The expression and significance of protease activated receptor 2 in ovarian epithelial carcinoma / 中华肿瘤杂志

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.

Design, synthesis and antiplatelet activity evaluation of novel quinoxaline antagonists of protease activated receptor 4 (PAR4) / 药学学报

Twenty-five compounds of novel quinoxaline-based scaffold with antiplatelet activity were designed and synthesized on the basis of previous quinoxaline analogues, and the structures were confirmed by 1H NMR, 13C NMR, and MS. The antiplatelet activity was evaluated, structure-activity relationship (SAR) study was summarized and the selectivity of PAR4 was confirmed by calcium mobilization assays. It was indicated that compound 14a, 14g, 13i, 13p showed moderate activity against PAR4, especially, the activity of compound 14g (IC50 = 0.26 μmol·L-1) was 6.7 times than the lead compound A (IC50 = 1.73 μmol·L-1). Therefore, 2,3-dihydro-[1,4]dioxino[2,3-g]quinoxaline and [1,3]dioxolo[4,5-g]quinoxaline derivatives are promising compounds for the discovery of novel antiplatelet agents. It is worthy of further research to develop highly effective and selective PAR4 antagonists.

Pharmacological blockade of protease-Activated Receptor 2 improves airway remodeling and lung inflammation in experimental allergic asthma

Abstract Protease-activated receptors (PARs) are metabotropic G-protein-coupled receptors that are activated via proteolytic cleavage of a specific sequence of amino acids in their N-terminal region. PAR2 has been implicated in mediating allergic airway inflammation. This study aims to study the effect of PAR2 antagonist ENMD1068in lung inflammation and airway remodeling in experimental asthma. Allergic lung inflammation was induced in sensitized BALB/c mice through intranasal instillations of ovalbumin (OVA), and mice were pretreated with ENMD1068 1 hour before each OVA challenge. Bronchoalveolar lavage fluid (BALF) was collected, and the lungs were removed at different time intervals after OVA challenge to analyze inflammation, airway remodeling and airway hyperresponsiveness. Ovalbumin promoted leukocyte infiltration into BALF in a PAR2-dependent manner. ENMD1068 impaired eosinophil peroxidase (EPO) and myeloperoxidase (MPO) activity in the lung parenchyma into BALF and reduced the loss of dynamic pulmonary compliance, lung resistance in response to methacholine, mucus production, collagen deposition and chemokine (C-C motif) ligand 5 expression compared to those in OVA-challenged mice. We propose that proteases released after an allergen challenge may be crucial to the development of allergic asthma in mice, and PAR2 blockade may be useful as a new pharmacological approach for the treatment of airway allergic diseases.

Ca mobilization and signaling pathways induced by rRgpB in human gingival fibroblast / 浙江大学学报·医学版

: To assess the () recombinant gingivalis gingipain R2 (rRgpB)-induced Ca mobilization in human gingival fibroblast (HGF) mediated by protease-activated receptor (PAR) and its downstream signal transduction pathways. : Flow cytometry was used to detect the expression of PAR in HGF. The proliferation of HGF was measured by CCK-8. The dynamic changes of intracellular Ca concentration in HGF induced by rRgpB and the blocking effect of PAR-1 antagonist were observed by laser confocal microscopy. Western blot was performed to determine the phosphorylation levels of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (p38 MAPK) and p65 in HGF. : PAR-1 and PAR-3 were expressed in HGF, and the rRgpB could promote the proliferation of HGF. rRgpB caused a transient increase in [Ca], which could be completely suppressed by vorapaxar, a PAR-1 antagonist. The phosphorylation levels of JNK, ERK1/2 and p65 were significantly up-regulated after the induction of rRgpB for and (all <0.05), which was completely inhibited by vorapaxar. However, the phosphorylation level of p38 MAPK had no significant change after rRgpB stimulation. : rRgpB causes an increase in [Ca] in HGF mediated by PAR-1. JNK, ERK1/2 and nuclear factor-κB may be involved in intracellular signal transduction after PAR-1 activation.

Protease-Activated Receptors 2-Antagonist Suppresses Asthma by Inhibiting Reactive Oxygen Species-Thymic Stromal Lymphopoietin Inflammation and Epithelial Tight Junction Degradation

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.

Chemical constituents from heartwoods of Caesalpinia sappan with antiplatelet aggregation activities / 中草药·英文版

Objective: To clarify the active constituents of the heartwoods of Caesalpinia sappan, a traditional Chinese medicine with the functions of promoting blood circulation (Huoxue in Chinese) and removing blood stasis (Quyu in Chinese). Methods: The chemical constituents were isolated and purified by combination of silica gel and Sephadex LH-20 column chromatography, along with semipreparative HPLC. Their chemical structures were established by multiple spectroscopic methods and comparison with literature data. The in vitro antiplatelet aggregation activities were evaluated using mouse platelet induced by AYPGKF-NH2, a gold agonist of protease-activated receptor 4 (PAR4). Results: Two new phenols, methyl 2-(4,4′,5′-trihydroxy-2′-(methoxymethyl) biphenyl-2-yloxy) acetate (1) and 1′-methylcaesalpin J (2), together with 24 known compounds (3–26), were isolated from the heartwoods of C. sappan. Among them, sappanchalcone (16) and brazilin (20) showed inhibitory activities against mouse platelet aggregation with IC50 values of 114.8 µmol/L and 100.8 µmol/L, respectively. Conclusion: Antiplatelet compounds from C. sappan targeting at PAR4 are reported for the first time.

Effect of protease activated receptor 2 on colonic motility in diabetes mice / 上海交通大学学报（医学版）

Objective • To observe the effect of protease activated receptor 2 (PAR2) on the colonic motility in diabetic mice and investigate the mechanism. Methods • The mouse model of type 1 diabetes mellitus was established by intraperitoneal injection of streptozotocin. The smooth muscle strips and segments of colons were isolated. The effects of PAR2 agonist on colonic motility were observed by muscle strip tension contraction and colonic migrating motor complex experiments. The effect of small conductance calcium-activated potassium channel (SK3 channel) antagonist on it was also observed. Results • PAR2 agonist inhibited colonic motility and colonic smooth muscle was more sensitive to PAR2 agonist in diabetic mice. PAR2 agonist-induced inhibition was inhibited by SK3 channel antagonist. Conclusion • PAR2 activity in diabetic mice colons is significantly enhanced, which may inhibit colonic motility through SK3 channel.

Expression of protease-activated receptor 2 in uveal melanoma and its effect on cell proliferation and invasion / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the expression of protease-activated receptor 2(PAR2)in uveal melanoma(UM), and the effects of silencing the expression of PAR2 gene on proliferation and invasion of human UM cell line M23. <p>METHODS: A total of 45 patients(45 eyes)with UM who underwent surgical treatment with complete information in our hospital were selected from February 2012 to December 2017. In the same period, 30 patients(30 eyes)who underwent eyeball removal due to ocular trauma and most of the uvea were normal were selected. Real-time quantitative PCR was used to detect the expressions of PAR2 gene in UM and normal choroidal tissues. M23 cells were cultured and divided into PAR2 interference group, negative control sequence group and blank group. Real-time quantitative PCR was used to detect the expression of PAR2 gene in cells. MTT assay was used to detect cell proliferation, and transwell assay was used to detect cell migration and invasion. <p>RESULTS: The relative expression level of PAR2 mRNA was 1.73±0.13 in UM tissues, and 1.06±0.10 in normal choroid tissues(<i>t</i>=23.732, <i>P</i><0.001). The relative expression level of PAR2 mRNA in UM tissues was associated with pathological type, scleral invasion, optic disc involvement and extraocular growth(<i>P</i><0.05). The relative expression level of PAR2 mRNA in PAR2 interference group was lower than that in negative control sequence group and blank group(<i>P</i><0.05). The absorbance <i>A</i> values at 24h, 48h, 72h and 96h in the PAR2 interference group cells were lower than those in negative control sequence group and blank group(<i>P</i><0.05). The number of migrated cells and the number of invasive cells in PAR2 interference group were lower than those in negative control sequence group and blank group(<i>P</i><0.05). <p>CONCLUSION: PAR2 was highly expressed in UM tissues and was associated with high risk of tumor metastasis. Specific silencing of PAR2 gene expression in M23 cells could effectively inhibit cell proliferation, migration and invasion.

The role of protease activated receptor 2 in visceral sensitivity of irritable bowel syndrome patients / 西安交通大学学报(医学版)

ABSTRACT:Objective To investigate the role of PAR2 in the visceral sensitivity of IBS patients by observing the expression of rectal PAR2 in patients with irritable bowel syndrome and the effect of exogenous PAR2 on visceral sensitivity.Methods Based on Rome III criteria,16 patients with constipation predominant IBS (IBS-C),18 patients with diarrhea predominant IBS (IBS-D ),and 1 8 controls were selected from our hospital inpatient and outpatient departments. All the patients received colonoscopic examination and rectal mucosa biopsies. The expression of rectal mucosa PAR2 was observed by immunohistochemistry and Real-time fluorescence quantitative PCR.We studied the abdominal reactions by administering the PAR2 agonist in the rectal mucosa of rats to explore whether PAR2 is involved in visceral sensitivity.Results The results of PAR2 immunohistochemistry showed that PAR2 was mainly expressed in intestinal epithelial cells,especially in the villi;in addition,endothelial cells were also found positive.While the integral optical density (IA)of PAR2 expression did not significantly differ between IBS-D or IBS-C patients and controls according to the IPP image analysis.The PAR2 mRNA level in IBS-D or IBS-C patients was not significantly different from that of the control group by Real-time PCR analysis.There was no significant difference between the IBS-D and IBS-C groups.The EMG activity significantly increased in a volume-dependent manner during the rectal balloon expansion in the PAR2 agonist group.However,there was little EMG activity when the balloon was not dilated.The area under the curve in the PAR2 agonist group with 0 mL,0.4 mL and 0.6 mL of distension volume did not differ compared with that of the vehicle group.When the balloon volume increased to 0.8 mL,1.0 mL and 1.2 mL,the EMG activity was statistically significant (P<0.01)in the PAR2 agonist group compared with the control group.Conclusion PAR2 is highly expressed in the rectal mucosa of IBS patients.Administration of exogenous PAR2 agonist increases visceral sensitivity,suggesting that PAR2 is involved in visceral sensitivity.

Effect of thrombin on malignant biological behavior of esophageal cancer cell line Eca109 / 肿瘤研究与临床

Objective To study the effect of thrombin on proliferation and invasion of esophageal cancer cell line Eca109, and to explore its possible mechanism. Methods The proliferation and invasion of Eca 109 cells treated with thrombin were detected by MTT and Transwell assay, respectively. The activity of matrix metalloproteinase 2 (MMP-2) and MMP-9 in the supernatant of Eca109 cells was detected by gelatin zymography. Reverse transcription polymerase chain reaction (PCR) and immunocytochemistry were used to study the mRNA expression of protease-activated receptor 1 (PAR-1), the important receptor of thrombin, and subcellular localization of PAR-1 protein in Eca109 cells, respectively. Results Thrombin could promote Eca109 cells proliferation in a dose-dependent manner. Cell proliferative rates of 0.5 U/ml and 1.0 U/ml thrombin were 34.38 % and 57.19 %, respectively (P< 0.05). Compared to that of control group, the number of Eca109 cells incubated with 1.0 U/ml thrombin invading through the basement membrane of Transwell was increased (303.33 ±6.66 vs. 116.33 ±11.51, P< 0.05). When treated with various concentrations of thrombin for 24 h, the activities of MMP-2 and MMP-9, especially MMP-9, in the supernatant of Eca109 cells were increased in a dose-dependent manner. Eca109 cells expressed PAR-1 mRNA, and PAR-1 protein was mainly located on the cellular membrane. Conclusion Thrombin increases proliferation and invasion of esophageal cancer Eca109 cells and enhances the activities of MMP-2 and MMP-9 in cells supernatant, which might be induced by activation of PAR-1 located on cellular membrane.

Research progress in medicines on protease-activated receptor 2 / 药学学报

Protease-activated receptor 2 (PAR2) is a member of protease-activated receptors (PARs). PAR2 distributed in tissues and cells (such as skin, airway epithelial cell, pancreas, etc.) has a broad biological effects, and is involved in pathogenesis of many diseases, such as mechanical pain, asthma, pain of pancreatic cancer, inflammation, pruritus, etc. Intervention of PAR2 will help us to identify the role of PAR2 in the mechanisms of diseases and in the development of new drugs. This article concentrates on the research progress of agonist, antagonist, and pepducin on PAR2.

Change in endogeous hydrogen sulfide in patients with acute pancreatitis and its relationship to coagulation function / 中华危重病急救医学

Objective To study the change in endogenous hydrogen sulfide (H2S) in patients with acute pancreatitis and its relationship to coagulation function. Methods A prospective case control study was conducted. Forty patients with mild acute pancreatitis (MAP group) and 40 with severe acute pancreatitis (SAP group) admitted to Yiwu Central Hospital in Zhejiang Province from December 2002 to March 2015 were enrolled. Forty healthy persons served as control (healthy control group). Blood was collected to determine the levels of H2S, blood coagulation factor Ⅷ (FⅧ), von Willebrand factor (vWF), plasminogen (PLG), antithrombin (AT), platelet count (PLT), tissue factor (TF), tumor necrosis factor-α (TNF-α), and protease activated receptor-1 (PAR-1). The correlations among the above parameters were analyzed. Results There was no statistical significance in sex, age, body weight and time of disease among three groups, indicating it was comparable among the groups. Compared with healthy control group, the levels of H2S, FⅧ, vWF, TF, TNF-α, and PAR-1 in MAP and SAP groups were significantly elevated [H2S (μmol/L): 67.42±6.34, 112.47±12.69 vs. 42.57±4.18, FⅧ: (67.5±5.8)%, (82.3±4.7)% vs. (57.2±6.4)%, vWF: (112.6±9.7)%, (142.5±12.5)% vs. (76.4±8.2)%, TF (ng/L): 45.27±4.34, 64.76±6.25 vs. 18.15±1.89, TNF-α (ng/L): 197.67±13.62, 324.72±25.54 vs. 20.08±2.57, PAR-1 (fluorescence intensity): 32.16±4.43, 56.12±7.07 vs. 12.27±2.12, all P < 0.01], and PLG and AT activity were significantly decreased [PLG: (52.4±4.7)%, (36.7±3.2)% vs. (62.1±5.6)%, AT: (43.2±6.9)%, (35.5±5.4)% vs. (53.6±6.1)%, all P < 0.01]. The changes in the parameters in SAP group were more remarkable than those in MAP group (all P < 0.01). PLT in SAP group was significantly lower than that in healthy control and MAP groups (×109/L: 8.5±1.1 vs. 15.7±2.8, 12.4±1.9, both P < 0.01). H2S was positively correlated with FⅧ, vWF, TF, TNF-α, and PAR-1 (r value was 0.56, 0.61, 0.72, 0.66, 0.64, respectively, all P < 0.01), and it was negatively correlated with PLG and AT (r value was -0.64, -0.57, both P < 0.01). Conclusion As an inflammatory factor, endogenous H2S deteriorates coagulation function in patients with acute pancreatitis by up-regulating TF, TNF-α, and PAR-1.

Role of Coagulation Factor 2 Receptor during Respiratory Pneumococcal Infections

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.

Vorapaxar, a novel oral antiplatelet drug.

Vorapaxor is first in the class of protease activated receptor 1 (PAR 1) antagonists. It acts by inhibiting the binding of thrombin to PAR 1 and thereby prevents platelet aggregation. USFDA approved it in May 2014 as the results of clinical trials showed that the benefit: risk ratio was high. It is to be used in a dose of 2.5 mg once daily as triple antiplatelet therapy with aspirin and clopidogrel for reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or peripheral arterial disease. Increase in the incidence of intracranial hemorrhage is the major side-effect seen.

Adding a New Anticoagulant of Antiplatelet Agent for Patient Receiving Aspirin after an Acute Coronary Syndrome?--Results form a Pairwise and Network Meta-Analysis of Randomized-Controlled Trials.

Objectives: To synthesize the efficacy and safety outcomes from randomized-controlled trials (RCTs) regarding new oral anticoagulant, protease-activated receptor-1 (PAR-1) antagonist, and warfarin adjunctive to aspirin for patients after acute coronary syndrome (ACS) via pair-wise and network meta-analyses. Methods: A comprehensive literature search was performed in Embase, Medline, Cochrane Library Web of Knowledge, and Scopus. The pair-wise meta-analysis was undertaken respectively to each agent/treatment category via Revmen 5.1. In order to estimate the relative efficacy of each agent/treatment category whilst preserving the randomized comparisons within each trial, a Bayesian network meta-analysis was conducted in WinBUGS using both fixed- and random-effects model. Covariate analysis was performed to explore the effects of length of follow-up and age of subject on the final results. Results: In total, 23 RCTs were included in the meta-analysis. As shown by the results (OR,95%CI) for the pair-wise meta-analysis, new oral anticoagulants (0.85, [0.78, 0.93] and 3.04, [2.21, 4.19]), PAR-1 antagonists (0.80, [0.52, 1.22] and 1.55, [1.25, 1.93]) and warfarin (0.87, [0.74, 1.02] and 1.77, [1.46, 2.14]) might be able to provide better outcome in the incidences of major adverse events (MAE) but with higher bleeding risk comparing to aspirin treatment alone. Based on the model fit assessment, the random-effects model was adopted. The network meta-analysis (treatment effect comparing to aspirin lone) identified ximelagatran (-0.3044, [-0.8601, 0.2502]), dabigatran (-0.2144, [-0.8666, 0.4525]), rivoroxaban (-0.2179, [-0.5986, 0.1628]) and vorapaxar (- 0.2272, [-0.81, 0.1664]) produced better improvements in MAE incidences whereas vorapaxar (0.3764, [-0.4444, 1.124]), warfarin (0.663, [0.3375, 1.037]), ximelagatran (0.7509, [-0.4164, 2.002]) and apixaban (0.8594, [-0.0049, 1.7]) produced less major bleeding events. The indirect comparisons among drug category (difference in incidence comparing to aspirin lone) showed new oral anticoagulants (-0.1974, [-0.284, -0.111]) and PAR-1 antagonists (-0.1239, [-0.215, -0.033]) to besuperior to warfarin (-0.1004, [-0.166, -0.035]) in the occurrences of MAE whereas PAR-1 antagonists (0.4292, [0.2123, 0.6476]) afforded better outcomes in major bleeding events against warfarin (0.5742, [0.3889, 0.7619]) and new oral anticoagulants (1.169, [0.8667, 1.485]). Conclusion: Based on the study results, we cannot recommend the routine administration of new oral anticoagulant as add-on treatment for patients after ACS. However, for ACS patients comorbid with atrial fibrillation, new oral anticoagulant might be superior to warfarin in both efficacy and safety outcomes.

Role of PAR-2 in tryptase mediated IEC-6 cell injury / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the role of protease activated receptor-2 ( PAR-2 ) in the process of tryptase mediated IEC-6 cell injury.METHODS:The rat intestinal epithelial cell line IEC-6 was treated with tryptase at different concentrations (1 μg/L, 10 μg/L, 100μg/L and 1 000μg/L) in the presence or absence of PAR-2 antagonist FSLLRY-NH2 for 12 h respectively.The cell survival rate was detected by MTT assay.The protein levels of PAR-2 and cleaved-caspase 3 were determined by Western blotting.The LDH activity was also measured.RESULTS:Compared with control group, the cell survival rates were significantly decreased in 100 μg/L and 1 000 μg/L tryptase treated groups, the LDH activities were significantly increased in 10 μg/L to 1 000 μg/L tryptase treated groups, and the protein levels of PAR-2 and cleaved caspase 3 were significantly increased in 100μg/L and 1 000μg/L tryptase treated groups (P<0.05).Com-pared with 1 000 μg/L tryptase treated group, the LDH activity and cleaved caspase 3 protein level were dramatically de-creased while the survival rate was significantly increased in the presence of PAR-2 antagonist FSLLRY-NH2 (P<0.05). CONCLUSION:Tryptase induces IEC-6 cell injury in a dose-dependent manner by activating PAR-2.

Protective effects of activated protein C on neurovascular unit in a rat model of intrauterine infection-induced neonatal white matter injury / 华中科技大学学报（医学）（英德文版）

Activated protein C (APC), a natural anticoagulant, has been reported to exert direct vasculoprotective, neural protective, anti-inflammatory, and proneurogenic activities in the central nervous system. This study was aimed to explore the neuroprotective effects and potential mechanisms of APC on the neurovascular unit of neonatal rats with intrauterine infection-induced white matter injury. Intraperitoneal injection of 300 μg/kg lipopolysaccharide (LPS) was administered consecutively to pregnant Sprague-Dawley rats at embryonic days 19 and 20 to establish the rat model of intrauterine infection- induced white matter injury. Control rats were injected with an equivalent amount of sterile saline on the same time. APC at the dosage of 0.2 mg/kg was intraperitoneally injected to neonatal rats immediately after birth. Brain tissues were collected at postnatal day 7 and stained with hematoxylin and eosin (H&E). Immunohistochemistry was used to evaluate myelin basic protein (MBP) expression in the periventricular white matter region. Blood-brain barrier (BBB) permeability and brain water content were measured using Evens Blue dye and wet/dry weight method. Double immunofluorescence staining and real-time quantitative PCR were performed to detect microglial activation and the expression of protease activated receptor 1 (PAR1). Typical pathological changes of white matter injury were observed in rat brains exposed to LPS, and MBP expression in the periventricular region was significantly decreased. BBB was disrupted and the brain water content was increased. Microglia were largely activated and the mRNA and protein levels of PAR1 were elevated. APC administration ameliorated the pathological lesions of the white matter and increased MBP expression. BBB permeability and brain water content were reduced. Microglia activation was inhibited and the PAR1 mRNA and protein expression levels were both down-regulated. Our results suggested that APC exerted neuroprotective effects on multiple components of the neurovascular unit in neonatal rats with intrauterine infection- induced white matter injury, and the underlying mechanisms might involve decreased expression of PAR1.

Suppression of Peripheral Sympathetic Activity Underlies Protease-Activated Receptor 2-Mediated Hypotension

Protease-activated receptor (PAR)-2 is expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although some reports have suggested involvement of a neurogenic mechanism in PAR-2-induced hypotension, the accurate mechanism remains to be elucidated. To examine this possibility, we investigated the effect of PAR-2 activation on smooth muscle contraction evoked by electrical field stimulation (EFS) in the superior mesenteric artery. In the present study, PAR-2 agonists suppressed neurogenic contractions evoked by EFS in endothelium-denuded superior mesenteric arterial strips but did not affect contraction elicited by the external application of noradrenaline (NA). However, thrombin, a potent PAR-1 agonist, had no effect on EFS-evoked contraction. Additionally, omega-conotoxin GVIA (CgTx), a selective N-type Ca2+ channel (I(Ca-N)) blocker, significantly inhibited EFS-evoked contraction, and this blockade almost completely occluded the suppression of EFS-evoked contraction by PAR-2 agonists. Finally, PAR-2 agonists suppressed the EFS-evoked overflow of NA in endothelium-denuded rat superior mesenteric arterial strips and this suppression was nearly completely occluded by omega-CgTx. These results suggest that activation of PAR-2 may suppress peripheral sympathetic outflow by modulating activity of I(Ca-N) which are located in peripheral sympathetic nerve terminals, which results in PAR-2-induced hypotension.

The hemostatic effect of hemocoagulase in lung cancer radical surgery analysis and its effect on PAR-1 / 中国生化药物杂志

Objective To investigate hemocoagulase hemostatic effect in lung cancer radical surgery and its effect on protease-activated receptor-1 (PAR-1 ).Methods 86 cases of pathologically confirmed lung cancer patients were randomly divided into control group and observation group,which controls were used etamsylate,observation group were used hemocoagulase,then to measure the amount of bleeding,clotting system-related factors before and after surgery,using ELISA assay to detect P-selectin,flow cytometry to measure platelet PAR-1 ,Western Blot to determine lung tissue expression of PAR-1,and the resulting data were statistically analyzed. Results The blood loss of observation group during surgery and postoperative 2d compared with the control group,had a significant difference (P<0.05 );flow cytometry results showed that,PAR-1-positive rate and P-selectin in different time periods in observation group were no statistically significant,while had a statistically significant compared with control group(P<0.05 );Western Blot detected hemocoagulase of lung tissue in patients with PAR-1 had no obvious effect,while control group had significant difference (P<0.05).Conclusion Venin hemocoagulase in lung cancer radical surgery can reduce the amount of bleeding,and be more stable for the role of the coagulation system,as well as less effect on the P-selectin and PAR-1.The results suggest that vonin hemocoagulase might not make patients with systemic hypercoagulable state in patients undergoing radical process with lung cancer,in other words it might not lead to PAR-1 highly expression to cause tumor cell metastasis.

Virtual and experimental screening of protease activated receptor 1 antagonist in plant ingredients / 中草药

Objective: To find the new type of structures with protease activated receptor 1 (PAR-1) inhibition from plant ingredients. Methods: Thirty ingredients were docked into PAR-1, and then, docking score, occupied space, hydrogen bonding, and other indicators were used for virtual screening. In vitro platelet aggregation experiments in guinea pig were performed to screen the activities of all ingredients. Results: Virtual screening suggested that T30 and T21 had the prospects to inhibit PAR-1. Experiment screening showed that T21, T5, T28, and T29 have the real inhibitory effects on PAR-1. Combination analyses of virtual and experimental screening suggested the following results. Residue 258 and area III had the key effects. Hydrogen matching was required at area II. Area IV and V regions mainly need hydrophobic match. The hydrogen bonding played an important role in improving the activity. Conclusion: According to the binding mode of control drug, T21 is found. To examine the binding mode of T5, T28, and T29, their experimental activities suggest a novel action mode which provides a new direction to find the PAR-1 antagonist.