P2Y12 and P2Y13 receptors involved in ADPßs induced the release of IL-1ß, IL-6 and TNF-α from cultured dorsal horn microglia.

OBJECTIVE: P2 receptors have been implicated in the release of neurotransmitter and pro-inflammatory cytokines due to their response to neuroexcitatory substances in the microglia. Dorsal horn P2Y12 and P2Y13 receptors are involved in the development of pain behavior induced by peripheral nerve injury. However, it is not known whether P2Y12 and P2Y13 receptors activation is associated with the expression and the release of interleukin-1B (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in cultured dorsal spinal cord microglia. For this reason, we examined the effects of ADPßs (ADP analog) on the expression and the release of IL-1ß, IL-6, and TNF-α. METHODS AND RESULTS: In this study, we observed the effect of P2Y receptor agonist ADPßs on the expression and release of IL-1ß, IL-6 and TNF-α by using real-time fluorescence quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). ADPßs induced the increased expression of Iba-1, IL-1ß, IL-6 and TNF-α at the level of messenger RNA (mRNA). ADPßs-evoked increase in Iba-1, IL-1ß, IL-6 and TNF-α mRNA expression was inhibited only partially by P2Y12 receptor antagonist MRS2395 or P2Y13 receptor antagonist MRS2211, respectively. Similarly, ADPßs-evoked release of IL-1ß, IL-6 and TNF-α was inhibited only partially by MRS2395 or MRS2211. Furthermore, ADPßs-evoked increased expression of Iba-1, IL-1ß, IL-6 and TNF-α mRNA, and release of IL-1ß, IL-6 and TNF-α were nearly all blocked after co-administration of MRS2395 plus MRS2179. Further evidence indicated that P2Y12 and P2Y13 receptor-evoked increased gene expression of IL-1ß, IL-6 and TNF-α were inhibited by Y-27632 (ROCK inhibitor), SB203580 (P38MAPK inhibitor) and PDTC (NF-κb inhibitor), respectively. Subsequently, P2Y12 and P2Y13 receptor-evoked release of IL-1ß, IL-6 and TNF-α, were also inhibited by Y-27632, SB203580 and PDTC, respectively. CONCLUSION: These observations suggest that P2Y12 and P2Y13 receptor-evoked gene expression and release of IL-1ß, IL-6 and TNF-α are associated with ROCK/P38MAPK/NF-κb signaling pathway.

A polymorphism (-455G>A) in the ß-fibrinogen gene is associated with an increased risk of cerebral infarction in the Chinese population: A meta-analysis.

BACKGROUND: Previous studies have investigated the association between a polymorphism (-455 G>A) in the ß-fibrinogen gene and the risk of cerebral infarction. However, these results are controversial. To shed light on these inconclusive findings, we performed a meta-analysis of studies relating the ß-fibrinogen genetic polymorphism (-455 G>A) to the risk of cerebral infarction. METHODS: We identified literature published before July 2013 by searching PubMed, EMBASE, ISI Web of Science, the Chinese National Knowledge Infrastructure database (CNKI) and the Wanfang database in China and by reviewing the references of retrieved articles. We included studies that reported odds ratio (OR) with 95% confidence interval (CI) for the association between the ß-fibrinogen genetic polymorphism and cerebral infarction risk. Publication bias was tested by a funnel plot, and the OR of all studies were combined dependent on the results of the heterogeneity tests among the individual studies. The software Review Manager (Version 5.2) was used for meta-analysis. RESULTS: Twenty independent case-control studies containing 9477 subjects were included. Our results showed that the -455 G>A polymorphism in the ß-fibrinogen gene was associated with the increased risk of cerebral infarction [(AA+GA) vs. GG, OR=1.17, 95%CI: 1.04-1.31, p=0.008; A vs. G, OR=1.12, 95%CI: 1.01-1.23, p=0.03] in the Chinese population by a meta-analysis. However, we did not find this association in the Caucasian population [(AA+GA) vs. GG, OR=0.99, 95%CI: 0.87-1.11, p=0.84; A vs. G, OR=0.97, 95%CI: 0.84-1.13, p=0.73, respectively]. CONCLUSION: The results of our meta-analysis indicate that the -455 G>A polymorphism in the ß-fibrinogen gene is a susceptibility marker of ischemic cerebral infarction in the Chinese population.

Serum Brain-derived neurotrophic factor levels in post-stroke depression.

BACKGROUND: Depression is a frequent mood disorder that affects around a third of stroke patients and has been associated with poorer outcome. Our aim was to determine whether there is a relationship between serum Brain-derived neurotrophic factor (BDNF) levels and post-stroke depression (PSD). METHODS: Two hundred and sixteen ischemic stroke patients admitted to the hospital within the first 24h after stroke onset were consecutively recruited and followed up for 3 months. Based on the symptoms, diagnoses of depression were made in accordance with DSM-IV criteria for post-stroke depression at day 90. Enzyme-linked immunosorbent assay (ELISA) was used to measure serum levels of BDNF at admission. Multivariate analyses were performed using logistic regression models. RESULTS: In our study, 59 patients (27.3%) were diagnosed as having major depression at 3 months. Patients with major depression showed lower levels of serum BDNF [8.1 (5.6-9.4) vs. 13.7 (10.4-16.5)ng/ml, P<0.0001] at admission. In multivariate analyses, serum BDNF was an independent predictor of PSD at 3 months [odds ratio (OR): 0.79(0.72-0.87), P=0.003]. Serum levels of BDNF≤10.2ng/ml were independently associated with post-stroke (OR, 11.5; 95% CI, 5.6-23.4, P<0.0001), after adjustment for possible variables. CONCLUSION: The present study demonstrates a strong relationship between serum BDNF levels at admission and the development of PSD within 3 months. Further studies are necessary to confirm this association, which may open the way to the proposal of new therapeutic options.

Plasma levels of glutamate during stroke is associated with development of post-stroke depression.

BACKGROUND: Depression is a frequent mood disorder that affects around 33% of stroke patient. Our aim was to test the possible association between plasma glutamate and the development of post-stroke depression (PSD) in Chinese patients. METHODS: The subjects were first-ever acute ischemic stroke (AIS) patients who were hospitalized during the period from November 2011 to September 2013. Clinical information and stroke severity was collected at admission. Neurological and neuropsychological evaluations were conducted at the 3-month follow-up. Plasma glutamate levels were analyzed at baseline using liquid chromatography followed by tandem mass spectrometry. Glutamate oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT) and blood markers were also tested. Multivariate analyses were performed using logistic regression models. RESULTS: During the study period, 209 patients were included in the analysis. Seventy patients (33.5%) were diagnosed as having major depression at 3 month. Patients with major depression showed higher levels of plasma glutamate [299 (235-353) vs. 157 (108-206) µM, P<0.0001] and lower GOT [14 (11-20) vs. 21 (15-32)U/L, P<0.0001] at admission. In multivariate analyses, plasma glutamate and GOT were independent predictors of PSD at 3 months [odds ratio (OR): 1.03 (1.02-1.04), P<0.0001; 0.84 (0.75-0.97), P=0.003]. Plasma levels of glutamate >205 µM were independently associated with PSD (OR, 21.3; 95% CI, 8.28-67.36, P<0.0001), after adjustment for possible variables. CONCLUSION: The present study demonstrates a strong relationship between plasma glutamate and GOT levels at admission and the development of PSD within 3 months. Further studies are necessary to confirm this association, which may open the way to the proposal of new therapeutic options.

Expression of P2X5 receptors in the rat, cat, mouse and guinea pig dorsal root ganglion.

P2X receptors are ATP-gated cationic channels composed of seven cloned subunits (P2X(1 -7)). P2X(3) homomultimer and P2X(2/3) heteromultimer receptors expressed by primary afferent dorsal root ganglion (DRG) neurons are involved in pain processing. The aim of the study was to investigate the expression of the P2X(5) receptor subunit in DRG in different species including mouse, rat, cat and guinea pig. Immunohistochemistry showed that P2X(5) receptors exhibited low levels of immunostaining in rat DRG, but high levels in mouse and guinea pig. Only a few neurons were immunoreactive for P2X(5) receptors in cat. In mouse DRG, the P2X(5) receptor was expressed largely by medium-diameter neurons (42.9 %), less in small (29.3 %) and large (27.8 %) neurons. In contrast, in the guinea pig DRG, P2X(5) receptor expression was greatest in small-diameter (42.6 %), less in medium- (36.3 %) and large-diameter (21.1 %) neurons. Colocalization experiments revealed that, in mouse DRG, 65.5, 10.9 and 27.1 % of P2X(5) receptors were immunoreactive for NF-200, CGRP and calbindin, while only a few P2X(5)-immunoreactive (IR) neurons were coexpressed with IB4 or with NOS. In guinea pig DRG, a total of 60.5 and 40.5 % of P2X(5)-IR neurons were coexpressed with IB4 or with CGRP, while 20.3 and 24.5 % of P2X(5) receptors were coexpressed with NF-200 or with NOS. Only a few P2X(5)-IR neurons were coexpressed with calbindin in guinea pig DRG. It will be of great interest to clarify the relative physiological and pathophysiological roles of P2X(5) receptors.

Role of P2Y1 receptor in astroglia-to-neuron signaling at dorsal spinal cord.

Several studies have shown that astrocytes release neurotransmitters into the extracellular space that may then activate receptors on nearby neurons. In the present study, the actions of adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS)-activated astrocyte conditioned medium (ADPbetaS-ACM) on cultured dorsal spinal cord neurons were evaluated by using confocal laser scanning microscopy and whole-cell patch-clamp recording. ADPbetaS caused astrocytic glutamate efflux (43 microM), which in turn induced inward currents in dorsal horn neurons with short time in culture. The inward currents were abolished by 2-amino-5-phosphonlanoicacid (AP-5; NMDAR antagonist) plus 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; non-NMDAR antagonist) but were unaffected by MRS2179 (selective P2Y(1) receptor antagonist). Furthermore, N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179) was used to block glutamate release from astrocytes. As a result, ADPbetaS-ACM-induced inward currents in neurons were significantly blocked. On the other hand, both NMDAR and non-NMDAR were involved in ADPbetaS-ACM (concentration was diluted to one-tenth)-evoked small [Ca(2+)](i) transients in neurons. Under this condition, the values of glutamate concentrations in the medium are close to values for extracellular glutamate concentrations under physiological conditions. For this reason, it is possible that astrocyte-derived glutamate is important for distant neuron under physiological conditions at dorsal spinal cord. These observations indicate that astrocytic P2Y(1) receptor activation triggered glutamate efflux, which acts on distant neurons to elevate calcium levels or acts on nearby neurons to evoke inward current. Finally, our results support the conclusion that the astrocytic P2Y(1) receptor plays an important role in bidirectional communication between astrocytes and neurons.

Inhibition of ATP-induced glutamate release by MRS2179 in cultured dorsal spinal cord astrocytes.

It was reported that ATP, an excitatory chemical mediator, exerts its effects by activation of the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) purinoceptors in the nervous system. In the present work, we used confocal laser scanning microscopy and high-performance liquid chromatography to assess the role of the P2Y1 receptor in ATP-evoked Ca2+ mobilization and glutamate release from cultured dorsal spinal cord astrocytes. ATP (0.01-100 micromol/l) produces a dose-dependent rise in the Ca2+ relative fluorescence intensity in cultured astrocytes. N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179, 0.01-100 micromol/l), a P2Y1-specific antagonist, could dose-dependently inhibit ATP-evoked Ca2+ mobilization. In addition, 100 micromol/l ATP caused glutamate efflux from cultured dorsal spinal cord astrocytes in a time-dependent manner. 100 micromol/l MRS2179 significantly inhibited the glutamate efflux induced by ATP, which suggests that P2Y1 receptor activation is responsible for the ATP-induced glutamate efflux from astrocytes. Taken together, our results demonstrate that P2Y1 receptor plays an important role in modulating the function of astrocytes, which raises the possibility that MRS2179, a potent P2Y1-specific antagonist, may become a potential drug in treating many chronic neurological diseases characterized by astrocytic activation in the nervous system.

Rapid inhibition of ATP-induced currents by corticosterone in rat dorsal root ganglion neurons.

The effects of corticosterone (CORT), a natural glucocorticoid hormone, on ATP-induced currents in rat dorsal root ganglion (DRG) neurons and the underlying signaling mechanism were studied by using patch-clamp techniques. Three types of currents (fast, slow and mixed) were evoked by ATP in cultured DRG neurons. Pretreatment with CORT (0.01-10 mumol/l) for 30 s could inhibit the fast current and the fast component of the mixed current. In contrast, CORT had no significant effect on the slow current evoked by ATP. The inhibitory effects were concentration dependent, reversible and could be blocked by glucocorticoid receptor antagonist RU38486 (10 micromol/l), but not by GDP-beta-S (0.2 mmol/l), a blocker of G protein activation. Membrane-impermeable bovine serum albumin-conjugated corticosterone failed to mimic the effects of CORT. The inhibitory effects of CORT on ATP-induced currents diminished after adding protein kinase A inhibitor H89 (10 micromol/l), but were not influenced by protein kinase C inhibitor chelerythrine chloride (10 micromol/l). These results suggest that glucocorticoid hormones might participate in the control of pain by modulating P2X(3) receptor-mediated events in sensory neurons, and the effect is mediated by glucocorticoid receptors and the downstream activation of protein kinase A.

P2Y1 receptor-mediated glutamate release from cultured dorsal spinal cord astrocytes.

P2 receptors have been implicated in the release of neurotransmitter and proinflammatory cytokines by the response to neuroexcitatory substances in astrocytes. In the present study, we examined the mechanisms of ADP and adenosine 5'-O-2-thiodiphosphate (ADPbetaS, ADP analogue) on glutamate release from cultured dorsal spinal cord astrocytes by using confocal laser scanning microscopy and HPLC. Immunofluorescence activity showed that P2Y(1) receptor protein is expressed in cultured astrocytes. ADP and ADPbetaS-induced [Ca(2+)](i) increase and glutamate release are mediated by P2Y(1) receptor. Ca(2+) release from IP(3)-sensitive calcium stores and protein kinase C (PKC) activation is important for glutamate release from astrocytes. Furthermore, P2Y(1) receptor-evoked glutamate release is regulated by volume-sensitive Cl(-) channels and anion co-transporter, which open up the possibility that P2Y(1) receptor activation causes the increase of cell volume. Release of glutamate by ADPbetaS was abolished by 5-nitro-2 (3-phenyl propy lamino)-benzoate plus furosemide but was unaffected by botulinum toxin A. These observations indicate that P2Y(1) receptor-evoked glutamate may be mediated via volume-sensitive Cl(-) channel but not via exocytosis of glutamate containing vesicles. We speculate that P2Y(1) receptors-evoked glutamate efflux, occurring under pathological condition, may modulate the activity of synapses in spinal cord.

Effect of rhubarb on contractile response of gallbladder smooth muscle strips isolated from guinea pigs.

AIM: To investigate the effect of rhubarb on contractile response of isolated gallbladder muscle strips from guinea pigs and its mechanism. METHODS: Guinea pigs were killed to remove the whole gallbladder. Two or three smooth muscle strips (8 mm x 3 mm) were cut along the longitudinal direction. The mucosa on each strip was carefully removed. Each longitudinal muscle strip was suspended in a tissue chamber containing 5 mL Krebs solution (37 degrees), bubbled continuously with 950 mL/L O(2) and 50 mL/L CO(2). The resting tension (g), mean contractile amplitude (mm), and contractile frequency (waves/min) were simultaneously recorded on recorders. After 2-h equilibration, rhubarb (10, 20, 70, 200, 700, 1,000 g/L) was added cumulatively to the tissue chamber in turns every 2 min to observe their effects on gallbladder. Antagonists were given 3 min before administration of rhubarb to investigate the possible mechanism. RESULTS: Rhubarb increased the resting tension (from 0 to 0.40+/-0.02, P<0.001), and decreased the mean contractile amplitude (from 5.22+/-0.71 to 2.73+/-0.41, P<0.001). It also increased the contractile frequency of the gallbladder muscle strips in guinea pigs (from 4.09+/-0.46 to 6.08+/-0.35, P<0.001). The stimulation of rhubarb on the resting tension decreased from 3.98+/-0.22 to 1.58+/-0.12 by atropine (P<0.001), from 3.98+/-0.22 to 2.09+/-0.19 by verapamil (P<0.001) and from 3.98+/-0.22 to 2.67+/-0.43 by phentolamine (P<0.005). But the effect was not inhibited by hexamethonium (P>0.05). In addition, the action of mean amplitude and frequency was not inhibited by the above antagonists. CONCLUSION: Rhubarb can stimulate the motility of isolated gallbladder muscle strips from guinea pigs. The stimulation of rhubarb might be relevant with M receptor, Ca(2+) channel and alpha receptor partly.