Edited by: Vittorio Maglione, Istituto Neurologico Mediterraneo (IRCCS), Italy
Reviewed by: Ilaria Bellezza, University of Perugia, Italy; Stefania Filosa, Istituto di Bioscienze e Biorisorse (IBBR), Italy
†These authors have equally contributed to this work.
Specialty section: This article was submitted to Neurodegeneration, a section of the journal Frontiers in Neurology
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Activated microglia secrete an array of pro-inflammatory factors, such as prostaglandins, whose accumulation contributes to neuronal damages. Prostaglandin endoperoxide synthases or cyclooxygenases (COX-1 and COX-2), which play a critical role in the inflammation, are the pharmacological targets of non-steroidal anti-inflammatory drugs, used to treat pain and inflammation. Since it was reported that COX-1 is the major player in mediating the brain inflammatory response, the aim of this study was to evaluate the effects of highly selective COX-1 inhibitors, such as P6 and mofezolac, in neuroinflammation models. Lipopolysaccharide (LPS)-activated mouse BV-2 microglial cells and LPS intracerebroventricular-injected mice as
Neuroinflammation is widely recognized as an inflammatory response originated in the central nervous system (CNS). It is a pathological condition mainly caused by the nervous tissue infiltration of host defense cells and molecules from the bloodstream. In addition, it implies a complex interplay of glia, in particular microglia, typically associated with neurological and neurodegenerative diseases, triggering several concerns from a nosological viewpoint (
Microglia are recognized as the innate immune cells of the CNS, where they mediate a number of tissue homeostatic functions, including immune surveillance (
Epidemiological data-based link between neuroinflammation and neurodegenerative diseases increased the worldwide scientific interest aimed to determine whether reducing inflammation would reverse neurodegeneration. Such data also indicate an inverse relationship between the use of traditional non-steroidal anti-inflammatory drugs (
Two COXs are known, COX-1 and COX-2. Upon inflammatory stimuli, COX-1, being constitutive in microglia, is responsible of the primary inflammatory response by inducing the production of PG, mainly PGE2. COX-2 is responsible, upon its induction, of a later and secondary response, with the exception of conditions in which the neurons are directly challenged (excitotoxicity and ischemia) (
Preliminarily, we accomplished an
In continuation of our investigations, herein, we report the validation of the study above mentioned by using both an
In particular, in this study, COX-1 role in neuroinflammation was explored by using P6 and mofezolac (Figure
Chemical structure of P6 and mofezolac. IC50 values refer to the human whole blood assay.
P6 was synthesized according to Di Nunno et al. (
Lipopolysaccharide from
BV2 microglia cells (ICLC HTL 03001-Interlab Cell Line Collection) were grown in high glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. They were maintained at 37°C in a humidified 5% CO2/95% environmental air.
Then, microglial cells were plated, at a density of 25 × 104/well in 6-well plates (Falcon) and treated with the chosen COX inhibitors (P6 and mofezolac) when they reached 80% confluence. Preliminary experiments were conducted to establish the optimal concentration and exposure times necessary for LPS (1 µg/mL) treatment, which were found to be in accordance with other reports (
Cell viability of microglial cells was quantified using the MTT reduction assay. The cells (8 × 103/well) were grown in 96-well plates (Becton Dickinson Labware) in complete medium and treated with different concentration of COX inhibitors, in presence or absence of LPS. Untreated cells were used as a control. A PBS 1× solution of MTT (5 mg/mL) was prepared and added to the cell medium at a final concentration of 0.5 mg/mL. Cells were incubated for 4 h at 37°C and 5% CO2 to allow the MTT metabolism. The formazan crystals formed (from MTT) into the cells were solubilized with DMSO (Sigma–Aldrich). The levels of MTT formazan were determined measuring the optical density at λ = 560 nm and subtracting the background (λ = 670 nm) with a Victor Multiplate Reader (Wallac). Optical density was directly correlated to cell quantity.
After treatment of cultures as previously described, cells were harvested and lysed by ice-cold lysis buffer [1% Triton X-100, 20 mM Tris–HCl, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin hemisulfate salt, and 0.2 U/mL aprotinin (all from Sigma–Aldrich)] for 30 min on an ice bath.
Substantia nigra pars compacta, hippocampus, frontal lobe, and caudate from mice brains were minced in ice-cold PBS, washed, and then homogenized in a buffer containing lysis buffer (50 mM Tris pH 8, 0.02 g/mL NaCl, 0.2% SDS, 1% Triton-X, 4 U/mL aprotinin, 2 mM leupeptin, and 100 mM phenylmethanesulfonyl fluoride).
The tissue and cell culture lysates were vortexed for 15–20 s and, then, centrifuged at 12,800 ×
After electrophoresis, the resolved proteins were transferred from the gel to nitrocellulose membranes. A blotting buffer [20 mM Tris/150 mM glycine, pH = 8.0, and 20% (v/v) methanol] was used for gel and membrane saturation and blotting. Then, membranes were incubated in the dark with the following specific primary Abs reported in reagents section for 60 min at room temperature. The membranes were washed with 0.1% Tween 20-PBS (for 20 min, three times) and then incubated with the secondary Ab diluted 1:2,000 for 60 min. Bands were visualized by chemiluminescence detection (Invitrogen, Milan, Italy). The β-actin level was used as a protein loading control. For tissue analysis, obtained bands were normalized to the level of β-actin performed for each cerebral area tested. For cell cultures, the bands were normalized to the β-actin level of each experimental condition. The bands obtained after immunoblotting were submitted to densitometric analysis using 1D Image Analysis Software (Kodak Digital Science). Results were expressed as arbitrary units.
This study was carried out in strict accordance with the European Council Directive 86/609/EEC and the Italian animal welfare legislation (art. 4 and 5 of D.L. 116/92). Seventy adult male 129S2/Sv mice (22–24 g body mass, 8–10 weeks of age) were purchased from Harlan—Italy and were kept under environmentally controlled conditions (20 ± 2°C, 50–80% humidity, 12 h light/dark cycle, food and water
Mice received either the selective COX-1 inhibitor mofezolac (6 mg/kg, i.p.; indicated as M in all the figures) or vehicle (40% DMSO in 0.1 M phosphate buffer, pH = 7.4; VM in all the figures) once a day for 10 days. Mofezolac amount to be injected was chosen taken into account previous study and its COXs IC50 values. On the seventh day, mice were anesthetized with tribromoethanol (250 mg/kg, i.p.) and positioned in a stereotactic apparatus (Kopf Instruments, Tujunga, CA, USA). Vehicle (sterile saline, 5 µL; V-LPS in all the figures) or 5 µg LPS in 5 µL of sterile saline (LPS in all the figures) was administered into the cerebral lateral ventricle using a fine needle glass syringe (Hamilton, Lyon, France) and a syringe pump (KD Scientific, Holliston, MA, USA) at a rate of 1 µL/min. This LPS dose and time point (72 h) induced the best neuroinflammatory response following a titration (24 up to 60 h) preliminary study (data not shown). Stereotaxic injections coordinates were 2.3 mm dorsal/ventral, 1.0 mm lateral, and 0.5 mm anterior/posterior from the bregma. Mofezolac was given 30 min prior to LPS injection (M + LPS in all the figures).
Mice were transcardially perfused with tris-buffered saline (pH = 7.6) followed by 4% paraformaldehyde in PBS pH = 7.4 at 4°C. Brains were subsequently postfixed in the same fixative, paraffin embedded, and 10 µm slices were obtained with a rotative microtome (Leica, Milan, Italy). Immunohistochemistry was performed following a standard avidin–biotin complex procedure. Briefly, specimens were incubated with mouse primary mAb anti-GFAP at a ratio of 1:1,000 (Merck Millipore, Milan, Italy), or a mouse mAb anti-Iba-1 at a ratio of 1:500 (Merck Millipore) overnight at 4°C and then with an anti-mouse biotinylated secondary Ab (Dako, Milan, Italy), at a 1:1,000 dilution for 1 h at room temperature. The antigen–Ab complexes were visualized by sections incubation for 1 h with extravidin peroxidase (Sigma-Aldrich) diluted 1:1,500 and 3,3′-diaminobenzidine oxidation in the presence of H2O2.
Microglial cells were cultured in 6-well plates at a density of 3 × 106 cells/well. Then, the cells were pretreated with selective COX-1 inhibitors P6 or mofezolac for 1 h and, subsequently stimulated with LPS (1 µg/mL). The cultures were maintained at 37°C for 24 and 48 h in a humidified air containing a 5% CO2. PGE2 levels were determined in the supernatant using a competitive binding immunoassay (Cayman Chemical, Ann Arbor, MI, USA) following the manufacturer’s instructions. Unstimulated cells were included as a control. PGE2 amount determination in the brain was performed in the tissue extracts, according to the manufacturer’s instructions. The optical density was measured at λ = 405–420 nm with precision microplate reader and the amount of PGE2 (ng/mL) was calculated using a PGE2 standard curve.
Student’s
MTT assay was used to quantitatively evaluate cell viability. This was performed to verify whether the tested selective COX-1 inhibitors (P6 and mofezolac) caused toxicity in LPS-treated BV2 cell line. Preliminarily, the effect of two different concentrations of P6 (0.5 and 1 µM) and mofezolac (0.1 and 0.5 µM) on BV2 microglial cell viability was evaluated. No cell toxicity was exerted by either P6, mofezolac, and LPS alone or a combination of LPS and each of the two inhibitors at 24 h. The two concentrations of P6 and mofezolac were chosen based on the basis of previous studies and their COXs IC50 values (
Cell viability was found to be significantly (
Astroglial activation was characterized by immunoreactivity and immunoblotting analysis of the GFAP expression, a marker used to distinguish astrocytes from other glial cells of the CNS. LPS treatment determined an increase of immunoreactive cell bodies in comparison to untreated mice suggesting astrocyte activation in different brain regions. In particular, the caudate, frontal lobe, hippocampus, and substantia nigra were selected to evaluate the astrocyte activation after different animal treatment (Figures
Glial fibrillary acidic protein immunoreactivity in the hippocampus
Ionized calcium-binding adapter molecule-1 immunoreactivity, a marker of activated microglia, was also evaluated (Figure
Ionized calcium-binding adapter molecule-1 immunoreactivity in the hippocampus
Immunoblotting analysis was also performed to semiquantitatively evaluate both astrocyte and microglia activation in samples derived from mice groups previously described. In LPS-treated mice, a significant increase of GFAP expression was detected in all the brain regions tested when compared to controls or vehicle-LPS (Figure
Effect of mofezolac on glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule-1 (Iba-1), cyclooxygenases (COXs), and pI
Conversely, in mofezolac-treated animals previously injected with LPS, the GFAP as well as Iba-1 levels resulted significantly reduced in comparison to the animals that received LPS alone (Figure
The effect of mofezolac and P6 on COX-1 expression in LPS-treated microglial cells was evaluated by western blotting analysis. After 24 h, no significant difference between LPS-stimulated cells in the presence or absence of both COX-1 inhibitors was observed (data not shown). Interestingly, LPS-stimulated BV2 cells exhibited, at 48 h, increased levels of COX-1 expression in comparison to untreated cells (Figure
Effects of cyclooxygenase (COX)-1 inhibitors on the COXs expression and NF-kB phosphorylation induced by lipopolysaccharide (LPS) in BV2 microglial cells. Total protein was subjected to SDS-PAGE, followed by immunoblotting using pI
The expression of COX-2 in the same cell lysates was also evaluated. Interestingly, COX-2 protein levels were unaffected by the presence of P6 or mofezolac. In these conditions, COX-2 expression resulted comparable to the level observed in cells stimulated with LPS alone (Figure
Therefore, in LPS-treated BV2 microglial cell line, P6 and mofezolac were able to reduce COX-1 expression without affecting COX-2.
Cyclooxygenases expression was also
Immunoblotting assay on tissues of the same brain areas was performed to test the
The PGE2 biosynthesis extent was evaluated in supernatants of cell cultures at 48 h incubation time (Table
(A) Effect of COX-1 inhibitors P6 and mofezolac (M) on PGE2 release (ng/mL) at 48 h in lipopolysaccharide (LPS)-stimulated BV2 microglial cells | |||||
---|---|---|---|---|---|
0.22 ± 0.025 | 0.97 ± 0.021 |
0.21 ± 0.010 |
0.12 ± 0.008 |
0.06 ± 0.004 |
0.03 ± 0.002 |
Caudate–putamen | 0.15 ± 0.050 | 0.73 ± 0.045 |
5.61 ± 0.136 |
1.84 ± 0.035 |
|
Hippocampus | 0.31 ± 0.036 | 0.69 ± 0.032 |
4.55 ± 0.046 |
2.33 ± 0.032 |
|
Frontal lobe | 0.33 ± 0.026 | 0.68 ± 0.026 |
4.60 ± 0.055 |
2.40 ± 0.046 |
|
Substantia nigra | 0.42 ± 0.021 | 0.74 ± 0.059 |
5.48 ± 0.115 |
3.31 ± 0.079 |
In the tested brain regions of LPS-treated mice, PGE2 levels were significantly increased with respect to control animals or mice receiving the vLPS (Table
Since the phosphorylation and degradation of I
Similar results were obtained in
p-I
Clinical data and basic research outcomes showed a strict correlation between neurodegeneration and neuroinflammation (
In physiological conditions, COX-1 is mainly expressed in microglia and perivascular cells, whereas COX-2 is found in postsynaptic dendrites and excitatory terminals, particularly in the cortex, hippocampus, and amygdala, with both neuronal and vascular associations. COX-1 recently has been recognized as a pivotal player in neuroinflammation (
In this regard, we previously reported that the COX-1 selective inhibitor P6 was able to control the inflammatory response in LPS-treated N13 microglial cells, an
Since the potential beneficial effect of COX-1 inhibition in the treatment of neuroinflammation has been considered, in the present study, we determined the modulatory effect of two selective COX-1 inhibitors—P6 and mofezolac—both in
More interestingly, these results were confirmed with those derived from the preclinical
In a previous work of Bosetti et al. carried out on a murine model, it was reported that evident neurodegeneration mediated by activated microglia and increased pro-inflammatory cytokines appeared in the hippocampus after LPS injection (
Hippocampus is a part of the encephalon, located at the temporal lobe, where it plays a crucial role in processing information selected for the long-term memory; therefore, it is important to check what happens at level of this brain area the neuroinflammation modulation (
Results from our experiments demonstrated that
Recently, it was reported that the potent PET probe highly selective for COX-1, [11C]-radiolabeled ketoprofen methyl ester was able to detect activated microglia associated with amyloid plaque progression, suggesting the involvement of COX-1 in the neuroinflammatory process in AD (
Our results demonstrated that both P6 and mofezolac were able to reduce COX-1-derived PGE2 release complemented with an anti-inflammatory activity at the level of the NF-kB, thus providing mechanistic insights into the suppressive effect of these COX-1 inhibitors on LPS-induced neuroinflammatory response by microglia.
In conclusion, this work consolidated the hypothesis that selective COX-1 inhibitors can positively modify the inflammatory response in LPS-induced neuroinflammatory models. Overall these results, from
This study was carried out in accordance with the recommendations of Ministero della Salute Decreto Ministeriale, Dott. Fabrizio Bertani. The protocol was approved by the Ministero della Salute Decreto Ministeriale no. 138/2014-B.
RC performed
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This work was supported by a grant from the Italian “Ministero dell’Istruzione, dell’Università e della Ricerca” (MIUR) under the project entitled “Research, Application, Innovation, Services in Bioimaging (R.A.I.S.E. in Bioimaging),” code PON01_03054. First AIRC Grant-MFAG2015 (Project Id. 17566).