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Furanodien-6-one from Commiphora erythraea inhibits the NF-κB signalling and attenuates LPS-induced neuroinflammation
Molecular Immunology 54 (2013) 347–354
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Furanodien-6-one from Commiphora erythraea inhibits the NF-B signalling and attenuates LPS-induced neuroinflammation Ilaria Bellezza a,∗ , Annalisa Mierla a , Silvia Grottelli a , Maria Carla Marcotullio b , Federica Messina b , Luca Roscini c , Gianluigi Cardinali c , Massimo Curini b , Alba Minelli a a
Dipartimento di Medicina Sperimentale Scienze Biochimiche, Università di Perugia, 06123 Perugia, Italy Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia, 06124 Perugia, Italy c Dipartimento di Biologia Applicata-Microbiologia, Università di Perugia, 06121 Perugia, Italy b
a r t i c l e
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Article history: Received 4 October 2012 Received in revised form 20 December 2012 Accepted 4 January 2013 Available online 26 January 2013 Keywords: Microglia In vivo model SIRT1 iNOS COX-2
a b s t r a c t We investigated the in vitro anti-inflammatory activity of 1(10),4-furanodien-6-one, one the most active compounds of the hexane extract of Commiphora erythraea (Ehrenb.) Engl., by exposing microglial BV2 cells to lipopolysaccharide. We showed that furanodien-6-one pre-treatment restored cell viability and ROS to control levels while halving NO generation. Production of pro-inflammatory IL-6, IL-23, IL-17, TGF-, and INF-␥, significantly induced by LPS, was also markedly reduced by furanodien-6one treatment. We further showed that furanodien-6-one protects primary neuronal cultures against the inflammatory/toxic insults of LPS-treated BV-2 conditioned media, indicating that furanodien-6one exerts anti-inflammatory/cytoprotective effects in neuronal cells. We then investigated whether furanodien-6-one exerts anti-inflammatory properties in an in vivo model of microglial activation. In adult mice ip-injected with LPS we found that furanodien-6-one had strong cerebral anti-inflammatory properties by inhibiting liver and brain TNF␣ as well as IL-1 expression. Results were not unexpected since FTIR-metabolomic analyses showed that furanodien-6-one-treated mice had a reduced dissimilarity to control animals and that the response to LPS treatment was markedly modified by furanodien-6-one. In conclusion our data provide strong evidence of the anti-inflammatory properties of furanodien-6-one that could be exploited to counteract degenerative pathologies based on neuroinflammation. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Microglial activation contributes to neuronal damage in neurodegenerative diseases (Block et al., 2007; Kim et al., 2000). In response to certain environmental toxins and endogenous proteins, microglia can become over-active and release inflammatory mediators and reactive oxygen/nitrogen species (RONS) that cause neurotoxicity. Neurodegenerative disorders, i.e. Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and AIDS dementia are linked to oxidative stress-mediated inflammation (Tansey and Goldberg, 2010; Wen et al., 2007; Chen et al., 2010; Markowitz et al., 2007). Therefore, blocking the activation of microglial cells and subsequent release of inflammatory mediators might be a promising therapeutic approach against a variety of neurodegenerative diseases. Persistently elevated reactive oxygen species (ROS) levels
∗ Corresponding author at: Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione di Biochimica Cellulare, Università di Perugia, Via del Giochetto, 06123, Perugia, Italy. Tel.: +39 075 5857440; fax: +39 075 5857442. E-mail address: [email protected] (I. Bellezza). 0161-5890/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2013.01.003
result in oxidative stress and cell death. They also activate the nuclear factor-B (NF-B) system, a crucial player in the inflammatory response, which is characterized by enhanced expression of pro-inflammatory cytokines, chemokines, adhesion molecules, growth factors, and enzymes that produce secondary inflammatory mediators, i.e. cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) (Bellezza et al., 2010; Brigelius-Flohe et al., 2011). NF-B-induced proteins can activate NF-B itself, thus leading to a self-propelling vicious loop that amplifies the initial response (Brigelius-Flohe et al., 2011). Lipopolysaccharide (LPS) is a common inflammogen that, by interacting with the membrane receptor Toll-like receptor 4 (TLR4), triggers an array of microglial responses leading to the production of pro-inflammatory mediators and NF-B self-activation (Kim et al., 2000; Meng and Lowell, 1997; Chung et al., 2010; Hinkerohe et al., 2010). Traditionally, Commiphora species have been used in southern Africa for the treatment of colds, fever, malaria, typhoid, wound healing, snake and scorpion bites, tumours, stomach aches, diseases of the gall bladder, chest ailments and skin infections. To date, Commiphora species have been a source of several novel and bio-active compounds (Lemenih et al., 2003; Hanuˇs et al., 2005; Paraskeva et al., 2008).
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1(10),4-Furanodien-6-one (FDN) is a furanosesquiterpenoid isolated and purified from resin hexan extract of Commiphora erythraea (Ehrenb.) Engl. and other Commiphora spp. Recently, it has been shown that the furanosesquiterpenoids possess antifungal, antioxidant and anti-inflammatory properties. In particular, topic application of FDN, contained in the H2 fraction of the resin hexan extract, reduces Croton oil-induced ear oedema in mice (Fraternale et al., 2011) and protects from LPS-induced cellular damage (Marcotullio et al., 2011). Microgliosis, as a common hallmark in neurodegenerative diseases (Block et al., 2007; Kim et al., 2000; Tansey and Goldberg, 2010; Wen et al., 2007; Chen et al., 2010; Markowitz et al., 2007), suggests an inflammatory mechanism in the progression of these disorders. Given that FDN reduces the pro-inflammatory action of LPS and the murine ear oedema, we hypothesized that FDN can also exert cerebral anti-inflammatory effects. Here, we investigated whether FDN can counteract the glial inflammation induced by a systemic LPS injection in mice and studied its molecular mechanism in LPS-activated microglial BV-2 cells. 2. Materials and methods 2.1. Materials All the reagents, unless otherwise stated, were from Sigma–Aldrich (St. Louis, MO). All the antibodies, unless otherwise stated, were from Santa Cruz Biotech (Santa Cruz, CA). Cell culture reagents were from Life Technologies (GibcoBRL, Gaithersburg, MD). 2.2. Plant materials The resin of C. erythraea (Agarsu grade I) was purchased from Agarsu Liben Cooperative (IPO, Increasing People Opportunities) association (www.ipoassociazione.org), Italy. A voucher specimen (# MCM-1) of the resin is deposited at Dipartimento di Chimica e Tecnologia del Farmaco, Sezione di Chimica Organica, University of Perugia (Italy). Ground resin (5 g) was extracted with 250 ml of n-hexane (4 h at RT), the suspension filtered under vacuum and the solvent removed under N2 . The composition of hexane extract by GC–MS technique has already been reported (Fraternale et al., 2011). Fractionation of the hexane extract (Marcotullio et al., 2011) resulted in the isolation of furanodien-6-one (C15 H20 O2 , Mw 232) (Dekebo et al., 2000, 2002), which, dissolved in DMSO (Stock solution 500 mM), was used in the study. 2.3. Animals Six/eight week-old male C57BL/6 mice (18–20 g) were housed at the Laboratory Animal Research Centre of Perugia University. The animals were maintained at a constant temperature of 24 ◦ C and 12-h light/dark cycle. 2.3.1. Ethics statement All experimental procedures were carried out in accordance with European Directives, approved by the Institutional Animal Care and Use Committee of Perugia University (N34/2009). Efforts were made to minimize animal stress/discomfort. 2.3.2. Animal Treatment Forty animals were divided into four groups. One group (10 mice) was ip injected with a single dose of LPS (5 mg/kg) (Qin et al., 2007). The second group received two ip injections of FDN (2.4 mg/kg): the first, 1 h prior to LPS injection and the second 30 min afterwards. Previous determinations of hepatic expression of Tnf˛ with different doses (1.2/2.4/3.6 mg/kg corresponding to
5/10/15 moles/kg) of FDN indicated 2.4 mg/kg as the minimal effective dose. The third group was treated with FDN alone and the fourth group with vehicle and used as control. Mice were sacrificed 3 h after LPS treatment, brain and liver used for real time PCR analyses and histological observations. 2.4. Cell culture and viability Murine macrophage RAW264.7 (ATCC, Rockville, MD) were cultured in DMEM supplemented with 2 mM glutamine,10% Foetal Bovine Serum (FBS), penicillin (100 U/ml), and streptomycin (100 g/ml) at 37 ◦ C in a humidified incubator under 5% CO2 . BV-2 cells were cultured in RPMI supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 g/ml) at 37 ◦ C in a humidified incubator under 5% CO2 . After 24 h subculture, cells (RAW 264.7: 4 × 104 , and BV-2: 9 × 104 ) were incubated for 24 h with FDN (50 M for BV-2 and 10 M for RAW 264.7), and then exposed to 10 g/ml lipopolysaccharide (LPS, Escherichia coli serotype 0111:B4) for the indicated times. Conditioned media (CCMs) were obtained from BV-2 cells after a 18 h exposure at LPS. Murine primary neuronal cultures were prepared according to Almeida and Medina (1998) in the presence of 10 M Ara C. Neuronal cells (8 × 104 ) were incubated for 24 h with 50 M FDN prior to exposure to BV-2-conditioned media (CCMs) for 24–72 h. Live cells were counted by hemocytometer, viability assessed by the (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) MTT reduction assay, and the absorbance at 550 nm was measured with a microplate reader (Seac, Florence, Italy). Results were expressed as the percentages of reduced MTT, assuming the absorbance of control cells as 100%. 2.5. Measurement of intracellular fluorescence The DCFH-DA method was used to detect the levels of intracellular reactive oxygen species (ROS) (Eruslanov and Kusmartsev, 2010). Cells (9 × 104 ) were treated as described and DCFH-DA (10 M) was added to the medium for a further 30 min at 37 ◦ C. The fluorescence of 2 ,7 -dichlorofluorescein was detected at 485 nm excitation and at 535 nm emission, using a plate reader Titertek Fluoroscan II (Flow Laboratories, Mc Lean, VA). Results were expressed as the percentage of the control DCF fluorescence, assumed as 100%. 2.6. Measurement of NO production Nitric oxide (NO) production was determined by the Griess reaction. Cells (9 × 104 ) were treated as described, then a 50 l aliquot of medium was mixed with an equal volume of Griess reagent and incubated for 20 min at RT. The absorbance was read at 550 nm using a microtiter plate reader (Seac, Florence, Italy) and results were expressed as the percentage of the control, assumed as 100%. Nitrite standard reference curve was prepared for each determination. 2.7. Measurement of cytokine levels The effects of FDN on cytokine production were measured with an enzyme-linked immunosorbent assay (ELISA) kit in CCMs collected from LPS-treated cells. Samples were analyzed according to the manufacturer’s protocols (BD Biosciences Pharmingen, San Diego, CA). 2.8. Electrophoretic mobility shift assay Synthetic double-stranded biotin 5 -oligonucleotide containing B responsive element (BRE) (5 -AGTTGAGGGGACTTTCCCAGGC3 and 3 -TCAACTCCCCTGAAAGGGTCCG-5 ) (M-Medical Srl, Milano,
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Italy) were used as probes. Nuclear lysates (8 g), obtained with NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL), were incubated with the labelled probe (15 fmol) for 20 min at RT, resolved on an 6% native polyacrylamide gel, and transferred to Hybond-N+ (Amersham Pharmacia). Signals were visualized with peroxidase-conjugated streptavidin (Pierce Biotechnology, Rockford, IL).
2.9. Western blotting analyses Whole cell extracts were obtained with 10% (w/v) of warm Laemmli sample buffer 1× and sonication for 15 s, nuclear/cytosolic extracts were obtained with NE-PER® Nuclear and Cytoplasmic Extraction Reagents. Extracts (20–50 g) were loaded on a 12% SDS–polyacrylamide gel and protein levels determined by Western blotting using NF-Bp65 (C-20) (1:100), COX-2 (C20) (1:200), iNOS (M19) (1:100), SIRT1(H300)(1:100) antibodies and horseradish peroxidase-conjugated-anti rabbit/mouse/goat
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IgG antibody (1:5000). Lamin B (H-90) and -actin (C-4) antibodies (1:400) were used as marker proteins for nuclear/cytosolic extracts. Immunocomplexes were visualized with an enhanced chemiluminescence kit (ECL, Pierce Biotechnology, Rockford, IL). 2.10. Glial fibrillar acidic protein immunohistochemistry Immediately after animal sacrifice, brains were removed and one hemisphere was immersion-fixed in Carnoy solution and embedded in paraffin. Cerebral sections at defined levels (10 m) were processed to evidence immunoreactivity for GFAP by Leica DM2500, DFC 420c and Application Suite V3.4 system microscope (Leica Microsystems Srl, Italy). 2.11. Real time PCR Total RNA was isolated with TRIZOL Reagent (Invitrogen Ltd, Paisley, UK) according to the manufacturer’s instructions and cDNA
Fig. 1. FDN reduces LPS toxic effects. (A) BV-2 cells were incubated with 50 M FDN for 24 h and then exposed to 10 g/ml LPS for 4 and 24 h; viability was detected by MTT assay (100% control MTT absorbance: 0.9 ± 0.07 at 4 h, 1.2 ± 0.1 at 24 h); NO production, detected by Griess Reagent (100% control NO absorbance: 0.20 ± 0.03) and ROS generation, detected by DCFH-DA (100% control ROS fluorescence: 0.8 ± 0.06 at 4 h, 0.90 ± 0.03 at 24 h). (B) iNos and Cox-2 gene expression by real time PCR analysis. Changes in gene expression, whose values were normalized to Gapdh expression, are presented as 2−Ct . Relative mRNA gene abundance in untreated cells was assumed to be 1.0 (control). (C) Time-course of iNOS and COX-2 protein levels. At each indicated time, cells were collected and whole cell lysates (40 g) were subjected to Western blotting analyses with the indicated antibodies. (D) iNOS and COX-2 protein levels in murine macrophage RAW264.7 cells. Cells were incubated with 10 M FDN for 24 h and then exposed to 10 g/ml LPS for 24 h, collected and whole cell lysates (20 g) were subjected to Western blotting analyses with the indicated antibodies. -actin was used as loading control. Data represent means ± S.D. of at least three experiments. *P < 0.05 vs control cells. #P < 0.05 vs LPS treated cells.
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was synthesized using iScript cDNA synthesis kit (Bio-Rad Lab, Hercules, CA). Real time PCR was performed using the iCycler iQ detection system (Bio-Rad) and SYBR Green chemistry. Mouse primers (Table S1) were obtained from Invitrogen (Invitrogen Ltd, Paisley, UK). Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.molimm.2013.01.003.
2.13. Statistical analysis All results were confirmed in at least three separate experiments and expressed as mean ±S.D. Data were analyzed for statistical significance by ANOVA followed by post hoc analysis. P-values <0.05 were considered significant. 3. Results 3.1. FDN reduces LPS-mediated toxic effects in BV-2 cells
2.12. Fourier transform infrared spectroscopy FTIR measurements were performed in transmission mode (Corte et al., 2010). All spectra were recorded in the range 4000 and 400 cm−1 with a TENSOR 27 FTIR spectrometer, equipped with HTS-XT accessory (BRUKER Optics GmbH, Ettlingen, Germany). OPUS version 6.5 software (BRUKER Optics GmbH, Ettlingen, Germany) was used to carry out the quality test, baseline correction, vector normalization and the calculation of the first and second derivatives of spectral values. Hierarchical cluster analysis, i.e. assigning a set of objects into groups (called clusters) so that the objects in the same cluster are more similar (in some sense or another) to each other than to those in other clusters, was performed using the software OPUS to highlight similarities or differences in terms of statistical distance. Spectra were divided into five spectral regions (fatty acids (W1) from 3000 to 2800 cm− 1, amides (W2) from 1800 to 1500 cm− 1, mixed region (W3) from 1500 to 1200 cm− 1, carbohydrates (W4) from 1200 to 900 cm− 1 and typing region (W5) from 900 to 700 cm− 1),exported as an ASCII file from OPUS and used in the “R” environment (http://cran.r-project.org/). Response spectra (RS) were calculated as the difference between each average spectrum and the average spectrum of control tissue, which is by definition a straight line with RS = 0. The intensity of the metabolomic response results from the difference between zero and the spectral line. Five mg of each liver were homogenized in saline, centrifuged at 2000 × g, washed twice and re-suspended in ultra pure water. For each sample, three/five repetitions were placed on the 96-multiwell, dried at 42 ◦ C for 20 min and used for the analysis.
We have already shown that FDN protects BV-2 cells from LPS toxicity (Marcotullio et al., 2011). Here we further analyzed the effects of FDN on BV-2 cells by investigating the molecular mechanism underlying this protection. LPS cytotoxicity was timedependent: a 4 h exposure to 10 g/ml LPS did not modify cell viability whereas a 24 h exposure caused a marked reduction in cell viability. NO and ROS generation were increased at each considered time. At 24 h, pre-treatment with 50 M FDN restored cell viability and ROS to control levels while halving NO production (Fig. 1A). FDN treatment maintained cell viability and decreased NO levels when added to microglial cells up to 1 h after LPS injury (data not shown). NF-B is a redox-sensitive transcription factor that plays a key role in the expression of a number of genes that are central to the inflammatory response (Brigelius-Flohe et al., 2011). To demonstrate the inhibition of the NF-B- inflammatory pathway by FDN, we performed a real time PCR analysis of NF-B-driven genes, i.e. iNos (inducible nitric oxide synthase) and Cox-2 (cyclooxygenase2) in LPS-exposed BV-2 (Fig. 1B). iNos and Cox-2 mRNA levels were strongly up regulated by LPS exposure and markedly decreased by FDN pre-treatment. Compatible with these results, the LPS-induced increases in iNOS and COX-2 protein expression were reduced by FDN pre-treatment (Fig. 1C). iNOS and COX-2 protein expression were reduced by FDN pre-treatment also in murine macrophage RAW264.7 cells thus confirming the FDN anti-inflammatory properties (Fig. 1D). Next we determined the levels of cytokines in LPS treated cells in the presence of FDN (Fig. 2). The production of pro-inflammatory IL-6, IL-23, IL-17A, TGF-, and INF-␥, significantly induced by LPS, was markedly reduced by FDN treatment while IL-27 production
Fig. 2. FDN reduces inflammatory cytokines levels. BV-2 cells were pre-treated with 50 M FDN for 24 h and then stimulated with 10 g/ml LPS for 18 h. Cytokines from BV-2 cell conditioned media were assayed by ELISA. Values represent mean ± S.D. of at least three experiments. *P < 0.05 vs control cells. #P < 0.05 vs LPS-treated cells.
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was not significantly modified. In all experiments, treatment with FDN alone did not modify control values (data not shown). Results show that FDN is responsible for the inhibition of NF-B signalling confirming its in vitro anti-inflammatory activity. 3.2. FDN protects primary neuronal cultures against glial over-activation Chronic glial activation is detrimental to neurons through sustained production of neurotoxic biomolecules (Block et al., 2007; Kim et al., 2000; Tansey and Goldberg, 2010; Wen et al., 2007; Chen et al., 2010; Markowitz et al., 2007). After showing that FDN attenuates BV-2 over-activation, we investigated whether the compound is also capable of protecting neuronal cells from glial-induced neurotoxicity. Thus we exposed murine primary neuronal cultures to LPS-activated BV-2 culture media (CCM). CCMs markedly reduced
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neuronal viability and increased NO generation. FDN pre-treatment attenuated the negative effects of CCMs by restoring neuronal viability to control values and significantly reducing NO production, indicating that FDN can protect primary neuronal cultures against over-activated microglial neurotoxicity (Fig. 3A). Next we investigated the effects of FDN on NF-B gene expression, nuclear translocation, DNA-binding activity and transcriptional efficiency by real time PCR, EMSA, and Western blotting (Fig. 3B–E). FDN reduced the mRNA levels, nuclear translocation and the binding capacity to BRE sequence of NF-B as well as COX-2 protein levels, confirming that the inflammatory pathway is inhibited by FDN. We then investigated whether FDN is also capable of modulating neuronal Sirt1 expression. Results of real time PCR and Western blotting analyses showed that in neuronal primary cultures Sirt1 expression as well as SIRT1 protein levels are significantly up regulated by FDN treatment suggesting the involvement of SIRT1 in the
Fig. 3. FDN protects primary neuronal cultures against microglial over-activation. (A) Primary neuronal cultures, pre-treated with 50 M FDN for 24 h, were exposed to CCMs for different times. Viability, detected by MTT assay (100% control MTT absorbance:0.89 ± 0.06), NO production, detected by Griess Reagent (100% control NO absorbance: 0.110 ± 0.09, 0.06 ± 0.001, 0.06 ± 0.005 at 24, 48, and 72 h). (B–E) FDN modulates the NF-B system. Primary neuronal cultures, pre-treated with 50 M FDN for 24 h, were exposed to CCMs for 48 h and collected for analyses: (B) real time PCR analysis of mRNA levels of the NF-B gene. (C) nuclear translocation of NF-B. Nuclear extracts (50 g proteins) were subjected to Western blotting analysis with NF-B antibody. Lamin B and -actin antibodies were used as markers of nuclear/cytosolic extracts. (D) DNA binding capacity of NF-B. EMSAs were performed by using 8 g nuclear protein lysate per lane and a biotin-labelled BRE probe (15 fmol). (E) transcriptional efficiency of NF-B. Whole cell lysates (40 g) were subjected to Western blotting analyses with the indicated antibody. -actin was used as loading control. (F) real time PCR analysis of mRNA levels of Sirt1 gene. (G) Western blotting analyses of SIRT1 protein levels. Whole cell lysates (40 g) were analyzed with the indicated antibody. -actin was used as loading control. Data represent means ± S.D. of at least three experiments. *P < 0.05 vs control cells. #P < 0.05 vs CCMs-treated cells.
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FDN causes a response markedly different from the response that follows the LPS treatment (Fig. 4B). Similar results were obtained by using average spectra of FDN-treated animals as a straight line with RS = 0 (data not shown). Results indicate that FDN markedly modifies the hepatic response to LPS. Systemic cytokines mediate the effects of peripheral inflammation on the brain and reactive gliosis underlies the progressive and long-lasting neuroinflammation and neurodegeneration (Block et al., 2007; Kim et al., 2000; Tansey and Goldberg, 2010; Wen et al., 2007; Chen et al., 2010; Markowitz et al., 2007; Meng and Lowell, 1997; Chung et al., 2010; Qin et al., 2007). Thus, at 3 h post LPS-treatment, we examined several brain regions, i.e. striatum, hippocampus, and cortex and observed an increase in GFAP-immunoreactivity compatible with glial overactivation. Treatment with LPS in the presence of FDN resulted in a decreased GFAP-immunoreactivity. FDN alone did not modify GFAP-immunoreactivity (Fig. 5A). The marked increases in mRNA levels of Tnf˛ and IL1B after LPS treatment were strongly reduced by FDN treatment. Only cortical up-regulation of IL1B expression was not counteracted by FDN, probably related to temporal regionspecific differential susceptibility to anti-inflammatory agents (Kim et al., 2000). FDN alone did not significantly alter gene expression of the control group. Results provide strong evidence of the in vivo antiinflammatory properties of the FDN (Fig. 5B).
4. Discussion
Fig. 4. FDN decreases LPS-induced hepatic metabolomic alterations. For each sample, three/five repetitions were placed on the 96-multiwell, dried at 42 ◦ C for 20 min and used for the analyses. (A) Hierarchical cluster analysis of the W1 region with the second derivative of sample averages showing a low grade heterogeneity between control and FDN-treated samples. (B) Response spectra (RS) analysis: variations by LPS in the positive range were abolished by FDN treatment.
attenuation of the NF-B system (Fig. 3F–G). Data provide evidence that FDN protects neuronal cells against over-activated microglial neurotoxicity. 3.3. FDN reduces neuroinflammation in response to systemic LPS administration Finally we investigated whether FDN shows anti-inflammatory properties in an in vivo model of glial activation. Because a single ip LPS injection results in liver TNF␣ production, that, in turn, initiates brain TNF␣ production, responsible for a chronic neuroinflammation (Qin et al., 2007), we first analyzed the hepatic metabolome profiles with FTIR spectroscopy. Hierarchical cluster analysis showed that the samples can be divided into three clades: one containing the samples treated with LPS, the second containing the control/FDN alone-treated samples and the third containing the samples treated with LPS plus FDN. This clade is very close to the control and clearly separated from the LPS-treated samples, since it shows a great statistical distance and a high level of heterogeneity compared to LPS-treated samples (Fig. 4A). Spectral data were then analyzed with an algorithm that allows different statistical treatments. Response spectra (RS) (Corte et al., 2010), where average spectra of control animals are assumed by definition as a straight line with RS = 0, showed that the treatment with
Here we showed for the first time that systemic administration of FDN exerts in vivo anti-inflammatory effects in the central nervous system by decreasing the expression of liver and brain Tnf˛ and IL1B. Our data also provide evidence that FDN can protect neurons exposed to microglia-produced inflammatory and neurotoxic factors thorough the inhibition of the NF-B system. Lipopolysaccharide (LPS), an endotoxin from gram-negative bacteria, is known to activate protein kinase C, protein–tyrosine kinases, mitogen-activated protein kinases, and NF-B, each protein/enzyme involved in the release of immune-related cytotoxic factors (Wen et al., 2007; Meng and Lowell, 1997; Chung et al., 2010; Hinkerohe et al., 2010). Given the influence of intracellular ROS signalling on several kinase-mediated pathways (Bellezza et al., 2010; Brigelius-Flohe et al., 2011; Lin et al., 2011), the LPS signalling pathway in microglia is a likely target for downstream ROS signalling, one of the key factors in LPS-mediated neurodegeneration. Indeed, LPS can damage neurons only in the presence of microglia (Gao et al., 2002; Gibbons and Dragunow, 2006; Rivest et al., 2000) by a reactive microgliosis process triggered by pro-inflammatory factors (Qin et al., 2007). An acute systemic injection of LPS is followed by a rapid increase in the hepatic expression of TNF␣ which, in turn, through TNF␣ receptors, activates the microglia thus initiating a sustained activation of brain cytokine synthesis and neuroinflammation. This sustained neuroinflammation leads to a delayed and progressive loss of dopaminergic neurons over a 7–10 months period (Qin et al., 2007). We showed that mice, treated with FDN, have decreased hepatic mRNA levels of Tnf˛, probably responsible for the attenuated microglia activation as well as marked brain Tnf˛ and IL1B down-regulation. FTIR spectroscopy analyses are mostly qualitative and do not quantify/characterize individual metabolites. However, both hierarchic cluster analyses and response spectra showed that the metabolomic profile of FDN plus LPS-treated animals strongly correlates with control animals and that FDN can suppress the hepatic response to LPS stress, hinting to beneficial effects of FDN. Oxidative damage activates redox-sensitive transcription factors and the increased production of reactive oxygen
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Fig. 5. FDN reduces neuroinflammation in response to systemic LPS administration. C57BL/6 mice were sacrificed 3 h following LPS (5 mg/kg) i.p. injection. (A) Immunohistochemical analyses. Brain sections were immunostained with GFAP specific antibody. The images are from one experiment that is representative of ten separate experiments. (B) real time PCR analysis of Tnf˛ and IL1B in cortex, striatum, hippocampus and liver. Changes in gene expression, whose values were normalized to Gapdh expression, are presented as 2−Ct . Data represent means ± S.D. of at least ten experiments. *P < 0.05 vs control cells. #P < 0.05 vs LPS-treated animals.
species is associated with up regulation of NF-B (Brigelius-Flohe et al., 2011). Activation of NF-B, in turn, induces the expression of pro-inflammatory genes, including cytokines, growth factors, and chemokines (Mattson and Meffert, 2006). Because some of the NF-B -induced proteins are also potent NF-B activators, the resulting vicious cycle may contribute to the establishment of a chronic inflammatory state and related pathologies. Prolonged innate immune responses, including activation of microglia and astrocytes, are seen in various neurodegenerative diseases (Block et al., 2007; Kim et al., 2000; Tansey and Goldberg, 2010; Wen et al., 2007; Chen et al., 2010; Markowitz et al., 2007). We showed that in vitro FDN protection is based on the inhibition of the NFB pro-inflammatory pathway and decreased expression of COX-2. NF-B activation is due to the liberation of the transcription factor from its cytosolic complex with IB thus allowing nuclear import and downstream gene activation (Bellezza et al., 2010; BrigeliusFlohe et al., 2011; Lin et al., 2011). In the nucleus NF-B, i.e. the RelAp65/p50 complex, binds to its promoter sequence together with the transcriptional co-activators CBP and p300, which have histone acetylase (HAT) activity. RelAp65, p50 and histones are
then acetylated leading to uncoiling of chromatin, thus allowing the access of NF-B to its canonical and other promoter regions (Gloire and Piette, 2009). SIRT1, the mammalian homologue of the evolutionarily conserved NAD+ -dependent deacetylase Sir2 (Michan and Sinclair, 2007), increases as a protective response to neurodegenerative conditions (Kim et al., 2007; Chen et al., 2005; Zhang et al., 2010). By deacetylating RelA/p65, it significantly inhibits the transactivation of NF-B and the expression of inflammatory cytokines (Kim et al., 2007; Chen et al., 2005; Lanzillotta et al., 2010; Yeung et al., 2004), thus blocking the neuropathogenic inflammatory loops (Michan and Sinclair, 2007; Zhang et al., 2010; Salminen et al., 2011). In this study we have provided evidence that primary neuronal cultures, treated with FDN prior to the neurotoxic insult, show a reduction in NF-B expression, nuclear translocation and binding activity as well as an up regulation in Sirt1 expression and SIRT1 protein levels. All data suggest that the inhibition of the proinflammatory pathway may be ascribed, at least partially, to the interference of SIRT1 with the complex interplay of the NF-B system. Thus FDN reduces glial activation and toxicity to neighbouring neurons which, in turn, by up-regulating Sirt1 expression, decrease
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glial activation and interrupt the vicious loop between neurons and glia. Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. Acknowledgements We thank Prof FR Donato (University of Perugia, Italy) for providing BV-2 and RAW264.7 cells. We also thank Dr M Kerrigan for helpful linguistic suggestions. IB, AM, GC, and MC contributed to conception, design, interpretation of data, and wrote the Ms. MCM and FM purified the compound. LR performed metabolomic analyses. ALM, SG performed the experiments. This work was supported by Fondazione Cassa di Risparmio, Perugia, Italy (N. 2010.011.0410) to MC. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References Almeida, A., Medina, J.M., 1998. A rapid method for the isolation of metabolically active mitochondria from rat neurons and astrocytes in primary culture. Brain Research Protocols 2, 209–214. Bellezza, I., Mierla, A.L., Minelli, A., 2010. Nrf2 NF-B and their concerted modulation in cancer pathogenesis and progression. Cancers 2, 483–487. Block, M.L., Zecca, L., Hong, J.S., 2007. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nature Reviews Neuroscience 8, 57–69. Brigelius-Flohe, R., Flohé, L., 2011. Basic principles and emerging concepts in the redox control of transcription factors. Antioxidants & Redox Signalling 15, 2335–2381. Chen, J., Zhou, Y., Mueller-Steiner, S., Chen, L.F., Kwon, H., Yi, S., Mucke, L., Gan, L., 2005. SIRT1 protects against microglia-dependent amyloid-beta toxicity through inhibiting NF-kappaB signaling. Journal of Biological Chemistry 280, 40364–40374. Chen, C.J., Ou, Y.C., Lin, S.Y., Raung, S.L., Liao, S.L., Lai, C.Y., Chen, S.Y., Chen, J.H., 2010. Glial activation involvement in neuronal death by Japanese encephalitis virus infection. Journal of General Virology 91, 1028–1037. Chung, D.W., Yoo, K.Y., Hwang, I.K., Kim, D.W., Chung, J.Y., Lee, C.H., Choi, J.H., Choi, S.Y., Youn, H.Y., Lee, I.S., Won, M.H., 2010. Systemic administration of lipopolysaccharide induces cyclooxygenase-2 immunoreactivity in endothelium and increases microglia in the mouse hippocampus. Cellular and Molecular Neurobiology 30, 531–541. Corte, L., Rellini, P., Roscini, L., Fatichenti, F., Cardinali, G., 2010. Development of a novel, FTIR (Fourier transform infrared spectroscopy) based, yeast bioassay for toxicity testing and stress response study. Analytica Chimica Acta 659, 258–265. Dekebo, A., Dagne, E., Hansen, L.K., Gauntun, O.R., Aasen, A.J., 2000. Crystal structures of two furanosesquiterpenes from Commiphora sphaerocarpa. Tetrahedron Letters 41, 9875–9878. Dekebo, A., Dagne, E., Sterner, O., 2002. Furanosesquiterpenes from Commiphora sphaerocarpa and related adulterants of true myrrh. Fitoterapia 73, 48–55. Eruslanov, E., Kusmartsev, S., 2010. Identification of ROS using oxidized DCFDA and flow-cytometry. Methods in Molecular Biology 594, 57–72. Fraternale, D., Sosa, S., Ricci, D., Genovese, S., Messina, F., Tomasini, S., Montanari, F., Marcotullio, M.C., 2011. Anti-inflammatory, antioxidant and antifungal furanosesquiterpenoids isolated from Commiphora erythraea (Ehrenb.) Engl. Resin. Fitoterapia 82, 654–661. Gao, H.M., Hong, J.S., Zhang, W., Liu, B., 2002. Distinct role for microglia in rotenoneinduced degeneration of dopaminergic neurons. Journal of Neuroscience 22, 782–790. Gibbons, H.M., Dragunow, M., 2006. Microglia induce neural cell death via a proximity-dependent mechanism involving nitric oxide. Brain Research 1084, 1–15.
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Furanodien-6-one from Commiphora erythraea inhibits the NF-B signalling and attenuates LPS-induced neuroinflammation Ilaria Bellezza a,∗ , Annalisa Mierla a , Silvia Grottelli a , Maria Carla Marcotullio b , Federica Messina b , Luca Roscini c , Gianluigi Cardinali c , Massimo Curini b , Alba Minelli a a
Dipartimento di Medicina Sperimentale Scienze Biochimiche, Università di Perugia, 06123 Perugia, Italy Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia, 06124 Perugia, Italy c Dipartimento di Biologia Applicata-Microbiologia, Università di Perugia, 06121 Perugia, Italy b
a r t i c l e
i n f o
Article history: Received 4 October 2012 Received in revised form 20 December 2012 Accepted 4 January 2013 Available online 26 January 2013 Keywords: Microglia In vivo model SIRT1 iNOS COX-2
a b s t r a c t We investigated the in vitro anti-inflammatory activity of 1(10),4-furanodien-6-one, one the most active compounds of the hexane extract of Commiphora erythraea (Ehrenb.) Engl., by exposing microglial BV2 cells to lipopolysaccharide. We showed that furanodien-6-one pre-treatment restored cell viability and ROS to control levels while halving NO generation. Production of pro-inflammatory IL-6, IL-23, IL-17, TGF-, and INF-␥, significantly induced by LPS, was also markedly reduced by furanodien-6one treatment. We further showed that furanodien-6-one protects primary neuronal cultures against the inflammatory/toxic insults of LPS-treated BV-2 conditioned media, indicating that furanodien-6one exerts anti-inflammatory/cytoprotective effects in neuronal cells. We then investigated whether furanodien-6-one exerts anti-inflammatory properties in an in vivo model of microglial activation. In adult mice ip-injected with LPS we found that furanodien-6-one had strong cerebral anti-inflammatory properties by inhibiting liver and brain TNF␣ as well as IL-1 expression. Results were not unexpected since FTIR-metabolomic analyses showed that furanodien-6-one-treated mice had a reduced dissimilarity to control animals and that the response to LPS treatment was markedly modified by furanodien-6-one. In conclusion our data provide strong evidence of the anti-inflammatory properties of furanodien-6-one that could be exploited to counteract degenerative pathologies based on neuroinflammation. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Microglial activation contributes to neuronal damage in neurodegenerative diseases (Block et al., 2007; Kim et al., 2000). In response to certain environmental toxins and endogenous proteins, microglia can become over-active and release inflammatory mediators and reactive oxygen/nitrogen species (RONS) that cause neurotoxicity. Neurodegenerative disorders, i.e. Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and AIDS dementia are linked to oxidative stress-mediated inflammation (Tansey and Goldberg, 2010; Wen et al., 2007; Chen et al., 2010; Markowitz et al., 2007). Therefore, blocking the activation of microglial cells and subsequent release of inflammatory mediators might be a promising therapeutic approach against a variety of neurodegenerative diseases. Persistently elevated reactive oxygen species (ROS) levels
∗ Corresponding author at: Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione di Biochimica Cellulare, Università di Perugia, Via del Giochetto, 06123, Perugia, Italy. Tel.: +39 075 5857440; fax: +39 075 5857442. E-mail address: [email protected] (I. Bellezza). 0161-5890/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2013.01.003
result in oxidative stress and cell death. They also activate the nuclear factor-B (NF-B) system, a crucial player in the inflammatory response, which is characterized by enhanced expression of pro-inflammatory cytokines, chemokines, adhesion molecules, growth factors, and enzymes that produce secondary inflammatory mediators, i.e. cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) (Bellezza et al., 2010; Brigelius-Flohe et al., 2011). NF-B-induced proteins can activate NF-B itself, thus leading to a self-propelling vicious loop that amplifies the initial response (Brigelius-Flohe et al., 2011). Lipopolysaccharide (LPS) is a common inflammogen that, by interacting with the membrane receptor Toll-like receptor 4 (TLR4), triggers an array of microglial responses leading to the production of pro-inflammatory mediators and NF-B self-activation (Kim et al., 2000; Meng and Lowell, 1997; Chung et al., 2010; Hinkerohe et al., 2010). Traditionally, Commiphora species have been used in southern Africa for the treatment of colds, fever, malaria, typhoid, wound healing, snake and scorpion bites, tumours, stomach aches, diseases of the gall bladder, chest ailments and skin infections. To date, Commiphora species have been a source of several novel and bio-active compounds (Lemenih et al., 2003; Hanuˇs et al., 2005; Paraskeva et al., 2008).
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1(10),4-Furanodien-6-one (FDN) is a furanosesquiterpenoid isolated and purified from resin hexan extract of Commiphora erythraea (Ehrenb.) Engl. and other Commiphora spp. Recently, it has been shown that the furanosesquiterpenoids possess antifungal, antioxidant and anti-inflammatory properties. In particular, topic application of FDN, contained in the H2 fraction of the resin hexan extract, reduces Croton oil-induced ear oedema in mice (Fraternale et al., 2011) and protects from LPS-induced cellular damage (Marcotullio et al., 2011). Microgliosis, as a common hallmark in neurodegenerative diseases (Block et al., 2007; Kim et al., 2000; Tansey and Goldberg, 2010; Wen et al., 2007; Chen et al., 2010; Markowitz et al., 2007), suggests an inflammatory mechanism in the progression of these disorders. Given that FDN reduces the pro-inflammatory action of LPS and the murine ear oedema, we hypothesized that FDN can also exert cerebral anti-inflammatory effects. Here, we investigated whether FDN can counteract the glial inflammation induced by a systemic LPS injection in mice and studied its molecular mechanism in LPS-activated microglial BV-2 cells. 2. Materials and methods 2.1. Materials All the reagents, unless otherwise stated, were from Sigma–Aldrich (St. Louis, MO). All the antibodies, unless otherwise stated, were from Santa Cruz Biotech (Santa Cruz, CA). Cell culture reagents were from Life Technologies (GibcoBRL, Gaithersburg, MD). 2.2. Plant materials The resin of C. erythraea (Agarsu grade I) was purchased from Agarsu Liben Cooperative (IPO, Increasing People Opportunities) association (www.ipoassociazione.org), Italy. A voucher specimen (# MCM-1) of the resin is deposited at Dipartimento di Chimica e Tecnologia del Farmaco, Sezione di Chimica Organica, University of Perugia (Italy). Ground resin (5 g) was extracted with 250 ml of n-hexane (4 h at RT), the suspension filtered under vacuum and the solvent removed under N2 . The composition of hexane extract by GC–MS technique has already been reported (Fraternale et al., 2011). Fractionation of the hexane extract (Marcotullio et al., 2011) resulted in the isolation of furanodien-6-one (C15 H20 O2 , Mw 232) (Dekebo et al., 2000, 2002), which, dissolved in DMSO (Stock solution 500 mM), was used in the study. 2.3. Animals Six/eight week-old male C57BL/6 mice (18–20 g) were housed at the Laboratory Animal Research Centre of Perugia University. The animals were maintained at a constant temperature of 24 ◦ C and 12-h light/dark cycle. 2.3.1. Ethics statement All experimental procedures were carried out in accordance with European Directives, approved by the Institutional Animal Care and Use Committee of Perugia University (N34/2009). Efforts were made to minimize animal stress/discomfort. 2.3.2. Animal Treatment Forty animals were divided into four groups. One group (10 mice) was ip injected with a single dose of LPS (5 mg/kg) (Qin et al., 2007). The second group received two ip injections of FDN (2.4 mg/kg): the first, 1 h prior to LPS injection and the second 30 min afterwards. Previous determinations of hepatic expression of Tnf˛ with different doses (1.2/2.4/3.6 mg/kg corresponding to
5/10/15 moles/kg) of FDN indicated 2.4 mg/kg as the minimal effective dose. The third group was treated with FDN alone and the fourth group with vehicle and used as control. Mice were sacrificed 3 h after LPS treatment, brain and liver used for real time PCR analyses and histological observations. 2.4. Cell culture and viability Murine macrophage RAW264.7 (ATCC, Rockville, MD) were cultured in DMEM supplemented with 2 mM glutamine,10% Foetal Bovine Serum (FBS), penicillin (100 U/ml), and streptomycin (100 g/ml) at 37 ◦ C in a humidified incubator under 5% CO2 . BV-2 cells were cultured in RPMI supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 g/ml) at 37 ◦ C in a humidified incubator under 5% CO2 . After 24 h subculture, cells (RAW 264.7: 4 × 104 , and BV-2: 9 × 104 ) were incubated for 24 h with FDN (50 M for BV-2 and 10 M for RAW 264.7), and then exposed to 10 g/ml lipopolysaccharide (LPS, Escherichia coli serotype 0111:B4) for the indicated times. Conditioned media (CCMs) were obtained from BV-2 cells after a 18 h exposure at LPS. Murine primary neuronal cultures were prepared according to Almeida and Medina (1998) in the presence of 10 M Ara C. Neuronal cells (8 × 104 ) were incubated for 24 h with 50 M FDN prior to exposure to BV-2-conditioned media (CCMs) for 24–72 h. Live cells were counted by hemocytometer, viability assessed by the (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) MTT reduction assay, and the absorbance at 550 nm was measured with a microplate reader (Seac, Florence, Italy). Results were expressed as the percentages of reduced MTT, assuming the absorbance of control cells as 100%. 2.5. Measurement of intracellular fluorescence The DCFH-DA method was used to detect the levels of intracellular reactive oxygen species (ROS) (Eruslanov and Kusmartsev, 2010). Cells (9 × 104 ) were treated as described and DCFH-DA (10 M) was added to the medium for a further 30 min at 37 ◦ C. The fluorescence of 2 ,7 -dichlorofluorescein was detected at 485 nm excitation and at 535 nm emission, using a plate reader Titertek Fluoroscan II (Flow Laboratories, Mc Lean, VA). Results were expressed as the percentage of the control DCF fluorescence, assumed as 100%. 2.6. Measurement of NO production Nitric oxide (NO) production was determined by the Griess reaction. Cells (9 × 104 ) were treated as described, then a 50 l aliquot of medium was mixed with an equal volume of Griess reagent and incubated for 20 min at RT. The absorbance was read at 550 nm using a microtiter plate reader (Seac, Florence, Italy) and results were expressed as the percentage of the control, assumed as 100%. Nitrite standard reference curve was prepared for each determination. 2.7. Measurement of cytokine levels The effects of FDN on cytokine production were measured with an enzyme-linked immunosorbent assay (ELISA) kit in CCMs collected from LPS-treated cells. Samples were analyzed according to the manufacturer’s protocols (BD Biosciences Pharmingen, San Diego, CA). 2.8. Electrophoretic mobility shift assay Synthetic double-stranded biotin 5 -oligonucleotide containing B responsive element (BRE) (5 -AGTTGAGGGGACTTTCCCAGGC3 and 3 -TCAACTCCCCTGAAAGGGTCCG-5 ) (M-Medical Srl, Milano,
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Italy) were used as probes. Nuclear lysates (8 g), obtained with NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL), were incubated with the labelled probe (15 fmol) for 20 min at RT, resolved on an 6% native polyacrylamide gel, and transferred to Hybond-N+ (Amersham Pharmacia). Signals were visualized with peroxidase-conjugated streptavidin (Pierce Biotechnology, Rockford, IL).
2.9. Western blotting analyses Whole cell extracts were obtained with 10% (w/v) of warm Laemmli sample buffer 1× and sonication for 15 s, nuclear/cytosolic extracts were obtained with NE-PER® Nuclear and Cytoplasmic Extraction Reagents. Extracts (20–50 g) were loaded on a 12% SDS–polyacrylamide gel and protein levels determined by Western blotting using NF-Bp65 (C-20) (1:100), COX-2 (C20) (1:200), iNOS (M19) (1:100), SIRT1(H300)(1:100) antibodies and horseradish peroxidase-conjugated-anti rabbit/mouse/goat
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IgG antibody (1:5000). Lamin B (H-90) and -actin (C-4) antibodies (1:400) were used as marker proteins for nuclear/cytosolic extracts. Immunocomplexes were visualized with an enhanced chemiluminescence kit (ECL, Pierce Biotechnology, Rockford, IL). 2.10. Glial fibrillar acidic protein immunohistochemistry Immediately after animal sacrifice, brains were removed and one hemisphere was immersion-fixed in Carnoy solution and embedded in paraffin. Cerebral sections at defined levels (10 m) were processed to evidence immunoreactivity for GFAP by Leica DM2500, DFC 420c and Application Suite V3.4 system microscope (Leica Microsystems Srl, Italy). 2.11. Real time PCR Total RNA was isolated with TRIZOL Reagent (Invitrogen Ltd, Paisley, UK) according to the manufacturer’s instructions and cDNA
Fig. 1. FDN reduces LPS toxic effects. (A) BV-2 cells were incubated with 50 M FDN for 24 h and then exposed to 10 g/ml LPS for 4 and 24 h; viability was detected by MTT assay (100% control MTT absorbance: 0.9 ± 0.07 at 4 h, 1.2 ± 0.1 at 24 h); NO production, detected by Griess Reagent (100% control NO absorbance: 0.20 ± 0.03) and ROS generation, detected by DCFH-DA (100% control ROS fluorescence: 0.8 ± 0.06 at 4 h, 0.90 ± 0.03 at 24 h). (B) iNos and Cox-2 gene expression by real time PCR analysis. Changes in gene expression, whose values were normalized to Gapdh expression, are presented as 2−Ct . Relative mRNA gene abundance in untreated cells was assumed to be 1.0 (control). (C) Time-course of iNOS and COX-2 protein levels. At each indicated time, cells were collected and whole cell lysates (40 g) were subjected to Western blotting analyses with the indicated antibodies. (D) iNOS and COX-2 protein levels in murine macrophage RAW264.7 cells. Cells were incubated with 10 M FDN for 24 h and then exposed to 10 g/ml LPS for 24 h, collected and whole cell lysates (20 g) were subjected to Western blotting analyses with the indicated antibodies. -actin was used as loading control. Data represent means ± S.D. of at least three experiments. *P < 0.05 vs control cells. #P < 0.05 vs LPS treated cells.
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was synthesized using iScript cDNA synthesis kit (Bio-Rad Lab, Hercules, CA). Real time PCR was performed using the iCycler iQ detection system (Bio-Rad) and SYBR Green chemistry. Mouse primers (Table S1) were obtained from Invitrogen (Invitrogen Ltd, Paisley, UK). Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.molimm.2013.01.003.
2.13. Statistical analysis All results were confirmed in at least three separate experiments and expressed as mean ±S.D. Data were analyzed for statistical significance by ANOVA followed by post hoc analysis. P-values <0.05 were considered significant. 3. Results 3.1. FDN reduces LPS-mediated toxic effects in BV-2 cells
2.12. Fourier transform infrared spectroscopy FTIR measurements were performed in transmission mode (Corte et al., 2010). All spectra were recorded in the range 4000 and 400 cm−1 with a TENSOR 27 FTIR spectrometer, equipped with HTS-XT accessory (BRUKER Optics GmbH, Ettlingen, Germany). OPUS version 6.5 software (BRUKER Optics GmbH, Ettlingen, Germany) was used to carry out the quality test, baseline correction, vector normalization and the calculation of the first and second derivatives of spectral values. Hierarchical cluster analysis, i.e. assigning a set of objects into groups (called clusters) so that the objects in the same cluster are more similar (in some sense or another) to each other than to those in other clusters, was performed using the software OPUS to highlight similarities or differences in terms of statistical distance. Spectra were divided into five spectral regions (fatty acids (W1) from 3000 to 2800 cm− 1, amides (W2) from 1800 to 1500 cm− 1, mixed region (W3) from 1500 to 1200 cm− 1, carbohydrates (W4) from 1200 to 900 cm− 1 and typing region (W5) from 900 to 700 cm− 1),exported as an ASCII file from OPUS and used in the “R” environment (http://cran.r-project.org/). Response spectra (RS) were calculated as the difference between each average spectrum and the average spectrum of control tissue, which is by definition a straight line with RS = 0. The intensity of the metabolomic response results from the difference between zero and the spectral line. Five mg of each liver were homogenized in saline, centrifuged at 2000 × g, washed twice and re-suspended in ultra pure water. For each sample, three/five repetitions were placed on the 96-multiwell, dried at 42 ◦ C for 20 min and used for the analysis.
We have already shown that FDN protects BV-2 cells from LPS toxicity (Marcotullio et al., 2011). Here we further analyzed the effects of FDN on BV-2 cells by investigating the molecular mechanism underlying this protection. LPS cytotoxicity was timedependent: a 4 h exposure to 10 g/ml LPS did not modify cell viability whereas a 24 h exposure caused a marked reduction in cell viability. NO and ROS generation were increased at each considered time. At 24 h, pre-treatment with 50 M FDN restored cell viability and ROS to control levels while halving NO production (Fig. 1A). FDN treatment maintained cell viability and decreased NO levels when added to microglial cells up to 1 h after LPS injury (data not shown). NF-B is a redox-sensitive transcription factor that plays a key role in the expression of a number of genes that are central to the inflammatory response (Brigelius-Flohe et al., 2011). To demonstrate the inhibition of the NF-B- inflammatory pathway by FDN, we performed a real time PCR analysis of NF-B-driven genes, i.e. iNos (inducible nitric oxide synthase) and Cox-2 (cyclooxygenase2) in LPS-exposed BV-2 (Fig. 1B). iNos and Cox-2 mRNA levels were strongly up regulated by LPS exposure and markedly decreased by FDN pre-treatment. Compatible with these results, the LPS-induced increases in iNOS and COX-2 protein expression were reduced by FDN pre-treatment (Fig. 1C). iNOS and COX-2 protein expression were reduced by FDN pre-treatment also in murine macrophage RAW264.7 cells thus confirming the FDN anti-inflammatory properties (Fig. 1D). Next we determined the levels of cytokines in LPS treated cells in the presence of FDN (Fig. 2). The production of pro-inflammatory IL-6, IL-23, IL-17A, TGF-, and INF-␥, significantly induced by LPS, was markedly reduced by FDN treatment while IL-27 production
Fig. 2. FDN reduces inflammatory cytokines levels. BV-2 cells were pre-treated with 50 M FDN for 24 h and then stimulated with 10 g/ml LPS for 18 h. Cytokines from BV-2 cell conditioned media were assayed by ELISA. Values represent mean ± S.D. of at least three experiments. *P < 0.05 vs control cells. #P < 0.05 vs LPS-treated cells.
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was not significantly modified. In all experiments, treatment with FDN alone did not modify control values (data not shown). Results show that FDN is responsible for the inhibition of NF-B signalling confirming its in vitro anti-inflammatory activity. 3.2. FDN protects primary neuronal cultures against glial over-activation Chronic glial activation is detrimental to neurons through sustained production of neurotoxic biomolecules (Block et al., 2007; Kim et al., 2000; Tansey and Goldberg, 2010; Wen et al., 2007; Chen et al., 2010; Markowitz et al., 2007). After showing that FDN attenuates BV-2 over-activation, we investigated whether the compound is also capable of protecting neuronal cells from glial-induced neurotoxicity. Thus we exposed murine primary neuronal cultures to LPS-activated BV-2 culture media (CCM). CCMs markedly reduced
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neuronal viability and increased NO generation. FDN pre-treatment attenuated the negative effects of CCMs by restoring neuronal viability to control values and significantly reducing NO production, indicating that FDN can protect primary neuronal cultures against over-activated microglial neurotoxicity (Fig. 3A). Next we investigated the effects of FDN on NF-B gene expression, nuclear translocation, DNA-binding activity and transcriptional efficiency by real time PCR, EMSA, and Western blotting (Fig. 3B–E). FDN reduced the mRNA levels, nuclear translocation and the binding capacity to BRE sequence of NF-B as well as COX-2 protein levels, confirming that the inflammatory pathway is inhibited by FDN. We then investigated whether FDN is also capable of modulating neuronal Sirt1 expression. Results of real time PCR and Western blotting analyses showed that in neuronal primary cultures Sirt1 expression as well as SIRT1 protein levels are significantly up regulated by FDN treatment suggesting the involvement of SIRT1 in the
Fig. 3. FDN protects primary neuronal cultures against microglial over-activation. (A) Primary neuronal cultures, pre-treated with 50 M FDN for 24 h, were exposed to CCMs for different times. Viability, detected by MTT assay (100% control MTT absorbance:0.89 ± 0.06), NO production, detected by Griess Reagent (100% control NO absorbance: 0.110 ± 0.09, 0.06 ± 0.001, 0.06 ± 0.005 at 24, 48, and 72 h). (B–E) FDN modulates the NF-B system. Primary neuronal cultures, pre-treated with 50 M FDN for 24 h, were exposed to CCMs for 48 h and collected for analyses: (B) real time PCR analysis of mRNA levels of the NF-B gene. (C) nuclear translocation of NF-B. Nuclear extracts (50 g proteins) were subjected to Western blotting analysis with NF-B antibody. Lamin B and -actin antibodies were used as markers of nuclear/cytosolic extracts. (D) DNA binding capacity of NF-B. EMSAs were performed by using 8 g nuclear protein lysate per lane and a biotin-labelled BRE probe (15 fmol). (E) transcriptional efficiency of NF-B. Whole cell lysates (40 g) were subjected to Western blotting analyses with the indicated antibody. -actin was used as loading control. (F) real time PCR analysis of mRNA levels of Sirt1 gene. (G) Western blotting analyses of SIRT1 protein levels. Whole cell lysates (40 g) were analyzed with the indicated antibody. -actin was used as loading control. Data represent means ± S.D. of at least three experiments. *P < 0.05 vs control cells. #P < 0.05 vs CCMs-treated cells.
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FDN causes a response markedly different from the response that follows the LPS treatment (Fig. 4B). Similar results were obtained by using average spectra of FDN-treated animals as a straight line with RS = 0 (data not shown). Results indicate that FDN markedly modifies the hepatic response to LPS. Systemic cytokines mediate the effects of peripheral inflammation on the brain and reactive gliosis underlies the progressive and long-lasting neuroinflammation and neurodegeneration (Block et al., 2007; Kim et al., 2000; Tansey and Goldberg, 2010; Wen et al., 2007; Chen et al., 2010; Markowitz et al., 2007; Meng and Lowell, 1997; Chung et al., 2010; Qin et al., 2007). Thus, at 3 h post LPS-treatment, we examined several brain regions, i.e. striatum, hippocampus, and cortex and observed an increase in GFAP-immunoreactivity compatible with glial overactivation. Treatment with LPS in the presence of FDN resulted in a decreased GFAP-immunoreactivity. FDN alone did not modify GFAP-immunoreactivity (Fig. 5A). The marked increases in mRNA levels of Tnf˛ and IL1B after LPS treatment were strongly reduced by FDN treatment. Only cortical up-regulation of IL1B expression was not counteracted by FDN, probably related to temporal regionspecific differential susceptibility to anti-inflammatory agents (Kim et al., 2000). FDN alone did not significantly alter gene expression of the control group. Results provide strong evidence of the in vivo antiinflammatory properties of the FDN (Fig. 5B).
4. Discussion
Fig. 4. FDN decreases LPS-induced hepatic metabolomic alterations. For each sample, three/five repetitions were placed on the 96-multiwell, dried at 42 ◦ C for 20 min and used for the analyses. (A) Hierarchical cluster analysis of the W1 region with the second derivative of sample averages showing a low grade heterogeneity between control and FDN-treated samples. (B) Response spectra (RS) analysis: variations by LPS in the positive range were abolished by FDN treatment.
attenuation of the NF-B system (Fig. 3F–G). Data provide evidence that FDN protects neuronal cells against over-activated microglial neurotoxicity. 3.3. FDN reduces neuroinflammation in response to systemic LPS administration Finally we investigated whether FDN shows anti-inflammatory properties in an in vivo model of glial activation. Because a single ip LPS injection results in liver TNF␣ production, that, in turn, initiates brain TNF␣ production, responsible for a chronic neuroinflammation (Qin et al., 2007), we first analyzed the hepatic metabolome profiles with FTIR spectroscopy. Hierarchical cluster analysis showed that the samples can be divided into three clades: one containing the samples treated with LPS, the second containing the control/FDN alone-treated samples and the third containing the samples treated with LPS plus FDN. This clade is very close to the control and clearly separated from the LPS-treated samples, since it shows a great statistical distance and a high level of heterogeneity compared to LPS-treated samples (Fig. 4A). Spectral data were then analyzed with an algorithm that allows different statistical treatments. Response spectra (RS) (Corte et al., 2010), where average spectra of control animals are assumed by definition as a straight line with RS = 0, showed that the treatment with
Here we showed for the first time that systemic administration of FDN exerts in vivo anti-inflammatory effects in the central nervous system by decreasing the expression of liver and brain Tnf˛ and IL1B. Our data also provide evidence that FDN can protect neurons exposed to microglia-produced inflammatory and neurotoxic factors thorough the inhibition of the NF-B system. Lipopolysaccharide (LPS), an endotoxin from gram-negative bacteria, is known to activate protein kinase C, protein–tyrosine kinases, mitogen-activated protein kinases, and NF-B, each protein/enzyme involved in the release of immune-related cytotoxic factors (Wen et al., 2007; Meng and Lowell, 1997; Chung et al., 2010; Hinkerohe et al., 2010). Given the influence of intracellular ROS signalling on several kinase-mediated pathways (Bellezza et al., 2010; Brigelius-Flohe et al., 2011; Lin et al., 2011), the LPS signalling pathway in microglia is a likely target for downstream ROS signalling, one of the key factors in LPS-mediated neurodegeneration. Indeed, LPS can damage neurons only in the presence of microglia (Gao et al., 2002; Gibbons and Dragunow, 2006; Rivest et al., 2000) by a reactive microgliosis process triggered by pro-inflammatory factors (Qin et al., 2007). An acute systemic injection of LPS is followed by a rapid increase in the hepatic expression of TNF␣ which, in turn, through TNF␣ receptors, activates the microglia thus initiating a sustained activation of brain cytokine synthesis and neuroinflammation. This sustained neuroinflammation leads to a delayed and progressive loss of dopaminergic neurons over a 7–10 months period (Qin et al., 2007). We showed that mice, treated with FDN, have decreased hepatic mRNA levels of Tnf˛, probably responsible for the attenuated microglia activation as well as marked brain Tnf˛ and IL1B down-regulation. FTIR spectroscopy analyses are mostly qualitative and do not quantify/characterize individual metabolites. However, both hierarchic cluster analyses and response spectra showed that the metabolomic profile of FDN plus LPS-treated animals strongly correlates with control animals and that FDN can suppress the hepatic response to LPS stress, hinting to beneficial effects of FDN. Oxidative damage activates redox-sensitive transcription factors and the increased production of reactive oxygen
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Fig. 5. FDN reduces neuroinflammation in response to systemic LPS administration. C57BL/6 mice were sacrificed 3 h following LPS (5 mg/kg) i.p. injection. (A) Immunohistochemical analyses. Brain sections were immunostained with GFAP specific antibody. The images are from one experiment that is representative of ten separate experiments. (B) real time PCR analysis of Tnf˛ and IL1B in cortex, striatum, hippocampus and liver. Changes in gene expression, whose values were normalized to Gapdh expression, are presented as 2−Ct . Data represent means ± S.D. of at least ten experiments. *P < 0.05 vs control cells. #P < 0.05 vs LPS-treated animals.
species is associated with up regulation of NF-B (Brigelius-Flohe et al., 2011). Activation of NF-B, in turn, induces the expression of pro-inflammatory genes, including cytokines, growth factors, and chemokines (Mattson and Meffert, 2006). Because some of the NF-B -induced proteins are also potent NF-B activators, the resulting vicious cycle may contribute to the establishment of a chronic inflammatory state and related pathologies. Prolonged innate immune responses, including activation of microglia and astrocytes, are seen in various neurodegenerative diseases (Block et al., 2007; Kim et al., 2000; Tansey and Goldberg, 2010; Wen et al., 2007; Chen et al., 2010; Markowitz et al., 2007). We showed that in vitro FDN protection is based on the inhibition of the NFB pro-inflammatory pathway and decreased expression of COX-2. NF-B activation is due to the liberation of the transcription factor from its cytosolic complex with IB thus allowing nuclear import and downstream gene activation (Bellezza et al., 2010; BrigeliusFlohe et al., 2011; Lin et al., 2011). In the nucleus NF-B, i.e. the RelAp65/p50 complex, binds to its promoter sequence together with the transcriptional co-activators CBP and p300, which have histone acetylase (HAT) activity. RelAp65, p50 and histones are
then acetylated leading to uncoiling of chromatin, thus allowing the access of NF-B to its canonical and other promoter regions (Gloire and Piette, 2009). SIRT1, the mammalian homologue of the evolutionarily conserved NAD+ -dependent deacetylase Sir2 (Michan and Sinclair, 2007), increases as a protective response to neurodegenerative conditions (Kim et al., 2007; Chen et al., 2005; Zhang et al., 2010). By deacetylating RelA/p65, it significantly inhibits the transactivation of NF-B and the expression of inflammatory cytokines (Kim et al., 2007; Chen et al., 2005; Lanzillotta et al., 2010; Yeung et al., 2004), thus blocking the neuropathogenic inflammatory loops (Michan and Sinclair, 2007; Zhang et al., 2010; Salminen et al., 2011). In this study we have provided evidence that primary neuronal cultures, treated with FDN prior to the neurotoxic insult, show a reduction in NF-B expression, nuclear translocation and binding activity as well as an up regulation in Sirt1 expression and SIRT1 protein levels. All data suggest that the inhibition of the proinflammatory pathway may be ascribed, at least partially, to the interference of SIRT1 with the complex interplay of the NF-B system. Thus FDN reduces glial activation and toxicity to neighbouring neurons which, in turn, by up-regulating Sirt1 expression, decrease
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