P2X7 Receptor Expression Research Articles

Analyzing the Role of the P2X7 Receptor in Epilepsy.

Purinergic signaling is increasingly recognized to play a role during the generation of hyperexcitable networks in the brain. Among the purinergic receptors, the ionotropic ATP-gated P2X7 receptor has attracted particular attention as a possible drug target for epilepsy. P2X7 receptor expression is increased in the brain of experimental models of epilepsy and in patients and, P2X7 receptor antagonism modulates seizure severity and epilepsy development. To date, studies analyzing the role of the P2X7 receptor during epilepsy have mainly focused on temporal lobe epilepsy, the most common form of acquired epilepsy in adults which is particularly prone to drug refractoriness.Animal models of seizures and epilepsy are an essential tool in the identification of novel anticonvulsive and antiepileptogenic drug targets and much data demonstrating a role for the P2X7 receptor during epilepsy have been obtained by using these models. The aim of the present book chapter is to provide a detailed description of two commonly used mouse models of temporal lobe epilepsy, which are the intra-amygdala kainic acid model of status epilepticus and the controlled cortical impact model of traumatic brain injury. This chapter concludes with a brief description of how these models can be used to investigate the impact of targeting the P2X7 receptor on acute seizures, epilepsy development and established epilepsy .

Effect of Regorafenib on P2X7 Receptor Expression and Different Oncogenic Signaling Pathways in a Human Breast Cancer Cell Line: A Potential of New Insight of the Antitumor Effects of Regorafenib.

Background: Breast cancer is the most common malignancy in women worldwide. P2X7 is a transmembrane receptor expressed in breast cancer and activated by the ATP tumor microenvironment, driving cell proliferation, angiogenesis, and metastasis via different signaling pathways. The role of the P2X7 receptor, hypoxia, and autophagy in regulating tumor progression is controversial. The multikinase inhibitor regorafenib prevents the activation of numerous kinases involved in angiogenesis, proliferation, and metastasis. The present study aimed to evaluate the modulatory effect of regorafenib on the hypoxia/angiogenesis/P2X7R/autophagy axis on the MCF7 breast cancer cell line and its impact on different signaling pathways involved in breast cancer pathogenesis. Methods: The levels of VEGF, VEGFR, PI3K, NF-κB, HIF-1α, and LC3-II were analyzed using ELISA, and caspase-3 activity was also assessed colorimetrically. Phosphorylated (p)-p38 MAPK and purinergic ligand-gated ion channel 7 (P2X7) receptor protein expression levels were analyzed via Western blotting. Reverse transcription-quantitative PCR was used to determine the mRNA expression levels of Beclin 1 (BECN1), LC3-II, and sequestosome 1 (p62). Results: Regorafenib reduced MCF7 cell viability in a dose-dependent manner. Furthermore, regorafenib significantly reduced levels of PI3K, NF-κB, VEGF, VEGFR, P2X7 receptor, and p-p38 MAPK protein expression, and markedly reduced p62 mRNA expression levels. However, regorafenib significantly increased caspase-3 activity, as well as BECN1 and LC3-II mRNA expression levels. Conclusions: Regorafenib was demonstrated to possibly exhibit antitumor activity on the breast cancer cell line via modulation of the P2X7/HIF-1α/VEGF, P2X7/P38, P2X7/ERK/NF-κB, and P2X7/beclin 1 pathways.

P2X7 receptor is essential for cross-dressing of bone marrow-derived dendritic cells

SummaryT cell activation requires the processing and presentation of antigenic peptides in the context of a major histocompatibility complex (MHC complex). Cross-dressing is a non-conventional antigen presentation mechanism, involving the transfer of preformed peptide/MHC complexes from whole cells, such as apoptotic cells (ACs) to the cell membrane of professional antigen-presenting cells (APCs), such as dendritic cells (DCs). This is an essential mechanism for the induction of immune response against viral antigens, tumors, and graft rejection, which until now has not been clarified. Here we show for first time that the P2X7 receptor (P2X7R) is crucial to induce cross-dressing between ACs and Bone-Marrow DCs (BMDCs). In controlled ex vivo assays, we found that the P2X7R in both ACs and BMDCs is required to induce membrane and fully functional peptide/MHC complex transfer to BMDCs. These findings show that acquisition of ACs-derived preformed antigen/MHC-I complexes by BMDCs requires P2X7R expression.

Betulin Targets Lipin1/2-Meidated P2X7 Receptor as a Therapeutic Approach to Attenuate Lipid Accumulation and Metaflammation.

The present study focused on the potential mechanism of betulin (BT), a pentacyclic triterpenoid isolated from the bark of white birch (Betula pubescens), against chronic alcohol-induced lipid accumulation and metaflammation. AML-12 and RAW 264.7 cells were administered ethanol (EtOH), lipopolysaccharide (LPS) or BT. Male C57BL/6 mice were fed Lieber-DeCarli liquid diets containing 5% EtOH for 4 weeks, followed by single EtOH gavage on the last day and simultaneous treatment with BT (20 or 50 mg/kg) by oral gavage once per day. In vitro, MTT showed that 0-25 mM EtOH and 0-25 μM BT had no toxic effect on AML-12 cells. BT could regulate sterolregulatory-element-binding protein 1 (SREBP1), lipin1/2, P2X7 receptor (P2X7r) and NOD-like receptor family, pyrin domains-containing protein 3 (NLRP3) expressions again EtOH-stimulation. Oil Red O staining also indicated that BT significantly reduced lipid accumulation in EtOH-stimulated AML-12 cells. Lipin1/2 deficiency indicated that BT might mediate lipin1/2 to regulate SREBP1 and P2X7r expression and further alleviate lipid accumulation and inflammation. In vivo, BT significantly alleviated histopathological changes, reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and triglyceride (TG) levels, and regulated lipin1/2, SREBP1, peroxisome proliferator activated receptor α/γ (PPARα/γ) and PGC-1α expression compared with the EtOH group. BT reduced the secretion of inflammatory factors and blocked the P2X7r-NLRP3 signaling pathway. Collectively, BT attenuated lipid accumulation and metaflammation by regulating the lipin1/2-mediated P2X7r signaling pathway.

Estrogen inhibits bladder overactivity in rats with cyclophosphamide-induced cystitis via downregulating the expression of P2X3 receptors in bladder epithelium cells.

The therapeutic effect of estrogen on interstitial cystitis/bladder pain syndrome is unclear. We aim to explore the effect of estrogen on bladder overactivity in rats with cyclophosphamide-induced cystitis and its underlying mechanism. In vivo cystometry was used to determine the effect of estrogen on bladder excitability. The effect of estrogen on the expression of P2X3 receptors in bladder epithelium was detected by real-time polymerase chain reaction and western blot. Effect of P2X3 receptors in bladder urothelium on stretch-released adenosine triphosphate was performed by a Flexcell FX5000 Compression system and an Enzyme-Linked Immunosorbent Assay Kit. Estrogen deprivation significantly increased the urinary frequency, while supplementation with diarylpropionitrile (DPN), an estrogen receptor β (ERβ) agonist, alleviated the urinary frequency. 17β-Estradiol and DPN decreased the expression of P2X3 receptors in urothelium cells which was partially inhibited by ERβ antagonist 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol. Meanwhile, inhibiting the expression of P2X3 receptors by ERβ agonist or antagonizing the function of P2X3 receptors by selective P2X3 receptor antagonist AF-353 or A-317491 significantly reduced the stretch-released ATP from urothelium cells. Estrogen has a direct effect on the regulation of bladder overactivity in rats with cyclophosphamide-induced cystitis by downregulating the expression of bladder epithelial P2X3 receptors through ERβ and reducing the adenosine triphosphate released from urothelium during bladder filling, thereby inhibiting the generation of the micturition reflex.

Neurotropin alleviates rat osteocarcinoma pain via P2X3 receptor activation in the midbrain periaqueductal gray.

Objective(s):Clinically effective analgesia treatment for patients afflicted with osteocarcinoma lessens the intensity of pain. The midbrain periaqueductal gray (PAG) plays a critical role in pain modulation, and activation of P2X3 receptors in this region mediates pain processing. Neurotropin is a small molecule drug used for analgesic treatment of a number of chronic pain conditions. The present study aims at determining whether P2X3 receptor activation in PAG is responsible for the analgesic effect of neurotropin in rats with osteocarcinoma pain. Materials and Methods:The tibia of female Sprague-Dawley rats was inoculated with breast carcinoma cells to establish the osteocarcinoma pain model. The effects of intraperitoneal injection of 6, 12, and 18 neurotropin units (NU)/kg on pain threshold and receptor expression of P2X3 in the ventrolateral PAG (vlPAG) were assessed. The P2X3 receptor antagonist A-317491 (1.5 nmol/0.3 µl) was administered into vlPAG with a high-dose neurotropin (18 NU/kg) to determine the role of this receptor in the analgesic effect. Results:The pain thresholds of the rats with osteocarcinoma pain continuously decreased, whereas P2X3 receptor expression in vlPAG only slightly increased after osteocarcinoma cell inoculation. Neurotropin substantially elevated the pain threshold and P2X3 receptor expression in vlPAG in a dose-dependent manner. A-317491 microinjection into vlPAG significantly reduced the analgesic effects of neurotropin in the rats with osteocarcinoma pain.Conclusion:Through these findings, it is shown that vlPAG P2X3 receptor activation participates in neurotropin-mediated analgesia mechanism in osteocarcinoma pain.

Circulating P2X7 Receptor Signaling Components as Diagnostic Biomarkers for Temporal Lobe Epilepsy.

Circulating molecules have potential as biomarkers to support the diagnosis of epilepsy and to assist with differential diagnosis, for example, in conditions resembling epilepsy, such as in psychogenic non-epileptic seizures (PNES). The P2X7 receptor (P2X7R) is an important regulator of inflammation and mounting evidence supports its activation in the brain during epilepsy. Whether the P2X7R or P2X7R-dependent signaling molecules can be used as biomarkers of epilepsy has not been reported. P2X7R levels were analyzed by quantitative ELISA using plasma samples from controls and patients with temporal lobe epilepsy (TLE) or PNES. Moreover, blood cell P2X7R expression and P2X7R-dependent cytokine signature was measured following status epilepticus in P2X7R-EGFP reporter, wildtype, and P2X7R-knockout mice. P2X7R plasma levels were higher in TLE patients when compared with controls and patients with PNES. Plasma levels of the broad inflammatory marker protein C-Reactive protein (CRP) were similar between the three groups. Using P2X7R-EGFP reporter mice, we identified monocytes as the main blood cell type expressing P2X7R after experimentally evoked seizures. Finally, cytokine array analysis in P2X7R-deficient mice identified KC/GRO as a potential P2X7R-dependent plasma biomarker following status epilepticus and during epilepsy. Our data suggest that P2X7R signaling components may be a promising subclass of circulating biomarkers to support the diagnosis of epilepsy.

Overexpression of P2X3 Receptors is Associated with the Painful Dental Pulp Tissue of Human Primary Teeth

ATP-gated P2X3 receptors play a crucial role in neurogenic inflammation and neuronal sensitization, which might is associated with pain. Our study was to investigate whether P2X3 receptors are involved in tooth pain. Dental pulp tissues were obtained from normal (n=14) and painful (n=16) pulps in permanent teeth as well as normal (n=10) and painful (n=14) pulps in primary teeth. Western blot and Real-time fluorescence quantitative PCR were used to detect the expression level of P2X3 receptors in dental pulp tissues. The results showed that P2X3 receptors were upregulated significantly in painful pulp when compared with normal pulp in primary teeth (P<0.05). However, there was no difference of the expression of P2X3 receptors between normal and painful permanent tooth pulp (P>0.05). Interestingly, we found that the intraneural expression of P2X3 receptors of primary tooth pulp was significantly higher than that of permanent tooth pulp (P<0.05). Our study shows for the first time that the P2X3 receptors may be related to primary dental pain, but not to permanent dental pain

P2X Receptor-Dependent Modulation of Mast Cell and Glial Cell Activities in Neuroinflammation.

Localisation of mast cells (MCs) at the abluminal side of blood vessels in the brain favours their interaction with glial cells, neurons, and endothelial cells, resulting in the activation of these cells and the release of pro-inflammatory mediators. In turn, stimulation of glial cells, such as microglia, astrocytes, and oligodendrocytes may result in the modulation of MC activities. MCs, microglia, astrocytes, and oligodendrocytes all express P2X receptors (P2XRs) family members that are selectively engaged by ATP. As increased concentrations of extracellular adenosine 5′-triphosphate (ATP) are present in the brain in neuropathological conditions, P2XR activation in MCs and glial cells contributes to the control of their communication and amplification of the inflammatory response. In this review we discuss P2XR-mediated MC activation, its bi-directional effect on microglia, astrocytes and oligodendrocytes and role in neuroinflammation.

The mechanism behind activation of the Nod-like receptor family protein 3 inflammasome in Parkinson's disease.

Previous studies have shown that the ATP-P2X4 receptor signaling pathway mediates the activation of the Nod-like receptor family protein 3 (NLRP3) inflammasome. The NLRP3 inflammasome may promote renal interstitial inflammation in diabetic nephropathy. As inflammation also plays an important role in the pathogenesis of Parkinson's disease, we hypothesized that the ATP-P2X4 receptor signaling pathway may activate the NLRP3 inflammasome in Parkinson's disease. A male rat model of Parkinson's disease was induced by stereotactic injection of 6-hydroxydopamine into the pars compacta of the substantia nigra. The P2X4 receptor and the NLRP3 inflammasome (interleukin-1β and interleukin-18) were activated. Intracerebroventricular injection of the selective P2X4 receptor antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) or knockdown of P2X4 receptor expression by siRNA inhibited the activation of the NLRP3 inflammasome and alleviated dopaminergic neurodegeneration and neuroinflammation. Our results suggest that the ATP-P2X4 receptor signaling pathway mediates NLRP3 inflammasome activation, dopaminergic neurodegeneration, and dopamine levels. These findings reveal a novel role of the ATP-P2X4 axis in the molecular mechanisms underlying Parkinson's disease, thus providing a new target for treatment. This study was approved by the Animal Ethics Committee of Qingdao University, China, on March 5, 2015 (approval No. QYFYWZLL 26119).

Purinergic P2Y2 and P2X4 Receptors Are Involved in the Epithelial-Mesenchymal Transition and Metastatic Potential of Gastric Cancer Derived Cell Lines.

Gastric cancer (GC) is a major health concern worldwide, presenting a complex pathophysiology that has hindered many therapeutic efforts so far. In this context, purinergic signaling emerges as a promising pathway for intervention due to its known role in cancer cell proliferation and migration. In this work, we explored in more detail the role of purinergic signaling in GC with several experimental approaches. First, we measured extracellular ATP concentrations on GC-derived cell lines (AGS, MKN-45, and MKN-74), finding higher levels of extracellular ATP than those obtained for the non-tumoral gastric cell line GES-1. Next, we established the P2Y2 and P2X4 receptors (P2Y2R and P2X4R) expression profile on these cells and evaluated their role on cell proliferation and migration after applying overexpression and knockdown strategies. In general, a P2Y2R overexpression and P2X4R downregulation pattern were observed on GC cell lines, and when these patterns were modified, concomitant changes in cell viability were observed. These modifications on gene expression also modified transepithelial electrical resistance (TEER), showing that higher P2Y2R levels decreased TEER, and high P2X4R expression had the opposite effect, suggesting that P2Y2R and P2X4R activation could promote and suppress epithelial-mesenchymal transition (EMT), respectively. These effects were confirmed after treating AGS cells with UTP, a P2Y2R-agonist that modified the expression patterns towards mesenchymal markers. To further characterize the effects of P2Y2R activation on EMT, we used cDNA microarrays and observed that UTP induced important transcriptional changes on several cell processes like cell proliferation induction, apoptosis inhibition, cell differentiation induction, and cell adhesion reduction. These results suggest that purinergic signaling plays a complex role in GC pathophysiology, and changes in purinergic balance can trigger tumorigenesis in non-tumoral gastric cells.

Inhibitory Effects of Palmatine on P2X7 Receptor Expression in Trigeminal Ganglion and Facial Pain in Trigeminal Neuralgia Rats

Trigeminal Neuralgia (TN) refers to recurrent severe paroxysmal pain in the distribution area of the trigeminal nerve, which seriously affects the quality of life of patients. This research applied the chronic constriction injury of the infraorbital nerve (CCI—ION) approach to induce an animal model of TN in rats. The mechanical pain threshold of each group of rats was determined postoperatively; the expression of P2X7 receptor in trigeminal ganglion (TG) was assessed by qRT-PCR, immunofluorescence and Western blot; and the changes of the proinflammatory cytokines IL-1β and TNF-α in serum of rats were detected by ELISA. The results showed that the administration of palmatine in the TN rats could reduce the mechanical pain threshold, significantly decrease the expression of P2X7 receptor in TG, and lower the serum concentrations of IL-1β and TNF-α, compared to the sham group. In addition, the phosphorylation level of p38 in TG of TN rats was significantly decreased after treatment with palmatine. Likewise, inhibition of P2X7 expression by shRNA treatment could effectively counteract the adversary changes of pain sensitivity, IL-1β and TNF-α production, and p38 phosphorylation in TN rats. Our data suggest that palmatine may alleviate mechanical facial pain in TN rats possibly by reducing the expression of P2X7 receptor in TG of TN rats, which may be attributable to inhibiting p38 phosphorylation and reducing the release of IL-1β and TNF-α.

P2X7R in Mast Cells is a Potential Target for Salicylic Acid and Aspirin in Treatment of Inflammatory Pain.

BackgroundMast cells are well known for their role in inflammatory pain. P2X7 receptor (P2X7R) has attracted much attention due to its prominent role in inflammatory diseases. Salicylates are commonly used anti-inflammatory and analgesic drugs. Until now, little has been known about whether P2X7R in mast cells is involved in inflammatory pain and whether it is a potential target for salicylates.MethodsFirst, the expression of P2X receptors in mouse peritoneal mast cells was detected by using RT-PCR, immunofluorescence, calcium imaging and electrophysiological technique. In addition, the functions of P2X receptors, especially P2X7R, in mast cells were studied by using QPCR, ELISA and behavioral tests. Furthermore, P2X7R was used as a target to screen for some anti-inflammatory monomers that could inhibit its activity. At last, the effect of salicylic acid (SA) and aspirin (ASA) on the activity of P2X7R was studied by using calcium imaging, electrophysiological technique, ELISA, real-time PCR, behavioral tests, immunofluorescence and molecular docking.ResultsWe found that P2X1, P2X3, P2X4 and P2X7 receptors were expressed in mouse peritoneal mast cells. The functions of different P2X receptors were various. Activation of P2X7R in mouse mast cells induced the release of inflammatory mediators, such as histamine, IL-1β, and CCL3. In addition, inflammation pain induced by high concentrations of ATP could be alleviated by P2X7R blockers or mast cell defects. Interestingly, SA or ASA could reduce high concentrations of ATP-induced inward current, P2X7R upregulation, mediators release, and inflammatory pain. SA or ASA also inhibited the inward current evoked by P2X7R agonist, BZATP. Molecular docking showed that SA or ASA had affinity for the cytoplasmic GDP-binding region of P2X7R.ConclusionP2X7R in mast cells was involved in inflammation pain by releasing inflammatory mediators, and P2X7R might be a potential target for SA and ASA analgesia.

Adipose-Derived Stem Cells and Their Derived Microvesicles Ameliorate Detrusor Overactivity Secondary to Bilateral Partial Iliac Arterial Occlusion-Induced Bladder Ischemia.

(1) Background: We established a new bladder ischemia rat model through bilateral partial iliac arterial occlusion (BPAO) and investigated the therapeutic effect of adipose-derived stem cells (ADSCs) and ADSC-derived microvesicles (MVs); (2) Methods: The study included four groups: (1) sham, (2) BPAO, (3) BPAO + ADSCs, and (4) BPAO + ADSC-derived MVs. Female Wistar rats with BPAO were injected with ADSCs or ADSC-derived MVs through the femoral artery. Doppler flowmetry and real-time laser speckle contrast imaging were performed to quantify blood flow in the common iliac arteries and bladder microcirculation. A 24-h behavior study and transcystometrogram were conducted after 2 weeks. Bladder histology, immunostaining, and lipid peroxidation assay were performed. The expressions of P2X2, P2X3, M2, and M3 receptors and nerve growth factor (NGF) were evaluated; (3) Results: BPAO significantly reduced bladder microcirculation, intercontraction interval (ICI), and bladder volume and increased the amplitude of nonvoiding contraction, neutrophil infiltration, and malondialdehyde and NGF levels. ADSCs and ADSC-derived MVs significantly ameliorated these effects. The results of Western blot showed that the BPAO group exhibited the highest expression of M3 and P2X2 receptors. ADSCs significantly attenuated the expressions of M2 and P2X2 receptors. ADSC-derived MVs significantly attenuated the expressions of M3 and P2X2 receptors; (4) Conclusions: ADSCs and ADSC-derived MVs ameliorated the adverse effects of BPAO including bladder overactivity, bladder ischemia, and oxidative stress. Inflammation, muscarinic signaling, purinergic signaling, and NGF might be involved in the therapeutic mechanism.

BAC transgenic mice to study the expression of P2X2 and P2Y1 receptors

Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y1R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y1R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice.

DOP21 Role of P2X7 in intestinal fibrosis and inflammasome activation: Relevance in Crohn′s Disease

Abstract Background Crohn’s disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract whose etiology is unknown. CD is associated with complications such as fibrosis or fistula, which cannot be pharmacologically reversed, requiring repeated surgery. Although a profibrotic effect of the P2X7 receptor has been described in some scenarios such as lung, heart and liver, its role in intestinal fibrosis has not been analysed yet. Given the crosstalk between fibrosis and inflammasome, we aim to analyze the relevance of P2X7 in intestinal fibrosis and inflammasome activation. Methods Surgical intestinal resections of CD patients and healthy ileum of carcinoma patients were obtained. Murine chronic colitis was induced by 4 cycles of DSS in wild-type (WT) or P2X7-/- (KO) mice. HT29 cells were treated 24 hours with an inflammasome activator cocktail (LPS, TNF-α and IFN-γ) and the P2X7 antagonist A-80. Gene and protein expression of P2X7, inflammasome markers (NLRP3, ASC, CASPASE1, IL1β and IL18) and alternative inflammasome pathways (APIs) (NLRP1, NLRC4 and AIM2) were analysed by qPCR and Western Blot. The collagen layer was analysed by Sirius Red Staining. Results are expressed by mean±SEM. Statistical analysis was performed with one-way ANOVA and correlations were analysed with Spearman coefficient. Results In CD patients, the expression of P2X7 (2.97±0.50), Nlrp3 (2.53±0.41), Asc (5.61±0.76), Caspase1 (6.90±1.41), IL18 (4.17±0.89) and APIs Nlrp1 (3.07±0.40), Nlrc4 (6.99±1.19) is significantly increased vs non-IBD patients. Moreover, P2X7 expression positively and significantly correlates with the expression of the inflammasome markers NLRP3 (r=0.51), ASC (r=0.38), CASPASE1 (r=0.46), IL18 (r=0.36) and API such as NLRP1 (r=0.73), NRLC4 (r=0.67) and AIM2 (r=0.51) in CD patients (n≥45). The chronic murine model of DSS revealed that: a) KO-DSS showed more aggravated colitis with lower survival and greater weight loss compared with WT-DSS; b) the expression of NLRP3, IL18, IL1β and NLRP1 were significantly increased in KO-DSS (101.00±16.33, 3.28±1.49, 327.50±113.90, 4.92±1.00 respectively) vs WT-DSS; c) the thickness of the collagen layer in KO-DSS was increased vs WT-DSS. As expected, HT29 cells treated with the inflammasome cocktail increased protein expression of caspase-1 and the treatment with the P2X7 antagonist A-80 impaired the inflammasome activation since it significantly reduced the protein expression of caspase-1. Conclusion An increased expression of P2X7 receptor, the inflammasome and its APIs is detected in CD patients. Lack of P2X7 worsens chronic colitis associated with an increased activation of the inflammasome. Additional studies are needed in order to elucidate this dual role of P2X7 in intestinal fibrosis.

Enhanced expression of P2X4 purinoceptors in pyramidal neurons of the rat hippocampal CA1 region may be involved ischemia-reperfusion injury.

Ischemic stroke is the most serious disease that harms human beings. In principle, its treatment is to restore blood flow supply as soon as possible. However, after the blood flow is restored, it will lead to secondary brain injury, that is, ischemia-reperfusion injury. The mechanism of ischemia-reperfusion injury is very complicated. This study showed that P2X4 receptors in the pyramidal neurons of rat hippocampus were significantly upregulated in the early stage of ischemia-reperfusion injury. Neurons with high expression of P2X4 receptors are neurons that are undergoing apoptosis. Intraventricular injection of the P2X4 receptor antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) and PSB-12062 can partially block neuronal apoptosis, to promote the survival of neurons, indicating that ATP through P2X4 receptors is involved in the process of cerebral ischemia-reperfusion injury. Therefore, identifying the mechanism of neuronal degeneration induced by extracellular ATP via P2X4 receptors after ischemia-reperfusion will likely find new targets for the treatment of ischemia-reperfusion injury, and will provide a useful theoretical basis for the treatment of ischemia-reperfusion injury.

Remodeling of Purinergic Receptor 2 Signaling in Podocytes In Response to Diabetic Kidney Disease

Growing evidence suggests that purinergic signaling undergoes significant changes in the kidney during pathophysiological conditions such as diabetes. The aim of this study was to determine the functional effect of both type 1 diabetes (T1D) and type 2 diabetes (T2D) with diabetic nephropathy (DN) on P2 receptor profile remodeling and purinergic calcium signaling in podocytes. To do this, we used a well-characterized T2DN model, which was compared to non-diabetic control rats (Wistar) and Goto-Kakizaki (GK) rats, which are diabetic but do not develop DN. As a T1D kidney model, we utilized streptozotocin (STZ)-injected Dahl Salt-Sensitive (SS) rats. The ATP-mediated calcium release in podocytes from intact glomeruli with diabetic kidney disease was measured using live confocal calcium imaging in combination with pharmacological inhibitors of P2 receptors. Both type 1 and type 2 diabetes resulted in a significant increase in the ATP-mediated [Ca2+]i flux in podocytes. Pharmacological inhibition of the ATP-mediated response indicated that ionotropic P2X4 and P2X7 are the major receptors contributing to the augmented ATP-mediated intracellular calcium signaling in diabetic podocytes, rather than metabotropic P2Y1, which was the predominant P2 signaling receptor observed in healthy adult Sprague Dawley rats (reported in an earlier study). The mRNA and protein expression profiles of several P2 receptors in the kidney cortex were also measured at different stages of disease. Ionotropic P2X4 and P2X7 receptor expression was elevated in the kidney cortex during early and late stages of T2D, compared to healthy kidneys. A decrease in metabotropic P2Y1 receptor abundance in the diabetic kidneys was also observed. Lastly, podocytes from T2DN rat glomeruli exhibited substantially elevated basal [Ca2+]i levels compared to controls (Wistar and GK). The observed changes in purinergic receptors’ expression and downstream effects may be a major contributor to podocyte loss and kidney injury in diabetes. This purinergic remodeling results in increased basal and ATP-stimulated [Ca2+]i, thereby exacerbating podocyte damage and further aggravating diabetic kidney disease progression.

Emodin Administration Depolarizes Tumor Associated M2‐Type Macrophages in the Colorectal Cancer Tumor Microenvironment

Natural anthraquinone emodin has been shown to have anti-inflammatory and anti-tumorigenic properties and may be an effective therapy for colorectal cancer (CRC). We evaluated the efficacy of emodin to be used as a preventative therapy in the AOM/DSS (n=20/group) mouse model of CRC cancer. Emodin successfully reduced tumor count (p<0.05) and size (p<0.05) in the Apcmin/+ mouse model and reduced tumor count (p<0.05) and size (p<0.05) in the AOM/DSS mouse model. AOM/DSS mice treated with 80mg/kg emodin had reduced circulating monocyte counts (p<0.05) and increased platelet counts (p<0.05) compared to vehicle treated controls. We further analyzed the colon microenvironment of AOM/DSS mice and observed decreased CD206-expressing M2-type macrophages in mice receiving both 40mg/kg (p<0.05) and 80mg/kg (p<0.05) emodin. Interestingly, when analyzing colon polyps, we discovered that despite reducing M2-type macrophages, 40mg/kg emodin did not significantly change local pro-or anti-inflammatory cytokine levels compared to control mice. Of note, only mice treated with 80mg/kg emodin had increased NOS2 expression (p<0.05) compared to control mice. We therefore investigated the bone marrow environment to attempt to explain our immunological findings. In the tibial bone marrow, we revealed that AOM/DSS mice had increased M1-type macrophages (p<0.05) compared to healthy controls, a phenotype that was completely reversed with both 40mg/kg (p<0.05) and 80mg/kg (p<0.05) emodin treatment. Analyzing the protein expression of the bone we discovered decreased P2X7 receptor expression (p<0.05) and reduced STAT3 phosphorylation (p<0.05) in AOM/DSS mice treated with both 40mg/kg and 80mg/kg emodin compared to controls. To observe the effect of emodin on macrophages; we isolated and cultured bone marrow derived macrophages and treated with tumor conditioned media from C-26 cells for 24hrs and then 50uM of emodin for another 24hrs. Utilizing flow-cytometry we discovered emodin treatment increased CD11c+/CD206- (M1-type macrophages) (p<0.05) and decreased CD11c-/CD206+ (M2-type macrophages) (p<0.05). Taken together, emodin treatment may work to ameliorate chronic inflammation present during CRC and improve CRC prognosis. Future investigations will interrogate the role of the P2X7 receptor to elucidate the mechanism of action of emodin on tumor conditioned macrophages.

The role of adenosine A1 receptor in the peripheral tramadol's effect in the temporomandibular joint of rats

Peripheral tramadol's delivery in the temporomandibular joint (TMJ) leads to significant analgesic outcomes and inflammatory process's resolvent actions. Mechanistically, these properties are apart from the opioid system. Nevertheless, the molecular mechanisms behind these effects are still unclear. Therefore, the present study investigated the hypothesis that adenosine A1 receptors are involved in the tramadol-induced analgesic and anti-inflammatory effects in the TMJ. Animals were pretreated with an intra-TMJ injection of DPCPX (antagonist of A1 receptor) or tramadol and subsequent nociceptive challenge with an intra-TMJ injection of 1.5% formalin. For over 45 min, the nociceptive behavior was quantitated, and by the end of this assessment, the animals were euthanized, and the periarticular tissue was collected. Lastly, an in vitro assay of BMDM (Bone Marrow-Derived Macrophages) was performed to investigate tramadol activity in macrophages. The intra-TMJ injection of tramadol ameliorates formalin-induced hypernociception along with inhibiting leukocyte migration. The tramadol's peripheral anti-inflammatory effect was mediated by the adenosine A1 receptor and was associated with increased protein expression of α2a-adrenoceptor in the periarticular tissues (p < 0.05: ANOVA, Tukey’s test). Also, tramadol inhibits formalin-induced leukocyte migration and protein expression of P2X7 receptors in the periarticular tissue (p < 0.05); however, DPCPX did not alter this effect (p > 0.05). Moreover, DPCPX significantly reduced the protein expression of the M2 macrophage marker, MRC1. In BMDM, tramadol significantly reduces inflammatory cytokines release, and DPCPX abrogated this effect (p < 0.05). We identify tramadol's peripheral effect is mediated by adenosine A1 receptor, possibly expressed in macrophages in the TMJ tissue. We also determined an important discovery related to the activation of A1R/α2a receptors in the tramadol action.