Novel neurological and immunological targets for salicylate-based phytopharmaceuticals and for the anti-depressant imipramine.
Keywords: Inflammation Neurological diseases Multi targeting Microarray Polyphenols Salicylates
Inflammatory processes are increasingly recognised to contribute to neurological and neuropsychatric disorders such as depression. Thus we investigated whether a standardized willow bark preparation (WB) which contains among other constituents salicin, the forerunner of non-steroidal antiphlogistic drugs, would have an effect in a standard model of depression, the forced swimming test (FST), compared to the antidepressant imipramine. Studies were accompanied by gene expression analyses. In order to allocate potential effects to the different constituents of WB, fractions of the extract with different compositions of salicyl alcohol derivative and polyphenols were also investigated.
Male Sprague Dawley rats (n = 12/group) were treated for 14 days (p.o.) with the WB preparation STW 33-I (group A) and its fractions (FR) (groups FR-B to E) in concentrations of 30 mg/kg. The FRs were characterized by a high content of flavone and chalcone glycosides (FR-B), flavonoid glycosides and salicyl alcohol derivatives (FR-C), salicin and related salicyl alcohol derivatives (FR-D) and proanthocyanidines (FR-E). The tricyclic antidepressant imipramine (20 mg/kg) (F) was used as positive control. The FST was performed on day 15. The cumulative immobility time was significantly (p < 0.05) reduced in group A (36%), group FR-D (44%) and by imipramine (16%) compared to untreated controls. RNA was isolated from peripheral blood. RNA samples (group A, group FR-D, and imipramine) were further analysed by rat whole genome microarray (Agilent) in comparison to untreated controls. Quantitative PCR for selected genes was performed.
Genes (>2 fold, p < 0.01), affected by WB and/or FR-D and imipramine, included both inflammatory (e.g. IL-3, IL-10) and neurologically relevant targets. Common genes regulated by WB, FR-D and imipramine were GRIA 2 [down arrow], SRP54 [down arrow], CYP26B [down arrow], DNM1L [up arrow] and KITLG [down arrow]. In addition, the hippocampus of rats treated (27 d) with WB (15-60 mg/kg WB) or imipramine (15 mg/kg bw) showed a slower serotonin turnover (5-hydroxyindol aceticacid/serotonin (p <0.05)) depending on the dosage. Thus WB (30 mg/kg), its ethanolic fraction rich in salicyl alcohol derivatives (FR-D) (30 mg/kg) and imipramine, by being effective in the FST, modulated known and new targets relevant for neuro- and immunofunctions in rats. These findings contribute to our understanding of the link between inflammation and neurological functions and may also support the scope for the development of co-medications from salicylate-containing phytopharma-ceuticals as multicomponent mixtures with single component synthetic drugs.
[c] 2012 Elsevier GmbH. All rights reserved.
Willow bark (WB) was already used by Hippocrates as an anti-inflammatory agent against fever and pain. It contains salicin and other salicylic acid derivatives which were designated as active principles since 1831. These compounds are prodrugs which are metabolised in the gut and the liver via salicylic alcohol to salicylic acid--the active compound. To improve tolerability compared to isolated salicyclic acid, Felix Hoffmann synthesised acetylsalicylic acid (ASA) in 1897, today one of the mostly consumed analgetic and anti-inflammatory drugs worldwide.
Salicylic acid is known to inhibit the cyclooxygenases (COX) 1 and 2 (Aronoff et al. 2003). Therefore, COX-inhibition was first regarded the main mechanism of the anti-inflammatory activity of WB. In the meanwhile it was shown that WB can also modulate relevant pro- and anti-inflammatory cytokines like TNF-[lapha], IL-6, IL-1, IL-10, and IL-8 (Bonaterra et al. 2009; Fiebich et al. 2005; Fiebich and Chrubasik 2004) and that not only salicyl alcohol derivatives, but also the polyphenols of WB contribute to this modulation (Khayyal et al. 2005; Nahrstedt et al. 2007). In addition, polyphenols are known to possess antioxidant and neuroprotective effects which can also interfere with inflammatory events (Kannappan et al. 2011). Very recently also a contribution of catechol to an antiin-flammatory effect of WB was discussed (Freischmidt et al. 2012; Knuth et al. 2011).
Recent clinical data propose that proinflammatory cytokines also influence the pathogenesis of depression. It was shown that the IL-1RA (interleukin-1 receptor antagonist) is increased in the plasma of middle aged and elderly patients (>65 years) suffering from depression (Milaneschi et al. 2009; Ovaskainen et al. 2009). Lindqvist et al. (2009) demonstrated increased IL-6 concentrations in the CSF of patients with depression. A recent meta-analysis summarising med-line studies on depression between 1967 and 2008 concluded that major markers of inflammation like CRP, IL-6 and IL-1 are significantly associated with depression (Howren et al. 2009). Even though the causal relationship is presently not clear, the link between the immune system via cytokines and the pathogenesis of psychiatric disorders led to the understanding that immunomodulatory drugs may also be beneficial for the treatment of psychiatric disorders (Berthold-Losleben et al. 2009).
In addition, it was observed that in clinical studies with willow bark extract the treatment groups had a lower number of adverse events (AEs) than in the placebo group (e.g. Schmid et al. 2000). Thus, it had been hypothesised by Winterhoff that willow bark might have an anxiolytic effect and could thereby be useful in depression. This was first evidenced by her working group (Hegger et al. 2005; Winterhoff et al. 2008) using the forced swimming test (FST) as an animal model of depression.
We now queried which molecular targets are affected by WB compared to the antidepressant imipramine and which components of the WB extract are active, especially considering that plant constituents have been repeatedly proposed for a possible future application in co-medications (Wagner 2011).
Materials and methods
Willow bark extract
The dried willow bark preparation STW 33-I (WB) was obtained from Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany. The extract was prepared from willow bark according to PhEur. 6.1, with a [DEV.sub.nati] of 16-23:1, total salicin content 23-26% (m/m) Europe C (2008). Imipramine hydrochloride was obtained from Sigma (Deisenhofen, Germany).
Preparation and characterization of the tested fractions
The investigated fractions were prepared as described in Freischmidt (2011) by application of partition and precipitation processes with ethyl acetate (Fr-B), butanol (Fr-C), ethanol (Fr-D) and water (FR-E) (Fig. 1).
As only for Fr-B an exact phytochemical characterization is described, revealing different flavanone and chalcone glycosides as well as catechol as most abundant phenols (Freischmidt 2011), a quantitative determination of different classes of compounds in the WB and the resulting fractions was done. The salicin and salicyl alcohol content was determined according Ph.Eur 6.4 (Europe C 2008), total polyphenol, tannin and rest phenol content was quantified according to Glasl (1983). As the flavonoid spectrum of willow bark mainly consists of flavanone and chalcone glycosides the common PhEur methods for determination of overall flavonoid content Is not applicable. Thus, it was determined according a newly developed method (Freischmidt 2011).
The WB is rich in salicin, salicin derivatives and polyphenols; the ethyl acetate fraction contains a high amount of polyphenols, whereas the ethanol fraction contains a high amount of salicin and salicin derivatives while having a comparatively low polyphenol content (Table 1).
Table 1 Characterization of WBE and its fractions (% [+ or -] SD, n=3). WB FR-B FR-C FR-D FR-E A Total 19.7 41.7 16.3 6.7 4.4 polyphenols [+ or -] [+ or -] [+ or -] [+or -] [+or -] 0.3 0.1 0.3 0.03 0.02 Tannin 6.8 8.7 4.3 2.7 2.8 polyphenols [+or -] [+or -] [+or -] [+or -] [+or -] 0.2 0.1 0.2 0.01 0.06 Rest 12.9 33.0 11.9 4.0 1.6 phenols [+ or -] [+ or -] [+ or -] [+or -] [+or -] 0.1 0.1 0.2 0.03 0.07 B Flavonoids 8.4 21.8 7.3 0.3 n.d. (b) (a) [+ or -] [+ or-] [+or -] [+or -] 0.2 0.1 0.5 0.02 C Salicin 12.8 5.2 18.2 19.7 3.9 [+ or -] [+ or -] [+ or -] [+ or -] [+ or -] 0.1 0.2 1.0 0.3 0.1 Salicyl 23.0 14.2 34.7 27.3 5.4 alcohol [+ or -] [+ or -] [+ or -] [+ or -] [+ or -] derivatives 0.8 0.2 1.8 0.9 0.3 (a.) Determined as nanngenin derivatives. (b.) Not detectable by TLC and thus not determined.
Male Sprague-Dawley (CD) rats (150-170 g, Charles River Laboratories, Sulzfeld, Germany) were housed in groups of two and kept in climatised rooms (24 [+ or -] 1 [degrees]C, light-dark cycle 12/12 h). Animals had free access to food (Altromin [R] 1324, Altromin, Lage, Germany) and tap water. The procedures used comply with the European Community's Council Directive of 24 November 1986 (86/609/EEC) and were officially approved by the local committee on animal care (Regierungsprasident Munster, AC/2004).
Animals (n= 12 per group) received the test solutions (WB, its fractions, or imipramine, suspended in water, 10 ml/kg bw) p.o. once daily. The positive control drug imipramine was first dissolved in 160 ill ethanol and then diluted with deionised water to a final volume of 10 ml (Butterweck et al. 2002). As negative control the solvent was used.
Forced swimming test
The forced swimming test (FST) was performed with 72 male CD-rats according to Porsolt et al. (1977, 1978). The animals (n = 12 per group), received the test solutions (p.o. 30 mg/kg bw/d over a period of 14 days) of WB (A), its ethyl acetate fraction (FRB), its n-butanol fraction (FR-C), its ethanol fraction (FR-D), its aqueous fraction (E), 30 mg/kg bw/d each, or the positive control imipramine, 20 mg/kg bw/d. One day before the test, the rats experienced a 15 min pre-test session, in which the animals were singly placed in a plexiglas cylinder that contained a 17-21 cm (depending on the body weight) high column of water at 25 [+ or -] 1 [degrees]C. Experimental testing was conducted 24h later by exposing the rats to the same test conditions for 5 min 12 h after last drug administration. Behaviour was recorded by video camera. The cumulative time rats persisted in an immobile position (despair behaviour) was evaluated afterwards by a blinded observer. A rat was judged to be immobile whenever it remained floating in the water in contrast to motor activities that represented attempts to escape from the situation. The time rats spent making small movements to keep the head above the water was assigned to time of immobility. A shortened immobility time indicates an antidepressant like effect. The FST was performed between 9.00 a.m. and 1.00 p.m.
Determination of serotonin and 5-hydroxyindol acetic acid in the hippocampus
CD-rats (n = 12 per group) received WB (15 mg, 30 mg, 60 mg and 125 mg/kg bw/d) or imipramine (15 mg/kg bw/d) over a period of 27 days. Animals were sacrificed by decapitation between 9.00 a.m. and 11.00 a.m. at the day after the last drug administration. Serotonin (5-hydroxytryptamine, 5-1-IT) and 5-hydroxyindol acetic acid (5-HIAA) concentrations were estimated in the hippocampus of 72 male CD-rats as described earlier (Butterweck et al. 2002).
For the statistical analysis of the results of the FST and the serotonin and 5-HIAA determinations, the STATVIEW statistical software package, version 5.0 (SAS[R], USA) was used. Data analysis was performed by analysis of variance (ANOVA) with the Student-Newman-Keuls post hoc test for multiple comparisons. PCR-data were analysed by Student's t-test with Sigma Stat Vers.1 statistical software. Data are expressed as means [+ or -] S.E.M. Statistical significance was set at p <0.05.
Gene microarrays and data processing
Blood samples (3 ml) of treated and untreated rats were collected and prepared as described (Ulrich-Merzenich et al. 2012). Only RNA of treatment groups showing significant responses in the FST compared to the untreated group were selected for detailed microarray analysis. For analysis single colour hybridization of the rat RNA on the Rat Agilent Whole Genome Oligo Micorarrays (41,013 genes) was performed (Miltenyi Biotec, Bergisch Gladbach, Germany). The ratios were computed using a common "artificial reference" (4 control samples combined). This common reference was used as baseline for all samples. The computed ratios were used for further analyses by Ingenuity systems Inc., Redwood City, USA as described (Ulrich-Merzenich et al. 2012).
Reverse transcription and gene specific polymerase chain reaction
In order to validate the data obtained by microarray, genes that were highly affected by the treatment and/or belonged to different biological pathways were selected for RT-PCR (glutaminergic system: Gria 2; protein secretory pathway: signal recognition protein (SRP) 54; neurogenesis: cytochrome P450 protein 26B1 (Cyp26B1); cytokines: IL-3; redox system: Lox-1). [beta]-Actin was used as internal reference control. In each group 4 samples were analysed if not mentioned otherwise. In brief, cDNA was synthesised from 1000 ng of RNA by Transcriptor High Fidelity cDNA Synthesis Kit (Roche). The gene specific primers are listed in Table 2. PCRs were performed on Perkin Elmer DNA Thermal Cycler 480 ([beta]-actin, Cyp26b1, 11-3, Lox-1) and Eppendorf Mastercycler (Srp54, Gria2). The thermocy-cler conditions were as follows: [beta]-actin: initial denaturing step: 94 eC for 7 min, amplification: 35 cycles of denaturation at 94[degrees]C for 1 min, annealing at 64[degrees]C for 1 min and elongation at 74[degrees]C for 3 min; and final elongation step: 74[degrees]C for 7 min; Cyp26b1: initial denaturing step: 95[degrees]C for 5 min, amplification: 30 cycles of denaturation at 94[degrees]C for 1 min, annealing at 54[degrees]C for 1 min and elongation at 74[degrees]C for 2 min; and final elongation step: 74[degrees]C for 12 min, Gria 2: initial denaturing step: 95[degrees]C for 2 min, amplification: 35 cycles of denaturation at 94[degrees]C for 30s, annealing at 51[degrees]C for 30s and elongation at 72[degrees]C for 1 min, and final elongation step: 72[degrees]C for 10 min; 11-3: initial denaturing step: 94[degrees]C for 7 min, amplification 30 cycles of denaturation at 94[degrees]C for 30s, annealing at 52[degrees]C for 90s and elongation at 72[degrees]C for 1 min, and final elongation step: 72[degrees]C for 10 min, Lox-1: initial denaturing step: 95[degrees]C for 4 min, amplification: 30 cycles of denaturation at 94[degrees]C for 45 s, annealing at 47[degrees]C for 1 min and elongation at 74[degrees]C for 2 min, and final elongation step: 74[degrees]C for 7 min; Srp54: initial denaturing step: 95 ''C for 5 min, amplification: 35 cycles of denaturation at 94[degrees]C for 1 min, annealing at 56[degrees]C for 1 min and elongation at 72[degrees]C for 40s; and final elongation step: 72[degtees]C for 5 min; Semi quantitative analysis was performed by densitometry employing the BioRAD GelDoc 100 system using the software Multi-Analyst as described earlier (Ulrich-Merzenich et al. 2007). Data were expressed as ratios to -actin. PCR-products were confirmed as the expected target genes via purification (Wizard[R] PCR Preps DNA Purification system, Promega) and product sequencing (data not shown) performed using standard techniques and the 3130/x Genetic Analyzer (Applied Biosystems).
Table 2 The sequences of the PCR primers. Gene Nucleotide sequence Product length bp [beta]-Actin F: 5'-AACCCCGAGAAGATGACCCAGATCATGnT-3' 350 R: 5'-AGCAGCCGTGGCCATCTCTTGCTCGAACTG-3' Cyp26b1 F: 5'-CAGCTAGTCACCACCCAGTG-3 345 R: 5'-CGGCAGAGAGAAGACATTCTC Cria 2 R: 5'-GCTCGAGAGAGGGTGATTG-3' 144 F: 5'-CTGTrGATGCCITGCGTC-3' II-3 F: 5'-GAGnTGATTCTCAGGACAC 360 R: 5'-GTCATCCAGATCnTATTGTAG Lox-1 F: 5'-GACTGGATCTCGCATAAAGA-3' 361 R: 5'-CCTTCTrCTCACATATGCTG-3' Srp54 F: 5'-CIIGTAGACCCTGGAGTrAAAG-3' 188 R: 5'-AGCTGGTCAAAACCTCCTGCTC-3' Gene Literature [beta]-Actin Park etal. (1997) Cyp26b1 Wang etal. (2010) Cria 2 Designed (NM_017261.2) II-3 Designed (NC.005109) Lox-1 Nagase etal. (2000) Srp54 Designed (NM.053871.1)
Forced swimming test (FST)
Table 1 shows a characterization of the WB and its different fractions with regard to the content of different polyphenols and salicyl alcohol derivatives. Besides WB (A) especially FR-D, a fraction with a low content of flavonoids and polyphenols but rich in salicin and other salicyl alcohol derivatives, proved to be active.
As shown in Fig. 2 the cumulative immobility time was significantly (p < 0.05) reduced in groups A (-36%), FR-D (-44%) and imipramine (-16%). Other fractions did not show a significant activity in a dosage of 30 mg/kg bw (Fig. 2).
Concentrations of 5-HT (serotonin) and 5-hydroxyindol acetic acid in the hippocampus
The four weeks treatment of CD-rats with WB (15 and 60 mg/kg bw) increased the serotonin concentrations in the hippocampus significantly, whereas the concentrations of 5-HIAA decreased (WB: 15 and 30 mg/kg bw) as depicted in Fig. 3. The turnover of 5-HT, calculated as the ratio of 5-HIAA/5-HT was significantly reduced in a concentration range between 15 and 60 mg/kg. The tricyclic antidepressant imipramine (15 mg/kg bw) as reference drug also reduced the ratio of 5-HIAA/5-HT, but did neither increase the serotonin concentrations nor decrease the 5-HIAA concentrations in the hippocampus significantly in the chosen dose of 15 mg/kg bw.
Whole genome micro-arrays
The analysis of the blood samples revealed that each group showed a distinct expression profile (data not shown). Data of groups, which had shown a significant response in the FST (group A, FR-D, imipramine) were further analysed. The top up- and down-regulated genes are given in the supplementary data.
Gene expression which was commonly modulated in the groups with a significant response in the FST is shown in Table 3. Four of these seven genes were commonly down-regulated. Gria2 encodes a subunit of the group of AMPA-glutamate receptors. They are activated in a variety of normal neurophysiological processes. Cyp26b1 codes for a protein belonging to the P450 family. Srp54 encodes a signal recognition particle (SRP) and Kit1g, a stem cell factor. In contrast, mRNA of Dnm 11 (dynamin like protein 1), a regulator of mitochondrial fission and distribution, was up-regulated in all three groups.
Table 3 Genes and fold changes regulated in all experiments in comparison to the control group. Molecule Group/fold changes Willow Ethanol Imipramine bark fraction SRP54 -19.3 -25.5 -22.9 CYP26B1 -12.6 -20.3 -16.1 GR1A2 -6.4 -10.6 -8.3 KITLG -4.9 -9.1 -7.0 DNM1L 3.6 2.8 3.1 ACAP1 4.6 -2.2 -2.9 EDNRB -2.5 6.1 -2.3 All values represent statistically significant results (p<0.01); Bold: different regulation compared to the other groups. SRP: signal recognition particle. CYP: Cytochrome P450. GRIA: glutamate receptor ionotropic AMPA. KITLG: stem cell factor; DNM1L: dynamin like protein. AGAP: CTPase activating protein. EDNRB: endothelin B receptor gene
The mRNA of Agapl, which belongs to the GTPase-activating protein family, was up-regulated by WB and down-regulated by FR-D and imipramine. Similarly, Ednrb, an endothelin B receptor gene, was differentially regulated--up-regulated by FR-D and down-regulated by WB and imipramine. The modulation of glutamatergic ionotropic or metabotropic receptors is shown in Table 4.
Table 4 Fold changes of genes coding for the group of glutamate receptors. Molecule Official full name Group/fold changes Willow EtOH-Fr. Imipramine bark GRIA2 Glutamate receptor, -6.4 -10.6 -83 ionotropic AMPA 2 GRID2 Glutamate receptor, 3.5 1.1 -2.3 ionotropic, delta 2 GRID2IP Glutamate receptor, -3.0 - -3.6 ionotropic, delta 2 (Grid2) interacting protein GRIK4 Glutamate receptor, 2.8 1.6 -1.2 ionotropic, kainate 4 GRIK5 Glutamate receptor, 2.0 1.1 -3.1 ionotropic kainate 5 GRIN2B Glutamate receptor, -2.2 1.1 -3.9 ionotropic, N-methyl D-aspartate 2B GRIN3B Glutamate receptor, -2.1 -1.0 -1.3 ionotropic, N-methyl-D-aspartate 3B GRINA Glutamate receptor, -3.8 -1.7 -1.5 ionotropic, N-methyl D-aspartate-associated protein 1 (glutamate binding) GRIP1 Glutamate receptor -2.4 1.3 -2.8 interacting protein 1 GRIP2 Glutamate receptor - - -2.4 interacting protein 2 CRM1 Glutamate receptor, -3.3 -1.2 -1.7 metabotropic 1 GRM2 Glutamate receptor, -2.6 -1.0 -1.9 metabotropic 2 GRM3 Glutamate receptor, -2.0 1.2 -1.7 metabotropic 3 GRM4 Glutamate receptor, -2.5 1.2 -7.0 metabotropic 4 Bold: statistically significant values (p < 0.01).
Cytokines, which were significantly (p<0.01) and more than twofold modulated in at least one of the groups are shown in Table 5. The treatment response was not uniform in the different treatment groups and considering the top fold changes in the groups A, FR-D and imipramine, the cytokine response was low. The imipramine treatment provoked in the interleukin- and TNF-families still the strongest response (changes between 1.5 and 9.1 fold). Interestingly, the lowest response with respect to the cytokine families was seen in FR-D (salicin rich ethanol fraction). This group differed from group A (WB) and group F (imipramine).
Table 5 Fold changes of inflammation associated genes. Molecule Group/fold changes Willow Ethanol Imipramine bark fraction IL3 -4.3 -1.1 -3.4 IL10 -2.2 1.1 -4.3 IL15 1.2 1.1 2.0 IL13RA1 2.6 1.8 2.5 IL13RA2 - - 14.8 IL17RA -2.7 -1.4 -1.5 IL1[beta] 2.3 -2.1 1.4 IL1RL2 - - -9.1 IL20RB -4.7 - -3.3 IL21R 2.1 1.2 1.5 IL22RA2 1.9 6.1 2.0 IL4R -1.9 1.1 -2.7 IL7R 3.1 1.3 2.3 TNFAIP2 - - -2.2 TNFRSF1A -2.7 -1.1 1.5 TNFSF12 1.5 -1.7 -2.1 TNFSF14 3.0 1.6 -1.0 Bold: statistically significant values (p <0.01), IL: interleukine, R: receptor, TNFSF: tumor necrosis factor a-superfamily. TNFALP: tumor necrosis factor, alpha-induced protein.
It is noteworthy, that the gene expression of one major proinflammatory mediator, 11-6 was down-regulated by WB and imipramine (1.4 and 1.3 fold respectively, p < 0.05). Equally, Nfkb 1 was down-regulated by WB and imipramine (1.9 and 1.5 fold, < 0.05). Genes encoding the extracellular Sod3 as well as catalase (Cat) were also down-regulated by WB (-1.8 and-1.7 fold respectively) and Sod3 alone by imipramine (-1.2 fold). Even though the magnitude of these regulations did not meet our initial selection criteria (2 fold/p < 0.01), it was stilt significant. The mitochondrial Sod2 met these criteria and was up-regulated by WB (2.2 folds) and down-regulated by imipramine (-2.3 fold).
In the imipramine group the transcripts for the 5-HT-receptors 1A, 1F, 3A and 5B were down-regulated; in the WB-group it was the 5-HTR-1A transcript. FR-D was not active on any RNA expression levels of these receptors (supplementary data).
RT-PCR for Gria2, Srp54, Cyp26b1, Lox-1 and 11-3
Microarray data were verified by RT-PCR for selected genes. Gria2, Srp54, Cyp26b1, Lox-1 and 11-3 were detected in the control group and in the three treatment groups A, FR-D and F (Fig. 4). Results correlated with the microarray data even though the fold changes detected in the PCR were substantially lower and not in all groups significant (Fig. 4).
Inflammatory events are increasingly recognised to contribute to neurospsychatric disorders like depression (Lindqvist et al. 2009; Howren et al. 2009). Thus, we investigated whether willow bark, the forerunner of nonsteroidal anti-inflammatory drugs, might show a beneficial effect in comparison to imipramine in the FST as animal model of depression.
This was indeed the case. The effect size in the groups A and FR-D (each 30 mg/kg bw) was more pronounced (-36% and -44%) compared to imipramine (-16%). The dose of 20 mg/kg imipramine had been chosen for the subacute treatment since in previous studies, doses > 15 mg proved to be active (Butterweck et al. 2000).
The fact that WB as well as FR-D contain high amounts of salicin and salicyl derivates and the latter exhibited a very low percentage of polyphenols and flavonoids allows the assumption that in this chosen dosage salicin and structurally related compounds are chiefly responsible for the reduction of the immobility time in the FST. However, FR-C showed a nearly identical content of salicin and its derivatives (Table 1) and was not active in the FST. Reasons may be a different bioavailability of the salicylates in both fractions or too high concentrations in the context of an U-shaped pharmacology (Kelber et al. 2011) and/or the relative proportions of the individual constituents which may represent a composition without a positive effect in the applied dose. Furthermore, the occurrence of different salicyl alcohol derivates with different pharmacological activity in both fractions can be discussed, a point which is currently under further evaluation.
Irrespective of the components in the WB preparations which are responsible for the effect, a four weeks treatment of the rats with WB regulated the 5-HT concentrations and its metabolism in the hippocampus comparable to "classical" antidepressiva. Serotonin concentrations were increased and its catabolism slowed down as observed in the decrease of the ratio of the serotonin metabolite 5-HIAA to 5-HT. Imipramine, a known non selective serotonin/noradrenalin-reuptake inhibitor, also decreased this ratio, confirming earlier results (Butterweck et al. 2002).
Imipramine blocks neurotransmitter receptors like [5-HT.sub.2A], 5 [HT.sub.-2c], [5-HT.sub.3], [alpha]l, [alpha]2, [H.sub.1], [D.sub.2] and others (Bandelow et al. 2002). Whether WB has also a receptor blocking activity requires further investigations. Since WB increases the serotonin concentration, an influence on the activity of the hypothalamic-pituitary-adrenocortical (HPA)-axis is likely. The HPA-axis is under the control of a variety of neurotransmitters, including serotonin and noradrenalin (Shah 2002), and is hyperactive in case of a 5-HT deficiency, which is well known for depression.
In agreement with the known mode of action of imipramine as serotonin-reuptake inhibitor in the CNS, the genes of four 5-HT receptors were down-regulated also in the periphery. WB obviously acts differently. Only the 5-HTR1 a was slightly down-regulated upon treatment.
The whole genome expression analysis of the peripheral blood cells of groups which showed a biological effect (A, FR-D, F) revealed five commonly regulated genes. Interestingly, these genes could be related to neurological functions and are discussed below.
WB, FR-D as well as imipramine targeted the group of peripheral glutamate receptors, including Gria2. Gria2 codes for a subunit of the alpha-amino-3-hydroxy-5-methy1-4-isoxazolepropionic acid (AMPA)-receptors, which belong to the group of glutamate receptors. They are the predominant excitatory neurotransmitter receptors in the mammalian brain and are activated in a variety of normal neurophysiologic processes (http://www.ncbi.nlm.nih.gov/gene/29627). Diseases which have been associated with an altered gene function of Gria2 including major depression and Alzheimer's disease are reviewed in Machado-Vieira et al. (2009).
In the brain, the tricyclic antidepressants, selective serotonin-reuptake inhibitors (SSRIs) as well as serotonin and noradrenalin-reuptake inhibitors (SNRIs) were shown to down-regulate the N-methyl-n-aspartate (NMDA) glutamate receptors and to potentiate the AMPA glutamate receptors (Novak et al. 1993; Paul et al. 1994; Skolnick et al. 1996; Machado-Vieira et al. 2009). Our data however, show a 6- to 10-fold peripheral down-regulation of Gria2 for imipramine as well as for WB treatment and a broad modulation of members of the glutamate receptor family. In the context of recent discoveries that platelets express AMPA-receptors for the excitatory neurotransmitter glutamate and influence platelet activation (Morrell et al. 2008), this peripheral down-regulation appears to be noteworthy. A modified platelet GLUR1 phosphorylation in major depressive disorders (MDD) has been hypothesised to contribute to the comorbidity of MDD and cardiovascular disorders (Chen 2009).
Further studies of these effects may increase the understanding of a contribution of the peripheral GluR family to the comorbidity of MDD and CVD and support improved drug developments.
Protein secretory pathways
One of the strongest regulated genes in the three groups is the signal regulating protein 54 (Srp54)gene (19-25-fold[down arrow] encoding a protein involved in the secretory pathway to guide proteins through the endoplasmatic reticulum (ER). Protein translocation across the ER-membrane requires signal sequence binding to Srp54 (Grudnik et al. 2009). As one of the two central elements of the so called SRP-system, human SRP54 has a guanosine triphosphatase (GTPase) activity and is universally conserved (Grudnik et al. 2009). A single nucleotide polymorphism (SNP) in 5RP54 has been associated with bipolar affective disorder (Baum et al. 2008). Watanuki et al. (2008) showed that the mRNA of SRP20, but not of SRP54 was increased in patients suffering from major depressive disorders. However, authors acknowledge limitations of their findings since the treatment of patients was not standardized. We can show here that in our animal model, WB, FR-D and imipramine down-regulate the central element of the SRP-system, Srp54, suggesting that protein secretion is decreased.
DNM1L (DLP1) is known to constrict and tubulate membranes and to divide mitochondria and peroxisomes. Recent data indicate that at the Golgi complex DNM1L is a component of the sorting/targeting machinery en route to the plasma membrane (Bonekamp et al. 2010). Mitochondrial dysfunction and synaptic loss are among the earliest events linked to Alzheimer's disease (AD) and might play a causative role in disease onset and progression (Bossy et al. 2010). Dnmll was up-regulated in all three groups suggesting that the regulation of mitochondrial fission and distribution will positively influence protein targeting and sorting.
Another strongly down-regulated gene (-12 to -20 fold) in all groups was Cyp26b1, encoding a cytochrome-P450 enzyme that catabolises retinoic acid (RA) (Maclean et at. 2009). All-trans-RA stimulates neurogenesis, dendritic growth of hippocampal neurons and higher cognitive functions. Genetic disruption of mouse Cyp26b1 affects the development of neural crest-derived central nervous system structures, but does not compromise hindbrain patterning (Maclean et at. 2009). A down-regulation of CYP26131 mRNA in brain motor cortex has been shown to be involved in Huntington's disease (Hodges et al. 2006). Brain CYP26 activity is considered a key effector inhibiting neuronal differentiations (Gonzalez-Quevedo et al. 2010). The relevance of a peripheral down-regulation is not known so far. However, we detected that imipramine as tricyclic antidepressant as well as WB and FR-D target this gene in the context of an antidepressant like activity in the FST. A very recent report also links CYP2681 to the regulation of RA-dependant signals in activated T-cells and thus to the immune system (Takeuchi et al. 2011).
The mRNA of Kitlg or stem cell factor (SCE) was 4- to 9-fold down-regulated in all three groups. SCE activates the c-kit ligand of the tyrosine-kinase receptor. It is also known as a haematopoietic growth factor (HGF) which promotes neuroprotective effects and supports neurogenesis in the brain (Laske et al. 2008; Zhao et al. 2007). Transcription of SCF is up-regulated in inflammatory conditions (Reber et al. 2009). Therefore, a down-regulation of SCF suggests an anti-inflammatory effect.
Modulation of cytokines in depression
WB is well known for its anti-inflammatory action. A down-regulation of the proinflammatory cytokines TNF-[alpha] and I1-6, as well as the proinflammatory transcription regulator NF-KB, has been shown in human cellular (Bonaterra et al. 2009; Fiebich and Chrubasik 2004; Khayyal et al. 2005) and animal models of inflammation (Khayyal et al. 2005) earlier. We stated under our experimental conditions a minor down-regulation of11-6 and Nf-Kb transcription through WB in agreement with these earlier findings. Interestingly, also imipramine as serotonin-uptake inhibitor did mildly down-regulate both molecules. However, before discussing the cytokine modulation, the relevance of a peripheral regulation of cytokines in the context of depression needs to be addressed.
Initial clinical data indicate that peripherally administered cytokines (IFN-[alpha]) can activate a CNS inflammatory response in humans which interacts with the 5-HT metabolism (Raison et al. 2009). Changes included increases in MCP-1 and IL-6 in the CSF. Further, several cytokines were reported to trigger the transcription of genes at cells of the blood-brain barrier including NF-KB and COX-2, and these proteins then promoted signalling within the CNS (Laflamme and Rivest 1999; Seguin et al. 2009). Himmerich et al. (2009) proposed that TNF-[alpha] and its soluble receptors might contribute to the pathogenesis of neurological diseases through an activation of the HPA axis, of neuronal 5-HT transporters and the stimulation of the indoleamine 2,3-dioxygenase leading to a tryptophan depletion, through an immunologically mediated destruction of neurons, or through the neurotoxic release of glutamate (Himmerich et al. 2009). It was further proposed that peripheral rather than central actions of TNF-[alpha] will influence the birth of new hippocampal dentate gyrus cells. Considering the relevance of the individual cytokines, other experiments of this group showed that IL-1 1[beta] generally produced more profound behavioural, neuroendocrine and neurotransmitter effects than TNF-[alpha] (Seguin et al. 2009).
In our gene expression analyses of peripheral blood cells I1-3-mRNA showed a down-regulation through WB and imipramine (-4.3 fold and -3.4 fold respectively). IL-3 is a growth promoting cytokine, supporting the proliferation of a broad range of haematopoietic cell types. It is proposed to be associated with neurological disorders (HGNC) and was shown to induce the activation of the JAK-STAT and MAP kinase pathways in microglial cells, the resident macrophages of the brain (Natarajan et al. 2004). A down-regulation, as seen in our experiments, could be favourable since the stress induced JAK-STAT pathway would not be activated.
Other down-regulated mRNA levels of cytokine receptors (>2 fold) were those of11-17ra and11-20rb. Human1L-17RA was shown to be expressed in the central nervous system glia and up-regulated in experimental autoimmune encephalomyelitis (Das Sarma et al. 2009), whereas not much is known so far on the IL-20 receptor beta except that the cytokine IL-20 is regarded to be a pleiotropic cytokine with potent inflammatory, angiogenic and chemoattrac-tive characteristics (Wei et al. 2006).
The I1-13-receptor, slightly down-regulated by WB, is present in human glioma cells and appears to be over expressed in solid brain tumors (Shimamura et al. 2006). However, the relevance of this finding is unclear.
WB up-regulated also the rat 11-21 receptor and 11-7r mRNA levels. In human, IL-21R has been attributed a critical role in the development of substantial memory B-cells (Rankin et al. 2011). Its presence on neurons has been demonstrated leading to the proposal that it plays a role in both, acute and chronic stages of multiple sclerosis (MS) via direct effects on T and B lymphocytes (Tzartos et al. 2011). Similarly IL-7R was shown to be involved in MS (Haas et al. 2011). Here, T-cells showed a reduced expression of the receptor and since IL-7 appears to play a critical role in Treg maturation (Haas et al. 2011), a relevance for the Treg homeostasis in MS was proposed.
The TNF-receptor superfamily member 1 A (TNFRSFI A) has been found to work in cancer cells as trigger molecule for the apoptotic cascade (Karabulut et al. 2010). WB lead to its down-regulation in the rat, whereas TNFSF14 (LIGAND), which was shown to be particularly involved in apoptosis and inflammation was slightly up-regulated here.
The proinflammatory cytokine transcript of Il-1[beta] was slightly up-regulated (2.3 fold) by WB. This is somewhat surprising, since in several models of inflammation the COX-inhibitor WB also reduced IL-1[beta] (Fiebich and Chrubasik 2004; Khayyal et al. 2005). In addition, mRNA of other proinflammatory cytokines like 11-3 or 11-6 was down-regulated in our experiments. In depression a peripheral up-regulation of IL-1 has been implicated (Milaneschi et al. 2009) and in the central nervous system IL-1[beta] was shown to induce COX-2, thereby contributing to inflammatory pain hypersensitivity (Howren et al. 2009). The explanation for this at first glance contradictory result may lay in the interplay of the cytokine network which still requires further investigations. The estimated expression profile of WB may represent just one profile for an anti-inflammatory activity.
There is an increasing evidence that mood disorders are associated with impairments in neuroplasticity and cellular resilience (Machado-Vieira et al. 2009). Alterations of the glutamatergic system (Machado-Vieira et al. 2009) as well as cytokines are attributed a major role in these impairments (Bret-Dibat et al. 1995; Rivest et al. 2000; Banks et al. 2001; Seguin et al. 2009). We show a low down-regulation of 11-3, 11-6 and Tnf-a associated receptor RNA in combination with a low up-regulation of II-1[beta]. At the same time, the diversity of glutamatergic receptors is targeted by all three groups (Table 4). IL-1[beta] and TNF-[alpha] were reported to alter hippocampal long-term potentiation and, together with IL-6, also influenced dendritic branching of hippocampal neurons. Seguin et al. (2009) proposed that they may differentially regulate hippocampal plasticity rather than proliferation. Thus, an effect on the plasticity can be expected for the three effective treatment groups.
Antioxidant status and depression
Recently, Zafir et al. (2009) proposed the antioxidant system as target for an antidepressive treatment. They showed that in rats identified by FST as depressive-like phenotypes the Sod, Cat, glutathione S-transferase (Gst) and glutathione reductase (Gr) were increased. These parameters declined following antidepressant treatment (fluoxetine as selective serotonin reuptake inhibitor, imipramine as tricyclic antidepressant; venlafaxine as dual sero-tonin/norepinephrine reuptake inhibitor (Zafir et al. 2009). In line with these findings, we observed a down-regulation of the mRNAs encoding extracellular Sod3 and the mitochondrial Sod2 under imipramine treatment. In addition, Cat and Sod3 were down-regulated by WB, supporting earlier studies that WB preparations have an antioxidant activity (Bonaterra et al. 2009; Khayyal et al. 2005). These data support the idea that regulation of the redox-state plays a role in depression.
Short term and long term administration
The time of administration will influence the mode of action especially with respect to the cytokine network and the neurotrans-mitter levels. For example, it was shown that a single infusion of IL-6 or IL-1[beta] was not a sufficiently potent challenge to influence the hippocampus (Seguin et al. 2009). Our cytokine data pertain to a treatment of 14 days which led to a positive FST and a measurable effect in the peripheral cytokine network for the treatments with WB, FR-D and imipramine (20 mg/kg). The 5-HT levels as well as its turn over were significantly affected by a WB treatment over 4 weeks, whereas imipramine changed the turn over, but not the serotonin or the 5-HIAA levels. These data for imipramine correspond to earlier studies of this group (Butterweck et al. 2002).
Interestingly, the native extract of Hypericum perforatum L yielded in comparable experiments (FST) undertaken by this group significant results in concentrations as high as 125 mg/kg bw and more (Winterhoff et al. 1995). The ratio of 5-HT/5-HIAA in the hippocampus was reduced after a two weeks and an eight weeks treatment with this preparation in concentrations of 500 mg/kg bw. Isolated fractions applied in combinations of procyanidines with naphtodianthrones, hypericin and pseudohypericin were, however, already significantly active in the FST at much lower concentrations (0.028 mg/kg and 0.166 mg/kg) (Butterweck et al. 2003). Also the biflavones of Hypericum perforatum L are regarded useful for the treatment of inflammation and depression (Michler et al. 2011).A positive response in the FST and increased 5-HT levels in the hippocampus caused by a treatment with the WB extract in concentrations of 30 mg/kg and 15 mg/kg bw allows the question of the relevance of inflammatory processes for the neurotransmitter metabolism.
A combinatorial treatment approach is supported by results of a recent double blind placebo controlled trial in patients with major depression. The treatment of celecoxib (COX-2 inhibitor) and fluoxetine had a significant superiority over fluoxetine alone in the treatment of symptoms of major depression (Akhondzadeh et al. 2009). Since both drugs have a good bioavailability, a pharmacokinetic interaction may not be the reason for this effect. We analysed our microarray data also for the prediction of AEs. The model analysis revealed less potential AEs for WB as multicomponent mixture compared to imipramine as single component drug (Ulrich-Merzenich et al. 2012).
In summary, these investigations identified novel neuro-and immunological targets associated with salicylate-containing phytopharmaceuticals and with the anti-depressant imipramine. The data contribute to our understanding of the link between inflammation and neurological functions and may support the development of co-medications from salicylate-containing phy-topharmaceuticals as multicomponent mixtures with synthetic single component drugs to improve therapeutic efficacy and the AEs-profile.
Authors are thankful to Steigerwald Arzneimittelwerk GmbH for financial support to undertake these studies. The willow bark extract was kindly provided by Dr. Ulrike Kroll from Steigerwald Arzneimittelwerk GmbH. Dr. Anna Koptina holds a scholarship of the German Academic Exchange Service. We would especially like to thank Prof. Dr. H. Wagner and Dr. S.N. Okpanyi for their critical comments and suggestions.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phymed.2012.05.004.
Abbreviations: 5-HT, serotonin; 5-HIAA, 5-hydroxyindol acetic acid; AD, Alzheimer's disease; AMPA, al pha-amino-3-hydroxy-5-methy1-4-isoxazolepropionic acid; ASA, acetylsalicylic acid; CAT, catalase; CD, Sprague-Dawley; CNS, central nervous system; COX, cyclooxygenases; CRH, corticotrophin-releasing hormone; CSF, cerebral spinal fluid; CYP26B1, cytochrome P450 protein 2681; DNM1L, dynamin like protein 1; EDNRB, endothelin B receptor gene; ER, endoplasmatic reticulum; EtOH-FR, ethanol fraction; FR, fraction; FST, Porsolt-Swimming Test; GR, glutathione reductase; GST, giutathione S-transferase; GTPase, guanosine triphosphatase; HGF. haematopoietic growth factor; HPA-axis, hypothalamic-pituitary-adrenocortical axis; MS, multiple sclerosis; NMDA, N-methyl-o-aspartate; RA, retinoic acid; SCF, stem cell factor; SNP, single nucleotide polymorphism; SNR1s, serotonin and noradrenalin-reuptake inhibitors; SOD, superoxidedismutase; SRP, signal recognition protein; SSRIs, selective serotonin-reuptake inhibitors; TNFRSF1A, TNF-receptor superfamily member 1A; WB, willow bark.
* Corresponding author. Tel.: +49 22828722674; fax: +49 22828722019.
E-mail address: Gudrun.Ulrich-Merzenich@ukb.uni-bonn.de (G. Ulrich-Merzenich).
0944-7113/$--see front matter [c] 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.05.004
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G. Ulrich-Merzenicha, (a), (*) O. Kelber, (b) A. Koptina (a), (h) A. Freischmidt, (c) J. Hellmann (c) J. Muller (b) H. Zeitler, (d) M.F. Seidel (e) M. Ludwig, (f) E.U. Heinrich (b) H. Winterhoff. (g), (1)
(a) Medizinische Poliklinik, Universittitsklinikum, Rheinische Friedrich-Wilhelms-Universittit Bonn, Germany
(b) Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany
(c) Pharrnazeutische Biologie, Universitiit Regensburg, Germany
(d) Medizinische Klinik I. Universittitsklinikum, Rheinische Friedrich-Wilhelms-Universittit Bonn, Germany
(e) Med izinische Klinik I. Rheumatology Unit, Universittitsklinikum, Rheinische Friedrich-Wilhelms-Universittit Bonn, Germany
(f) Department of Clinical Chemistry and Clinical Pharmacology, Universittitsklinikum, Rheinische Fried rich-Wilhelms Universittit Bonn, Germany
(g) Institut fur Pharrnakologie und Toxikologie, Westftilische Wilhelms-Universittit, Munster. Germany
(h) Mari State Technical University, Yoshkar Ola, Russia
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|Author:||Ulrich-Merzenich, G.; Kelber, O.; Koptina, A.; Freischmidt, A.; Hellmann, J.; Muller, J.; Zeitler, H|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Jul 15, 2012|
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