P2X7 Receptor mRNA Research Articles

Implication of lncRNA MSTRG.81401 in Hippocampal Pyroptosis Induced by P2X7 Receptor in Type 2 Diabetic Rats with Neuropathic Pain Combined with Depression.

Major depressive disorder (MDD) is a common complication of diabetes and is often observed alongside diabetic neuropathic pain (DNP) as a comorbidity in diabetic patients. Long non-coding RNA (lncRNA) plays an important role in various pathophysiological processes. The P2X7 receptor is responsible for triggering inflammatory responses, such as pyroptosis, linked to pain and depression. The aim of this study was to investigate the effect of lncRNA MSTRG.81401 on hippocampal pyroptosis induced by the P2X7 receptor in diabetic rats with DNP combined with MDD (DNP + MDD). Our results showed that the expression of lncRNA MSTRG.81401 was significantly elevated in the hippocampus of DNP + MDD rats compared with the control group. Following the administration of shRNA targeting lncRNA MSTRG.81401, a notable elevation in mechanical and thermal pain thresholds was observed in rats with comorbid DNP and MDD. Additionally, significant improvements in depression-like behaviors were evident in the open-field test (OFT), sucrose preference test (SPT), and forced swim test (FST). In the DNP + MDD rats, elevated levels in hippocampal P2X7 receptor mRNA and protein were observed, along with increased co-expression of P2X7 and the astrocytic marker glial fibrillary acidic protein (GFAP). Meanwhile, in DNP + MDD rats, the heightened mRNA expression of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), pyroptosis-related protein Gasdermin D (GSDMD), caspase-1, IL-1β, IL-18, and TNF-α was detected, in addition to increased serum levels of IL-1β, IL-18 and TNF-α. After shRNA treatment with lncRNA MSTRG.81401, the above abnormal changes in indicators for pyroptosis and inflammation were improved. Therefore, our study demonstrates that shRNA of lncRNA MSTRG.81401 can alleviate the pain and depression-like behaviors in diabetic rats associated with the comorbidity of DNP and MDD by inhibiting the hippocampal P2X7 receptor-mediated pyroptosis pathway and pro-inflammatory responses. This suggests that the P2X7R/NLRP3/caspase-1 implicated pyroptosis and inflammatory scenario may serve as a potential target for the management of comorbid DNP and MDD in diabetes.

P2X7R promotes migration and invasion of Lewis lung cancer cells by activating the AKT signaling pathway

To explore the role of P2X7 receptor (P2X7R) in migration and invasion of mouse Lewis lung cancer (LLC) cells and examine the tumorigenic ability of LLC cells in P2X7R-knockout mice. RT-PCR was used to examine P2X7R mRNA expression in LLC cells. LLC cells were treated with ATP (as a P2X7R agonist) or 2'- 3'- O- (4-benzoyl- benzoyl)-ATP (BzATP) (a P2X7R agonist) with or without pretreatment with P2X7R antagonist oxATP or A438079. The changes in migration and invasive abilities of the cells were evaluated using wound healing assay and Transwell assay; Western blotting was performed to determine the activation level of the key proteins in the AKT signaling pathway. The effects of BzATP, A438079, and LY294002 (a inhibitor of the PI3K/AKT pathway) on migration and invasion of LLC cells were also examined. In wild-type (WT) and P2X7R knockout (P2X7-/-) C57BL/6 mice, the growth of subcutaneous LLC cell xenografts were observed by measuring tumor volume and weight. P2X7R expression was detected in LLC cells. Treatment with P2X7R agonist significantly enhanced migration and invasion abilities of LLC cells, and this effect was inhibited by application of P2X7R antagonists (P < 0.001). Western blotting showed that BzATP treatment of LLC cells significantly increased the expression level of p-AKT protein, which was obviously lowered by treatment with P2X7R antagonist (P < 0.01). P2X7R antagonist strongly inhibited BzATP-induced enhancement of LLC cell migration and invasion (P < 0.001). In the tumor- bearing mice, the tumor volume and weight were significantly lower in P2X7-/- mice than in WT mice (P < 0.05). P2X7R promotes migration and invasion of LLC cells by activating the AKT signaling pathway, and LLC cells show lowered tumorigenic capacity in P2X7-/- mice.

MiR-106b-5p Attenuates Neuropathic Pain by Regulating the P2X4 Receptor in the Spinal Cord in Mice.

The P2X4 receptor (P2X4R) can be upregulated after nerve injury, and its mediated spinal microglial activation makes a critical contribution to pathologically enhanced pain processing in the dorsal horn. Although some studies have partly clarified the mechanism underlying altered P2X4R expression, the specific mechanism is not well understood. MicroRNAs (miRNAs) are small noncoding RNAs which control gene expression by binding with their target mRNAs. Thus, in the present study, we investigated whether miRNA is involved in the pathogenesis of neuropathic pain by regulating P2X4R. Our results showed that P2X4R was upregulated in the spinal dorsal horn of mice following spared nerve injury (SNI), and 69 miRNAs (46 upregulated and 23 downregulated miRNAs) were differentially expressed (fold change > 2.0, P < 0.05). P2X4R was found to be a major target of miR-106b-5p (one of the downregulated miRNAs) using bioinformatics technology; quantitative real-time PCR analysis confirmed the change in expression of miR-106b-5p, and dual-luciferase reporter assays confirmed the correlation between them. Fluorescence in situ hybridization was used to show cell co-localization of P2X4R and miR-106b-5p in the spinal dorsal horn. Transfection with miR-106b-5p mimic into BV2 cells reversed the upregulation of P2X4R induced by lipopolysaccharide (LPS). Moreover, miR-106b-5p overexpression significantly attenuated neuropathic pain induced by SNI, with decreased expression of P2X4R mRNA and protein in the spinal dorsal horn; intrathecal miR-106b-5p antagomirinduced pain behaviors, and increased expression of P2X4R in the spinal dorsal horn of naïve mice. These data suggest that miR-106b-5p can serve as an important regulator of neuropathic pain development by targeting P2X4R.

The potential of the P2X7 receptor as a therapeutic target in a sub-chronic PCP-induced rodent model of schizophrenia

ObjectiveWe studied the role of the P2X7 receptor on cognitive dysfunction in a mouse model of schizophrenia. MethodsAn adult mouse model was established by treatment with phencyclidine (PCP), an N-methyl-D-aspartate (NMDA) receptor antagonist. Young mice were divided into three groups: 1) the control (saline-injected) group; 2) experimental 5 mg/kg PCP-injected group; and 3) experimental 10 mg/kg PCP-injected group. The mice were subjected to the open-field and Morris water maze tests at 7 weeks. After intraperitoneal injection of the P2X7 receptor antagonist JNJ-47965567, the behaviour tests were performed again. Samples were taken after testing. The P2X7 receptor protein and mRNA expression levels were detected by immunohistochemistry, Western blotting and PCR. ResultsThis study revealed that the infant sub-chronic PCP mice model showed severe spatial learning and memory impairment in the Morris water maze and schizophrenia-like symptoms (hypermotor behaviour) in the open-field test. The P2X7 receptor protein was highly expressed in the sub-chronic PCP mouse model and more highly expressed in the hippocampus than the prefrontal lobe. After the P2X7 receptor was blocked with JNJ-47965567, P2X7 receptor protein and mRNA expression in the frontal lobe were significantly increased, and the spatial memory impairment and hypermotor behaviour induced by PCP were reversed. ConclusionPCP-induced cognitive impairment can be significantly improved by antagonizing the P2X7 receptor. Therefore, we believe that the P2X7 receptor plays an important role in the cognition of schizophrenic-like mice.

Elevated circulating level of P2X7 receptor is related to severity of coronary artery stenosis and prognosis of acute myocardial infarction.

Acute myocardial infarction (AMI) is a severely life-threatening cardiovascular disease. Previous research has identified an association between the P2X7 receptor (P2X7R) and the development of atherosclerosis. However, the correlation of its expression with the clinical prognosis of patients with AMI remains unclear. The present study aimed to investigate the potential role of P2X7R in Chinese patients with AMI. Seventy-nine patients with AMI and 48 controls were consecutively enrolled in this prospective observational study. Circulating P2X7R mRNA expression levels and other clinical variables were determined upon admission to the hospital. Patients were followed up for 360 days, and the end-point was considered as the occurrence of major adverse cardiovascular events (MACE). Circulating P2X7R mRNA expression level in peripheral blood mononuclear cells of patients with AMI were significantly higher than those in controls and had promising diagnostic ability of AMI with an area under the curve of 0.928. Furthermore, P2X7R was demonstrated to be correlated positively with the severity of coronary artery stenosis. Additionally, this is the first study to indicate that higher P2X7R mRNA expression is associated with a higher rate of MACE within 360 days after AMI. The present study showed that the circulating level of P2X7R was elevated in AMI patients and was closely associated with the severity of coronary artery stenosis and prognosis of AMI.

LncRNA uc.48+ regulates immune and inflammatory reactions mediated by the P2X7 receptor in type 2 diabetic mice.

Diabetes and non-coding RNAs are receiving increasing attention in contemporary medical research. The present study aimed to explore the role of the long non-coding RNA uc.48+ in the pathological changes of type 2 diabetes mellitus (T2DM) by observing the effects of uc.48+ small interfering RNA (siRNA) on the abdominal cells of a mouse model of T2DM. Mice with T2DM (DM group) were established by feeding with a high-sugar and -fat diet combined with intraperitoneal injections of low-dose streptozotocin. An intraperitoneal injection of uc.48+ siRNA was administered to the diabetic mice, and the serum levels of cytokines together with other clinical parameters, namely blood pressure, heart rate, mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were examined. Following the collection and identification of abdominal cells from the mice, the mRNA levels of uc.48+, mRNA and protein levels of the P2X7 receptor, and phosphorylation levels of ERK1/2 were evaluated by reverse transcription-PCR and western blotting, respectively. The MWT and TWL were significantly decreased in the DM group compared with the non-diabetic control group. However, the reductions in MWT and TWL were significantly attenuated following uc.48+ siRNA injection. The systolic and diastolic blood pressure, as well as the serum levels of tumor necrosis factor α and interleukin 1β of mice in the DM group were significantly increased compared with those in the control group, whereas these changes were significantly attenuated following the injection of uc.48+ siRNA. In addition, the expression levels of P2X7 receptor mRNA and protein, and the degree of phosphorylation of ERK1/2 in the abdominal cells were significantly increased in the DM group compared with the control group. These changes were also significantly attenuated following transfection with uc.48+ siRNA in vivo. In conclusion, these data suggest that uc.48+ may play an important role in the pathological changes of blood pressure, neurology and abdominal cell function in T2DM via interaction with the P2X7 receptor.

Transplantation of olfactory ensheathing cells combined with chitosan down-regulates the expression of P2X7 receptor in the spinal cord and inhibits neuropathic pain

BackgroundNeuropathic pain (NPP) is the common symptom of most clinical diseases, and its treatment has always been a difficult problem at present. Therefore, the purpose of this study is to explore a new method for the treatment of NPP by transplanting olfactory ensheathing cells combined with chitosan (OECs-CS). MethodsAnimal model of chronic compression sciatic nerve injury (CCI) was made, olfactory ensheathing cells (OECs) were cultured, chitosan (CS) biomaterials were prepared, and biocompatibility of OECs and CS were detected by MTT method, OECs and OECs-CS were transplanted into the site of the injured sciatic nerve respectively, behavioral method was used to measured the mechanical withdrawal thresholds (MWT) and thermal withdrawal latency (TWL) of rats. On days 7 and 14 after surgery, the expression level of P2X7 receptor (P2X7R) in the L4-5 spinal cord was measured by using in situ hybridization, western-blotting and qRT-PCR. To explore the therapeutic effect of OECs-CS transplantation on pain suppression. ResultsAfter chronic compression sciatic nerve injury, the MWT and TWL of rats were significantly reduced, and the expression levels of P2X7R protein and mRNA in the L4-5 spinal cord was significantly increased. After the transplantation of OECs and OECs-CS, the expression levels of P2X7R was significantly reduced, and the MWT and TWL of rats were significantly increased. Importantly, compared with the transplantation of OECs, OECs-CS transplantation could better reduce the expression levels of P2X7R, and relieve hyperalgesia in rats. Moreover, compared with the CCI + OECs-CS group on days 7 after surgery, the expression levels of P2X7R in the CCI + OECs-CS group was reduced on days 14 after surgery, and the pain in rats was relieved. ConclusionOECs and OECs-CS transplantation can inhibit P2X7R overexpression mediated NPP, while OECs-CS transplantation has better therapeutic effect than OECs transplantation alone. Our results provide a novel method and theoretical basis for the treatment of NPP.

Downregulating the P2X3 receptor in the carotid body to reduce blood pressure via acoustic gene delivery in canines

The purinergic P2X3 receptor in the carotid body (CB) is considered a new target for treating hypertension, although approaches for targeted regulating P2X3 receptor expression are lacking. Here, we explored the feasibility of targeted P2X3 receptor down-regulation in CBs by localized low-intensity focused ultrasound (LIFU)-mediated gene delivery to reduce the blood pressure. Thirty-two Kunming canines were randomly assigned to the treatment group (n = 14), negative control group (n = 10), LIFU + cationic microbubbles group (n = 4), and LIFU-only group (n = 4). Plasmid-loaded cationic microbubbles were injected and bilateral CBs were irradiated with a LIFU-based transducer. Flow cytometry showed that 33.15% of transfected cells expressed the green fluorescent protein reporter gene. T7 endonuclease I assays showed an insertion-deletion rate of 8.30%. The P2X3 receptor mRNA- and protein-expression levels in CBs decreased by 56.31% and 45.10%, respectively, in the treatment group. Mean systolic (152.5 ± 3.0 vs 138.0 ± 2.9 mm Hg, P = 0.003) and diastolic (97.8 ± 1.5 vs 87.2 ± 2.3 mm Hg, P= 0.002) blood pressures reduced on day 14 in the treatment group, compared with the baseline values, whereas no effects were observed with LIFU treatment or cationic microbubbles injection alone. Canines treated with this strategy exhibited no local or systemic adverse events. Thus, LIFU-mediated gene delivery to CBs successfully modulated CB function and reduced blood pressure in a canine model, suggesting a new possibility for treating hypertension and further clinical translation.

Comparison of mRNA Expression of P2X Receptor Subtypes in Different Arterial Tissues of Rats.

This study aims to compare the expression of P2X receptor subtype mRNA in different arterial tissues of rats. After the rats were sacrificed, the internal carotid, pulmonary, thoracic aorta, mesenteric and caudal arteries were dissected out. Then, the P2X receptor mRNA expression in different blood vessels was detected by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction. The P2X1, P2X4 and P2X7 receptor mRNA amplification products revealed specific bands of the same size as the amplified target fragment in their respective lanes, while the P2X2, P2X3, P2X5 and P2X6 receptor mRNA amplification products did not reveal significant specific bands in their respective lanes by RT-PCR. Based on the P2X1 receptor mRNA expression of the mesenteric artery, there were no significant differences in the internal carotid, pulmonary and thoracic aorta (0.64 ± 0.07, 0.17 ± 0.11 and 1.49 ± 0.65, respectively). However, the P2X1 receptor mRNA expression level in the caudal artery significantly increased (11.06 ± 1.99, P < 0.01). Furthermore, there was no difference in P2X4 receptor mRNA expression among these five blood vessels (P > 0.05). The P2X7 receptor mRNA expression level was significantly different: pulmonary artery < tail artery = thoracic aorta < internal carotid artery < mesenteric artery. The relative P2X1 receptor mRNA expression in the caudal artery was observed to be elevated when compared to that of the internal carotid, pulmonary and thoracic aorta as well as the mesenteric arteries. The P2X7 receptor mRNA expression level is pulmonary artery < caudal artery = thoracic aorta < internal carotid artery < mesenteric artery. P2X4 receptor mRNA expression was not significantly different among these five blood vessels.

Effect of olfactory ensheathing cells combined with chitosan on inhibition of P2×4 receptor over-expression-mediated neuropathic pain

Neuropathic pain (NPP) is a clinically refractory disease that causes pain to patients, and its treatment has always been an urgent problem to be solved. P2X4 receptor (P2X4R) plays a key role in the pathogenesis of neuropathic pain. Therefore, the aim of this studies to explore the effect of olfactory ensheathing cells combined with chitosan (OECs+CS) transplantation on NPP caused by sciatic nerve injury in rats, and its relationship with the expression levels of P2X4R in the L4-5 dorsal root ganglion (DRG). In this study, olfactory ensheathing cells (OECs) were cultured, chitosan (CS) was prepared, and the compatibility of CS and OECs was detected by MTT method. Animal model of chronic constrictive sciatic nerve injury (CCI) was made, OECs and OECs+CS were transplanted to the region surrounding the chronic sciatic nerve injury, and the difference between the two groups in the treatment of NPP was compared. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured by using behavioral method. in situ hybridization and Western-blotting were used to detect the expression of P2X4R mRNA and protein in the DRG. These results showed that OECs had good biocompatibility with CS. Compared with the CCI, the MWT and TWL were significantly increased (P＜0.05), the expression levels of P2X4R mRNA and protein in the OECs and OECs+CS group were significantly reduced (P＜0.05). Compared with the OECs, the expression levels of P2X4R mRNA and protein in the OECs+CS group were significantly reduced (P＜0.05), the MWT and TWL were significantly increased (P＜0.05). We conclude that OECs+CS can better inhibit P2X4R over-expression-mediated NPP, and its therapeutic effect was superior to simple OECs transplantation, which may become another potential method for the treatment of NPP.

P2 receptors mRNA expression profiles in macrophages from ankylosing spondylitis patients and healthy individuals.

Ankylosing spondylitis (AS) is a multifactorial rheumatic disease which mainly involves the axial skeleton. Macrophages and extracellular nucleotides have been shown to contribute to the inflammation process in autoimmune diseases. Membrane-bound purinergic P2 receptors might be involved in the modulation of immune cells in AS. Therefore, we aimed to analyze the messenger RNA (mRNA) expression of P2 receptors in the macrophages of AS patients and healthy controls. Twenty-three AS patients and 23 age- and sex-matched healthy individuals were included in our study. Whole blood-separated monocytes of study participants were stimulated by macrophage colony-stimulating factor for 7days and differentiated to macrophages. Monocyte and macrophage markers were analyzed by flow cytometry. SYBR green real-time polymerase chain reaction was used to measure the relative expression levels of P2RX1 , P2RX2 , P2RX3 , P2RX4 , P2RX5 , P2RX6 , P2RX7 , P2RY1 , P2RY2 , P2RY4 , P2RY6 , P2RY11 , P2RY12 , P2RY13 , P2RY14 , and PANX1 genes. P2RY13 and P2RY6 genes had the highest expression levels in macrophagesamong P2RY genes. P2RY1 mRNA expression was significantly down-regulated (-1.75 fold) and P2RY14 was up-regulated (2.6 fold) in macrophages of AS patients compared to healthy individuals. P2RX4 gene had the highest expression in monocyte-derived macrophages, followed by P2RX7 andP2RX1 genes. There was no significant difference in P2X receptor mRNA expression level between macrophages of AS patients and healthy individuals. Our results indicate that AS patients show altered expression levels of P2 receptor genes. Moreover, these changes might be associated with disease activity and patients' status.

Microencapsulated olfactory ensheathing cell transplantation reduces P2X4 receptor overexpression and inhibits neuropathic pain in rats

ObjectivesThe aim of this study was to determine the role of microencapsulated olfactory ensheathing cells (MC-OECs) transplantation in rats with sciatic nerve injury-induced neuropathic pain, and its relationship with P2X4 receptor expression in the L4-5 spinal cord segment. MethodsOlfactory bulb tissues of healthy Sprague Dawley (SD) rats were collected to culture olfactory ensheathing cells (OECs) using differential attachment methods. Seventy-two healthy SD rats were randomly assigned to four groups: the sham, chronic constriction injury (CCI), OECs, and MC-OECs groups. Mechanical paw withdrawal thresholds were measured on days 7, 14 and 21 after surgery. The expression of P2X4 receptor genes in the L4-5 spinal cord segment was detected by in situ hybridization, western blotting and RT-PCR. ResultsOn post-surgical days 7, 14 and 21. The mechanical paw withdrawal thresholds of rats from low to high were the CCI, OECs, MC-OECs and sham groups. The expression level of P2X4 receptor mRNA and protein from low to high were the sham, MC-OECs, OECs and CCI groups. Compared with the OECs group, the mechanical paw withdrawal thresholds of rats were increased, and the expression level of P2X4 receptor mRNA and protein were decreased in the MC-OECs group. In addition, the mechanical paw withdrawal thresholds of rats were higher and the expression level of P2X4 receptor were lower in the MC-OECs group on post-surgical days 21 than that on days 7 and 14. All differences between groups were statistically significant (P value < 0.05). ConclusionsOECs and MC-OECs transplantation can reduce the expression level of P2X4 receptor and relieve pain. The therapeutic efficacy was better in the MC-OECs group than in the OECs group. MC-OECs transplantation have the better effects of treatment with the increase of MC-OECs transplantation time.

Abnormal sympathetic activity after myocardial ischemia involving P2X4 in dorsal root ganglia

The satellite glial cells (SGCs) of the dorsal root ganglia (DRG) expressed P2X4 receptor. In this study, we investigated the abnormal sympathetic activity after myocardial ischemia (MI) involving P2X4 receptor in the cervical DRG SGC. The results showed that MI injury upregulated the P2X4 receptor mRNA and protein in DRG, and the upregulated P2X4 receptor was co-localized with glial fibrillary acidic protein (GFAP) in DRG SGCs. P2X4 short hairpin RNA (shRNA) treatment decreased the expression of P2X4 receptor, counteracted the upregulation of GFAP and IL-1β and inhibited P38MAPK phosphorylation in DRG of MI rats. These results indicate that application of P2X4 shRNA may reduce P2X4-mediated nociceptive signal via inhibiting DRG afferents to alleviate the abnormal sympathetic activity induced by MI.

Role of spinal P2X4 receptor in remifentanil-induced postoperative hyperalgesia

To explore the role of P2X4 receptor in opioid-induced hyperalgesia (OIH). Methods: A total of 30 Sprague-Dawley (SD) male rats were randomly divided into 5 groups: a saline (N0) group, a remifentanil at 0.5 μg/(kg.min) (R1) group, a remifentanil at 1.0 μg/(kg.min) (R2) group, a remifentanil at 1.5 μg/(kg.min) (R3) group, and a remifentanil at 5.0 μg/(kg.min) (R4) group. The paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured at follow time points to optimize the dosages: the day before treatment (T1), 30 min after tail intravenous catheterization (T2), and 30 min (T3), 1 h (T4), 2 h (T5), 24 h (T6) after withdrawal from remifentanil. Then, the rats were randomly divided into 2 groups: a saline group (N group), a remifentanil at 1.0 μg/(kg.min) group (R group). The PWMT and PWTL were measured at follow time points: T1, T2, and T4. The lumbar enlargement of spine was selected at 1 h after withdrawal from remifentanil, and the expression of P2X4 receptor mRNA and protein was examined in OIH. Additional male rats were selected and randomly divided into 2 groups: a plantar incision surgery followed by saline treatment group (I+N group), a plantar incision surgery followed by remifentanil treatment group (I+R group). The PWMT and PWTL were measured at follow time points: T1, T2, T3, T4, T5, T6, 48 h (T7) and 72 h (T8) after withdrawal from remifentanil. The lumbar enlargement of spine was selected at 1 h after withdrawal from remifentanil, the expression of P2X4 receptor mRNA and protein was examined by PCR and Western blotting, and the microglial activation in spine 1 h after withdrawal from remifentanil were assessed by immunofluorescence. Results: The pain thresholds including PWMT and PWTL in different groups were as follows: R4 group<R3 group<R2 group<R1 group at T3 and T5 (all P<0.05). At T4, the PWMT and PWTL in the R4 group were lower than those in the R2 group, while in the R4 group they were lower than those in the R3 group (both P<0.05). Meanwhile, the PWMT and PWTL in the R2 group, the R3 group and the R4 group at T4 were lower than those in the other time points (all P<0.05). Compared with the N group, the rats in the R group had a lower PWMT and PWTL at T4 (both P<0.05), but there was no significant difference in P2X4 receptor protein and mRNA expression in the lumbar enlargement of spine between the R group and the N group. Compared with the I+N group, the pain threshold including PWMT and PWTL at T4, T5, and T6 in the I+R group was significantly decreased (all P<0.05). Meanwhile, compared with the I+N group, the expressions of P2X4 receptor mRNA and protein in rat spinal cord in the I+R group were significantly up-regulated, and the microglia was activated (all P<0.05). Conclusion: Remifentanil pumped via tail intravenous can result in the hyperalgesia, but the spinal P2X4 receptor might be not relevant to remifentanil-induced hyperalgesia. Remifentanil incision pain can cause significant hyperalgesia in rats, and the spinal P2X4 receptors and microglial activation is involved in the remifentanil-induced postoperative hyperalgesia.

Diagnostic value of P2X7 receptor and its role in inflammatory reaction in rheumatoid arthritis

To study the diagnostic value of P2X7 receptor for rheumatoid arthritis (RA) and its role in the inflammatory response. With the synovial tissues from 25 patients with bone and joint replacement as the control,the synovial tissues of 25 RA patients were examined for the relative expression of P2X7 receptor mRNA using qRT-PCR.In an immortalized RA synovial cell line (MH7A),the effect of P2X7 receptor knockdown via a small interfering RNA were examined on the productions of the inflammatory cytokines including interleukin-1β(IL-1β),IL-6,and IL-8 using ELISA. The RA patients showed significantly higher levels of P2X7 receptor mRNA expression in the synovial tissue than the control patients.P2X7 receptor had a good diagnostic value for RA.The expression levels of IL-1β,IL-6,and IL-8 were positively correlated with the levels of P2X7 receptor in the synovial tissues of RA patients (P&lt;0.001).In MH7A cells,P2X7 receptor knockdown obviously reduced the secretion of IL-1β and IL-6. RA patients show elevated P2X7 receptor level in the synovial tissue, which has a good diagnostic value for RA.Blocking P2X7 receptor can inhibit inflammatory factor secretion and suppress inflammatory reactions.

P2X4R promotes airway remodeling by acting on the phenotype switching of bronchial smooth muscle cells in rats.

The P2X4 receptor (P2X4R) contributes to airway inflammation and airway remodeling in mice with allergic asthma. However, the molecular mechanism by which P2X4R affects the airway remodeling in allergic asthma remains largely unknown. We established an allergic asthma model by ovalbumin (OVA) inhalation in BALB/c mice. Compared with the mice in the control group, the expression of proliferating cell nuclear antigen (PCNA) increased and that of alpha-smooth muscle actin (α-SMA) decreased in the OVA-challenged mice. 5-BDBD, a P2X4R antagonist, alleviated the OVA-induced changes. To clarify the role of P2X4R in the phenotype switching of the bronchial smooth muscle, bronchial smooth muscle contractility and p38MAPK expression were investigated. Platelet-derived growth factor-BB (PDGF-BB) was used to activate the proliferation of primary-cultured rat bronchial smooth muscle cells (BSMCs). P2X4R, p38MAPK, and phenotype markers were evaluated using Western blotting or immunofluorescence. PDGF-BB administration increased the P2X4R and phospho-p38MAPK expression in BSMCs, and the increased phospho-p38MAPK expression was downregulated by silencing of the P2X4R mRNA. PDGF-BB stimulated the proliferation and synthetic phenotype of BSMCs, which was aggravated by a P2X4R agonist and alleviated by a P2X4R antagonist or silencing the P2X4R mRNA. The decreased contractile phenotype induced by PDGF-BB was alleviated by a P2X4R antagonist or by silencing the P2X4R mRNA. SB203580, p38MAPK inhibitor, inhibited the PDGF-BB-induced increasing of synthetic phenotype and the proliferation of BSMCs. These findings indicate that P2X4R acts directly on the phenotype switching of BSMCs. Inhibiting P2X4R can promote the contractile differentiation of BSMCs via p38MAPK signaling. Thus, the effect of P2X4R on airway remodeling indicates that this receptor could be a target for future drug candidates.

1,8-cineole decreases neuropathic pain probably via a mechanism mediating P2X3 receptor in the dorsal root ganglion

1,8-cineole is a natural monoterpene cyclic ether present in eucalyptus and has been reported to exhibit anti-inflammatory and antioxidant effects. The therapeutic effects of 1,8-cineole on neuropathic pain and the molecular mechanisms of its pharmacological actions remain largely unknown. In the present study, we investigated the analgesic mechanisms of orally administered 1,8-cineole in a rat model of chronic constriction injury (CCI) and examined the drug-induced modulation of P2X3 receptor expression in dorsal root ganglia. The mechanical withdrawal threshold and thermal withdrawal latency were measured in rats to assess behavioural changes 7 and 14 days after CCI surgery. Changes in P2X3 receptor mRNA expression of L4–5 dorsal root ganglia were analysed using quantitative real-time polymerase chain reaction at the 7th and 14th postoperative day. Additionally, we examined the expression of P2X3 receptor protein in L4–5 dorsal root ganglia 7 and 14 days after surgery using immunohistochemistry and western blots. We found that 1,8-cineole can alleviate pathological pain caused by P2X3 receptor stimulation and explored new methods for the prevention and treatment of neuropathic pain.

Andrographolide Inhibits Mechanical and Thermal Hyperalgesia in a Rat Model of HIV-Induced Neuropathic Pain.

Aim: In this study, we investigated whether andrographolide (Andro) can alleviate neuropathic pain induced by HIV gp120 plus ddC treatment and the mechanism of its action.Methods: The paw withdrawal threshold and the paw withdrawal latency were observed to assess pain behaviors in all groups of the rats, including control group, control combined with Andro treatment group, sham group, gp120 combined with ddC treatment group, gp120 plus ddC combined with A438079 treatment group, and gp120 plus ddC combined with Andro treatment by intrathecally injecting at a dose of 25 μg/20 μl group. The protein expression levels of the P2X7 receptor, tumor necrosis factor-α-receptor (TNFα-R), interleukin-1β (IL-1β), IL-10, phospho-extracellular regulated protein kinases (ERK) (p-ERK) in the L4–L6 dorsal root ganglia (DRG) were measured by western blotting. Real-time quantitative polymerase chain reaction was used to test the mRNA expression level of the P2X7 receptor. Double-labeling immunofluorescence was used to identify the co-localization of the P2X7 receptor with glial fibrillary acidic protein (GFAP) in DRG. Molecular docking was performed to identify whether the Andro interacted perfectly with the rat P2X7 (rP2X7) receptor.Results: Andro attenuated the mechanical and thermal hyperalgesia in gp120+ddC-treated rats and down-regulated the P2X7 receptor mRNA and protein expression in the L4–L6 DRGs of gp120+ddC-treated rats. Additionally, Andro simultaneously decreased the expression of TNFα-R and IL-1β protein, increased the expression of IL-10 protein in L4–L6 DRGs, and inhibited the activation of ERK signaling pathways. Moreover, Andro decreased the co-expression of GFAP and the P2X7 receptor in the SGCs of L4–L6 DRG on 14th day after surgery.Conclusion: Andro decreased the hyperalgesia induced by gp120 plus ddC.

Effects of dexamethasone on purinergic signaling in murine mast cells: Selective suppression of P2X7 receptor expression

Mast cells express many different purinergic receptors, including ionotropic P2X4 and P2X7, which recognize the accumulation of extracellular ATP released from activated and/or damaged cells. This results in the stimulation of mast cell functions. In this study, we investigated the effects of dexamethasone (Dex), an anti-inflammatory glucocorticoid widely used for the treatment of allergic disease, on purinergic receptor expression in mouse bone marrow-derived mast cells (BMMCs). Treatment of BMMCs with Dex decreased P2X7 receptor mRNA levels in a time- and concentration-dependent manner without affecting the expression of other purinergic receptor subtypes. Accordingly, fluorescence-activated cell sorting analysis revealed that Dex treatment also decreased P2X7 receptor protein levels. This effect was mimicked by prednisolone, another anti-inflammatory glucocorticoid, and was inhibited by the glucocorticoid receptor antagonist mifepristone. Functionally, treatment of BMMCs with Dex impaired the P2X7-mediated rise in intracellular Ca2+ concentration, degranulation, and ethidium uptake, a response relevant to receptor-pore formation. Finally, oral administration of Dex to C57BL/6 mice in vivo resulted in a significant decrease in P2X7 receptor expression in peritoneal mast cells. These results suggest that reduction of P2X7 receptor expression in mast cells might be one of the anti-allergic mechanisms of Dex.

Neuronal P2X7 Receptor: Involvement in Neuronal Physiology and Pathology.

The proposed presence of P2X7 receptor (P2X7R) in neurons has been the source of some contention. Initial studies suggested an absence of P2X7R mRNA in neurons, and the apparent nonspecificity of the antibodies used to identify P2X7R raised further doubts. However, subsequent studies using new pharmacological and biomolecular tools provided conclusive evidence supporting the existence of functional P2X7Rs in neurons. The P2X7 receptor has since been shown to play a leading role in multiple aspects of neuronal physiology, including axonal elongation and branching and neurotransmitter release. P2X7R has also been implicated in neuronal pathologies, in which it may influence neuronal survival. Together, this body of research suggests that P2X7R may constitute an important therapeutic target for a variety of neurological disorders.