UNIVERSIDADE FEDERAL DE SANTA CATARINA
CURSO DE FISIOTERAPIA
GRAZIELA VIEIRA
TERPINEOL INIBIU O COMPORTAMENTO TIPO-DEPRESSIVO INDUZIDO PELA
INFLAMAÇÃO PERIFÉRICA
Araranguá
2018
GRAZIELA VIEIRA
TERPINEOL INIBIU O COMPORTAMENTO TIPO-DEPRESSIVO INDUZIDO PELA
INFLAMAÇÃO PERIFÉRICA
Artigo
apresentado
ao
Curso
de
Graduação
em
Fisioterapia,
da
Universidade Federal de Santa Catarina,
como requisito parcial da disciplina de
Trabalho de Conclusão de Curso II.
Orientador: Professor Rafael Cypriano
Dutra.
Araranguá
2018
DEDICATÓRIA
Dedico este trabalho a minha maior incentivadora,
minha amada filha, Isabeli Vieira Calegari.
AGRADECIMENTOS
A Deus pela oportunidade de concluir esse trabalho. Aos meus amados pais que acreditaram em mim e sempre apoiaram meus sonhos. Ao curso de Fisioterapia da Universidade Federal de Santa Catarina, aos professores e colegas que convivi durante esses anos nessa longa jornada de muito estudo e dedicação. Ao meu orientador que compartilhou vários conhecimentos comigo. Ao Laboratório de Autoimunidade e Imunofarmacologia (LAIF) que proporcionou ferramentas que permitiram a conclusão desse trabalho. As minhas queridas colegas de laboratório Tainara, Larissa, Elaine e a todos outros que fazem do LAIF um lugar de crescimento e muito conhecimento.
“Por vezes sentimos que aquilo que fazemos não é senão uma gota de água no mar. Mas o mar seria menor se lhe faltasse uma gota.”
RESUMO
De acordo com a Organização Mundial da Saúde (OMS), a depressão é um transtorno mental comum e uma das principais causas de incapacidade em todo o mundo A depressão apresenta etiologia multifatorial decorrente de fatores ambientais, psicológicos, genéticos e biológicos. Diferentes estudos relataram efeitos farmacológicos do Tepineol (TPN), tais como anti-alérgicos, anti-nociceptivo, anti-inflamatório, anti-hiperálgico, antidiabético, anticonvulsivante, efeitos protetores da dependência, tolerância a morfina e isquemia cerebral. No entanto, até o presente momento não existem relatos na literatura demonstrando o efeito antidepressivo do TPN, assim o presente estudo investigou a ação do TPN em comportamento tipo-depressivo em camundongos induzido pela administração sistêmica de lipopolissacarídeo (LPS). Além disso, investigou-se o efeito do TPN na ativação da enzima acetilcolinesterase (AChE), assim como a citotoxicidade do TPN na viabilidade celular de fibroblastos murinos L929. Os resultados do presente estudo demonstram que o TPN inibiu significativamente o comportamento tipo-depressivo, através da modulação dos receptores canabinóides CB1 e CB2, receptores dopaminérgicos e, possivelmente, pela modulação do sistema colinérgico. O TPN exibiu alta atividade de inibição da AChE com 91,11%, quando comparado a rivastigmina – fármaco padrão-ouro na inibição da AchE. Além disso, o TPN apresentou baixa citotoxidade em células normas, demonstrando assim segurança em fibroblastos murinos. Em conjunto, nossos resultados sugerem que o TPN surge como ferramenta terapêutica alternativa para o controle da depressão e suas respectivas comorbidades, embora estudos adicionais sejam necessários para melhor detalhamento do mecanismo de ação deste composto.
Palavras-chave: Neuroinflamação. Transtornos do humor. Lipopolissacarídeo. Terpineol. Citotoxicidade. Acetilcolinesterase.
LISTA DE ILUSTRAÇÕES
Table 1: Antagonists………..………32
Graphical Abstract……….……….………...33
Figure 1: Schematic diagram demonstrating a trial of the first experiment with the
TPN antidepressant preventive treatment………….………...……….34
Figure 2: Schematic diagram demonstrating a trial of the first experiment with the
TPN antidepressant therapeutic treatment effects.……...………..……..………..34
Figure 3: Schematic diagram demonstrating a trial of the second experiment with the
TPN antidepressant effects……….……34
Figure 4: Effects of TPN preventive treatment on the LPS-induced depression-like
behavior in mice………..………...35
Figure 5: Effects of TPN therapeutic treatment on the LPS-induced depression-like
behavior in mice………..…...………36
Figure 6: Locomotor activity on the open field test with TPN preventive treatment and
therapeutic treatment………....37
Figure 7: Cannabinoid receptors involvement underlying the antidepressant-like
effects of TPN on tail suspension tests and splash test……….……..…….….38
Figure 8: Dopaminergic system involvement in the mechanisms underlying the
antidepressant-like effects of TPN on tail suspension tests and splash test…...39
Figure 9: Adenosine A1 and A2 receptors Involvement in the antidepressant-like
effects of TPN (TPN 200 mg/kg, p.o.) on tail suspension tests and splash test…….40
Figure 10: Adrenergic receptors involvement underlying the antidepressant-like
effects of TPN (200 mg/kg, p.o.) on tail suspension tests and splash test…………...41
Figure 11: TPN L929 fibroblast cell viability 48 h post-exposure to TPN in different
concentrations………....42
LISTA DE ABREVIATURA E SIGLAS
AChE
Acetylcholinesterase
CAPES
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
CNPq
Conselho Nacional de Desenvolvimento Científico e Tecnológico
FAPESC
Fundação de Apoio a Pesquisa do Estado de Santa Catarina
HPA
Hypothalamus-pituitary-adrenal
LD
light–dark
LPS
Lipopolysaccharide
OFT
Open Field Test
PGN
Programa de Pós-Graduação em Neurociências
SFB
Fetal Bovine Serum
SPSS
Statistical Package for the Social Sciences
ST
Splash Test
TNFα
Tumor Necrosis Factor-α
TPN
Terpineol
TST
Tail Suspension Test
UFSC
Universidade Federal de Santa Catarina
WHO
According to the World Health Organization
SUMÁRIO
1. INTRODUCTION……….……….……….…...13
2. MATERIALS AND METHODS………...14
2.1. Animals……….………...15
2.2. Drugs and Reagents……….………15
2.3. Experimental Design……….……….…..….15
2.4. Behavioral analysis………..………..……15
2.4.1. Tail Suspension Test (TST).……….………..………...…15
2.4.2. Splash Test (ST)...16
2.4.3. Open Field Test (OFT)………..………16
2.5. Enzyme Activity Assay………...………...…16
2.6. Cytotoxicity Measurement………...…...………...….17
2.7. Statistical analysis……….……….…...17
3. RESULTS………...………...….17
3.1. Antidepressant-like effect of the TPN in the TST and ST……….………..17
3.1.1. Preventive treatment……….………..17
3.1.2. Therapeutic treatment………...18
3.2. Effect of the LPS in the locomotor activity in the OFT………...18
3.3. Investigation of the mechanisms underlying the antidepressant-like effect of the
TPN in the TST and ST………...18
3.3.1. Involvement of the cannabinoid system………...18
3.3.2. Involvement of the dopaminergic system……….…..19
3.3.3. Involvement of the adenosinergic system………...….……...…..19
3.3.4. Involvement of the noradrenergic system……….……...…..19
3.8. Acetylcholinesterase inhibitor……….….19
3.9. Cytotoxicity of murine L929 fibroblast cells………...20
4. DISCUSSION……...….20
REFERENCES………...…21
APÊNDICES…………...30
APÊNDICE A – Legenda das figuras…………...29
APÊNDICE B – Tabela…………...…31
APÊNDICE C – Figuras...32
ANEXOS...43
ANEXO A – Normas da revista...43
DATA IN BRIEF TEMPLATE
*Title: Terpineol ameliorates inflammation-induced depressive-like behavior
*Authors: Graziela Vieira a, Elaine C. D. Gonçalves a,b, Tainara R. Gonçalves a, Larissa R. Laurindo a, Nádia. R. B. Raposoc, and Rafael C. Dutra a,b.
*Affiliations: a Laboratory of Autoimmunity and Immunopharmacology (LAIF), Department of Health Sciences, Campus Araranguá, Federal University of Santa Catarina, Araranguá, Brazil
b Post-Graduate Program of Neuroscience, Center of Biological Sciences, Federal University of Santa Catarina, Florianópolis, Brazil
c Center for Research and Innovation in Health Sciences (NUPICS), Faculty of Pharmacy, Federal University of Juiz de Fora, Rua José Lourenço Kelmer, s / n, 36036-330, Juiz de Fora-MG, Brazil.
*Contact email: Rafael C. Dutra (rafaelcdutra@gmail.com)
*Co-authors: Graziela Vieira (graziela.v@grad.ufsc.br)
Elaine C. D. Gonçalves (nanidalazen@hotmail.com),
Tainara R. Gonçalves(tainararibeirogoncalves@hotmail.com), Larissa R. Laurindo (larissa-laurindo@hotmail.com) and Rafael C. Dutra (rafaelcdutra@gmail.com).
*CATEGORY: Behavioral Neuroscience
Title:
Authors:
Graziela Vieira a, Elaine C. D. Gonçalves a,b, Tainara R. Gonçalves a, Larissa R. Laurindo a, Nádia. R. B. Raposoc, and Rafael C. Dutra a,b.
Affiliations:
a Laboratory of Autoimmunity and Immunopharmacology (LAIF), Department of Health Sciences, Campus Araranguá, Federal University of Santa Catarina, Araranguá, Brazil b Post-Graduate Program of Neuroscience, Center of Biological Sciences, Federal University of Santa Catarina, Florianópolis, Brazil
c Center for Research and Innovation in Health Sciences (NUPICS), Faculty of Pharmacy, Federal University of Juiz de Fora, Rua José Lourenço Kelmer, s / n, 36036-330 Juiz de Fora-MG, Brazil.
Contact email: Rafael C. Dutra (rafaelcdutra@gmail.com)
Abstract
As stablished by the World Health Organization (WHO), depression is a mental disorder and a leading cause of disability worldwide. Its etiology emerges from environmental, psychological, genetic and biological factors. Though no reports of its antidepressant properties were found, several analyzed studies have reported the pharmacological properties of Terpineol - such as allergic, antinociceptive, inflammatory, anti-hyperalgesia, anti-diabetic, anticonvulsants, protective dependence effects, morphine tolerance and cerebral ischemia. This study researched the effects of Terpineol on acetylcholinesterase activation, the cytotoxicity of Terpineol on cell viability of L929 murine fibroblast, as well as its effects on depressive-like behavior in mice by systemic administration of lipopolysaccharide. The results found indicated that through modulation of cannabinoid CB1 and CB2 receptors, dopaminergic receptors, and possibly through cholinergic system modulation, Terpineol inhibited the depression-like behavior significantly. Once compared to Rivastigmine, Terpineol demonstrated effective acetylcholinesterase inhibition (91,11%), also including low cytotoxicity in healthy cells and proving being safe on murine fibroblast. Our findings further suggest Terpineol as an alternative therapeutic depression agent and depression related comorbidities controler. Further studies are necessary in order to explain the mechanism of action in this compound.
Keywords: neuroinflammation, mood disorders, lipopolysaccharide, terpineol, acetylcholinesterase.
1. INTRODUCTION
According to the World Health Organization (WHO) depression is a common mental disorder and a leading cause of disability worldwide [1]. Recent data suggest that depression is the disorder that induces more lifetime suicide risk [2]. A systematic review examined the results of psychological autopsy in suicide studies that showed that the majority of mental disorder cases were of depression [3]. Depression has a multi-factorial etiology arising from environmental, psychological, genetic and biological factors [4]. Environmental stress and genetic factors acting through immunologic and endocrine responses initiate structural and functional changes in brain regions inducing neurogenesis and neurotransmission dysfunction resulting in specific symptoms such as sadness, loss of interest, anhedonia (loss of interest or pleasure), lack of appetite, feelings of guilt, low self-esteem or self-worth, sleep deprivation, feelings of tiredness, and poor concentration [5]. Modification of monoaminergic activity, immune-inflammation, oxidative stress, hypothalamus-pituitary-adrenal (HPA) activity and neuroprogression contribute to the pathology of depression [6]. Chronic cytokine levels stimulates neurotransmitter changes, resulting to stress experiences [7]. The cholinergic system is known to powerfully modulate depression symptomology and administration of cholinergic receptor antagonists induces antidepressant-like effects [8]. Pre-clinical studies of cholinergic mechanisms in depression underscore roles for cholinergic activity in several dopaminergic brain regions as the prefrontal cortex [9,10], the nucleus accumbens [11,12] and the hippocampus [13].
Through the administration of bacterial lipopolysaccharide (LPS; the active component of endotoxin from gram-negative rod), a strong production of both peripheral and brain proinflammatory cytokines was induced, such as interleukin-1β 1β, interleukin-6 (IL-6) and tumor-necrosis factor-α (TNFα) [14–17], and brain IL-1β is a key cytokine for the development of sickness behavior [18–20]. The proinflammatory cytokines produced in the periphery by activated innate immune cells induce the production of the same molecular signals by microglial cells and macrophage-like cells in the brain [20]. The communication between the brain and the immune system happens by a neural pathway when peripherally produced pathogen-associated molecular patterns (PAMPs) and cytokines activate primary afferent nerves [21–23], by humoral pathway toll-like receptors (TLRs) on macrophage-like
cells respond to circulating pathogen-associated molecular patterns [24], these cytokines produced by circumventricular organs cross the blood–brain barrier (BBB) by volume diffusion, overflowing the systemic circulation with cytokines[25]. Recently, it has been suggested that pro-inflammatory cytokines induce not only symptoms of sickness, but also true major depressive disorders in physically ill patients with no previous history of mental disorders [26].
Moreover, monoterpenes are the main terpenoids compounds found in plant species. Those are compounds of small molecular weight and non-polar nature [27]. Terpineol (TPN) is an important monoterpenoid alcohol present in the essential oils of several species of plants, such as Eucalyptus genus [28]. Some TPN pharmacological effects have been reported, such as anti-allergy [29], antinociceptive [30], anti-inflammatory [31], antihyperalgesic [32], anti-diabetic [27], morphine dependence and tolerance protective effects [33], cerebral ischemia improvement [34] and anticonvulsant [35]. Since TPN has a small molecular weight and non-polar nature it is possible that it plays a hippocampal memory system modulatory effect directly [34]. Considering the correlation between hippocampus size reductions and depression previously reported [36], we therefore investigated the depressive-like effects of TPN and furthermore the relationship between TPN and acetylcholinesterase (AChE), as well as TPN cytotoxicity cell therapy viability of murine L929 fibroblasts.
2. MATERIALS AND METHODS
2.1. Animals
Male Swiss mice weighing 20-30 g were supplied by the Federal University of Santa Catarina. The mice went through a period of acclimation of at least a week in the facility prior to the start of the experiment, being also allowed free access to tap water and chow. They were maintained in a 12:12-h light–dark (LD) cycle, the onset of light at 07:00, and at constant temperature (22°C ± 2°C) with between 40-60% relative humidity (%rH) in groups of 10 mice per group. During experiments, animals were given a period of at least an H to acclimate to the laboratory settings before testing and were used only once throughout the experiments. The mice were randomly assigned before treatment or behavioral evaluation. All procedures used in the present study followed the “Principles of laboratory animal care” (NIH publication no. 85–23) and were approved by the Animal Ethics
Committee of the Universidade Federal de Santa Catarina (CEUA-UFSC, protocol number PP00956).
2.2. Drugs and Reagents
The following drugs were used: LPS from Escherichia coli 0127:B8, TPN (mixture of isomers, ≥96%, FG), sulpiride, haloperidol, imipramine and propranolol were obtained from Sigma Chemical Company (St. Louis,MO, U.S.A.). The AM281 and AM630 were purchased from Tocris Bioscience (Ellisville, Missouri, USA). All drugs were administered by intraperitoneal (i.p) route, whereas TPN was administered by oral gavage (p.o.). Drugs were dissolved in saline, except sulpiride that was diluted in saline with 5% DMSO [37] and TPN diluted in saline using Tween 80 0.5%, v/v [38].
2.3. Experimental Design
To assess the TPN antidepressant agent different dosages TPN 100 mg/kg, p.o., TPN 200 mg/kg, p.o., TPN 100 mg/kg, i.p. and imipramine (20 mg/kg, i.p.) [39] were used as positive control. TPN has been administered 1h prior to LPS (0.5 mg/kg, i.p.) in preventive treatment (Figure 1) and 1h after LPS (0.5 mg/kg, i.p.) in therapeutic treatment (Figure 2). The behavioral analysis was recorded 24h after the administration of LPS.
In the second part of this study, we investigated the underlying antidepressant-like effects of TPN associated with haloperidol antagonists [37,40], sulpiride [37,40], propranolol [41], caffeine [42]. AM281 and AM630 [43], dosages are shown in Table 1. After 30 min of administrating two antagonists, they received a dose of TPN (200 mg/kg, p.o.) and after 1h LPS (0.5 mg/kg, i.p.) was administered and the behavioral analysis was then performed 24h after as illustrated in Figure 3.
2.4. Behavioral analysis
2.4.1. Tail Suspension Test (TST)
In this behavioral test mice are suspended by tip of the tail for a period of 6 min using adhesive tape, and the time of immobility, latency to immobility and latency of immobility are recorded [44] . The immobility time is considered depression-like behavior and
antidepressant medications reverse this immobility and promote escape-related behavior [45].
2.4.2. Splash Test (ST)
This test consisted of squirting 200 μl of a 10% sucrose solution on the animal dorsal coat. Because of its viscosity, the sucrose solution dirties the mouse fur and animals initiate grooming behavior. After applying the sucrose solution, the time spent grooming was recorded throughout a span of 5 min as an index of self-care and motivational behavior [28,46].
2.4.3. Open Field Test (OFT)
To investigate locomotion activity and exploratory behavior, the mice were individually placed in a wooden box (40 x 60 x 50 cm) with the floor divided into 12 squares. The crossing number (number of squares crossed by the animal using all paws) was used to evaluate locomotion activity, whereas the rearing behavior (number of times the mice stood on its hind legs or engaged in vertical exploratory activity) was used to assess exploratory behavior [47].
2.5. Enzyme Activity Assay
The inhibition quantitative evaluation of acetylcholinesterase was tested using TPN dispensed into wells of a 96-well microplate in triplicate. Afterwards, mixed with 190 μL Ellman’s reagent containing 20 μL enzyme soluiton, 140 μL to phosphate buffer, 10 μL of 0.5 mM of 5, 5’- dithio-bis-(2-nitrobenzoic acid) (DTNB, Sigma-Aldrich, Germany) and 20 μL acetylthiocholine iodide (ATCI, Sigma-Aldrich, Germany), pH 8. The control wells contained ethanol instead of the TPN. The enzymatic activity was monitored at 412 nm every 30 s for 3 min (linear reaction). The enzyme activity was calculated from the slope of absorbance vs. time curve. As screening strategy, final concentration of 1000 μg/mL from TPN was examined and the average percentage inhibition was calculated in relations to the enzyme activity at the control wells according to the following equation [48]:
%Inhibition = 100 − {(Ac − At)/Ac} × 100
2.6. Cytotoxicity Measurement
To evaluate the TPN cytotoxicity, L929 mouse fibroblast cells viability (Federal University of Minas Gerais) was evaluated after DMEM culture (Nutricell, Campinas, São Paulo, Brazil) supplemented with 10% fetal bovine serum (SFB) (Invitrogen, Waltham, MA, USA), 100 U/Ml penicillin, 100 U/Ml streptomycin and 10 Mm 4-(2-HydroxyEthyl)-1-PiperazinEethaneSulfonic buffer (HEPES) [49]. The L929 cells at density of 5 × 103 cells per well were cultured in sterile 96-well plates with level depths (Sarstedt, Nümbrecht, Germany) and were then incubated at 37 ± 2 °C in an atmosphere containing 5% CO2 for 48 h. Subsequently, culture media were replaced with sample solutions at concentrations of 7.81– 1,000 μg/Ml and controls were not inoculated and contained 2 Μl of 70% (v/v) ethanol solution. After 48 Hs, culture media were removed and 100 Μl aliquots of 10% [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT; 5 mg/Ml) in DMEM were added to all wells. Plates were immediately incubated at 37 ± 2 °C in an atmosphere containing 5% CO2 for 3h. Finally, the resulting formazan crystals were dissolved in DMSO and absorbance was evaluated using a spectrophotometer (SpectraMax 190; Molecular Devices, San Jose, CA, USA) at 540 nm [50–52].
2.7. Statistical analysis
Values will be expressed as mean ± standard error of the mean (SEM); p < 0.05 and p < 0.001 were considered statistically significant. Statistical analysis was performed by the Analysis of Variance (ANOVA) test, by the Newman-Keuls procedure for multiple comparisons and by the Bonferroni test with (Graphpad Prism version six and IBM SPSS Statistics 21 (Statistical Package for the Social Sciences, Inc., Chicago, IL, USA)) and were considered significant when p < 0.05.
3. RESULTS
3.1. Antidepressant-like effect of the TPN on the TST and ST
3.1.1. Preventive treatment
The results in Figure 4 (A, B and C) show the antidepressant-like effects of TPN (200 mg / kg v.o.) significantly decreased the immobility time (P<0.005) and immobility number stops (P<0.001) against the naïve control group, as well as TPN (100 mg / kg v.o.)
and the conventional antidepressant imipramine (20 mg / kg i.p.) significantly decreased immobility index (P<0.005) vs. LPS group on the TST.
The results presented in Figure 4 (D, E and F) show that TPN (100 mg / kg v.o.) significantly increased in the latency to grooming (P<0.005) and grooming time (P<0.001) as well as imipramine (20 mg / kg i.p.) significantly increased in the grooming time (P<0.001) vs. LPS group on the ST.
As presented in Figure 4 (B, C and E), the peripheral administration of LPS (0,5 mg / kg i.p.) induces the depressant-like behavior because they significantly increased the immobility time (P<0.005), immobility number (P<0.005) on the TST and significantly decreased the grooming time (P<0.001) on the ST vs. the naive group.
3.1.2. Therapeutic treatment
The results in Figure 5D show the antidepressive-like effects of TPN significantly increasing the grooming time in the following dosages: 100 mg / kg v.o. (P<0.005) and 200 mg / kg v.o. (P<0.001) on the ST.
3.2. LPS Effects in the locomotor activity on the OFT
No global change of locomotion was observed after the LPS (0.5 mg / kg i.p.), TPN (100 mg / kg, p.o., 200 mg / kg, p.o., 100 mg / kg, i.p.) or Imipramine (20 mg / kg i.p.) treatments as depicted in Figure 6. This confirms the specific TPN antidepressant-like effects on the TST and ST. Post hoc Tuky HSD test could not detect any significant difference between any pair of groups on the OFT as shown in Figure 6.
3.3. Investigation of the mechanisms underlying the antidepressant-like effects of the TPN on the TST and ST
3.3.1. Involvement of the cannabinoid system
The results presented in Figure 7 show the effect of TPN preventive treatment (200 mg / kg, p.o.) with cannabinoid-receptor antagonists on TST and ST. Figure 7A demonstrates the AM281 (a selective CB1 cannabinoid receptor antagonist/inverse agonist) and the AM630 (a selective inverse agonist for the CB2 cannabinoid receptor) by inhibiting the increase in immobility latency time (P < 0.05) and in figure 7B by inhibiting the decrease in number of stops (P < 0.001) of TPN preventive treatment (200 mg / kg, p.o.) on the TST.
In Figures 7c and 7b no relevant difference between the TPN (200 mg/kg, p.o.) group and the TPN (200 mg/kg, p.o.) + AM281 (1 mg/kg, i.p.) or TPN (200 mg/kg, p.o.) + AM630 (1 mg/kg, i.p.) group were found.
3.3.2. Involvement of the dopaminergic system
The results depicted in Figure 8 show the dopaminergic antagonists effect in the TPN antidepressant-like (200 mg / kg, v.o.) agent on the TST and ST. Figure 8A shows that the pretreatment of mice with a non-selective dopamine receptor antagonist haloperidol (0.2 mg / kg, i.p.) significantly prevented the increase in the latency to immobility time (P<0.05) and the decrease in the immobility number (P < 0.001) in Figure 8A and 8B on the TST. Moreover, the pretreatment of mice with sulpiride (50 mg / kg, i.p.) prevented the decrease in immobility time (P < 0.001) elicited by the TPN (200 mg / kg, v.o.) in Figure 8B on the TST.
3.3.3. The Adenosinergic system Involvement
Figure 9 (A and B) illustrate the results of the pretreatment of mice with Caffeine (3 mg / kg, i.p., is a nonselective adenosine receptor antagonist in vitro) on the TST and ST. As expected, the pretreatment with TPN (220 mg / kg v.o.) increased in immobility latency time (P < 0.01) and decreased in immobility number (P < 0.05) in TST. Also, decreased in grooming latency time (P < 0.05) and increased in grooming time (P < 0.01) on ST. Caffeine (3 mg / kg, i.p.) has not been able to inhibit the antidepressant-like effect of TPN (200 mg/kg, v.o.) on the TST and ST.
3.3.4. Noradrenergic system Involvement
Figure 10 (B and D) show the propranolol (2 mg/kg, a β-adrenergic receptor antagonist, i.p.) pretreatment, reducing the time of immobility (P <0.01) produced on the TTS and the increase of grooming time (P <0.05) on TS produced by NPT (200 mg/kg, v.o.) in mice. The nonselective adenosine receptor antagonist propranolol (2 mg / kg, i.p.) pretreatment has not been able to inhibit the antidepressant-like effect of TPN (200 mg/kg, v.o.) on the TST and ST.
3.4. Acetylcholinesterase inhibitor
In the current study, TPN exhibited high AChEI action with a total of 91.11% IC50 0,4352 ± 0.00 μg/ml or 2,82 ± 0.00 nMol activity.
3.5. Cytotoxicity of murine L929 fibroblast cells
The graphic in figure 9 shows L929 fibroblast cells viability, 48 h post-exposure to TPN in different concentrations. The cytotoxicity of murine L929 fibroblast cells exposed to TPN for 48 h showed a dose-dependent increase in TPN concentration, causing significant cell death at the concentrations of 125, 250 and 500 μg mL.
4. DISCUSSION
In this study it was shown that TPN (200 mg/kg, p.o) administrated 1h before LPS (0.5 mg/kg, i.p.) produced a significant antidepressant-like response on the TST and ST. Both commonly used behavioral tests that predict antidepressant treatments efficacy and those effects were comparable to the one found on the classical antidepressant Imipramine (20 mg/kg, i.p.).
The cannabinoid receptors relation with symptoms of anxiety and depression previously explained in clinical trials demonstrate that cannabinoid receptors have an important role on understanding depression physiopathology [53–56]. Furthermore, we investigated a selective CB1 cannabinoid receptor antagonist/inverse agonist and a potent and selective inverse agonist of the cannabinoid receptor CB2, and the endocannabinoid system involvement in antidepressant-like activity of TPN. Both CB1 antagonist AM281 and CB2 antagonist AM630 significantly reversed the effects of TPN immobility reduction time supporting the hypothesis that TPN is linked to CB1 and CB2 cannabinoid receptors.
Among the receptors that modulate the monoaminergic system, the dopamine is the most abundant monoamine capable of regulating the anhedonia that is a core symptom of depression[57]. In the present study, the dopaminergic system was investigated by using haloperidol and sulpiride, which are respectively a nonselective dopaminergic receptor antagonist and a selective dopamine D2 receptor antagonist [37]. Supporting the findings of the presented study, Shewale et al. in 2012 [58] reported that the pre-treated haloperidol group exhibited significant improvement in immobility time on the TST. The treatment with TPN has not managed to reverse the rise in immobility time on the TST suggesting that both bind ligand on the same receptor. Administration of sulpiride also prevented TPN of acting in in the immobility time reduction, though at a lower ratio, but no significant difference between the TPN and TPN + sulpiride groups. This shows a tendency of TPN acting more when exposed to sulpiride than to when haloperidol and it may be justified by the fact that TPN
binds to other dopaminergic receptors in addition to the D2 dopaminergic receptors. Considering that compounds that enhance dopaminergic neurotransmission are used to treat depression[37] the results indicate that TPN exerts synergistic effects with dopaminergic and cannabinoid agonists supporting notion that this monoterpene exerts antidepressant properties.
Previous studies measured the modulation nicotinic acetylcholine receptor as well as the muscarinic acetylcholine receptor. The investigations show that the cholinergic system modulation might represent an alternative therapy for treating mood disorders as the major depressive disorder and anxiety [8,59]. Clinical trials show positive antidepressant effects with the acetylcholinesterase inhibitors [60–62] supporting the hypothesis that TPN could act as an antidepressant through the cholinergic system by presenting a potential inhibition power (91%) of the acetyl cholinesterase.
The cytotoxicity of alpha Terpineol towards different tumor cell lines was evaluated in vitro by Hassan and his team, and has shown antitumor activity, a rise in cellular viability [63] as well as in our findings concentrations below 62,5 μg mL show greater cell viability.
5. CONCLUSIONS
In summary, the present study show that TPN significantly inhibited depressive-like behavior through modulation of CB1 and CB2 cannabinoid receptors, dopaminergic receptors and also possibly through modulation of cholinergic system. Additionally, TPN showed significantly inhibition of AChE activity. Moreover, MTT assay demonstrated that TPN was safe fibroblast cells. Taken together, our results suggest that TPN represents an alternative therapeutic to treatment of depression and comorbidities.
Conflict of Interest Statement
There are no conflicts of interest.
Acknowledgments
Grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Apoio a Pesquisa do Estado de Santa Catarina (FAPESC), Programa INCT-INOVAMED (grant 465430/2014-7), and Programa de Pós-Graduação em
Neurociências (PGN), all from Brazil, supported this work. G.V. and T.R.G are undergraduate students receiving grants from CNPq. E.C.D.G. is a PhD student of the Neuroscience Program receiving grants from CAPES/FAPESC. R.C.D. is recipient of a research productivity fellowship from the CNPq.
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APÊNDICE A – Legenda das figuras
Figure 1. Schematic diagram demonstrating a trial of the first experiment with the TPN antidepressant preventive treatment. 1h after LPS was administered animals received the treatment and then the behavioral analysis was performed 24h after.
Figure 2. Schematic diagram demonstrating a trial of the first experiment with the TPN antidepressant therapeutic treatment effects. 1h after LPS was administered animals received the treatment and then the behavioral analysis was performed 24 h after.
Figure 3. Schematic diagram demonstrating a trial of the second experiment with the TPN antidepressant effects. 30 min after administering two antagonists, animals received dosage of TPN, LPS was administered 1h after that and then the behavioral analysis was performed 24h after.
Figure 4. Effects of TPN preventive treatment on the LPS-induced depression-like behavior in mice. Many different dosages and administration routes were measured (TPN 100 mg/kg, p.o., TPN 200 mg/kg, p.o., TPN 100 mg/kg, i.p.) and Imipramine (20 mg/kg, i.p.). TPN (100 mg/kg, p.o., 200 mg/kg, p.o., 100 mg/kg, i.p.) was managed 1h prior to LPS (0.5 mg/kg, i.p.) as a preventive treatment. Data are presented as mean ± SEM of six to ten mice/group. Differences between groups are indicated: *p < 0.05, **p < 0.01 compared to control group; # p < 0.05, ## p < 0.01, compared to all other groups.
Figure 5. Effects of TPN therapeutic treatment on the LPS-induced depression-like behavior in mice. The therapeutic treatment of the TPN (100 mg/kg, p.o., 200 mg/kg, p.o., 100 mg/kg, i.p.) was administered 1h after LPS (0.5 mg/kg, i.p.). The behavioral analysis was recorded 24 hours after the administration of TPN. Data are presented as mean ± SEM of six to ten mice/group. Differences between groups are indicated: *p < 0.05, **p < 0.01 compared to control group; # p < 0.05, ## p < 0.01, compared to all other groups.
Figure 6. Locomotor activity on the open field test with TPN preventive treatment and therapeutic treatment. TPN (100 mg/kg, p.o., 200 mg/kg, p.o., 100 mg/kg, i.p.) and Imipramine (20 mg/kg, i.p.) was administered 1h before LPS (0.5 mg/kg, i.p.) as a preventive treatment. Figure 6 A shows the TPN (100 mg/kg, p.o., 200 mg/kg, p.o., 100 mg/kg, i.p.) administration 1h after LPS (0.5 mg/kg, i.p.) as a therapeutic treatment (Figure 6 B). The behavioral analysis was performed 24h later and no significant difference between groups was found.
Figure 7. Cannabinoid receptors involvement underlying the antidepressant-like effects of TPN on tail suspension tests and splash test. Cannabinoid receptors involvement were evaluated in separate groups. Mice were pretreated with a CB1 cannabinoid receptor selective antagonist inverse agonist (AM281, 1 mg/kg, i.p.) or a potent and selective inverse agonist for the CB2 cannabinoid receptor (AM630, 1 mg/kg, i.p.). After 30 min of administrating two antagonists, they received a dosage of TPN (200 mg/kg, p.o.) and
after 1h LPS (0.5 mg/kg, i.p.) was administered and the behavioral analysis was performed 24h later. Differences between groups are indicated: *p < 0.05, **p < 0.01, compared to control group; # p < 0.05, ## p < 0.01, compared to all other groups and Δp < 0.05, ΔΔ p < 0.01 compared to the TPN group.
Figure 8. Dopaminergic system involvement in the mechanisms underlying the antidepressant-like effects of TPN on tail suspension tests and splash test. Dopaminergic system involvement in the mechanisms underlying the antidepressant-like effects of TPN (200 mg/kg, p.o.) on tail suspension tests and splash test. Mice were pretreated with the vehicle (control group), haloperidol (0.2 mg/kg, i.p., a nonselective dopaminergic receptor antagonist) [25,28], sulpiride (50 mg/kg, i.p., a selective dopamine D2 receptor antagonist). Data are presented as mean ± SEM of six to ten mice/group. After 30 min of administrating two antagonists, they received a dose of TPN (200 mg/kg, p.o.) and 1h after that, LPS (0.5 mg/kg, i.p.) was administered and the behavioral analysis was performed 24h later. Differences between groups are indicated by *p < 0.05, **p < 0.01, compared to control group; # p < 0.05, ## p < 0.01, compared to all other groups and Δp < 0.05, ΔΔ p < 0.01 compared to the TPN group.
Figure 9. Adenosine A1 and A2 receptors Involvement in the antidepressant-like effects of TPN (TPN 200 mg/kg, p.o.) on tail suspension tests and splash test. Animals were pretreated with caffeine (3 mg/kg, i.p., a nonselective adenosine receptor antagonist). After 30 min of administrating two antagonists, they received a dosage of TPN (200 mg/kg, p.o.) and 1h after that, LPS (0.5 mg/kg, i.p.) was administered and the behavioral analysis was performed 24h later. Data are presented as mean ± SEM of six to ten mice/group. Differences between groups are indicated: *p < 0.05, **p < 0.01, compared to control group; # p < 0.05, ## p < 0.01, compared to all other groups.
Figure 10. Adrenergic receptors involvement underlying the antidepressant-like effects of TPN (200 mg/kg, p.o.) on tail suspension tests and splash test. Mice were pretreated with the vehicle (control group), propranolol (2 mg/kg i.p., a β-adrenoceptor antagonist). After 30 min of administrating two antagonists, they received a dose of TPN (200 mg/kg, p.o.) and 1h after that, LPS (0.5 mg/kg, i.p.) was administered and the behavioral analysis was performed 24h later. Differences between groups are indicated: *p < 0.05, **p < 0.01, compared to control group; # p < 0.05, ## p < 0.01, compared to all other groups.
Figure 11. TPN L929 fibroblast cell viability 48 h post-exposure to TPN in different concentrations. Data are expressed as mean ± standard errors of the mean versus control. Analysis of variance (ANOVA) is followed by Bonferroni’s test ***p< 0.0001.
APÊNDICE B – Tabela Table 1
Reference Antagonist Dose
[38,41] haloperidol 0.2 mg/kg nonselective dopaminergic receptor antagonist [38,41] Sulpiride 50 mg/kg selective dopamine D2 receptor antagonist [42] propranolol 2 mg/kg β-adrenoceptor antagonist
[43] Caffeine 3 mg/kg nonselective adenosine receptor antagonist
[44] AM281 1 mg/kg selective CB1 cannabinoid receptor antagonista / inverse agonist [44] AM630 1 mg/kg selective inverse agonist for the CB2 cannabinoid receptor
APÊNDICE C - Figuras
Figure 1
Figure 2
Figure 4
Figure 11
C
e
ll
V
ia
b
il
it
y
(
%
)
C S 5 0 0 2 5 0 1 2 5 6 2 .5 3 1 .2 5 1 5 .6 2 7 .8 10
5 0
1 0 0
1 5 0
g /m l
***
***
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