J Mol Neurosci DOI 10.1007/s12031-014-0246-2
Endocannabinoid 2-Arachidonylglycerol Protects Primary Cultured Neurons Against LPS-Induced Impairments in Rat Caudate Nucleus Yongli Lu & Fang Peng & Manman Dong & Hongwei Yang
Received: 19 December 2013 / Accepted: 21 January 2014 # Springer Science+Business Media New York 2014
Abstract Inflammation plays a pivotal role in the pathogenesis of many diseases in the central nervous system. Caudate nucleus (CN), the largest nucleus in the brain, is also implicated in many neurological disorders. 2-Arachidonoylglycerol (2-AG), the most abundant endogenous cannabinoid and the true natural ligand for CB1 receptors, has been shown to exhibit neuroprotective effects through its anti-inflammatory action from proinflammatory stimuli in hippocampus. However, it is still not clear whether 2-AG is also able to protect CN neurons from proinflammation stimuli. In the present study, we discovered that 2-AG significantly protects CN neurons in culture against lipopolysaccharide (LPS)-induced inflammatory response. 2-AG is capable of suppressing elevation of LPS-induced cyclooxygenase-2 expression associated with ERK/p38MAPK/NF-κB signaling pathway in CB1 receptor-dependant manner in primary cultured CN neurons. Moreover, 2-AG inhibits LPS-induced increase in voltagegated sodium channel currents and hyperpolarizing shift of activation curves through CB1 receptor-dependant pathway. Our study suggests the therapeutic potential of 2-AG for the treatment of some inflammation-induced neurological disorders and pain.
Keywords Endocannabinoid . Caudate nucleus (CN) . 2-Arachidonoylglycerol (2-AG) . Lipopolysaccharide (LPS) . Cannabinoid receptor . Sodium channel Y. Lu : F. Peng : M. Dong : H. Yang (*) Department of Physiology, College of Medical Science, China Three Gorges University, 8 University Road, 443002 Yichang, Hubei, People’s Republic of China e-mail: [email protected]
Introduction Endocannabinoids (eCBs) are important endogenous lipid mediators capable of binding to cannabinoid (CB) receptors (Sugiura and Waku 2002; Freund et al. 2003) and some channels (Oz 2006; Pertwee 2010) to produce extensive biological effects on body. 2-Arachidonoylglycerol (2-AG), the most abundant endogenous cannabinoid and the true natural ligand for both CB1 and CB2 receptors (Sugiura et al. 2006), has been demonstrated to be involved in many physiological and pathological effects, including neuroprotective effects (Panikashvili et al. 2001; Sugiura et al. 2006). Particularly, accumulating evidence shows that 2-AG rather than arachidonoylethanolamide (or anandamide, another most well-known eCB) plays a variety of roles as a messenger molecule in the nervous system (Sugiura et al. 2006). Neuroinflammation is the inflammation in the central nervous system and has been implicated in many neurological disorders, such as Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s chorea. Recent evidence indicates that anti-inflammatory properties of eCBs are important factors in the eCB-mediated neuroprotective effect (Walter and Stella 2004; Eljaschewitsch et al. 2006). Moreover, recent researches suggest that the anti-inflammatory effect of 2-AG was associated with the suppression of the cyclooxygenase-2 (COX-2) expression via CB1 receptors in hippocampal neurons (Zhang and Chen. 2008; Chen et al. 2011). Caudate nucleus (CN), the largest nucleus in the brain, is an important component of basal ganglia. Neuroanatomical and imaging studies indicate that CN has rich and diverse anatomical connectivity with other parts of the brain (Kotz et al. 2013). CN plays a vital role in the regulation of cognition and motor function and has been implicated in several
J Mol Neurosci
neurodegenerative diseases, such as AD (Sprengelmeyer et al. 1995; Brück et al. 2001). There are abundance of the cannabinoid CB1 receptors and 2-AG in the basal ganglia (Sugiura et al. 2006). However, little is known about the evidences for the involvement of 2-AG on pro-inflammatory stimulus in CN. Voltage-gated sodium channels (VGSCs), which participate in many physiological and pathological processes, are associated with eCBs modulating the functional properties of voltage-gated ion channels (Okada 2005; Oz et al. 2006; Duan et al. 2008). Whether VGSCs are involved in the function of 2-AG in CN remains unknown. In the present study, we investigated the anti-inflammatory effects of 2-AG on lipopolysaccharide (LPS, a proinflammatory stimulus) underlying its molecular mechanism by studying primary cultured CN neurons. We discovered for the first time that 2-AG is capable of suppressing elevated COX-2 expression induced by LPS associated with extracellular signal-regulated kinase (ERK)/p38MAPK/nuclear factor-κB (NF-κB) signaling pathway and facilitating VGSC currents in response to LPS in primary CN neurons.
Materials and Methods Materials Neurobasal A, B27, 0.25 % trypsinase-EDTA solution, fetal bovine serum, and Dulbecco’s Modified Eagle’s Medium (DMEM) dry powder were obtained from Gibco BRL (Grand Island, NY, USA). LPS, AM630, poly-L-lysine, L-glutamine, L-glutamate, and cytosine arabinoside were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Antibodies for detecting nuclear factor-κB (NF-κB), phospho-p38 mitogenactivated protein kinase (p-p38MAPK), ERK1/2, and pp38MAPK were purchased from Beyotime Institute of Biotechnology (Haimen, China). COX-2 polyclonal antibody, 2AG, NS398, and Rimonabant (SR141716) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Cell Culture The methods for obtaining primary cultured caudate nucleus neurons have been described in detail elsewhere (Peng et al. 2012). Briefly, primary caudate nucleus cultures were prepared from neonatal Sprague–Dawley rats (within 24 h) which were provided by the Experimental Animal Research Center of Hubei Province. The caudate nucleuses were taken out and washed several times in a cold (4 °C) DMEM solution. After caudate nucleuses were cut to small pieces (1××1 mm) with spring scissors, they were moved into 5 mL 0.25 % trypsinEDTA sterile solution and incubated at 37 °C for 10∼15 min with 5 % CO2. Then, they were triturated with a flamed and
siliconized Pasteur pipette to separate cells gently. Subsequently, they were placed in a centrifuge tube with 5 mL Dulbecco’s modified Eagle’s medium solution containing 10 % fetal bovine serum and centrifuged two times for 5 min at 800 rpm/min. Cells were spun down and resuspended in neurobasal A/B27 medium (Gibco) supplemented with 1 mM L-glutamine, penicillin/streptomycin, and 25 μM glutamate. Cells (0.5×106∼1×106/mL) were loaded into poly-Llysine-coated 35-mm culture dishes for electrophysiological recordings and Western blot analysis. After 5 days in culture, the cells were fed with the medium supplemented with 2.5 μg/ mL cytosine arabinoside to prevent glial cell division. Medium was replaced every 3 days with the same medium without glutamate until use. Cells were used in experiments between 7 and 12 days in culture. Electrophysiological Recording Whole-cell recordings were performed at room temperature (21∼26 °C). Cultured cells in the glass cover slip were placed in a recording chamber and visualized under the phase contrast on an inverted microscope (IX-71, Olympus, Tokyo, Japan). The currents were measured with an EPC10 patchclamp amplifier (Heka Electronic, Lambrecht, Germany). Patch pipettes were prepared from glass capillary tubes (Liuhe Laboratory Apparatus Factory, Nanjing, China) using a P-97 Flaming/Brown Micropipette Puller (Sutter Instrument Co., San Rafael, CA, USA). The pipette resistances were 3∼6 MΩ after filling with the internal solution. For whole-cell recordings, the electrodes were filled with a solution containing 140 mM CsF, 8 mM NaCl, 2 mM MgCl2·6H2O, 1 mM EGTA, 10 mM HEPES, and 2 mM Na-ATP (pH 7.2 with Tris base). The bath solution for whole-cell recordings contained 120 mM NaCl, 20 mM TEA-Cl, 5 mM CsCl, 1 mM MgCl2·6H2O, 1 mM CaCl2, 0.1 mM CdCl2, 10 mM HEPES, and 10 mM glucose (pH 7.3∼7.4 with NaOH). The adjustment of capacitance compensations and series resistance compensations were done before recording the membrane currents. Currents were filtered at 3 kHz and sampled at 10 kHz. Current recordings were obtained on a PC with the PatchMaster software (Heka Electronic, Lambrecht, Germany). For current density measurements, the currents were divided by the cell capacitance which was read from the PatchMaster software. Western Blot CN neurons in cultures were extracted and immediately homogenized in a one-to-one volume of modified radioimmune precipitation assay lysis buffer consisting of a number of protease inhibitors. SDS–polyacrylamide gel electrophoresis (PAGE) was carried out on 5 % stacking and 12 % resolving gel with low range molecular weight standards (Solarbio,
J Mol Neurosci
Beijing, China). Supernatants were fractionated on SDSPAGE and transferred onto polyvinylidene difluoride membranes (Beyotime, Haimen, China). The membrane was incubated with ERKl/2 and phospho-ERKl/2, p38 and phosphop38MAPK, NF-κB65 and phospho-NF-κB antibodies (dilution of 1:1,000, Beyotime, Haimen, China), and COX-2 polyclonal antibodies (dilution of 1:1,000, Cayman, Ann Arbor, MI, USA) at 4 °C overnight. The blot was washed and incubated with a secondary antibody (goat anti-rabbit/mouse 1:2,000, Beyotime, Haimen, China) at room temperature for 1.5 h. Proteins were visualized by enhanced chemiluminescence (Beyotime, Haimen, China). The densities of specific bands were quantified by densitometry using ImageJ 1.46i software. Band densities were normalized to the total amount of protein loaded in each well as determined by mouse anti-βactin (1:4,000, Beyotime, Haimen, China). Data Analysis All values are presented as mean ± SEM unless stated otherwise. Student’s t test and analysis of variance with Student– Newman–Keuls test were used for statistical comparison when appropriate. Differences were considered significant when P
Endocannabinoid 2-Arachidonylglycerol Protects Primary Cultured Neurons Against LPS-Induced Impairments in Rat Caudate Nucleus Yongli Lu & Fang Peng & Manman Dong & Hongwei Yang
Received: 19 December 2013 / Accepted: 21 January 2014 # Springer Science+Business Media New York 2014
Abstract Inflammation plays a pivotal role in the pathogenesis of many diseases in the central nervous system. Caudate nucleus (CN), the largest nucleus in the brain, is also implicated in many neurological disorders. 2-Arachidonoylglycerol (2-AG), the most abundant endogenous cannabinoid and the true natural ligand for CB1 receptors, has been shown to exhibit neuroprotective effects through its anti-inflammatory action from proinflammatory stimuli in hippocampus. However, it is still not clear whether 2-AG is also able to protect CN neurons from proinflammation stimuli. In the present study, we discovered that 2-AG significantly protects CN neurons in culture against lipopolysaccharide (LPS)-induced inflammatory response. 2-AG is capable of suppressing elevation of LPS-induced cyclooxygenase-2 expression associated with ERK/p38MAPK/NF-κB signaling pathway in CB1 receptor-dependant manner in primary cultured CN neurons. Moreover, 2-AG inhibits LPS-induced increase in voltagegated sodium channel currents and hyperpolarizing shift of activation curves through CB1 receptor-dependant pathway. Our study suggests the therapeutic potential of 2-AG for the treatment of some inflammation-induced neurological disorders and pain.
Keywords Endocannabinoid . Caudate nucleus (CN) . 2-Arachidonoylglycerol (2-AG) . Lipopolysaccharide (LPS) . Cannabinoid receptor . Sodium channel Y. Lu : F. Peng : M. Dong : H. Yang (*) Department of Physiology, College of Medical Science, China Three Gorges University, 8 University Road, 443002 Yichang, Hubei, People’s Republic of China e-mail: [email protected]
Introduction Endocannabinoids (eCBs) are important endogenous lipid mediators capable of binding to cannabinoid (CB) receptors (Sugiura and Waku 2002; Freund et al. 2003) and some channels (Oz 2006; Pertwee 2010) to produce extensive biological effects on body. 2-Arachidonoylglycerol (2-AG), the most abundant endogenous cannabinoid and the true natural ligand for both CB1 and CB2 receptors (Sugiura et al. 2006), has been demonstrated to be involved in many physiological and pathological effects, including neuroprotective effects (Panikashvili et al. 2001; Sugiura et al. 2006). Particularly, accumulating evidence shows that 2-AG rather than arachidonoylethanolamide (or anandamide, another most well-known eCB) plays a variety of roles as a messenger molecule in the nervous system (Sugiura et al. 2006). Neuroinflammation is the inflammation in the central nervous system and has been implicated in many neurological disorders, such as Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s chorea. Recent evidence indicates that anti-inflammatory properties of eCBs are important factors in the eCB-mediated neuroprotective effect (Walter and Stella 2004; Eljaschewitsch et al. 2006). Moreover, recent researches suggest that the anti-inflammatory effect of 2-AG was associated with the suppression of the cyclooxygenase-2 (COX-2) expression via CB1 receptors in hippocampal neurons (Zhang and Chen. 2008; Chen et al. 2011). Caudate nucleus (CN), the largest nucleus in the brain, is an important component of basal ganglia. Neuroanatomical and imaging studies indicate that CN has rich and diverse anatomical connectivity with other parts of the brain (Kotz et al. 2013). CN plays a vital role in the regulation of cognition and motor function and has been implicated in several
J Mol Neurosci
neurodegenerative diseases, such as AD (Sprengelmeyer et al. 1995; Brück et al. 2001). There are abundance of the cannabinoid CB1 receptors and 2-AG in the basal ganglia (Sugiura et al. 2006). However, little is known about the evidences for the involvement of 2-AG on pro-inflammatory stimulus in CN. Voltage-gated sodium channels (VGSCs), which participate in many physiological and pathological processes, are associated with eCBs modulating the functional properties of voltage-gated ion channels (Okada 2005; Oz et al. 2006; Duan et al. 2008). Whether VGSCs are involved in the function of 2-AG in CN remains unknown. In the present study, we investigated the anti-inflammatory effects of 2-AG on lipopolysaccharide (LPS, a proinflammatory stimulus) underlying its molecular mechanism by studying primary cultured CN neurons. We discovered for the first time that 2-AG is capable of suppressing elevated COX-2 expression induced by LPS associated with extracellular signal-regulated kinase (ERK)/p38MAPK/nuclear factor-κB (NF-κB) signaling pathway and facilitating VGSC currents in response to LPS in primary CN neurons.
Materials and Methods Materials Neurobasal A, B27, 0.25 % trypsinase-EDTA solution, fetal bovine serum, and Dulbecco’s Modified Eagle’s Medium (DMEM) dry powder were obtained from Gibco BRL (Grand Island, NY, USA). LPS, AM630, poly-L-lysine, L-glutamine, L-glutamate, and cytosine arabinoside were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Antibodies for detecting nuclear factor-κB (NF-κB), phospho-p38 mitogenactivated protein kinase (p-p38MAPK), ERK1/2, and pp38MAPK were purchased from Beyotime Institute of Biotechnology (Haimen, China). COX-2 polyclonal antibody, 2AG, NS398, and Rimonabant (SR141716) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Cell Culture The methods for obtaining primary cultured caudate nucleus neurons have been described in detail elsewhere (Peng et al. 2012). Briefly, primary caudate nucleus cultures were prepared from neonatal Sprague–Dawley rats (within 24 h) which were provided by the Experimental Animal Research Center of Hubei Province. The caudate nucleuses were taken out and washed several times in a cold (4 °C) DMEM solution. After caudate nucleuses were cut to small pieces (1××1 mm) with spring scissors, they were moved into 5 mL 0.25 % trypsinEDTA sterile solution and incubated at 37 °C for 10∼15 min with 5 % CO2. Then, they were triturated with a flamed and
siliconized Pasteur pipette to separate cells gently. Subsequently, they were placed in a centrifuge tube with 5 mL Dulbecco’s modified Eagle’s medium solution containing 10 % fetal bovine serum and centrifuged two times for 5 min at 800 rpm/min. Cells were spun down and resuspended in neurobasal A/B27 medium (Gibco) supplemented with 1 mM L-glutamine, penicillin/streptomycin, and 25 μM glutamate. Cells (0.5×106∼1×106/mL) were loaded into poly-Llysine-coated 35-mm culture dishes for electrophysiological recordings and Western blot analysis. After 5 days in culture, the cells were fed with the medium supplemented with 2.5 μg/ mL cytosine arabinoside to prevent glial cell division. Medium was replaced every 3 days with the same medium without glutamate until use. Cells were used in experiments between 7 and 12 days in culture. Electrophysiological Recording Whole-cell recordings were performed at room temperature (21∼26 °C). Cultured cells in the glass cover slip were placed in a recording chamber and visualized under the phase contrast on an inverted microscope (IX-71, Olympus, Tokyo, Japan). The currents were measured with an EPC10 patchclamp amplifier (Heka Electronic, Lambrecht, Germany). Patch pipettes were prepared from glass capillary tubes (Liuhe Laboratory Apparatus Factory, Nanjing, China) using a P-97 Flaming/Brown Micropipette Puller (Sutter Instrument Co., San Rafael, CA, USA). The pipette resistances were 3∼6 MΩ after filling with the internal solution. For whole-cell recordings, the electrodes were filled with a solution containing 140 mM CsF, 8 mM NaCl, 2 mM MgCl2·6H2O, 1 mM EGTA, 10 mM HEPES, and 2 mM Na-ATP (pH 7.2 with Tris base). The bath solution for whole-cell recordings contained 120 mM NaCl, 20 mM TEA-Cl, 5 mM CsCl, 1 mM MgCl2·6H2O, 1 mM CaCl2, 0.1 mM CdCl2, 10 mM HEPES, and 10 mM glucose (pH 7.3∼7.4 with NaOH). The adjustment of capacitance compensations and series resistance compensations were done before recording the membrane currents. Currents were filtered at 3 kHz and sampled at 10 kHz. Current recordings were obtained on a PC with the PatchMaster software (Heka Electronic, Lambrecht, Germany). For current density measurements, the currents were divided by the cell capacitance which was read from the PatchMaster software. Western Blot CN neurons in cultures were extracted and immediately homogenized in a one-to-one volume of modified radioimmune precipitation assay lysis buffer consisting of a number of protease inhibitors. SDS–polyacrylamide gel electrophoresis (PAGE) was carried out on 5 % stacking and 12 % resolving gel with low range molecular weight standards (Solarbio,
J Mol Neurosci
Beijing, China). Supernatants were fractionated on SDSPAGE and transferred onto polyvinylidene difluoride membranes (Beyotime, Haimen, China). The membrane was incubated with ERKl/2 and phospho-ERKl/2, p38 and phosphop38MAPK, NF-κB65 and phospho-NF-κB antibodies (dilution of 1:1,000, Beyotime, Haimen, China), and COX-2 polyclonal antibodies (dilution of 1:1,000, Cayman, Ann Arbor, MI, USA) at 4 °C overnight. The blot was washed and incubated with a secondary antibody (goat anti-rabbit/mouse 1:2,000, Beyotime, Haimen, China) at room temperature for 1.5 h. Proteins were visualized by enhanced chemiluminescence (Beyotime, Haimen, China). The densities of specific bands were quantified by densitometry using ImageJ 1.46i software. Band densities were normalized to the total amount of protein loaded in each well as determined by mouse anti-βactin (1:4,000, Beyotime, Haimen, China). Data Analysis All values are presented as mean ± SEM unless stated otherwise. Student’s t test and analysis of variance with Student– Newman–Keuls test were used for statistical comparison when appropriate. Differences were considered significant when P
