P2Y Receptors Research Articles

Abstract WMP120: MicroRNAs as a Therapeutic Target to Reduce Microglial Activation After Post-Stroke Social Isolation

Introduction: Loneliness and social isolation (SI) are associated with increased risk of stroke and other vascular disorders, along with all-cause mortality. However, it is unknown what mechanisms mediate how social factors affect stroke recovery. Methods: To investigate the negative effects of post-stroke SI on brain miRNA profiles, aged (18-20 months) C57BL/6 male mice were utilized. Three days following a 60-minute transient right middle cerebral artery occlusion (MCAO), mice were randomly assigned to pair housing (PH) or single housing (SI). At post-stroke timepoints (specifically day 3 after the stroke), the infarct was equal between groups, reducing the possibility of differential effects resulting from different infarct sizes. At two-time intervals (post-stroke SI D4 and D27), temporal miRNA profiling of the ipsilateral hemisphere was performed. Results: Distinct miRNA candidates were significantly altered in post-stroke SI, within the brain across both acute and chronic time points (n=4/grp, FDR adjusted *p<0.05). MiR-10a-5p and miR-10b-5p were observed to have crucial nodes in the pool of miRNAs that interacted with the largest group of miRNAs for post-stroke at SI D4 and D27, respectively. KEGG pathway analysis showed 4 days of isolation resulted in the enrichment of pathways related to microglial activation and 27 days of isolation lead to the activation of neuronal-specific pathways that regulate cognition and motivation (FDR adjusted *p<0.05). At post-stroke SI D4, significant microglial activation was observed in independent validation cohorts as assessed by the median fluorescence intensity (MFI) of purinergic receptor P2Y12 (P2RY12), in CD45 int CD11b + P2RY12 + cells in the brain. MFI of P2RY12 was significantly downregulated in post-stroke SI mice at D4 (n=7-8/grp, *p<0.05) compared to PH mice. Conclusions: These findings validate our hypothesis that post-stroke SI exacerbates microglia activation and leads to differential expression of miRNAs involved in regulating microglial pathways. Taken together, utilizing miRNA-based therapies to restore miRNA-profiles in the brain may be able to alleviate the detrimental impact of post-stroke SI on chronic depressive-like phenotypes.

Blood oxygen regulation via P2Y12R expressed in the carotid body

BackgroundPeripheral blood oxygen monitoring via chemoreceptors in the carotid body (CB) is an integral function of the autonomic cardiorespiratory regulation. The presence of the purinergic P2Y12 receptor (P2Y12R) has been implicated in CB; however, the exact role of the receptor in O2 sensing and signal transduction is unknown.MethodsThe presence of P2Y12R was established by immunoblotting, RT qPCR and immunohistochemistry. Primary glomus cells were used to assess P2Y12R function during hypoxia and hypercapnia, where monoamines were measured by HPLC; calcium signal was recorded utilizing OGB-1 and N-STORM Super-Resolution System. Ingravescent hypoxia model was tested in anaesthetized mice of mixed gender and cardiorespiratory parameters were recorded in control and receptor-deficient or drug-treated experimental animals.ResultsInitially, the expression of P2Y12R in adult murine CB was confirmed. Hypoxia induced a P2Y12R-dependent release of monoamine transmitters from isolated CB cells. Receptor activation with the endogenous ligand ADP promoted release of neurotransmitters under normoxic conditions, while blockade disrupted the amplitude and duration of the intracellular calcium concentration. In anaesthetised mice, blockade of P2Y12R expressed in the CB abrogated the initiation of compensatory cardiorespiratory changes in hypoxic environment, while centrally inhibited receptors (i.e. microglial receptors) or receptor-deficiency induced by platelet depletion had limited influence on the physiological adjustment to hypoxia.ConclusionsPeripheral P2Y12R inhibition interfere with the complex mechanisms of acute oxygen sensing by influencing the calcium signalling and the release of neurotransmitter molecules to evoke compensatory response to hypoxia. Prospectively, the irreversible blockade of glomic receptors by anti-platelet drugs targeting P2Y12Rs, propose a potential, formerly unrecognized side-effect to anti-platelet medications in patients with pulmonary morbidities.

P2Y1 RECEPTOR SIGNALING INFLUENCES EPITHELIAL RESPONSE TO INTESTINAL INFLAMMATION

Abstract Purines are among the most influential and ancient biochemical molecules in evolutionary history. In recent years, several studies have identified that nucleotides are released from cells during infection and tissue injury and serve as paracrine signaling molecules that help coordinate host defense and repair responses. These important extracellular signaling molecules activate purinergic receptors, of which there are 19 different purinergic receptor subtypes, which are classified into P1 and P2 receptors. Several studies have revealed crucial roles for P2 purinergic receptors during inflammatory and infectious diseases; however, these studies have largely examined purinergic signaling in immune cells and the enteric nervous system. Yet, while epithelial cells serve as the first barrier against infection and inflammation, the role of purinergic signaling within the gastrointestinal tract remains largely unknown. Our group recently discovered a novel and prominent role for P2Y1 in mediating host responses during rotavirus infection. To extend these studies, we sought to elucidate the P2 purinergic receptor repertoire expressed within gut epithelium and assess their functional role during insult. Using the Human Protein Atlas, we queried single-cell RNA sequencing data for P2 purinergic receptor expression in small intestine, colon, and rectum. In silico analysis revealed high expression of P2Y1, P2Y2, P2Y11 and P2X4 among the purinergic receptors throughout the gastrointestinal tract. Interestingly, P2Y1 showed highest expression in intestinal enterocytes and goblet cells. Further, we compared P2 receptor expression between commonly used, cancer-derived intestinal cell lines (T84, HT29 and Caco2) and crypt-derived human intestinal organoids (HIOs). Both HIOs and T84 and Caco2 cell lines had similar mRNA expressions of P2Y1, P2Y2, P2Y11 and P2X4. However, live calcium imaging studies showed robust responses to P2Y receptor-specific agonists in HIOs, particularly P2Y1 and P2Y2 agonists, but the cancer-derived cell lines failed to respond to these agonists, suggesting these cell lines have defects in P2Y signaling. Functionally, blocking P2Y1, but not P2Y2, significantly delayed wound healing in epithelial scratch assays, indicating a potential role in epithelial injury repair. To delineate the role of epithelial P2Y1 in vivo, we generated VillinCre P2Y1flex/flox mice. At baseline, the knockout animals did not exhibit any overt intestinal defects; however, in the DSS-induced colitis model, the epithelial P2Y1 KO mice had significantly greater weight loss and epithelial damage than their wildtype littermates. These findings confirm that the gastrointestinal tract express P2Y1, P2Y2, P2Y11 and P2X4 purinergic receptors and P2Y1 signaling plays important functional roles in both gastrointestinal epithelial homeostasis and repair of injury.

Microglia regulate sleep through calcium-dependent modulation of norepinephrine transmission.

Sleep interacts reciprocally with immune system activity, but its specific relationship with microglia-the resident immune cells in the brain-remains poorly understood. Here, we show in mice that microglia can regulate sleep through a mechanism involving Gi-coupled GPCRs, intracellular Ca2+ signaling and suppression of norepinephrine transmission. Chemogenetic activation of microglia Gi signaling strongly promoted sleep, whereas pharmacological blockade of Gi-coupled P2Y12 receptors decreased sleep. Two-photon imaging in the cortex showed that P2Y12-Gi activation elevated microglia intracellular Ca2+, and blockade of this Ca2+ elevation largely abolished the Gi-induced sleep increase. Microglia Ca2+ level also increased at natural wake-to-sleep transitions, caused partly by reduced norepinephrine levels. Furthermore, imaging of norepinephrine with its biosensor in the cortex showed that microglia P2Y12-Gi activation significantly reduced norepinephrine levels, partly by increasing the adenosine concentration. These findings indicate that microglia can regulate sleep through reciprocal interactions with norepinephrine transmission.

Raman spectra simulation of antiplatelet drug-platelet interaction using DFT

The paper reflects the results of molecular docking and mathematical DFT simulation for antiplatelet drugs and the target platelet receptor/ferment interaction in the limited area. The results of Raman spectra simulation are implemented and obtained from the interaction of the clopidogrel metabolite of the P2Y12 receptor. The interaction of aspirin with the COX-1 enzyme was also investigated. As a result, theoretical Raman spectra of the drug-receptor area were obtained. The theoretical data were compared with the experimental SERS results. The characteristic bands corresponding to metabolite/ferment and antiplatelet drug vibrations were clarified. The prospects of obtaining results for pathologies based on platelet conformations during cardiovascular diseases have been demonstrated.

Impact of Preloading Strategy With Ticagrelor on Periprocedural Myocardial Injury in Patients With Non-ST Elevation Myocardial Infarction Undergoing Early Invasive Strategy.

Pretreatment with an oral P2Y12 receptor blocker (before coronary angiography) versus treatment in the catheterization laboratory has been a matter of debate in patients presenting with non-ST segment elevation myocardial infarction (NSTEMI). The primary aim of this study was to assess the impact of an immediate preloading strategy with ticagrelor on periprocedural myocardial injury in patients with NSTEMI treated with an early invasive strategy. NSTEMI patients who underwent coronary angiography and subsequent percutaneous coronary intervention (PCI) within 24 hours after hospital admission were divided into 2 groups: the first group (pretreatment group) included patients who received ticagrelor pretreatment as soon as possible after admission and the second group (no pretreatment group) included patients who received a loading dose of ticagrelor after coronary angiography. The pretreatment group included 232 patients, and the no pretreatment group included 87 patients. Male patients represented the majority of the patients. The 2 groups were similar in baseline characteristics, except for a greater incidence of hypertension ( P = 0.014) and higher hemoglobin levels ( P = 0.01) in the pretreatment group in comparison with the no pretreatment group. Patients in the ticagrelor pretreatment group had less myocardial injury until coronary angiography based on troponin measurements collected at 12 hours after admission ( P = 0.025). Patients in the ticagrelor pretreatment group also had fewer periprocedural myocardial injuries based on troponin measurements taken between 12 and 24 hours after the PCI ( P = 0.026 and P = 0.022, respectively). Our findings suggested that ticagrelor pretreatment reduces periprocedural myocardial injury in NSTEMI patients who underwent PCI within 24 hours after admission.

The P2Y1 receptor in the colonic myenteric plexus of rats and its correlation with opioid-induced constipation

This study was designed to explore the expression changes of P2Y1 receptors in the distal colonic myenteric layer of rats. An opioid induced constipation(OIC) rat model was generated by intraperitoneal (i.p) injection of loperamide. At 7 days post-treatment, the model rats were assessed by calculating the fecal water content and the gastrointestinal transit ratio. The immunofluorescence (IF)-based histochemical study was used to observe the distribution of P2Y1 receptors in the distal colonic myenteric plexus. Western blotting (WB) was performed to evaluate the expression changes of P2Y1 proteins in the myenteric layer, and the electrophysiological approaches were carried out to determine the regulatory roles of P2Y1 receptors on distal colonic motor function. IF showed that P2Y1 receptors are co-expressed MOR in the enteric nerve cells of the distal colonic myenteric plexus. Moreover, the WB revealed that the protein levels of P2Y1 were significantly decreased in the distal colonic myenteric layer of OIC rats. In vitro tension experiments exhibited that the P2Y1 receptor antagonist MRS2500 enhanced the spontaneous contraction amplitude, adding EM2 and β-FNA did not have any effect on MRS2500. Therefore, P2Y1 receptor expression could be associated with the occurrence of OIC in this rat model and the regulation of colonic motility by MOR may be related to the release of purine neurotransmitters such as ATP in the colonic nervous system.

The role of P2Y6 receptor in the pathogenesis of cardiovascular and inflammatory diseases

The purinergic receptor P2Y6 receptor (P2Y6R) is a member of the G protein-coupled receptors (GPCR) family. P2Y6R is widely expressed in various cell types and plays a critical role in physiological processes, where it is activated by extracellular uridine diphosphate (UDP) and mobilizes Ca2+ via the Gαq/11 protein pathway. We have recently discovered the pathophysiological role of P2Y6R in cardiovascular and inflammatory diseases, including inflammatory bowel disease and non-alcoholic fatty liver disease. Furthermore, we uncovered the redox-dependent internalization of P2Y6R. In this review, we provide a comprehensive overview of the pathophysiological activity of P2Y6R in cardiovascular and inflammatory diseases. Additionally, we discuss the concept of atypical internalization control of GPCRs, which may be applied in the prevention and treatment of intestinal inflammation and cardiovascular remodeling.

P2Y2-P2X7 receptors cross-talk in primed mesenteric endothelial cells upregulates NF-κB signaling favoring mononuclear cell adhesion in schistosomiasis.

Schistosomiasis is an intravascular infectious disease that impacts over 200 million people globally. In its chronic stage, it leads to mesenteric inflammation with significant involvement of monocytes/macrophages. Endothelial cells lining the vessel lumens play a crucial role, and mount of evidence links this disease to a downregulation of endoprotective cell signaling favoring a primed and proinflammatory endothelial cell phenotype and therefore the loss of immunovascular homeostasis. One hallmark of infectious and inflammatory conditions is the release of nucleotides into the extracellular milieu, which, in turn, act as innate messengers, activating purinergic receptors and triggering cell-to-cell communication. ATP influences the progression of various diseases through P2X and P2Y purinergic receptor subtypes. Among these receptors, P2Y2 (P2Y2R) and P2X7 (P2X7R) receptors stand out, known for their roles in inflammation. However, their specific role in schistosomiasis has remained largely unexplored. Therefore, we hypothesized that endothelial P2Y2R and P2X7R could contribute to monocyte adhesion to mesenteric endothelial cells in schistosomiasis. Using a preclinical murine model of schistosomiasis associated with endothelial dysfunction and age-matched control mice, we showed that endothelial P2Y2R and P2X7R activation increased monocyte adhesion to cultured primary endothelial cells in both groups. However, a distinct upregulation of endothelial P2Y2R-driven canonical Ca2+ signaling was observed in the infected group, amplifying adhesion. In the control group, the coactivation of endothelial P2Y2R and P2X7R did not alter the maximal monocyte adhesion induced by each receptor individually. However, in the infected group, this coactivation induced a distinct upregulation of P2Y2R-P2X7R-driven canonical signaling, IL-1β release, and VCAM-1 expression, with underlying mechanisms involving inflammasome and NF-κB signaling. Therefore, current data suggest that schistosomiasis alters endothelial cell P2Y2R/P2X7R signaling during inflammation. These discoveries advance our understanding of schistosomiasis. This intricate interplay, driven by PAMP-triggered endothelial P2Y2R/P2X7R cross-talk, emerges as a potential key player in the mesenteric inflammation during schistosomiasis.

Clopidogrel, ticagrelor, prasugrel or an alternation of two P2Y12 in patients with acute myocardial infarction with cardiogenic shock.

Data are lacking on the effects of the alternation of P2Y12 receptor antagonists (P2Y12) on bleeding and outcome in patients with myocardial infarction (MI) with cardiogenic shock (CS). We compared the effects of different P2Y12 and alternation of P2Y12 (combination) on bleeding and outcome in patients with MI and CS. Data from 247 patients divided into four groups: clopidogrel, ticagrelor, prasugrel, and the combination group, were analyzed. The association between P2Y12 and bleeding as well as 30-day and one-year mortality was examined. The highest bleeding rate was observed in patients in the combination group, followed by the clopidogrel, ticagrelor, and prasugrel groups [12(50%) patients, 22(28.2%), 21(18.3%) and 4(13.3%), respectively; p = 0.003]. Bleeding occurred with a similar frequency in the combination and clopidogrel groups (p = 0.081), but more frequently than in the ticagrelor and prasugrel groups (p = 0.002 and p = 0.006, respectively). Bleeding rates were similar in patients receiving P2Y12 alone (p = 0.13). Compared to clopidogrel, both ticagrelor and prasugrel had a lower bleeding risk (aOR: 0.40; 95% CI: 0.18-0.92; p = 0.032 and aOR: 0.20; 95% CI: 0.05-0.85; p = 0.029, respectively) and the combination had a similar bleeding risk (aOR: 2.31; 95% CI: 0.71-7.48; p = 0.16). The ticagrelor and prasugrel groups had more than an 80% and 90% lower bleeding risk than the combination group (aOR: 0.17; 95% CI: 0.06-0.55; p = 0.003 and aOR: 0.09; 95% CI: 0.02-0.44; p = 0.003, respectively). The unadjusted 30-day and one-year mortality were highest in the clopidogrel group, followed by the ticagrelor, prasugrel, and combination groups (44(56.4%) and 55(70.5%) patients died in the clopidogrel group, 53(46.1%) and 56(48.7%) in the ticagrelor group, 12(40%) and 14(46.7%) patients died in the prasugrel, and 6(25%) and 9(37.5%) patients died in the combination group; p = 0.045 and p < 0.0001. After adjustment for confounders, the P2Y12 groups were not independently associated with either 30-day (p = 0.23) or one-year (p = 0.17) mortality risk. Our results suggest that the choice of P2Y12 was not associated with treatment outcome. The combination of P2Y12 increased bleeding risk compared with ticagrelor and prasugrel and was comparable to clopidogrel in patients with MI and CS. However, these higher bleeding rates did not result in worse treatment outcomes.

Interaction of Tau with G-Protein-Coupled Purinergic P2Y12 Receptor by Molecular Docking and Molecular Dynamic Simulation.

Alzheimer's disease, a progressive neurological disorder, is characterized by the accumulation of neurofibrillary tangles and senile plaques by Tau and amyloid-β, respectively, in the brain microenvironment. The misfolded protein aggregates interact with several components of neuronal and glial cells such as membrane lipids, receptors, transporters, enzymes, cytoskeletal proteins, etc. Under pathological conditions, Tau interacts with several G-protein-coupled receptors (GPCRs), which undergoes either receptor signaling or desensitization followed by internalization of the protein complex. The purinergic GPCR, P2Y12 which is expressed in microglial cells, plays a key role in its activation and migration. Microglial cells sense and migrate to the site of injury aided by P2Y12 receptor that interacts with ADP released from damaged cells. P2Y12 receptor also interacts with misfolded Tau accumulated at the extracellular space and promotes receptor-mediated internalization. Immunocolocalization and co-immunoprecipitation studies demonstrated the interaction of Tau species with the P2Y12 receptor. Later, in-silico analyses were carried out with the repeat domain of Tau (TauRD), which has been identified as the interacting partner of P2Y12 receptor by in-vitro studies. Molecular docking and molecular dynamics simulation studies show the stability and the type of interaction in TauRD-receptor complex. Tau interaction with P2Y12 receptor plays a significant role in maintaining the active state of microglia which could lead to neuroinflammation and neuronal damage in AD brain. Hence, blocking P2Y12-Tau interaction and P2Y12-mediated Tau internalization in microglial cells could be possible therapeutic strategies in downregulating the severity of neuroinflammation in AD.

The protective effect of silver nanoparticles' on epithelial cornea cells against ultraviolet is accompanied by changes in calcium homeostasis and a decrease of the P2X7 and P2Y2 receptors

PurposeThe aim of the study was to evaluate the effect of silver nanoparticles hydrocolloids (AgNPs) on human corneal epithelial cells. Epithelial cells form the outermost and the most vulnerable to environmental stimuli layer of the cornea in the eye. Mechanical stress, UV radiation, and pathogens such as bacteria, viruses, and parasites challenge the fragile homeostasis of the eye. To help combat stress, infection, and inflammation wide portfolio of interventions is available. One of the oldest treatments is colloidal silver. Silver nanoparticle suspension in water is known for its anti-bacterial anti-viral and antiprotozoal action. However, AgNPs interact also with host cells, and the character of the interplay between corneal cells and silver seeks investigation. MethodsThe human epithelial corneal cell line (HCE-2) was cultured in vitro, treated with AgNPs, and subjected to UV. The cell’s viability, migration, calcium concentration, and expression/protein level of selected proteins were investigated by appropriate methods including cytotoxicity tests, “wound healing” assay, Fluo8/Fura2 AM staining, qRT-PCR, and western blot. ResultsIncubation of human corneal cells (HCE-2) with AgNP did not affect cells viability but limited cells migration and resulted in altered calcium homeostasis, decreased the presence of ATP-activated P2X7, P2Y2 receptors, and enhanced the expression of PACAP. Furthermore, AgNPs pretreatment helped restrain some of the deleterious effects of UV irradiation. Interestingly, AgNPs had no impact on the protein level of ACE2, which is important in light of potential SARS-CoV-2 entrance through the cornea. ConclusionsSilver nanoparticles are safe for corneal epithelial cells in vitro.

Purinergic Receptor P2Y12-Mediated Tau Internalization in Microglia.

Microglia are the resident brain macrophage cells that are involved in constant surveillance of brain microenvironment. In Alzheimer's disease, microglia get over activated upon the accumulation of Tau and amyloid-β species in the extracellular space, ultimately leading to neurodegeneration. Microglia phagocytose the extracellular Tau species by several mechanisms among which P2Y12 receptor-mediated internalization of extracellular Tau is recently studied. Extracellular Tau activates microglia and directly interacts with the P2Y12 receptor. Tau-receptor complex is then internalized followed by perinuclear accumulation and lysosomal degradation. Upon microglial activation by extracellular Tau, P2Y12 receptor is also involved in membrane-associated actin remodeling which has its key role in active migration and phagocytosis.

COVID-19 Diarrhea Is Inflammatory, Caused by Direct Viral Effects Plus Major Role of Virus-induced Cytokines

BACKGROUND AND AIMSDiarrhea occurs in up to 50% of cases of COVID-19. Nonetheless, the pathophysiologic mechanism(s) have not been determined. METHODSThis was examined using normal human enteroid monolayers exposed apically to live SARS-CoV-2 or non-replicating virus like particles (VLPs) bearing the four SARS-CoV-2 structural proteins or irradiated virus, all of which bound and entered enterocytes. RESULTSLive virus and VLPs increased secretion of multiple cytokines and reduced mRNAs of ACE2, NHE3 and DRA. IL-6 plus IL-8 alone reduced NHE3 mRNA and protein and DRA mRNA. Neither VLPs nor IL-6 plus IL-8 alone altered Cl- secretion, but together they caused Cl- secretion, which was Ca2+ dependent, CFTR independent, blocked partially by a specific TMEM16 A inhibitor, and entirely by a general TMEM16 family inhibitor. VLPs and irradiated virus, but not IL-6 plus IL-8, produced Ca2+ waves that began within minutes of VLP exposure, lasted for at least 60 min, and were prevented by pretreatment with apyrase; a P2Y1 receptor antagonist; and general TMEM16 family inhibitor but NOT by the specific TMEM16A inhibitor. CONCLUSIONSThe pathophysiology of COVID-19 diarrhea appears to be a unique example of a calcium dependent inflammatory diarrhea, that is caused by direct viral effects plus the virus-induced intestinal epithelial cytokine secretion.

Antiplatelet Treatment Preferences of a Group of Cardiologists from Türkiye: A Survey Research Study.

Deciding on the optimal duration of dual antiplatelet treatment (DAPT) remains a complex decision. This survey aims to explore the preferences for antiplatelet therapy and the daily routine regarding DAPT duration in coronary artery disease among a group of cardiologists in Türkiye. Using an online questionnaire with 38 questions, the preferences of 314 cardiologists were collected. Qualitative descriptive characteristics of the answers received from the participants were examined. Participating cardiologists mostly worked in training and research hospitals (51.59%) and university hospitals (21.66%). Participants primarily favored ticagrelor in patients undergoing PCI with a diagnosis of STEMI and NSTE-ACS (69.75% and 55.73% respectively). Clopidogrel was the most preferred P2Y12 treatment in patients with chronic coronary syndrome (CCS) after PCI (94.90%). Pre-treatment with a loading dose of a P2Y12 receptor inhibitor was administered to 57.01% of patients with NSTE-ACS, irrespective of the planned treatment strategy. In NSTE-ACS patients with low bleeding risk treated with PCI, 83.12% of participants recommended DAPT for 12 months and 14.65% for >12 months. In high-bleeding-risk NSTE-ACS patients treated with PCI, DAPT durations of six months (74.52%), three months (19.75%), and one month (5.73%) were chosen. Among CCS patients treated with PCI without an increased risk of bleeding, 12 months of DAPT was preferred by 68.15% of participants. Most participants (70.70%) were switching to a more potent P2Y12 receptor inhibitor therapy in emergency department clopidogrel-loaded patients with ACS. The aim of this survey to capture a snapshot of the preferences of a group of cardiologists in Türkiye regarding DAPT treatment and duration. The responses were both in accordance and in conflict with the current guidelines.

Characterization of Prasugrel Degradation Profile by Several Oxidative Reagents using HPLC and LC-MS/MS Technique

Prasugrel, a derivative of thienopyridine functions as a prodrug that exerts irreversible inhibition on platelet ADP receptors by specifically targeting the P2Y12 receptor. In pharmaceuticals, the stability of the product plays a crucial role in establishing the shelf life of product. The second common degradation pathway next to hydrolysis is the oxidation. In practice, the oxidative stress studies were carried out using hydrogen peroxide (H2O2), free radicals, oxygen purging, transition metals, singlet oxygen, Fenton reagent, etc. This study compares the oxidative degradation behaviour of prasugrel hydrochloride using different oxidative stressors and was analyzed by HPLC using a method that could separate degradation products individually and from the drug. The degradation products formed under different stress conditions were characterized using LC-TOF-MS/MS. Analysis of accelerated stability samples of prasugrel formulation revealed that the degradation products formed under azobisisobutyronitrile (AIBN) and H2O2 stress conditions were simulated with the stability data. This study on prasugrel showed that the degradation products formed with H2O2 were also observed under hydrolytic conditions. In the pharmaceutical industry, H2O2 is the only oxidative stressor (&lt; 95%) used to study the oxidation degradation of the sample. The present work has demonstrated that AIBN serves as a valuable oxidative stressor for accurately assessing the oxidative stability of drug.

P2 purinergic receptors regulate the progression of colorectal cancer.

More and more studies have revealed that P2 purinergic receptors play a key role in the progression of colorectal cancer (CRC). P2X and P2Y purinergic receptors can be used as promoters and regulators of CRC and play a dual role in the progression of CRC. CRC microenvironment is rich in ATP and its cleavage products (ADP, AMP, Ado), which act as activators of P2X and P2Y purinergic receptors. The activation of P2X and P2Y purinergic receptors regulates the progression of CRC mainly by regulating the function of immune cells and mediating different signal pathways. In this paper, we focus on the specific mechanisms and functional roles of P2X7, P2Y12, and P2Y2 receptors in the growth and progression of CRC.The antagonistic effects of these selective antagonists of P2X purinergic receptors on the growth, invasion, and metastasis of CRC were further discussed. Moreover, different studies have reported that P2X7 receptor can be used as an effective predictor of patients with CRC.All these indicate that P2 purinergic receptors are a key regulator of CRC.Therefore, antagonizing P2 purinergic receptors may be an innovative treatment for CRC.

The P2Y2 receptor mediates terminal adipocyte differentiation and insulin resistance: Evidence for a dual G-protein coupling mode

Several P2Y nucleotide receptors have been shown to be involved in the early stage of adipocyte differentiation in vitro and insulin resistance in obese mice; however, the exact receptor subtype(s) and its underlying molecular mechanism in relevant human cells are unclear. Here, using human primary visceral preadipocytes as a model, we found that during preadipocyte-to-mature adipocyte differentiation, the P2Y2 nucleotide receptor (P2Y2R) was the most upregulated subtype among the eight known P2Y receptors and the only one further dramatically upregulated after inflammatory TNFα treatment. Functional studies indicated that the P2Y2R induced intracellular Ca2+, ERK1/2, and JNK signaling but not the p38 pathway. In addition, stimulation of the P2Y2R suppressed basal and insulin-induced phosphorylation of AKT, accompanied by decreased GLUT4 membrane translocation and glucose uptake in mature adipocytes, suggesting a role of P2Y2R in insulin resistance. Mechanistically, we found that activation of P2Y2R did not increase lipolysis but suppressed PIP3 generation. Interestingly, activation of P2Y2R triggered Gi-protein coupling, and pertussis toxin pretreatment largely inhibited P2Y2R-mediated ERK1/2 signaling and cAMP suppression. Further, treatment of the cells with AR-C 118925XX, a selective P2Y2R antagonist, significantly inhibited adipogenesis, and P2Y2R knockout decreased mouse body weight gain with smaller eWAT mass infiltrated with fewer macrophages as compared to wild-type mice in response to a Western diet. Thus, we revealed that terminal adipocyte differentiation and inflammation selectively upregulate P2Y2R expression and that P2Y2R mediates insulin resistance by suppressing the AKT signaling pathway, highlighting P2Y2R as a potential new drug target to combat obesity and type-2 diabetes.

Club cell CREB regulates the goblet cell transcriptional network and pro-mucin effects of IL-1B.

Introduction: Club cells are precursors for mucus-producing goblet cells. Interleukin 1β (IL-1B) is an inflammatory mediator with pro-mucin activities that increases the number of mucus-producing goblet cells. IL-1B-mediated mucin production in alveolar adenocarcinoma cells requires activation of the cAMP response element-binding protein (CREB). Whether the pro-mucin activities of IL-1B require club cell CREB is unknown. Methods: We challenged male mice with conditional loss of club cell Creb1 and wild type littermates with intra-airway IL-1B or vehicle. Secondarily, we studied human "club cell-like" H322 cells. Results: IL-1B increased whole lung mRNA of secreted (Mucin 5ac, Mucin 5b) and tethered (Mucin 1, Mucin 4) mucins independent of genotype. However, loss of club cell Creb1 increased whole lung mRNA of member RAS oncogene family (Rab3D), decreased mRNA of the muscarinic receptor 3 (M3R) and prevented IL-1B mediated increases in purinergic receptor P2Y, (P2ry2) mRNA. IL-1B increased the density of goblet cells containing neutral mucins in wildtype mice but not in mice with loss of club cell Creb1. These findings suggested that club cell Creb1 regulated mucin secretion. Loss of club cell Creb1 also prevented IL-1B-mediated impairments in airway mechanics. Four days of pharmacologic CREB inhibition in H322 cells increased mRNA abundance of forkhead box A2 (FOXA2), a repressor of goblet cell expansion, and decreased mRNA expression of SAM pointed domain containing ETS transcription factor (SPDEF), a driver of goblet cell expansion. Chromatin immunoprecipitation demonstrated that CREB directly bound to the promoter region of FOXA2, but not to the promoter region of SPDEF. Treatment of H322 cells with IL-1B increased cAMP levels, providing a direct link between IL-1B and CREB signaling. Conclusion: Our findings suggest that club cell Creb1 regulates the pro-mucin properties of IL-1B through pathways likely involving FOXA2.

Natural history of superior mesenteric artery in-stent restenosis

ObjectiveSuperior mesenteric artery (SMA) stenting is the preferred approach for patients with symptomatic SMA-associated chronic mesenteric ischemia (CMI). The durability of this modality is impacted by in-stent restenosis (ISR). Duplex ultrasound (DUS) and computed tomographic angiography (CTA)-measured ISR may be weakly correlated and not uniformly associated with recurrence of presenting symptoms. This study aims to analyze the association between the degree of ISR for patients with CMI and to develop a predictive model for symptom recurrence. MethodsSingle center, retrospective study included all patients with CMI with SMA stents from the period of 2003 to 2020. Follow-up period analysis included patients’ symptoms recurrence, DUS, CTA, and angiography. A receiver operating characteristic (ROC) analysis was used to evaluate whether peak systolic velocity (PSV) was predictive of symptom recurrence. A subgroup analysis of patients (asymptomatic and symptomatic) with SMA ISR was identified; restenosis defined by DUS with peak systolic velocity (PSV) ≥350. ResultsThe study included 186 patients with the ROC analysis obtained from 503 postoperative visits. PSV was not a predictor of symptoms return with area under the curve (AUC) = 0.49 (95% confidence interval [CI], 0.40-0.57). Agreement analysis between imaging modalities showed higher agreement between CTA and angiogram (AUC, 0.769; 95% CI, 0.688-0.849) vs CTA and DUS (AUC, 0.650; 95% CI, 0.589-0.711). The subgroup analysis of patients with ISR included 99 patients (asymptomatic n = 67; symptomatic n = 32). There was no statistical difference between median time (months) to ISR between both groups: 4.5 (asymptomatic group) and 7.6 (symptomatic group). The use of preoperative antiplatelet (86% vs 65%; P = .015) and P2Y12 receptor blockers (36% vs 13%; P = .016) was more prevalent in the asymptomatic group. There was no difference between the type or number of stents placed, stent diameter, or concomitant celiac artery intervention between both groups. ConclusionsThe natural history of SMA and multimodality defined ISR in CMI has not previously been described. Elevated PSV was a poor predictor of symptoms recurrence. Both asymptomatic and symptomatic patients with ISR did not differ in type of stent placed, time to ISR, or involvement of celiac artery. Antiplatelet use pre- and postoperatively appears protective against symptoms recurrence. Our findings underscore the need for long-term surveillance integrating clinical evaluation and multimodality imaging when indicated.