JM-20, a novel hybrid molecule, protects against rotenone-induced neurotoxicity in experimental model of Parkinson’s disease

Accepted Manuscript Title: JM-20, a novel hybrid molecule, protects against rotenone-induced neurotoxicity in experimental model of Parkinson’s disease Authors: Luis Arturo Fonseca-Fonseca, Maylin Wong-Guerra, Jeney Ram´ırez-S´anchez, Yanay Montano-Peguero, Alejandro Sa´ul Padr´on Yaquis, Abel Mondelo Rodr´ıguez, V´ıctor Di´ogenes Amaral da Silva, Silvia Lima Costa, Gilberto L. Pardo-Andreu, Yanier N´un˜ ez-Figueredo PII: DOI: Reference:

S0304-3940(18)30678-5 https://doi.org/10.1016/j.neulet.2018.10.008 NSL 33863

To appear in:

Neuroscience Letters

Received date: Revised date: Accepted date:

30-7-2018 24-9-2018 5-10-2018

Please cite this article as: Fonseca-Fonseca LA, Wong-Guerra M, Ram´ırez-S´anchez J, Montano-Peguero Y, Padr´on Yaquis AS, Rodr´ıguez AM, da Silva VDA, Costa SL, Pardo-Andreu GL, N´un˜ ez-Figueredo Y, JM-20, a novel hybrid molecule, protects against rotenone-induced neurotoxicity in experimental model of Parkinson’s disease, Neuroscience Letters (2018), https://doi.org/10.1016/j.neulet.2018.10.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

JM-20,

a

novel

hybrid

molecule,

protects

against

rotenone-induced

neurotoxicity in experimental model of Parkinson’s disease Luis Arturo Fonseca-Fonseca 1, Maylin Wong-Guerra 1, Jeney Ramírez-Sánchez 1, Yanay MontanoPeguero 1, Alejandro Saúl Padrón Yaquis 1, Abel Mondelo Rodríguez 1, Víctor Diógenes Amaral da Silva 2, Silvia Lima Costa 2, Gilberto L. Pardo-Andreu 3, Yanier Núñez-Figueredo

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Centro de Investigación y Desarrollo de Medicamentos (CIDEM), Ave 26, No. 1605 Boyeros y

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Puentes Grandes, CP 10600, La Habana, Cuba. 2

1*.

Laboratório de Neuroquímica e Biologia Celular, Instituto de Ciências da Saúde, Universidade

Federal da Bahia (UFBA), Av. Reitor Miguel Calmon s/n, Vale do Canela, CEP 41100-100, Salvador,

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Bahia, Brazil.

Centro de Estudio para las Investigaciones y Evaluaciones Biológicas, Instituto de Farmacia y

Alimentos, Universidad de La Habana, Ave 23 No. 21425 e/214 y 222, La Coronela, La Lisa, CP

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13600, Ciudad Habana, Cuba

* Corresponding author. Centro de Investigación y Desarrollo de Medicamentos, Ave 26, No. 1605

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Boyeros y Puentes Grandes, CP 10600, La Habana, Cuba.

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E-mail address: [email protected] (Yanier Núñez-Figueredo).

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Highlights:

Novel hybrid molecule (JM-20) prevents rotenone-induced cell death.



JM-20 reduced oxidative stress and improved mitochondrial functions in

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



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rotenone-treated rats.

JM-20 prevents the increase in sensory indifference (apathy) in rats chronically

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treated with rotenone. 

JM-20 increased survival and body weight gain of animals treated with rotenone.

Abstract

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Oxidative stress and mitochondrial dysfunction are two pathophysiological factors often associated with the neurodegenerative process involved in Parkinson’s disease (PD). The aim of this study was to investigate the effects of a novel hybrid molecule, named JM-20, in different in vitro and in vivo models of PD induced by rotenone. To perform in vitro studies, SHSY-5Y cells were exposed to rotenone and/or treated with JM-20. To perform in vivo studies male Wistar rats were intoxicated with rotenone (2.5 mg/kg) via intraperitoneal injection and/or treated with JM-20 (40 mg/kg) administered via oral (for 25 days, both

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treatment). Rats were evaluated for global motor activity by measurement of locomotor activity. In addition, the effects on mortality, general behavior and redox parameters were also investigated. JM-20 protected SHSY-5Y cells against rotenone-induced cytotoxicity, evidenced by a significant diminution of cell death. In in vivo studies, JM-20 prevented rotenone-induced vertical exploration and locomotion frequency reductions, moreover prevented body weight loss and mortality induced by rotenone. It also improved the redox state of rotenone-exposured animals by increasing superoxide dismutase and catalase

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activities, total tissue-SH levels and decreasing malondialdehyde concentrations. Finally, JM-20 inhibited spontaneous mitochondrial swelling and membrane potential dissipation in

isolated rats brain mitochondria. These results demonstrate that JM-20 is a potential

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neuroprotective agent against rotenone-induced damage in both in vitro and in vivo models, resulting in reduced neuronal oxidative injury and protection of mitochondria from impairment.

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Keywords: JM-20, SHSY-5Y, Parkinson’s disease, rotenone, mitochondria, oxidative

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stress

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1. Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. It is characterized by degeneration of nigrostriatal dopaminergic neurons that are critically involved in the control of voluntary movements. Several pathophysiological mechanisms have been implicated in the progressive loss of dopaminergic neurons of the substantia nigra pars compacta (SNpc), including oxidative stress, mitochondrial dysfunction, protein misfolding and aggregation, neuroinflammation,

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excitotoxicity, altered intracellular calcium (Ca2+) homeostasis and apoptotic cell death [1].

Accumulating evidence from both animal models and human post-mortem studies suggests

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that oxidative stress and mitochondrial dysfunction are hallmarks of PD [1]. Despite the variety of preclinical models of PD, rotenone model is capable to reproducing some of the

main motors and pathophysiological features described for this disease, such as (1) Lewy bodies, (2) mitochondrial dysfunction via inhibition of complex I, (3) it induces oxidative

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damage to several brain areas, (4) induces apoptosis [2], (5) reproduces PD gastrointestinal

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neuropathology [3] and (6) provide an excellent tool to test new neuroprotective strategies. In recent years, the use of multiple drug therapies or the combination in a molecule of

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several structural groups with varied pharmacological targets (multiple mechanism drugs)

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has been suggested. In fact, specifically for the treatment of PD, some multiligand drugs have been designed, fallowing the approach of design and rationality mentioned above [4].

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In this context, and based on a multimodal drug design paradigm, Organic Synthesis Laboratory (Havana University) has developed a new molecule, with a 1,5 benzodiazepine

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ring fused to a 1,4 dihydropyridine moiety, named JM-20 (3-ethoxycarbonyl-2-methyl-4-(2nitrophenyl)-4,11-dihydro-1H-pyrido[2,3-b][1,5]benzodiazepine)

[5].

Considering

the

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combination of more than one neuroprotective pharmacophore into one structure, our group initially hypothesize that JM-20 could be a suitable compound for neuroprotection in experimental models related to cerebral ischemia. Different in vitro and in vivo models were performed [6-8] and multitarget mechanisms were demonstrated [6-11], summarized in the review paper published by Nuñes-Figueredo and colleagues [12]. Therefore the aim of this

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study was to investigate the effect of JM-20, in in vitro and in vivo rotenone models of PD in view to elucidate a possible protective role associated to mitochondrial and antioxidants function.

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2. Materials and Methods 2.1. Compounds and reagents All chemicals used were of the highest grade available and were purchased from SigmaAldrich (St. Louis, MO, USA), unless otherwise specified. JM-20 was synthesized, purified and characterized as previously reported [5]. 2.2. SHSY-5Y cell culture

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Human SHSY-5Y (ATCC® CRL-2266TM) were grown in culture medium containing Roswell

Park Memorial Institute (RPMI-1640), 10% inactivated fetal bovine serum, supplemented

with 2 mM L-glutamine, 1% penicillin and 1% streptomycin (Gibco, USA), 1% non-essential

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amino acids and 1 mM sodium pyruvate (Gibco, USA). SHSY-5Y cells were cultured until confluence in 75 cm 2 (Nunc, USA), trypsinized and replated on 96-well plates (Costar, USA)

at a density of 10,000 cells/well. Cells were maintained in an incubator with humidified

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atmosphere with 5% of CO 2 and 37°C.

(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide)

is

a

water

soluble

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MTT

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2.3. Cell treatments and MTT Assay

tetrazolium salt that is converted to an insoluble purple formazan by mitochondrial

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succinate dehydrogenase. Control and treated cells were incubated with MTT at a final concentration of 0.5 mg/ml for 3 h, in a controlled environment atmosphere (37°C / 5%

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CO2). Thereafter, cells were lysed with 3% (w/v) sodium dodecyl sulfate and the formazan crystals dissolved in acidified (0.04 M HCl) isopropanol .The absorbance of each sample

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was measured at 570 nm using a POLARstar Omega spectrophotometer (BMG Labtech, Germany). Stock solutions of JM-20 (1 mM) were daily prepared in dimethyl sulfoxide

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(DMSO) and the final concentration was prepared by dilution in RPMI-1640. To investigate the neuroprotective effect of JM-20 against rotenone induced cytotoxicity, cells were treated with different concentrations of JM-20 (0.001, 0.01, 0.1, and 1 μM) and incubated with rotenone (20 μM) for 24 h. The control group only contained DMSO diluted in the culture medium at the higher equivalent volume used in the treated groups (1/1,000 (v/v) dilutions).

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Three independent experiments were carried out with six replicate wells for each analysis. 2.4. Experimental animals Male Wistar rats (CENPALAB, Havana, Cuba) weighing 220–240 g were housed in a temperature-controlled environment (22 ± 2°C) with a 12 h light/dark cycle, with water and commercial food ad libitum. Animal housing care and the application of experimental procedures were in accordance with national and institutional guidelines (Animal Care

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Committee from CIDEM). All efforts were made to minimize the number of animals used and their suffering. 2.5. Rotenone/JM-20 administration and experimental design Rats were injected with either rotenone (2.5 mg/kg, intraperitoneally (ip)) suspended in sunflower oil (1 ml/kg) (rotenone vehicle) or vehicle alone [3], daily for 25 consecutive days (from 9:00 a.m to 11:00 a.m.). After one week, the rats were randomly divided into four

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groups: (1) vehicle group (n=10), carboxymethyl cellulose (CMC) + sunflower oil (1 ml/kg); (2) rotenone + JM-20 group (n=12), rotenone (2.5 mg/kg) + JM-20 (40 mg/kg); (3) rotenone group (n=12), rotenone (2.5 mg/kg) + CMC and (4) JM-20 group (n=10), JM-20 (40 mg/kg)

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+ sunflower oil. Immediately before use, JM-20 was suspended in a 0.05% CMC solution

and administered as a single dose. JM-20 (40 mg/kg) or CMC 0.05% (10 ml/kg), were orally administrated daily by gastric gavage (i.g), for 25 days, beginning the same day of the first rotenone administration. During the experimental study period, animals from each group

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were weighed on days 0, 3, 6, 9, 12, 15, 18, 21, 23 and 25.

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2.6. Behavioral studies

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Twenty six days following treatment rats were placed in a clean open field square box (100 x 100 cm) with 40 cm high plastic walls. Rats were video-recorded (Webcam, Logitech) for

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6 min, 1 min for habituation and 5 min for behavioral analyzes. Two motor parameters were quantified throughout this test: locomotion frequency (number of squares crossings, defined

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as the number of quadrant crossings with the four paws) and rearing frequency (times the animal rise for at least 2 s on their rear paws in the air or against the walls). The open-field

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was cleaned with a 70% ethanol between each trial and allowed to air dry [13].

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2.7. Isolation of rats brain mitochondria Rat brain mitochondria were isolated as previously described [7, 14]. Briefly, after behavioral test was concluded, rats were sacrificed by decapitation, and their brains were rapidly removed (within 1 min) and placed into 10 ml of ice-cold isolation buffer, containing: 225 mM mannitol, 75 mM sucrose, 1 mM K-EGTA, 0.1% bovine serum albumin (fatty-acid free)

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and 10 mM HEPES-KOH, pH 7.2. The cerebellum and underlying structures were removed and the remaining tissue was used to isolate brains mitochondria. 2.7.1. Continuous-monitoring mitochondrial assays For all assays, rat brain mitochondria (0.5 mg/ml) were energized with 5 mM potassium succinate in a standard incubation medium consisting of 125 mM sucrose, 65 mM KCl and 10 mM HEPES–KOH, pH 7.4 at 37ºC. Mitochondrial membrane potential (ΔΨ) was

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determined spectrofluorimetrically (POLARstar Omega) using 10 μM safranine O as a probe at 495/586 nm excitation/emission wavelengths. These assays were performed in the presence of 0.1 mM EGTA and 2 mM K2HPO4 [7]. Spontaneous mitochondrial swelling was estimated spectrophotometrically from the decrease in the absorbance at 540 nm measured using a POLARstar Omega spectrophotometer [10]. 2.8. Study of oxidative stress markers

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After behavioral test was concluded, the effects of rotenone and JM-20 treatment on redox status in SNpc and striatum were evaluated. The animals were sacrificed by decapitation,

previously anesthetized with a lethal dose of chloral hydrate (480 mg/kg, i.p.). For the nigral

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tissue extraction was followed the protocol described by Jimenez-Martin et al. [15] and for the striatum extraction was followed the protocol described by Chiu et al. [16]. The samples were manually homogenized as we previously reported [17].

2.8.1. Measurement of lipid peroxidation, total tissue –SH (T-SH), superoxide dismutase

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(SOD) and catalase (CAT) activity

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The extent of lipid peroxidation (malondialdehyde (MDA)), total T-SH groups, and the

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activity of SOD and CAT enzymes in the brain, was assayed as we previously reported [17].

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2.9. Statistical Analysis

Significant differences among groups were determined by one-way analysis of variance

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(ANOVA), followed by Tukey’s post hoc analysis or two-way ANOVA (factors: treatment × time as repeated measures) followed by Bonferroni's post hoc analysis (body weight

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change among groups, during the 25 days). Survival was analyzed using Kaplan-Meier curves and then log-rank (Mantel-Cox) test for statistical differences in survival rates.

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GraphPad Prism 7.00 (Software Inc., USA) was used for all statistical analyses. All values were expressed as the means ± standard error of the mean (SEM). Differences were

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considered statistically significant at p<0.05.

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3. Results 3.1. Cytoprotective effects of JM-20 against rotenone-mediated damage in SHSY-5Y cells MTT test demonstrated that 24 h exposure to rotenone-induced a dose-dependent reduction in SHSY-5Y cell viability (Fig. 1A). Cellular viability was significantly decreased in cells exposed to 20 µM of rotenone for 24 h, when compared with untreated control cells (F (6,41)

= 43 p<0.05) (Fig. 1A). This concentration was used for all subsequent in vitro

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experiments. JM-20 alone (0.001–1 μM) did not affect SHSY-5Y cell viability, demonstrating it is not toxic for these cells at the concentration range evaluated (Fig. 1B). Furthermore, JM-20 at concentrations of 0.01 μM (p<0.05), 0.1 μM (p<0.05) and 1 μM JM-20 (F

(6,54)

=

23.66 p<0.05) inhibited SHSY-5Y cells death induced by rotenone after 24 h co-treatment

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(Fig. 1C). The EC50 value, estimated by a non-linear regression algorithm, was 2.2 nM. 3.2. JM-20 preserves global motor function in rotenone-exposed rat

p<0.05) (Fig. 2A) and locomotion frequency (F

(3,33)

(3,33)

= 8.352

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The present results revealed a significant decrease in rearing frequency (F

= 8.321 p<0.05) (Fig. 2B) in rotenone

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group as compared to vehicle group. Moreover, rotenone + JM-20 group showed an

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increase in locomotion frequency (p<0.05) (Fig. 2A) and in the rearing number when compared with rotenone group (Fig. 2B). JM-20 at 40 mg/kg (JM-20 group) did not interfere

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either in the locomotion frequency nor rearing frequency in the open field test (Fig. 2AB). No significant difference was observed between the two control groups (Fig. 2AB).

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3.3. JM-20 improved the redox state in SNpc and striatal tissue of rotenone-poisoned rats

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3.3.1. Effects of JM-20 treatment on brain lipid peroxidation induced by rotenone MDA brain levels were found to be significantly increased in the SNpc (F (3,15)

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p<0.05) and striatum (F

(3,15)

= 7.957

= 14.16 p<0.05) of rotenone-treated rats as compared with

vehicle group. In rotenone + JM-20 group, the brain levels of MDA were found to be significantly lower in the SNpc (p<0.05) and the striatum (p<0.05), as compared with levels found in the rotenone group. JM-20 alone (JM-20 group), did not modify brain levels of

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MDA, in both evaluated regions (Table 1). 3.3.2. Effects of JM-20 treatment on SOD and CAT enzymatic activities and T-SH levels SOD (F

(3,11)

= 4.732 p<0.05) and CAT (F

(3,12)

= 5.809 p<0.05) activities were found to

decrease in striatum of rotenone-treated rats when compared with vehicle group. Interestingly, SOD (p<0.05) and CAT (p<0.05) activities were significantly higher in striatum of rotenone + JM-20 group when compared with rotenone-treated rats. None statistically

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difference was observed in SOD and CAT activity in the SNpc of rotenone-treated rats and/or JM-20 treated rats when compared with vehicle group (Table 2). T-SH levels were found to be significantly decreased following rotenone administration for 25 days in the striatum (F

(3,13)

= 5.027 p<0.05), but not in the SNpc of rotenone-treated rats

when compared with vehicle group. In rotenone + JM-20 group, T-SH levels were found to be significantly higher in the striatum (p<0.05), when compared with rotenone-treated evaluated brain regions (Table 2). 3.4. JM-20 prevented rotenone-induced mitochondrial dysfunction

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group. JM-20 alone (JM-20 group) did not modify levels of T-SH, CAT or SOD, in both

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To analyze the mitochondrial protective effects of JM-20 against rotenone-induced toxicity in brain, was evaluated the effects of JM-20 in isolated rat brain mitochondria from animals subjected to rotenone treatment during 25 days. It was observed that these type of mitochondria (rotenone group) were more susceptible to spontaneous swelling (F (3,14)

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6.213 p<0.05) (Fig. 3A) and membrane potential dissipation (F

(3,14)

=

= 33.26 p<0.05) (Fig.

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3B) when compared with mitochondria from vehicle group. The mitochondrial membrane potential dissipation occurred even in the presence of EGTA, which discard the permeability

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transition pore, strictly dependent on calcium, as a plausabile cause of the organelle

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impairment. Instead, the unespecific membrane permeabilization due to the oxidative stress evidenced by the results on table 1, seems to be involved. JM-20 treatment improved mitochondrial function, as evidenced by decreased spontaneous organellar swelling

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(p<0.05) (Fig. 3A) and preservation of membrane potential (p<0.05) (Fig. 3B). No

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mitochondrial afectations were observed in JM-20 group (Fig. 3AB). 3.5. JM-20 prevents body weight loss in rotenone -exposed rats

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Two-way repeated ANOVA (Fig. 4A) (treatment × time as repeated measures) showed significant main effects of days (F (9,

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) = 124.8 p<0.0001), treatment (F (3,

p=0.0055) and a significant interaction between these two factors (F (27,

) = 5.175

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) = 2.041

p=0.0024) on accumulated body weight. As shown in the figure 4A, the body weight of rats markedly decreased following rotenone treatment. Significant differences were observed in

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rotenone-treated group, from 15th day of rotenone administration, when compared with control groups (p<0.05). Already on 21th day, weight differences were also observed between rotenone group (p<0.05) and rotenone + JM-20 group (p<0.05). This difference was maintained until 25th day. Also, a significant decrease in the body weight gain (Fig. 4B) was observed in rotenonetreated rats, at 25th day following rotenone treatment, when compared with vehicle group 8

(p<0.05), JM-20 treated group (p<0.05) and JM-20 + rotenone-treated group (F (3,23) = 10.13 p<0.05). 3.6. Effects of JM-20 on survival rates in rats exposed to rotenone During the experimental procedure, there was no mortality in controls groups (100% survival). In contrast, repeated administration of rotenone resulted in a low survival rate (5 deaths of 12 animals). Treatment with JM-20 improved the survival rate (2 deaths of 12

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animals) when compared with rotenone group (Fig. 5).

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4. Discussion Here, the rotenone-induced PD models (in vitro and in vivo) was used to investigate the neuroprotective potential of the JM-20. The major limitation of the rotenone model has been its variability, both in terms of the percentage of animals that develop a clear-cut nigrostriatal lesion [18]. Despite these limitations, rotenone model has been widely used to explore the mechanisms of cell death in PD and possible therapeutic interventions. Our group has obtained several results concerning the neuroprotective mechanisms of JM-20,

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highlighting its antioxidant [10] and mitochondrial protective capacity [6]. The neurotoxicity mechanism of rotenone is primarily mediated by its potent complex I inhibition, inducing

mitochondrial damage and oxidative stress, both hallmarks of PD pathogenesis. Thus it can

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be hypothesized that JM-20 could protect against rotenone-induced neuronal damage.

The neurotoxicity of rotenone was demonstrated by MTT assay in 5HSY-5Y cell culture. The SHSY-5Y cells in both undifferentiated and differentiated states express a number of

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dopaminergic neuronal markers. These cells express tyrosine hydroxylase (TH), an enzyme critical for the catalysis of dopamine. Importantly, SHSY-5Y cells also express dopamine

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dopaminergic neurons as well as for drugs [19].

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transporter, making them an exemplary in vitro system for the study of neurotoxicity in

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Previous in vitro studies have demonstrated that JM-20 exerts a direct protective effect in PC-12 cells against neurotoxic conditions induced by glutamate and H2O2 excess [6]. In the

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present study, we highlight the potential of JM-20 to attenuate the cellular death induced by rotenone, in SHSY-5Y cells. Our group previously demonstrated that JM-20 is an

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antioxidant that acts at both synaptosomal and mitochondrial levels [6, 10]. Its antioxidant action was not related to its ability to scavenge superoxide anion radicals or H2O2, but to its

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electron affinity given by its low redox potential similar to that of oxygen. This electrochemical characteristic endows JM-20 with the ability to scavenge electrons from the mitochondrial electron transport chain or even to compete with oxygen for the electrons released from mitochondria, thus preventing reactive oxygen species (ROS) formation, which could explain, at least in part, the cytoprotective effects observed in SHSY-5Y cell

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culture exposed to rotenone. In the present work, rotenone administered during 25 days was used to induce dopaminergic neurodegeneration. In the open-field test, hypoactivity in rotenone-treated rats was observed in active sitting, rearing and line crossing behavior, compared with control experimental subjects. Not only the global motor response of the animals was affected, also an increase in depression, anxiety-related behavior and decreased motivation to explore this new open arena for them was observed, related to the sensory component of 10

the test. In agreement with our results, other investigators support that rotenone-treated rats exhibit loss of motor skills and less exploratory behavior. [20]. Co-treatment with JM-20 successfully attenuated the rotenone-induced behavior alteration, increasing rearing behavior and crossed quadrants. Based on the important role of oxidative stress in the generation of rotenone-induced PD [21], the present study also aimed to explore the antioxidant effects of JM-20 in the brain of

rotenone-exposed rats. According to our results, rotenone injection was able to modify the

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redox balance, in the nigroestriatal pathway. Treatment with rotenone, during 25 days,

caused a significant enhancement in the MDA levels, in the striatum and SNpc. It is widely

documented that rotenone, upon systemic administration, causes complex I inhibition and to the rotenone-injured animals, in both evaluated regions.

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ROS production [21]. JM-20 administration significantly lowered the MDA levels compared

The endogenous cellular defense network against ROS and free radicals constitutes

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enzymatic (SOD and CAT) and non-enzymatic (T-SH) molecules to scavenge the oxygenfree radicals, which otherwise lead to oxidative damage [22]. Here, we found that these

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components, which provide cell protection against oxidative damage, were statistically

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diminished in the striatum of animals treated with rotenone, when compared to control rats,

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but not in the SNpc.

Betarbet et al. found that animals with partial reductions in TH staining in the striatum

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presented dopaminergic neurons in SNpc that looked relatively normal (7 days of rotenone administration); however, in animals with near complete striatal denervation (35 days of

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rotenone administration), there were obvious reductions in TH-stained cells in SNpc, being more severely in the striatum. These results suggest that striatal nerve terminals were

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affected earlier and more severely by rotenone than nigral cell bodies [21]. Also, Ferrante et al., find that the basal ganglia and striatum appear to be more particularly vulnerable to the toxic effects of several mitochondrial toxins, than SNpc. The explanation for the vulnerability of basal ganglia and striatum neurons to rotenone is also unknown, but one can theorize that there may be differences in energy requirements or tissue-specific isoenzymes of

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complex I [23].

This could explain the results obtained, that antioxidants parameters were only altered in the striatum of rotenone-treated animals without affecting the SNpc, at least at the evaluated time point. This does not mean that rotenone has not affected the SNpc of the animals, since as was previously discussed, MDA levels were significantly increased in both studied regions.

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Our results showed that animals treated with JM-20 reduced oxidative damage in the nigrostriatal pathway following rotenone administration. The precise mechanisms underlying the JM-20 induced reduction in oxidative stress observed here are currently not fully known. However, as discussed above, our group had obtained preclinical evidence about its antioxidant mechanism, elicited mainly at mitochondrial level [10]. Severe defects in complex I activity induce mitochondria depolarization and Ca2+ disregulation [2]. The excessive mitochondrial Ca2+ uptake can cause non-selective

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permeabilization of the inner mitochondrial membrane, that could also contribute to mitochondrial swelling and membrane potential dissipation from massive proton leakage

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[24].

Our results support this mechanism because brain mitochondria isolated from rotenonetreated rats spontaneously lost their membrane potential and were more prone to membrane permeabilization/swelling when compared with the vehicle group. On the other

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hand, we confirmed the JM-20 protective effect against rotenone-induced mitochondrial dysfunction, similarly to previous results [6, 7, 10]. Interesting, the contribution of its

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antioxidant effects seems to be important to the overall mitochondrial protection, since the

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oxidative damage to the mitochondrial membranes instead of mitochondrial permeability transition appears to mediate the rotenone-induced damage to the organelles in our

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experimental conditions.

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Here we also observed that rotenone-treated rats showed a tendency to weight loss and increased mortality rate. The possible causes related to this decline in body weight could be

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related to a delay in gastric emptying and the damage to gastrointestinal neurons, occurred during rotenone intoxication [3].These findings indicate that we successfully replicated

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rotenone-treated rats as model of PD [20]. We also observed that rotenone-treated animals administered with JM-20 were less prone to weight loss and to die, arguing in favor of the protective effects of JM-20. In conclusion, our results show the neuroprotective potential of JM-20 against rotenoneinduced impairment in both in vitro and in vivo models, by preserving SH5Y-SY cell viability

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and by attenuating behavioral alterations, improving striatal antioxidant enzymes, avoiding brain mitochondrial alteration, loss of body weight and mortality induced by this neurotoxin. More studies must be developing to characterize JM-20 as a new possible drug against PD.

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Acknowledgments This work was partially supported by CAPES/MES (Brazil-Cuba) project 178/12 2 and from NonGovernmental Organization MEDICUBA-SPAIN.

Conflict of Interests

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The authors declare that they have no conflict of interest.

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References

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[1] W. Poewe, et al., Parkinson disease, Nat Rev Dis Primers 3 (2017) 17013. [2] V.N. Uversky, Neurotoxicant-induced animal models of Parkinson's disease: understanding the role of rotenone, maneb and paraquat in neurodegeneration, Cell Tissue Res 318 (2004) 225-241. [3] L.H. Morais, et al., Characterization of motor, depressive-like and neurochemical alterations induced by a short-term rotenone administration, Pharmacol Rep 64 (2012) 1081-1090. [4] W.J. Geldenhuys, et al., The emergence of designed multiple ligands for neurodegenerative disorders, Prog Neurobiol 94 (2011) 347-359. [5] Y.N. Figueredo, et al., Characterization of the anxiolytic and sedative profile of JM -20: a novel benzodiazepine-dihydropyridine hybrid molecule, Neurol Res 35 (2013) 804-812. [6] Y. Nunez-Figueredo, et al., JM-20, a novel benzodiazepine-dihydropyridine hybrid molecule, protects mitochondria and prevents ischemic insult-mediated neural cell death in vitro, Eur J Pharmacol 726 (2014) 57-65. [7] Y. Nunez-Figueredo, et al., A novel multi-target ligand (JM-20) protects mitochondrial integrity, inhibits brain excitatory amino acid release and reduces cerebral ischemia injury in vitro and in vivo, Neuropharmacology 85 (2014) 517-527. [8] J. Ramirez-Sanchez, et al., Neuroprotection by JM-20 against oxygen-glucose deprivation in rat hippocampal slices: Involvement of the Akt/GSK-3beta pathway, Neurochem Int 90 (2015) 215-223. [9] Y. Nunez-Figueredo, et al., The effects of JM-20 on the glutamatergic system in synaptic vesicles, synaptosomes and neural cells cultured from rat brain, Neurochem Int 81 (2015) 41-47. [10] Y. Nunez-Figueredo, et al., Antioxidant effects of JM-20 on rat brain mitochondria and synaptosomes: mitoprotection against Ca(2)(+)-induced mitochondrial impairment, Brain Res Bull 109 (2014) 68-76. [11] Y. Nunez-Figueredo, et al., Therapeutic potential of the novel hybrid molecule JM -20 against focal cortical ischemia in rats, J Pharm Pharmacogn Res 4 (2016) 153-158. [12] Y. Nuñez-Figueredo, et al., Multi-targeting effects of a new synthetic molecule (JM-20) in experimental models of cerebral ischemia, Pharmacol Rep 70 (2018) 699-704. [13] J. Bures, et al., Techniques and Basic Experiments for the Study of Brain and Behavior, Elsevier, 1976, pp. 37-89. [14] L.A. Fonseca-Fonseca, et al., KM-34, a Novel Antioxidant Compound, Protects against 6Hydroxydopamine-Induced Mitochondrial Damage and Neurotoxicity, Neurotox Res doi: 10.1007/s12640-017-9851-5 (2018). [15] J. Jimenez-Martin, et al., Effect of neurotoxic lesion of pedunculopontine nucleus in nigral and striatal redox balance and motor performance in rats, Neuroscience 289 (2015) 300-314. [16] K. Chiu, et al., Micro-dissection of rat brain for RNA or protein extraction from specific brain region, J Vis Exp 7 (2007) 269-271. [17] M. Wong-Guerra, et al., Mitochondrial involvement in memory impairment induced by scopolamine in rats, Neurol Res 39 (2017) 649-659. [18] J.R. Cannon, et al., A highly reproducible rotenone model of Parkinson's disease, Neurobiol Dis 34 (2009) 279-290. [19] P. Arun, et al., Antipsychotic drugs increase N-acetylaspartate and N-acetylaspartylglutamate in SH-SY5Y human neuroblastoma cells, J Neurochem 106 (2008) 1669-1680. [20] M. Sonia Angeline, et al., Rotenone-induced parkinsonism elicits behavioral impairments and differential expression of parkin, heat shock proteins and caspases in the rat, Neuroscience 220 (2012) 291-301. [21] R. Betarbet, et al., Chronic systemic pesticide exposure reproduces features of Parkinson's disease, Nat Neurosci 3 (2000) 1301-1306. [22] J.J. Sutachan, et al., Cellular and molecular mechanisms of antioxidants in Parkinson's disease, Nutr Neurosci 15 (2012) 120-126. [23] R.J. Ferrante, et al., Systemic administration of rotenone produces selective damage in the striatum and globus pallidus, but not in the substantia nigra, Brain Res 753 (1997) 157-162. [24] M. Crompton, The mitochondrial permeability transition pore and its role in cell death, Biochem J 341 ( Pt 2) (1999) 233-249.

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Figure legends

Fig. 1. Protective effects of JM-20 against rotenone induced cytotoxicity in SHSY-5Y cells. The cells were seeded at a density of 1 x 10 5 cells/ml in 96 multiwell flat bottom plates and were incubated with different rotenone (Rot) concentrations (0.4–20 μM) (A) or with different JM-20 concentrations (0.001–1 μM) for 24 h (B). Finally, cells were co-treated with 20 μM of Rot and with different JM-20 concentrations, for 24 h (C). Cellular viability was assessed using MTT assay and

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expressed as a percentage of the untreated control (Control) cells. The data are presented as the means ± S.E.M. Different letters: p<0.05, by one way ANOVA analysis, followed by Tukey’s multiple

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comparison post hoc test.

Fig. 2. Protective effects of JM-20 against rotenone-induced motor impairment.

Rats were administered with rotenone (2.5 mg/kg, ip_Rot) (n=7), or concomitantly with JM-20 (40

U

mg/kg, i.g_Rot+JM-20) (n=10), daily for 25 consecutive days. Experimental groups without Rot. :

N

rotenone vehicle (Control) (n=10) and JM-20 (JM-20) (n=10) were also included. Two motor parameters were quantified throughout this test, using an open field cage: rearing frequency (A) and

A

locomotion frequency (B). The data are presented as the means ± S.E.M. Different letters: p<0.05,

M

by one way ANOVA analysis, followed by Tukey’s multiple comparison post hoc test.

D

Fig. 3. Protective effects of JM-20 against rotenone-induced mitochondrial impairment.

TE

Rats were administered with rotenone (2.5 mg/kg, ip_Rot) (n=3), or concomitantly with JM-20 (40 mg/kg, i.g_Rot+JM-20) (n=5), daily for 25 consecutive days. Experimental groups without Rot.: rotenone vehicle (Control) (n=5) and JM-20 (JM-20) (n=5) were also included. Rat brain

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mitochondria (0.5 mg/ml) were isolated after behavioral test, (day 26), and assess ed for mitochondria swelling (A) and mitochondria membrane potential dissipation (B). The relative fluorescent units (RFU) or absorbance were recorded for 600 seconds. The data are presented as the means ± S.E.M. of three experiments that were conducted us ing different mitochondrial preparations. Different letters: p<0.05, by one way ANOVA analysis, followed by Tukey’s multiple

A

comparison post hoc test.

Fig. 4. Effects of JM-20 treatment on body weight curve in rotenone -poisoned rats. Rats were administered with rotenone (2.5 mg/kg, ip_Rot) (n=7), or concomitantly with JM -20 (40 mg/kg, i.g_Rot+JM-20) (n=10), daily for 25 consecutive days. Experimental groups without Rot.: rotenone vehicle (Control) (n=10) and JM-20 (JM-20) (n=10) were also included. The respective

15

weights of rats were measured prior to each treatment and every three days. Body weight time course (by two ANOVA analysis, factors: treatment × time as repeated measures) (A) and the net body weight gain at day 25 (by one way ANOVA analysis) (B) were recorded. The data are presented as the means ± S.E.M. Different letters: p<0.05.

Fig. 5. Kaplan-Meier survival curve after Rot and JM-20 treatments. Rats were administered with rotenone (2.5 mg/kg, ip_Rot) (n=7), or concomitantly with JM-20 (40

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mg/kg, i.g_Rot+JM-20) (n=10), daily for 25 consecutive days. Experimental groups without Rot.:

rotenone vehicle (Control) (n=10) and JM-20 (JM-20) (n=10) were also included. For statistical

A

CC EP

TE

D

M

A

N

U

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differences in survival rates log-rank (Mantel-Cox) test was used.

16

17

CC EP

A D

TE

IP T

SC R

U

N

A

M

18

CC EP

A D

TE

IP T

SC R

U

N

A

M

19

CC EP

A D

TE

IP T

SC R

U

N

A

M

Table legends Table 1. Effect of JM-20 on lipid peroxidation in the striatum (ST) and substantia nigra pars compacta (SNpc) of rotenone-treated rats

Table 1

Control

SNpc

ST

0.05 ± 0.01 b

0.04 ± 0.004

b

(n=5)

Rot.

0.14 ± 0.03

a

0.08 ± 0.001 a

(n=4)

0.06 ± 0.004 b

0.045 ± 0.005

0.04 ± 0.025 b

0.03 ± 0.002 b

A

N

(n=5)

JM-20

b

U

Rot.+JM-20

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Groups

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MDA (nmol/mg protein)

M

(n=5)

The data are presented as mean ± S.E.M. Different letters: p< 0.05, by one way ANOVA analysis,

A

CC EP

TE

D

followed by Tukey’s multiple comparison post hoc test.

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Table 2. Effect of JM-20 on antioxidant defense in the striatum (ST) and substantia nigra pars compacta (SNpc) of rotenone-treated rats

Groups

SOD (U/min/mg protein)

CAT (U/min/mg protein)

T-SH (μmol/mg protein)

SNpc

ST

SNpc

ST

SNpc

ST

19.04 ± 1.37 a

14.66 ± 0.79 a

3.68 ± 0.4 a

2.34 ± 0.19 a

1.08 ± 0.04 a

1.68± 0.04 a

13.94 ± 1.09 a

10.49 ± 0.37 b

3.19 ± 0.46 a

1.53 ± 0.04 b

0.98 ± 0.06 a

16.18 ± 0.1 a

14.94 ± 0.87 a

3.58 ± 0.3 a

2.32 ± 0.14 a

0.96 ± 0.02 a

1.54 ± 0.08 a

15.73 ± 1.45 a

13.82 ± 0.98 a

4.21 ± 0.2 a

2.17 ± 0.1 a

1.05 ± 0.06 a

1.46 ± 0.07 a

Control

Rot. (n=4)

20

SC R

Rot.+JM-

IP T

(n=5)

(n=5) JM-20

N

U

(n=5)

1.07 ± 0.02 b

A

The data are presented as the means ± S.E.M. Different letters: p<0.05, by one way ANOVA

A

CC EP

TE

D

M

analysis, followed by Tukey’s multiple comparison post hoc test.

21