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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.
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Objective To explore the effects of combining tumor necrosis factor-α (TNF-αt) inhibitor with adenosine A2b receptor antagonist CVT-6883 on asthmatic lung irflammation in mice.Methods A total of 40 female Balb/c mice were evenly randomized into 5 groups,including normal control group,asthma group,CVT-6883 group,CVT-6883 + etanercept group,and etanercept group.The pathological changes in the lungs were determined and the number of white blood cells(WBC) and eosinophil(EOS) in the bronchoalveolar lavage fluid(BALF) was counted by cell count in each group.The levels of TNF-α in BALF were evaluated by enzyme-linked immunosorbent assay (ELISA).The expression of adenosine A2b receptor mRNA in the lung tissues were measured by reverse transcriptionpolymerase chain reaction (RT-PCR).Results 1.The lung tissue in asthma group,dyed by HE,was found to have a large number of airway inflammatory cell infiltration,thickening of the bronchial mucosa,the alveolar septa widened and fracture.In the CVT-6883,CVT-6883 + etanercept and etanercept group,the pathological changes were relieved.2.The WBC and EOS counts in BALF of the asthma group[(413.8 ±5.8)/L,(139.3 ± 1.4)/L] were higher than those of the normal control group [(24.0 ± 1.3)/L,(1.8 ± 0.1)/L,P ＜ 0.05].The WBC and EOS counts of the CVT-6883 group[(111.5 ±3.8)/L,(3.3 ±0.1)/L],the etanercept group + the CVT-6883 group[(173.8 ±3.9)/L,(10.4 ± 0.2)/L],and the etanercept group[(138.4 ± 3.0)/L,(4.1 ± 0.1)/L] were lower than those of the asthma group (P ＜0.05).3.Compared with the control group(100.4 ± 5.7) ng/L,the TNF-α concentration of the asthma group (145.2 ± 8.8) ng/L was significantly higher (P ＜ 0.05) ; the TNF-α concentration of CVT-6883 group (130.9 ± 5.9) ng/L,CVT-6883 + etanercept group(115.7 ± 8.2) ng/L and the etanercept group(122.0 ± 8.7) ng/L,were significantly decreased compared with asthma group (P ＜ 0.05).4.In asthma group (8.9 ± 1.1) compared with the control group(0.6 ± 0.2),the A2bAR (adenosine A2b receptor) mRNA expression was upregulated (P ＜ 0.05) ; CVT-6883 group(1.6 ±0,3),CVT-6883 + etanercept group(2.5 ±0.6) and the etanercept group(5.3 ±0.4),the A2bAR(adenosine A2b receptor) mRNA expression was significantly decreased compared with asthma group (all P ＜0,05).Conclusion Combination of TNF-α inhibitor with adenosine A2b receptor antagonist can reduce asthmatic lung inflammation.