Uridine adenosine tetraphosphate acts as a pro-angiogenic factor in vitrothrough purinergic P2Y receptors

OBJECTIVE Uridine adenosine tetraphosphate(Up4A),a dinucleotide,contains both purine and pyrimidine moieties,and exerts its vascular influence via activation of purinergic receptors.Here,we aimed to investigate the effects of Up4 A on angiogenesis and the putative purinergic receptors(PR)involved in this process.METHODS Tubule formation assay was performed in 3D matrix system.In this assay,human umbilical vein endothelial cells(HUVECs)were co-cultured with pericytes with various Up4 A doses(0,1,2.5,5,10 and 20μmol·L-1)in the absence and presence of P2Y6 R antagonist MRS2578(10μmol·L-1)for 5d.Expression profile of PR subtypes and angiogenic factors was assessed in HUVECs by q-PCR with and without P2Y6 R antagonist.RESULTS No difference in initial tubule formation was detected between Up4 A stimulation and control conditions at day 2.In contrast,a significant increase in vascular density in response to Up4 A was observed at day 5.Up4 A at a dose of 2.5and 5μmol·L-1 promoted total tubule length(by-1.89 fold and-2.23fold),number of tubules(by-1.71 fold and-1.89fold)as well as number of junctions(by-2.24 fold and-2.80fold),all of which were inhibited by MRS2578.Further increase in Up4 A dose to10 and 20μmol·L-1 did not induce an increase in these vascular parameters as compared to non-treated controls.Moreover,Up4 A increased mRNA level of P2YRs(P2Y2R,P2Y4 R and P2Y6R)but not P2XR(P2X4R and P2X7R)or P1R(A2AR and A2BR),while Up4 A upregulated VEGFA and ANGPT1 but not VEGFR2,ANGPT2,Tie1 and Tie2at mRNA level.Transcriptional upregulation of P2 YRs and angiogenic factors by Up4 A was inhibited by MRS2578.CONCLUSION Up4 A is functionally capable of promoting tubule formation in vitro co-culture system.This process is likely mediated by activation of pyrimidine-favored P2 YRs but not P2 XR or P1 Rs,and involves stimulation of well known angiogenic factors.

P2Y1 receptor in Alzheimer’s disease

Alzheimer’s disease is the most frequent form of dementia characterized by the deposition of amyloid-beta plaques and neurofibrillary tangles consisting of hyperphosphorylated tau.Targeting amyloid-beta plaques has been a primary direction for developing Alzheimer’s disease treatments in the last decades.However,existing drugs targeting amyloid-beta plaques have not fully yielded the expected results in the clinic,necessitating the exploration of alternative therapeutic strategies.Increasing evidence unravels that astrocyte morphology and function alter in the brain of Alzheimer’s disease patients,with dysregulated astrocytic purinergic receptors,particularly the P2Y1 receptor,all of which constitute the pathophysiology of Alzheimer’s disease.These receptors are not only crucial for maintaining normal astrocyte function but are also highly implicated in neuroinflammation in Alzheimer’s disease.This review delves into recent insights into the association between P2Y1 receptor and Alzheimer’s disease to underscore the potential neuroprotective role of P2Y1 receptor in Alzheimer’s disease by mitigating neuroinflammation,thus offering promising avenues for developing drugs for Alzheimer’s disease and potentially contributing to the development of more effective treatments.

Electroacupuncture diminishes P2X_2 and P2X_3 purinergic receptor expression in dorsal root ganglia of rats with visceral hypersensitivity

Electroacupuncture at Shangjuxu （ST37） and Tianshu （ST25） can improve visceral hypersensitivity in rats. Colorectal distension was used to establish a rat model of chronic visceral hypersensitivity. Immunohistochemistry was used to detect P2X2 and P2X3 receptor expression in dorsal root ganglia from rats with chronic visceral hypersensitivity. Results demonstrated that abdominal withdrawal reflex scores obviously increased following establishment of the model, indicating visceral hypersensitivity. Simultaneously, P2X2 and P2X3 receptor expression increased in dorsal root ganglia. After bilateral electroacupuncture at Shangjuxu and Tianshu, abdominal withdrawal reflex scores and P2X2 and P2X3 receptor expression decreased in rats with visceral hypersensitivity. These results indicated that electroacupuncture treatment improved visceral hypersensitivity in rats with irritable bowel syndrome by reducing P2X2 and P2X3 receptor expression in dorsal root ganglia.

Study of the roles of caspase-3 and nuclear factor kappa B in myenteric neurons in a P2X7 receptor knockout mouse model of ulcerative colitis

BACKGROUND The literature indicates that the enteric nervous system is affected in inflammatory bowel diseases(IBDs)and that the P2X7 receptor triggers neuronal death.However,the mechanism by which enteric neurons are lost in IBDs is unknown.AIM To study the role of the caspase-3 and nuclear factor kappa B(NF-κB)pathways in myenteric neurons in a P2X7 receptor knockout(KO)mouse model of IBDs.METHODS Forty male wild-type(WT)C57BL/6 and P2X7 receptor KO mice were euthanized 24 h or 4 d after colitis induction by 2,4,6-trinitrobenzene sulfonic acid(colitis group).Mice in the sham groups were injected with vehicle.The mice were divided into eight groups(n=5):The WT sham 24 h and 4 d groups,the WT colitis 24 h and 4 d groups,the KO sham 24 h and 4 d groups,and the KO colitis 24 h and 4 d groups.The disease activity index(DAI)was analyzed,the distal colon was collected for immunohistochemistry analyses,and immunofluorescence was performed to identify neurons immunoreactive(ir)for calretinin,P2X7 receptor,cleaved caspase-3,total caspase-3,phospho-NF-κB,and total NF-κB.We analyzed the number of calretinin-ir and P2X7 receptor-ir neurons per ganglion,the neuronal profile area(μm^(2)),and corrected total cell fluorescence(CTCF).RESULTS Cells double labeled for calretinin and P2X7 receptor,cleaved caspase-3,total caspase-3,phospho-NF-κB,or total NF-κB were observed in the WT colitis 24 h and 4 d groups.The number of calretinin-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups,respectively(2.10±0.13 vs 3.33±0.17,P<0.001;2.92±0.12 vs 3.70±0.11,P<0.05),but was not significantly different between the KO groups.The calretinin-ir neuronal profile area was increased in the WT colitis 24 h group compared to the WT sham 24 h group(312.60±7.85 vs 278.41±6.65,P<0.05),and the nuclear profile area was decreased in the WT colitis 4 d group compared to the WT sham 4 d group(104.63±2.49 vs 117.41±1.14,P<0.01).The number of P2X7 receptor-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups,respectively(19.49±0.35 vs 22.21±0.18,P<0.001;20.35±0.14 vs 22.75±0.51,P<0.001),and no P2X7 receptor-ir neurons were observed in the KO groups.Myenteric neurons showed ultrastructural changes in the WT colitis 24 h and 4 d groups and in the KO colitis 24 h group.The cleaved caspase-3 CTCF was increased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups,respectively(485949±14140 vs 371371±16426,P<0.001;480381±11336 vs 378365±4053,P<0.001),but was not significantly different between the KO groups.The total caspase-3 CTCF,phospho-NF-κB CTCF,and total NF-κB CTCF were not significantly different among the groups.The DAI was recovered in the KO groups.Furthermore,we demonstrated that the absence of the P2X7 receptor attenuated inflammatory infiltration,tissue damage,collagen deposition,and the decrease in the number of goblet cells in the distal colon.CONCLUSION Ulcerative colitis affects myenteric neurons in WT mice but has a weaker effect in P2X7 receptor KO mice,and neuronal death may be associated with P2X7 receptor-mediated caspase-3 activation.The P2X7 receptor can be a therapeutic target for IBDs.

Enteric nervous system and inflammatory bowel diseases:Correlated impacts and therapeutic approaches through the P2X7 receptor

The enteric nervous system(ENS)consists of thousands of small ganglia arranged in the submucosal and myenteric plexuses,which can be negatively affected by Crohn’s disease and ulcerative colitis-inflammatory bowel diseases(IBDs).IBDs are complex and multifactorial disorders characterized by chronic and recurrent inflammation of the intestine,and the symptoms of IBDs may include abdominal pain,diarrhea,rectal bleeding,and weight loss.The P2X7 receptor has become a promising therapeutic target for IBDs,especially owing to its wide expression and,in the case of other purinergic receptors,in both human and model animal enteric cells.However,little is known about the actual involvement between the activation of the P2X7 receptor and the cascade of subsequent events and how all these activities associated with chemical signals interfere with the functionality of the affected or treated intestine.In this review,an integrated view is provided,correlating the structural organization of the ENS and the effects of IBDs,focusing on cellular constituents and how therapeutic approaches through the P2X7 receptor can assist in both protection from damage and tissue preservation.

Isoflurane-induced neuronal apoptosis in developing hippocampal neurons

We hypothesized that the P2X7 receptor may be the target of isoflurane, so we investigated the roles of the P2X7 receptor and inositol triphosphate receptor in calcium overload and neuronal apoptosis induced by isoflurane in cultured embryonic rat hippocampal neurons. Results showed that isoflurane induced widespread neuronal apoptosis and significantly increased cytoplasmic Ca^2＋ Blockade of P2X7 receptors or removal of extracellular Ca^2＋ combined with blockade of inositol triphosphate receptors completely inhibited apoptosis or increase in cytoplasmic Ca^2＋. Removal of extracellular Ca^2＋ or blockade of inositol triphosphate receptor alone could partly inhibit these effects of isoflurane. Isoflurane could directly activate P2X7-gated channels and induce inward currents, but did not affect the expression of P2X7 receptor protein in neurons. These findings indicate that the mechanism by which isoflurane induced neuronal apoptosis in rat developing brain was mediated by intracellular calcium overload, which was caused by P2X7 receptor mediated calcium influx and inositol triphosphate receptor mediated calcium release.

Purinergic contraction of the rat vas deferens in L-NAME-induced hypertension：effect of sildenafil

Hypertension （HTN） is a risk factor for erectile dysfunction, but its effect on vas deferens （VD） contractility and the ejaculatory response has not been delineated. NG-nitro-L-arginine methyl ester （L-NAME）, a nitric oxide synthase inhibitor, was used for induction of nitric oxide （NO）-deficient HTN. Our aim was to evaluate the effects of L-NAME-induced HTN on rat VD contractility and to determine whether sildenafil affects VD contractility. A total of 36 male rats were divided into （1） control, （2）L-NAME-HTN, （3） sildenafil treated L-NAME-HTN groups. Group 2 was treated with L-NAME （40 mg kgI per day） in drinking water for 4 weeks. Group 3 received sildenafil （1.5 mg kg^-1 per day, by oral gavage） concomitantly with L-NAME. The prostatic portion of the VD was subjected to electrical field stimulation （EFS, 1-20 Hz）, and the P2X1 agonist α,β-methylene ATP （α,β meATP, 100 μmol L^-1-1 μmol L-1） and the al-adrenoceptor agonist phenylephrine （Phe, 100 μmol L^-1-1 mmol L^-1） were used to construct concentration-response curves. These experiments were repeated in the presence of P2X receptor antagonist, pyridoxalphosphate-6-azophenyl-2＇,4＇-disulfonic acid （PPADS, 30 μmol L-1）. VD contractions in response to EFS, a,β-meATP and Phe were significantly enhanced by L-NAME. Sildenafil treatment in the L-NAME group improved the contractile response of VD to EFS （20 Hz）. In the presence of PPADS, the enhanced contractile response of VD to EFS and a,β-meATP in hypertensive rats was reversed. In the rat model of chronic NO depletion, the purinergic and adrenergic components and EFS affect VD contractility. The VD contractile response may be mediated more by the purinergic system than the adrenergic system, and sildenafil may alter the ejaculatory response in men with PE.

THE CORRELATIVITY ANALYSIS OF SIX METHODS OF DETECTING APOPTOSIS

The aim of this study was to compare six methods of detecting apoptosis induced by extracellular adenosine triphosphate (ATP) in human leukemic lymphocytes with purinergic P2Z receptors.These methods used were electron microscopy(EM), detection of internucleosomal DNA fragmentation by agarose gel electrophoresis, autoradiographic analysis of DNA fragmentation, in situ labeling of DNA strand breaks with fluorescein dUTP and exogenous terminal deoxynucleotidyl transferase (TUNEL), quantitation of 3 ends of DNA breaks by labeling with α 32 PdCTP(TdT assay), and quantitation of apoptotic cells with fluorescein annexin V using flow cytometry(FCA).We found EM and detection of DNA ladder pattern by agarose gel electrophoresis to be specific,but lacking in sensitivity. The combination of autoradiography and gel electrophoresis gave an increase in sensitivity of at least 50 fold although, of all the methods, the TdT assay was shown to be most sensitive. The four methods for quantifying apoptosis EM, FCA, TUNEL and TdT assay proved to be reliable and gave statistically similar results on apoptotic lymphocytes. These observations indicate it is essential to combine specific, sensitive and quantitative techniques in detecting apoptosis.

P2X7 Receptor Mediated Growth-Inhibitory Effect in KG1a Cell Line

OBJECTIVE This study was conducted to investigate ATP- induced growth inhibition in human leukemic cells KG1a. METHODS ATP inhibited cell growth was analyzed by MTS assay. Externalization of phosphatidylserine could be detected by Annexin-V-FITC apoptosis staining after activation of the P2X7 receptor. P2X7 mediated pore formation was detected in KGla cells by Yo-Pro-1 uptake assay. RESULTS ATP inhibited cell growth in a dose-dependent manner. The cytotoxic effect could be blocked by P2X7 antagonists, oxidized ATP （oATP） and KN62. Externalization of phosphatidylserine could be detected in a time-dependent manner. P2X7 mediated pore formation could be detected in KG1a cells. These effects could not be observed in P2X7 null Ramos cells. CONCLUSION The results and our previously reports that mRNA, protein expression and calcium response of the P2X7 receptor in KGla cells, suggested that extracellular ATP effectively induces growth inhibition through apoptosis in KGla cells by activation of P2X7 receptor, and that may be mediated by extracellular Ca^2＋ in ux and pore formation.

P2X7 receptor activation causes phosphatidylserine exposure in canine erythrocytes

AIM To determine if activation of the ATP-gated P2X7 receptor channel induces phosphatidylserine(PS) exposure in erythrocytes from multiple dog breeds.METHODS Peripheral blood was collected from 25 dogs representing 13 pedigrees and seven crossbreeds. ATP-induced PS exposure on canine erythrocytes in vitro was assessed using a flow cytometric Annexin V binding assay.RESULTS ATP induced PS exposure in erythrocytes from all dogs studied. ATP caused PS exposure in a concentrationdependent manner with an EC50 value of 395 μmol/L. The non-P2X7 agonists, ADP or AMP, did not cause PS exposure. The P2X7 antagonist, AZ10606120, but not the P2X1 antagonist, NF449, blocked ATP-induced PS exposure.CONCLUSION The results indicate that ATP induces PS exposure in erythrocytes from various dog breeds and that this process is mediated by P2X7 activation.

P2X7 receptor in skin biology and diseases

The P2X7 receptor is a trimeric ligand-gated cation channel present on immune and other cells. Activation of this receptor by its natural ligand extracellular adenosine triphosphate results in a variety of downstream responses, including the release of pro-inflammatory mediators and cell death. In normal skin, P2X7 is present on keratinocytes, Langerhans cells and fibroblasts, while the presence of this receptor on other cutaneous cells is mainly inferred from studies of equivalent cell types present in other tissues. Mast cells in normal skin however express negligible amounts of P2X7, which can be upregulated in cutaneous disease. This review discusses the potential significance of P2X7 in skin biology, and the role of this receptor in inflammatory skin disorders such as irritant and chronic dermatitis, psoriasis, graft-versus-host disease, as well is in wound healing, transplantation and skin cancer.

The bumpy road of purinergic inhibitors to clinical application in immune-mediated diseases

Purinergic signaling plays important roles throughout the body in the regulation of organ functions during and following the disruption of homeostasis.This is also reflected by the widespread expression of two families of purinergic receptors(P1 and P2)with numerous subtypes.In the last few decades,there has been increasing evidence that purinergic signaling plays an important role in the regulation of immune functions.Mainly,signals mediated by P2 receptors have been shown to contribute to immune system-mediated pathologies.Thus,interference with P2 receptors may be a promising strategy for the modulation of immune responses.Although only a few clinical studies have been conducted in isolated entities with limited success,preclinical work suggests that the use of P2 receptor inhibitors may bear some promise in various autoimmune diseases.Despite the association of P2 receptors with several disorders from this field,the use of P2 receptor antagonists in clinical therapy is still very scarce.In this narrative review,we briefly review the involvement of the purinergic system in immunological responses and clinical studies on the effect of purinergic inhibition on autoimmune processes.We then open the aperture a bit and show some preclinical studies demonstrating a potential effect of purinergic blockade on autoimmune events.Using suramin,a non-specific purinergic inhibitor,as an example,we further show that off-target effects could be responsible for observed effects in immunological settings,which may have interesting implications.Overall,we believe that it is worthwhile to further investigate this hitherto underexplored area.

Silencing miRNA-324-3p protects against cerebral ischemic injury via regulation of the GATA2/A1R axis

Previous studies have suggested that miR-324-3p is related to the pathophysiology of cerebral ischemia,but the mechanism underlying this relationship is unclea r.In this study,we found that miR-324-3p expression was decreased in patients with acute ischemic stroke and in in vitro and in vivo models of ischemic stro ke.miR-324-3p agomir potentiated ischemic brain damage in rats subjected to middle cerebral artery occlusion,as indicated by increased infarct volumes and cell apoptosis rates and greater neurological deficits.In a PC12 cell oxygen-glucose deprivation/reoxygenation model,a miR-324-3 p mimic decreased cell viability and expression of the anti-apoptotic protein BCL2 and increased expression of the pro-apoptotic protein BAX and rates of cell apoptosis,whereas treatment with a miR-324-3p inhibitor had the opposite effects.Silencing miR-324-3p increased adenosine A1 receptor(A1R)expression thro ugh regulation of GATA binding protein 2(GATA2).These findings suggest that silencing miR-324-3p reduces ischemic brain damage via the GATA2/A1R axis.

Ecto-F_1-ATPase: A moonlighting protein complex and an unexpected apoA-I receptor

Mitochondrial ATP synthase has been recently detected at the surface of different cell types, where it is a high affinity receptor for apoA-I, the major protein component in high density lipoproteins (HDL). Cell surface ATP synthase (namely ecto-F1-ATPase) expression is related to different biological effects, such as regulation of HDL uptake by hepatocytes, endothelial cell proliferation or antitumor activity of Vγ9/Vδ2 T lymphocytes. This paper reviews the recently discovered functions and regulations of ecto-F1-ATPase. Particularly, the role of the F1-ATPase pathway(s) in HDL-cholesterol uptake and apoA-Imediated endothelial protection suggests its potential importance in reverse cholesterol transport and its regulation might represent a potential therapeutic target for HDL-related therapy for cardiovascular diseases. Therefore, it is timely for us to better understand how this ecto-enzyme and downstream pathways are regulated and to develop pharmacologic interventions.

Regulatory effect of mild moxibustion on P2X3 receptors in spinal cord,anterior cingulate cortex and thalamic ventral posterolateral nucleus of rats with IBS visceral hyperalgesia

Objective To observe the therapeutic effect of mild moxibustion on irritable bowel syndrome(IBS)visceral hyperalgesia model rats and its regulatory effect on P2X3 receptors in the spinal cord,anterior cingutate cortex(ACC)and thalamic ventral posterolateral nucleus(VPL).Methods Thirty 8-day-old newborn rats were randomly divided into a normal group(n=6)and a modeling group(n=24)according to the completely random number table method.Rats in the normal group were bred routinely,and those in the modeling group were subjected to preparing IBS chronic visceral hyperalgesia model using colorectal distention(CRD)in stimulation method.Rats successfully modelled were re-divided into a model group,a mild moxibustion group,a P2X3 receptor antagonist group,and a normal saline group according to the completely random number table method with 6 rats in each group.Rats in each group received corresponding interventions from the 37-day old,once a day for 7 consecutive days.Immunohistochemistry and Western blot assays were used to detect P2X3 protein expressions in the spinal cord,ACC and VPL of rats.Results Under different intensities of CRD stimulation,the abdominal withdrawal reflex(AWR)scores of the model group were significantly increased versus the normal group(all P<0.05);the AWR scores of the mild moxibustion group and the P2X3 receptor antagonist group were significantly reduced versus the model group(all P<0.01).The P2X3 protein expressions in rat spinal cord,ACC and VPL tissues of the model group were significantly increased versus the normal group(all P<0.01);the P2X3 protein expressions in rat spinal cord,ACC and VPL tissues of the mild moxibustion group and the P2X3 receptor antagonist group were significantly reduced versus the model group(all P<0.01).Conclusion Mild moxibustion can inhibit the P2X3 receptor expressions in the spinal cord,ACC,and VPL tissues of IBS visceral hyperalgesia model rats,which may be the mechanism of mild moxibustion in relieving the central sensitization of rats with IBS visceral hyperalgesia.

高压氧后处理对神经病理性痛大鼠脊髓背角及海马P2X4受体表达的影响

目的 评价高压氧后处理对神经病理性痛大鼠脊髓背角及海马P2X4受体表达的影响.方法 雄性SD大鼠72只,8～10周龄,体重300 ～ 350 g,采用随机数字表法,将其分为3组（n=24）：假手术组（S组）、神经病理性痛组（NP组）和高压氧后处理组（H组）.采用坐骨神经慢性压迫性损伤法制备大鼠神经病理性痛模型.H组于模型制备后1d时行高压氧处理,以10 kPa/min的速率向舱内加压至2个标准大气压时,保持压力60 min,随后以10 kPa/min的速率减压至正常,1次/d,连续7d.于模型制备前1d及模型制备后1、3、7、14d时测定机械缩足反应阈和热缩足反应潜伏期,测定结束后,每组取6只大鼠,取L4-6节段脊髓及海马组织,采用免疫组化法测定P2X4受体表达.结果 与S组比较,NP组和H组MWT降低,TWL缩短,脊髓背角及海马P2X4受体表达上调（P＜0.05）;与NP组比较,H组MWT升高,TWL延长,脊髓背角及海马P2X4受体表达下调（P＜0.05）.结论 高压氧后处理通过下调脊髓背角及海马P2X4受体表达,减轻大鼠神经病理性痛.

Adenosine triphosphate mediates the pain tolerance effect of manual acupuncture at Zusanli(ST36) in mice

OBJECTIVE:To investigate the mechanisms behind the effects of acupuncture in Traditional Chinese Medicine,we delved into the adenosine triphosphate/peripheral purinergic P2X receptor 3(ATP/P2X3)receptor signaling system as an indicator of the body's energy state,commonly referred to as"Qi".METHODS:The tail-flick test was utilized to explore the impact of acupuncture on pain tolerance threshold(PTT)in mice,while also assessing adenosine(ADO)levels and adenylate energy charge(EC)at Zusanli(ST36).The study further investigated the dose-dependent effects of acupuncture on PTT and ADO levels at Zusanli(ST36).To shed light on the underlying mechanisms of acupuncture's effects,the study examined the impact of ATP,a P2X3 receptor antagonist,and adenosine disodium on PTT following acupuncture administration.RESULTS:Acupuncture at Zusanli(ST36)led to significant improvements in PTT in mice,with the most effective interventions being twirling for 2 min and needle retention for 28 min.These interventions also resulted in significant increases in ATP levels.The effects of acupuncture were further augmented by administration of different doses of ATP at Zusanli(ST36),and pretreatment with a P2X3 receptor antagonist decreased PTT.Adenylate EC peaked at 30 min after intraperitoneal injection of ATP,and pretreatment with various doses of i.p.ATP 30 min prior to acupuncture increased PTT in a dose-dependent manner.Additionally,pretreatment with an i.p.or intramuscular injection of adenosine disodium enhanced the effects of acupuncture.CONCLUSION:This research provides compelling evidence that ATP is involved in the regulation of PTT through acupuncture,revealing new avenues for achieving enhanced clinical outcomes.

Effects of moxibustion on the P2X7R/STAT3/VEGF pathway in rats with colitis-associated colon cancer

Objective:To observe the effects of herb-partitioned moxibustion and ginger-partitioned moxibustion on the growth of colon tumors in rats with colitis-associated colon cancer(CACC),and explore the mechanism of moxibustion intervening CACC through the purinergic receptor P2X ligand-gated ion channel 7(P2X7R)/signal transducer and activator of transcription 3(STAT3)/vascular endothelial growth factor(VEGF)pathway.Methods:A total of 26 male Sprague-Dawley rats were selected.According to the random number table method,6 rats were selected as the normal group.The remaining 20 rats were injected intraperitoneally with azoxymethane(AOM)combined with oral dextran sodium sulfate(DSS)to prepare the CACC model.After the model was successfully established,2 rats were randomly selected for model identification.The remaining 18 rats which were successfully modeled were randomly divided into a model group,a herb-partitioned moxibustion group and a ginger-partitioned moxibustion group,with 6 rats in each group.Moxibustion intervention was performed in the herb-partitioned moxibustion group and the ginger-partitioned moxibustion group at Qihai(CV 6)and bilateral Tianshu(ST 25).Moxibustion was performed twice at each point each time,once a day,at a 1-day interval after 6 consecutive interventions,for a total of 30 interventions.After intervention,the colon tumor load,pathological change and histopathological score were observed.Immunohistochemistry was used to detect the expressions of VEGF,P2X7R,phospho-STAT3(p-STAT3),and nuclear factor-kappa B p65(NF-κB p65)proteins in rat colon tissue.Western blot was used to detect the levels of p-STAT3 and NF-κB p65 proteins in rat colon tissue.Results:Compared with the normal group,the colon tumor load and histopathological score in the model group were significantly increased(both P<0.001),and different grades of dysplasia were observed in colon tissue from the model group,reaching the degree of adenocarcinoma;the expression level of P2X7R protein in colon tissue was significantly decreased(P<0.001),and the expression levels of p-STAT3,NF-κB p65 and VEGF proteins were significantly increased(all P<0.001)in the model group.Compared with the model group,the colon tumor load,colon histopathological score and the levels of p-STAT3,NF-κB p65 and VEGF proteins in colon tissue were significantly decreased(all P<0.05)in the herb-partitioned moxibustion group and the ginger-partitioned moxibustion group while the expression levels of P2X7R protein in colon tissue were significantly increased(both P<0.05).Conclusion:Both herb-partitioned moxibustion and ginger-partitioned moxibustion can reduce the colon tumor load in CACC rats and delay the progression of colon adenomas.The mechanism may be mediated by the P2X7R/STAT3 pathway to inhibit STAT3 phosphorylation,thereby reducing VEGF protein expression.

Effect of electroacupuncture on inflammatory signal expression in local tissues of rats with chronic pelvic pain syndrome based on purinergic 2X7 receptor/NOD-like receptor pyrin domaincontaining 3 signal pathway

OBJECTIVES: To study the expression of inflammatory signal in local prostate tissue of chronic pelvic pain syndrome(CPPS) rats by electroacupuncture(EA) of Guanyuan(CV4), Zhongji(CV3), Huiyang(BL35) and Sanyinjiao(SP6), and to explore the possible mechanism of anti-inflammatory and analgesic effects of EA. METHODS : A total of 36 Sprague-Dawley male rats were randomly divided into three groups: control, model and EA(n=12 rats/group). The CPPS model was made by injection of CFA into ventral lobes of the prostate(0.1 m L). Electric acupuncture apparatus was applied to stimulate Guanyuan(CV4), Zhongji(CV3), bilateral Huiyang(BL35) and Sanyinjiao(SP6) acupoints in EA group. The general condition of rats was observed and the prostate index(PI) was calculated. The thermal pain threshold was collected after each therapeutic course. Histopathological changes of the prostate tissue were examined by hematoxylin-eosin staining method. The expression levels of tumor necrosis factor α(TNF-α), interleukin-1β(IL-1β) and prostaglandin E2(PGE2) in prostatic homogenates were measured by enzyme linked immunosorbent assay(ELISA). Moreover, the expression levels of purinergic 2X7 receptor(P2X7R), NOD-like receptor pyrin domain-containing 3(NLRP3), caspase-1 and interleukin-18(IL-18) m RNA were quantified by quantitative real-time polymerase chain reaction. RESULTS: Compared with control group, the PI of rats increased, and the thermal pain threshold decreased significantly in model group. The morphological structure of prostate tissues of rats in model group was severely damaged with a large number of inflammatory cells infiltration. Additionally, the levels of TNF-α, IL-1β and PGE2 were higher, and the expressions of P2X7R, NLRP3, caspase-1 and IL-18 m RNA were higher than those in control group. After EA treatment, the PI was significantly decreased, the thermal pain threshold was significantly increased, and the tissue damage was significantly improved. The expressions of inflammatory cytokines were lower in EA group, and expression of P2X7R/NLRP3 pathway was down-regulated. CONCLUSION: The effect of EA at Guanyuan(CV4), Zhongji(CV3), Huiyang(BL35) and Sanyinjiao(SP6) can improve inflammation and pain symptoms of CPPS rats induced by Complete Freund’s adjuvant(CFA). This suggests that EA at Guanyuan(CV4), Zhongji(CV3), Huiyang(BL35) and Sanyinjiao(SP6) can produce antiinflammatory analgesia effect by preventing the activation of P2X7R/NLRP3 signal pathway, inhibit the release of inflammatory cytokines in CPPS rats, which may provide a putative novel target for the treatment of CPPS.