Background
Astrocytes are the main glial cells in the central nerve system (CNS) where they play a central role in neurotrophic support, regulation of the concentration of extracellular ions, antioxidant defense, and neurotransmitter metabolism [
1]. In astrocytes, glutamine synthetase (GS), an ATP-dependent enzyme, could modulate the extracellular level of glutamate, an essential but neurotoxic excitatory neurotransmitter, by converting glutamate to nontoxic amino acid glutamine [
2]. Changes in GS expression have been identified in a number of neurological disorders, including traumatic brain injury, neurodegenerative diseases, and various models of nociceptive pain, while modulating GS expression could diminish relative clinical features [
3‐
8]. Endocannabinoids are endogenous lipid signaling mediators capable of modulating synaptic function and producing neuroprotection and anti-inflammation [
9,
10]. 2-arachidonoyl glycerol (2-AG), the most abundant endocannabinoid, has been illustrated to play a significant role in protecting neurocytes from inflammatory injuries, such as stimuli of interleukine-1 beta (IL-1β), lipopolysaccharide (LPS), and β-amyloid in models of neurodegenerative diseases [
11,
12]. Besides, 2-AG has been shown to have the ability of controlling neuropathic pain and mechanical hyperalgesia in several preclinical models of chronic pain [
13‐
16]. Although the role of 2-AG has been extensively investigated, it is not clear whether 2-AG could directly modulate the GS expression in astrocytes, and the exact molecular mechanism remains unknown.
The mitogen-activated protein kinase (MAPK) cascades are a family of serine/threonine kinases that can mediate a wide variety of extracellular stimuli into the cytoplasm and nuclei and regulate cellular gene expression and protein synthesis [
17]. Previous studies have indicated that MAPK members, extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 pathways, in astrocytes may be involved in the process of various neurological disorders [
18]. Accumulating evidence showed that activation (phosphorylation) of ERK1/2 and p38 in spinal astrocytes under different persistent pain conditions results in the generation and maintenance of pain hypersensitivity via distinct molecular and cellular mechanisms [
19]. In addition, activation of ERK1/2 and p38 in cortical astrocytes has been identified in several neurodegenerative diseases, while blockade of ERK1/2 and p38 pathways has been shown to alleviate inflammation and clinical features in different animal models [
20‐
22].
It has been verified that 2-AG could modulate synaptic function, produce neuroprotection, and stimulate MAPK family by bonding to and activating two receptors, CB
1R and CB
2R, which are two G-protein-coupled receptors and have been grossly identified in astrocytes [
23]. Previous studies have shown that activation of CB
1R or CB
2R results in anti-inflammation, prevention of neurodegeneration, and inhibition of nociceptive signaling pathways [
24‐
26], as well as stimulation of the MAPK cascade [
27‐
29]. Interestingly, activation of CB
1R or CB
2R also could inhibit stress-induced activation of the MAPK cascade. Based on these findings, it is proposed that 2-AG may exert neuroprotection and analgesia via activating CB
1R or CB
2R and regulating activation of the MAPK cascade.
Methods
Primary astrocyte cultures
Primary astrocytes from the cerebral cortex of neonatal Sprague–Dawley rats (postnatal 1~3 days) were cultured as described preciously [
30]. The neonatal Sprague–Dawley rats were provided by the Experimental Animal Center of Gansu University of Chinese Medicine, China. All efforts were performed to minimize the number of neonatal rats used and their suffering. The procedures were approved by the Animal Care and the Ethic Committee of Animal Usage of Lanzhou University Second Hospital. Briefly, the newborn rats were decapitated and the cerebral hemispheres were aseptically removed into HBSS (Hank’s Balanced Salt Solution). After removal of the meninges, the cerebral cortices were cut into small pieces, digested with 0.25% Trypsin-EDTA (Gibco Life Technology, CA, USA), mechanically dissociated by gentle pipetting with Pasteur pipette, and then centrifuged at 400
g for 5 min. The cells were resuspended in complete culture medium containing 90% DMEM/F12 (Gibco Life Technology, CA, USA) and 10% FBS (PAN-Biotech, Germany), and plated at a density of 3~5 × 10
5 cells/cm
2 in 25-cm
2 flasks. Cells in flasks were cultured at 37 °C in CO
2 incubator for 5~7 days to reach the first confluence. To obtain quite pure astrocytes (more than 95%), the confluent cultures in flasks were shaken at 200 rpm overnight to diminish microglia contamination. Afterward, the astrocytes were evenly passaged into 35-mm dishes for western blot analysis, on coverslips pre-coated with poly-
l-lysine for immunocytochemistry analysis, and into 96-well plates for methyl thiazolyl tetrazolium (MTT) analysis after different treatments. Astrocytes were cultured with serum-free medium for 6 h before different treatments.
Drugs treatment
All drugs were dissolved and/or diluted with serum-free DMEM/F12 into final concentration. To study the effect of 2-AG, astrocytes were incubated with 0.01 μM 2-AG for 2 h and then stimulated with 1 μg/ml LPS. To study the roles of ERK1/2 and p38 in LPS-induced inflammation, astrocytes were pretreated with PD98059 or SB203580 for 1 h before LPS exposure. To study the roles of CB1R and CB2R on effects of 2-AG, astrocytes were pretreated with 1 μM AM281 or AM630 for 1 h before the treatment of 2-AG and LPS.
Hoechst 33342 staining
After various treatments, astrocytes were stained with Hoechst 33342 kit (St. Louis, MO, USA) and counted blindly as described previously [
31]. Briefly, astrocytes on coverslips were rinsed with PBS and fixed with 4% paraformaldehyde for 30 min. After being rinsed three times with PBS, astrocytes were incubated with 0.4% Triton X-100 for 20 min, and then stained with Hoechst 33342 for 10 min in the dark. After washing cells with PBS, the nuclear morphological changes of apoptosis were observed using a fluorescence microscope (Olympus, Japan). Astrocytes with bright staining, highly condensed and fragmented nuclei were defined as apoptotic cells. The number of apoptotic cells and total cells were counted, and then the cell apoptosis rate was calculated by the following equation:
$$ \mathrm{Cell}\ \mathrm{apoptosis}\ \mathrm{rate}\ \left(\%\right)=\left({N}_{\mathrm{apoptotic}\ \mathrm{cells}}/{N}_{\mathrm{total}\ \mathrm{cells}}\right)\times 100 $$
Methyl thiazolyl tetrazolium (MTT) assay
Cell viability was detected using 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium (MTT) assay as described previously [
31]. In brief, astrocytes were cultured in 96-well plates at a density of 3 × 10
4 cells/well. After different treatments, astrocytes were incubated with 1 mg/ml substrate MTT at 37 °C for 4 h. Then the culture medium was replaced by 100 μl dimethyl sulfoxide (DMSO) to dissolve the formazan crystals. The amount of formazan was measured at 570 nm using a Universal Microplate Reader (Elx 800; Bio-TEK instruments, Winooski, VT, USA). The cell viability was expressed as a percentage of viable cells in treated groups versus a control group using the following formula:
$$ \mathrm{Cell}\ \mathrm{viability}\ \left(\%\right)=\left({\mathrm{Opticaldensity}}_{\mathrm{treatment}}/{\mathrm{Opticaldensity}}_{\mathrm{control}}\right)\times 100 $$
Protein isolation and western blotting
Western blotting was performed according to a previous report [
32]. Briefly, astrocytes in 35-mm dishes were lysed with 100 μl radioimmunoprecipitation assay (RIPA) lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF) after different treatments. The lysates were centrifuged at 12,000 rpm for 10 min to clear cell debris and further diluted with 30 μl sample buffer. Total protein in lysates was loaded onto 10% SDS-polyacrylamide gels at 5–20 μg per lane (as measured by BCA), then separated by electrophoresis and transferred to PVDF membranes. Following the blockade of nonspecific binding sites with 5% non-fat milk in Tris-buffered saline with Tween-20 (TBST) for 2 h at room temperature (RT), the membranes were incubated overnight at 4 °C with primary antibodies according to the manufacturer’s instruction (anti-MAPK antibody, 1:1000, Cell Signaling Technology, MA, USA; anti-GS antibody, 1:10,000, St. Louis, MO, USA) and then washed extensively with TBST three times at 10-min intervals and incubated with appropriate second antibodies (1:10,000; Danvers, MA, USA) at RT for 2 h. The membranes were washed three times with TBST at 10-min intervals, and immunolabeled protein bands on membranes were detected using an enhanced chemiluminescence kit.
Immunocytochemistry
After different treatments, the astrocytes on coverslips were fixed with 4% paraformaldehyde for 30 min and washed with PBS. The fixed cells were permeabilized with 0.4% TritonX-100 for 20 min, washed again with PBS, blocked with 3% normal goat serum for 30 min, and then incubated with different primary antibodies (GS, 1:5000; ERK1/2, 1:500; p38, 1:500) overnight at 4 °C, respectively. After 24 h, the coverslips were washed and incubated with appropriate second antibodies (Invitrogen, UK) conjugated with Alexa Fluor® 488 (green staining) or 594 (red staining) for 2 h at RT. All cells were stained with DAPI for nuclei observation, and cells were visualized by an immunofluorescence microscope (Olympus, Japan).
Statistics analysis
All experiments were carried out in triplicate and repeated at least three times, and all measurements were performed by blinded evaluators. The data were expressed as mean ± standard deviation (SD), and STATA software (version 14.2, Stata Corp, College Station, TX, USA) was used for statistical analysis. One-way ANOVA followed by Neuman Keuls test was mainly performed, and two-way ANOVA followed by Dennett test was performed in Fig.
4;
p < 0.05 was set as the level of significant difference.
Discussion
GS plays a key role through preventing the excessive accumulation of ammonia and glutamate in synaptic surroundings, and thereby suppressing the development of glutamate/ammonia neurotoxicity [
2,
34,
35]. Accumulating evidence demonstrated that alterations of GS expression in astrocytes are involved in a number of neurological disorders, including neuropathic pain [
3,
5,
7], inflammatory pain [
36], Alzheimer’s disease, and Parkinson’s disease [
6,
8]. Intriguingly, some diseases, such as hepatic encephalopathy, traumatic brain injury, and epilepsy, demonstrated both increase and decrease of GS while controlling GS expression can alleviate these diseases [
4]. Consistently, the present study confirmed a biphasic change of GS expression in astrocytes after treatment with LPS. In addition, the present study found that LPS exposure induced increased of apoptosis and decrease of cell viability in the late stage of LPS exposure. These studies suggest that LPS induces a dynamic change of GS expression in astrocytes following the prolongation of LPS exposure time, which could modulate the survival of astrocytes. An improved understanding of the exact role of GS and mechanism in these neurological disorders and its involvement in pathology of diseases could enable the identification of innovative drugs to treat these diseases.
MAPK signaling, including p38 and ERK1/2, is a family of kinases involved in multiple physiological and pathological processes, including pain and neurodegenerative diseases [
17‐
19]. In addition to neurons, MAPK signaling also exists in astrocytes and is activated under pathological stimulation [
20‐
22]. The present study indicated that p38 and ERK1/2 were activated and translocated into nucleus in astrocytes by LPS exposure with different patterns, i.e., p38 was firstly activated at 30 min and reached peak at 2 h while ERK1/2 activated at 1 h and reached peak at 3 h, while inhibition of p38 and ERK1/2 attenuated the changes of GS expression induced by LPS. These results suggested that there is a sequential activation of MPAK signaling in astrocytes, which plays an important role in modulating the GS expression. In addition, the present results were also consistent with previous study indicating that MAPK signaling is involved in the LPS-induced changes of GS in astrocytes [
21].
2-AG is an endocannabinoid playing its roles in binding to CB
1R and CB
2R which are found to be expressed in astrocytes [
23]. The present study indicated that 2-AG could modulate the GS expression and MAPK activation induced by LPS exposure through different CB receptors. Pascal et al. observed that 2-AG could activate p38 while blockade of CB
1R could inhibit the effect of 2-AG on p38 [
27], suggesting that 2-AG could modulate MAPK signaling in astrocytes. The results of present study indicated that, under the condition of the early phase of LPS exposure, activated p38 could translocate into nucleus, resulting in the increase of GS expression, and 2-AG could suppress the increase of GS by inhibiting the phosphorylation level and translocation of p38. While under the condition of late LPS exposure, activated ERK1/2 translocated into nucleus resulting in decrease of GS expression and 2-AG reversed the decrease of GS expression through reducing the activation and translocation of ERK1/2. It should be noted that although ERK1/2 and p38 were activated by early LPS exposure and late LPS exposure, respectively, the activation was relatively weaker when compared to late and early LPS exposure, respectively. These results indicated that 2-AG modulates the astrocyte survival by modulating p38 and ERK1/2 activation through different CB receptors, which is supported by the results that 2-AG could prevent the apoptosis of astrocytes induced by LPS exposure.
Acknowledgements
We thank Dr. Shengjun Fu, Dr. Jianzhong Lu, and Dr. Yan Tao from Key Laboratory of Urological Disease of Gansu Province for the equipment support.
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