Elmann et al. BMC Complementary and Alternative Medicine 2011, 11:98
http://www.biomedcentral.com/1472-6882/11/98
RESEARCH ARTICLE
Open Access
Anti-Neuroinflammatory effects of the extract of
Achillea fragrantissima
Anat Elmann1*, Sharon Mordechay1, Hilla Erlank1, Alona Telerman1, Miriam Rindner1 and Rivka Ofir2
Abstract
Background: The neuroinflammatory process plays a central role in the initiation and progression of
neurodegenerative diseases such as Parkinson’s and Alzheimer’s diseases, and involves the activation of brain
microglial cells. During the neuroinflammatory process, microglial cells release proinflammatory mediators such as
cytokines, matrix metalloproteinases (MMP), Reactive oxygen species (ROS) and nitric oxide (NO). In the present
study, extracts from 66 different desert plants were tested for their effect on lipopolysaccharide (LPS) - induced
production of NO by primary microglial cells. The extract of Achillea fragrantissima (Af), which is a desert plant that
has been used for many years in traditional medicine for the treatment of various diseases, was the most efficient
extract, and was further studied for additional anti-neuroinflammatory effects in these cells.
Methods: In the present study, the ethanolic extract prepared from Af was tested for its anti-inflammatory effects
on lipopolysaccharide (LPS)-activated primary cultures of brain microglial cells. The levels of the proinflammatory
cytokines interleukin1b (IL-1b) and tumor necrosis factor-a (TNFa) secreted by the cells were determined by
reverse transcriptase-PCR and Enzyme-linked immunosorbent assay (ELISA), respectively. NO levels secreted by the
activate cells were measured using Griess reagent, ROS levels were measured by 2’7’-dichlorofluorescein diacetate
(DCF-DA), MMP-9 activity was measured using gel zymography, and the protein levels of the proinflammatory
enzymes cyclooxygenase-2 (COX-2) and induced nitric oxide synthase (iNOS) were measured by Western blot
analysis. Cell viability was assessed using Lactate dehydrogenase (LDH) activity in the media conditioned by the
cells or by the crystal violet cell staining.
Results: We have found that out of the 66 desert plants tested, the extract of Af was the most efficient extract and
inhibited ~70% of the NO produced by the LPS-activated microglial cells, without affecting cell viability. In addition,
this extract inhibited the LPS - elicited expression of the proinflammatory mediators IL-1b, TNFa, MMP-9, COX-2
and iNOS in these cells.
Conclusions: Thus, phytochemicals present in the Af extract could be beneficial in preventing/treating
neurodegenerative diseases in which neuroinflammation is part of the pathophysiology.
Keywords: Achillea fragrantissima, microglial cells, neuroinflammation, nitric oxide, matrix metalloproteinase-9,
cyclooxygenase-2
Background
The increase in the life span of populations in the Western world has been accompanied by an elevation in the
frequencies of neurodegenerative diseases, e.g., Alzheimer’s and Parkinson’s diseases. In these diseases, a gradual and progressive neuronal cell death occurs,
* Correspondence: aelmann@volcani.agri.gov.il
1
Department of Food Quality and Safety, Volcani Center, Agricultural
Research Organization, Bet Dagan, 50250, Israel
Full list of author information is available at the end of the article
amongst other, as a consequence of increased nitrosative
and oxidative stress and an uncontrolled neuroinflammatory response [1-3]. These processes play a pivotal
role in the initiation and progression of various neurodegenerative diseases and involve the activation of
microglial cells [4]. Microglial cells, are cells of the
macrophage lineage in the central nervous system
(CNS), and are quiescent in the normal brain. However,
they can be activated by the cytokines produced by infiltrating immune effector cells in response to CNS injury
© 2011 Elmann et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Elmann et al. BMC Complementary and Alternative Medicine 2011, 11:98
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or to the lipopolysaccharide (LPS) excreted during bacterial infection. Activated microglial cells release either
neurotrophic factors, supporting neuronal cell survival,
or neurotoxic factors, such as oxygen radicals, nitric
oxide (NO) and proinflammatory cytokines [4]. While
microglial activation is necessary and critical for host
defense, prolonged and excessive stimulation of these
cells initiates an inflammatory cascade in the CNS that
contributes to the pathogenesis of several neurodegenerative diseases. Therefore, controlling microglial activation is regarded as a promising therapeutic target to
combat neurodegenerative diseases.
Cyclooxygenase-2 (COX-2) and induced NO-synthase
(iNOS) are inducible forms of enzymes which are up
regulated in activated microglia in response to inflammatory challenge. The induction and regulation of these
enzymes are tightly coupled and thought to contribute
to the pathogenesis of various diseases, including neurodegenerative diseases [5]. The excessive amounts of NO,
a free radical produced by iNOS, and of prostaglandin
E, an arachidonic acid metabolite produced by COX-2,
which are secreted by activated microglial cells during
the neuroinflammatory process, cause nitrosative stress
and brain cell death [5,6]. NO is a free radical, and high
levels of NO have been implicated in the pathogenesis
of stroke, trauma, demyelinating, and neurodegenerative
diseases [7]. iNOS and COX-2 are upregulated in activated microglia in response to inflammatory stimuli
such as Alzheimer’s amyloid peptide, interferon gamma
(IFNg) and bacterial LPS. Co-induction and co-regulation of iNOS and COX-2 have also been demonstrated
in a number of cell culture studies and in inflammatory
animal model systems [8].
Other molecules that are secreted by stimulated
microglial cells include tumor necrosis factor alpha
(TNFa) and interleukin 1b (IL-1b) [4], both of which
can cause neuronal cell death both directly and indirectly via the induction of NO and free radicals in microglial cells [9].
Matrix metalloproteinase-9 (MMP-9) is a zinc-dependent enzyme, that belongs to the family of MMPs and
contributes to the neuroinflammatory response in neuroinflammation and in neurodegenerative diseases such
as amyotrophic lateral sclerosis [10], and Alzheimer’s
disease [11,12]. MMP-9 is also upregulated in rodent
models of cerebral ischemia, hemorrhage and trauma
[13-15] and after its activation by proteases and ROS
[16,17] can disrupt the blood brain barrier (BBB), a disruption that leads to extravasation of blood proteins, to
brain edema, to cerebral hypoperfusion, and ultimately
to neuronal damage [18-20]. A deleterious role for
MMP-9 is indicated because MMP-9 knockout mice are
protected against focal cerebral ischemia [21,22] brain
trauma [23] and experimental encephalomyelitis [24].
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Brain microglial cells and endothelial cells have been
shown to be a source of MMP-9 [12]. Microglial cells
serve as important source of MMP-9, and lipopolysaccharide (LPS), IL-1b and TNF-a were shown to stimulate its production from these cells [12].
Thus, elevated activity and/or expression of iNOS,
COX-2, MMP-9, IL-1b and TNF-a in brain cells have
been implicated in the cascade of events leading to neurodegenerative diseases.
Many herb and plant extracts are used as folk medicines for various kinds of inflammatory diseases, organ
dysfunctions and systemic disorders. In the present
study we screened ethanolic extracts prepared from 66
different desert plants for their capacity to inhibit NO
production from LPS-activated microglial cells. The
extract of Achillea fragrantissima (Af; Asteraceae)
exerted the most potent inhibitory activity.
Achillea fragrantissima (Af) is a desert plant that has
been used for many years in traditional medicine in the
Arabia region for the treatment of respiratory diseases
and gastrointestinal disturbances [25-28]. It was therefore thought worthwhile to investigate the effects of Af
on neurodegenerative diseases, effects that have not
been studied to date. The present study describes the
anti-neuroinflammatory activities of this plant.
Methods
Reagents
Dulbecco’s modified Eagle’s medium (DMEM), RPMI1640 (with or without phenol red), Leibovitz-15 medium, glutamine, antibiotics (10,000 IU/ml penicillin and
10,000 μg/ml streptomycin), soybean trypsin inhibitor,
fetal bovine serum (FBS) and Dulbecco’s phosphate buffered saline (PBS) (without calcium and magnesium)
were purchased from Biological Industries (Beit Haemek, Israel); Griess reagent and rabbit anti COX-2 polyclonal antibody were obtained from Cayman chemical,
Ml, USA; DreamTaq Green PCR master Mix (2x) and
ReverAid First Strand cDNA Synthesis Kit were purchased from Fermentas life sciences (Eisenberg Bros.
Ltd, Israel). iNOS polyclonal antibody was purchased
from AbD Serotec, Ox, UK; Horseradish peroxidase
(HRP)-conjugated anti-rabbit IgG was obtained from
Jackson ImmunoResearch Laboratories Inc. Baltimore,
USA; Monoclonal mouse anti-b-actin was purchased
from MP Biomedicals, Ohio, USA; LPS (Escherichia coli
0127 B:8), 2-mercaptoethanol, L-NMMA (NG-Methyl-Larginine acetate salt), Gelatin, Crystal violet and protease
inhibitor cocktail were purchased from Sigma Chemical
Co. (St Louis, MO, USA). 2,2’-Azobis(amidinopropane)
(ABAP) was obtained from Wako chemicals (Richmond,
VA), and 2’7’-dichlorofluorescein diacetate (DCF) were
purchased from Sigma Chemical Co. (St Louis, MO,
USA).
Elmann et al. BMC Complementary and Alternative Medicine 2011, 11:98
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Preparation of Plant Extracts
The plants were collected in the Arava Valley, and the
voucher specimens have been kept and authenticated as
part of the Arava Rift Valley Plant Collection; VPC
(Dead Sea & Arava Science Center, Central Arava
Branch, Israel, http://www.deadseaarava-rd.co.il/
_Uploads/dbsAttachedFiles/Arava_Rift_Valley_Plant_Collection.xls) under the accession code AVPC0040.
Freshly collected plants were dried at 40°C for three
days and extracted in ethanol (96%). The liquid phase
was then evaporated off, and the dry material was dissolved in DMSO to a concentration of 100 mg/mL to
produce the various extracts, including Af extract.
Preparation of Primary Microglial Cell Cultures
Cultures of primary rat microglial cells were prepared
from cerebral cortices of 1- to 2-day-old neonatal Wistar rats as described [29]. The research was conducted
in accordance with the internationally accepted principles for laboratory animal use and care, as found in the
US guidelines, and was approved by the Institutional
Animal Care and Use Committee of The Volcani Center, Agricultural Research Organization.
Nitrite Quantification
For NO measurements, 1 × 10 5 microglial cells/well
were plated in a 24-well tissue culture plate. After 36 h
of incubation in RPMI-1640 (without phenol red), containing 2% FBS, 2 mM glutamine, 100 U/mL penicilin,
100 μg/mL streptomycin, 1 mM sodium pyruvate, and
50 μM b-mercaptoethanol, cells were stimulated with
LPS (4.5 ng/mL). NO levels in the culture medium were
estimated by measuring the concentration of nitrite, its
stable metabolite, with Griess reagent as described [29].
Fresh culture medium was used as the blank in all the
experiments.
Determination of Cell Viability
Cell viability was determined using a commercial colorimetric assay (Roche Applied Science, Germany), based
on the measurement of lactate dehydrogenase (LDH)
activity released from the cytosol of damaged cells into
the supernatant, according to the manufacturer’s
instructions. In MMP-9 assay, cell viability was determined by a modification of the crystal violet assay [30].
At the end of cell treatments, cells were fixed with 150
μL of 5% (v/v) formaldehyde (in PBS) for 15 min at
room temperature. Plates were washed by submersion in
de-ionized water, dried and stained for 15 min with 150
μL of a 1% crystal violet solution. After careful aspiration of the crystal violet solution the plates were washed
with de-ionized water, and dried prior to the solubilization of the bound dye with 150 μL of a 33% aqueous
glacial acetic acid solution. The optical density of the
Page 3 of 10
plates was measured at 540 nm (with a 690 nm reference filter) in a microplate spectrophotometer.
Western Blot Analysis
Microglial cells were plated at a concentration of 4 ×
106 /10 mL and treated as described above. Following
treatment, the cells were processed and subjected to
Western blot analysis as described [29].
Measurement of TNFa Levels in Conditioned Media
For TNFa measurements, 3.5 × 104 cells/well were plated on a 24-well tissue culture plate. After 24 h of incubation in DMEM containing 10% FBS, cells were
stimulated with LPS (100 ng/mL). Five hours later, conditioned media from duplicate wells per sample were
collected and tested for cytokine levels with a rat TNFa
ELISA kit (Diaclone ® ; Gen-Probe Life Sciences Ltd.
France), used according to the manufacturer’s
instructions.
Cellular Antioxidant Activity of Af Extract
Intracellular ROS production was detected using the
non-fluorescent cell permeating compound, 2’7’-dichlorofluorescein diacetate (DCF-DA). DCF-DA is hydrolyzed
by intracellular esterases and then oxidized by ROS to a
fluorescent compound 2’-7’-DCF. Peroxyl radicals are
generated by thermolysis of 2,2’-Azobis(amidinopropane) (ABAP) at physiological temperature. ABAP
decomposes at approximately 1.36 × 10 -6 s -1 at 37°C,
producing at most 1 × 10 12 radicals/ml/s [31-33].
Microglial cells were plated in DMEM containing 2%
FBS, 2 mM glutamine, 100 U/mL penicillin and 100 μg/
mL streptomycin, onto 24 wells plates (300,000 cells/
well) and were incubated for 1 hr with Af extract. Then
microglial cells were preloaded with DCF-DA for 30
min, washed twice with PBS, and ABAP (0.6 mM final
concentration) was then added. The fluorescence, which
indicates ROS levels, was measured in a plate reader
with excitation at 485 nm and emission at 520 nm.
Determination of MMP Activities in Conditioned Media of
Microglial Cells
MMP-9 was quantified by gelatin zymography [18]. For
the determination of MMP activities in conditioned
media of microglial cells, 1 × 105 cells/well were plated
in a 24-well tissue culture plate in (DMEM containing 2
mM glutamine, 100 U/mL penicilin, and 100 μg/mL
streptomycin). After 24 h of incubation the medium was
replaced with fresh medium and cells were stimulated
with LPS (4.5 ng/mL). The medium conditioned by the
cells was collected 24 h after an LPS challenge, and was
concentrated x3. Samples (21 μL) of CM were mixed
with non-reduced sample buffer and were loaded on 8%
SDS-polyacrylamide gels (SDS-PAGE) that contains 1
Elmann et al. BMC Complementary and Alternative Medicine 2011, 11:98
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A.
40
NO production
(Fold change)
mg/mL gelatin type A. Electrophoresis was performed
under non-reducing conditions. Gels were washed (30
min) in 2.5% Triton X-100 to remove SDS and then for
30 min in reaction buffer (50 mM Tris-HCl, pH 7.5,
0.02% Brij 35, 10 mM CaCl2, 200 mM NaCl). The reaction buffer was then changed to a fresh one and the gels
were incubated (24 h, 37°C) in a shaking incubator.
Gelatinolytic activity was visualized by staining the gels
with 0.5% Coomassie brilliant blue. The densities of the
specific protein bands were quantified by the ImajJ
image analysis and processing program.
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30
20
10
0
0
1
2
3
4
5
6
LPS (ng/mL)
RNA was prepared using TRI reagent (Molecular
Research Center, Inc., Cincinnati, OH) according to
manufacturer instructions. Genomic DNA was removed
from the RNA samples by using 50 units of RNase-free
DNaseI at 37°C for 1 h. For cDNA preparation, RNA
(20 μg) was incubated with reverse transcriptase and
Oligo (dT) 18 primer (0.5 μg/uL) for 1 h at 42°C followed by 10 min at 72°C. For PCR, reaction mixture
contained the following: 1 μL of cDNA, 100 ng of each
primer, 12.5 μL of DreamTaq PCR Mix (2X) and doubly
distilled water to 20 μL. The following conditions were
used for IL-1b and for the control gene b-actin: 5 min
at 95°C; 30 s at 94°C, 30 s at 50°C and at 53°C, respectively, and 30 s at 72°C for 35 cycles and 25 cycles,
respectively. Products were examined by agarose gel
electrophoresis. The primers used were: IL-1b: 5’TTGCCCGTGGAGCTTC-3’ and 5’-CGGGTTCCATG
GTGAAC-3’; a-tubulin: 5’-CTCCATCCTCACCACCCACAC-3’ and 5’-CAGGGTCACATTTCACCATCT.
The densities of the specific RNA bands were quantified
by the ImajJ image processing and analysis in Java
program.
Data Analysis
Statistical analyses were performed with one-way
ANOVA followed by Tukey-Kramer multiple comparison tests using Graph Pad InStat 3 for windows (GraphPad Software, San Diego, CA, USA).
Results
Extracts of various desert plants affect NO production by
LPS-activated microglial cells
In order to conduct a first selection for prospective antineuroinflammatory activity, extracts from 66 different
desert plants, which belong to 23 different plant
families, were tested for their ability to down regulate
NO production by activated microglial cells. For that
purpose, we used a system in which stimulation of primary microglial cells with LPS induced significant
increase of NO production (Figure 1A). Induction of
NO production from LPS-activated microglial cells was
B.
120
NO production
Cytotoxicity
100
Activity (%)
RNA Extraction and Two-Step RT-PCR
80
60
40
20
0
0
20
40
60
L-NMMA (μM)
80
100
Figure 1 NO production by LPS-activated primary microglial
cells. (A) Microglial cells were stimulated with different
concentrations of LPS. (B) L-NMMA was added concomitant with
activation by LPS. NO levels in cell conditioned supernatants were
measured 20 h later. The results represent means ± SEM of 3
separate experiments (n = 9).
specifically inhibited (90%) by L-NMMA (NG-Methyl-Larginine acetate salt), a specific inhibitor of NOS (Figure
1B).
To exclude the possibility that reduction in NO
secreted by microglial cells was due to the direct toxicity
of the plant extracts to the cells, we tested cell toxicity
by LDH release into culture media. For the 46 plants
presented in Table 1 the extract-induced cytotoxicity
was negligible at concentrations of 100 μg/mL used in
this screening procedure (data not shown). Twenty
plant extracts that exhibited cytotoxic effect to the
microglial cells were excluded from the study. The distribution of the 46 non-toxic extracts according to their
extent (%) of inhibition of NO release is presented in
Table 1. It can be seen that 10 extracts (21% of the
tested plants) upregulated NO production from activated microglial cells, and the other plant extracts inhibited the NO production to various degrees.
The extract of Achilea fragrantissima was the most
efficient extract and inhibited ~70% of the NO released
with respect to the LPS-activated cells. Therefore we
have further characterized the anti-neuroinflammatory
effects of this extract.
Elmann et al. BMC Complementary and Alternative Medicine 2011, 11:98
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Table 1 The effect of different desert plant extracts on
NO production by LPS-activated microglial cells a.
Attenuation by the Af Extract of NO release in LPSStimulated Microglial cells
Plant family
Af extract was tested for its ability to downregulate NO
production from primary cultures of LPS-activated
microglial cells. Figure 2A demonstrates that the LPSelicited nitrite accumulation was markedly inhibited by
the Af extract in a dose-dependent manner. To exclude
the possibility that reduction in NO secreted by the activated microglial cells was due to the direct toxicity of
the plant extract to the cells, we tested cell toxicity following treatment with Af extract by measuring Lactate
dehydrogenase (LDH) release into culture media. The
LDH assay showed that the extract-induced cytotoxicity
was negligible at concentrations below 150 μg/mL. Viability of cells was tested also by the crystal violet assay
and showed similar results (Figure 3B). To elucidate the
Asteraceae
Achillea fragrantissima
30 ± 5 **
Papilionaceae
Crotalaria algyptiaca
63 ± 2 **
Polygonaceae
Emex spinosa
63 ± 6 **
Polygonaceae
Capparaceae
Rumex vesicarius
Cleome droserifolia
64 ± 6 **
66 ± 5 **
Asteraceae
Launala nudicaulis
69 ± 7 **
Amaranthaceae
Aerva javanica
71 ± 4 **
Asclepiadaceae
Calotropis procera
72 ± 7 **
Tamaricaceae
Tamarix nilotica
73 ± 6 **
Chenopodiaceae
Hammada salicornica
74 ± 6 **
Asclepiadaceae
Pergularia tomentosa
77 ± 4 *
Cruciferae
Malvaceae
Matthiola livida
Abutilon hirtum
77 ± 8 *
79 ± 8 *
Gramineae
Schismus arabicus
80 ± 8
Brassicaceae
Moricandia nitens
83 ± 6
Brassicaceae
Brassica tournefortii
84 ± 7
Asclepiadaceae
Pentatropis nivalis
84 ± 5
Gramineae
Stipagrostis ciliate
85 ± 3
Chenopodiaceae
Salsola cyclophylla
87 ± 4
Solanaceae
Zygophyllaceae
Solanum incanum
Tribulus bimucronatus
87 ± 4
88 ± 5
Chenopodiaceae
Atriplex holocarpa
89 ± 3
Molluginaceae
Glinus lotoides
92 ± 9
Tamaricaceae
Tamarix aphylla
94 ± 6
Chenopodiaceae
Hammaola salicornica
94 ± 3
Chenopodiaceae
Salsola gaetula
94 ± 8
Salvadoraceae
Salvadora persica
94 ± 6
Ephedraceae
Chenopodiaceae
Ephedra aphylla
Hammada scoparia
96 ± 7
96 ± 8
Solanaceae
Lycium shawii
97 ± 4
Chenopodiaceae
Salsola cyclophylla
97 ± 5
Capparaceae
Capparis spinosa
98 ± 7
Euphorbiaceae
Thymelaeaceae
Apiaceae
Ricinus communis
Thymelaea hirsute
Deverra triradiata
99 ± 5
99 ± 7
94 ± 7
Chenopodiaceae
Gramineae
Salsola vermiculata
Typha Domingensis
100 ± 6
101 ± 5
Chenopodiaceae
Anabasis setifera
102 ± 7
Gramineae
Stipagrostis plumosa
102 ± 4
Solanaceae
Solanum nigrum
109 ± 9
Chenopodiaceae
Atriplex leucoclada
113 ± 8
Mimosoideae
Acacia raddiana
114 ± 7
Mimosoideae
Acacia tortilis
117 ± 6
Zygophyllaceae
Gramineae
Zygophyllum album
Lasiurus scindicus
140 ± 9 **
157 ± 9 **
Plantaginaceae
Plantago cylindrica
157 ± 9 **
a
The results represent the mean ± SEM of 2-3 experiments (and of 8-9
experiments for Af) performed in triplicated or tetraplicates. The ethanolic
extracts (100 μg/ml) were added to the microglial cells concomitant with the
addition of LPS. * p < 0.05, ** p < 0.01
A.
120
NO production
100
Cytotoxicity
80
Activity (%)
NO production (%)
Mean ± SEM
60
40
20
0
0
25
50
75
100
125
150
175
Af extract (μg/mL)
B.
NO production
(%of LPS-activated cells)
Plant Species
120
100
80
60
*
40
*
*
*
20
0
w/o
LPS
LPS
w/o
extract
-2
-1
0
1
2
LPS+Af extract
Time (hr) of extract administration relative to
LPS challenge
Figure 2 Inhibition of NO production by activated microglial
cells in response to Af extract. (A) Microglial cells were treated
with different concentrations of the extract and concomitantly
activated by LPS (4.5 ng/mL) for 20 h. (B) Af extract was added
before, concomitant with, or after activation by LPS (4.5 ng/mL). NO
levels in cell conditioned supernatants were measured. The results
represent means ± SEM of 3 separate experiments (A, n = 9) or 2
separate experiments (B, n = 6). * p < 0.01; ** p < 0.001.
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A.
10000
ROS production (FU)
ABAP only
8000
ABAP+Af
6000
4000
2000
**
*
0
0
3
6
9
12
15
18
21
Time (h)
B.
Figure 3 Down-regulation of MMP-9 activity in activated
microglial cells by Af extract. Microglial cells were treated with the
indicated concentrations of Af extract, followed by stimulation with
LPS (4.5 ng/mL). After 24 h: A. conditioned media were collected and
tested for MMP-9 activity by gel zymography. The zymogram
represents two independent experiments. B. Cell viability was
determined by the crystal violet assay. The histogram represents the
means ± SD of two independent experiments (n = 2).
ROS production (%)
120
100
80
**
60
**
40
**
20
**
0
0
20
40
60
80
100
120
Af extract (μg/mL)
optimal time for the addition of Af extract with respect
to LPS addition, three different regimes were tested: the
cells were pre-incubated in the presence of the extract
for 1 or 2 h before the addition of LPS; the extract was
added concomitantly with LPS; or the extract was added
1 or 2 h after cell activation. The most effective times
for the addition of Af extract were concomitant with or
after cell stimulation (Figure 2B).
Figure 4 Af extract inhibits the peroxyl radical - induced
oxidation of DCFH in primary microglial cells. Microglial cells
were incubated for 1 h with Af extract. They were then preloaded
with DCF-DA for 30 min and washed with PBS, after which, 0.6 mM
ABAP was added and ROS levels were measured at the indicated
time points. Each point represents mean ± SEM of 2 experiments (n
= 8). A. Af extract at 50 μg/mL. B. ROS production was measured 22
h after the addition of ABAP. * p < 0.05; ** p < 0.001.
Af extract reduces 2,2’-azobis(amidinopropane) (ABAP)mediated peroxyl radicals levels in microglial cells
Inhibition of LPS-Induced iNOS and COX-2 Expression by
the Af Extract
The cellular antioxidant activity assay was used in order
to measure the ability of compounds present in the Af
extract to enter the cells and prevent the formation of
DCF by ABAP-generated peroxyl radicals [34]. In this
assay, the efficiency of cellular uptake, combined with
the radical-scavenging activity dictates the efficacy of the
tested compounds. The kinetics of DCFH oxidation in
microglial cells by peroxyl radicals generated from
ABAP is shown in Figure 4A, where it can be seen that
ABAP generated radicals in a time-dependent manner,
and that treatment of cells with Af extract moderated
this induction. Figure 4B shows that the increase in
ROS-induced fluorescence was inhibited by Af extract in
a dose-dependent manner. This indicates that compounds present in the Af extract entered the cells and
acted as efficient intracellular hydroperoxyl radical
scavengers.
Cells were activated with LPS in the presence or
absence of Af extract. Twenty hours later, cells were
harvested and levels of iNOS and COX-2 were determined by Western blot analysis. While the expression
levels of iNOS and COX-2 proteins were barely detectable in untreated control cells, they were markedly
increased in response to LPS. Treatment with Af extract
markedly inhibited the LPS-elicited iNOS and COX-2
expression in microglial cells (Figure 5). Expression of
the internal control, b-actin, was not affected by the different treatments (Figure 5A).
Attenuation by Af Extract of IL-1b Transcription and TNFa
Secretion in LPS-Stimulated Microglial Cells
To test whether the Af extract reduced the release of the
inflammatory cytokines TNFa and IL-1b from microglial cells, LPS was added to the culture media of the
cells in the presence or absence of the Af extract. In
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TNFĮ secretion
(% of LPS-activated cells)
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120
100
*
80
60
**
40
20
0
0
W/O LPS
0
100
200
With LPS
Af extract (ȝg/mL)
Figure 6 Down-regulation of TNFa secretion from activated
microglial cells by Af extract. Microglial cells were treated with
the indicated concentrations of Af extract, followed by stimulation
with LPS (100 ng/mL). After 5 h, conditioned media were collected
and tested for cytokine levels by ELISA. TNFa levels in the activated
cells (designated as 100%) were 900 pg/mL. Data represent the
means ± SEM of two independent experiments (n = 4). * p < 0.01;
** p < 0.001.
Figure 5 Inhibition of iNOS and COX-2 expression in LPSstimulated microglial cells by Af extract. Microglial cells were
treated with 100 μg/mL of Af extract, followed by stimulation with
LPS for 20 h. Equal amounts of cell lysates were separated by SDSPAGE and immunoblotted with antibodies to iNOS, COX-2, and bactin. A. The immunoblot represents one of three different
experiments with similar results. B. Data represent the means ± SEM
of three independent experiments (n = 3). The levels of each
protein were normalized to the levels of b-actin protein.
unstimulated microglial cells, only a small amount of
TNFa could be detected in the medium conditioned by
the cells (Figure 6). However, stimulation of the cells
with LPS resulted in a remarkable increase in TNFa
release, which was reduced (50%) by the Af extract in a
dose-dependent manner (Figure 6). Similarly, IL-1b
transcription that had been induced by LPS in the activated microglial cells was significantly inhibited when
cell activation was performed in the presence of Af
extract (Figure 7).
Attenuation by Af Extract of MMP-9 activity in LPSStimulated Microglial Cells
To study the effect of the Af extract on MMP-9 activity
in LPS-activated microglial cells, LPS was added to the
culture media of microglial cells in the presence or
absence of the Af extract, and the media conditioned by
the cells was collected after 24 hr. As MMP-9 degrades
denatured collagen (gelatin) in addition to collagen,
MMP-9 activity was measured using gelatin zymography. As shown in Figure 3, MMP-9 activity in unstimulated microglial cells is very low. However, stimulation
Figure 7 Down-regulation of IL-1b expression by LPSstimulated microglial cells by Af extract. Microglial cells (5 × 106
cells) were treated with 100 μg/mL of Af extract, followed by
stimulation with LPS (4.5 ng/mL) for 20 h. The products following
RT-PCR were separated on agarose gel. A. The gel represents one of
three different experiments with similar results. The levels of b-actin
transcripts were similar in all samples. B. Data represent the means
± SD of three independent experiments (n = 3). The levels of IL-1b
transcripts were normalized to the levels of a-Tubulin transcripts.
Elmann et al. BMC Complementary and Alternative Medicine 2011, 11:98
http://www.biomedcentral.com/1472-6882/11/98
of the cells with LPS resulted in a remarkable increase
in MMP-9 activity compared to control cells. MMP-9
activity was markedly reduced by the Af extract in a
dose-dependent manner (Figure 3). The reduction was
not a result of cell death as no toxicity was observed
using the crystal violet assay for cell viability (data not
shown).
Discussion
The main findings of this study are that out of the 66
desert plant extracts which were tested, the extract of
Achillea fragrantissima was the most active extract, and
inhibited 70% of the NO produced by the activated
cells. This reduction was dose dependent and did not
result from a cytotoxic effect of the extract. In addition,
Af extract inhibited the LPS-elicited expression of the
proinflammatory cytokines IL-1b and TNFa and of the
proinflammatory enzymes COX-2, iNOS and MMP-9
and down-regulated NO and ROS production from primary cultures of activated microglial cells. This inhibition did not result from a cytotoxic effect of the extract.
It seems that the Af extract is a polyvalent cocktail
which contains compounds that interferes with the LPS
signal as well as compounds with radical-scavenging
activity that can enter the cells and react with ROS
intracellularly.
Previous studies have shown that there is a complex
relationship between the various anti-inflammatory
compounds tested in this study; for example, activation
of iNOS and COX-2 via TNFa and IL-1b stimulate the
coupled release of NO and PGE2, while NO modulates
the TNFa- and IL-1b-dependent elevation of PGE 2
levels in astrocytes [35]. In addition, the expression of
MMPs is regulated, amongst others, by inflammatory
cytokines [36]. Moreover, S-nitrosylation [37] and tyrosine nitration [38] activates MMP-9 and NO is known
to stimulate the enzymatic activity of COX-2 both in
vitro [39] and in vivo [40].
The expression of the inflammatory molecules TNFa,
IL-1b, iNOS, COX-2 and MMP-9 can be regulated
through the activation of NF-B by activators such as
LPS and IL-1b [41,42]. Therefore, the inhibitory effect
of the Af extract on the expression of these molecules
might be attributed to inhibition of NF-B activation or
to other signaling events leading to the production of
proinflammatory molecules in microglial cells such as
protein kinase C (PKC) [43], p38 mitogen-activated protein kinase (MAPK) or p42/44 MAPK [41,44,45].
The proinflammatory molecules tested in this research
are produced, not only by activated microglial cells but
also by activated macrophages and many other cell
types. Thus, the Af extract might also be beneficial in
many other inflammatory diseases that are not related
Page 8 of 10
to neurodegenerative disease. Also, MMPs and ROS
have been shown to be involved in blood brain barrier
breakdown and in brain damage in bacterial meningitis
[19,20].
The importance of all of these proteins in the neuroinflammatory response in various animal models of
brain pathologies was demonstrated by specific inhibitors and knockout strategies of the relevant genes that
could protect against brain damage in experimental
pathology [18,46-49].
Thus, COX-2, iNOS and MMP-9 activities, as well as
TNFa, IL-1b and NO generation have become accepted
as markers and therapeutic targets in neurodegenerative
diseases, and thus their down-regulation might assist in
preventing or delaying the onset of these diseases.
To the best of our knowledge, the effects of Af in the
context of neurodegenerative diseases have not been
studied in the past, and this is the first study characterizing the anti-neuroinflammatory activities of this plant.
Conclusions
On the basis of the current results, we suggest that various compounds present in the Af extract might have
complementary beneficial bioactivities, and thus propose
that Af extracts should be further studied as polyvalent
cocktails for nutraceutical development for the prevention or treatment of neurodegenerative diseases.
List of Abbreviations
Af: Achillea fragrantissima; COX: Cyclooxygenase; IL-1β: Interleukin 1 beta;
iNOS: inducible nitric oxide synthase; LDH: Lactate dehydrogenase; LPS:
Lipopolysaccharide; MMP: matrix metalloproteinases; NO: Nitric oxide; TNFα:
Tumor necrosis factor alpha
Acknowledgements and Funding
This work was supported by the Chief Scientist of the Ministry of Science,
Israel, and by THE ISRAEL SCIENCE FOUNDATION (grant No. 600/08).
Author details
Department of Food Quality and Safety, Volcani Center, Agricultural
Research Organization, Bet Dagan, 50250, Israel. 2Dead Sea & Arava Science
Center and Department of Microbiology & Immunology, Ben-Gurion
University of the Negev, Beer-Sheva, 84105, Israel.
1
Authors’ contributions
AE carried out the study design, some of the experiments, literature search
and manuscript preparation. SM, HE, AT and MR carried out the cell culture
and biochemical experiments. RO collected the plants, prepared the extracts,
performed the RT-PCR experiments, and contributed in drafting the
manuscript. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 24 May 2011 Accepted: 21 October 2011
Published: 21 October 2011
References
1. Halliwell B: Oxidativ stress and neurodegeneration: where are we now? J
Neurochem 2006, 97:1634-1658.
Elmann et al. BMC Complementary and Alternative Medicine 2011, 11:98
http://www.biomedcentral.com/1472-6882/11/98
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
Ischiropoulos H, Beckman JS: Oxidativ stress and nitration in
neurodegeneration: Cause, effect, or association? J Clin Invest 2003,
111:163-169.
Minghetti L: Role of inflammation in neurodegenerative diseases. Curr
Opin Neurol 2005, 18:315-321.
Block ML, Hong JS: Microglia and inflammation-mediated
neurodegeneration: multiple triggers with a common mechanism. Prog
Neurobiol 2005, 76:77-98.
Minghetti L: Cyclooxygenase-2 (COX-2) in inflammatory and
degenerative brain diseases. J Neuropathol Expl Neurol 2004, 63:901-910.
Calabrese V, Mancuso C, Calvani M, Rizzarelli E, Butterfield DA, Stella AM:
Nitric oxide in the central nervous system: neuroprotection versus
neurotoxicity. Nat Rev Neurosci 2007, 8:766-775.
Saha RN, Pahan K: Regulation of inducible nitric oxide synthase gene in
glial cells. Antiox Redox Signaling 2006, 8:929-947.
Cuzzocrea S, Salvemini D: Molecular mechanisms involved in the
reciprocal regulation of cyclooxygenase and nitric oxide synthase
enzymes. Kidney Int 2007, 71:290-297.
Gosselin D, Rivest S: Role of IL-1 and TNF in the brain: Twenty years of
progress on a Dr. Jekyll/Mr. Hyde duality of the innate immune system.
Brain, Behav Immun 2007, 21:281-289.
Lim GP, Backstrom JR, Cullen MJ, Miller CA, Atkinson RD, Tokes ZA: Matrix
metalloproteinases in the neocortex and spinal cord of amyotrophic
lateral sclerosis patients. J Neurochem 1996, 67:251-259.
Deb S, Gottschall PE: Increased production of matrix metalloproteinases
in enriched astrocyte and mixed hippocampal cultures treated with
beta-amyloid peptides. J Neurochem 1996, 66:1641-1647.
Rosenberg GA: Matrix metalloproteinases in neuroinflammation. Glia
2002, 39:279-291.
Gasche Y, Fujimura M, Morita-Fujimura Y, Copin JC, Kawase M,
Massengale J, Chan PH: Early appearance of activated matrix
metalloproteinase-9 after focal cerebral ischemia in mice: a possible role
in blood-brain barrier dysfunction. J Cereb Blood Flow Metab 1999,
19:1020-1028.
Morita-Fujimura Y, Fujimura M, Gasche Y, Copin JC, Chan PH:
Overexpression of copper and zinc superoxide dismutase in transgenic
mice prevents the induction and activation of matrix metalloproteinases
after cold injury-induced brain trauma. J Cereb Blood Flow Metab 2000,
20:130-138.
Montaner J, Alvarez-Sabin J, Molina C, Angles A, Abilleira S, Arenillas J,
Gonzalez MA, Monasterio J: Matrix metalloproteinase expression after
human cardioembolic stroke: temporal profile and relation to
neurological impairment. Stroke 2001, 32:1759-1766.
Kim GW, Gasche Y, Grzeschik S, Copin JC, Maier CM, Chan PH:
Neurodegeneratio in striatum induced by the mitochondrial toxin 3nitropropionic acid: Role of matrix metalloproteinase-9 in early bloodbrain barrier disruption? J Neurosci 2003, 23:8733-8742.
Meli DN, Christen S, Leib SL: Matrix metalloproteinase-9 in pneumococcal
meningitis: Activation via an oxidative pathway. J Infect Dis 2003,
187:1411-1415.
Leib SL, Clements JM, Lindberg RL, Heimgartner C, Loeffler JM, Pfister LA,
Tauber MG, Leppert D: Inhibition of matrix metalloproteinases and
tumour necrosis factor alpha converting enzyme as adjuvant therapy in
pneumococcal meningitis. Brain 2001, 124:1734-1742.
Leppert D, Leib SL, Grygar C, Miller KM, Schaad UB, Hollander GA: Matrix
metalloproteinase (MMP)-8 and MMP-9 in cerebrospinal fluid during
bacterial meningitis: association with blood-brain barrier damage and
neurological sequelae. Clin Infect Dis 2000, 31:80-84.
Leib SL, Leppert D, Clements J, Tauber MG: Matrix metalloproteinases
contribute to brain damage in experimental pneumococcal meningitis.
Infect Immun 2000, 68:615-620.
Asahi M, Asahi K, Jung JC, del Zoppo GJ, Fini ME, Lo EH: Role for matrix
metalloproteinase 9 after focal cerebral ischemia: effects of gene
knockout and enzyme inhibition with BB-94. J Cereb Blood Flow Metab
2000, 20:1681-1689.
Asahi M, Wang X, Mori T, Sumii T, Jung JC, Moskowitz MA, Fini ME, Lo EH:
Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis
of blood-brain barrier and white matter components after cerebral
ischemia. J Neurosci 2001, 21:7724-7732.
Wang X, Jung J, Asahi M, Chwang W, Russo L, Moskowitz MA, Dixon CE,
Fini ME, Lo EH: Effects of matrix metalloproteinase-9 gene knock-out on
Page 9 of 10
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
morphological and motor outcomes after traumatic brain injury. J
Neurosci 2000, 20:7037-7042.
Dubois B, Masure S, Hurtenbach U, Paemen L, Heremans H, van den
Oord J, Sciot R, Meinhardt T, Hammerling G, Opdenakker G, Arnold B:
Resistance of young gelatinase B-deficient mice to experimental
autoimmune encephalomyelitis and necrotizing tail lesions. J Clin Invest
1999, 104:1507-1515.
Shabana MM, Mirhom YW, Genenah AA, Aboutabl EA, Amer HA: Study Into
Wild Egyptian Plants Of Potential Medicinal Activity. Ninth
communication: Hypoglycemic Activity Of Some Selected Plants In
Normal Fasting And Alloxanized Rats. Arch Exp Veterinarmedizin 1990,
44:389-394.
Mustafa EH, Abu Zarga M, Abdalla S: Effects of cirsiliol, a flavone isolated
from Achillea fragrantissima, on rat isolated ileum. Gen Pharmacol 1992,
23:555-560.
Yaniv Z, Dafni A, Friedman J, Palevitch D: Plants used for the treatment of
diabetes in Israel. J Ethnopharmacol 1987, 19:145-151.
Hamdan I, Afifi FU: Studies on the in vitro and in vivo hypoglycemic
activities of some medicinal plants used in treatment of diabetes in
Jordanian traditional medicine. J Ethnopharmacol 2004, 93:117-121.
Elmann A, Mordechay S, Rindner M, Ravid U: Anti-neuroinflammatory
effects of the essential oil from Pelargonium graveolens in microglial
cells. J Func Foods 2009, 2:17-22.
Kueng W, Silber E, Eppenberger U: Quantification of cells cultured on 96well plates. Anal Biochem 1989, 182:16-19.
Bowry VW, Stocker R: Tochoferol-mediated oxidation. The prooxidant
effect of vitamin E on the radical-initiated oxidation of human low
density lipoproteins. J Am Chem Soc 1993, 115:6029-6044.
Niki E, Saito M, Yoshikawa Y, Yamamoto Y, Kamiya Y: Oxidation of lipids
XII. Inhibition of oxidation of soybean phosphatidylcholine and methyl
linoleate in aqueous dispersions by uric acid. Bull Chem Soc Jpn 1986,
59:471-477.
Thomas MJ, Chen Q, Franklin C, Rudel LL: A comparison of the kinetics of
low-density lipoprotein oxidation initiated by copper or by azobis (2amidinopropane). Free Radic Biol Med 1997, 23:927-935.
Wolfe KL, Liu RH: Cellular antioxidant activity (CAA) assay for assessing
antioxidants, foods, and dietary supplements. J Agric Food Chem 2007,
55:8896-8897.
Mollace V, Colasanti M, Muscoli C, Lauro G, Iannone M, Rotiroti D, Nistico G:
The effect of nitric oxide on cytokine-induced release of PGE2 by
human cultured astroglial cells. Br J Pharmacol 1998, 124:742-746.
Yong VW, Krekoski CA, Forsyth PA, Bell R, Edwards DR: Matrix
metalloproteinases and diseases of the CNS. Trends Neurosci 1998,
21:75-80.
Gu Z, Kaul M, Yan B, Kridel SJ, Cui J, Strongin A, Smith JW, Liddington RC,
Lipton SA: S-nitrosylation of matrix metalloproteinases: signaling
pathway to neuronal cell death. Science (New York, N.Y.) 2002,
297:1186-1190.
Wang HH, Hsieh HL, Yang CM: Nitric oxide production by endothelin-1
enhances astrocytic migration via the tyrosine nitration of matrix
metalloproteinase-9. J Cell Physiol 2011, 226:2244-2256.
Salvemini D, Seibert K, Masferrer JL, Settle SL, Currie MG, Needleman P:
Nitric Oxide Activates the Cyclooxygenase Pathway in Inflammation. Am
J Ther 1995, 2:616-619.
Marnett LJ, Wright TL, Crews BC, Tannenbaum SR, Morrow JD: Regulation
of prostaglandin biosynthesis by nitric oxide is revealed by targeted
deletion of inducible nitric-oxide synthase. J Biol Chem 2000,
275:13427-13430.
Wu CY, Hsieh HL, Jou MJ, Yang CM: Involvement of p42/p44 MAPK, p38
MAPK, JNK and nuclear factor-kappa B in interleukin-1beta-induced
matrix metalloproteinase-9 expression in rat brain astrocytes. J
Neurochem 2004, 90:1477-1488.
Liu SF, Malik B: NF-kB activation as a pathological mechanism of septic
shock and inflammation. Am J Physiol Lung Cell Mol Physiol 2006, 290:
L622-L645.
Fiebich BL, Butcher RD, Gebicke-Haerter PJ: Protein kinase C-mediated
regulation of inducible nitric oxide synthase expression in cultured
microglial cells. J Neuroimmunol 1998, 92:170-178.
Bhat NR, Zhang P, Lee JC, Hogan EL: Extracellular signal-regulated kinase
and p38 subgroups of mitogen-activated protein kinases regulate
inducible nitric oxide synthase and tumor necrosis factor-alpha gene
Elmann et al. BMC Complementary and Alternative Medicine 2011, 11:98
http://www.biomedcentral.com/1472-6882/11/98
45.
46.
47.
48.
49.
Page 10 of 10
expression in endotoxin-stimulated primary glial cultures. J Neurosci
1998, 18:1633-1641.
Sondergaard BC, Schultz N, Madsen SH, Bay-Jensen AC, Kassem M,
Karsdal MA: MAPKs are essential upstream signaling pathways in
proteolytic cartilage degradation–divergence in pathways leading to
aggrecanase and MMP-mediated articular cartilage degradation.
Osteoarthritis Cartilage 2010, 18:279-288.
Auer M, Pfister LA, Leppert D, Tauber MG, Leib SL: Effects of clinically used
antioxidants in experimental pneumococcal meningitis. J Infect Dis 2000,
182:347-350.
Aid S, Bosetti F: Targeting cyclooxygenases-1 and -2 in
neuroinflammation: Therapeutic implications. Biochimie 2011, 93:46-51.
Allan SM, Pinteaux E: The interleukin-1 system: an attractive and viable
therapeutic target in neurodegenerative disease. Curr Drug Targets: CNS
Neurol Disord 2003, 2:293-302.
Rothwell NJ, Luheshi GN: Interleukin 1 in the brain: biology, pathology
and therapeutic target. Trends neurosci 2000, 23:618-625.
Pre-publication history
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doi:10.1186/1472-6882-11-98
Cite this article as: Elmann et al.: Anti-Neuroinflammatory effects of the
extract of Achillea fragrantissima. BMC Complementary and Alternative
Medicine 2011 11:98.
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