Adenosine A 2A receptor deficiency prevents p38MAPK activation and apoptosis of prefrontal cortex cells in mice with chronic hypoxic hypercapnia / 中华物理医学与康复杂志

Objective:To observe the effect of genetic inactivation of adenosine A 2A receptor on apoptosis in the prefrontal cortex and on the expression of phosphorylated p38 mitogen-active protein kinase (p38MAPK) in mice with chronic hypoxic hypercapnia. Methods:Sixteen male wild-type mice and 16 male mice in which the adenosine A 2A receptor gene had been knocked out were randomly divided into a 4 weeks group (including 4HH+ /+ and 4HH-/- subgroups) and a normal control group (including NC+ /+ and NC-/- subgroups). The 4HH+ /+ and 4HH-/- group mice were exposed to an atmosphere containing 9-11% O 2 and 5-6% CO 2 8 hours a day, 6 days a week for 4 weeks. The apoptosis index (AI) in their prefrontal cortices was then evaluated using terminal-deoxynucleoitide transferase mediated nick end labelling (TUNEL) staining. The expression of p38MAPK protein in the prefrontal cortices was measured using western blotting. Results:The average AI had increased significantly in the 4HH+ /+ and 4HH-/- groups compared with the controls, with significantly more apoptotic cells in the 4HH+ /+ group than in the 4HH-/- group. In the 4HH+ /+ and 4HH-/- groups the average expression of p38 protein in the prefrontal cortex was significantly higher than among their controls. Moreover, the average expression of p-p38MAPK protein in the prefrontal cortex of the 4HH-/- group was significantly lower than in the 4HH+ /+ group.Conclusion:Adenosine A 2A receptor knockout inhibits apoptosis in the prefrontal cortex and down-regulates the p38MAPK activation of mice after exposure to chronic hypoxic hypercapnia.

Sleep Promoting Effect of Luteolin in Mice via Adenosine A1 and A2A Receptors

Luteolin, a widespread flavonoid, has been known to have neuroprotective activity against various neurologic diseases such as epilepsy, and Alzheimer’s disease. However, little information is available regarding the hypnotic effect of luteolin. In this study, we evaluated the hypnotic effect of luteolin and its underlying mechanism. In pentobarbital-induced sleeping mice model, luteolin (1, and 3 mg/kg, p.o.) decreased sleep latency and increased the total sleep time. Through electroencephalogram (EEG) and electromyogram (EMG) recording, we demonstrated that luteolin increased non-rapid eye movement (NREM) sleep time and decreased wake time. To evaluate the underlying mechanism, we examined the effects of various pharmacological antagonists on the hypnotic effect of luteolin. The hypnotic effect of 3 mg/kg of luteolin was not affected by flumazenil, a GABAA receptor-benzodiazepine (GABAAR-BDZ) binding site antagonist, and bicuculine, a GABAAR-GABA binding site antagonist. On the other hand, the hypnotic effect of 3 mg/kg of luteolin was almost completely blocked by caffeine, an antagonist for both adenosine A1 and A2A receptor (A1R and A2AR), 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), an A1R antagonist, and SCH-58261, an A2AR antagonist. From the binding affinity assay, we have found that luteolin significantly binds to not only A1R but also A2AR with IC₅₀ of 1.19, 0.84 μg/kg, respectively. However, luteolin did not bind to either BDZ-receptor or GABAAR. From these results, it has been suggested that luteolin has hypnotic efficacy through A1R and A2AR binding.

A2AR activation after traumatic brain injury in the role of excessive tau protein phosphorylation and mechanism research / 中国生化药物杂志

Objective To explore the A2AR activation after traumatic brain injury mechanisms and the role of excessive tau protein phosphorylation.Methods With no specific mice experiment research of specific pathogens, position in the left parietal cortex in mice, by the method of controllable cortical against brain trauma model model, 15 min after injury in mice abdominal injection of A2AR specific inhibitors ZM241385 or use A2AR knockout mice, testing the brain neuron loss and tau protein phosphorylation level;Use specific agonists CGS21680 activate the original generation of nerve cells in the hippocampus and the A2AR human neuroblastoma cells, using immunocytochemistry and immunofluorescence test tua protein phosphorylation level of change, to observe axon transport function of mitochondria.Results Immunohistochemical results accumulation of optical density analysis showed that inhibition of A2AR activation can significantly reduce after cerebral trauma Ser404 tua protein loci phosphorylation levels, reduce excessive tua protein phosphorylation with nerve pathological change;A2AR activation after tua phosphorylation of proteins at a Ser404 site level increased significantly, nerve axons per unit length processes in the mitochondria number decreased significantly, resulting in axoplasmic transport dysfunction;To activate the original generation of nerve cells in the hippocampus and after the A2AR human neuroblastoma cells, tua protein phosphorylation Ser404 locus levels increased significantly.Conclusion A2AR activation after cerebral trauma has obvious influence on tua protein phosphorylation levels, may be a function by influencing the axoplasmic transport, eventually forming cognitive dysfunction.

Effect of chronic intermittent hypoxia on AMPK pathway in young rats / 中国病理生理杂志

[ ABSTRACT] AIM: To investigate the effect of chronic intermittent hypoxia on AMP-activated protein kinase ( AMPK) pathway in the brain of young rats.METHODS:Part one:SD mice (3~4 weeks old) were randomly divided into 4 groups (n=8): simulated air control group for 2 weeks (2AC), chronic intermittent hypoxia group for 2 weeks (2IH), simulated air control group for 4 weeks (4AC) and chronic intermittent hypoxia group for 4 weeks (4IH).Part two:SD mice (3~4 weeks old) were randomly divided into 2 groups (n=8): chronic intermittent hypoxia group for 4 weeks (4IH) and chronic intermittent hypoxia group treated with AMPK inhibitor for 4 weeks (4IHI).After modeling, the eight-arm maze test was performed.TUNEL method was used to detect the neuronal apoptosis in the hippocampal and pre-frontal cortical tissues.The mRNA expression of adenosine A2a receptor was examined by RT-qPCR, and the protein levels of phosphorylated AMPK (p-AMPK) and mammalian target of rapamycin (p-mTOR) were determined by Western blot. RESULTS:Compared with control group, the numbers of reference memory error ( RME) , working memory error ( WME) and total error (TE) in 2IH group and 4IH group significantly increased (P<0.01).Compared with 2IH group, the num-bers of errors in 4IH group also increased significantly (P<0.01).Compared with 4IH group, the values in 4IHI group significantly decreased.Compared with control group, the neuronal apoptosis of hippocampus and prefrontal cortex in 2IH group and 4IH group increased, and that in 4IH group was more evident (P<0.05).In 4IHI group, the neuronal apopto-sis decreased.The mRNA expression of adenosine A2a receptor in the hippocampal and cortical tissues in 2IH group and 4IH group was higher than that in control group.The protein level of p-AMPK was higher, and p-mTOR was lower in 2IH group and 4IH group, and those in 4IH group were more evident (P<0.05).Compared with 4IH group, the protein level of p-AMPK was lower, and p-mTOR was higher in 4IHI group.CONCLUSION: Chronic intermittent hypoxia induces neuronal apoptosis, resulting in impairment of learning and memory in a time-dependent manner by upregulating adenosine A2a receptor, activating AMPK activity, and inhibiting mTOR phosphorylation in rats.

Adenosine A2 A receptor antagonist SCH58261 attenuates hypoxic-ischemic brain damage in a fetal rabbit model / 中国病理生理杂志

[ ABSTRACT] AIM: To study the effect of adenosine A 2A receptor antagonist SCH58261 on hypoxic-ischemic brain damage ( HIBD) in a mature fetal rabbit model .METHODS:Pregnant New Zealand white rabbits at gestational day 29 were selected and were randomly divided into sham-operated group, hypoxic-ischemic group, SCH58261 0.04 mg/kg group, SCH58261 0.12 mg/kg group and DMSO group.The intrauterine rabbit HIBD model was established .All pregnant rabbits were subjected to cesarean section 24 h after the sham operation or experimental procedure to induce hypoxic-ische-mic injury in the fetus .The survival neonatal rabbits were kept in a neonatal incubator at 35℃.The general conditions of the newborn rabbits were recorded .The degree of neurobehavioral damage in the newborn rabbits was estimated by a neu -robehavioral scoring protocol .The concentration of SCH 58261 in the serum of pregnant rabbits , the serum of neonatal rab-bits and the brain tissues of neonatal rabbits was measured by mass spectrometry .The mRNA expression of Bcl-2/Bax and protein levels of p-P38 mitogen-activated protein kinase (MAPK) in the cortex, hippocampus and striatum area in the brain of the neonatal rabbits were determined by real-time PCR and Western blot .RESULTS:SCH58261 was detected in the se-rum and brain tissues of the newborn rabbits .The SCH58261 concentration was approximately 40 μg/L in the brain tissue of the newborn rabbits .The mRNA expression of Bcl-2 in the cortex , hippocampus and striatum of brain tissues in SCH58261 0.04 mg/kg group and SCH58261 0.12 mg/kg group was higher , and the mRNA expression of Bax was lower than those in HI group (P<0.05).The protein level of p-P38 MAPK in the cortex, hippocampus and striatum of brain tis-sues was reduced in SCH58261 0.04 mg/kg group and SCH58261 0.12 mg/kg group compared with HI group (P<0.05). The protein level of p-P38 MAPK in SCH58261 0.12 mg/kg group was a little lower than that in SCH 58261 0.04 mg/kg group (P<0.05).CONCLUSION: Adenosine A2A receptor antagonist SCH58261 attenuates hypoxia-ischemia induced neonatal brain injury by blocking adenosine A 2A receptor, subsequently inhibiting p-P38 MAPK phosphorylation to reduce neuronal apoptosis .

Proliferation and activation of acetaldehyde-induced HSC-T6 cells through RNA inference targeting adenosine A1 and A2 A receptors / 中国药理学通报

Aim To investigate the influence of down-regulating adenosine A1 receptor and adenosine A2 A receptor gene expression on proliferation and activation of acetaldehyde-induced hepatic stellate cell-T6 cells through siRNA. Methods Alcoholic liver fibrosis in vitro model was constructed by inducing HSC-T6 cells with acetaldehyde. siRNA targeting A1R and A2AR were designed and synthesized according to its mRNA. The siRNA was transfected into rat HSC-T6 cells by li-posome LipofectamineTM 2000. HSC cell proliferation was measured by MTT. The mRNA levels of A1R, A2AR, α-SMA, Collagen I in the supernatant of the cell culture were measured by Quantitative Real-Time PCR. The protein levels of A1R, A2AR, α-SMA, Collagen I were measured by Western blot. Results A1 R and A2 AR siRNA effectively inhibited the cell proliferation, and they also significantly decreased the levels of A1R, A2AR,α-SMA, Collagen I, suggesting that A1 R and A2 AR might be potential target genes in the alcoholic liver fibrosis. Conclusions Silencing A1 R or A2 AR by RNAi can significantly inhibit the HSC proliferation, A1R and A2AR may be potential therapeutic target genes for alcoholic liver fibrosis.

Roles of the unselective adenosine receptor agonist 5'-(N-ethylcarboxamido) adenosine-induced cardio-protection in vitro / 中国药学杂志

OBJECTIVE: To study the roles of adenosine A2A and A2B receptor in 5'-(N-ethylcarboxamido) adenosine (NECA)-induced cardioprotection in vitro against reperfusion injury, and to explore the underlying mechanism. METHODS: Simulated ischemia/reperfusion injury model was developed in cardiac H9c2 cells. NECA, an unselective adenosine receptor agonist, the selective antagonists of adenosine A2A receptor antagonist SCH58261 (SCH) and the selective A2B receptor antagonist MRS1706 (MRS) were used. CCK-8 assay was used to evaluate cell viability. Mitochondrial membrane potential (ΔΨm) was measured with fluorescence microscope using JC-1. Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit was used to detect the level of intracellular H2O2. Intracellular reactive oxygen species (ROS) levels were determined with DCFH-DA. Mitochondrial ROS were detected with MitoSox Red. RESULTS: NECA applied at reperfusion reduced cell death in cells subjected to simulated ischemia/reperfusion. Compared with the ischemia/reperfusion injury group, NECA inhibited the reduction of cell viability and ΔΨm, and the elevation of intracellular and mitochondrial ROS, which were all abolished by adenosine A2A and A2B receptor antagonists(P < 0.01 or P < 0.05). CONCLUSION: Adenosine A2A and A2B receptors work in concert to mediate the cardioprotective effect of NECA presumably by modulating the mPTP opening and mitochondrial ROS generation.

A novel therapy for PD-adenosine A_ (2A) receptor antagonist / 中国药理学通报

Adenosine A_ 2A receptors are selectively localized in basal ganglia and can affect the locomotor activity. Epidemiological and laboratory data have suggested that A_ 2A blockade may confer neuroprotection against the underlying dopaminergic neuron degeneration. A_ 2A receptor antagonist may ameliorate the symptom of Parkinsons disease (PD) and block the disease progress. Thus, the data suggested that A_ 2A receptor antagonist might be effective as a novel therapy for the management of PD.