ABSTRACT
Traumatic brain injury (TBI) triggers the activation of the endogenous coagulation mechanism, and a large amount of thrombin is released to curb uncontrollable bleeding through thrombin receptors, also known as protease-activated receptors (PARs). However, thrombin is one of the most critical factors in secondary brain injury. Thus, the PARs may be effective targets against hemorrhagic brain injury. Since the PAR1 antagonist has an increased bleeding risk in clinical practice, PAR4 blockade has been suggested as a more promising treatment. Here, we explored the expression pattern of PAR4 in the brain of mice after TBI, and explored the effect and possible mechanism of BMS-986120 (BMS), a novel selective and reversible PAR4 antagonist on secondary brain injury. Treatment with BMS protected against TBI in mice. mRNA-seq analysis, Western blot, and qRT-PCR verification in vitro showed that BMS significantly inhibited thrombin-induced inflammation in astrocytes, and suggested that the Tab2/ERK/NF-κB signaling pathway plays a key role in this process. Our findings provide reliable evidence that blocking PAR4 is a safe and effective intervention for TBI, and suggest that BMS has a potential clinical application in the management of TBI.
ABSTRACT
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.
Subject(s)
Animals , Dogs , Humans , Adenosine Diphosphate , Blood Platelets , GTP-Binding Proteins , Hemostasis , Pets , Phosphorylation , Platelet Activation , Platelet Aggregation , Receptors, Proteinase-Activated , Thrombin , ThrombosisABSTRACT
Objective:To investigate the modulatory effect of IL-29 on trypsin-induced protease activated receptors (PARs) ex-pression on P815 mast cell.Methods:After P815 mast cells were challenged with different concentrations of IL-29 alone or combined with trypsin for 2 h, 6 h and 16 h, the challenged cells were collected and analysed by flow cytometry to detect the protein expression of PARs on P815 cells, and analysed by real time RT-PCR to detect the mRNA expression of PARs on P 815 cells.Results:Compared with the corresponding control , IL-29 induced significantly decreased expression of PAR-1 at protein and mRNA level on P815 cells, and upregulated PAR-3, PAR-4 mRNA level on P815 cells, whereas IL-29 did little effect on the expressions of PAR-2,3,4 at protein level on P815 cells accordingly.Preincubation of mast cell with IL-29 did not alter trypsin-induced PAR-1 expression on P815 cells, whereas up-regulated expression of PAR-2, 3, 4 were detected when P815 cell were pre-treated with IL-29 before being challenged with trypsin compared with the corresponding control .Conclusion: IL-29 can upregulate trypsin-induced PARs expression on mast cells through which participated in mast cell related inflammation .
ABSTRACT
Among cockroaches (CR) that live in people’shomes, two species, i.e., German CR (Blattella germanica) and American CR (Periplaneta americana) predominate in temperate and tropical areas, respectively. CR is an important source of inhalant indoor allergens that sensitize atopic subjects to (localized) type I hypersensitivity or atopy including allergic rhinitis and atopic asthma. In Thailand the predominant CR species is P. americana. CR allergens are found throughout CR infested houses; the number found in kitchens correlates with the degree of CR infestation while sensitization and reactivation of the allergic morbidity are likely to occur in the living room and bedroom. Levels of the CR allergens in homes of CR allergic Thais, measured by using locally made quantification test kits, revealed that the highest levels occur in dust samples collected from the wooden houses of urban slums and in the cool and dry season. CR allergens are proteins that may be derived from any anatomical part of the insect at any developmental stage. The allergens may be also from CR secretions, excretions, body washes or frass. The proteins may be the insect structural proteins, enzymes or hormones. They may exist as dimers/multimers and/or in different isoforms. Exposure to CR allergens in infancy leads to allergic morbidity later in life. Clinical symptoms of CR allergy are usually more severe and prolonged than those caused by other indoor allergens. The mechanisms of acute and chronic airway inflammation and airway hyper-responsiveness (AHR) have been addressed including specific IgE- and non-IgEmediated mechanisms, i.e., role of proteaseactivated receptor-2 (PAR2). Participation of various allergen activated-CD4+ T cells of different sublineages, i.e., Th2, Th17, Th22, Th9, Th25, Tregs/Th3 as well as invariant NKT cells, in asthma pathogenesis have been mentioned. The diagnosis of CR allergy and the allergy intervention by CR population control are also discussed.
ABSTRACT
Objective To investigate the expression of protease activated receptors(PARs)in mast cells.Methods Reverse transcription polymerase chain reaction(RT-PCR),flowcytometry and immunofluorescent cell staining were used to detect the expression of PARs in mast cell lines P815 and MC/9 at the levels of protein and mRNA.Results Both the P815 and MC/9 of mast cell lines expressed PAR-1,PAR-2,PAR-3 and PAR-4 at either protein or mRNA level.Conclusion The expression of all the four PARs in mast cells were detectable,which may be of significance for the further study on the function of PARs in mast cells.
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AIM: To study the dynamic expression of protease - activated receptors - 1 (PAR- 1 ) after intracerebral hemorrhage (ICH) and the influence of Nao Xue Kang Tablet (NXKT) on it's expression. METHODS: 72 Wistar rats were divided into normal group, ICH model groups (ICH, 6 h, 24 h, 3 d, 7 d), Nao Xue Kang groups (NXKT, 6 h, 24 h, 3 d,7 d).ICH models were produced with the induction of collagenase typeⅦ - S, except normal group. Immunohistochemical method was used to detect PAR- 1 protein and RT- PCR technique was used to detect PAR- 1 mRNA in brain tissues around the haematoma at different time points of different groups. RESULTS: PAR - 1 protein and mRNA were mildly positive in normal group. In ICH model groups, intensity of PAR - 1 expression started to increase at 6 h, and further increased at 24 h. PAR - 1 expression reached the peak at 3 d and began to descend. At 7 d the decent was obvious. At 6 h, 24 h, 3 d, and 7 d time points, the PAR-1 protein positive cell number and PAR- 1 mRNA absorbance ratio in ICH model and NXKT groups were significantly higher than those in normal group ( P ＜0.05 or P ＜ 0.01). The PAR- 1 protein positive cell number and PAR- 1 mRNA absorbance ratio in NXKT group were significantly lower than in ICH model group ( P ＜ 0.05 or P ＜ 0.01 ). CONCLUSION: After ICH,PAR - 1 is continuously activated because of the stimulation of thrombin. Action of thrombin after ICH may be mediated by PAR- 1; NXKT may inhibit the activation of PAR- 1, so the praxiology is improved. This may be one of the main mechanisms that NXKT could facilitate the recovery of nervous function.
ABSTRACT
Objective To explore the expression of protease activated receptors (PARs) on human monocytes. Methods Flow cytometry and RT - PCR was used to detect the expressions of PARs on human monocytes that purified by MACS. Results FACS analysis showed that monocytes expressed PAR - 1 , PAR - 3 , PAR - 4, but not PAR - 2.RT - PCR revealed that monocytes expressed PAR - 1 and PAR -3, but not PAR -2 and PAR -4 mRNA. Conclusion PARs is expressed on human monocyte cells,which may facilitate further investigation of the potential functions of PARs on monocytes.
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Objective:To study the effect of RNA interference(RNAi) on PAR1 gene mRNA expression and the invasive petential of human colon cancer cell lines.Methods:Chemically synthesizeddouble stranded RNA(dsRNA) targeting PAR1 was transfect- ed into human colon cancer cell Lovo using SiPORT Lipid.The transfection eficiency was observed under fluorescence confocal microscopy.Expression of PAR1 mRNA and protein was detected by reverse transcription polymerase chain reaction(RT-PCR) and Western blot.Cell penetrate matrigel capacity were determined by invitro experiment.Results:PAR1-siRNA effectively in- hibited PAR1 mRNA and protion expression(P
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Objective To investigate the actions of PAR1 agonists and thrombin on the secretion of monocyte chemoattractant protein (MCP)-1 from human lung epithelial cells. Methods A549 cells were cultured in a 12-well culture plate. The challenge was performed by addition of various concentrations of PAR1 agonist peptides SFLLR and its reverse peptides RLLFS, thrombin or thrombin inhibitor named hirudin into each well, respectively. After 2 h or 16 h, the reactions were terminated by removal of the supernatant from each well. A sandwich ELISA was used to determine the levels of MCP-1 in supernatants. Results Following 16 h incubation, SFLLR could induce concentration-dependent secretion of MCP-1. The maximum release of MCP-1 was nearly 12-fold more than baseline release. The reverse PAR1 agonists had little effects on MCP-1 release. Thrombin could induce concentration-dependent secretion of MCP-1. As low as 3 000 U/L thrombin could induce MCP-1 release from epithelial cells, and the maximum of accumulated release of MCP-1 was observed with 10 000 U/L thrombin, which was 5-fold more than baseline release. Thrombin inhibitor hirudin could inhibit thrombin induced secretion of MCP-1. The time course showed that the actions of PAR1 agonist peptides SFLLR and thrombin initiated at 2 h and reached their peak at 16 h. Conclusion PAR1 agonist peptides and thrombin are potent secretogogue of MCP-1 release from cultured human lung epithelial cells, and PAR1 antagonists and thrombin inhibitor may possess the ability to inhibit airway inflammation.
ABSTRACT
AIM: To study the dynamic expression of protease-activated receptors-1 (PAR-1) after intracerebral hemorrhage (ICH) and the influence of Nao Xue Kang Tablet (NXKT) on it's expression. METHODS: 72 Wistar rats were divided into normal group, ICH model groups (ICH, 6 h, 24 h, 3 d, 7 d), Nao Xue Kang groups (NXKT, 6 h, 24 h, 3 d,7 d). ICH models were produced with the induction of collagenase typeⅦ-S, except normal group. Immunohistochemical method was used to detect PAR-1 protein and RT-PCR technique was used to detect PAR-1 mRNA in brain tissues around the haematoma at different time points of different groups. RESULTS: PAR-1 protein and mRNA were mildly positive in normal group. In ICH model groups, intensity of PAR-1 expression started to increase at 6 h, and further increased at 24 h. PAR-1 expression reached the peak at 3 d and began to descend. At 7 d the decent was obvious. At 6 h, 24 h, 3 d, and 7 d time points, the PAR-1 protein positive cell number and PAR-1 mRNA absorbance ratio in ICH model and NXKT groups were significantly higher than those in normal group (P