Effect of electroacupuncture on P2X4R-p38 MAPK-BDNF signaling pathway in trigeminal ganglion of rats with trigeminal neuralgia / 中华麻醉学杂志

Objective:To evaluate the effect of electroacupuncture on P2X4R-p38 mitogen-activated protein kinase (p38 MAPK)-brain-derived neurotrophic factor (BDNF) signaling pathway in trigeminal ganglion of rats with trigeminal neuralgia.Methods:Thirty-six clean-grade healthy adult male Sprague-Dawley rats, weighing 190-230 g, aged 2-3 months, were divided into 3 groups ( n=12 each) using a random number table method: sham operation group (S group), trigeminal neuralgia group (TN group), and electroacupuncture group (E group). The model was developed by chronic constriction of the infraorbital nerve in anesthetized animals. The infraorbital nerve was only exposed without ligation in group S. Rats received electroacupuncture stimulation at the Baihui and Xiaguan acupoints on the affected side for 20 min after developing the model, with a frequency of 80 Hz, twice a day, for 14 consecutive days in E group. Facial mechanical pain threshold (FMT) was measured at 1 day before developing the model and 3, 7, 14, 21 and 28 days after developing the model. The rats were sacrificed after the last behavioral testing, and the trigeminal ganglia were taken for examination of histopathological changes of trigeminal ganglion (by HE staining) and for determination of the expression of P2X4R, p38 MAPK, phosphorylated p38 MAPK (p-p38 MAPK) and BDNF (by Western blot) and contents of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and IL-6 (by enzyme-linked immunosorbent assay). Results:Compared with group S, the FMT was significantly decreased at each time point after developing the model, the expression of P2X4R, p-p38 MAPK and BDNF in trigeminal ganglion was up-regulated, and the contents of TNF-α, IL-1β and IL-6 were increased ( P<0.05), the pathological changes of the trigeminal ganglion were obvious in group TN. Compared with group TN, the FMT was significantly increased at each time point after developing the model, and the expression of P2X4R, p-p38 MAPK and BDNF in trigeminal ganglion was down-regulated, and the contents of TNF-α, IL-1β and IL-6 were decreased ( P<0.05), and the pathological changes of the trigeminal ganglion were significantly attenuated in group E. Conclusions:The mechanism by which electroacupuncture alleviates trigeminal neuralgia may be related to inhibiting the activity of P2X4R-p38MAPK-BDNF signaling pathway and reducing neuroinflammation in rats.

Role of P2X4R in maintenance of trigeminal neuralgia in rats: relationship with p38 MAPK/BDNF signaling pathway / 中华麻醉学杂志

Objective:To evaluate the role of P2X4 receptor (P2X4R) in the maintenance of trigeminal neuralgia and the relationship with p38 mitogen-activated protein kinase (p38 MAPK)/brain-derived neurotrophic factor (BDNF) signaling pathway in rats.Methods:Forty-eight clean-grade healthy adult male Sprague-Dawley rats, weighing 190-230 g, aged 2-3 months, were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (S group), trigeminal neuralgia group (TN group), trigeminal neuralgia+ dimethylsulfoxide (DMSO) group (TN+ DMSO group), and trigeminal neuralgia+ P2X4R specific antagonist 5-BDBD group (TN+ 5-BDBD group). The model was developed by chronic constriction of the infraorbital nerve. The infraorbital nerve was only exposed without ligation in group S. At 3, 7, 10 and 14 days after developing the model, 5 μg/μl 5-BDBD 10 μl was intrathecally injected in TN+ 5-BDBD group, and 2% DMSO 10 μl was intrathecally injected in TN+ DMSO group. The facial mechanical pain withdrawal threshold (MWT) was measured at 1 day before developing the model and 1, 3, 7, 10, 14 and 28 days after developing the model (T 0-6). The rats were sacrificed and the trigeminal ganglia were taken for determination of the expression of P2X4R, p38 MAPK, phosphorylated p38 MAPK (p-p38 MAPK) and BDNF (by Western blot) and contents of tumor necrosis factor (TNF)-α and interleukin (IL)-1β and IL-6 (by enzyme-linked immunosorbent assay). Results:Compared with group S, the MWT was significantly decreased at T 1-6, the expression of P2X4R, p-p38 MAPK and BDNF in trigeminal ganglion was up-regulated, and the contents of TNF-α, IL-1β and IL-6 were increased in TN group ( P<0.05). Compared with TN group, the MWT was significantly increased at T 3-6, and the expression of P2X4R, p-p38 MAPK and BDNF in trigeminal ganglion was down-regulated, and the contents of TNF-α, IL-1β and IL-6 were decreased in TN+ 5-BDBD group ( P<0.05), and no significant change was found in the indexes mentioned above in TN+ DMSO group ( P>0.05). Conclusions:P2X4R is involved in the maintenance of trigeminal neuralgia in rats, which may be related to the activation of p38 MAPK/BDNF signaling pathway and the increase in inflammatory mediator release.

Study on the mechanism of P2X receptors involved in electroacupuncture treatment of neuropathic pain in dorsal root ganglion and spinal cord / 针灸推拿医学（英文版）

Neuropathic pain(NP)is directly caused by lesions or diseases of the somatosensory system.It can be attributed to a variety of causes and has a complex pathological mechanism.Purinergic P2X receptors play a critical role in NP.P2X receptors will be activated by the extracellular adenosine triphosphate(ATP)to mediate the pain when the tissue is damaged.In vertebrates,there are 7 subtypes of P2X receptors(P2X1-7)encoded by genes,among which the receptors of P2X3,P2X4,and P2X7 are closely related to NP.Acupuncture produces good therapeutic efficacy on NP.To explore the mechanism of purinergic signaling involved in acupuncture analgesia,a review of the actions of P2X3,P2X4,and P2X7 receptors in acupuncture for NP was conducted.The P2X3 receptor is specifically expressed in small-and moderate-sized neurons in the dorsal root ganglion of the primary afferent nerve and trigeminal ganglion.Acupuncture can directly mediate analgesia by down-regulating the expression of P2X3 receptor in neurons.P2X4 and P2X7 receptors mainly exist in the spinal microglia.Acupuncture can inhibit the nociceptive transmission of substantia gelatinosa neurons by regulating P2X4 receptor in spinal microglia.Moreover,acupuncture may alleviate NP by inhibiting the secretion of inflammatory factors by reducing P2X7 receptor in the spinal microglia.

P2X7 receptor-nitric oxide interaction mediates apoptosis in mouse immortalized mesangial cells exposed to high glucose / A interação do receptor P2X7 com o óxido nítrico medeia a apoptose em células mesangiais imortalizadas de camundongos expostas a níveis elevados de glicose

Abstract Introduction: Diabetes mellitus (DM) is a chronic disease characterized by hyperglycemia that leads to diabetic nephropathy (DN). We showed that P2X7, a purinergic receptor, was highly expressed in DM; however, when oxidative stress was controlled, renal NO recovered, and the activation of this receptor remained significantly reduced. The aim of this study was to assess the influence of NO on the P2X7 and apoptosis in mouse immortalized mesangial cells (MiMC) cultured in high glucose (HG) medium. Methods: MiMCs were cultured with DMEM and exposed to normal glucose (NG), mannitol (MA), or HG. Cell viability was assessed by an automated counter. Supernatants were collected for NO quantification, and proteins were extracted for analysis of NO synthases (iNOS and eNOS), caspase-3, and P2X7. Results: Cell viability remained above 90% in all groups. There was a significant increase in the proliferation of cells in HG compared to MA and NG. NO, iNOS, caspase-3, and P2X7 were significantly increased in HG compared to NG and MA, with no changes in eNOS. We observed that there was a strong and significant correlation between P2X7 and NO. Discussion: The main finding was that the production of NO by iNOS was positively correlated with the increase of P2X7 in MCs under HG conditions, showing that there is a common stimulus between them and that NO interacts with the P2X7 pathway, contributing to apoptosis in experimental DM. These findings could be relevant to studies of therapeutic targets for the prevention and/or treatment of hyperglycemia-induced kidney damage to delay DN progression.

Resumo Introdução: Diabetes mellitus (DM) é uma doença crônica caracterizada por hiperglicemia levando à nefropatia diabética (ND). Mostramos que P2X7, um receptor purinérgico, foi altamente expresso na DM; entretanto, quando o estresse oxidativo foi controlado, o NO renal recuperou-se, e a ativação deste receptor permaneceu significativamente reduzida. Este estudo objetivou avaliar a influência do NO no P2X7 e a apoptose em células mesangiais imortalizadas de camundongos (CMiC) cultivadas em meio de glicose elevada (GE). Métodos: CMiCs foram cultivadas em meio DMEM e expostas à glicose normal (GN), manitol (MA), ou GE. A viabilidade celular foi avaliada por contador automático. Sobrenadantes foram coletados para quantificação de NO, e foram extraídas proteínas para análise de NO sintases (iNOS e eNOS), caspase-3, e P2X7. Resultados: A viabilidade celular permaneceu acima de 90% em todos os grupos. Houve aumento significativo na proliferação de células na GE comparado com MA e GN. NO, iNOS, caspase-3 e P2X7 foram significativamente aumentados na GE comparados com GN e MA, sem alterações na eNOS. Observamos que houve correlação forte e significativa entre P2X7 e NO. Discussão: O principal achado foi que a produção de NO pela iNOS foi positivamente correlacionada com aumento de P2X7 em CMs sob condições de GE, mostrando que existe um estímulo comum entre eles e que o NO interage com a via do P2X7, contribuindo para apoptose na DM experimental. Estes achados podem ser relevantes para estudos de alvos terapêuticos para a prevenção e/ou tratamento de danos renais induzidos por hiperglicemia para retardar a progressão da ND.

Effect of lappaconitine on neuropathic pain mediated by retrograde transport of P2X3 receptor in dorsal root ganglion neurons of rats with chronic constriction injury of the sciatic nerve / 中华创伤杂志

Objective:To investigate the effect of lappaconitine (LA) on neuropathic pain (NPP) mediated by retrograde transport of purinergic P2X3 receptor (P2X3R) in dorsal root ganglion (DRG) neurons of rats with chronic constriction injury (CCI) of the sciatic nerve.Methods:Seventy-two male healthy SD rats were selected to construct the NPP model following CCI of the sciatic nerve by ligating the right sciatic nerve. according to the random number table method. The rats were divided into CCI group, CCI+LA group and normal control group according to the random number table method, with 24 rats in each group. In normal control group, the right sciatic nerve was exposed without ligation. In CCI+LA group, the rats were given 2 g/L LA (ie, 4 mg/kg intravenously for once a day for one day only) after the same treatment as CCI group. Other two groups were injected with the identical amount of normal saline in the same way. The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were evaluated before injury and at 2, 6, 12 and 24 hours after injury to evaluate the symptoms of neuralgia caused by nerve injury. The proximal and distal nerve fragments were collected in the three groups at 2, 6, 12 and 24 hours after injury. Western blotting was applied to analyze the expression of P2X3R at 2, 6, 12 and 24 hours after injury and the expression of neurotrophic factor (NGF) and tyrosine kinase receptor A (TrkA) at 24 hours after injury to evaluate the effect of LA on P2X3R, NGF and TrkA.Results:There were insignificant differences in MWT and TWL among all groups before injury (all P>0.05). Compared with normal control group, MWT and TWL were significantly decreased in CCI group and CCI+LA group at 2, 6, 12 and 24 hours after injury (all P<0.05 or 0.01). There were insignificant differences in MWT and TWL between CCI group and CCI+LA group at 2 and 6 hours after injury (all P>0.05), while MWT and TWL were significantly higher in CCI+LA group than those in CCI group at 12 and 24 hours after injury (all P<0.05 or 0.01). In the proximal sciatic nerve segment, Western blotting showed similar levels of P2X3R among all groups at 2, 6, 12 and 24 hours after injury (all P>0.05). In the distal sciatic nerve segment, Western blotting showed higher expression of P2X3R in CCI group than that in normal control group at 2, 6, 12, 24 hours after injury (all P<0.01), higher expression of P2X3R in CCI+LA group than that in normal control group at 2, 6 and 12 hours after injury (all P<0.05), similar expression of P2X3R expression between CCI+LA group and normal control group at 24 hours after injury ( P>0.05), similar expression of P2X3R between CCI group and CCI+LA group at 2 and 6 hours after injury (all P>0.05), and lower expression of P2X3R in CCI+LA group than that in CCI group at 12 and 24 hours after injury ( P<0.05 or 0.01). In the proximal and distal nerve fragments, the expression of NGF was lower in normal control group than that in CCI group and CCI+LA group ( P<0.05 or 0.01), but was similar between CCI group and CCI+LA group at 24 hours after injury ( P>0.05). In the proximal and distal nerve fragments, there were insignificant differences in the expression of TrkA among all groups at 24 hours after injury (all P>0.05). Conclusion:Early LA treatment after injury can alleviate mechanical and thermal hyperalgesia in NPP rats, which may be related to the reduction of P2X3R retrograde transport in DRG neuron axonal.

Role of P2X 7 receptor in microglia in medial prefrontal cortex in neuropathic pain in rats: relationship with autophagy / 中华麻醉学杂志

Objective:To evaluate the role of P2X 7 receptor in microglia in the medial prefrontal cortex (mPFC) in neuropathic pain (NP) and the relationship with autophagy in rats. Methods:Sixty-four healthy SPF male Sprague-Dawley rats, aged 6-8 weeks, weighing 200-250 g, were divided into 4 groups ( n=16 each) using a random number table method: sham operation group (S group), NP group, sham operation+ P2X 7 receptor blocking group (SP group), and NP+ P2X 7 receptor blocking group (NP+ P group). The NP model was established by ligation of the sciatic nerve.Fourteen days later a cannula was placed in the mPFC with a brain stereotactic instrument, P2X 7 receptor blocker A-740003 0.5 μg/0.5 μl was injected into bilateral mPFC for 3 consecutive days starting from the 14th day in SP and NP+ P groups, and DMSO 0.5 μl was injected instead of A-740003 in S and NP groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 3, 7 and 10 days after establishing the model and 14, 15 and 16 days after administration.Then the rats were sacrificed, and the mPFC was removed for determination of the expression of P2X 7 receptor and mRNA and autophagy-related proteins Beclin1 and LC3Ⅱ/Ⅰ (by quantitative real-time polymerase chain reaction or by Western blot) and co-expression of P2X 7R and microglia (by immunofluorescence) and the number of autophagosomes in mPFC (with a transmission electron microscope). Results:Compared with group S, MWT was significantly decreased, and TWL was shortened at 3, 7 and 10 days after establishing the model, the expression of P2X 7 receptor and mRNA, Beclin1 and LC3Ⅱ/Ⅰ was up-regulated at 30 min after administration on 16 days after establishing the model, and the number of cells co-expressing P2X 7 receptor and IBA-1 and the number of autophagosomes were increased in NP and NP+ P groups ( P<0.05), and no significant change was found in the indexes mentioned above in group SP ( P>0.05). Compared with group NP, MWT was significantly increased, and TWL was prolonged at 30 min after administration on 14, 15 and 16 days after establishing the model, the expression of P2X 7 receptor and mRNA, Beclin1 and LC3Ⅱ/Ⅰ was down-regulated, and the number of cells co-expressing P2X 7 receptor and Iba-1 and the number of autophagosome were decreased in group NP+ P ( P<0.05). Conclusion:Up-regulation of P2X 7 receptor expression in microglia in mPFC is involved in the process of NP in rats, which is associated with the promotion of autophagy.

Role of P2X7 receptor in colon cancer progression / 国际肿瘤学杂志

P2X7 receptor is an ion channel receptor with adenosine triphosphate (ATP) as its ligand, which is widely expressed in various immune cells and tissues. Activated P2X7 receptor is involved in a variety of physiological and pathological processes. P2X7 receptor is abnormally expressed in colon cancer, and plays a duel role of cancer-promoting and cancer-suppressing in colon cancer progression. When P2X7 receptor is activated by extracellular ATP, it can effectively inhibit proliferation and induce apoptosis of colon cancer cells through various mechanisms. In addition, P2X7 receptor can also promote the growth, invasion and metastasis of colon cancer. Understanding the activation of P2X7 receptor and its effect mechanism is of great significance for the treatment of colon cancer.

Role of spinal P2Y1R in development of remifentanil-induced hyperalgesia in rats with incisional pain: relationship with function of NR1 and NR2B in spinal cord / 中华麻醉学杂志

Objective:To evaluate the role of spinal P2Y1R in the development of remifentanil-induced hyperalgesia in rats with incisional pain (IP) and the relationship with the function of NR1 and NR2B in spinal cord.Methods:Forty-eight healthy adult male Sprague-Dawley rats, aged 10-12 weeks, weighing 250-280 g, in which intrathecal catheters were successfully placed, were divided into 6 groups ( n=8 each) using a random number table method: control group (group C), P2Y1R antagonist MRS2179 group (group M), remifentanil group (group R), remifentanil plus MRS2179 group (group R+ M), IP plus remifentanil group (group I+ R) and IP plus remifentanil plus MRS2179 group (group I+ R+ M). In group C, normal saline 10 μl was intrathecally injected, and 10 min later normal saline was infused for 60 min via the tail vein at a rate of 0.1 ml·kg -1·min -1.In group M, MRS2179 0.6 nmol/kg was intrathecally injected, and 10 min later normal saline was infused for 60 min via the tail vein at a rate of 0.1 ml·kg -1·min -1.In group R, normal saline 10 μl was intrathecally injected, and 10 min later remifentanil was infused for 60 min via the tail vein at a rate of 1 μg·kg -1·min -1.In group R+ M, MRS2179 0.6 nmol/kg was intrathecally injected, and 10 min later remifentanil was infused for 60 min at a rate of 1 μg·kg -1·min -1 via the tail vein.In group I+ R, normal saline 10 μl was intrathecally injected, 10 min later remifentanil was infused for 60 min via the tail vein at a rate of 1 μg·kg -1·min -1, and IP was established at 10 min after onset of remifentanil infusion.In group I+ R+ M, MRS2179 0.6 nmol/kg was intrathecally injected, 10 min later remifentanil was infused via the tail vein for 60 min at a rate of 1 μg·kg -1·min -1, and IP was established at 10 min after onset of remifentanil infusion.The mechanical paw withdrawal threshold (MWT), thermal paw withdrawal latency (TWL), and the number of paw lifts on the cold plate were measured at 24 h before infusion of remifentanil or normal saline and at 2, 6, 24, and 48 h after the end of infusion.The animals were sacrificed after the last measurement of the pain threshold, L 4-6 segments of the spinal cord were removed for determination of the expression of P2Y1R, phosphorylated NR1 (p-NR1), NR1, phosphorylated NR2B (p-NR2B) and NR2B (by Western blot), for calculation of the ratios of p-NR1/NR1 and p-NR2B/NR2B, and for detection of expression of P2Y1R mRNA, NR1 mRNA and NR2B mRNA (by real-time polymerase chain reaction). Results:Compared with group C, MWT was significantly decreased, TWL was shortened, the number of paw lifts on the cold plate was increased, the expression of P2Y1R protein and mRNA, NR1 protein and mRNA, p-NR1, NR2B protein and mRNA and p-NR2B was up-regulated, and p-NR1/NR1 ratio and p-NR2B/NR2B ratio were increased in group R ( P<0.01). Compared with group R, MWT was significantly increased, TWL was prolonged, the number of paw lifts on the cold plate was decreased, the expression of P2Y1R, p-NR1, NR1 protein and mRNA, p-NR2B, NR2B protein and mRNA was down-regulated, and p-NR1/NR1 ratio and p-NR2B/NR2B ratio were decreased in group R+ M ( P<0.05 or 0.01). Compared with group I+ R, MWT was significantly increased, TWL was prolonged, the number of paw lifts on the cold plate was decreased, the expression of P2Y1R, p-NR1, NR1 protein and mRNA, p-NR2B, NR2B protein and mRNA was down-regulated, and p-NR1/NR1 ratio and p-NR2B/NR2B ratio were decreased in group I+ R+ M ( P<0.01). Conclusion:Spinal P2Y1R can enhance the function of NR1 and NR2B, which may be involved in the development of remifentanil-induced hyperalgesia in rats with IP.

Dexmedetomidine attenuates P2X4 and NLRP3 expression in the spine of rats with diabetic neuropathic pain

Abstract Purpose: To evaluate the effects of Dexmedetomidine (Dex) on spinal pathology and inflammatory factor in a rat model of Diabetic neuropathic pain (DNP). Methods: The rats were divided into 3 groups (eight in each group): normal group (N group), diabetic neuropathic pain model group (DNP group), and DNP model with dexmedetomidine (Dex group). The rat model of diabetes was established with intraperitoneal streptozotocin (STZ) injections. Nerve cell ultrastructure was evaluated with transmission electron microscopy (TEM). The mechanical withdrawal threshold (MWT) and motor nerve conduction velocity (MNCV) tests documented that DNP rat model was characterized by a decreased pain threshold and nerve conduction velocity. Results: Dex restored the phenotype of neurocytes, reduced the extent of demyelination and improved MWT and MNCV of DNP-treated rats (P=0.01, P=0.038, respectively). The expression of three pain-and inflammation-associated factors (P2X4, NLRP3, and IL-IP) was significantly upregulated at the protein level in DNP rats, and this change was reversed by Dex administration (P=0.0022, P=0.0092, P=0.0028, respectively). Conclusion: The P2X4/NLRP3 signaling pathway is implicated in the development and presence of DNP in vivo, and Dex protects from this disorder.

Asociación entre el volumen plaquetario medio y la resistencia a aspirina e inhibidores del receptor p2y12 en pacientes ancianos con síndrome coronario agudo / Association between Mean Platelet Volume and Resistance to Aspirin and P2Y12 Receptor Inhibitors in Elderly Patients with Acute Coronary Syndrome

RESUMEN Introducción: La resistencia a antiagregantes y el volumen plaquetario medio (VPM) son predictores de eventos en el síndrome coronario agudo (SCA). La asociación entre ambos ha sido poco estudiada. Objetivos: Evaluar si existe asociación entre la resistencia a la aspirina (AAS) e inhibidores del receptor P2Y12 (iP2Y12) y el VPM en pacientes mayores de 65 años con SCA. Material y métodos: Se incluyeron pacientes mayores de 65 años con diagnóstico de SCA. Se dividieron en: grupo 1 (resistencia a ambos antiagregantes), grupo 2 (a uno de los antiagregantes) y grupo 3 (a ningún antiagregante). Se midió la agregación plaquetaria entre las 12 y 24 horas poscarga (por light transmission aggregometry). Se consideró resistencia a iP2Y12 a un porcentaje máximo de agregación (PMA) con ADP > 60% y a la AAS a un PMA con ARA > 20%. En el seguimiento se consi-deró el punto final combinado de muerte global y reinternación cardiovascular. Resultados: Se incluyeron 195 pacientes que recibieron AAS e iP2y12 (120 recibieron clopidogrel y 75 ticagrelor); grupo 1 (19%), grupo 2 (34,4%) y grupo 3 (46,6%). El VPM se asoció a la resistencia a ambos antiagregantes (OR 1,02 (IC 95% 1,01-1,05), p = 0,03. A su vez, el VPM y el GRACE fueron predictores independientes del punto combinado (HR 1,03 (IC 95% 1,01-1,07), p = 0,04 y HR 1,02 (IC 95% 1,01-1,04), p = 0,02), respectivamente. Conclusiones: El VPM se asoció a la presencia de resistencia a ambos antiagregantes. En el seguimiento el VPM y el score GRACE fueron predictores del punto combinado.

ABSTRACT Background: Antiplatelet resistance and mean platelet volume (MPV) are event predictors in acute coronary syndrome (ACS). However, the association between both has been poorly studied. Objective: The aim of this study was to evaluate the association between MPV and resistance to aspirin (ASA) and P2Y12 receptor inhibitors (P2Y12i) in elderly patients with ACS. Methods: Patients over 65 years old with diagnosis of ACS were included in the study. They were divided into group 1 (re-sistance to both antiplatelet agents), group 2 (resistance to one antiplatelet agent) and group 3 (no resistance to antiplatelet agents). Platelet aggregation was measured between 12 and 24 hours postload (by light transmission aggregometry). Resis-tance to P2Y12i was considered as maximum percentage of aggregation (MPA) with adenosine diphosphate (ADP) >60% and resistance to ASA as MPA with arachidonic acid (ARA) >20%. The composite endpoint of global death and cardiovascular re-hospitalization was considered during follow-up. Results: One hundred and ninety five patients included in the study received ASA and P2Y12i (120 received clopidogrel and 75 ticagrelor). Nineteen percent of patients belonged to group 1, 34.4% to group 2 and 46.6% to group 3. Mean platelet volume was associated with resistance to both antiplatelet agents [OR 1.02 (95% CI 1.01-1.05), p=0.03], while MPV and the GRACE score were independent predictors of the composite endpoint [HR 1.03 (95% CI 1.01-1.07), p=0.04] and [HR 1.02 (95% CI 1.01-1.04), p=0.02], respectively. Conclusions: Mean platelet volume was associated with the presence of resistance to both antiplatelet agents. During follow-up, MPV and the GRACE score were predictors of the composite endpoint.

Relationship between hydrogen sulfide and P2X3 receptors in dorsal root ganglion of rats with neuropathic pain / 中华麻醉学杂志

Objective To evaluate the relationship between hydrogen sulfide (H2S) and P2X3 receptors in dorsal root ganglions (DRGs) of rats with neuropathic pain (NP).Methods Thirty-six healthy male Sprague-Dawley rats,aged 4-6 weeks,weighing 180-200 g,in which IT catheters were successfully implanted,were divided into 3 groups (n=12 each) using a random number table method:sham operation group (group S),group NP and endogenous H2S synthase (cystathionine beta-syntheses [CBS]) inhibitor animooxyacetic acid (AOAA) group (group A).NP was induced by chronic constriction injury (CCI) to the sciatic nerve at 3 days after IT catheters were successfully implanted.AOAA (10 μg/kg) 10 μl and normal saline 10 μl were intrathecally injected once a day for 14 consecutive days starting from 1 day after CCI in group A,and normal saline 20 μl was intrathecally injected instead in S and NP groups.At 1 day before CCI and 1,3,7,10 and 14 days after CCI,the thermal paw withdrawal latency (TWL) and mechanical paw withdrawal threshold (MWT) were measured at 30 min after intrathecal injection.The animals were sacrificed at 7 and 14 days after CCI,and ipsilateral DRGs of the lumbar segment (L4-6) were removed for detection of the expression of CBS and P2X3 receptors by Western blot.Results Compared with group S,the TWL was significantly shortened,MWT was decreased,and the expression of CBS and P2X3 receptors in DRGs was up-regulated at each time point after CCI in group NP (P＜0.05).Compared with group NP,the TWL was significantly prolonged,the MWT was increased,and the expression of CBS and P2X3 receptors in DRG s was down-regulated at each time point after CCI in group A (P＜0.05).Conclusion H2S in DRG s can up-regulate the expression of P2X3 receptors,which may be involved in the pathophysiological mechanism of NP in rats.

Study on the best concentration of miRNA-22 agomir in the treatment of temporal lobe epilepsy by single injection of lateral ventricle / 中国医师杂志

Objective To investigate the optimal initial concentration of microRNA22 agomir in epilepsy model induced by lithium chloride-pilocarpine after single injection of lateral ventricle.Methods 36 rats with acute temporal lobe epilepsy were randomly divided into 6 groups:the control group and the other five groups were the experimental group.All epilepsy rats were selected for right lateral ventricle injection.The control group was given negative control reagent,while the experimental group were given 0.1 mmol/L,2.5 mmol/L,5 mmol/L,10 mmol/L,20 mmol/L different concentrations of miRNA22agomir reagent.6 rats in each group were randomly selected for acute phase experiment after 3 days of administration.The expression of P2X7 in hippocampus of epilepsy rats was determined by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR).Results Compared with control group,the protein and mRNA expression of P2X7 reduced in all of the model group.The protein and mRNA expression level of P2X7 protein in hippocampus of rats injected with 2.5 mmol/L,5 mmol/L and 10 mmol/L in each experimental group were significantly lower than that in the other two groups (P ＜ 0.05).Moreover,the protein and mRNA expression level of P2X7 were the lowest at 2.5 mmol/L injection and 10 mmol/L,and there was no significant difference between the two groups (P ＞ 0.05).Conclusions The optimal onset concentration for unilateral lateral ventricle injection miRNA22 agomir treatment of temporal lobe epilepsy is 2.5 mmol/L.

Terapia fotodinâmica na artrite experimental induzida por Paracoccidioides brasiliensis / Photodynamic therapy in experimental arthritis induced by Paracoccidioides brasiliensis

OBJETIVOS: Avaliar a eficácia da terapia fotodinâmica com Brilliant Blue G no tratamento de um modelo experimental de artrite por Paracoccidioides brasiliensis (P. brasiliensis). MÉTODOS: Após a indução de artrite experimental com isolado de P. brasiliensis da linhagem Pb18 nos joelhos de ratos Wistar, os animais foram divididos em grupos e submetidos a terapia fotodinâmica com fotossensibilizador Brilliant Blue G intra-articular e a laserterapia apenas, sem o Brilliant Blue G. Todos os grupos receberam seus respectivos tratamentos do sétimo ao 11º dia. Para análise do edema foi mensurado o diâmetro latero-lateral do joelho de cada animal diariamente e após o período de tratamento os animais foram sacrificados para dissecação do joelho experimental e coleta de sangue para análise por ELISA, a fim de quantificar os níveis de anticorpos anti-P. brasiliensis. RESULTADOS: A aplicação da terapia fotodinâmica foi capaz de impedir a formação de edema quando comparado ao controle (p>0,005), bem como a produção de anticorpos anti-Gp-43 de P. brasiliensis (p=0,001). No exame anatomopatológico foi possível observar maior grau de sinovite e maior presença de granulomas com o fungo em seu interior no grupo que não recebeu tratamento quando comparado aos grupos que receberam a terapia fotodinâmica. CONCLUSÕES: A terapia fotodinâmica foi eficaz para atenuar a artrite experimental induzida por P. brasiliensis no modelo articular proposto.

AIMS: To evaluate the effectiveness of photodynamic therapy with Brilliant Blue G in the treatment of an experimental model of arthritis by Paracoccidioides brasiliensis (P. brasiliensis). METHODS: After the induction of experimental arthritis with isolated from P. brasiliensis of lineage Pb18 in the knees of Wistar rats, the animals were divided into groups and submitted to photodynamic therapy with intra-articular Brilliant Blue G photosensitizer and laser therapy only, without Brilliant Blue G. All groups received their respective treatments from the seventh to the 11th day. For edema analysis, the knee lateral-lateral diameter of each animal was measured daily and after the treatment period the animals were sacrificed for experimental knee dissection and blood collection for analysis by ELISA, in order to quantify levels of anti-P. brasiliensis antibodies. RESULTS: The results showed that the application of photodynamic therapy was able to prevent the formation of edema when compared to the control (p>0.005), as well as the production of anti-Gp-43 antibodies from P. brasiliensis (p=0.001). In the anatomopathological examination it was possible to observe a higher degree of synovitis and a greater presence of granulomas with the fungus inside the group that did not receive treatment when compared to the groups that received the photodynamic therapy. CONCLUSIONS: Photodynamic therapy was effective in attenuating the experimental arthritis induced by P. brasiliensis in the proposed joint model.

Effects of ATP on Pacemaker Activity of Interstitial Cells of Cajal from the Mouse Small Intestine / 전남의대학술지

Purinergic receptors play an important role in regulating gastrointestinal (GI) motility. Interstitial cells of Cajal (ICCs) are pacemaker cells that regulate GI smooth muscle activity. We studied the functional roles of external adenosine 5′-triphosphate (ATP) on pacemaker activity in cultured ICCs from mouse small intestines by using the whole-cell patch clamp technique and intracellular Ca²⁺ ([Ca²⁺]ᵢ) imaging. External ATP dose-dependently depolarized the resting membrane and produced tonic inward pacemaker currents, and these effects were antagonized by suramin, a purinergic P2 receptor antagonist. ATP-induced effects on pacemaker currents were suppressed by an external Na⁺-free solution and inhibited by the nonselective cation channel blockers, flufenamic acid and niflumic acid. The removal of external Ca²⁺ or treatment with thapsigargin (inhibitor of Ca²⁺ uptake into endoplasmic reticulum) inhibited the ATP-induced effects on pacemaker currents. Spontaneous [Ca²⁺]ᵢ oscillations were enhanced by external ATP. These results suggest that external ATP modulates pacemaker activity by activating nonselective cation channels via external Ca²⁺ influx and [Ca²⁺]ᵢ release from the endoplasmic reticulum. Thus, it seems that activating the purinergic P2 receptor may modulate GI motility by acting on ICCs in the small intestine.

Correlations of P2Y12 gene polymorphism with clopidogrel resistance and long -term outcome in patients with acute ischemic stroke / 国际脑血管病杂志

Objective To investigate the correlations of P2Y12 gene polymorphisms with clopidogrel resistance and long-term outcome in patients with acute ischemic stroke. Methods From June 2015 to June 2017, consecutive patients with acute ischemic stroke admitted to the Department of Neurology, Nanjing Jiangbei People's Hospital were enrolled. Thromboelastography was used to measure platelet inhibition rate and assess clopidogrel resistance. Polymerase chain reaction was used to assay C34T and G52T polymorphisms of P2Y12 gene. The patients were followed up at 12 months after discharge. The primary outcome was combined outcome of stroke recurrence, myocardial infarction, and death due to cardiocerebrovascular events. Results A total of 214 patients were enrolled, 51 (23.8%) had clopidogrel re-sistance and 29 (13.4%) had major outcome events. One hundred twenty-eight (59.8%) patients were C34T CC genotype and 86 (40.2%) were CT+TT genotype. The proportion of clopidogrel resistance in patients with CT+TT genotype was significantly higher than that with CC genotype ( 76.5% vs.28.8%;χ2=25.672, P=0.001). There were 131 patients (61.2%) with G52T GG genotype and 83 (38.8%) with GT+TT genotype. There was no significant difference in the proportion of clopidogrel resistance between the GT+TT genotype and the GG genotype (43.1% vs.37.4%; χ2=0.534, P=0.465). Multiple logistic regression analysis indicated that age (odds ratio [OR] 1.064, 95%confidence interval [CI] 1.009-1.115;P=0.021), diabetes ( OR 3.773, 95%CI 1.672-8.475; P=0.004), and C34T CT+TT genotype ( OR 9.087, 95%CI 4.416-22.665; P=0.002) were the independent risk factors fot clopidogrel resistance. Cox proportional hazards model analysis showed that age (Hazard ratio [HR] 1.058, 95%CI 1.001-1.121; P=0.049), hypertension ( HR 3.105, 95%CI 1.149-9.523; P=0.028), homocysteine ( HR 1.101, 95%CI 1.020-1.190; P=0.014), and C34T CT+TT genotype ( HR 2.588, 95%CI 1.121-5.967; P=0.026) were independent risk factors for the composite outcome. Conclusion C34T polymorphism of P2Y12 gene in patients with acute ischemic stroke may be a risk factor for clopidogrel resistance and is independently associated with the risk of long-term recurrence of vascular events.

The influence of adenosine receptor A1 subtype on the immune regulatory function of retinal pigment epithelium cells / 天津医药

Objective To clarify which adenosine receptor subtype is the most powerful one on controlling retinal pigment epithelial cell (RPE) binding adenosine, and what is its function in RPE. Methods Total mRNA was isolated, and membrane protein was extracted from in vitro cultured human ARPE-19 cells. For all four kinds of adenosine receptors, ARA1, ARA2A, ARA2B and ARA3, their gene expressions were tested by real-time PCR while their molecules in the membrane protein were detected by Western blot assay. To check the influence of each adenosine receptor subtype on ARPE-19 cell binging ability to adenosine the cultured cells were divided into five groups, named A-E. A group was set up as untreated control, while, groups B-E were separately treated by ARA1 agonist DPCPX (50 nmol/L), ARA2A agonist SCH58261 (100 nmol/L), ARA2B agonist MRS1754 (100 nmol/L) or ARA3 agonist MRS1220 (5μmol/L). H3-adenosine a radioactive ligand binding assay was performed and the maximum binding capacities (Bmax) were calculated in groups A-E of ARPE-19 cells. Then, ARPE-19 cells were all treated by the combination of TNF-αand IFN-γbut with or without CCPA (100 nmol/L), an ARA1 agonist. MCP-1, IP-10, IL-6, IL-10 and TGF-β in their mediums were determined by ELISA. Results Either mRNA expression or membrane localization of ARA1, ARA2A, ARA2B and ARA3 were verified by real-time PCR and Western blot assay respectively. For A-E groups of ARPE-19 cells the Bmax of adenosine binding were (2.04± 0.31), (0.44 ± 0.06), (1.82 ± 0.28), (2.01 ± 0.42) and (2.06 ± 0.44) fmol respectively;and which were statistically decreased in group B than those of all other groups (P<0.01). Compared with control RPE, the contents of IL-6, MCP-1 and IP-10 were decreased after treatment with CCPA, and the content of IL-10 increased in RPE group (P<0.01). There was no significant difference in TGF-β content between the two groups. Conclusion APRE-19 cells predominantly use ARA1 to absorb adenosine, and the activation of ARA1 in ARPE-19 cells inhibits its IL-6, MCP-1, and IP-10 production, which have potentially immunosuppressive effects to APRE-19 cells.

Expression of P2X7 receptor in RGC-5 cell line and selection of an effective siRNA sequence down-regulating P2X7 receptor / 中国医师杂志

Objective This study aims to investigate the gene expression of P2X7 receptor in retinal ganglion cell line (RGC-5),and to design and select the small interfering RNA (siRNA) which can specifically and effectively downregulate P2X7 receptor expression.Methods The mRNA and protein expressions of P2X7 receptor in RGC-5 cells were examined by real-time polymerase chain reaction (qRT-PCR),Western blot and Immunofluorescence,and compared with those of mouse retina and other tissues.Three P2X7 receptor-specific double stranded siRNAs (P2X7-siRNA-1-3) were designed and delivered into RGC-5 cells by riboFECTTM CP.60 hours after transfection,the expression level of P2X7 receptor was analyzed by qRT-PCR,Western blot and Immunofluorescence.Results The expression of P2X7 receptor was detected in both RGC-5 cell line and mouse retina,and the receptor was mainly expressed in the nucleus and cytoplasm of RGC-5 cells.P2X7-siRNA-3 can effectively down-regulate the mRNA and protein expression of P2X7 at the same time.Conclusions RGC-5 cell line can serve as a good model for studying the biological function of P2X7 receptor in retinal ganglion cells in vitro.We have managed to design and synthesize the siRNA pair that specifically and effectively lowering P2X7 receptor expression.The current study has laid the foundation for future investigations on treating glaucomatous retinopathy by downregulation of P2X7 receptor.

Role of P2X7 receptor in ventrolateral periaqueductal gray in tramadol-induced reduction of neuropathic pain in rats / 中华麻醉学杂志

Objective To evaluate the role of P2X7 receptor in the ventrolateral periaqueductal gray (vlPAG) in tramadol-induced reduction of neuropathic pain (NP) in rats.Methods Fifty-four male clean-grade healthy Sprague-Dawley rats,aged 7 days,weighing 190-230 g,were studied.NP was induced by chronic constrictive injury (CCI) to sciatic nerve.Experiment Ⅰ Thirty-six rats were divided into 3 groups (n =12 each) using a random number table method:sham operation group (group S),group NP1 and NP plus tramadol group (group NP1 +T).Tramadol 15 mg/kg was intraperitoneally injected once a day from day 7 to day 14 after CCI in group NP1+T.The mechanical and thermal pain thresholds of the nerve-injured hindlimb were measured before CCI and on 1,5,7,10,12 and 14 days after CCI.Rats were sacrificed after measurement of pain threshold on day 14 after CCI,and the expression of P2X7 receptor in vlPAG was detected by immunohistochemistry and Western blot assay.Experiment Ⅱ Eighteen rats were divided into 3 groups (n =6 each) using a random number table method:group NP2,NP plus tramadol group (group NP2+T) and NP plus tramadol plus a specific P2X7 receptor antagonist A-438079 group (group NP2+T+A).In NP2+T+A group,a catheter was implanted in vlPAG,and the NP model was established on 5th day after successful catheterization.Tramadol 15 mg/kg was intraperitoneally injected once a day from day 7 to day 14 after CCI in group NP2+T.In group NP2+T+A,tramadol 15 mg/kg was intraperitoneally injected once a day from day 7 to day 14 after CCI,followed by a microinjection of A-438079 100 pmol (0.3 μl) via vlPAG before giving tramadol on day 14.The mechanical and thermal pain thresholds were measured at the end of the last tramadol administration and within 1 h after the end of the last tramadol administration.Results Experiment Ⅰ Compared with group S,the mechanical and thermal pain thresholds were significantly decreased at each time point after CCI,the number of P2X7 receptor positive cells was increased,and the expression of P2X7 receptor was up-regulated in the other two groups (P＜0.01).Compared with group NP1,the mechanical and thermal pain thresholds were significantly increased at days 7-14 after CCI,the number of P2X7 receptor positive cells was increased,and the expression of P2X7 receptor was up-regulated in group NP1 +T (P＜0.01).Experiment Ⅱ Compared with group NP2,the mechanical and thermal pain threshold were significantly increased at each time point after CCI in NP2+T and NP2 +T+A groups (P＜0.01).Compared with group NP2 +T,the mechanical and thermal pain thresholds were significantly decreased at each time point after CCI in group NP2+T+A (P＜ 0.01).Conclusion The mechanism by which tramadol mitigates NP is partially related to enhanced function of P2X7 receptors in vlPAG of rats.

Comparison of the Infarct Size between the Loading of Ticagrelor and Clopidogrel in Patients with Acute Myocardial Infarction Undergoing Primary Percutaneous Coronary Intervention

BACKGROUND AND OBJECTIVES: Ticagrelor is considered a potent antiplatelet agent compared to clopidogrel. However, there are no studies regarding the effect of ticagrelor loading on infarct size in patients with ST-segment elevation myocardial infarction (STEMI) in a primary percutaneous coronary intervention (PCI) setting. SUBJECTS AND METHODS: In this single-center, randomized, open-label study, 188 patients who underwent primary PCI for STEMI were enrolled (92 patients in the clopidogrel group and 96 in the ticagrelor group) and compared the infarct size by technetium-99m (Tc-99m) tetrofosmin single-photon emission computed tomography (SPECT) and serial cardiac biomarker levels between the groups. SPECT was performed at a median of 2 days after PCI. RESULTS: Baseline clinical and procedural characteristics were similar between the groups. Infarct size on SPECT, was similar between the 2 groups (28.1%±34.5% vs. 32.8%±29.2%; p=0.169). At all time-points after PCI (8, 24, and 48 hours), the peak levels of creatine kinase-myocardial band (CK-MB) and troponin T were lower in the clopidogrel group. The clopidogrel group showed lower cumulative troponin T levels than the ticagrelor group (12.59±10.66 vs. 17.67±19.51 ng/mL; p=0.029). CONCLUSION: Ticagrelor loading before primary PCI was not associated with reduced myocardial infarct size during the first 48 hours, compared to clopidogrel loading.

Correlations of P2Y1 and ITGB3 polymorphisms with aspirin resistance in patients with large artery atherosclerotic stroke / 国际脑血管病杂志

Objective To investigate the correlations of P2Y1 and ITGB3 single nucleotide polymorphisms (SNP) with aspirin resistance (AR) in patients with large atherosclerotic stroke (LAA) in a Chinese Han population.Methods Patients with first-ever LAA from Anhui stroke registration system were enrolled.Thrombus elasticity diagram was used to detect the platelet function.TaqMan technology was used to detect the P2Y1 and ITGB3 genotypes.Results A total of 206 patients with LAA were enrolled.Thirty-one patients (15.0％) had AR and 175 (85.0％) were aspirin sensitive (AS).The frequency of P2Y1 rs701265 G allele in the AR group was significantly higher than that in the AS group (43.5％ vs.26.9％;x2 =7.074,P=0.008).The frequency of P2Y1 rs701265 AA genotype in the AR group was significantly lower than that in the AS group (32.3％ vs.53.7％;x2 =4.850,P=0.028).There were no significant significances in the frequencies of P2Y1 rs1065776 and ITGB3 rs5918 alleles and genotypes between the AR group and the AS group.Multivariate logistic regression analysis showed that P2Y1 rs701265 G allele was an independent risk factor for AR in patients with LAA (odds ratio 2.186,95％ confidence interval 1.190-4.016;P=0.012).Conclusion The P2Y1 rs701265 polymorphism is associated with AR in Chinese Han patients with LAA,while the P2Y1 rs1065776 and ITGB3 rs5918 polymorphisms are not.