Neohesperidin Alleviates the Neuropathic Pain Behavior of Rats by Downregulating the P2X4 Receptor.

Neuropathic pain (NP) is a type of chronic pain affecting 6-8% of human health as no effective drug exists. The purinergic 2X4 receptor (P2X4R) is involved in NP. Neohesperidin (NH) is a dihydroflavonoside compound, which has anti-inflammatory and antioxidative properties. This study aimed to investigate whether NH has an effect on P2X4R-mediated NP induced by chronic constriction injury (CCI) of the sciatic nerve in rats. In this study, the CCI rat model was established to observe the changes of pain behaviors, P2X4R, and satellite glial cells (SGCs) activation in dorsal root ganglion (DRG) after NH treatment by using RT-PCR, immunofluorescence double labeling and Western blotting. Our results showed CCI rats had mechanical and thermal hyperalgesia with an increased level of P2X4R. Furthermore, SGCs were activated as indicated by increased expression of glial fibrillary acidic protein and increased tumor necrosis factor-alpha receptor 1and interleukin-1ß. In addition, phosphorylated extracellular regulated protein kinases and interferon regulatory factor 5 in CCI rats increased. After NH treatment in CCI rats, the levels of above protein decreased, and the pain reduced. Overall, NH can markedly alleviate NP by reducing P2X4R expression and SGCs activation in DRG.

Purinergic signaling: a potential therapeutic target for depression and chronic pain.

The comorbid mechanism of depression and chronic pain has been a research hotspot in recent years. Until now, the role of purinergic signals in the comorbid mechanism of depression and chronic pain has not been fully understood. This review mainly summarizes the research results published in PubMed during the past 5 years and concludes that purinergic signaling is a potential therapeutic target for comorbid depression and chronic pain, and the purinergic receptors A1, A2A, P2X3, P2X4, and P2X7and P2Y6, P2Y1, and P2Y12 may be important factors. The main potential pathways are as follows: A1 receptor-related G protein-dependent activation of introverted K+ channels (GIRKs), A2A receptor-related effects on the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) and MAPK/nuclear factor-κB (NF-κB) pathways, P2X3 receptor-related effects on dorsal root ganglia (DRG) excitability, P2X4 receptor-related effects on proinflammatory cytokines and inflammasome activation, P2X7 receptor-related effects on ion channels, the NLRP3 inflammasome and brain-derived neurotrophic factor (BDNF), and P2Y receptor-related effects on the phospholipase C (PLC)/inositol triphosphate (IP3)/Ca2+ signaling pathway. We hope that the conclusions of this review will provide key ideas for future research on the role of purinergic signaling in the comorbid mechanism of depression and chronic pain.

Imperatorin Improves Obesity-Induced Cardiac Sympathetic Nerve Injury Mediated by P2X4 Receptor in Stellate Sympathetic Ganglion.

Obesity can activate the inflammatory signal pathway, induce in the body a state of chronic inflammation, and increase the excitability of the sympathetic nervous system, which may induce sympathetic neuropathic injury. The stellate sympathetic ganglia (SG) can express the P2X4 receptor, and the abnormal expression of the P2X4 receptor is related to inflammation. Imperatorin (IMP) is a kind of furan coumarin plant which has anti-inflammatory effects. This project aimed to investigate whether IMP can affect the expression of P2X4 receptors in the SG of obese rats to display a protective effect from high-fat-triggered cardiac sympathetic neuropathic injury. Molecular docking through homology modelling revealed that IMP had good affinity for the P2X4 receptor. Our results showed that compared with the normal group, the administration of IMP or P2X4 shRNA decreased sympathetic excitement; reduced the serum levels of triglyceride, total cholesterol, and lactate dehydrogenase; downregulated the expression of P2X4 receptors in SG; and inhibited the expression of inflammatory factors in the SG and serum of obese rats significantly. In addition, the expression of factors associated with the cell pyroptosis GSDMD, caspase-1, NLRP-3, and IL-18 in obese rats were significantly higher than those of the normal rats, and such effects were decreased after treatment with IMP or P2X4 shRNA. Furthermore, IMP significantly reduced the ATP-activated currents in HEK293 cells transfected with P2X4 receptor. Thus, the P2X4 receptor may be a key target for the treatment of obesity-induced cardiac sympathetic excitement. IMP can improve obesity-induced cardiac sympathetic excitement, and its mechanism of action may be related to the inhibition of P2X4 receptor expression and activity in the SG, suppression of cellular pyroptosis in the SG, and reduction of inflammatory factor levels.

Correction to: LncRNA NONRATT021972 involved the pathophysiologic processes mediated by P2X7 receptors in stellate ganglia after myocardial ischemic injury.

In the original article, Figs. 2, 3b, and 4b were published incorrectly.

Long non coding RNAs involved in MAPK pathway mechanism mediates diabetic neuropathic pain.

Diabetes is the largest global epidemic of the 21st century, and the cost of diabetes and its complications comprise about 12% of global health expenditure. Diabetic neuropathy is the most common complication of diabetes, affecting up to 50% of patients over the course of their disease. Among them, 30%-50% develop neuropathic pain, which has typical symptoms that originate from the toes and progress to foot ulcers and seriously influence quality of life. The pathogenesis of diabetic neuropathic pain (DNP) is complicated and incompletely understood and there is no effective treatment except supportive treatment. Long noncoding RNAs (lncRNAs), a class of noncoding RNAs exceeding 200 nucleotides in length, have been shown to play key roles in fundamental cellular processes, and are considered to be potential targets for treatment. Recent research indicates that lncRNA is involved in the pathogenesis of DNP. Certain overexpressed lncRNAs can enhance the purinergic receptor-mediated neuropathic pain in peripheral ganglia and inflammatory cytokines are released due to receptors activated by adenosine triphosphate. In recent years, our laboratory also has been exploring the relationship and pathogenesis between lncRNAs and DNP. In this review, we focus on the recent progress in functional lncRNAs associated with DNP and investigate their roles related to respective receptors.

Noncoding transcribed ultraconserved region (T-UCR) UC.48+ is a novel regulator of high-fat diet induced myocardial ischemia/reperfusion injury.

Increasing evidence has suggested high-fat diet (HFD) is an independent risk factor for myocardial ischemia/reperfusion (MI/R) injury. Long noncoding RNAs (lncRNAs) recently attracted much attraction in the study of MI/R injury. However, the functional questions of specific lncRNAs in HFD-induced MI/R injury have not been well elucidated. Uc.48+ is a lncRNA from a transcribed ultraconserved region (T-UCR) of human, mouse, and rat genomes. Here, we explored the aggravating role of uc.48+and identified purinergic P2X7 receptor (P2X7R) as a downstream regulator of uc.48+ in HFD-induced MI/R vulnerability. We demonstrated uc.48+ expression was upregulated, accompanied by the corresponding upregulation of P2X7R in HFD I/R myocardium and HFD-induced MI/R vulnerability. Overexpression of uc.48+enhanced, whereas silencing of uc.48 + decreased the expression of P2X7R, cardiomyocyte apoptosis, and MI/R injury. The functional relevance of uc.48+ regulated P2X7R expression and the subsequent NF-κB signaling to promote cardiomyocyte apoptosis was supported by inhibition of P2X7R with its specific antagonist (A438079) as well as the inhibitor of NF-κB signaling (pyrrolidine dithiocarbamate, PDTC) in H9c2 hypoxia/reoxygenation (H/R) cells transfected with pcDNA3.0-uc.48 + plasmid, and RNA immunoprecipitation (RIP) suggested uc.48+ could interact with transcription factor Sp1. Importantly, Sp1 inhibitor (mithramycin, MIT) was found to suppress uc.48+ -induced P2X7R expression and the NF-κB signaling and cardiomyocyte apoptosis. Our findings provide a potential novel mechanism through which uc.48+ boosts cardiomyocyte apoptosis and MI/R vulnerability to HFD. Thus, uc.48+ is a novel regulator of HFD-induced MI/R injury; targeting uc.48+ may be a novel therapeutic approach of MI/R vulnerability to HFD.

Quercetin relieved diabetic neuropathic pain by inhibiting upregulated P2X4 receptor in dorsal root ganglia.

The upregulation of nociceptive ion channels expressed in dorsal root ganglia (DRG) contributes to the development and retaining of diabetic pain symptoms. The flavonoid quercetin (3,3',4',5,7-pentahydroxyflavone) is a component extracted from various fruits and vegetables and exerts anti-inflammatory, analgesic, anticarcinogenic, antiulcer, and antihypertensive effects. However, the exact mechanism underlying quercetin's analgesic action remains poorly understood. The aim of this study was to investigate the effects of quercetin on diabetic neuropathic pain related to the P2X4 receptor in the DRG of type 2 diabetic rat model. Our data showed that both mechanical withdrawal threshold and thermal withdrawal latency in diabetic rats treated with quercetin were higher compared with those in untreated diabetic rats. The expression levels of P2X4 messenger RNA and protein in the DRG of diabetic rats were increased compared with the control rats, while quercetin treatment significantly inhibited such enhanced P2X4 expression in diabetic rats. The satellite glial cells (SGCs) enwrap the neuronal soma in the DRG. Quercetin treatment also lowered the elevated coexpression of P2X4 and glial fibrillary acidic protein (a marker of SGCs) and decreased the upregulation of phosphorylated p38 mitogen-activated protein kinase (p38MAPK) in the DRG of diabetic rats. Quercetin significantly reduced the P2X4 agonist adenosine triphosphate-activated currents in HEK293 cells transfected with P2X4 receptors. Thus, our data demonstrate that quercetin may decrease the upregulation of the P2X4 receptor in DRG SGCs, and consequently inhibit P2X4 receptor-mediated p38MAPK activation to relieve the mechanical and thermal hyperalgesia in diabetic rats.

Involvement of purinergic 2X4 receptor in glycoprotein 120-induced pyroptosis in dorsal root ganglia.

Pyroptosis is a type of programmed cell death, displaying caspase-1-dependent and pro-inflammatory features. Purinergic 2X4 (P2X4 ) receptor activation in response to high-adenosine triphosphate release can induce inflammation. Envelope glycoprotein 120 (gp120) of human immunodeficiency virus type 1 is considered one of the primary pathogens leading to neuronal injury. In this study, we investigated the possible role of P2X4 receptor activation in gp120-triggered pyroptosis in cultured satellite glial cells (SGCs) of rat dorsal root ganglia (DRG). MTS assay, TdT-mediated dUTP Nick-end labeling assay, real-time RT-PCR, and western blotting et al. methods were used. The results indicated that the expression of P2X4 receptor in SGCs of DRG was up-regulated upon cultured with gp120 for 24 h. The highest decrease in viability of SGCs due to gp120 treatment was accompanied by marked increases of positive pyroptosis cells and cellular lactate dehydrogenase release, elevated levels of interleukin-1ß, interleukin-18, active caspase-1 and NOD-like receptor family, pyrin domain containing 1, and enhanced phosphorylation of p38MAPK. These abnormal changes because of gp120 were significantly inhibited and cell viability was markedly improved when SGCs of DRG were treated with short hairpin RNAs targeting P2X4 receptor. Our data suggest that silencing of P2X4 receptor may act effectively against gp120-induced pyroptosis mediated by the activation of NOD-like receptor family, pyrin domain containing 1 inflammasome and caspase-1 signaling in SGCs of DRG.

Electroacupuncture inhibits visceral pain via adenosine receptors in mice with inflammatory bowel disease.

To investigate the involvement of peripheral adenosine receptors in the effect of electroacupuncture (EA) on visceral pain in mice with inflammatory bowel disease (IBD). 2,4,6-Trinitrobenzene sulfonic acid (TNBS) was used to induce the visceral pain model. EA (1 mA, 2 Hz, 30 min) treatment was applied to bilateral acupoints "Dachangshu" (BL25) 1 day after TNBS injection once daily for 7 consecutive days. Von Frey filaments were used to measure the mechanical pain threshold. Western blot was used to detect the protein expression levels of adenosine 1 receptor (A1R), adenosine 2a receptor (A2aR), adenosine 2b receptor (A2bR), adenosine 3 receptor (A3R), substance P (SP), and interleukin 1 beta (IL-1ß) in colon tissue. EA significantly ameliorated the disease-related indices and reduced the expression of SP and IL-1ß in the colon tissues of mice with IBD. EA increased the expression of A1R, A2aR, and A3R and decreased the expression of A2bR in the colon tissue. Furthermore, the administration of adenosine receptor antagonists influenced the effect of EA. EA can inhibit the expression of the inflammatory factors SP and IL-1ß by regulating peripheral A1, A2a, A2b, and A3 receptors, thus inhibiting visceral pain in IBD mice.

P2X receptors mediated abnormal interaction between satellite glial cells and neurons in visceral pathological changes.

The adenosine triphosphate (ATP)-gated P2X receptor cation channel family consists of permeable ligand-gated ion channels that expand on the binding of extracellular adenosine 5'-ATP. ATP-gated P2X receptors are trimer ion channels that assemble homo or isomer from seven cloned subunits. P2X receptors are discovered mostly in mammalian and are being found in an increasing number of non-vertebrates, such as zebrafish, bullfrog, and ameba. P2X receptors are involved in many physiological processes, including regulation of heart rhythm and contractility, and regulation of pain, especially chronic pain and glia integration. This review summarizes the current studies on the regulation of P2X receptors in abnormal neuronal-glial interaction and the pathological changes in viscera, especially in myocardial ischemia.

Expression and Regulation of Brain Natriuretic Peptide and Natriuretic Peptide Receptor A (NPR-A) in L6-S1 Dorsal Root Ganglia in a Rat Model of Chronic Nonbacterial Prostatitis.

BACKGROUND The study aimed to investigate the expression of brain natriuretic peptide (BNP) and natriuretic peptide receptor A (NPR-A) in L6-S1 dorsal root ganglia (DRG) in a rat model of chronic nonbacterial prostatitis (CNP). MATERIAL AND METHODS One hundred specific pathogen-free (SPF) male Sprague-Dawley rats were randomly divided into a control group (N=50) and a study group (N=50). The control group underwent prostatic injection of 0.1 ml of normal saline on days 3, 7, 10, 14, and 28. The study group, or rat model of CNP, underwent prostatic injection of 0.1 ml of complete Freund's adjuvant on days 3, 7, 10, 14, and 28. At the end of the study, the rats were euthanized, and the prostate tissues and L6-S1 DRG were removed. Histology was performed on the prostate tissue from the rats in the study group and control group. Real-time fluorescence-based quantitative polymerase chain reaction (PCR) and Western blot were used to study the expression of BNP and NPR-A mRNA and protein in the DRG from the rats in the study group and control group. RESULTS In the rat model of CNP, the expression of BNP and NPR-A were significantly increased in L6-S1 DRG compared with the controls. CONCLUSIONS In a rat model of CNP, the increased expression of BNP and NPR-A in L6-S1 DRG may have a role in pain signaling pathways associated with chronic prostatitis.

Role of hesperidin in P2X3 receptor-mediated neuropathic pain in the dorsal root ganglia.

Aim: This study investigated whether the neuronal P2X3 receptor in rat dorsal root ganglia (DRG) mediated the effects of hesperidin on neuropathic pain. Materials and methods: The chronic constriction injury (CCI) model was used as a model of neuropathic pain. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured. The mRNA and protein expression levels were assayed by real-time RT-PCR and Western blotting. Results: The results showed that mechanical and thermal hyperalgesia in the CCI rats were increased as compared to those in the sham group. The expression levels of P2X3 mRNA and protein in CCI rats were higher than those in the sham group. Dual-labelling immunofluorescence showed that the elevated P2X3 receptor was co-expressed with the neuronal marker NeuN in the DRG of CCI rats. Hesperidin treatment decreased both the mechanical and thermal hyperalgesia, and upregulated P2X3 expression in the CCI rats. Hesperidin treatment also reduced the ERK1/2 phosphorylation in the DRG of CCI rats. Moreover, hesperidin inhibited the P2X3 agonist ATP-induced currents in HEK293 cells transfected with the P2X3 plasmid. Therefore, hesperidin treatment could reverse the elevated expression of neuronal P2X3 receptor and reduce the activation of ERK1/2 in the DRG of CCI rats. Conclusions: Our findings suggested that hesperidin inhibited the nociceptive transmission mediated by the P2X3 receptor in neurons of DRG, and thus, relieved the mechanical and thermal hyperalgesia in CCI rats.

Downregulation of P2Y12 in the superior cervical ganglia alleviates abnormal sympathetic activity after myocardial ischemia.

Superior cervical ganglia (SCG) innervate the myocardium and participate in sympathoexcitatory transmission. P2Y12 receptor is expressed in satellite glial cells (SGCs). This study seeks to clarify whether the P2Y12 receptor is involved in the sympathoexcitation reflex after myocardial ischemia (MI). MI model was induced by occlusion of the left coronary artery. P2Y12 were assayed by real time PCR and Western blotting. Our results showed that expression levels of P2Y12 mRNA and protein were significantly higher in the MI group than in the sham group. Administration of P2Y12 short hairpin RNA (shRNA) caused downregulation of the P2Y12 receptor in the SCG. In MI rats plus P2Y12 shRNA treatment group, the abnormal changes in diastolic blood pressure (DBP), systolic blood pressure (SBP), heart rate (HR), electrocardiograms (ECGs), and cardiac tissue structures were alleviated. When the treatment of P2Y12 shRNA in MI rats, upregulated co-expression values of P2Y12 and glial fibrillary acidic protein (GFAP), the upregulation of tumor necrosis factor α (TNF-α) and phosphorylated P38 mitogen activated protein kinase (p-P38 MAPK) in the SCG were decreased. Downregulation of the P2Y12 receptor in the SCG after MI may improve cardiac function by alleviating the sympathoexcitatory reflex.

P2Y12 shRNA treatment decreases SGC activation to relieve diabetic neuropathic pain in type 2 diabetes mellitus rats.

Diabetic neuropathic pain is a common complication of type 2 diabetes mellitus (DM). Activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRG) plays a crucial role in neuropathic pain through the release of proinflammatory cytokines. The P2Y12 receptor is expressed in SGCs of the DRG. In this study, our aim was to investigate the role of the P2Y12 receptor on the pathological changes in diabetic neuropathic pain. The present study showed that diabetic neuropathic pain increased mechanical and thermal hyperalgesia in type 2 DM model rats. The results showed that the expression levels of P2Y12 messenger RNA (mRNA) and protein in DRG SGCs were increased in DM model rats compared with control rats. Glial fibrillary acidic protein (GFAP) and interleukin-1ß (IL-1ß) expression levels in the DRG were increased in DM rats. Upregulation of GFAP is a marker of SGC activation. Targeting the P2Y12 receptor by short hairpin RNA (shRNA) decreased the upregulated expression of P2Y12 mRNA and protein, coexpression of P2Y12 and GFAP, the expression of GFAP, IL-1ß, and tumor necrosis factor-receptor 1 in the DRG of DM rats, and relieved mechanical and thermal hyperalgesia in DM rats. After treatment with the P2Y12 receptor shRNA, the enhancing integrated OPTICAL density (IOD) ratios of p-P38 MAPK to P38 mitogen activated protein kinase (MAPK) in the DM rats treated with P2Y12 shRNA were significantly lower than that in the untreated DM rats. Therefore, P2Y12 shRNA treatment decreased SGC activation to relieve mechanical and thermal hyperalgesia in DM rats.

Protective Effects of Oxymatrine on Vascular Endothelial Cells from High-Glucose-Induced Cytotoxicity by Inhibiting the Expression of A2B Receptor.

BACKGROUND/AIMS: Diabetes mellitus (DM) has become an increasingly epidemic metabolic disease. Vascular endothelial cells play a key role in developing the cardiovascular complications of DM. The A2B receptor is expressed in vascular endothelial cells, and may help regulate the function of endothelial cells. The aim of this study was to investigate the protective effects of oxymatrine (OMT) on human umbilical vein endothelial cells (HUVECs) from high glucose-induced cytotoxicity. METHODS: Homology modeling and molecular docking analysis were used to detect the binding sites between the adenosine A2B receptor and OMT. HUVECs were cultured with control (5.5 mM) or elevated glucose (22.2 mM) in the presence or absence of 3 µM OMT or A2B siRNA for 3 days. The MTS cell viability assay was used to measure the toxicity of high glucose on HUVECs and the protective effect of OMT or A2B siRNA. The expression of the adenosine A2B receptor and CCL5 in HUVECs was detected with real-time quantitative PCR (qPCR) and Western blotting methods in each group. Levels of IL-1ß and TNF-α were measured using an enzyme-linked immunosorbent assay (ELISA) kit, and the concentration of NO was detected with the nitrate reductase method. Monocyte chemotactic activity in each group was detected using Transwell chambers. Furthermore, the phosphorylation of p38 and ERK1/2 in each group was observed through the Western blotting method. RESULTS: Homology modeling and molecular docking analysis showed that OMT contains well-fitted binding sites to the A2B receptor. After chronic culture at high glucose, the rate of cell viability was significantly lower than that of the control group. After co-treatment with OMT or A2B siRNA, cell viability was significantly increased compared with the high-glucose group. The results from real-time quantitative RT-PCR (qRT-PCR) and Western blotting indicated that high glucose could increase the expression of A2B receptors in HUVECs, an effect that was inhibited by OMT. In addition, the results revealed that the expression of CCL5, IL-1ß and TNF-α was increased in the high-glucose group, and that the NO produced by HUVECs decreased due to hyperglycemia; however, co-culture with OMT or A2B siRNA abolished these effects. Meanwhile, the chemotaxis activity of monocytes to HUVECs cultured in high-glucose medium was enhanced 2.59-fold compared to the control cells. However, the inflammatory reactions in HUVECs were completely relieved by co-treatment with OMT or A2B siRNA. Moreover, the phosphorylation of p38 and ERK1/2 in HUVECs in the high-glucose group was significantly higher than that of the control group; these effects were reversed after co-treatment with OMT or A2B siRNA. CONCLUSION: OMT may protect the HUVECs from high glucose-induced cytotoxicity through inhibitting the expression of A2B receptor and inflammatory factors as well as decreasing the phosphorylation of p38 and ERK1/2.

Effects of Baicalin on Diabetic Cardiac Autonomic Neuropathy Mediated by the P2Y12 Receptor in Rat Stellate Ganglia.

BACKGROUND/AIMS: Chronic diabetic hyperglycemia can damage various of organ systems and cause serious complications. Although diabetic cardiac autonomic neuropathy (DCAN) is the primary cause of death in diabetic patients, its pathogenesis remains to be fully elucidated. Baicalin is a flavonoid extracted from Scutellaria baicalensis root and has antibacterial, diuretic, anti-inflammatory, anti- metamorphotic, and antispasmodic effects. Our study explored the effects of baicalin on enhancing sympathoexcitatory response induced by DCAN via the P2Y12 receptor. METHODS: A type 2 diabetes mellitus rat model was induced by a combination of diet and streptozotocin. Serum epinephrine was measured by enzyme-linked immunosorbent assay. Blood pressure and heart rate were measured using the indirect tail-cuff method. Heart rate variability was analyzed using the frequency-domain of electrocardiogram recordings. The expression levels of P2Y12, interleukin-1beta (IL-1ß), tumor necrosis factor alpha (TNF-α), and connexin 43 (Cx43) were determined by quantitative real-time reverse transcription-polymerase chain reaction and western blotting. The interaction between baicalin and P2Y12 determined using by molecular docking. RESULTS: Baicalin alleviated elevated blood pressure and heart rate, improved heart rate variability, and decreased the elevated expression levels of P2Y12, IL-1ß, TNF-α, and Cx43 in the stellate ganglia of diabetic rats. Baicalin also reduced the elevated concentration of serum epinephrine and the phosphorylation of p38 mitogen-activated protein kinase in diabetic rats. CONCLUSION: Baicalin decreases sympathetic activity by inhibiting the P2Y12 receptor in stellate ganglia satellite glial cells to maintain the balance between sympathetic and parasympathetic nerves and relieves DCAN in the rat.

Involvement of P2Y12 receptor of stellate ganglion in diabetic cardiovascular autonomic neuropathy.

Diabetes as a chronic epidemic disease with obvious symptom of hyperglycemia is seriously affecting human health globally due to the diverse diabetic complications. Diabetic cardiovascular autonomic neuropathy (DCAN) is a common complication of both type 1 and type 2 diabetes and incurs high morbidity and mortality. However, the underlying mechanism for DCAN is unclear. It is well known that purinergic signaling is involved in the regulation of cardiovascular function. In this study, we examined whether the P2Y12 receptor could mediate DCAN-induced sympathetic reflexes. Our results revealed that the abnormal changes of blood pressure, heart rate, heart rate variability, and sympathetic nerve discharge were improved in diabetic rats treated with P2Y12 short hairpin RNA (shRNA). Meanwhile, the expression of P2Y12 receptor, interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and connexin 43 (Cx43) in stellate ganglia (SG) was decreased in P2Y12 shRNA-treated diabetic rats. In addition, knocking down the P2Y12 receptor also inhibited the activation of p38 MARK in the SG of diabetic rats. Taken together, these findings demonstrated that P2Y12 receptor in the SG may participate in developing diabetic autonomic neuropathy, suggesting that the P2Y12 receptor could be a potential therapeutic target for the treatment of DCAN.

P2Y12 receptor upregulation in satellite glial cells is involved in neuropathic pain induced by HIV glycoprotein 120 and 2',3'-dideoxycytidine.

The direct neurotoxicity of HIV and neurotoxicity of combination antiretroviral therapy medications both contribute to the development of neuropathic pain. Activation of satellite glial cells (SGCs) in the dorsal root ganglia (DRG) plays a crucial role in mechanical and thermal hyperalgesia. The P2Y12 receptor expressed in SGCs of the DRG is involved in pain transmission. In this study, we explored the role of the P2Y12 receptor in neuropathic pain induced by HIV envelope glycoprotein 120 (gp120) combined with ddC (2',3'-dideoxycytidine). A rat model of gp120+ddC-induced neuropathic pain was used. Peripheral nerve exposure to HIV-gp120+ddC increased mechanical and thermal hyperalgesia in gp120+ddC-treated model rats. The gp120+ddC treatment increased expression of P2Y12 receptor mRNA and protein in DRG SGCs. In primary cultured DRG SGCs treated with gp120+ddC, the levels of [Ca2+]i activated by the P2Y12 receptor agonist 2-(Methylthio) adenosine 5'-diphosphate trisodium salt (2-MeSADP) were significantly increased. P2Y12 receptor shRNA treatment inhibited 2-MeSADP-induced [Ca2+]i in primary cultured DRG SGCs treated with gp120+ddC. Intrathecal treatment with a shRNA against P2Y12 receptor in DRG SGCs reduced the release of pro-inflammatory cytokines, decreased phosphorylation of p38 MAPK in the DRG of gp120+ddC-treated rats. Thus, downregulating the P2Y12 receptor relieved mechanical and thermal hyperalgesia in gp120+ddC-treated rats.

Genetic interaction of purinergic P2X7 receptor and ER-α polymorphisms in susceptibility to osteoporosis in Chinese postmenopausal women.

Osteoporosis (OP) is an increasing public health problem worldwide. Genetic factors are considered to be major contributors to the pathogenesis of OP. The aim of this study was to investigate the association of the purinergic P2X7 receptor (P2X7R) and estrogen receptor-α (ER-α) genes with OP risk, and the effect of the possible interaction between the two genes on predisposition to OP in Chinese postmenopausal women. A total of 596 subjects, including 350 OP patients and 246 controls, were recruited in this case-control study. Five functional single-nucleotide polymorphisms (SNPs) in the P2X7R gene (rs2393799, rs7958311, rs1718119, rs2230911, rs3751143) and two ER-α PvuII and XbaI polymorphisms were genotyped and analyzed. Single-gene variant analysis showed that the carriers of the CC genotype of P2X7R rs3751143 revealed an increased OP risk. Haplotype rs1718119G-rs2230911G-rs3751143C also appeared to be a significant 'risk' haplotype with OP. For the ER-α gene, no evidence of significant association of PvuII or XbaI polymorphism with OP risk was found. Moreover, there was a significant gene-gene interaction between P2X7R rs3751143 and ER-α PvuII; the cross-validation consistency was 10/10 and the testing accuracy was 0.5818 (P = 0.0107). A 1.67-fold-increased risk for OP was detected in individuals carrying the genotypes of AC or CC of rs3751143 and Pp or PP of PvuII compared to subjects with AA of rs3751143 and pp of PvuII. Our findings suggest an important association of the P2X7R rs3751143CC genotype and the rs1718119G-rs2230911G-rs3751143C haplotype with an increased OP risk. Also, the P2X7R rs3751143 and ER-α PvuII two-locus interaction confers a significantly high susceptibility to OP in Chinese postmenopausal women.

Chronic lead exposure enhances the sympathoexcitatory response associated with P2X4 receptor in rat stellate ganglia.

Chronic lead exposure causes peripheral sympathetic nerve stimulation, including increased blood pressure and heart rate. Purinergic receptors are involved in the sympathoexcitatory response induced by myocardial ischemia injury. However, whether P2X4 receptor participates in sympathoexcitatory response induced by chronic lead exposure and the possible mechanisms are still unknown. The aim of this study was to explore the change of the sympathoexcitatory response induced by chronic lead exposure via the P2X4 receptor in the stellate ganglion (SG). Rats were given lead acetate through drinking water freely at doses of 0 g/L (control group), 0.5 g/L (low lead group), and 2 g/L (high lead group) for 1 year. Our results demonstrated that lead exposure caused autonomic nervous dysfunction, including blood pressure and heart rate increased and heart rate variability (HRV) decreased. Western blotting results indicated that after lead exposure, the protein expression levels in the SG of P2X4 receptor, IL-1ß and Cx43 were up-regulated, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was activated. Real-time PCR results showed that the mRNA expression of P2X4 receptor in the SG was higher in lead exposure group than that in the control group. Double-labeled immunofluorescence results showed that P2X4 receptor was co-expressed with glutamine synthetase (GS), the marker of satellite glial cells (SGCs). These changes were positively correlated with the dose of lead exposure. The up-regulated expression of P2X4 receptor in SGCs of the SG maybe enhance the sympathoexcitatory response induced by chronic lead exposure.