[8,9]. However, various studies have shown the involve-ment of alpha(7)-nicotinic acetylcholine receptor ( α 7-nAChR) in the modulation of neuroinfl ammation associated to the neurodegenerative diseases but the expression of α 7-nicotinic receptor associated to the astroglial cells activation is not properly known [8,9]. However, accumulating evidences suggest that astroglial activation may be the key response for neurodegeneration but the exact mechanism of action in deleterious events ultimately leading to neuronal dysfunctions remains elu-sive and is of great importance towards development of noble therapeutics.

Therefore, the present study was undertaken primarily, to investigate the mechanism of action of melatonin on LPS-induced neuroinfl ammatory cascade of events asso-ciated to the nitrative and oxidative stress, intracellular Ca 2� ion level, mRNA expression profi le of pro-infl am-matory cytokine genes, expression profi le of glial fi bril-lary acidic protein (GFAP), cyclooxygenase-2 (COX-2), P-p38, inducible nitric oxide synthase (iNOS), C/EBP homologous protein 10 (CHOP) proteins and nuclear fac-tor kappa-B (NF- κ B) translocation in rat astrocytoma C6, cells.

Melatonin attenuated mediators of neuroinfl ammation and alpha-7 nicotinic acetylcholine receptor mRNA expression in lipopolysaccharide (LPS) stimulated rat astrocytoma cells, C6 Rituraj Niranjan , Chandishwar Nath & Rakesh Shukla

Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow, India

Abstract Melatonin has been known to aff ect a variety of astrocytes functions in many neurological disorders but its mechanism of action on neuroin-fl ammatory cascade and alpha-7 nicotinic acetylcholine receptor ( α 7-nAChR) expression are still not properly understood. Present study demonstrated that treatment of C6 cells with melatonin for 24 hours signifi cantly decreased lipopolysaccharide (LPS) induced nitrative and oxidative stress, expressions of cyclooxigenase-2 (COX-2), inducible nitric-oxide synthase (iNOS) and glial fi brillary acidic protein (GFAP). Melatonin also modulated LPS-induced mRNA expressions of α 7-nAChR and infl ammatory cytokine genes. Furthermore, mela-tonin reversed LPS-induced changes in C/EBP homologous protein 10 (CHOP), microsomal prostaglandin E synthase-1(mPGES-1) and phosphorylated p38 mitogen activated protein kinase (P-p38). Treatment with pyrrolidine dithiocarbamate (PDTC) inhibited α 7-nAChR mRNA expression in LPS-induced C6 cells. Our fi ndings explored anti-neuroinfl ammatory action of melatonin, which may suggests its benefi cial roles in the neuroinfl ammation associated disorders.

Keywords: Neuroinfl ammation , rat astrocytoma cells (C6) , alpha-7 nicotinic acetylcholine receptor , NF- κ B and CHOP activation , melatonin

Abbreviations: ROS , reactive oxygen species; TNF- α , tumour necrosis factor- α NF- κ B , nuclear factor κ -B COX-2 , cyclooxygenase-2; GFAP , glial fi brillary acidic protein; CHOP , C/EBP homologous protein 10; DCF-DA , dichlorofl uorescein diacetate; iNOS , inducible nitric oxide synthase; MDA , melondialdehyde; IL-1 α , interleukin-1 α , IL-1β , interleukin-1 β ; IL-6 , interleukin-6; Mela , melatonin; P-p38 MAPK , phosphorylated p38 mitogen activated protein kinase; NO , nitric oxide; α 7-nAChR , alpha(7)-nicotinic acetylcholine receptor; PDTC , pyrrolidine dithiocarbamate; mPGES-1 , microsomal prostaglandin E synthase-1

Introduction

Melatonin (a neurohormone), a good antioxidant and anti-infl ammatory agent, has shown benefi cial eff ect in various CNS related disorders [1,2]. Melatonin ( N -acetyl-5-meth-oxytryptamine) is an indolamine in nature and mostly produced by the pineal gland during the dark phase [3]. However, several reports insist on the neuroprotective ability of the melatonin in brain related disorders but the eff ect of melatonin in the cascade of events associated to the LPS induced neuroinfl ammatory signal transduction pathway particularly in astrocytes is still less clearly explored [4,5].

Although the neuroinfl ammation is an integral part of pathophysiology of neurodegenerative disease and other CNS disorders but the exact mechanism of action may be diff erent in each disease resulting in the specifi c neuronal dysfunction [6]. In brain, LPS induces reactive gliosis, the cellular manifestation of neuroinfl ammation in response to neuronal insult, but the mechanism associated to the astroglial functions is still poorly understood [7]. Nicotinic anti-infl ammatory pathway is one of the main pathways involved in the regulation of neuroinfl ammation

Correspondence: Dr. Rakesh Shukla, Sc.-F, Division of Pharmacology, Central Drug Research Institute, Lucknow, U.P., India. Tel: � 91 – 522-2612411-18, ext 4420. Fax: � 91 – 522-2623405. E-mail: rakeshshukla_cdri@rediff mail.com

(Received date: 15 April 2012 ; Accepted 23 May 2012 ; Published online: 18 June 2012 )

Free Radical Research, September 2012; 46(9): 1167–1177© 2012 Informa UK, Ltd.ISSN 1071-5762 print/ISSN 1029-2470 onlineDOI: 10.3109/10715762.2012.697626

ORIGINAL ARTICLE

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Materials and methods

Materials

Primary antibodies, rabbit polyclonal specifi c for anti-COX-2, anti-NF- κ B (p-65), anti-iNOS, anti-CHOP, aniti-P-p38, anti-mPGES-1, anti- β -actin and goat anti-rabbit HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. Secondary goat anti-rabbit fl uorescent-conjugated (Alexa-fl uor-546) antibody was purchased from Invitrogen (CA, USA). RT-PCR (two steps) kit was purchased from Fermentas and primers spe-cifi c for tumour necrosis factor- α (TNF- α ), GAPDH, interleukin (IL)-1 α , IL-1 β and IL-6 were purchased from Metabion International AG (Hamburg, Germany). Rabbit polyclonal anti-GFAP and all other chemicals were pur-chased from Sigma – Aldrich (St. Louis, MO, USA). LPS (Sigma – Aldrich) and melatonin (Sigma – Aldrich) were dissolved in sterile water and subsequent dilutions were made in medium as per the required concentrations. Pyr-rolidine dithiocarbamate (PDTC), a nuclear factor kappa B (NF- κ B inhibitor) (Sigma – Aldrich) was dissolved in the PBS and subsequent dilutions were made in the medium.

Cell culture and treatment

Rat astrocytoma cell line (C6) was initially procured from National Centre for Cell Sciences, Pune, India, and main-tained in CDRI tissue culture facility. C6 cells were cul-tured in DMEM nutrient mixture medium supplemented with 10% heat-inactivated FBS (fetal bovine serum), at 37C in a humidifi ed atmosphere of 5% CO

2 [10]. C6 cells

were incubated with LPS in the absence or presence of melatonin and melatonin alone for 24 hours. Measure-ments for nitrite, MDA formation, ROS generation, intra-cellular glutathione (GSH), Ca 2� ion level and NF- κ B translocation were done. Measurements for expressions of mRNA of pro-infl ammatory genes (TNF- α , IL-1 α , IL-1 β and IL-6) and protein expressions of COX-2, microsomal prostaglandin E synthase-1 (mPGES-1), GFAP, iNOS, CHOP and phosphorylated p38 mitogen activated protein kinase (P-p38 MAPK) were also done in respective groups.

Nitrite estimation by Griess reagent

Estimations of nitrite in culture supernatant from diff erent groups were done by the Griess reagent [11,12]. C6 cells, at the cell density of 1 � 10 4 cells/well, were seeded in 96 well plates. After the incubation, an equal amount of culture supernatant (100 μ l) from treated groups were mixed with equal amount (100 μ l) of Griess reagent (1% p-amino-benzene sulphonamide, 0.01% naphthylethylene-diamide in 2.5% phosphoric acid) and were incubated for a period of 20 minutes in dark. Absorbance was read at 570 nm. Nitrite release was expressed as per cent increase from basal level.

Measurement of reactive oxygen species

Reactive oxygen species (ROS) generation was measured by fl uorescence dye dichlorofl uorescein diacetate (DCF-DA) [13]. C6 cells, at the cell density of 1 � 10 4 cells/well in the 96 well, were seeded plates. After treat-ment medium was aspirated and DCF-DA 100 μ l/well (10 μ M fi nal concentrations) in phenol red free HBSS buff er was added to wells containing cells. An incubation period of 30 minutes in dark at 37°C was given to the cells in CO

2 incubator. Fluorescence was read at wave-

length excitation � 485 nm and emission at � 530 nm, by micro plate fl uorescence reader (using Cary Eclipse soft-ware, VARIAN Optical Spectroscopy Instruments, Aus-tralia). ROS generation was expressed as per cent increase from control.

Melondialdehyde estimation

The level of melondialdehyde (MDA) in C6 cells was estimated according to the method of Colado [12,14]. C6 cells, at the cell density of 4 � 10 5 cells/well, were seeded in six well plates and were left for 24 hours for the proper attachment. After incubation, cells were scraped in 100 μ l of the sodium phosphate buff er (pH 7.0). Lysis of cells was done by sonication (a pulse of 30 seconds for each) and then centrifuged at 10,000g for 5 minutes. Pellet was then discarded and supernatant was collected. In 100 μ l of supernatant 60 μ l of TCA (trichlo-roacetic acid) and 30 μ l of 5 N HCl was added. After 5 minutes of incubation, 30 μ l of TBA (thiobarbituric acid) in 1 N NaOH was added and then samples were heated at 90°C. After heating samples were again centrifuged at 10,000 RPM for 10 minutes. Supernatants were then transferred to a micro plate and absorbance was mea-sured at 532 nm. The level of MDA was expressed as per cent increase in basal.

Glutathione estimation

Level of reduced glutathione (GSH) was accomplished by the method of Anderson with micro plate modifi ca-tion [12,15]. C6 cells, at the cell density of 4 � 10 5 cells/well, were seeded in six well plates. Following treat-ments, cells were scraped in 100 μ l of PBS (ice cold). After lysis of C6 cells (by ultrasonication), lysate was then centrifuged at 10,000g for 5 minutes at 4°C. Col-lected cell supernatants were then deproteinised by add-ing pre-cooled 10% trichloroacetic acid (100 μ l) with an incubation period of 1 hour at 4°C. Samples were re-centrifuged at 5000g for 5 minutes. Supernatants were collected and pellets were then discarded. Supernatants (75 μ l) were then mixed with 25 μ l of the distilled water � 100 μ l of buff er (0.25 M of Tris base � 20 mM EDTA) � 50 μ l of DTNB (0.1%). After 10 minutes of incubation, OD was read at 412 nm. Results were presented as relative GSH level compared to control.

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The eff ect of melatonin on LPS stimulated α 7-nAChR activation 1169

Estimation of intracellular calcium (Ca 2� ) ion by FURA-2AM

Measurement of intracellular calcium level was accom-plished by fl uorescence probe FURA-2 AM following the method of Grynkiewicz [16]. Rat astrocytoma cells, C6, at the cell density of 100,000 cells/ml in 96 well plates were seeded. After treatment for 24 hours, medium was aspirated and cells were incubated with HBSS buff er (137 mM NaCl, 5.4 mM KCl, 0.49 mM MgCl

2 , 0.44 mM

KH 2 PO

4 , 0.64 mM Na

2 HPO

4 , 3 mM NaHCO

3 , 5.5 mM

glucose,1.26 mM CaCl 2 , 20 mM HEPES) for 1 hour at

37°C in incubator containing 5 μ M FURA-2AM as a fi nal concentration. HBSS buff er was removed from wells and replaced with the fresh HBSS buff er. Fluorescence was measured using a spectrofl uoremeter (using Cary Eclipse software, VARIAN optical spectroscopy instruments, Australia) at dual wave length ex � 340/380 nm and em � 510 nm. The level of calcium was calculated according to Grynkiewicz et al [16]. Calcium infl ux was expressed as per cent of control.

Immunocytochemistry for NF- k B translocation

A batch of C6 cells was harvested and then cells were seeded at the cell density of 2 10 5 /well in six well plates [17]. Cover slips at the bottom of wells were placed prior to seeding of cells. C6 cells were then treated with diff er-ent combinations of LPS and melatonin for 24 hours. After fi xing in PFA, C6 cells were then treated with 0.05% H

2 O

2

in methanol for 1 hour with mild agitation to make them permeable. Plate was blocked with blocking buff er (0.02% BSA � 0.002% triton-X 100) for 30 minutes at room tem-perature. Primary antibody of NF- κ B (p65 subunit), 1:100 dilutions for 1 hour with mild agitation was used for stain-ing. Cells were again treated with secondary fl uorescent-conjugated Alexa-fl our 546 antibody at 1:200 dilutions for 1 hour with mild agitation at room temperature. Cells were washed with PBS at each step. Images of cells were captured by upright fl uorescence microscope at 100 magnifi cation.

Western blotting for COX-2, iNOS, CHOP, GFAP, mPGES-1 and P-p38 expressional analysis

Cell lysates of glial cells from diff erent treatment groups were prepared in the 200 μ l of the lysis buff er, containing 50 mM Tris – HCl, 100 mM NaCl, 1 mM EDTA, 100 μ g/ml phenylmethylsulfonyl fl uoride (PMSF), 1 μ g/ml aprotinin, 10 μ g/ml pepstatin for non-phosphorylated proteins and for phosphorylated proteins 2 mM NaF, 2 mM Na

3 Vo

4, 1%

triton-X 100 were additionally added. Cell lysate was cen-trifuged at 10,000g for 5 minutes at 4C. Protein estimation in supernatants were done by standard Lowry method [18]. Samples were mixed with the 3X loading buff er con-taining 100 mM Tris – HCl (pH 6.8), 4% sodiumdodecyl sulphate (SDS), 200 mM dithiothritol (DTT), 0.2% bro-mophenol blue, 20% glycerol and were boiled for 5 min-utes at 100C. Proteins (50 – 100 μ g) were separated on

8 – 15% SDS – PAGE, transferred to PVDF membrane. Membranes were then blocked by blocking buff er (10 mM Tris, pH 7.5, 5% non-fat dry milk, 100 mM NaCl, 0.1% tween-20) over night at 4°C and washed with wash buff er. Membranes were treated with primary anti-COX-2, anti-CHOP, anti-iNOS, anti-GFAP, anti-mPGES-1 or anti-P-p38 antibodies at 1:1000 dilutions at room temperature for a period of 2 hours. After washing, membranes were again incubated with HRP-conjugated secondary antibodies in a 1:2000 dilution for 1 hour at room temperature. Blots were then developed using the ECL (enhanced chemilumines-cence) system provided by the Amersham Biosciences. Densitometry analyses of bands were done by the Alpha Image gel documentation system (Alpha Innotech, USA). Results have been expressed as a fold change to control.

Reverse transcription-polymerase chain reaction (RT-PCR) for transcriptional analysis of TNF- α , IL-1 b , IL-1 α , IL-6 and alpha-7 nicotinic acetylcholine receptor genes

RNA was isolated by standard tri-reagent provided by Sigma – Aldrich. RNA (2 μ g) was quantifi ed spectrophoto-metrically and was reverse transcribed using oligo-(dT) primers by kit according to the manufacture ’ s protocol (Fermentas). Equal amount of cDNA was subjected to subsequent PCR analysis in a total volume of 50 μ l con-taining 0.5 μ M of primers specifi c for TNF- α , IL-1 β , IL-1 α , IL-6, α -7 nAChER and GAPDH (glyceraldehyde phosphate dehydrogenase). Details of primers are given in Table I. PCR was accomplished in following conditions: (1) 5 minutes at 94°C, (2) 45 seconds at 94°C, 45 seconds at 60°C for TNF- α or 68°C for GAPDH or 60°C for α 7-nAChR or 58°C for IL-1 β or 65°C for IL-6 and IL-1 α , 45 seconds at 72°C for 35 cycles to all except GAPDH (30 cycles) and (3) 10 minutes at 70°C. Controls are taken of RNA subjected to the RT-PCR procedure without addition of reverse transcriptase and PCR performed in the absence of cDNA, which always yielded negative results. Results have been expressed as a fold change to control.

Statistical analysis

All data are expressed as means � SEM and are represen-tative of at least three separate experiments. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by Newman – Keuls test as post-hoc test. GraphPad prism (version-3) was used to accomplish statistical tests. The value p � 0.05 was considered statis-tically signifi cant.

Results

Eff ect of LPS alone and in combination with melatonin on nitrite release, ROS generation, MDA formation, GSH level and intracellular calcium ion level in C6 cells

To assess the eff ect of melatonin on nitrative and oxida-tive stress, we measured the LPS-induced nitrite release

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melatonin (50 – 200 μ M) dose dependently. However, melatonin (200 μ M), per se , did not show any signifi -cant diff erence on these parameters. Moreover, we have also estimated intracellular calcium ion level after 24 hours of incubation with LPS and melatonin. In this study, LPS signifi cantly upregulated intracellular Ca 2�

ion level, which was downregulated by melatonin in dose dependent manner (Figure 2). However, melatonin per se does not infl uence the intracellular calcium ion level.

and ROS generation in C6 cells after 24 hours of incu-bation period. As shown in Figure 1, LPS signifi cantly increased nitrite release and ROS generation in C6 cells which were signifi cantly inhibited by the melatonin (50, 100 and 200 μ M) in a dose dependent manner. We also measured LPS-induced MDA formation and GSH level in C6 cells. As shown in Figure 1, LPS signifi cantly increased MDA formation and a signifi cant decrease in GSH level. LPS-induced MDA formation was signifi -cantly attenuated with increase in GSH level by the

Figure 1. Eff ect of melatonin on LPS-induced nitrite release, ROS generation, MDA formation and GSH level. (A) # p � 0.001 signifi cant with control, * * * p � 0.001 signifi cant with LPS treated group. (B) # p � 0.01 signifi cant with control, * p � 0.05 and * * p � 0.01 signifi cant with LPS treated group. (C) # p � 0.001 signifi cant with control, * * * p � 0.001 signifi cant with LPS treated group. (D) # p � 0.001 signifi cant with control, * * p � 0.01 and * * * p � 0.001 signifi cant with LPS treated group.

Table I. Details of PCR conditions used.

Probe Cycles Orientation Sequence Length (bp) Temperature (°C)

GAPDH 30 Forward 5 ′ -TGAAGGTCGGTGTGAACGGATTTGGC-3 ′ 983 68Reverse 5 ′ -CATGTAGGCCATGAGGTCCACCAC-3 ′

TNF- α 35 Forward 5 ′ -TTCTGTCTACTGAACTTCGGGTGATCGGTCC-3 ′ 111 58

Reverse 5 ′ -TATGAGATAGCAAATCGGCTGACGGTGTGGG-3 ′

IL-1 β 35 Forward 5 ′ -CACCTCTCAAGCAGAGCACAG-3 ′ 79 58

Reverse 5 ′ -GGGTTCCATGGTGAAGTCAAC-3 ′

IL-1 α 35 Forward 5 ′ -AAGACAAGCCTGTGTTGCTGAAGG-3 85 65

Reverse 5 ′ -TCCCAGAAGAAAATGAGGTCGGTC-3IL-6 35 Forward 5 ′ -TCCTACCCCAACTTCCAATGCTC-3 ′ 79 65

Reverse 5 ′ -TTGGATGGTCTTGGTCCTTAGCC-3 ′

α -7nAChE 35 Forward 5 ′ -GTGGAACATGTCTGAGTACCCCGGAGTGAA-3 ′ 510 60

Reverse 5 ′ -GAGTCTGCAGGCAGCAAGAATACCAGCA-3 ′

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The eff ect of melatonin on LPS stimulated α 7-nAChR activation 1171

Eff ect of melatonin on LPS-induced P-p38 MAPK, CHOP and mPGES-1 proteins expressions in C6 cells

Since P-p38 kinase, transcription factor CHOP and mPGES-1 are the key signalling molecules involved in the regulation of number of infl ammatory mediators there-fore, to explore the mechanisms underlying the protective eff ect of melatonin against LPS, measurements for P-p38 kinase, CHOP and mPGES-1 proteins expressions were done. Treatment of C6 cells with LPS signifi cantly upregulated the expression of phosphorylated form of p38 kinase (P-p38), which was downregulated by the mela-tonin. To unravel the further downstream targets of mela-tonin and LPS actions, we measured CHOP and mPGES-1 expressions by western blotting. Treatment of C6 cells with LPS signifi cantly increased CHOP expression which was attenuated by melatonin (Figure 5). In addition to P-38 and CHOP proteins, we also measured the expression of mPGES-1 protein in response to LPS and melatonin. Treatment of C6 cells with LPS for 24 signifi cantly down-regulated mPGES-1 protein expression, which was upreg-ulated by the melatonin (Figure 5).

Eff ect of melatonin on LPS-induced nuclear factor kappa B (NF-kB) translocation

To explore the mechanisms underlying the protective eff ect of melatonin against LPS, translocation of NF- κ B was studied. Treatment of C6 cells with LPS (10 μ g/ml) caused NF- κ B (p65) translocation, from cytosol to nucleus which was inhibited by melatonin (Figure 6). Treatment of melatonin with LPS signifi cantly inhibited NF- κ B translocation in C6 cells (Figure 6).

Eff ect of melatonin and PTDC on LPS-induced α 7-nAChR mRNA expression in C6 cells

As it is known that α 7-nAChR mediated infl ammatory path-way plays an important role in the regulation of astroglial

Infl uence of LPS alone and in combination with melatonin on mRNA expressions of pro-infl ammatory cytokine genes in C6 cells

Further, we also measured the mRNA expression profi le of infl ammatory cytokine genes (IL1 α , IL-1 β , TNF- α , and IL-6), because they have widely been involved in the initiation and progression of neuroinfl ammation associ-ated neurodegeneration. LPS (10 μ g/ml) signifi cantly downregulated IL-1 α and IL-1 β expressions (Figure 3A). Melatonin (200 μ M), signifi cantly upregulated LPS-in-duced defi cit in IL-1 β expression with no signifi cant eff ect of IL-1 α expression. However, melatonin (200 μ M), per se , did not show any change in the IL-1 α and IL-1 β expressions. As illustrated in Figure 3B, LPS (10 μ g/ml) signifi cantly upregulated IL-6 and TNF- α expressions. Melatonin (200 μ M) signifi cantly downregulated LPS-induced increase in IL-6 and TNF- α expressions. How-ever, melatonin (200 μ M), per se , did not show any signifi cant eff ect on IL-6 and TNF- α mRNA expressions.

Infl uence of melatonin on LPS-induced cyclooxigenase-2 (COX-2), inducible nitric-oxide synthase (iNOS) and GFAP expressions in C6 cells

We also assessed the eff ect of melatonin on LPS-induced COX-2, iNOS and GFAP protein expressions in C6 cells. LPS (10 μ g/ml) signifi cantly upregulated COX-2 protein expression (Figure 4). Melatonin (200 μ M) signifi cantly downregulated LPS-induced COX-2 expression. Mela-tonin (200 μ M), per se , also signifi cantly downregulated COX-2 expression. LPS alone showed signifi cant change in GFAP protein expression (Figure 5). Melatonin alone and in combination with LPS signifi cantly downregulated GFAP protein expression. LPS (10 μ g/ml) signifi cantly upregulated iNOS protein expression (Figure 4). Mela-tonin (200 μ M) signifi cantly downregulated LPS-induced iNOS expression. Melatonin (200 μ M), per se , also signifi cantly downregulated iNOS expression.

Figure 2. Eff ect of melatonin on LPS-induced Ca 2� ion level in C6 cells. # p � 0.01 signifi cant with control, * * p � 0.01 signifi cant with LPS treated group.

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IL-1β

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PD

H

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00.5

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Mela200μM

Rela

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/G

AP

DH

Concentrations

#

*****

***

+ LPS µg/ml

Figure 3. Eff ect of melatonin on LPS-induced mRNA expressions of pro-infl ammatory cytokine genes (IL-1 α , IL-1 β , IL-6 and TNF- α ). Lane-1 � control, Lane-2 � LPS 10 μ g/ml, Lane-3 � LPS 10 μ g/ml � Mela 50 μ M, Lane-4 � LPS 10 μ g/ml � Mela 100 μ M, Lane-5 � LPS 10 μ g/ml � Mela 200 μ M, Lane-6 � Mela 200 μ M. (a) IL-1 α , # p � 0.01 signifi cant with control, * * p � 0.001 signifi cant with LPS, IL-1 β , # p � 0.001 signifi cant with control, * p � 0.05, * * * p � 0.001 signifi cant with LPS. (b) IL-6: # p � 0.001 signifi cant with control, * * p � 0.01 signifi cant with LPS treated group. TNF- α , # p � 0.001 signifi cant with control, * * p � 0.01, * * * p � 0.001 signifi cant with LPS.

response [8,9]. Therefore, further to assess the correlation of infl ammation and α 7-nAChR mRNA expression , we measured the eff ect of LPS alone and in combination with melatonin or PTDC on α 7-nAChR expression. LPS sig-nifi cantly increased the expression of α 7nAChR expres-sion and melatonin signifi cantly downregulated the LPS induced α 7nAChR expression in dose dependent manner. PTDC also signifi cantly downregulated α 7nAChR expres-sion (Figure 7).

Discussion

It has now widely been accepted that neuroinfl ammation is an inherent part of Alzheimer ’ s disease [19 – 21]. LPS induces glial cells activation by toll like receptor mediated mechanism. Earlier studies on various models show that LPS induces a characteristic pattern of neuroinfl ammation,

which resembles to the neurodegeneration in AD [22]. Evidence, which described that LPS receptor (CD14) is a receptor for amyloid beta protein phagocytes supported the involvement of LPS induced neuroinfl ammation simi-lar in AD [23]. Another important study descried that LPS increases intracellular accumulation of amyloid beta pro-tein and amyloid beta peptide in an animal model of Alzheimer ’ s disease [24]. A transgenic model of AD fur-ther emphasized that LPS increases the amyloid beta pro-tein deposition [25]. Although, accumulating evidences suggest that astroglial activation may be the key response for LPS-induced neurodegeneration [26] but the exact mechanism of action in deleterious events ultimately lead-ing to neuronal cell death remains elusive and is of great importance towards development of novel therapeutics.

As oxidative stress and infl ammatory mediators are main executors of neurodegeneration. Therefore, anti-oxidants and anti-infl ammatory agents can provide the

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Figure 4. Eff ect of melatonin on LPS-induced expression of COX-2, iNOS and GFAP proteins. Lane-1 � control, Lane-2 � LPS 10 μ g/ml, Lane-3 � LPS 10 μ g/ml � Mela 50 μ M, Lane-4 � LPS 10 μ g/ml � Mela 100 μ M, Lane-5 � LPS 10 μ g/ml � Mela 200 μ M, Lane-6 � Mela 200 μ M. COX-2: # p � 0.001 signifi cant with control, * p � 0.05, * * p � 0.001 signifi cant with LPS, iNOS: # p � 0.001 signifi cant with control, * * * p � 0.001 signifi cant with LPS. GFAP: # p � 0.001 signifi cant with control, * p � 0.05, * * * p � 0.001 signifi cant with LPS.

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Figure 5. Eff ect of melatonin on LPS-induced expressions of P-p38, CHOP, mPGES-1 proteins. Lane-1 � control, Lane-2 � LPS 10 μ g/ml, Lane-3 � LPS 10 μ g/ml � Mela 200 μ M. P-p38: # p � 0.01 signifi cant with control, * * p � 0.01 signifi cant with LPS, CHOP: # p � 0.01 signifi cant with control, * * p � 0.001 signifi cant with LPS. mPGES-1: # p � 0.001 signifi cant with control, * * p � 0.001 signifi cant with LPS.

neuroprotection possibly by attenuating neuroinfl amma-tion. Melatonin showed the neuroprotective eff ect in vari-ous models of neurological diseases but their mechanism of action on astrocytes mediated neuroinfl ammation is also not yet clears [27]. Even though the multiple factors

such as cytokines, fatty acid metabolites produced from activated astroglial glial cells infl uence neurodegenera-tion, but the ROS may be the key mediator of glia-facili-tated neurotoxicity of neighbour neurons [28]. In addition to direct oxidative damage, ROS may trigger intracellular

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Figure 7. Eff ect of melatonin and PTDC on LPS-induced α 7-nAChR mRNA expression. Lane-1 � control, Lane-2 � LPS 10 μ g/ml, Lane-3 � LPS 10 μ g/ml � Mela 50 μ M, Lane-4 � LPS 10 μ g/ml � Mela 100 μ M, Lane-5 � LPS 10 μ g/ml � Mela 200 μ M, Lane-6 � Mela 200 μ M. Left panel: # p � 0.05 signifi cant with control, * p � 0.05, * * * p � 0.001 signifi cant with LPS. Right panel: # p � 0.001 signifi cant with control, * * p � 0.001 signifi cant with LPS.

Figure 6. Eff ect of melatonin on LPS-induced NF- κ B translocation: (A) control; (B) LPS 10 μ g/ml and (C) LPS 10 μ g/ml � Mela 200 μ M.

signalling pathways resulting in the increased release of neurotoxins and decreased release of trophic factors by astrocytes resulting in neurodegeneration. Robinson et al. have described that redox-sensitive protein phosphatase activity regulates the phosphorylation state of p38 protein kinase in primary astrocyte culture and then p38 associ-ated release of neurotoxins [29]. Van Muiswinkel et al. have reported that ROS interrupt Nrf2-ARE signalling pathway, the key signalling pathway involved in the regu-lation of neurotrophic factor release by astrocytes. We observed that LPS is producing oxidative stress (enhanced formation of ROS and MDA and GSH defi cit), which may interrupt P-p38 and NrF2-ARE signalling in astrocytes and subsequent release of neurotoxins causing neurode-generation [30]. Melatonin and protects these changes in astroglial cells. The antioxidant property of melatonin has also shown in various animal models, which supported the present fi ndings [31].

Toll like receptor activation and oxidative stress may be associated in the Ca 2� ion release in astrocytes, which is an important event and involved in the regulation of

number of mechanism including release of S100B and other neurotoxins [32 – 34]. However, earlier studies have reported alteration in the calcium ion homeostasis in neu-rological disorders [35] but the state of intracellular Ca 2�

ion level in astrocytes in response to LPS and melatonin was not demonstrated. This study showed that LPS ele-vated Ca 2� ion level, which was downregulated by the melatonin and possibly subsequent Ca 2� ion-mediated release of neurotoxins.

In addition to release of neurotrophic or neurotoxic molecules, astrocytes also perform immune functions by releasing infl ammatory cytokines, which can act on micro-glial or neurons modulating their functions in the direction of neuronal survival or loss. One immune function of astrocytes is IL-6 production [6,36]. Synthesis of IL-6 within the central nervous system (CNS) can produce sev-eral diff erent responses, acting on glia, neurons and lym-phocytes infi ltrating brain tissue and some of these eff ects are associated with CNS autoimmune disease [37]. IL-6 gene expression in astrocytes is regulated by cytokines, infectious agents, neuropeptides and neurotransmitters

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The eff ect of melatonin on LPS stimulated α 7-nAChR activation 1175

and most of these stimuli interact synergistically [38]. More recently, it has been showed that LPS signifi cantly increased IL-6 expression in an animal model of neuro-logical disorder [38]. The more strong evidence for IL-6 functions comes from the knock out study in which it was shown that LPS could cause memory impairment by the IL-6 mediated mechanism [39]. In the present study, LPS also upregulated IL-6 expression, which supports that in vivo fi nding and insists that upregulation in astroglial mediated IL-6 production may cause memory impairment. The IL-6 along with soluble IL-6 receptor signifi cantly regulates VCAM-1 gene expression, which is understood as a resultant of glial cell activation [40]. Melatonin sig-nifi cantly attenuated LPS-induced IL-6 expression, explor-ing its mechanism of protective role, emphasizing on astroglial mediated IL-6 release. Another study showed that IL-1 α plays a crucial role in modulating glia cells proliferation and thereby guidance and trophic factors for new fi bres, in response to brain injury [36]. A more recent study showed that sustained IL- β overexpression mediate chronic neuroinfl ammation and ameliorates Alzheimer ’ s plaque pathology describing that IL-1 β over expression is a benefi cial event in Alzheimer ’ s disease [41]. Here mela-tonin upregulated IL-1 β expression suggesting its vital role in ameliorating Alzheimer ’ s plaque pathology. Mela-tonin signifi cantly increased LPS-induced decrease in IL-1 α and IL-1 β expression in these astrocytoma cells C6 suggesting its benefi cial role in regulating these cytokines in the states of infection or brain injury. It has widely been known that TNF- α is responsible for the neurodegenera-tion in the neurodegenerative diseases and other neuroin-fl ammation associated disorders. In our study, LPS signifi cantly upregulated TNF- α expression, which was down-regulated by the dose dependent concentration of melatonin depicting its regulatory mechanism behind its neuroprotective eff ects [42].

Activation of p38 MAPK and NF- κ B translocation is an important events involved in the process of toll like receptor mediated intra cellular signalling. An upregulated levels of P-p38 MAPK expression and increase in NF- κ B translocation are evident in various LPS induced models of neurological disorders but their states are unknown in LPS-induced astrocytes [4,5,43]. Importantly, melatonin also showed its potent NF- κ B inhibiting activity in cell culture models [44] but its astrocytes specifi c action was not known. In the present study, LPS signifi cantly caused NF- κ B translocation and increased P-p38 expression revealing the intracellular mechanism of action in the release of infl ammatory mediators by C6 cells. Melatonin has signifi cantly inhibited LPS-induced NF- κ B transloca-tion and P-p38 activation, which reveals that the inhibition of NF- κ B and p38 activation may be involved in the astrocytes-facilitated neuroprotective action of melatonin. Activation of CHOP transcription factor, a downstream target of P-p38 MAPK kinase activation was unknown in response to LPS and melatonin with respect to astrocytes. However, previously it has been reported that CHOP upregulation is involved in the apoptotic events in the ani-mal model [45,46] astroglial mediated expression was still

unclear with special aspects of regulation of infl ammatory mediators release. Our results demonstrated that LPS upregulated CHOP expression which was subsequently downregulated by the melatonin emphasized CHOP a mere target for melatonin in LPS stimulated astrocytes.

A large amount of data have described the role of iNOS in neurodegenerative diseases and in the other CNS dis-orders which produces NO, that in turn form neurotoxin peroxynitrate causing neurodegeneration [47]. Reports also described that melatonin downregulated the iNOS induction in various models [48]. However, these studies showed iNOS inhibiting activity of melatonin, but the exact role of melatonin on LPS-induced astroglial cells was not clearly understood [49]. We demonstrated that melatonin signifi cantly downregulated the LPS-induced iNOS expression and subsequent nitrite release supporting that neuroprotection by melatonin may be mediated by the inhibition of nitrite release by astroglial cells.

Several reports have described the vital role of COX-2 enzyme in the LPS-induced neurodegeration in the animal models and in patients of neurological diseases. [50]. COX-2 upregulation is thought to mediate neuronal dam-age presumably by producing excessive amount of harm-ful prostanoids and free radicals [51]. LPS elevated COX-2 expression in animal models [48,51]. Melatonin also showed downregulation in LPS-induced COX-2 expres-sion in a cell culture model [52]. However, these reports described the reciprocal expression of COX-2 in response to LPS and melatonin but their cell specifi c actions with regard to astroglia were largely unknown. In present study, LPS signifi cantly upregulated the COX-2 expres-sion, which may represent prostaglandins mediated neu-rodegeneration. Melatonin signifi cantly downregulated LPS-induced COX-2 expression, thus inhibiting the prostaglandins synthesis and subsequent inhibition in neurodegeneration.

Reactive astrogliosis, the cellular manifestation of neu-roinfl ammation, has been suspected to cause neurodegen-eration, is characterized by formation of glial cell soma and upregulated level of GFAP expression [53]. Our study hereby demonstrates that LPS signifi cantly enhanced GFAP expression and melatonin has signifi cantly down-regulated the LPS-induced GFAP expression in dose dependent manner. Melatonin could be eff ective enough in attenuating GFAP expression and thus subsequent inhibition in reactive gliosis depicting its mechanisms of neuroprotective functions.

α 7-nAchR mediated nicotinic infl ammatory pathway regulates many functions in the neuroinfl ammation associ-ated to the neurodegeneration [8,9]. Recently, it has been demonstrated that α 7-nAchR is expressed in the glial cells in additions to the neurons and thereby modulate immune functions in the brain associated to the neurodegeneration [54]. The present study has demonstrated that LPS signifi cantly increased the α 7-nAChR expression and melatonin has signifi cantly inhibited the LPS induced α 7-nAChR expression. Yu et al. have also reported upregula-tion in α 7-nAchR receptor expression in Alzheimer patients [9]. Recently, Chi et al. have reported that LPS

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increases α 7-nAchR expression in uveitis in rats gives strong support to the present fi ndings [8]. These present results indicate that, in activated astroglial, α 7-nAchR expression is increased that may * * be one of the mecha-nism of neuroinfl ammation. Furthermore, the eff ect of melatonin to α 7-nAchR expression points towards one of its protective action [55]. PTDC (an inhibiter of NF- κ B) signifi cantly inhibited α 7-nAchR mRNA expression, which indicates involvement of NF- κ B in its regulation. On the other hand melatonin inhibited NF- κ B and α 7-nAchR expressions, which indicate that melatonin down-regulate α 7-nAchR expression via an NF- κ B dependent mechanism.

In conclusion, the present study demonstrates that LPS activates astrocytes cells, resulting in a sustained NF- κ B and CHOP activation and the release of potentially neu-rotoxic molecules including ROS and nitrite. The regula-tion of pro-infl ammatory cytokine genes expression profi le by melatonin could focus astroglial mediated neuroinfl am-mation. Further, GFAP expression pattern explored the understanding of reactive astrogliosis. Decrease in α 7-nAChR mRNA expression by PTDC provided the possible involvement of NF- κ B in its regulation. The modulatory eff ect of melatonin on these LPS-induced neuroinfl amma-tory mediators elucidated its mechanism of protective eff ects. Collectively, the present study demonstrated that melatonin attenuated mediators of neuroinfl ammation and alpha-7 nicotinic acetylcholine receptor mRNA expression in LPS-stimulated C6 cells, which may suggest its benefi -cial roles in neuroinfl ammation associated disorders.

Acknowledgement

Senior Research Fellowship of Indian Council of Medical Research, New Delhi, India to Mr. Rituraj Niranjan is gratefully acknowledged.

Confl ict of interest

All authors declare that they have no confl ict of interest. The authors alone are responsible for the content and writ-ing of the paper.

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