Activation of Cannabinoid CB2 Receptor–Mediated AMPK/CREB Pathway Reduces Cerebral Ischemic Injury
2013; Elsevier BV; Volume: 182; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2012.11.024
ISSN1525-2191
AutoresIn Young Choi, Chung Ju, Angela M. A. Anthony Jalin, Da In Lee, Paul L. Prather, Won-Ki Kim,
Tópico(s)Biochemical effects in animals
ResumoThe type 2 cannabinoid receptor (CB2R) was recently shown to mediate neuroprotection in ischemic injury. However, the role of CB2Rs in the central nervous system, especially neuronal and glial CB2Rs in the cortex, remains unclear. We, therefore, investigated anti-ischemic mechanisms of cortical CB2R activation in various ischemic models. In rat cortical neurons/glia mixed cultures, a CB2R agonist, trans-caryophyllene (TC), decreased neuronal injury and mitochondrial depolarization caused by oxygen-glucose deprivation/re-oxygenation (OGD/R); these effects were reversed by the selective CB2R antagonist, AM630, but not by a type 1 cannabinoid receptor antagonist, AM251. Although it lacked free radical scavenging and antioxidant enzyme induction activities, TC reduced OGD/R-evoked mitochondrial dysfunction and intracellular oxidative stress. Western blot analysis demonstrated that TC enhanced phosphorylation of AMP-activated protein kinase (AMPK) and cAMP responsive element-binding protein (CREB), and increased expression of the CREB target gene product, brain-derived neurotrophic factor. However, TC failed to alter the activity of either Akt or extracellular signal–regulated kinase, two major CB2R signaling pathways. Selective AMPK and CREB inhibitors abolished the neuroprotective effects of TC. In rats, post-ischemic treatment with TC decreased cerebral infarct size and edema, and increased phosphorylated CREB and brain-derived neurotrophic factor expression in neurons. All protective effects of TC were reversed by co-administration with AM630. Collectively, these data demonstrate that cortical CB2R activation by TC ameliorates ischemic injury, potentially through modulation of AMPK/CREB signaling, and suggest that cortical CB2Rs might serve as a putative therapeutic target for cerebral ischemia. The type 2 cannabinoid receptor (CB2R) was recently shown to mediate neuroprotection in ischemic injury. However, the role of CB2Rs in the central nervous system, especially neuronal and glial CB2Rs in the cortex, remains unclear. We, therefore, investigated anti-ischemic mechanisms of cortical CB2R activation in various ischemic models. In rat cortical neurons/glia mixed cultures, a CB2R agonist, trans-caryophyllene (TC), decreased neuronal injury and mitochondrial depolarization caused by oxygen-glucose deprivation/re-oxygenation (OGD/R); these effects were reversed by the selective CB2R antagonist, AM630, but not by a type 1 cannabinoid receptor antagonist, AM251. Although it lacked free radical scavenging and antioxidant enzyme induction activities, TC reduced OGD/R-evoked mitochondrial dysfunction and intracellular oxidative stress. Western blot analysis demonstrated that TC enhanced phosphorylation of AMP-activated protein kinase (AMPK) and cAMP responsive element-binding protein (CREB), and increased expression of the CREB target gene product, brain-derived neurotrophic factor. However, TC failed to alter the activity of either Akt or extracellular signal–regulated kinase, two major CB2R signaling pathways. Selective AMPK and CREB inhibitors abolished the neuroprotective effects of TC. In rats, post-ischemic treatment with TC decreased cerebral infarct size and edema, and increased phosphorylated CREB and brain-derived neurotrophic factor expression in neurons. All protective effects of TC were reversed by co-administration with AM630. Collectively, these data demonstrate that cortical CB2R activation by TC ameliorates ischemic injury, potentially through modulation of AMPK/CREB signaling, and suggest that cortical CB2Rs might serve as a putative therapeutic target for cerebral ischemia. CME Accreditation Statement: This activity ("ASIP 2013 AJP CME Program in Pathogenesis") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("ASIP 2013 AJP CME Program in Pathogenesis") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("ASIP 2013 AJP CME Program in Pathogenesis") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("ASIP 2013 AJP CME Program in Pathogenesis") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. 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The anti-ischemic effects of TC in mixed cortical cultures is mediated by activation of CB2Rs, most likely leading to an increase in the levels of AMP-activated protein kinase (AMPK) and phosphorylation of one of its substrates, cAMP responsive element-binding protein (CREB). In an in vivo ischemic rat model, it was further found that post-ischemic treatment of TC increases the levels of phosphorylated CREB and brain-derived neurotrophic factor (BDNF), one of the target gene products of CREB. TC was purchased from Sigma-Aldrich (St. Louis, MO). AM251 and AM630 were purchased from Tocris (Ellisville, MO), and compound C (CC) and CREB inhibitor were obtained from Calbiochem (Darmstadt, Germany). Tetramethylrhodamine methyl ester (TMRM), 5-(and 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCF-DA), and secondary antibodies (conjugated Alexa fluorophores) were obtained from Molecular Probes (Eugene, OR). The following primary antibodies were used: CB1R (Cayman Chemicals, Ann Arbor, MI); CB2R (an N-terminus antibody from Cayman Chemicals); mitogen-activated protein (MAP)-2 (Sigma-Aldrich); neuronal nuclei (NeuN; Chemicon, Temecula, CA); phosphorylated AMPK (pAMPK), AMPK, phosphorylated CREB (pCREB), and CREB antibodies (Cell Signaling, Danvers, MA); and BDNF (Abcam, Cambridge, MA). Neurobasal medium and B27 supplement were purchased from Invitrogen (Grand Island, NY). Fetal bovine serum was obtained from Hyclone (Logan, UT). All other chemicals were purchased from Sigma-Aldrich. Dissociated cultures of rat cortical neurons/glia were prepared from embryonic 17- to 18-day-old Sprague-Dawley rat embryos. In brief, meninges-free cortexes were dissociated by triturating through a Pasteur pipette (Poulten & Graf Gmbh, Wertheim, Germany). Neuronal cells (1.5 × 103 cells/mm2) were initially plated in plates precoated with poly-d-lysine (100 μg/mL) and laminin (4 μg/mL) in neurobasal medium containing 10% fetal bovine serum. Cells were then maintained in a B27-supplemented neurobasal medium in humidified 95% air/5% CO2 at 37°C. Experiments were performed on cultures 14 to 15 days after initial plating. Cultures contain 40% to 50% of neurons, as assessed by immunostaining with cell type–specific markers. To induce ischemic insult in vitro, cells were replenished with glucose-free Dulbecco's modified Eagle's medium and transferred to an anaerobic chamber containing 5% CO2 and 10% H2 atmosphere, balanced with N2 (partial pressure of oxygen, <2 mm Hg), for 1.5 hours at 37°C. OGD was terminated by returning plates to the original growth conditions, in oxygenated Dulbecco's modified Eagle's medium supplemented with 25 mmol/L glucose under normoxic conditions. TC was applied immediately before initiation of OGD and maintained throughout OGD/re-oxygenation (OGD/R). In a subset of experiments to determine a potential mechanism of action for TC, cells were exposed to a 30-minute pretreatment with AM251 (a selective CB1R antagonist), AM630 (a selective CB2R antagonist), CC (an AMPK inhibitor), or CREB–CREB-binding protein interaction inhibitor (CREB signaling inhibitor) before the addition of TC and maintained throughout OGD/R. Five hours after re-oxygenation, lactate dehydrogenase (LDH) release was measured to assess cell injury or death. Cell injury or death was assessed by morphological examination using a phase-contrast microscope (DM IL; Leica, Nussloch, Germany) or by measuring the amount of LDH released into the culture medium using a diagnostic kit (Sigma-Aldrich), as previously described.35Choi I.Y. Lee J.C. Ju C. Hwang S. Cho G.S. Lee H.W. Choi W.J. Jeong L.S. Kim W.K. 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Dichlorofluorescein then reacts with intracellular free radicals, such as peroxyl radical, peroxynitrite, or hydrogen peroxide, to form dichlorofluorescein, a green fluorescent dye. Two hours after loading, cells were washed with Earle's balanced salt solution buffer containing 0.1% bovine serum albumin and 2.5 mmol/L probenecid. Fluorescent intensities were then measured at 488 nm of excitation wavelength and 525 nm of emission wavelength using a fluorescence microscope (DM IL HC Fluo; Leica) equipped with a digital camera (DFC420C; Leica). The intensity of fluorescence was quantified by an image analyzer (TOMORO ScopeEye 3.5; Techsan Digital Imaging, Seoul, Korea). Mitochondrial membrane potential was measured by quantifying the accumulation of TMRM, as previously described.37Choi Y. Kim S.K. Choi I.Y. Ju C. Nam K.W. Hwang S. Kim B.W. Yoon M.J. Won M.H. Park Y.K. Kim W.K. 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Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly methylated beta-cyclodextrin as the solubility enhancer.J Agric Food Chem. 2002; 50: 1815-1821Crossref PubMed Scopus (435) Google Scholar In brief, for the DPPH reduction assay, an organic nitrogen radical generator, DPPH (23.6 μg/mL in ethanol), was incubated for 30 minutes at 37°C in the presence of test drugs at various concentrations. The de-crease in absorbance was then measured at 517 nm by a microplate reader (SPECTRAmax 340PC; Molecular Devices). The scavenging activity of free radicals was expressed as the percentage of maximum inhibition obtained from a standard curve generated by using vitamin C. For the ORAC assay, various concentrations of antioxidants react to peroxyl radicals generated from 2,2′-azobis-(2-methylpropionamide)-dihydrochloride (60 mmol/L) in a competitive manner with a fluorescent indicator, fluorescein (50 nmol/L). A fluorescence decay curve was measured every 5 minutes for 3 hours at 37°C by a fluorescence microplate reader (SpectraMax GeminiEM; Molecular Devices) using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. For quantification of scavenging capacity of the peroxyl radical, the area under the curve (AUC) was calculated based on kinetic curves: AUC = (0.5 + f1/f0 + f2/f0 + f3/f0 + … + fn-2/f0 + fn-1/f0 + fn/f0) × 5, where fi is the fluorescence reading at time i (in minutes). The net AUC = AUCsample − AUCblank. To examine putative downstream signaling molecules of TC, the amount of total and phosphorylated proteins for AMPK, CREB, or BDNF was analyzed using Western blot analysis, as previously described.40Cai Y. Cho G.S. Ju C. Wang S.L. Ryu J.H. Shin C.Y. Kim H.S. Nam K.W. Jalin A.M. Sun W. Choi I.Y. Kim W.K. 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