Effects of a pulsed electromagnetic field on intervertebral disc degeneration and A2A adenosine receptor-mediated reactive oxygen signaling / 中华物理医学与康复杂志

Objective:To observe any regulatory effect of a pulsed electromagnetic field (PEMF) on A2A adenosine receptors (A2ARs) in the nucleus pulposus of rats with intervertebral disc degeneration (IDD), and to explore any combination with the A2ARs′ agonist-mediating ROS/PI3K/Akt signaling pathway.Methods:Fifty Sprague-Dawley rats were randomly divided into a control group, an intervertebral disc degeneration group (the model group), an A2AR agonist CGS-21680 treatment group (the agonist group), a PEMF group and a PEMF combined with CGS-21680 treatment group (the observation group). IDD was modeled in all except the rats in the control group. 100μL of CGS-21680 (100μg/kg) was injected into the L 5-6 intervertebral discs of the agonist group, while the PEMF group was given 30 minutes of PEMF intervention daily for 14 days at 1.5mT and 75Hz with a pulse width of 150μs. The observation group was injected with CGS-21680 and then given the same PEMF intervention. Primary nucleus pulposus cells from each group (50ng/mL) were cultured and the expressions of 8-OHDG, SOD, MDA and ROS were detected by immunohistochemistry, immunofluorescence or with an ELISA kit. The A2AR, PI3K, AKT and p-AKT protein levels were detected using western blotting. Results:The nucleus pulposus cells and the annulus fibrosus were obviously wrinkled, necrotic and broken in the model group but the annulus fibrosus was intact and the nucleus pulposus was almost normal in the observation group. Compared with the model group, the levels of SOD and A2AR, PI3K, p-AKT and AKT protein were higher in the agonist, PEMF and observation groups, while the expressions of MDA, ROS and 8-OHDG were weaker. The ROS level in the observation group was significantly lower than that in the agonist and PEMF groups, and the phosphorylation level of p-AKT in the observation group was significantly higher than in the agonist and PEMF groups. The average levels of SOD, A2AR, PI3K, p-AKT and AKT protein in the nucleus pulposus cells of the agonist, PEMF and observation groups were significantly higher than the IL-1β group′s average, while the average levels of MDA, ROS and 8-OHDG were significantly lower. The ROS levels in the observation group were significantly lower than in the agonist and PEMF groups, while the A2AR protein content and p-AKT phosphorylation in the observation group were significantly greater. The average Bax levels in the nucleus pulposus cells of the agonist, PEMF and observation groups were significantly lower than that in the IL-1β group, and the expression of Bcl-2 was significantly increased. There was significantly less apoptosis observed in the observation group than in the agonist and PEMF groups, while the expression of Bcl-2 was significantly higher.Conclusions:PEMF plays an anti-oxidative stress role by up-regulating A2AR activity and reducing ROS generation. Treatment with PEMF and A2AR agonist could further activate the phosphorylation of PI3K/Akt, down-regulate Bax and up-regulate Bcl-2, thus inhibiting the apoptosis of nucleus pulposus cells and alleviating the malignant progression of IDD.

Progress for targeting adenosine A2A receptors in cancer immunotherapy / 药学学报

Immunotherapy has completely changed the paradigm of clinical tumor treatment, but immune checkpoint inhibitors still have low objective response rates and are prone to drug resistance for most solid tumors. The immune suppression tumor microenvironment and complicated tumor immune escape mechanisms are key factors that affect the clinical outcome and response rates. Therefore, it is critical to reverse the obstacle of the tumor microenvironment to improve immunotherapy efficacy. The immune suppression caused by the increased level of adenosine in the tumor microenvironment raises the attention of people. Targeting adenosine receptors, especially A2AR, will be an effective strategy to improve immunotherapy efficacy. Targeting the adenosine-A2A pathway can increase immune infiltration, enhance immune cell function, and partially reverse immunotherapy-insensitive "cold tumors" to "hot tumors" to enhance treatment response rates and improve the efficacy of current immunotherapy. At present, many adenosine receptor inhibitors have shown good results in clinical trials, especially in combination with immune checkpoint inhibitors, chemotherapy, and adoptive cell transfer therapeutic drugs, which are expected to be used for tumor immunotherapy to bring new breakthroughs. This article reviews the accumulation mode of adenosine in the tumor microenvironment, the role of A2AR and their regulatory mechanism in immune response, the progress of A2AR inhibitors in clinical trials, potential risks to target A2AR, and the prospects for therapeutic targeting A2AR.

Blocking Adenosine/A2AR Pathway for Cancer Therapy / 中国肺癌杂志

Adenosine is a metabolite produced abundantly in the tumor microenvironment, dampening immune response in inflamed tissues via adenosine A2A receptor (A2AR) which is widely expressed on immune cells, inhibiting anti-tumor immune response accordingly. Therefore, blocking adenosine signaling pathway is of potential to promote anti-tumor immunity. This review briefly introduces adenosine signaling pathway, describes its role in regulating tumor immunity and highlights A2AR blockade in cancer therapy. Prospective anti-tumor activity of adenosine/A2AR inhibition has been revealed by preclinical data, and a number of clinical trials of A2AR antagonists are under way. Primary results from clinical trials suggest that A2AR antagonists are well tolerated in cancer patients and are effective both as monotherapy and in combination with other therapies. In the future, finding predictive biomarkers are critical to identify patients most likely to benefit from adenosine pathway blockade, and further researches are needed to rationally combine A2AR antagonists with other anti-tumor therapies.
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The effects of combining pulsed electromagnetic field stimulation with A 2A adenosine receptor agonist CGS-21680 on apoptosis and inflammation of nucleus pulposus cells in rats with intervertebral disc degeneration / 中华物理医学与康复杂志

Objective:To explore the effect of combining pulsed electromagnetic field (PEMF) stimulation with A 2A adenosine receptor agonist CGS-21680 on apoptosis and inflammation of nucleus pulposus cells in cases of intervertebral disc degeneration. Methods:Forty-eight Sprague-Dawley rats were randomly divided into a sham operation group (Sham group), an intervertebral disc degenerative disease group (Model group), an A 2A adenosine receptor agonist CGS-21680 group (Agonist group), and a group in which PEMF was combined with CGS-21680 (Observation group). The rat model of intervertebral disc degeneration (IDD) was established in all other groups than the sham operation group. The rats in the Agonist group were injected with 100μL of CGS-21680 (100μg/kg) at the L 5-6 intervertebral disc. The Observation group was injected with CGS-21680 similarly but then received 14 conse-cutive days of PEMF stimulation (30min/time). The Sham and Model groups were injected with the same amount of normal saline solution. Eight weeks later, HE staining was used to evaluate the pathological changes in the intervertebral disc tissues. The expression of type II collagen was determined by immunohistochemistry. The content and mRNA expression of inflammatory factors interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined using enzyme-linked immunosorbent assays and reverse transcription-polymerase chain reactions (RT-PCRs). The protein and mRNA levels of A 2A, NLRP3 and caspase-3 were determined by western blotting and RT-PCR. Results:The degeneration in the nucleus pulposus and annulus fibrosus in the Model group was significant, while significantly less shrinkage, necrosis and fibrous annulus rupture was observed in the Observation group. Compared with Model and Agonist groups, the positive expression of Col Ⅱ in the nucleus pulposus, A 2AR protein levels and relative expression of its mRNA had all increased significantly in the Observation group, while the average levels and mRNA expression of pro-inflammatory cytokines IL-6 and TNF-α had decreased significantly. The average protein level and mRNA expression of NLRP3 and caspase-3 in the intervertebral disc tissues of the Observation group were significantly lower than in the Model and Agonist groups. Conclusions:Combining pulsed electromagnetic field stimulation with A 2A adenosine receptor agonist CGS-21680 can inhibit the apoptosis of nucleus pulposus cells, alleviate disease response and delay IDD by up-regulating the activity of A 2A receptors and down-regulating the expressions of pro-inflammatory factors IL-6, TNF-α, NLRP3 and caspase-3 in nucleus pulposus cells.

Research status and prospects on adenosine receptor mechanism of acupuncture for myocardial ischemia / 中国针灸

The relationship between adenosine receptor (AdoR) and myocardial ischemia (MI), effect of acupuncture for MI and action mechanism of acupuncture improving MI by regulating AdoR are summarized. The existing researches have preliminarily reflected that the improvement of MI treated with acupuncture may be achieved by influencing the expression of AdoR. However, there are still some limitations, e.g. most of the research regimens are single-acupoint, the research results are not entirely consistent and the interaction of AdoRs are ignored, all these need to be further verified and supplemented.

Clostridium difficile toxins or infection induce upregulation of adenosine receptors and IL-6 with early pro-inflammatory and late anti-inflammatory pattern

Clostridium difficile causes intestinal inflammation, which increases adenosine. We compared the expression of adenosine receptors (AR) subtypes A1, A2A, A2B, and A3 in HCT-8, IEC-6 cells, and isolated intestinal epithelial cells, challenged or not with Clostridium difficile toxin A and B (TcdA and TcdB) or infection (CDI). In HCT-8, TcdB induced an early A2BR expression at 6 h and a late A2AR expression at 6 and 24 h. In addition, both TcdA and TcdB increased IL-6 expression at all time-points (peak at 6 h) and PSB603, an A2BR antagonist, decreased IL-6 expression and production. In isolated cecum epithelial cells, TcdA induced an early expression of A2BR at 2s and 6 h, followed by a late expression of A2AR at 6 and 24 h and of A1R at 24 h. In CDI, A2AR and A2BR expressions were increased at day 3, but not at day 7. ARs play a role in regulating inflammation during CDI by inducing an early pro-inflammatory and a late anti-inflammatory response. The timing of interventions with AR antagonist or agonists may be of relevance in treatment of CDI.

Role of adenosine A2b receptors in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide / 中华危重病急救医学

Objective To explore the role of the low-affinity A2b adenosine receptors (Adora2b) in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide and its mechanism. Methods Rat pulmonary microvascular endothelial cells (PMVECs) were isolated and cultured in vitro. After serum deprivation for 24 hours, cells were pretreated with Adora2b specific agonist BAY60-6583 (0.1, 1, 10 μmol/L) or Adora2b specific antagonist PSB1115 (1 μmol/L) for 1 hour, respectively, and then challenged with LPS (100 μg/L). Cells without treatment were served as the control group, and those treated with LPS, BAY60-6583 or PSB1115 alone were served as single challenge groups. After incubation with specific drugs for 24 hours, the apoptosis of PMVECs was analyzed by flow cytometry using Annexin V/propidium iodide (PI) technique. The levels of early inflammatory factors in cultured medium were measured using enzyme linked immunosorbent assay (ELISA). The mRNA expressions of chemotactic factors and adhesion molecules were determined by real-time quantitative-polymerase chain reaction (RT-qPCR). Polymorph nuclear neutrophils (PMNs) from venous blood of healthy rats were isolated, and PMN migration through PMVECs monolayer under stimulation of drugs was observed in transwell inserts. The monolayer permeability of PMVECs after adhesion of PMNs was determined by fluorescein isothiocyanate (FITC)-albumin assay. Oxidative stress was detected by DCFH-DA assay. Results Compared with the control group, more cells entered into the apoptosis stage after LPS challenge. Meanwhile, the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in cultured medium were significantly increased, as well as the mRNA expressions of chemotactic factors [C-X-C motif chemokine ligand 1 (CXCL-1), CXCL-3 and monocyte chemoattractant protein-1 (MCP-1)] and adhesion molecules [E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. More PMNs migrated through PMVECs following adhesion and the monolayer permeability of PMVECs was rapidly enhanced. The oxidative stress was upregulated. Compared with LPS group, BAY60-6583 pretreatment could dose-dependently decrease the rate of apoptosis, attenuate trans-endothelial migration of PMNs and decrease the endothelial cell barrier leakage. There were significant differences even after incubation of 0.1 μmol/L BAY60-6583 [apoptosis rate: (21.12±2.12)％vs. (27.66±3.57)％, number of migrated PMNs/HP: 260.60±18.24 vs. 290.20±16.48, permeability coefficient (Pd, ×10-6 cm/s): 28.28±2.04 vs. 32.55±2.13, all P < 0.05]. Meanwhile, BAY60-6583 pretreatment also downregulated the levels of early proinflammatory factors in a dose-dependent manner as well as the mRNA expressions of chemotactic factors and adhesion molecules. The statistic difference was significant while treated with 1 μmol/L BAY60-6583 [IL-1β(ng/L): 475.75±63.15 vs. 755.25±67.42, TNF-α (ng/L): 560.25±69.96 vs. 818.75±60.92, CXCL-1 mRNA (2-ΔΔCt):3.57±0.28 vs. 5.27±0.69, CXCL-3 mRNA (2-ΔΔCt): 4.56±0.48 vs. 7.32±0.54, MCP-1 mRNA (2-ΔΔCt): 2.21±0.31 vs. 3.35±0.21, E-selectin mRNA (2-ΔΔCt): 4.64±0.09 vs. 7.28±0.73, ICAM-1 mRNA (2-ΔΔCt): 4.14±0.30 vs. 5.89±0.25, VCAM-1 mRNA (2-ΔΔCt): 2.23±0.19 vs. 2.92±0.33, all P < 0.05]. Furthermore, pretreatment of 10 μmol/L BAY60-6583 could decrease the oxidative stress [reactive oxygen species (RFU): 629.05±33.10 vs. 781.45±64.59, P < 0.05]. Contrast, PSB1115 pretreatment aggravated apoptosis of PMVECs after LPS incubation [(34.36±4.57)％ vs. (27.66±3.57)％], upregulated expressions of proinflammatory and chemotactic factors as well as adhesion molecules [IL-1β (ng/L): 889.00±63.11 vs. 755.25±67.42, TNF-α (ng/L): 939.00±43.44 vs. 818.75±60.92, CXCL-1 mRNA (2-ΔΔCt): 6.66±0.65 vs. 5.27±0.69, CXCL-3 mRNA (2-ΔΔCt): 10.42±0.51 vs. 7.32±0.54, MCP-1 mRNA (2-ΔΔCt):4.85±0.34 vs. 3.35±0.21, E-selectin mRNA (2-ΔΔCt): 8.42±0.47 vs. 7.28±0.73, ICAM-1 mRNA (2-ΔΔCt): 7.46±0.72 vs. 5.89±0.25, VCAM-1 mRNA (2-ΔΔCt): 4.35±0.26 vs. 2.92±0.33], aggravated trans-endothelial migration of PMNs (cells/HP: 348.40±22.68 vs. 290.20±16.48), enhanced the leakage of PMVECs monolayer [Pd (×10-6 cm/s):39.65±2.69 vs. 32.55±2.13] and increased oxidative stress in PMVECs [reactive oxygen species (RFU): 847.04±29.26 vs. 781.45±64.59], with statistically significant difference (all P < 0.05). Conclusion Activation of endothelial Adora2b attenuates LPS-induced pulmonary microvascular inflammation by decreasing the release of early inflammatory factors, downregulating expressions of chemotactic factors and adhesion molecules, attenuating trans-endothelial migration of PMNs and oxidative stress in PMVECs, which suggest endothelial Adora2b is apotential anti-inflammatory target in the treatment of LPS-induced acute lung injury.

Mechanism of acupoint selection along meridians to improve adenosine receptor of myocardial ischemia based on acupoint specificity / 中国针灸

OBJECTIVE@#To explore the action mechanism of acupoint selection along meridians to improve adenosine receptor in myocardial ischemia (MI) rats by comparing the effects of acupoint selection along meridians, acupoint selection at other meridians and non-acupoint on expression of adenosine receptor.@*METHODS@#A total of 120 SD rats were randomly divided into a blank group, a sham operation group, a model group, an acupoint-selection-along-meridians (ASAM) group, an acupoint-selection-at-other-meridians (ASAOM) group and a non-acupoint group, 20 rats in each group. The model of MI was not made in the blank group; the left anterior descending coronary artery was not ligated after thoracotomy in the sham operation group; the model of MI was made but acupuncture was not given in the model group. After the model of MI was made, electroacupuncture (EA) was applied at "Neiguan" (PC 6) in the ASAM group, at "Hegu" (LI 4) in the ASAOM group, and at the area between the third and fourth metatarsal bone in the non-acupoint group. EA was given 20 min per treatment, once a day for 5 days. After treatment, the TTC staining was used to detect myocardial infarction, the Tunel method was used to detect cardiomyocyte apoptosis, and the immunohistochemistry was used to detect the expression of adenosine receptors A1, A2a and A2b.@*RESULTS@#Compared with the blank group and the sham operation group, the percentage of myocardial infarction and apoptotic rate of myocardial cells in the model group were increased significantly (<0.01). After EA treatment, compared with the model group, the percentage of myocardial infarction and apoptotic rate of myocardial cells in the ASAM group were decreased significantly (<0.01), and the expression levels of adenosine receptors A1, A2a and A2b were increased significantly (<0.01). The percentage of myocardial infarction and apoptotic rate of myocardial cells in the ASAM group were significantly lower than those in the ASAOM group and the non-acupoint group (<0.01), and the expression levels of adenosine receptors A1, A2a and A2b in the ASAM group were significantly higher than those in the ASAOM group and non-acupoint group (<0.01).@*CONCLUSION@#Compared with acupoint selection at other meridians or non-acupoints, acupoint selection along meridians can effectively regulate the expression of adenosine receptors A1, A2a and A2b, improve the condition of myocardial infarction, inhibit myocardial cell apoptosis, and consequently protect ischemic myocardium.

Role of adenosine A2b receptors in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide / 中华危重病急救医学

Objective@#To explore the role of the low-affinity A2b adenosine receptors (Adora2b) in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide and its mechanism.@*Methods@#Rat pulmonary microvascular endothelial cells (PMVECs) were isolated and cultured in vitro. After serum deprivation for 24 hours, cells were pretreated with Adora2b specific agonist BAY60-6583 (0.1, 1, 10 μmol/L) or Adora2b specific antagonist PSB1115 (1 μmol/L) for 1 hour, respectively, and then challenged with LPS (100 μg/L). Cells without treatment were served as the control group, and those treated with LPS, BAY60-6583 or PSB1115 alone were served as single challenge groups. After incubation with specific drugs for 24 hours, the apoptosis of PMVECs was analyzed by flow cytometry using Annexin V/propidium iodide (PI) technique. The levels of early inflammatory factors in cultured medium were measured using enzyme linked immunosorbent assay (ELISA). The mRNA expressions of chemotactic factors and adhesion molecules were determined by real-time quantitative-polymerase chain reaction (RT-qPCR). Polymorph nuclear neutrophils (PMNs) from venous blood of healthy rats were isolated, and PMN migration through PMVECs monolayer under stimulation of drugs was observed in transwell inserts. The monolayer permeability of PMVECs after adhesion of PMNs was determined by fluorescein isothiocyanate (FITC)-albumin assay. Oxidative stress was detected by DCFH-DA assay.@*Results@#Compared with the control group, more cells entered into the apoptosis stage after LPS challenge. Meanwhile, the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in cultured medium were significantly increased, as well as the mRNA expressions of chemotactic factors [C-X-C motif chemokine ligand 1 (CXCL-1), CXCL-3 and monocyte chemoattractant protein-1 (MCP-1)] and adhesion molecules [E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. More PMNs migrated through PMVECs following adhesion and the monolayer permeability of PMVECs was rapidly enhanced. The oxidative stress was upregulated. Compared with LPS group, BAY60-6583 pretreatment could dose-dependently decrease the rate of apoptosis, attenuate trans-endothelial migration of PMNs and decrease the endothelial cell barrier leakage. There were significant differences even after incubation of 0.1 μmol/L BAY60-6583 [apoptosis rate: (21.12±2.12)% vs. (27.66±3.57)%, number of migrated PMNs/HP: 260.60±18.24 vs. 290.20±16.48, permeability coefficient (Pd, ×10-6 cm/s): 28.28±2.04 vs. 32.55±2.13, all P < 0.05]. Meanwhile, BAY60-6583 pretreatment also downregulated the levels of early proinflammatory factors in a dose-dependent manner as well as the mRNA expressions of chemotactic factors and adhesion molecules. The statistic difference was significant while treated with 1 μmol/L BAY60-6583 [IL-1β (ng/L): 475.75±63.15 vs. 755.25±67.42, TNF-α (ng/L): 560.25±69.96 vs. 818.75±60.92, CXCL-1 mRNA (2-ΔΔCt): 3.57±0.28 vs. 5.27±0.69, CXCL-3 mRNA (2-ΔΔCt): 4.56±0.48 vs. 7.32±0.54, MCP-1 mRNA (2-ΔΔCt): 2.21±0.31 vs. 3.35±0.21, E-selectin mRNA (2-ΔΔCt): 4.64±0.09 vs. 7.28±0.73, ICAM-1 mRNA (2-ΔΔCt): 4.14±0.30 vs. 5.89±0.25, VCAM-1 mRNA (2-ΔΔCt): 2.23±0.19 vs. 2.92±0.33, all P < 0.05]. Furthermore, pretreatment of 10 μmol/L BAY60-6583 could decrease the oxidative stress [reactive oxygen species (RFU): 629.05±33.10 vs. 781.45±64.59, P < 0.05]. Contrast, PSB1115 pretreatment aggravated apoptosis of PMVECs after LPS incubation [(34.36±4.57)% vs. (27.66±3.57)%], upregulated expressions of proinflammatory and chemotactic factors as well as adhesion molecules [IL-1β (ng/L): 889.00±63.11 vs. 755.25±67.42, TNF-α (ng/L): 939.00±43.44 vs. 818.75±60.92, CXCL-1 mRNA (2-ΔΔCt): 6.66±0.65 vs. 5.27±0.69, CXCL-3 mRNA (2-ΔΔCt): 10.42±0.51 vs. 7.32±0.54, MCP-1 mRNA (2-ΔΔCt): 4.85±0.34 vs. 3.35±0.21, E-selectin mRNA (2-ΔΔCt): 8.42±0.47 vs. 7.28±0.73, ICAM-1 mRNA (2-ΔΔCt): 7.46±0.72 vs. 5.89±0.25, VCAM-1 mRNA (2-ΔΔCt): 4.35±0.26 vs. 2.92±0.33], aggravated trans-endothelial migration of PMNs (cells/HP: 348.40±22.68 vs. 290.20±16.48), enhanced the leakage of PMVECs monolayer [Pd (×10-6 cm/s): 39.65±2.69 vs. 32.55±2.13] and increased oxidative stress in PMVECs [reactive oxygen species (RFU): 847.04±29.26 vs. 781.45±64.59], with statistically significant difference (all P < 0.05).@*Conclusion@#Activation of endothelial Adora2b attenuates LPS-induced pulmonary microvascular inflammation by decreasing the release of early inflammatory factors, downregulating expressions of chemotactic factors and adhesion molecules, attenuating trans-endothelial migration of PMNs and oxidative stress in PMVECs, which suggest endothelial Adora2b is apotential anti-inflammatory target in the treatment of LPS-induced acute lung injury.

Activation of A2b adenosine receptor decreases lipopolysaccharide-induced pulmonary microvascular permeability / 中华危重病急救医学

Objective To explore the role of the A2b adenosine receptor (Adora2b) in lipopolysaccharide (LPS)-induced injury of human pulmonary microvascular endothelial cells (HPMECs), and its mechanism. Methods HPMECs were cultured in vitro. The LPS dose-effect experiment, time-effect experiment and the Adora2b agonist/antagonist intervention experiment were performed respectively. ① Dose-effect and time-effect experiments: HPMECs were stimulated with 1, 10, 100, 1 000 μg/L LPS for 24 hours, or 100 μg/L LPS for 4, 8, 12, 16, 24 hours. Cell viability was measured by cell counting kit-8 (CCK8). The protein and mRNA expressions of Adora2b were determined by Western Blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) respectively. ② Adora2b agonist/antagonist intervention experiment: serum-starved HPMECs were pretreated with Adora2b specific agonist BAY60-6583 (0.1, 1, 10 μmol/L) or Adora2b specific antagonist PSB1115 (1 μmol/L) for 1 hour, respectively, and then incubated with 100 μg/L of LPS for 24 hours. The HPMECs without treatment were served as blank control group, and those treated with LPS, BAY60-6583 or PSB1115 alone were served as single challenge groups. The monolayer permeability of HPMECs was determined by fluorescein isothiocyanate (FITC)-dextran. Cell cycle was analyzed by flow cytometry. The mRNA expressions of VE-cadherin, occludin, vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANGPT1) were determined by RT-PCR. Results ① Dose-effect and time-effect experiments: LPS induced the decreased cell viability of HPMECs in dose and time-dependent manner. Compared with the control group, the protein expression of Adora2b was sharply up-regulated after 100 μg/L or 1 000 μg/L LPS stimulation. Meanwhile, LPS was shown to cause a dose and time-dependent induction of Adora2b transcript level. ② Adora2b agonist/antagonist intervention experiments: compared with the control group, the monolayer permeability of HPMECs was rapidly enhanced after LPS treatment, and lower cell viability and proliferation, as well as the expression of cell junction and angiogenic factors were downregulated. Compared with LPS group, 0.1, 1, 10 μmol/L BAY 60-6583 pretreatment could decrease the endothelial cell barrier leakage in a dose-dependent manner [Pd: (203.06±15.24)%, (164.15± 17.82)%, (125.69±10.38)% vs. (218.53±12.05)%], and promote cell proliferation of HPMECs [the proportion of S and G2 phases: (24.36±1.40)%, (32.37±0.95)%, (40.05±2.99)% vs. (18.83±0.73)%]. Pretreatment of 10 μmol/L BAY60-6583 also upregulated the mRNA expressions of cell junction and angiogenic factors [VE-cadherin (2-ΔΔCt):2.17±0.23 vs. 0.56±0.10, occludin (2-ΔΔCt): 5.32±0.28 vs. 0.48±0.11, VEGF (2-ΔΔCt): 4.44±0.34 vs. 0.58±0.09, ANGPT-1 (2-ΔΔCt): 5.98±0.73 vs. 0.66±0.10, all P < 0.01]. PSB1115 pretreatment aggravated injury of microvascular endothelial cells after LPS incubation, with lower cell viability, slower proliferation and less expression of VEGF and ANGPT1. There was no influence of BAY 60-6583 or PSB1115 single treatment on cell viability, cell cycle and the expression of angiogenic factors in HPMECs. Conclusions In vitro studies of cultured HPMECs exposed to LPS are identified as dose and time-dependent induction of Adora2b transcript and corresponding protein induction. Activation of Adora2b attenuates LPS-induced pulmonary microvascular endothelial cell barrier enhancement by regulating intercellular junction and promoting angiogenesis, suggesting Adora2b as potential therapeutic target in the treatment of LPS-induced forms of acute lung injury.

Renoprotective Effects of a Highly Selective A3 Adenosine Receptor Antagonist in a Mouse Model of Adriamycin-induced Nephropathy

The concentration of adenosine in the normal kidney increases markedly during renal hypoxia, ischemia, and inflammation. A recent study reported that an A3 adenosine receptor (A3AR) antagonist attenuated the progression of renal fibrosis. The adriamycin (ADX)-induced nephropathy model induces podocyte injury, which results in severe proteinuria and progressive glomerulosclerosis. In this study, we investigated the preventive effect of a highly selective A3AR antagonist (LJ1888) in ADX-induced nephropathy. Three groups of six-week-old Balb/c mice were treated with ADX (11 mg/kg) for four weeks and LJ1888 (10 mg/kg) for two weeks as following: 1) control; 2) ADX; and 3) ADX + LJ1888. ADX treatment decreased body weight without a change in water and food intake, but this was ameliorated by LJ1888 treatment. Interestingly, LJ1888 lowered plasma creatinine level, proteinuria, and albuminuria, which had increased during ADX treatment. Furthermore, LJ1888 inhibited urinary nephrin excretion as a podocyte injury marker, and urine 8-isoprostane and kidney lipid peroxide concentration, which are markers of oxidative stress, increased after injection of ADX. ADX also induced the activation of proinflammatory and profibrotic molecules such as TGF-β1, MCP-1, PAI-1, type IV collagen, NF-κB, NOX4, TLR4, TNFα, IL-1β, and IFN-γ, but they were remarkably suppressed after LJ1888 treatment. In conclusion, our results suggest that LJ1888 has a renoprotective effect in ADX-induced nephropathy, which might be associated with podocyte injury through oxidative stress. Therefore, LJ1888, a selective A3AR antagonist, could be considered as a potential therapeutic agent in renal glomerular diseases which include podocyte injury and proteinuria.

The effect of non -xanthine adenosine receptor antagonist on ventricular remodeling and RAAS in chronic heart failure / 中国基层医药

Objective To analyze the effect of non -xanthine adenosine receptor antagonist (SLV320)on the ventricular remodeling and renin -angiotensin -aldosterone system (RAAS)in animal experimental models of chronic heart failure (CHF).Methods The 40 healthy male New Zealand rabbits were received adriamycin by intra-venous injection to establishing the experimental animal models and were randomly divided into 4 groups,which were high -dosage group (injected with SLV320,10.0μg·kg -1 ·d -1 ),medium -dosage group (injected with SLV320, 5.0μg·kg -1 ·d -1 ),low -dosage group (injected with SLV320,2.5μg·kg -1 ·d -1 )and furosemide group (fed with furosemide,2.0mg·kg -1 ·d -1 ).Each group had 10 rabbits and continuous treated for one week.The indexes of plasma renin activity (PRA),angiotensinⅡ (AngⅡ),aldosterone (ALD)and beta ntriuretic peptide (BNP)were detected at pre -and post -treatment,and compared among 4 groups.The indexes of left ventricular end -systolic dimension (LVESD),left ventricular end -diastolic dimension (LVEDD),left ventricular posterior wall (LVPW), left ventricle ejection fraction (LVEF),left ventricular fractional shortening (LVFS)and E /A at pre -and post -treatment were detected by echocardiography and compared among 4 groups.The wet weigh of the left and right ventri-cle were weigh accurately.And the indexes of left ventricle weight index (LVWI)and the body weight index (BWI) were calculated and compared among 4 groups.Results The plasma levels of PRA,AngⅡ,ALD and BNP were no different among 4 groups before study (all P >0.05).The sequence of 4 groups on the plasma levels of RAAS indexes was high -dosage group 0.05).The sequence of 4 groups on the levels of LVEF and LVFS was high -dosage group >medium -dosage group >low -dosage group >furosemide group,and the sequences of LVESD,LVEDD,LVPW and E /A were high -dosage group

Research progress of adenosine receptors in fibrosis diseases / 中国药理学通报

The fibrosis can occur in many kinds of organs,and its sustained progress may lead to organ structural damage and functional decline,and even the organ failure,which threatens the human health and the life seriously.Adenosine is an endogenous purine nucleoside that can be generated in various tissues of the body and regulate a multitude of body functions via the combina-tion with four different kinds of G protein-coupled receptors.Re-cent studies have found that adenosine receptors play an impor-tant role in regeneration tissue and fibrosis process.To under-stand the processes may be helpful to the treatment of fibrosis diseases.This review makes a summary on latest research pro-gress of adenosine receptors in fibrosis diseases.

Highly expressed adenosine receptor A2B in mucosa dendritic cells is associated with enhanced pathogenicity of Crohn's disease / 中华微生物学和免疫学杂志

Objective To investigate the expression of adenosine receptor (ADOR) subtypes (A2A and A2B subtypes) in the mucosal dendritic cells (DCs) from patients with Crohn's disease and their pathogenic roles.Methods Mucosal DCs (mDCs) were isolated from resected intestine of patients with or without Crohn's disease.Some of the mDCs were cultured in vitro and others were used to extract RNA.The expression of ador-a2a and ador-a2b were detected by real-time qPCR.mDCs in culture were treated with selective ADOR-A2A and ADOR-A2B agonists (CGS 21680 and BAY 60-6583) and then the concentration of IL-1,IL-6 and IL-12 in the medium were measured by ELISA.The binding affinities of ADOR-A2A and ADOR-A2B to adenosine were determined by 3H-adenosine in combination with selective ADOR-A2A and A2B antagonists (SCH58261 and MRS1706).Na(i)ve CD4+ cells were collected from human umbilical cord blood and co-cultured with mDCs treated by different ADOR agonists to observe T cell responses.The production of cytokines in culture was measured by ELISA.The polarization of CD4+ cells was analyzed by intracellular cytokine staining and FACs analysis.Peripheral blood mononuclear cells (PBMCs) were treated with IL-4 and GMCSF to induce the expression of monocyte-derived DCs (Mo-DCs).Mo-DCs were treated with different toll-like receptor ligands to investigate their effects on the expression of ador-a2a and adora2b.Moreover,Mo-DCs were treated with LPS and BAY 60-6583 individually or in the combination to stimulate CD4+ cells.Then the production of cytokines and the polarization of CD4+ cells were evaluated.Results Compared with patients without Crohn's disease,patients with Crohn's disease showed no change in the expression of ador-a2a but a significantly increased expression of ador-a2b in mCDs (CD-mDCs),enabling to bind more adenosines.Activated ADOR-A2B signaling pathway induced CD-mDCs to secret more proinflammatory cytokines and to promote polarization of CD4+ cells toward Th1 and Th17 cells.Toll-like receptor ligands,pam3csk4 and LPS could intensively augment the expression of ador-a2b in Mo-DCs.The pathogenicity of Mo-DCs was strengthened upon a combined stimulation with BAY 60-6583 and LPS.Conclusion The significantly increased expression of ador-a2b in mDCs might be involved in the pathogenesis of Crohn's disease by promoting mDCs to secret more pro-inflammatory cytokines and enhancing the polarization of CD4+ cells.Moreover,the expression of ador-a2b in DCs could be regulated by certain toll like receptors.

Antiallodynic effects of intrathecal tianeptine in a neuropathic pain rat

BACKGROUND: Tianeptine is an antidepressant drug which is used for treating depression. Interestingly, the tianeptine has shown antinociceptive effects within a variety of nociceptions. The aim of this study is to investigate the antiallodynic effects of tianeptine in neuropathic pain rats and also determine the involvements of serotonergic, alpha-2 adrenergic and adenosine receptors at the spinal level. METHODS: Neuropathic pain was induced by ligation of left lumbar at 5th and 6th spinal nerves in male Sprague-Dawley rats. PE-10 catheters were placed into the thoracolumbar subarachnoid space for drug injections. Mechanical allodynia was evaluated by measuring the withdrawal threshold to von Frey filament when applying on the plantar surface of rats. The effects of intrathecal tianeptine were observed at 15, 30, 60, 90, 120, 150, 180 minutes after delivery. Antagonists for serotonergic (dihydroergocristine), alpha-2 adrenergic (yohimbine) and adenosine (CGS 15943) receptors were intrathecally administered 10 minutes prior to tianeptine in order to evaluate the involvement of both receptors. RESULTS: Intrathecal tianeptine increased dose-dependently at the withdrawal threshold in the ligated paw. Pretreatment with intrathecal dihydroergocristine, yohimbine and CGS 15943 antagonized the antiallodynic effects of tianeptine. CONCLUSIONS: These results suggested that intrathecal tianeptine attenuates the spinal nerve ligation induced tactile allodynia. Serotonergic, alpha-2 adrenergic and adenosine receptors are all involved in the antiallodynic effects of tianeptine at the spinal level.

Deoxyadenosine antagonized methotrexate-induced suppression of osteoclastogenesis / 实用口腔医学杂志

Objective:To study the mechanism of the effectiveness loss of methotrexate(MTX)in the treatment of rheumatoid arthri-tis.Methods:The culture system of rat whole bone marrow cells(WBMCs)and tartrateresistant acid phosphatase(TRAP)staining were utilized to evaluate osteoclastogenesis.The mRNA expression of osteoclastogenesis factors in the WBMCs culture system was examined by semi-quantitative RT-PCR.Results:Deoxyadenosine(dAdo)decreased MTX-induced suppression of osteoclastogenesis.The recov-ery effect of dAdo on MTX was partially prevented by caffeine.MTX significantly reduced mRNA expression of receptor activator of nu-clear factor kappa-B ligand(RANKL),dAdo partially recovered RANKL mRNA expression and inhibited osteoprotegerin(OPG)expres-sion.Conclusion:The accumulation of dAdo may induce the effectiveness loss of methotrexate in rheumatoid arthritis treatment.Com-bination of MTX and caffeine can be a potential therapeutic strategy.

Therapeutic approaches for tumor necrosis factor inhibition

Tumor necrosis factor (TNF) consists of an inflammatory cytokine essential for homeostasis and organism defense. Despite its physiological relevance, both increased biosynthesis and release of TNF lead to the exacerbation of inflammatory and oxidative responses, which are related to the pathogenesis of a host of diseases of an inflammatory, autoimmune and/or infectious nature. In this context, effective therapeutic approaches for the modulation of TNF have been the focus of research efforts. Approximately one million individuals worldwide have been treated with biotechnological inhibitors of this cytokine, the so-called anti-TNF biopharmaceuticals. However, given the high risk of infection and the limitations related to cost and administration routes, new therapeutic approaches aimed at biological targets that directly or indirectly modulate the production and/or activation of TNF appear promising alternatives for the discovery of new anti-inflammatory and immunomodulatory orally active drugs and are therefore discussed in this paper.

O fator de necrose tumoral (do inglês, tumor necrosis factor - TNF) consiste em uma citocina inflamatória essencial para a homeostase e defesa do organismo. A despeito de sua relevância fisiológica, o aumento da biossíntese e liberação do TNF conduzem à exacerbação das respostas inflamatória e oxidativa, as quais estão relacionadas à patogênese de várias doenças de natureza inflamatória, auto-imune e/ou infecciosa. A busca por abordagens terapêuticas eficientes na modulação do TNF tem sido alvo de diversos esforços de pesquisa. Aproximadamente um milhão de pessoas ao redor do mundo já foi tratado com inibidores biotecnológicos desta citocina, os chamados biofármacos anti-TNF. Entretanto, em face ao elevado risco de infecções e as limitações relacionadas ao custo e a via de administração, novas abordagens terapêuticas com foco em alvos que modulem, de forma direta ou indireta, a produção e/ou ativação do TNF surgem como alternativas promissoras para a descoberta de novos fármacos antiinflamatórios e imunomoduladores ativos por via oral e serão discutidas neste trabalho.

Effects of A3 adenosine receptor antagonist on lung injury after cardiopulmonary bypass in rabbits / 上海交通大学学报（医学版）

Objective To investigate the effects of A3 adenosine receptor ( A3AR) agonist on lung injury after cardiopulmonary bypass ( CPB) in rabbits. Methods Twenty-four rabbits were randomly divided into 3 groups, with 8 in each group. Rabbits in control group only received CPB, those in agonist group were given selective A3AR agonist IB-MECA intravenously 15 min before aorta clamp, and those in agonist + antagonist group were managed with selective A3AR receptor antagonist MRS-1191 intravenously before IB-MECA infusion. After CPB, serum concentrations of tumor necrosis factor-α ( TNF-α) and interleukin-8 ( IL-8), concentrations of malondialdehyde ( MDA) and myeloperoxidase ( MPO) in lung tissues, lung wet/dry weight ratio ( W/D), lung function related indexes of PaO_2/FiO_2, airway pressure (AWP) and pulmonary vascular resistance ( PVR), and histological changes of lung tissues were observed. Results Concentrations of serum TNF-a and IL-8 were significantly lower in agonist group than in control group and agonist + antagonist group (P <0.05). Compared with control group and agonist + antagonist group, W/D was much smaller, and concentrations of MDA and MPO were significantly lower in agonist group after CPB (P <0.05). PaO_2/FiO_2 was significantly higher, while AWP and PVR were significantly lower in agonist group than in control group and agonist + antagonist group (P <0.05). It was revealed by histological examinations that the pathological changes were less severe in agonist group than in control group and agonist + antagonist group. Conclusion A3AR agonist IB-MECA can reduce lung injury after CPB.

Cardioprotective signaling cascade of A2 adenosine receptor agonist 5'-N-ethylcarboxaminidoadenosine against myocardial reperfusion injury / 대한마취과학회지

BACKGROUND: This experiments investigated the signaling cascade responsible for anti-infarct effect by an A2 adenosine receptor (AR) agonist 5'-N-Ethylcarboxaminidoadenosine (NECA). METHODS: Langendorff perfused isolated rat hearts were subjected to 30 minutes of regional ischemia and 120 minutes of reperfusion. Drugs were perfused for a period of 5 minutes before and 60 minutes after reperfusion. For comparison of cardioprotection among groups, area at necrosis (AN) and area at risk (AAR) were measured by triphenyltetrazolium chloride staining. RESULTS: NECA significantly attenuated AN/AAR (14.1 +/- 1.9%, P < 0.001) compared with control hearts (30.7 +/- 2.8%). Anti-infarct effect by NECA was attenuated by an A(2A)AR antagonist 8-(3-chlorostyryl)caffeine (23.7 +/- 3.4%, P < 0.05) and an A(2B)AR antagonist MRS1706 (29.9 +/- 3.3%, P < 0.001). Cardioprotection by NECA was blocked by a guanylyl cyclase inhibitor (23.1 +/- 2.9%, P < 0.05) and a protein kinase G (PKG) inhibitor KT5823 (30.3 +/- 3.2%, P < 0.001). Glycogen synthase kinase-3beta (GSK-3beta) inhibitor SB216763 attenuated the AN/AAR in both NECA with MRS (17.8 +/- 2.7%, P < 0.01 vs. control) and NECA with KT5823 treated hearts (8.2 +/- 1.8%, P < 0.001 vs. control). The mitochondrial permeability transition pore (mPTP) opener atractyloside also aborted NECA's anti-infarct effect (24.7 +/- 2.4% P < 0.05). CONCLUSIONS: The signaling pathway by NECA administered at reperfusion involves the activation of both A2AAR and A2BAR and cGMP/PKG pathway, which in turn depends on inactivation of GSK-3beta and inhibition of mPTP opening.

The Mechanism of R-PIA Induced Mechanical Antiallodynia in a Peripheral Neuropathic Rat / 대한마취과학회지

BACKGROUND: The injury by a nerve ligation produces a mechanical allodynia. The antiallodynic effect resulted from intrathecal administration of the adenosine analogues has been well known. ATP-sensitive potassium channel blockers have been known to reverse the effect of some antinociceptive drugs in animal and human studies. Therefore, the present study is to assess the relationship between antiallodynic effect of N6-(R)-phenylisopropyl adenosine (R-PIA) and mitochondrial ATP-sensitive potassium (mKATP) channel in a neuropathic pain model. METHODS: Allodynia was induced in male Sprague Dawley rats by the tight ligation of the left lumbar 5th and 6th spinal nerves. We tested the mechanical allodynia by pricking von Frey filaments to the left hind paw and assessed withdrawal thresholds of paw with up-down method. For the estimation of the antiallodynic effect of R-PIA, R-PIA (0.5, 1 and 2microgram) or saline were administered intrathecally.To investigate the reversal effect on antiallodynic effect of R-PIA, variable amounts of 5-hydroxydecanoate (5-HD, 20, 30 and 40 mg), mKATP channel blocker were administered intraperitoneally at 5 min prior to the intrathecal injection of 2microgram of R-PIA, and the degree of allodynia was assessed. RESULTS: The paw withdrawal threshold was gradually increased with increased dose of R-PIA and reached the maximum level with 2microgram R-PIA (P < 0.05). The increase of paw withdrawal threshold with 2microgram R-PIA was significantly reversed dose-dependently by intraperitoneal pretreatment of 20, 30 and 40 mg/kg 5-HD (P < 0.05). CONCLUSIONS: In our results, intraperitoneal injection of 5-HD before intrathecal injection of R-PIA had reversed the antiallodynic effect of R-PIA. This results suggest that the mechanism of mechanical antiallodynia induced by intrathecal injection of R-PIA may relate with the mK(ATP) channel in a rat model of nerve ligation injury.