species (ROS) and thymic stromal lymphopoietin (TSLP) were significantly increased by GCE treatment, but were suppressed by PAR2-antagonist (PAR2-ant) or N-acetylcysteine (NAC) treatment. Claudin-1 was degraded by GCE, and was restored by PAR2-ant or NAC in the cells. In the mouse model, the clinical appearance including bronchial hyperresponsiveness, bronchoalveolar lavage fluid analysis and total immunoglobulin E were significantly suppressed by PAR2-ant or NAC. Moreover, TSLP levels in the lung were suppressed by the same treatments in the lung. Claudin-1 was also degraded by GCE, and was restored by PAR2-ant or NAC. CONCLUSIONS: ROS generation and epidermal tight junction degradation are triggered by protease, followed by the induction of TSLP in allergic asthma. Our findings could suggest that PAR2-ant or anti-oxidants could be considered for allergic diseases as preventive alternatives.
Acetylcysteine ; Animals ; Asthma ; Blattellidae ; Bronchoalveolar Lavage Fluid ; Claudin-1 ; Epithelial Cells ; Humans ; Immunoglobulin E ; Immunoglobulins ; In Vitro Techniques ; Inflammation ; Lung ; Mice ; Oxygen ; Reactive Oxygen Species ; Receptor, PAR-2 ; Receptors, Proteinase-Activated ; Tight Junctions

BACKGROUND/AIMS: Postoperative ileus (POI) is characterized by impaired propulsive function of the gastrointestinal tract after surgery. Although inflammation is considered to be an important pathogenesis of POI, significant data are lacking. We aim to correlate the recovery time of postoperative dysmotility with that of inflammation and mucosal permeability. METHODS: An experimental POI model of guinea pig was used. Contractile activity of the circular muscle of the stomach, jejunum, ileum, and proximal colon was measured through a tissue bath study. Inflammatory cells were counted, and the expression of calprotectin and tryptase were analyzed. The expression of protease-activated receptor 2 (PAR-2), claudin-1, and claudin-2 were analyzed with immunofluorescence. RESULTS: The small bowel and colon showed decreased contractile amplitude in the POI groups compared to control. In contrast to the colon, the contractile amplitude of the small bowel significantly recovered in the POI group at 6 hours after the operation compared to the control group. Inflammation was highly significant in the POI groups compared to the control and sham groups, especially in the colon. Immunofluorescence showed increased PAR-2 expression in the POI groups compared to sham. The decreased claudin-1 expression and increased claudin-2 expression may suggest increased mucosal permeability of the small bowel and colon in the POI groups. CONCLUSIONS: Increased inflammation and mucosal permeability may play an important role in the differential recovery stages in POI. These data may provide further insights into the pathophysiology and potential new therapeutic prospects of POI.
Animals ; Baths ; Claudin-1 ; Claudin-2 ; Colon ; Fluorescent Antibody Technique ; Gastrointestinal Tract ; Guinea Pigs ; Guinea ; Ileum ; Ileus ; Inflammation ; Jejunum ; Leukocyte L1 Antigen Complex ; Permeability ; Receptor, PAR-2 ; Stomach ; Tryptases

Guanxinshutong capsule (GXSTC) is an effective and safe traditional Chinese medicine used in the treatment of cardiovascular diseases (CVDs) for many years. However, the targets of this herbal formula and the underlying molecular mechanisms of action involved in the treatment of CVDs are still unclear. In the present study, we used a systems pharmacology approach to identify the active ingredients of GXSTC and their corresponding targets in the calcium signaling pathway with respect to the treatment of CVDs. This method integrated chromatographic techniques, prediction of absorption, distribution, metabolism, and excretion, analysis using Kyoto Encyclopedia of Genes and Genomes, network construction, and pharmacological experiments. 12 active compounds and 33 targets were found to have a role in the treatment of CVDs, and four main active ingredients, including protocatechuic acid, cryptotanshinone, eugenol, and borneol were selected to verify the effect of (GXSTC) on calcium signaling system in cardiomyocyte injury induced by hypoxia and reoxygenation. The results from the present study revealed the active components and targets of GXSTC in the treatment of CVDs, providing a new perspective to enhance the understanding of the role of the calcium signaling pathway in the therapeutic effect of GXSTC.
Animals ; Animals, Newborn ; Camphanes ; chemistry ; Cardiotonic Agents ; chemistry ; pharmacology ; Cells, Cultured ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Eugenol ; chemistry ; Gene Expression ; drug effects ; Hydroxybenzoates ; chemistry ; Mass Spectrometry ; Models, Biological ; Myocytes, Cardiac ; drug effects ; Nitric Oxide Synthase Type III ; genetics ; Phenanthrenes ; chemistry ; Rats ; Rats, Sprague-Dawley ; Receptor, PAR-1 ; genetics ; Systems Biology

BACKGROUND/AIMS: Although epigallocatechin-3-gallate (EGCG), which is found in high contents in the dried leaves of green tea, has been reported to have an anti-platelet effect, synergistic effects of EGCG in addition to current anti-platelet medications remains to be elucidated. METHODS: Blood samples were obtained from 40 participants who took aspirin (ASA, n = 10), clopidogrel (CPD, n = 10), ticagrelor (TCG, n = 10) and no anti-platelet medication (Control, n = 10). Ex vivo platelet aggregation and adhesion under various stimulators were analyzed by multiple electrode aggregometry (MEA) and Impact-R systems. PAC-1 and P-selectin expressions in human platelets were analyzed by flow cytometry. RESULTS: In MEA analysis, adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP)-induced platelet aggregations were lower in the CPD and the TCG groups; arachidonic acid (AA)-induced platelet aggregation was lower in the ASA group, whereas collagen (COL)-induced platelet aggregations were comparable among four groups. EGCG significantly reduced ADP- and COL-induced platelet aggregation in dose-dependent manner (ADP, p = 0.04; COL, p < 0.01). There were no additional suppressions of platelet aggregation stimulated by AA in the ASA group, and by ADP in the CPD and TCG groups. Moreover, EGCG suppressed shear stress-induced platelet adhesion on Impact-R, and had no effect on P-selectin and PAC-1 expressions. CONCLUSIONS: Ex vivo treatment of EGCG inhibited platelet adhesion and aggregation without changes in P-selectin and PAC-1 expression. There was no additional suppressions in platelet aggregation stimulated by AA in the ASA group and ADP in the CPD and TCG groups.
Adenosine Diphosphate ; Arachidonic Acid ; Aspirin* ; Blood Platelets ; Catechin ; Collagen ; Electrodes ; Flow Cytometry ; Humans ; P-Selectin ; Platelet Aggregation ; Platelet Aggregation Inhibitors ; Receptors, Thrombin ; Tea

Epithelial attachment via the basal lamina on the tooth surface provides an important structural defence mechanism against bacterial invasion in combating periodontal disease. However, when considering dental implants, strong epithelial attachment does not exist throughout the titanium-soft tissue interface, making soft tissues more susceptible to peri-implant disease. This study introduced a novel synthetic peptide (A10) to enhance epithelial attachment. A10 was identified from a bacterial peptide display library and synthesized. A10 and protease-activated receptor 4-activating peptide (PAR4-AP, positive control) were immobilized on commercially pure titanium. The peptide-treated titanium showed high epithelial cell migration ability during incubation in platelet-rich plasma. We confirmed the development of dense and expanded BL (stained by Ln5) with pericellular junctions (stained by ZO1) on the peptide-treated titanium surface. In an adhesion assay of epithelial cells on A10-treated titanium, PAR4-AP-treated titanium, bovine root and non-treated titanium, A10-treated titanium and PAR4-AP-treated titanium showed significantly stronger adhesion than non-treated titanium. PAR4-AP-treated titanium showed significantly higher inflammatory cytokine release than non-treated titanium. There was no significant difference in inflammatory cytokine release between A10-treated and non-treated titanium. These results indicated that A10 could induce the adhesion and migration of epithelial cells with low inflammatory cytokine release. This novel peptide has a potentially useful application that could improve clinical outcomes with titanium implants and abutments by reducing or preventing peri-implant disease.
Amino Acid Sequence ; Animals ; Benzeneacetamides ; chemical synthesis ; pharmacology ; Cattle ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Cytokines ; metabolism ; Dental Implants ; Enzyme-Linked Immunosorbent Assay ; Epithelial Attachment ; drug effects ; Epithelial Cells ; cytology ; metabolism ; Microscopy, Confocal ; Microscopy, Electron, Scanning ; Piperidones ; chemical synthesis ; pharmacology ; Platelet-Rich Plasma ; Receptors, Thrombin ; Surface Properties ; Titanium ; chemistry

Coagulation factor 2 receptor (F2R), also well-known as a protease-activated receptor 1 (PAR1), is the first known thrombin receptor and plays a critical role in transmitting thrombin-mediated activation of intracellular signaling in many types of cells. It has been known that bacterial infections lead to activation of coagulation systems, and recent studies suggest that PAR1 may be critically involved not only in mediating bacteria-induced detrimental coagulation, but also in innate immune and inflammatory responses. Community-acquired pneumonia, which is frequently caused by Streptococcus pneumoniae (S. pneumoniae), is characterized as an intra-alveolar coagulation and an interstitial neutrophilic inflammation. Recently, the role of PAR1 in regulating pneumococcal infections has been proposed. However, the role of PAR1 in pneumococcal infections has not been clearly understood yet. In this review, recent findings on the role of PAR1 in pneumococcal infections and possible underlying molecular mechanisms by which S. pneumoniae regulates PAR1-mediated immune and inflammatory responses will be discussed.
Bacterial Infections ; Blood Coagulation Factors* ; Inflammation ; Negotiating ; Neutrophils ; Pneumococcal Infections* ; Pneumonia ; Receptor, PAR-1 ; Receptors, Thrombin ; Streptococcus pneumoniae

BACKGROUND/AIMS: Previous studies have revealed that mast cells (MCs) may activate the protease-activated receptors and release of neuropeptides involved in the pathogenesis of irritable bowel syndrome (IBS). The levels of protease-activated receptor 2 (PAR-2) and tryptase can contribute to understanding the pathogenesis of IBS. METHODS: Colonoscopic biopsies were performed of 38 subjects (20 with IBS-diarrhea [IBS-D], eight with IBS-constipation [IBS-C], and 10 healthy volunteers). The mRNA and protein levels of tryptase and PAR-2 were assessed by real-time PCR and Western blot. The levels of vasoactive intestinal peptide (VIP), substance P (SP), and calcitonin gene-related peptide (CGRP) were measured by immunohistochemistry, and MCs were counted by toluidine blue staining. RESULTS: Significant increases in the mRNA expression of tryptase (p<0.05, IBS-D, IBS-C vs control) and PAR-2 (p<0.05, IBS-D, IBS-C vs control) and in the tryptase protein level (p<0.05, IBS-D, IBS-C vs control) were detected in IBS. Elevations of MCs, CGRP, VIP and SP (p<0.05, IBS-D vs control) were observed for IBS-D only. CONCLUSIONS: Tryptase levels may upregulate the function of PAR-2, resulting in the release of neuropeptide and they were correlated with clinical symptoms associated with IBS.
Biopsy ; Blotting, Western ; Calcitonin Gene-Related Peptide ; Immunohistochemistry ; Inflammation ; Irritable Bowel Syndrome* ; Mast Cells ; Neuropeptides ; Real-Time Polymerase Chain Reaction ; Receptor, PAR-2* ; Receptors, Proteinase-Activated ; RNA, Messenger ; Substance P ; Tolonium Chloride ; Tryptases* ; Vasoactive Intestinal Peptide

Irritable bowel syndrome (IBS) is the most common disorder referred to gastroenterologists and is characterized by altered bowel habits, abdominal pain, and bloating. Visceral hypersensitivity (VH) is a multifactorial process that may occur within the peripheral or central nervous systems and plays a principal role in the etiology of IBS symptoms. The pharmacological studies on selective drugs based on targeting specific ligands can provide novel therapies for modulation of persistent visceral hyperalgesia. The current paper reviews the cellular and molecular mechanisms underlying therapeutic targeting for providing future drugs to protect or treat visceroperception and pain sensitization in IBS patients. There are a wide range of mediators and receptors participating in visceral pain perception amongst which substances targeting afferent receptors are attractive sources of novel drugs. Novel therapeutic targets for the management of VH include compounds which alter gut-brain pathways and local neuroimmune pathways. Molecular mediators and receptors participating in pain perception and visceroperception include histamine-1 receptors, serotonin (5-hydrodytryptamine) receptors, transient receptor potential vanilloid type I, tachykinins ligands, opioid receptors, voltage-gated channels, tyrosine receptor kinase receptors, protease-activated receptors, adrenergic system ligands, cannabinoid receptors, sex hormones, and glutamate receptors which are discussed in the current review. Moreover, several plant-derived natural compounds with potential to alleviate VH in IBS have been highlighted. VH has an important role in the pathology and severity of complications in IBS. Therefore, managing VH can remarkably modulate the symptoms of IBS. More preclinical and clinical investigations are needed to provide efficacious and targeted medicines for the management of VH.
Abdominal Pain ; Central Nervous System ; Gonadal Steroid Hormones ; Humans ; Hyperalgesia ; Hypersensitivity* ; Irritable Bowel Syndrome* ; Ligands ; Pain Perception ; Pathology ; Phosphotransferases ; Receptors, Adrenergic ; Receptors, Cannabinoid ; Receptors, Glutamate ; Receptors, Opioid ; Receptors, Proteinase-Activated ; Receptors, Serotonin ; Tachykinins ; Tyrosine ; Visceral Pain