Expression Of Protease-activated Receptors Research Articles

Protease-activated receptor 2 (PAR2) inhibition reduces complement-associated cardiac remodelling in obesity-related heart failure

Abstract Background and Aims Obesity is considered a major risk factor for the development of heart failure (HF) due to lipid accumulation, oxidative stress and inflammation which results in cardiac remodeling. Protease activated receptor 2 (PAR2) has been linked to various cardiovascular and inflammatory diseases. The role of PAR2 has not yet been studied in obesity-related HF. Methods In the experimental mouse study, we used 1-year-old ApoE knockout (ApoE-/-) and ApoE PAR2 double knockout (ApoE-/-PAR2-/-) mice with a C57BL/6J background on a high fat (HF) diet (n=5/ group). Cell culture experiments were performed with human primary cardiac fibroblasts and HepG2 hepatocytes. Patients (n=50) included in the clinical study had a BMI > 25kg/m², symptoms of heart failure (HF), coronary angiography (CA) that excluded coronary artery disease (CAD) and endomyocardial biopsy (EMB) that confirmed the absence of active myocarditis or primary cardiac amyloidosis. Patients were divided into PAR2low (n=22) and PAR2high (n=22) based on the median PAR2 expression measured via qPCR in the EMB of the cohort, and six patients served as healthy controls. Results Evaluation of endomyocardial biopsies (EMB) from overweight/ obese non-ischemic cardiomyopathy (NICM) patients with heart failure (HF) (n=44) revealed that a reduced cardiac PAR2 expression is associated with less cardiac C3 (p≤0.054), C4 (p≤0.041) and C5 gene expression (p≤0.045), as well as plasma levels of complement C3 (1.1g/l± 0.7 vs 2.1g/l±0.2; p≤0.016) and C5b-9 (303.4ng/ml± 215.7 vs 992.7ng/ml± 756.2; p≤0.0002) and as a result, better systolic function i.e. LVEF (46.1%±17.1 vs 36.8%±15.2; p≤0.046) and LV GLS (-14.9%± 5.7 vs -9.5%± 4.6; p≤0.009), less cardiac remodeling and damage. Correspondingly, aged ApoE-/-PAR2-/-mice on a Western diet showed less hepatic and cardiac complement expression (i.e. C1q, C3, C4, C5, C9), cardiac C3 deposition (p≤0.047) and C5b-9 plasma levels (p≤0.049), as well as less cardiac necrosis (p≤0.046) and lower BNP (p≤0.016) than ApoE-/-. PAR2 overexpression in human cardiac fibroblasts (HCFs) independently enhanced and PAR2 antagonism reduced interferon γ (IFNγ)-mediated STAT1-dependent complement C3, C4 and C5 expression. Conclusions Protease-activated receptor 2 regulates complement-mediated cardiac damage and remodeling in HF. The PAR2-IFNγ/STAT1/C3-C4-C5 complement-axis might be a promising prognostic and therapeutic approach to identify and treat high-risk cases of HF in overweight/obese patients.

Protease activated receptor 2 deficiency retards progression of abdominal aortic aneurysms by modulating phenotypic transformation of vascular smooth muscle cells via ERK signaling

Abdominal aortic aneurysm (AAA) is characterized by localized structural deterioration of the aortic wall, leading to progressive dilatation and rupture. Protease activated receptor 2 (PAR2) dependent signaling has been implicated in the pathophysiology of atherosclerosis through the regulation of smooth muscle cell function. However, its role in AAA remains unclear. This study investigates the function and potential mechanism of PAR2 in AAA progression. Angiotensin II (Ang II) and β-aminopropionitrile (BAPN) were administered to wild type (WT) mice to induce AAA. Increased PAR2 expression was observed in the aneurysmal tissues of these mice and in Ang II-treated vascular smooth muscle cells (VSMCs). We demonstrated that PAR2 deficiency markedly inhibited aorta dilatation and vascular remodeling in the AAA model relative to WT mice. Immunohistochemical staining showed significant upregulation of contractile markers and a reduction in synthetic markers in PAR2 knockout mice. Consistent with in vivo results, PAR2 knockdown diminished the effects of Ang II on VSMCs phenotypic switching, resulting in reduced proliferation and migration. Conversely, a PAR2 agonist (SLIGRL) induced the opposite effect, which was partially mitigated by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor (PD98059). This study suggests that PAR2 deficiency restrains aortic expansion and mitigates adverse vascular remodeling in AAA models, mediated in part by the ERK signaling pathway, indicating that PAR2 could be a potential therapeutic target for mitigating AAA development or progression.

Targeting PAR2-mediated inflammation in osteoarthritis: a comprehensive in vitro evaluation of oleocanthal’s potential as a functional food intervention for chondrocyte protection and anti-inflammatory effects

BackgroundOsteoarthritis (OA) is a prevalent degenerative joint disease characterized by chronic inflammation and progressive cartilage degradation, ultimately leading to joint dysfunction and disability. Oleocanthal (OC), a bioactive phenolic compound derived from extra virgin olive oil, has garnered significant attention due to its potent anti-inflammatory properties, which are comparable to those of non-steroidal anti-inflammatory drugs (NSAIDs). This study pioneers the investigation into the effects of OC on the Protease-Activated Receptor-2 (PAR-2) mediated inflammatory pathway in OA, aiming to validate its efficacy as a functional food-based therapeutic intervention.MethodsTo simulate cartilage tissue in vitro, human bone marrow-derived mesenchymal stem cells (BMSCs) were differentiated into chondrocytes. An inflammatory OA-like environment was induced in these chondrocytes using lipopolysaccharide (LPS) to mimic the pathological conditions of OA. The therapeutic effects of OC were evaluated by treating these inflamed chondrocytes with various concentrations of OC. The study focused on assessing key inflammatory markers, catabolic enzymes, and mitochondrial function to elucidate the protective mechanisms of OC. Mitochondrial function, specifically mitochondrial membrane potential (ΔΨm), was assessed using Rhodamine 123 staining, a fluorescent dye that selectively accumulates in active mitochondria. The integrity of ΔΨm serves as an indicator of mitochondrial and bioenergetic function. Additionally, Western blotting was employed to analyze protein expression levels, while real-time polymerase chain reaction (RT-PCR) was used to quantify gene expression of inflammatory cytokines and catabolic enzymes. Flow cytometry was utilized to measure cell viability and apoptosis, providing a comprehensive evaluation of OC’s therapeutic effects on chondrocytes.ResultsThe results demonstrated that OC significantly downregulated PAR-2 expression in a dose-dependent manner, leading to a substantial reduction in pro-inflammatory cytokines, including TNF-α, IL-1β, and MCP-1. Furthermore, OC attenuated the expression of catabolic markers such as SOX4 and ADAMTS5, which are critically involved in cartilage matrix degradation. Importantly, OC was found to preserve mitochondrial membrane potential (ΔΨm) in chondrocytes subjected to inflammatory stress, as evidenced by Rhodamine 123 staining, indicating a protective effect on cellular bioenergetics. Additionally, OC modulated the Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL)/Receptor Activator of Nuclear Factor Kappa-Β (RANK) pathway, suggesting a broader therapeutic action against the multifactorial pathogenesis of OA.ConclusionsThis study is the first to elucidate the modulatory effects of OC on the PAR-2 mediated inflammatory pathway in OA, revealing its potential as a multifaceted therapeutic agent that not only mitigates inflammation but also protects cartilage integrity. The preservation of mitochondrial function and modulation of the RANKL/RANK pathway further underscores OC’s comprehensive therapeutic potential in counteracting the complex pathogenesis of OA. These findings position OC as a promising candidate for integration into nutritional interventions aimed at managing OA. However, further research is warranted to fully explore OC’s therapeutic potential across different stages of OA and its long-term effects in musculoskeletal disorders.

Inhibition of Protease-Activated Receptor-2 Activation in Parkinson's Disease Using 1-Piperidin Propionic Acid.

In Parkinson's disease, neuroinflammation is a double-edged sword; when inflammation occurs it can have harmful effects, despite its important role in battling infections and healing tissue. Once triggered by microglia, astrocytes acquire a reactive state and shift from supporting the survival of neurons to causing their destruction. Activated microglia and Proteinase-activated receptor-2 (PAR2) are key points in the regulation of neuroinflammation. 1-Piperidin Propionic Acid (1-PPA) has been recently described as a novel inhibitor of PAR2. The aim of our study was to evaluate the effect of 1-PPA in neuroinflammation and microglial activation in Parkinson's disease. Protein aggregates and PAR2 expression were analyzed using Thioflavin S assay and immunofluorescence in cultured human fibroblasts from Parkinson's patients, treated or untreated with 1-PPA. A significant decrease in amyloid aggregates was observed after 1-PPA treatment in all patients. A parallel decrease in PAR2 expression, which was higher in sporadic Parkinson's patients, was also observed both at the transcriptional and protein level. In addition, in mouse LPS-activated microglia, the inflammatory profile was significantly downregulated after 1-PPA treatment, with a remarkable decrease in IL-1β, IL-6, and TNF-α, together with a decreased expression of PAR2. In conclusion, 1-PPA determines the reduction in neuroglia inflammation and amyloid aggregates formation, suggesting that the pharmacological inhibition of PAR2 could be proposed as a novel strategy to control neuroinflammation.

Detrimental interactions of hypoxia and complement MASP-1 in endothelial cells as a model for atherosclerosis-related diseases

Both hypoxia and the complement lectin pathway (CLP) are involved in atherosclerosis and atherosclerosis-related stroke and acute myocardial infarction (AMI). We have previously shown that mannose-binding lectin-associated serine protease-1 (MASP-1), the most abundant enzyme of CLP, induces an inflammatory phenotype of endothelial cells (ECs) by cleaving protease activated receptors (PARs). In the absence of data, we aimed to investigate whether hypoxia and MASP-1 interact at the level of ECs, to better understand their role in atherosclerosis-related diseases. Hypoxia attenuated the wound healing ability of ECs, increased ICAM-1 and decreased ICAM-2 expression and upregulated PAR2 gene expression. Hypoxia and MASP-1 increased GROα and IL-8 production, and endothelial permeability without potentiating each other’s effects, whereas they cooperatively disrupted vascular network integrity, activated the Ca2+, CREB and NFκB signaling pathways, and upregulated the expression of E-selectin, a crucial adhesion molecule in neutrophil homing. VCAM-1 expression was not influenced either by hypoxia, or by MASP-1. In summary, hypoxia potentiates the effect of MASP-1 on ECs, at least partially by increasing PAR expression, resulting in interaction at several levels, which may altogether exacerbate stroke and AMI progression. Our findings suggest that MASP-1 is a potential drug target in the acute phase of atherosclerosis-related diseases.

The effect of GnRH-a on the angiogenesis of endometriosis

PurposeNeoangiogenesis is necessary for adhesion and invasiveness of endometriotic lesions in women affected by endometriosis. Vascular endothelial growth factor (VEGF) is one of the main components of angiogenesis and is part of the major pathway tissue factor (TF)-protease activated receptor-2 (PAR-2)-VEGF that leads to neoangiogenesis. Specificity protein 1 (SP1) is a transcriptional factor that has recently been studied for its crucial role in angiogenesis via a specific pathway. We hypothesize that by blocking angiogenetic pathways we can suppress endometriotic lesions. Gonadotrophin-releasing hormone-agonists (GnRH-a) are routinely used, especially preoperatively, in endometriosis. It would be of great interest to clarify which angiogenetic pathways are affected and, thereby, pave the way for further research into antiangiogenetic effects on endometriosis.MethodsWe used quantitative real-time polymerase chain reaction (qRT-PCR) to study mRNA expression levels of TF, PAR-2, VEGF, and SP1 in endometriotic tissues of women who underwent surgery for endometriosis and received GnRH-a (leuprolide acetate) preoperatively.ResultsVEGF, TF, and PAR-2 expression is significantly lower in patients who received treatment (p < 0,001) compared to those who did not, whereas SP1 expression is not altered (p = 0.779).ConclusionsGnRH-a administration does affect some pathways of angiogenesis in endometriotic lesions, but not all of them. Therefore, supplementary treatments that affect the SP1 pathway of angiogenesis should be developed to enhance the antiangiogenetic effect of GnRH-a in patients with endometriosis.Trial registrationClinicaltrial.gov ID: NCT06106932.

Ex Vivo Antiplatelet Effects of Oral Anticoagulants.

The impact of non-vitamin K antagonist oral anticoagulants (NOACs) on platelet function is still unclear. We conducted a comprehensive ex vivo study aimed at assessing the effect of the four currently marketed NOACs on platelet function. We incubated blood samples from healthy donors with concentrations of NOACs (50, 150 and 250 ng/mL), in the range of those achieved in the plasma of patients during therapy. We evaluated generation of thrombin; light transmittance platelet aggregation (LTA) in response to adenosine diphosphate (ADP), thrombin receptor-activating peptide (TRAP), human γ-thrombin (THR) and tissue factor (TF); generation of thromboxane (TX)B2; and expression of protease-activated receptor (PAR)-1 and P-selectin on the platelet surface. All NOACs concentration-dependently reduced thrombin generation compared with control. THR-induced LTA was suppressed by the addition of dabigatran at any concentration, while TF-induced LTA was reduced by factor-Xa inhibitors. ADP- and TRAP-induced LTA was not modified by NOACs. TXB2 generation was reduced by all NOACs, particularly at the highest concentrations. We found a concentration-dependent increase in PAR-1 expression after incubation with dabigatran, mainly at the highest concentrations, but not with FXa inhibitors; P-selectin expression was not changed by any drugs. Treatment with the NOACs is associated with measurable ex vivo changes in platelet function, arguing for antiplatelet effects beyond the well-known anticoagulant activities of these drugs. There are differences, however, among the NOACs, especially between dabigatran and the FXa inhibitors.

Effect of moxibustion on colonic low-grade inflammatory response in rats with diarrhea-predominant irritable bowel syndrome based on mast cell degranulation.

To observe the effects of moxibustion on colonic mast cell degranulation and inflammatory factor expression in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), and explore the potential mechanism of moxibustion in treating IBS-D. Forty-five rat pups born from 5 healthy SPF-grade pregnant SD rats, with 8 rats were randomly selected as the normal group. The remaining 37 rats were intervened with maternal separation, acetic acid enema, and chronic restraint stress to establish the IBS-D model. The successfully modeled 32 rats were then randomly assigned to a model group, a ketotifen group, a moxibustion group, and a moxibustion-medication group, with 8 rats in each group. The rats in the ketotifen group were intervened with intragastric administration of ketotifen solution (10 mL/kg); the rats in the moxibustion group were intervened with suspended moxibustion on bilateral "Tianshu" (ST 25) and "Shangjuxu" (ST 37); the rats in the moxibustion-medication group were intervened with suspended moxibustion combined with intragastric administration of ketotifen solution. All interventions were administered once daily for 7 consecutive days. The diarrhea rate and minimum volume threshold of abdominal withdrawal reflex (AWR) were calculated before and after modeling, as well as after intervention. After intervention, colonic tissue morphology was observed using HE staining; colonic mucosal ultrastructure was examined by scanning electron microscopy; colonic mast cell ultrastructure was observed using transmission electron microscopy; mast cell degranulation was assessed by toluidine blue staining; serum and colonic levels of histamine, interleukin (IL)-1β, IL-6, IL-1α, trypsin-like enzyme, and protease-activated receptor 2 (PAR-2) were measured by ELISA; the Western blot and real-time quantitative PCR were employed to evaluate the protein and mRNA expression of colonic IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2; the immunofluorescence was used to detect the positive expression of histamine, IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colonic tissue. Compared to the normal group, the rats in the model group exhibited extensive infiltration of inflammatory cells in colonic tissue, severe damage to the colonic mucosa, disordered arrangement of villi, reduced electron density, and a significant decrease in granule quantity within mast cells. The diarrhea rate and mast cell degranulation rate were increased (P<0.01), AWR minimum volume threshold was decreased (P<0.01); the serum and colonic levels of histamine, IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 were elevated (P<0.01); the positive expression of histamine, as well as protein, mRNA and positive expression of IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colon were all elevated (P<0.01). Compared to the model group, the rats in the ketotifen group, the moxibustion group, and the moxibustion-medication group exhibited significantly reduced infiltration of inflammatory cells in colonic tissue, relatively intact colonic mucosa, orderly arranged villi, increased electron density, and an augmented number of mast cell granules; the diarrhea rate and mast cell degranulation rate were decreased (P<0.01), and AWR minimum volume threshold was increased (P<0.01); the serum and colonic levels of histamine, IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 were reduced (P<0.01); the positive expression of histamine, as well as protein, mRNA and positive expression of IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 in the colon were all decreased (P<0.01). Compared to the ketotifen group, the moxibustion group showed decreased serum levels of histamine, IL-6, and trypsin-like enzyme (P<0.01, P<0.05), as well as reduced colonic levels of IL-1β and IL-6 (P<0.01, P<0.05); the protein expression of colonic IL-1β, IL-1α, and PAR-2 was reduced (P<0.05), and the positive expression of colonic IL-1β and trypsin-like enzyme was reduced (P<0.01, P<0.05). Compared to both the ketotifen group and the moxibustion group, the moxibustion-medication group exhibited decreased diarrhea rate and mast cell degranulation rate (P<0.01), an increased AWR minimum volume threshold (P<0.01), reduced serum and colonic levels of histamine, IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 (P<0.01), decreased protein expression of colonic IL-1β, trypsin-like enzyme, and PAR-2 (P<0.01, P<0.05), reduced mRNA and positive expression of colonic IL-1β, IL-6, IL-1α, trypsin-like enzyme, and PAR-2 (P<0.01, P<0.05), and decreased positive expression of colonic histamine (P<0.01). Moxibustion on "Tianshu" (ST 25) and "Shangjuxu" (ST 37) might inhibit low-grade inflammatory reactions in the colon of IBS-D model rats. The mechanism may be related to the inhibition of histamine and trypsin-like enzyme secreted by mast cell, thereby reducing the expression of related inflammatory factors.

Involvement of Protease-Activated Receptor2 Pleckstrin Homology Binding Domain in Ovarian Cancer: Expression in Fallopian Tubes and Drug Design.

Studying primordial events in cancer is pivotal for identifying predictive molecular indicators and for targeted intervention. While the involvement of G-protein-coupled receptors (GPCRs) in cancer is growing, GPCR-based therapies are yet rare. Here, we demonstrate the overexpression of protease-activated receptor 2 (PAR2), a GPCR member in the fallopian tubes (FTs) of high-risk BRCA carriers as compared to null in healthy tissues of FT. FTs, the origin of ovarian cancer, are known to express genes of serous tubal intraepithelial carcinoma (STICs), a precursor lesion of high-grade serous carcinoma (HGSC). PAR2 expression in FTs may serve as an early prediction sensor for ovarian cancer. We show now that knocking down Par2 inhibits ovarian cancer peritoneal dissemination in vivo, pointing to the central role of PAR2. Previously we identified pleckstrin homology (PH) binding domains within PAR1,2&4 as critical sites for cancer-growth. These motifs associate with PH-signal proteins via launching a discrete signaling network in cancer. Subsequently, we selected a compound from a library of backbone cyclic peptides generated toward the PAR PH binding motif, namely the lead compound, Pc(4-4). Pc(4-4) binds to the PAR PH binding domain and blocks the association of PH-signal proteins, such as Akt or Etk/Bmx with PAR2. It attenuates PAR2 oncogenic activity. The potent inhibitory function of Pc(4-4) is demonstrated via inhibition of ovarian cancer peritoneal spread in mice. While the detection of PAR2 may serve as a predictor for ovarian cancer, the novel Pc(4-4) compound may serve as a powerful medicament in STICs and ovarian cancer. This is the first demonstration of the involvement of PAR PH binding motif signaling in ovarian cancer and Pc(4-4) as a potential therapy treatment.

Inhibition of proteinase-activated receptor 2 (PAR2) decreased the malignant progression of lung cancer cells and increased the sensitivity to chemotherapy

ObjectivesThis study aimed to study the effect of protease-activated receptor 2 (PAR2) on the proliferation, invasion, and clone formation of lung cancer cells. It also aimed to evaluate the inhibitory effect of melittin on PAR2 and the anti-lung cancer effect of melittin combined with gefitinib.MethodsThe correlation between the co-expression of PAR2 and epithelial–mesenchymal transition (EMT) markers was analyzed. PAR2 in A549 and NCI-H1299 cells was knocked down using siRNA. MTT assay, Transwell assay, and colony formation assay were used to detect the effects of PAR2 on cell proliferation, invasion, and clone formation. The anti-cancer effect of PAR2 knockdown on gefitinib treatment was analyzed. The synergistic effect of melittin on gefitinib treatment by inhibiting PAR2 and the underlying molecular mechanism were further analyzed and tested.ResultsThe expression of PAR2 was upregulated in lung cancer, which was associated with the poor prognosis of lung cancer. PAR2 knockdown inhibited the stemness and EMT of lung cancer cells. It also inhibited the proliferation, invasion, and colony formation of A549 and NCI-H1299 cells. Moreover, PAR2 knockdown increased the chemotherapeutic sensitivity of gefitinib in lung cancer. Melittin inhibited PAR2 and the malignant progression of lung cancer cells. Melittin increased the chemotherapeutic sensitivity of gefitinib in lung cancer by inhibiting PAR2.ConclusionPAR2 may promote the proliferation, invasion, and colony formation of lung cancer cells by promoting EMT. Patients with a high expression of PAR2 have a poor prognosis. Inhibition of PAR2 increased the chemotherapeutic sensitivity of gefitinib. PAR2 may be a potential therapeutic target and diagnostic marker for lung cancer.

MicroRNA-26a alleviates tubulointerstitial fibrosis in diabetic kidney disease by targeting PAR4.

Our previous study found that miR-26a alleviates aldosterone-induced tubulointerstitial fibrosis (TIF). However, the effect of miR-26a on TIF in diabetic kidney disease (DKD) remains unclear. This study clarifies the role and possible mechanism of exogenous miR-26a in controlling the progression of TIF in DKD models. Firstly, we showed that miR-26a was markedly decreased in type 2 diabetic db/db mice and mouse tubular epithelial cells (mTECs) treated with high glucose (HG, 30 mM) using RT-qPCR. We then used adeno-associated virus carrying miR-26a and adenovirus miR-26a to enhance the expression of miR-26a invivo and invitro. Overexpressing miR-26a alleviated the TIF in db/db mice and the extracellular matrix (ECM) deposition in HG-stimulated mTECs. These protective effects were caused by reducing expression of protease-activated receptor 4 (PAR4), which involved in multiple pro-fibrotic pathways. The rescue of PAR4 expression reversed the anti-fibrosis activity of miR-26a. We conclude that miR-26a alleviates TIF in DKD models by directly targeting PAR4, which may provide a novel molecular strategy for DKD therapy.

Evaluating the potential of Vitamin D and curcumin to alleviate inflammation and mitigate the progression of osteoarthritis through their effects on human chondrocytes: A proof-of-concept investigation.

Osteoarthritis (OA) is a chronic degenerative joint disorder primarily affecting the elderly, characterized by a prominent inflammatory component. The long-term side effects associated with current therapeutic approaches necessitate the development of safer and more efficacious alternatives. Nutraceuticals, such as Vitamin D and curcumin, present promising therapeutic potentials due to their safety, efficacy, and cost-effectiveness. In this study, we utilized a proinflammatory human chondrocyte model of OA to assess the anti-inflammatory properties of Vitamin D and curcumin, with a particular focus on the Protease-Activated Receptor-2 (PAR-2) mediated inflammatory pathway. Employing a robust siRNA approach, we effectively modulated the expression of PAR-2 to understand its role in the inflammatory process. Our results reveal that both Vitamin D and curcumin attenuate the expression of PAR-2, leading to a reduction in the downstream proinflammatory cytokines, such as Tumor Necrosis Factor-alpha (TNF-α), Interleukin 6 (IL-6), and Interleukin 8 (IL-8), implicated in the OA pathogenesis. Concurrently, these compounds suppressed the expression of Receptor Activator of Nuclear Factor kappa-Β Ligand (RANKL) and its receptor RANK, which are associated with PAR-2 mediated TNF-α stimulation. Additionally, Vitamin D and curcumin downregulated the expression of Interferon gamma (IFN-γ), known to elevate RANKL levels, underscoring their potential therapeutic implications in OA. This study, for the first time, provides evidence of the mitigating effect of Vitamin D and curcumin on PAR-2 mediated inflammation, employing an siRNA approach in OA. Thus, our findings pave the way for future research and the development of novel, safer, and more effective therapeutic strategies for managing OA.

Coagulation Factor Xa Has No Effects on the Expression of PAR1, PAR2, and PAR4 and No Proinflammatory Effects on HL-1 Cells.

Atrial fibrillation (AF), characterised by irregular high-frequency contractions of the atria of the heart, is of increasing clinical importance. The reasons are the increasing prevalence and thromboembolic complications caused by AF. So-called atrial remodelling is characterised, among other things, by atrial dilatation and fibrotic remodelling. As a result, AF is self-sustaining and forms a procoagulant state. But hypercoagulation not only appears to be the consequence of AF. Coagulation factors can exert influence on cells via protease-activated receptors (PAR) and thereby the procoagulation state could contribute to the development and maintenance of AF. In this work, the influence of FXa on Heart Like-1 (HL-1) cells, which are murine adult atrial cardiomyocytes (immortalized), was investigated. PAR1, PAR2, and PAR4 expression was detected. After incubations with FXa (5-50 nM; 4-24 h) or PAR1- and PAR2-agonists (20 µM; 4-24 h), no changes occurred in PAR expression or in the inflammatory signalling cascade. There were no time- or concentration-dependent changes in the phosphorylation of the MAP kinases ERK1/2 or the p65 subunit of NF-κB. In addition, there was no change in the mRNA expression of the cell adhesion molecules (ICAM-1, VCAM-1, fibronectin). Thus, FXa has no direct PAR-dependent effects on HL-1 cells. Future studies should investigate the influence of FXa on human cardiomyocytes or on other cardiac cell types like fibroblasts.

Expression and clinical implications of PARs in the stenotic tissue of ureteropelvic junction obstruction.

To explore the expression and clinical implications of protease activated receptors (PARs) in the pathogenesis of children with ureteropelvic junction obstruction (UPJO). Immunohistochemistry was employed to investigate the distribution of PARs in both normal human ureteropelvic junction (UPJ) and cases of UPJO. Furthermore, PAR gene expression levels were assessed using real-time PCR (RT-PCR), and the patients in the UPJO group were stratified according to the Onen grading system. Subsequently, the clinical implications of PARs in UPJO were explored through RT-PCR analysis. Immunofluorescence showed robust PAR2 expression in the control group compared with the UPJO group. The results of RT-PCR analysis revealed a significant decrease in the relative mRNA expression of PAR2 in the UPJO group compared to the control group. Notably, the relative RNA expression of PAR1 was significantly lower in the Onen-4 group compared to the control group. Furthermore, the relative mRNA expression of PAR2 exhibited a statistically significant difference among the Onen-3 group, Onen-4 group, and control group. PARs are widely distributed throughout the SIP syncytium of the UPJ and play a role in maintaining smooth muscle cells (SMCs) membrane potential by interacting with interstitial cells of Cajal (ICCs), as well as platelet-derived growth factor receptor alpha-positive cells (PDGFR α+ cells). The decreased expression of PAR1 suggests a higher preoperative Onen grade in UPJO patients. Furthermore, the downregulation of PAR2 effects at the UPJ may be involved in the loss of inhibitory neuromuscular transmission, disrupting the rhythmic peristalsis of the UPJ.

Clinically uninvolved but not healthy-The skin of patients with atopic dermatitis is primed for itch and inflammation.

Atopic dermatitis (AD) is a highly prevalent inflammatory skin disorder characterized by episodic exacerbations and remissions. Why the clinically healthy skin of AD patients becomes rapidly inflamed and very pruritic is poorly understood. To investigate cowhage- and histamine-induced itch and skin expression levels of their target receptors in lesional and non-lesional skin of AD, compared to the skin of patients with psoriasis, chronic spontaneous urticaria (CSU) and healthy subjects. Patients with AD, psoriasis and chronic spontaneous urticaria (CSU) as well as healthy control subjects (HC) (n = 20 each) were assessed for differences in itch parameters, neurogenic flare reaction and local blood flow responses to skin provocations with cowhage and histamine. Skin biopsies from 10 AD, 10 psoriasis,11 CSU and 12 HC were obtained to assess expression of protease-activated receptors 2 and 4 (PAR-2, PAR-4), histamine H1 and H4 receptors (H1R, H4R), and mast cells. Provocation of non-lesional skin of AD patients with cowhage resulted in prolonged itch (p = 0.020), which was not observed in psoriasis and CSU. Significantly prolonged and more intense cowhage- and histamine-induced itch (for duration, peak and overall intensity) was also observed in lesional AD skin. Diminished neurogenic flare reaction and blood flow after histamine provocation were shown in AD and psoriasis patients. Non-lesional AD skin along with lesional AD and psoriasis skin showed an increased expression of PAR-2 and PAR-4, H1R and H4R. Mast cell number was higher in lesional AD and psoriasis skin (p = 0.006 and p = 0.006, respectively). The non-lesional skin of AD patients markedly differs from healthy skin in cowhage-induced itch responses and the expression of receptors for proteases and histamine. Proactive therapeutic interventions that downregulate these receptors may prevent episodic exacerbation in AD.

Platelet-activating factor and protease-activated receptor 2 cooperate to promote neutrophil recruitment and lung inflammation through nuclear factor-kappa B transactivation

Although it is well established that platelet-activated receptor (PAF) and protease-activated receptor 2 (PAR2) play a pivotal role in the pathophysiology of lung and airway inflammatory diseases, a role for a PAR2-PAFR cooperation in lung inflammation has not been investigated. Here, we investigated the role of PAR2 in PAF-induced lung inflammation and neutrophil recruitment in lungs of BALB/c mice. Mice were pretreated with the PAR2 antagonist ENMD1068, PAF receptor (PAFR) antagonist WEB2086, or aprotinin prior to intranasal instillation of carbamyl-PAF (C-PAF) or the PAR2 agonist peptide SLIGRL-NH2 (PAR2-AP). Leukocyte infiltration in bronchoalveolar lavage fluid (BALF), C-X-C motif ligand 1 (CXCL)1 and CXCL2 chemokines, myeloperoxidase (MPO), and N-acetyl-glycosaminidase (NAG) levels in BALF, or lung inflammation were evaluated. Intracellular calcium signaling, PAFR/PAR2 physical interaction, and the expression of PAR2 and nuclear factor-kappa B (NF-КB, p65) transcription factor were investigated in RAW 264.7 cells stimulated with C-PAF in the presence or absence of ENMD1068. C-PAF- or PAR2-AP-induced neutrophil recruitment into lungs was inhibited in mice pretreated with ENMD1068 and aprotinin or WEB2086, respectively. PAR2 blockade impaired C-PAF-induced neutrophil rolling and adhesion, lung inflammation, and production of MPO, NAG, CXCL1, and CXCL2 production in lungs of mice. PAFR activation reduced PAR2 expression and physical interaction of PAR2 and PAFR; co-activation is required for PAFR/PAR2 physical interaction. PAR2 blockade impaired C-PAF-induced calcium signal and NF-κB p65 translocation in RAW 264.7 murine macrophages. This study provides the first evidence for a cooperation between PAFR and PAR2 mediating neutrophil recruitment, lung inflammation, and macrophage activation.

Jiawei guomin decoction regulates the degranulation of mast cells in atopic dermatitis mice via the HIS/PAR-2 pathway

Ethnopharmacological relevanceGuomin decoction (GMD) is a traditional Chinese medicine commonly used in clinical practice. It has traditionally been used to treat all allergic diseases. Currently, Jiawei Guomin Decoction (JWGMD) is used to treat sensitive skin after initial therapy. Although it has a significant clinical therapeutic effect, the exact role of mast cell degranulation in treating atopic dermatitis (AD) is still unclear. Aim of the studyGMD and JWGMD can both treat allergic diseases, while JWGMD focuses on skin allergies. This study aims to explore the potential effect of JWGMD on the degranulation of mast cells in an AD mouse model induced by 2,4-dinitrofluorobenzene (DNFB) and investigate the effectiveness of JWGMD in alleviating disease progression to further provide specific therapeutic targets for treating AD. Materials and methodsThe scratching times and skin lesions of model mice induced by DNFB were observed, and skin tissues were collected for subsequent measurement. Histopathological changes in the back skin of mice were observed by haematoxylin eosin (H&E) staining, Toluidine blue staining was used to detect the degranulation of mouse skin mast cells, and the relationship between the expression of histamine (HIS), mast cell tryptase (MCT) and mast cell degranulation was analysed by enzyme-linked immunosorbent assay (ELISA). The expression of protease-activated receptor-2 (PAR-2), histamine 1 receptor (H1R), H2R, H4R and MCT proteins in AD mice was detected by Western blot (WB). Immunofluorescence assay (IFA) further confirmed the localization of PAR-2, H1R, H2R, H4R, and MCT proteins in the skin. Quantitative real-time PCR (qPCR) was used to determine PAR-2, H1R, H2R and H4R mRNA levels in skin lesions to further clarify the mechanism by which JWGMD amplifies mast cell degranulation in AD. In addition, a reliable ultrahigh-performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry (UPLC-QE-MS) nontargeted metabolomics analysis was performed to analyse the differences in metabolite abundance between GMD and JWGMD, and these results were used to identify the active components in JWGMD that may have antipruritic and anti-inflammatory properties and inhibit mast cell degranulation. ResultsAfter intermittent stimulation with DNFB, the skin lesions showed extensive desquamation, dryness, scabbing, skin thickening, and slight bleeding. Both treatments alleviated this phenomenon and reduced the number of scratches, with JWGMD being the most effective. JWGMD can significantly reduce inflammatory cell infiltration, oedema, and some capillary neogenesis in mice and reduce the degranulation of mast cells. The ELISA results showed that JWGMD can increase the levels of MCT and HIS proteins. The WB and IFA results demonstrated that JWGMD reduced the expression levels of PAR-2, H1R, H4R, and MCT proteins in skin lesions, with protein localization mainly in the epidermal layer, while H2R protein levels were increased and mainly localized in the dermis. In addition, JWGMD downregulates the mRNA expression of PAR-2, H1R, H2R, and H4R. Interestingly, through UPLC-QE-MS nontargeted metabolomic analysis, we detected the anti-inflammatory and antiallergy active substances in JWGMD, such as methyl eugenol, dictamnine and sinapine. ConclusionsJWGMD may alleviate itching through methyl syringol, dictamnine, sinapine and other substances, and its mechanism may be related to inhibiting the HIS/PAR-2 pathway in AD model mice and further regulating the self-amplification of mast cell degranulation. JWGMD is a potential drug for treating AD. Therefore, it deserves continuous attention and research.

Lactobacillus plantarum AR495 improves stress-induced irritable bowel syndrome in rats by targeting gut microbiota and Mast cell-PAR2-TRPV1 signaling pathway

Probiotics have great potential in regulating intestinal pain. In this study, the effects of Lactobacillus plantarum AR495 on the visceral sensitivity and gut microbiota of irritable bowel syndrome (IBS) rats were studied. The results showed that tryptase released after mast cell activation and degranulation plays a key role in visceral pain, and L. plantarum AR495 reduced the stimulation of colonic mast cells and the expression of protease-activated receptor 2 (PAR2) and TRPV1 in dorsal root ganglia. Research further showed that supplementation with L. plantarum AR495 increased the level of short-chain fatty acids (SCFAs) and enhanced the barrier function of the colon. In addition, the microbiota analysis of the colon indicated that L. plantarum AR495 promoted the proliferation of Bifidobacterium and inhibited the proliferation of Lachnospiraceae, which alleviated the imbalance of the intestinal microbiota caused by IBS to a certain extent. In total, L. plantarum AR495 might reduce visceral sensitivity through the Mast cell-PAR2-TRPV1 signaling pathway by maintaining the homeostasis of the intestinal barrier.

PAR1 regulates sepsis-induced vascular endothelial barrier dysfunction by mediating ERM phosphorylation via the RhoA/ROCK signaling pathway

Sepsis begins with vascular endothelial barrier breakdown and causes widespread organ failure. Protease-activated receptor 1 (PAR1) is an important target for modulating vascular endothelial permeability; however, little research has been undertaken in sepsis, and its putative molecular mechanism remains unknown. The vascular endothelial permeability was examined by detecting FITC-dextran flux. F-actin was examined by immunofluorescence (IF). PAR1, ERM phosphorylation, and RhoA/ROCK signaling pathway expression in lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs) line were examined by IF and Western blot. To develop the sepsis model, cecal ligation and puncture (CLP) were conducted. The PAR1 inhibitor SCH79797 was utilized to inhibit PAR1 expression in vivo. Vascular permeability in main organs weres measured by Evans blue dye extravasation. The pathological changes in main organs were examined by HE staining. The expression of PAR1, ERM phosphorylation, and the RhoA/ROCK signaling pathway was examined using IF, immunohistochemical and WB in CLP mice. In vitro, in response to LPS stimulation of HUVECs, PAR1 mediated the phosphorylation of ERM, promoted F-actin rearrangement, and increased endothelial hyperpermeability, all of which were prevented by inhibiting PAR1 or RhoA. Additionally, inhibiting PAR1 expression reduced RhoA and ROCK expression. In vivo, we showed that inhibiting PAR1 expression will reduce ezrin/radixin/moesin (ERM) phosphorylation to relieve vascular endothelial barrier dysfunction and thereby ameliorate multiorgan dysfunction syndrome (MODS) in CLP-induced septic mice. This study revealed that PAR1-mediated phosphorylation of ERM induced endothelial barrier dysfunction, which in turn led to MODS in sepsis, and that the RhoA/ROCK signaling pathway underlay these effects.

Genome-wide DNA methylation mediates the resistance to vibriosis in Cynoglossus semilaevis

Chinese tongue sole (Cynoglossus semilaevis) is an economically important marine fish in China. However, vibriosis has caused huge mortality and economic losses in its culturing industry. To reveal the effect of DNA methylation on the resistance to vibriosis in tongue sole, we conducted RNA sequencing and whole genome bisulfite sequencing (WGBS), and compared the gene expressions and DNA methylation patterns between the resistant and susceptible families. We identified a total of 741 significantly differentially expressed genes (DEGs) in kidney and 17460 differentially methylated genes (DMGs), which were both enriched in immune-related pathways, such as “cAMP signaling pathway” and “NOD-like receptor signaling pathway”. Through the correlation analysis of DEGs and DMGs, we identified two important immune pathways, including “complement and coagulation cascades”, and “cytokine-cytokine receptor interaction”, which played important roles in regulating the inflammation level and immune homeostasis. For example, the expression of proinflammatory cytokine il17c was down-regulated under the regulation of DNA methylation; in addition, the expression of protease-activated receptor 3 (par3) was up-regulated, which could induce the up-expressionof il8. These results demonstrated that the regulation of DNA methylation on the genes involved in immune responses might contribute to the resistance to vibriosis in tongue sole, and provided a basis for the control of diseases in fish aquaculture.