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

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

OBJECTIVETo assess the relationship between protease-activated receptor 1 (PAR1) expression in the basilar artery and cerebral vasospasm (CVS) in a rat model of subarachnoid hemorrhage (SAH).

METHODSTwenty-four SD rats were randomized into normal control group, SAH 3-days group, SAH 5-days group and SAH 7-days group. Rat models of SAH were established by two injections of blood into the cisterna magna and the behavioral changes of the rats were observed. The basilar arteries were taken at 3, 5, or 7 days following the modeling for measuring the cross-sectional area of the basilar artery and for immunohistochemical detection of PAR1 expression.

RESULTSThe SAH model rats, especially those in SAH 3-days group, presented with obvious neurological deficits, which was not found in the normal control group. CVS was not observed in the normal control group but occurred in the SAH model rats, which showed reduced cross-sectional area of the basilar artery and worsening spasm over time. The expression level of PAR1 tended to increase gradually in SAH 3-days, SAH 5-days and SAH 7-days groups. Pearson correlation analysis showed an inverse correlation between the expression of PAR1 and the cross-sectional area of the basilar artery (r=-0.779, P<0.01).

CONCLUSIONSThe expression of PAR1 increases significantly in rat basilar artery wall following SAH in positive correlation with the severity of CVS, suggesting the role of thrombin in the pathological process of CVS after SAH.

Animals ; Basilar Artery ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, PAR-1 ; metabolism ; Subarachnoid Hemorrhage ; metabolism ; Vasospasm, Intracranial ; metabolism

OBJECTIVETo investigate the neuro-protective effects of baicalin in Wistar rats with focal cerebral ischemic reperfusion injury.

METHODSNinety adult male Wistar rats weighing 320-350 g were randomly divided into the following groups (n=5): (a) sham control group; (b) vehicle group, subjected to middle cerebral artery occlusion and received vehicle intraperitoneally; (c-e) baicalin groups, which were subjected to the middle cerebral artery occlusion and treated with baicalin 25, 50 and 100 mg/kg, respectively. The neurological scores were determined at postoperative 1, 3 and 7 d after the treatment. The expression of protease-activated receptor-1 (PAR-1), PAR-1 mRNA and Caspase-3 were determined using Western blot, reverse transcription polymerase chain reaction (RTPCR) analysis and immunohistochemistry, respectively.

RESULTSSignificant decrease was noted in the neurological score in the baicalin group compared with that of the vehicle group (P<0.01). Additionally, down-regulation of PAR-1 mRNA, PAR-1 and Caspase-3 was observed in the baicalin groups compared with those obtained from the vehicle group (P<0.01). Compared with the low-dose baicalin group (25 mg/kg), remarkable decrease was noted in neurological score, and the expression of PAR-1 mRNA, PAR-1 as well as Caspase-3 in the high-dose group (P<0.05).

CONCLUSIONBaicalin showed neuro-protective effects in focal cerebral ischemic reperfusion injury through inhibiting the expression of PAR-1 and apoptosis.

Animals ; Apoptosis ; drug effects ; Brain Ischemia ; complications ; drug therapy ; genetics ; pathology ; Caspase 3 ; metabolism ; Flavonoids ; administration & dosage ; pharmacology ; therapeutic use ; Gene Expression Regulation ; drug effects ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Receptor, PAR-1 ; antagonists & inhibitors ; genetics ; metabolism ; Reperfusion Injury ; complications ; drug therapy ; genetics ; pathology

OBJECTIVETo investigate the roles of protease-activated receptors (PARs) in thrombin-induced brain injury and neurogenesis in rats.

METHODSNinety male SD rats were randomly assigned to receive intra-hippocampus injection of NS, thrombin or specific agonists of 3 protease-activated receptors (PAR-1, PAR-3 and PAR-4), respectively. At 1,3 and 7 d after injection, the area of the hippocampus was determined with HE staining, the density and morphology of astrocyte were detected with GFAP staining, degenerated neurons were detected with Fluoro-Jade C staining, and the neurogenesis was examined with DCX staining.

RESULTSCompared to NS injection, the area of the hippocampus significantly increased at 1-3 d and decreased at 7 d after the injection of thrombin and PAR-1 agonist (P<0.05). In addition, injection of thrombin and PAR-1 agonist significantly increased the density of astrocyte and Fluoro-Jade C positive cells at 1-7 d after injection (P<0.05), and significantly increased the density of DCX positive cells at 3-7 d after injection(P<0.05). The injection of PAR-3 agonist and PAR-4 agonist had no affect on the area of the hippocampus, the density of astrocyte, Fluoro-Jade C positive cells and DCX positive cells.

CONCLUSIONThe activation of protease-activated receptor-1 may be related to the thrombin-induced brain injury and neurogenesis in rat hippocampus.

Animals ; Brain Injuries ; chemically induced ; metabolism ; pathology ; Hippocampus ; pathology ; Male ; Neurogenesis ; drug effects ; Rats ; Rats, Sprague-Dawley ; Receptor, PAR-1 ; agonists ; physiology ; Receptors, Thrombin ; agonists ; Thrombin ; toxicity

Animals ; Cardiovascular Diseases ; etiology ; metabolism ; Humans ; Inflammation ; metabolism ; Periodontitis ; complications ; metabolism ; microbiology ; Platelet Aggregation ; physiology ; Porphyromonas gingivalis ; pathogenicity ; RNA, Messenger ; metabolism ; Receptor, PAR-1 ; metabolism ; Receptor, PAR-2 ; genetics ; metabolism ; Receptors, Proteinase-Activated ; metabolism ; Receptors, Thrombin ; metabolism

OBJECTIVETo evaluate the relationship between PAR1 (Protease-Activated Receptor 1) expression and the clinicopathologic features and to investigate the prognostic value of PAR1 expression in hepatocellular carcinoma (HCC) in early stage after curative resection.

METHODSReal-time PCR was used to detect PAR1 expression in 41 pairs of tumors and matched peritumoral samples of HCC in early stage. Prognostic value of PAR1 mRNA expression was evaluated. Meanwhile, another 49 tissue paraffin slices of HCC were tested using immunohistochemistry (Envision) and the prognostic value of PAR1 expression and other clinicopathologic factors were evaluated.

RESULTSPeritumoral PAR1 mRNA expression was significantly increased in HCCs from the patients with tumor recurrence as compared with those without recurrence (P < 0.05). Peritumoral PAR1 protein expression was related to tumor differentiation (P < 0.05). Kaplan-Meier analysis showed that Peritumoral PAR1 protein expression was associated with the overall survival (OS) (P < 0.05) of HCC patients and the time to recurrence (TTR) (P < 0.05). The 1, 3 and 5 -year overall survival time and the cumulative recurrence time in the high PAR1 protein expression group were significantly lower as compared to the low PAR1 expression group in the peritumoral liver tissue.

CONCLUSIONSPeritumoral PAR1 expression is closely associated with the prognosis of early stage HCC patients after curable surgery. PAR1 may be involved in thrombin-mediated invasion process and may be used as a prognostic marker for HCC.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Female ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Postoperative Period ; Prognosis ; Receptor, PAR-1 ; metabolism

OBJECTIVESTo investigate molecular mechanisms of PAR-1 regulation on intracellular Ca²(+) mobilization in lung giant cell carcinoma cells in vitro and its involvement in tumor metastasis.

METHODSFree intracellular Ca²(+) ([Ca²(+)]i) was measured in lung giant cell carcinoma PLA801C and PLA801D cells by confocal microscopy. Sense and anti-sense PAR-1 expression vectors were transfected into PLA801C (C+)and PLA801D(D-) cells, respectively. The effects of PAR-1 expression were investigated by thrombin and TRAP-induced mobilization of [Ca²(+)]i in the C+ and D-cells.

RESULTSThere were significant differences of the mean values of [Ca²(+)]i between PLA801D (59.55) and PLA801C cells (35.46, P < 0.01). The mean [Ca²(+)]i of C+ cells (45.77) was significantly higher than that of its control CV cells (35.46, P < 0.05), and the mean [Ca²(+)]i of D-cells (48.42) was significantly lower than that of its control DV cells (59.55, P < 0.05). The peaks of [Ca²(+)]i of C+ and CV cells were 48.19 ± 9.84 and 45.64 ± 9.87 (P < 0.05) respectively at 80 s and 100 s after thrombin treatment, but were 111.31 ± 25.00 and 52.93 ± 11.21 (P < 0.05) respectively at 60 s after TRAP treatment. The peaks of [Ca²(+)]i of D- and DV cells were 40.71 ± 5.89 and 61.07 ± 21.36 (P < 0.05) respectively at 60 s after thrombin treatment, but were 84.98 ± 11.23 and 102.58 ± 21.48 (P < 0.05) respectively at 40 s after TRAP treatment.

CONCLUSIONSThe high metastatic potential of PLA801D and PLA801C may be related to [Ca²(+)]i of the tumor cells. PAR-1 may play an important role in the metastasis of lung giant cell carcinoma cells by up-regulating the intracellular Ca²(+).

Calcium ; metabolism ; Calcium Signaling ; drug effects ; Carcinoma, Giant Cell ; metabolism ; pathology ; Cell Line, Tumor ; DNA, Antisense ; genetics ; Humans ; Lung Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Receptor, PAR-1 ; genetics ; metabolism ; physiology ; Receptors, Thrombin ; metabolism ; Thrombin ; pharmacology ; Transfection ; Up-Regulation