OBJECTIVE: To observe the effect of moxibustion at "Guanyuan" (CV 4) and "Shenque" (CV 8) on acetylcholine (Ach), adenosine triphosphate (ATP) and muscarinic-type choline receptor (M2) and purine receptor P2X3 in bladder tissue in the rats with neurogenic bladder (NB) of detrusor areflexia after lumbar-sacral spinal cord injury and explore the underlying mechanism of moxibustion for promoting detrusor contraction. METHODS: Sixty SD rats were randomly divided into a model preparation group (n=45) and a sham-operation group (n=15). In the model preparation group, the modified Hassan Shaker spinal cord transection method was used to prepare the model of NB. In the sham-operation group, the spinal cord transection was not exerted except laminectomy and spinal cord exposure. Among the rats with successfully modeled, 30 rats were selected and divided randomly into a model group and a moxibustion group, with 15 rats in each one. On the 15th day after the operation, moxibustion was applied at "Guanyuan" (CV 4) and "Shenque" (CV 8) in the moxibustion group, 10 min at each acupoint, once a day. The consecutive 7-day treatment was as one course and the intervention for 2 courses was required. Urodynamic test was adopted to evaluate bladder function in rats. Using HE staining, the morphological changes in bladder tissue were observed. The content of Ach and ATP in bladder tissue was measured with biochemical method, and the protein and mRNA expression levels of M2 and P2X3 receptors in bladder tissue were detected with Western blot and real-time fluorescence quantification PCR method. RESULTS: Compared with the sham-operation group, the maximum bladder capacity, leakage point pressure and bladder compliance were increased in the rats of the model group (P<0.05). Compared with the model group, the maximum bladder capacity, the leakage point pressure and bladder compliance were decreased in the rats of the moxibustion group (P<0.05). In the model group, the detrusor fibres were arranged irregularly, bladder epithelial tissues were not tightly connected and cell arrangement was disordered, combined with a large number of vacuolar cells. In the moxibustion group, compared with the model group, the detrusor fibres were arranged regularly, bladder epithelial cells were well distributed and vacuolar cells were reduced. Compared with the sham-operation group, the content of Ach and ATP in bladder tissue was decreased (P<0.05), the protein and mRNA expression levels of M2 and P2X3 receptors were reduced (P<0.05) in the model group. In the moxibustion group, the content of Ach and ATP in bladder tissue was increased (P<0.05) and the protein and mRNA expression levels of M2 and P2X3 receptors were increased (P<0.05) as compared with the model group. CONCLUSION Moxibustion at "Guanyuan" (CV 4) and "Shenque" (CV 8) may effectively improve bladder function in the rats with NB of detrusor areflexia after lumbar-sacral spinal cord injury and its underlying mechanism is related to promoting the release of Ach and up-regulating the expression of M2 receptor, thereby enhancing the release of ATP and increasing the expression of P2X3 receptor. Eventually, detrusor contraction is improved.
Animals ; Moxibustion/methods* ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic P2X3/metabolism* ; Spinal Cord Injuries/therapy* ; Urinary Bladder ; Urinary Bladder, Neurogenic/therapy*

Irritable bowel syndrome is a gastrointestinal disorder of unknown etiology characterized by widespread, chronic abdominal pain associated with altered bowel movements. Increasing amounts of evidence indicate that injury and inflammation during the neonatal period have long-term effects on tissue structure and function in the adult that may predispose to gastrointestinal diseases. In this study we aimed to investigate how the epigenetic regulation of DNA demethylation of the p2x7r locus guided by the transcription factor GATA binding protein 1 (GATA1) in spinal astrocytes affects chronic visceral pain in adult rats with neonatal colonic inflammation (NCI). The spinal GATA1 targeting to DNA demethylation of p2x7r locus in these rats was assessed by assessing GATA1 function with luciferase assay, chromatin immunoprecipitation, patch clamp, and interference in vitro and in vivo. In addition, a decoy oligodeoxynucleotide was designed and applied to determine the influence of GATA1 on the DNA methylation of a p2x7r CpG island. We showed that NCI caused the induction of GATA1, Ten-eleven translocation 3 (TET3), and purinergic receptors (P2X7Rs) in astrocytes of the spinal dorsal horn, and demonstrated that inhibiting these molecules markedly increased the pain threshold, inhibited the activation of astrocytes, and decreased the spinal sEPSC frequency. NCI also markedly demethylated the p2x7r locus in a manner dependent on the enhancement of both a GATA1-TET3 physical interaction and GATA1 binding at the p2x7r promoter. Importantly, we showed that demethylation of the p2x7r locus (and the attendant increase in P2X7R expression) was reversed upon knockdown of GATA1 or TET3 expression, and demonstrated that a decoy oligodeoxynucleotide that selectively blocked the GATA1 binding site increased the methylation of a CpG island in the p2x7r promoter. These results demonstrate that chronic visceral pain is mediated synergistically by GATA1 and TET3 via a DNA-demethylation mechanism that controls p2x7r transcription in spinal dorsal horn astrocytes, and provide a potential therapeutic strategy by targeting GATA1 and p2x7r locus binding.
Animals ; Astrocytes/metabolism* ; DNA Demethylation ; Epigenesis, Genetic ; GATA1 Transcription Factor/metabolism* ; Inflammation/metabolism* ; Oligodeoxyribonucleotides/metabolism* ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic P2X7/metabolism* ; Visceral Pain/metabolism*

OBJECTIVE: To investigate the effect of interference of P2X4 receptor expression in tumor-associated macrophages (TAMs) on invasion and migration of glioma cells. METHODS: C57BL/6 mouse models bearing gliomas in the caudate nucleus were examined for glioma pathology with HE staining and expressions of Iba-1 and P2X4 receptor with immunofluorescence assay. RAW264.7 cells were induced into TAMs using conditioned medium from GL261 cells, and the changes in mRNA expressions of macrophage polarization-related markers and the mRNA and protein expressions of P2X4 receptor were detected with RT-qPCR and Western blotting. The effect of siRNA-mediated P2X4 interference on IL-1β and IL-18 mRNA and protein expressions in the TAMs was detected with RT-qPCR and Western blotting. GL261 cells were cultured in the conditioned medium from the transfected TAMs, and the invasion and migration abilities of the cells were assessed with Transwell invasion and migration experiment. RESULTS: The glioma tissues from the tumor-bearing mice showed a significantly greater number of Iba-1-positive cells, where an obviously increased P2X4 receptor expression was detected (P=0.001), than the brain tissues of the control mice (P < 0.001). The M2 macrophage markers (Arg-1 and IL-10) and M1 macrophage markers (iNOS and TNF-α) were both significantly up-regulated in the TAMs derived from RAW264.7 cells (all P < 0.01), but the up-regulation of the M2 macrophage markers was more prominent; the expression levels of P2X4 receptor protein and mRNA were both increased in the TAMs (P < 0.05). Interference of P2X4 receptor expression significantly lowered the mRNA(P < 0.01)and protein (P < 0.01, P < 0.05)expression levels of IL-1β and IL-18 in the TAMs and obviously inhibited the ability of the TAMs to promote invasion and migration of the glioma cells (P < 0.05). CONCLUSION Interference of P2X4 receptor in the TAMs suppresses the migration and invasion of glioma cells possibly by lowering the expressions of IL-1β and IL-18.
Animals ; Culture Media, Conditioned ; Glioma ; Interleukin-18 ; Mice ; Mice, Inbred C57BL ; RNA, Messenger ; Receptors, Purinergic P2X4/metabolism* ; Tumor-Associated Macrophages

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION P2X7R promotes migration and invasion of LLC cells by activating the AKT signaling pathway, and LLC cells show lowered tumorigenic capacity in P2X7^-/- mice.
Humans ; Mice ; Animals ; Receptors, Purinergic P2X7 ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Mice, Inbred C57BL ; Signal Transduction ; Adenosine Triphosphate/metabolism* ; Lung Neoplasms

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Ciliao" (BL 32) and "Huiyang" (BL 35) on the pain, urodynamic and the expressions of transient receptor poteintial vanilloid 1 (TRPV1) and P2X3 receptors in bladder of rats with interstitial bladder (IC), and to explore the possible mechanism on EA for IC. METHODS: A total of 24 Wistar female rats were randomly divided into a blank group, a model group and an EA group, 8 rats in each group. In the model group and the EA group, IC model was established by intraperitoneal injection of cyclophosphamide by 150 mg/kg at once. EA was applied at "Ciliao" (BL 32) and "Huiyang" (BL 35) in the EA group for 20 min, with continuous wave, 30 Hz in frequency, once a day for 3 consecutive days. Mechanical pain threshold of bladder and urodynamic indexes (first urination time, bladder effective volume and urination pressure) were observed after model establishment and after intervention, the expressions of TRPV1 and P2X3 receptors in the bladder were detected by Western blot. RESULTS: After model establishment, the mechanical pain threshold of bladder was decreased in the model group and the EA group compared with that in the blank group (P<0.01). After intervention, the mechanical pain threshold of bladder in the model group was lower than the blank group (P<0.01), and that in the EA group was higher than the model group (P<0.01). The urodynamic of the rats in the blank group was normal, obvious abnormal contraction during the filling period of bladder was found in the rats of the model group, while no abnormal contraction during the filling period was found in the rats of the EA group. After model establishment, in the model group and the EA group, the first urination time was earlier than the blank group (P<0.01), while bladder effective volume and urination pressure were lower than the blank group (P<0.01). After intervention, in the model group, the first urination time was earlier than the blank group (P<0.01), while bladder effective volume and urination pressure were lower than the blank group (P<0.05); in the EA group, the first urination time was later than the model group (P<0.05), while bladder effective volume and urination pressure were higher than the model group (P<0.05). Compared with the blank group, the protein expressions of TRPV1 and P2X3 receptors in bladder were up-regulated in the model group (P<0.01); compared with the model group, the protein expressions of TRPV1 and P2X3 receptors in bladder were down-regulated in the EA group (P<0.05). CONCLUSION EA can relieve bladder pain and improve urodynamic in IC rats. The mechanism may be related to the down-regulation on the expressions of TRPV1 and P2X3 receptors and the further inhibition on the abnormal input of bladder signal.
Rats ; Female ; Animals ; Cystitis, Interstitial/therapy* ; Electroacupuncture ; Urinary Bladder ; Receptors, Purinergic P2X3/metabolism* ; Rats, Sprague-Dawley ; Rats, Wistar ; Pain ; Antineoplastic Agents ; TRPV Cation Channels/metabolism*

Microglia are involved in the inflammatory response and retinal ganglion cell damage in glaucoma. Here, we investigated how microglia proliferate and migrate in a mouse model of chronic ocular hypertension (COH). In COH retinas, the microglial proliferation that occurred was inhibited by the P2X7 receptor (P2X7R) blocker BBG or P2X7R knockout, but not by the P2X4R blocker 5-BDBD. Treatment of primary cultured microglia with BzATP, a P2X7R agonist, mimicked the effects of cell proliferation and migration in COH retinas through the intracellular MEK/ERK signaling pathway. Transwell migration assays showed that the P2X4R agonist CTP induced microglial migration, which was completely blocked by 5-BDBD. In vivo and in vitro experiments demonstrated that ATP, released from activated Müller cells through connexin43 hemichannels, acted on P2X7R to induce microglial proliferation, and acted on P2X4R/P2X7R (mainly P2X4R) to induce microglial migration. Our results suggest that inhibiting the interaction of Müller cells and microglia may attenuate microglial proliferation and migration in glaucoma.
Adenosine Triphosphate/pharmacology* ; Animals ; Cell Proliferation ; Glaucoma/metabolism* ; Mice ; Microglia/metabolism* ; Receptors, Purinergic P2X4/metabolism* ; Receptors, Purinergic P2X7/metabolism* ; Retinal Ganglion Cells/metabolism*

OBJECTIVE: To investigate the correlation of receptor gene (P2X7, VDR and SLC19A1) polymorphisms with risk suffering from acute leukemia (AL) in Fujian area. METHODS: Ninety-three cases of newly diagnosed AL as AL group and 90 persons not suffered from hematologic and other tumors as control group were selected and used for comparative analysis of receptor gene polymorphisms and risk suffering from AL between case and control groups. The bone marrow and peripheral blood were collected, from which the DNA was extracted. The PCR-RFLP was used to detect 8 SNP sites (P2X7: rs208294, rs2230911, rs3751143; VDR: rs2228570, rs7975232; SLC194A1: rs1051266, rs1131596, rs3788200) of receptor genes related with the environment response, and the genotypes analysis was used to the correlation of receptor gene polymorphisms with risk suffering from adult AL. RESULTS: The unvariate logistic analysis showed that as compared with control group, P2X7 rs208294 T>C mutation and rs3751143 A>C mutation in codominant model, dominant model and over-dominant model were higher in case group, moreover the differences were statistically significant (P<0.01, P<0.05 and P<0.05, respectively), which suggested that they could reduce the risk suffering from AL. The recessive inheritance model showed that SLC1941 rs1131596 G>A mutation could increase the risk suffering from AL (P<0.05). The stepwise multivariate logistic regression analysis showed that there was still statistically significant difference in P2X7 rs208294 mutation between case group and control group (P<0.05), moreover, the heterozygous mutation (CT) could decrease the risk suffering from AL, showing the better protective effect, compared with homozygous mutation(CC). CONCLUSION The P2X7 rs208294 T>C mutation is one of protective factors against adult acute leukemia.
Adult ; Case-Control Studies ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Homozygote ; Humans ; Leukemia, Myeloid, Acute ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Receptors, Purinergic P2X7

Painful diabetic neuropathy (PDN) is a diabetes mellitus complication. Unfortunately, the mechanisms underlying PDN are still poorly understood. Adenosine triphosphate (ATP)-gated P2X7 receptor (P2X7R) plays a pivotal role in non-diabetic neuropathic pain, but little is known about its effects on streptozotocin (STZ)-induced peripheral neuropathy. Here, we explored whether spinal cord P2X7R was correlated with the generation of mechanical allodynia (MA) in STZ-induced type 1 diabetic neuropathy in mice. MA was assessed by measuring paw withdrawal thresholds and western blotting. Immunohistochemistry was applied to analyze the protein expression levels and localization of P2X7R. STZ-induced mice expressed increased P2X7R in the dorsal horn of the lumbar spinal cord during MA. Mice injected intrathecally with a selective antagonist of P2X7R and P2X7R knockout (KO) mice both presented attenuated progression of MA. Double-immunofluorescent labeling demonstrated that P2X7R-positive cells were mostly co-expressed with Iba1 (a microglia marker). Our results suggest that P2X7R plays an important role in the development of MA and could be used as a cellular target for treating PDN.
Acetamides/pharmacology* ; Animals ; Diabetes Mellitus, Experimental/complications* ; Diabetes Mellitus, Type 1/complications* ; Diabetic Neuropathies/etiology* ; Hyperalgesia/etiology* ; Male ; Mice ; Mice, Inbred C57BL ; Quinolines/pharmacology* ; Receptors, Purinergic P2X7/physiology* ; Spinal Cord/physiology* ; Streptozocin/pharmacology*

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 groupP2X4 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.
Animals ; Hyperalgesia ; Male ; Pain, Postoperative ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic P2X4 ; Remifentanil ; Spinal Cord

OBJECTIVE: Purinergic 2X7 receptor (P2X7R) activation is known to be involved in pathogenesis of depression. Our aims were to investigate P2X7R-activated inflammasome pathways in parallel with induction of depression and to test the antidepressant-like effects of the selective P2X7R antagonist Brilliant Blue G (BBG) in a rat model of chronic unpredictable mild stress (CUMS). METHODS: Male Wistar albino rats were divided into control, CUMS, CUMS+BBG25 (25 mg/kg/day) and CUMS+BBG50 (50 mg/kg/day) groups (n=10 for each group). Various stressors were applied to rats for 6 weeks to establish the CUMS model and daily BBG treatment was started at the end of 3rd week. Sucrose preference test and forced swim test (FST) were performed to assess antidepressant-like effects. Brain samples were obtained for real-time polymerase chain reaction and immunohistochemistry analysis. RESULTS: In FST, duration of immobility was reduced in the CUMS+BBG50 group. Also, BBG treatment significantly enhanced sucrose preference. While NLRP3 gene expression levels were unchanged in rats exposed to the CUMS protocol, expression levels of other inflammasome pathway factors NLRP1, caspase-1, ASC, NF-κB, IL-1β, IL-6 and P2X7R were increased. BBG treatment reduced expression levels of these factors. Likewise, Iba-1 and GFAP immunoreactivities were enhanced by the CUMS protocol and this action was reversed by BBG treatment. CONCLUSION: Chronic administration of BBG in CUMS model results in antidepressant-like activity in a dose dependent manner. Molecular and histological results show that these effects might be at least partially related to the suppression of inflammasome-related neuroinflammatory responses and suggest involvement of NLRP1 in depression.
Animals ; Brain ; Depression ; Gene Expression ; Humans ; Immunohistochemistry ; Inflammasomes ; Interleukin-6 ; Male ; Models, Animal ; Rats ; Real-Time Polymerase Chain Reaction ; Receptors, Purinergic P2 ; Receptors, Purinergic P2X7 ; Sucrose