Therapeutic Effects of Curcuma longa against Rotenone-Induced Gross Motor Skill Deficits in Rats

Syeda Madiha, Zehra Batool, Saiqa Tabassum, Laraib Liaquat, Sadia Sadir, Tahira Perveen and Saida Haider*

Neurochemistry and Biochemical Neuropharmacology Research Unit, Department of Biochemistry, University of Karachi, Karachi 75270, Pakistan

ABSTRACT

Rotenone (pesticide and mitochondrial complex I inhibitor) has been associated to induce toxicity in humans and as well as in animals. The present study evaluated the protective role of curcumin against rotenone-induced motor deficits, biochemical and neurochemical alterations. Rotenone was injected subcutaneously at the dose of 1.5 mg/kg for 8 days. Supplementation of curcumin (100 mg/kg/day, p.o.) was started before 15 days of rotenone injection. The effects of curcumin pre-treatment were evaluated by different motor behavioral parameters (pole test, Kondziela’s inverted screen test, inclined plane test, open field test, beam walking test and footprint test). Biochemical and neurochemical alterations were also monitored. Pre-treatment with curcumin reversed the motor impairments, biochemical and neurochemical alterations which were produced by rotenone. Furthermore, curcumin treatment restored the dopaminergic and cholinergic functions. We conclude that curcumin prevent against toxic effect of rotenone and can be beneficial against different neurodegenerative diseases.

Article Information

Received 25 June 2017

Revised 30 August 2017

Accepted 02 October 2017

Available online 11 May 2018

Authors’ Contribution

SM and SH designed the research. LL, SS and SM conducted aboratory work. ZB and ST performed statistical analyses. LL, SS, SM and TP wrote the article. SH supervised the research.

Key words

Curcuma longa, Rotenone, Dopamine, Acetylcholine, Kondziela’s inverted screen test, Footprint test.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.4.1245.1256

* Corresponding author: [email protected]

0030-9923/2018/0004-1245 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan

Introduction

Environmental factors are the main contributor to the development of neurodegenerative diseases in humans (Freire and Koifman, 2012; Gatto et al., 2009). There are several environmental factors among them the exposure to agricultural chemicals such as pesticide (Zeb et al., 2016) gaining more focus in recent years for the development of human diseases. Rotenone is one of the toxins which extensively used as pesticide; primarily it was used as a fish poison. Epidemiological studies suggested that exposure to environmental toxin such as rotenone has been associated to increase Parkinson’s disease (Greenamyre et al., 2011; Freire and Koifman, 2012; Gatto et al., 2009). Rotenone is lipophilic in nature which easily crosses the blood brain barrier and gathers in mitochondria thus, inhibits the mitochondrial complex I (NADH-ubiquinone oxidoreductase) (Betarbet et al., 2000). Complex I function is to translocate proton which is used for maintenance of energy (Hatefi, 1985). Rotenone toxicity causes complex I inhibition which results in the energy crisis in cells and provide important impending into the mechanism of mitochondrial related human diseases.

Rotenone causes systemic inhibition of mitochondrial complex I, which results in the formation of reactive oxygen species (ROS) that block the electron transport chain and cause neurodegeneration (Moon et al., 2005). Rotenone can enters into dopaminergic neurons because it does not depend on dopamine (DA) transporters, resulting in complex I inhibition (Betarbet et al., 2000). Although, rotenone is widely distributed in the brain, it can cause selective neurodegeneration in specific regions (Greenamyre et al., 2011), and has been directly associated with the motor impairments in rats. It is reported that rotenone administration in rats caused the death of dopaminergic neurons (Betarbet et al., 2000). The bulk of the data are suggesting that rotenone exposure contributes to toxic effects in both human and animals and that can be used to test potential compounds for possible therapeutics.

Curcumin is an active polyphenolic compound of turmeric (Curcuma longa) which is commonly used as food additives in Asian cuisine. Curcumin is lipophilic in nature and has multiple characteristics such as anti-inflammatory, antioxidant, anticarcinogenic, and anti-protein aggregate activities (Fu and Kurzrock, 2010; Hatcher et al., 2008). Curcumin provides protection against different toxins. Various studies reported that pre and post-treatment with curcumin cause a significant attenuation of toxin induced depletion in striatal dopamine level and neuronal cell loss (Rajeswari, 2006; Vajragupta et al., 2003; Zbarsky et al., 2005). A number of pharmacological effects of curcumin have been studied, however effects of curcumin on rotenone-induced toxicity is still not clear. Therefore the main goal of the study was to investigate the protective effect of curcumin in ameliorating the rotenone-induced motor deficits by using different behavioral parameters (pole test, kondziela inverted screen test, inclined plane test, open field test, beam walking and footprint test) and biochemical alterations in rats. Furthermore, dopaminergic neurotransmission was also analyzed. The emphasis in our study was to find out the cholinergic and dopaminergic activity following rotenone administration and further to determine the protective role of curcumin in restoring the cholinergic and dopaminergic functions in rotenone intoxicated rat.

Materials and methods

Animals

Twenty four (24) Albino-Wistar rats (150-200g) were used in the study. Rats were confined separately with access to rodent diet and water (Bocarsly et al., 2012). Animals were allowed to acclimatize for 3 days in order to cancel out the environmental psychological suffering.

Reagents and chemicals

Rotenone, curcumin and rest of the chemicals were obtained from Sigma Chemical Co. (St. Louis, USA).

Experimental protocol

After three days of acclimation period, rats (n=24) were divided into four groups and named as control (sunflower oil), curcumin (Cur), rotenone (Rot) and Curcumin+Rotenone (Cur+Rot) each group containing six rats (n=6). Treatment was started with the administration of curcumin dissolved in oil at the dose of 100 mg/kg to Cur and Cur+Rot groups. Sunflower oil was given to control group. Remaining groups were given tap water orally with the same procedure. After 15 days of curcumin administration, rotenone was injected subcutaneously (s.c) to Rot and Cur+Rot groups at the dose of 1.5 mg/kg dissolved in sunflower oil. Curcumin was given for 15 days prior to the administration of rotenone and along with rotenone for eight days (Madiha et al., 2017). Body weight and food intake was measured throughout the pre-treatment of curcumin and rotenone administration. Rotenone injection was continued for eight days during which curcumin treatment was continued. After the rotenone injection, rats were subjected to open field test, beam walking test, and pole test to observe the motor coordination and balance. Kondziela’s inverted screen test and inclined plane test were also carried out to determine the muscular strength and cataleptic effects respectively following rotenone administration. Footprint test was used to attain the foot step pattern of rats (Madiha et al., 2017). After behavioral procedures rats were decapitated in order to collect their brains. Whole brain was taken out from the skull within 30 s after decapitation. The dissection procedure for dissecting brain regions was carried out as described by Haleem (1990) and Haleem and Perveen (1994). For neurochemical assay all brain samples were kept at −70°C (Fig. 1). Dopamine (DA), dihydroxyphenylacetic acid (DOPAC), ACh (acetylcholine) and AChE (acetylcholinesterase) were estimated in striatum. Reduced glutathione (GSH), malondialdehyde (MDA) levels and enzyme activities were also monitored including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in rat brain.

The pole test used to assess basal ganglia-related movement disorders in rodents. In brief, rats were placed head-up on top of a vertical wooden pole (2.5 cm in diameter and 100 cm in height) and base of the pole was positioned in the home cage. The time to come down from pole to the floor was monitored in rats (Yanpallewar et al., 2004).

Kondziela’s inverted screen test

Kondziela the inverted screen test has been used previously for measure of muscular strength of animals using all four limbs (Kondziela, 1964). The test was done by placing the rat in the center of screen which was inverted over 120 seconds with the rats head declining first. The time when the rat fell off from the screen was noted.

Inclined plane test

The cataleptic effect was investigated according to previously described method by Costall and Naylor (1974). The test was done by gently placing the animal on an inclined surface for three minutes and the duration of time the animal remained in one position in seconds was recorded. Cataleptic score was calculated (Batool et al., 2010).

Open field test

The open field test is used to monitor motor activity. The apparatus consists of a square (76 × 76 cm) with walls 42 cm high. In the central square of the open field animals were placed individually. Latency to move and numbers of square crossed were monitored for five minutes (Haleem et al., 2007).

Beam walking test

Beam walking is a test of motor coordination. The rats have to cross a beam which is suspended between a start platform and their home cage at a height of 50 cm and is supported by two pillars. A cushion was placed under the beam in order to protect the animals from the bang into the floor. The difficulty of this task can be assorted by using beams with different shapes and widths (Jover et al., 2006). Motor activity was assessed by the ability of a rat to crossways a different size of beams. Three circular beams of different diameters were used in this study such as 3 cm, 2 cm, 1 cm and length of 100 cm. In the training phase, animals were trained to traverse the three beams (from widest to narrowest). This helps to make certain that the behavior during testing is more stable and more precisely reflects motor coordination as opposed to the rodent’s natural aversion to crossing over unprotected spaces. After training session testing phase was done and the time taken to cross the beam was recorded.

Footprint test

To take the footprints, the rat hindlimb and forelimb were colored with green and red paints. Animals walk on a runway having following dimensions (100 cm length, 10 cm wide). A white paper was positioned on the floor of the runaway for each rat run. The footprints were evaluated for three step parameters: stride length, base width and overlap between forelimb and hindlimb in centimeters. Four successive steps were chosen for evaluation (Nascimento-Ferreira et al., 2013).

Neurochemical analysis

Determination of monoamines

Concentrations of DA and its metabolite DOPAC in striatum was assessed by means of high performance liquid chromatography with electrochemical detector (HPLC-EC) as described by Haider et al. (2014).

Determination of striatal acetylcholine (ACh) content and acetylcholinesterase (AChE)

The method as explained by Batool et al. (2016) was used to estimate acetylcholine (ACh) content and the method as illustrated by Haider et al. (2015) was used to determine acetylcholinesterase (AChE) activity in striatum.

Biochemical analysis

Activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and lipid peroxidation (LPO were estimated in the same way as explained by Haider et al. (2015) whereas reduced glutathione (GSH) was determined as explained by Haider et al. (2016).

Statistical analysis

Data are presented as mean±SD. Mean differences were evaluated by one-way ANOVA followed by Tukey’s post-hoc test using SPSS version 20.

Food intake and growth rate (Fig. 2A, B) were monitored daily to assess the general health condition of rats after rotenone administration. Following rotenone administration general health was not changed.

Muscular strength

Pole test (Fig. 3) and Kondziela’s inverted screen (Fig. 4) were used to analyze motor and muscular strength following the administration of rotenone and curcumin. One-way ANOVA revealed considerable effects of treatment in pole test (F=10.91, p<0.01) and inverted screen test (F=30.67, p<0.01). Post-hoc analysis by Tukey’s test showed a significantly decrease in time to descend the pole in rats injected with rotenone as compared to controls (p<0.01). However, Time to descend was significantly increased in Cur+Rot group (p<0.01) when it compared to rotenone injected group. Kondziela’s inverted screen test also showed impaired muscular strength by rotenone administration as evident by significantly reduced time of falling (p<0.01) as compared to that of controls. Whereas, prior administration of curcumin drastically increased the time of falling (p<0.01) from inverted screen in Cur+Rot group when compared to rotenone administered group.

Figure 5 shows the effects of rotenone, curcumin and their co-administration on the induction of catalepsy. Statistical analysis by one-way ANOVA revealed significant effects of treatment on % cataleptic score (F=143.66, p<0.01). Compared to control rats injected with rotenone exhibited a significant increase in cataleptic score (p<0.01). Cur+Rot group showed significantly reduced cataleptic score (p<0.01) compared to the group injected with rotenone alone. Rotenone also affected the ambulatory activity of rats as depicted in open field test (Fig. 6). One-way ANOVA for open field test showed significant effects of treatment on latency to move (F=73.98, p<0.01) and number of square crossed (F=7.89, p<0.01) in an open arena. Post-hoc analysis revealed that rotenone administration considerably increased latency to move (p<0.01) and reduced the number of squares crossed

Rotenone administration exhibited a prominent loss of motor coordination in beam walking test (Fig. 7). In this test three beams of varying diameters (3, 2, 1 cm) were used and time to traverse the beam was monitored after the training session. One-way ANOVA revealed significant effects on the latency to cross the 3 cm beam (F=20.17, p<0.01), 2 cm beam (F=19.42, p<0.01) as well as 1 cm beam (F=39.72, p<0.01). Rotenone administration significantly (p<0.01) increased latency to cross the beam tested on all three sizes of beams showed by Post-hoc analysis. Whereas, latency to cross all the three sizes of beams was (p<0.01) significantly reduced in Cur+Rot group as compared to that of rotenone injected group.

Walking pattern was also monitored by footprint test (Fig. 8A). There were significant effects of treatment on stride length of forelimb (F=67.73, p<0.01) and hindlimb (F=33.40, p<0.01), hind base width (F=9.96, p<0.01) and paw overlapping (F=27.30, p<0.01). In the present study administration of rotenone showed gait abnormalities as evident by significant shortened

(p<0.01) stride length for both forelimb and hindlimb as compared to that of controls (Fig. 8B). Hind base width (Fig. 8C) and paw overlapping (Fig. 8D) were also significantly increased (p<0.01) in rotenone injected rats. Pre-administration of curcumin in rotenone injected rats showed normal walking pattern. In Cur+Rot group, forelimb stride and hindlimb stride were considerably increased (p<0.01) than that of rotenone injected animals. Moreover, in Cur+Rot group hind base width and paw overlapping were significantly (p<0.01) lower than rotenone group.

Striatal monoamine levels

Striatal DA and DOPAC levels were also determined in the present study. Data of standard and striatal brain samples were analyzed by the HPLC (Fig. 9A, B). DA (F=68.56, p<0.01) and DOPAC (F=23.30, p<0.01) levels were significantly affected by rotenone and curcumin treatment (Fig. 9C). Rotenone administration significantly reduced the DA (p<0.01) and DOPAC (p<0.01) levels while compared to control rats. Curcumin pre-treatment significantly attenuated the rotenone-induced decreased dopaminergic function in striatum as evident by a notably increase in DA (p<0.01) and DOPAC (p<0.01) levels as compared to the rats injected with rotenone alone.

Brain cholinergic function

In the current study striatal AChE activity and ACh levels were also estimated following the administration of rotenone and curcumin (Fig. 10). One-way ANOVA revealed significant effects of treatment on AChE activity (F=33.41, p<0.01) and ACh levels (F=33.41, p<0.01). Post-hoc analysis by Tukey’s test showed that the rotenone administration significantly reduced (p<0.01) AChE activity in brain. Whereas ACh content was significantly increased (p<0.01) following the administration of rotenone. On the other hand, a significant increase in AChE activity (p<0.01) and decrease in ACh levels (p<0.01) was observed in Cur+Rot group as compared to that of rotenone injected rats.

Oxidative stress

Another potential consequence of rotenone-induced toxicity is oxidative stress (Fig. 11). Statistical analysis showed significant effects of treatment on brain MDA (F=29.05, p<0.01) and GSH (F=57.12, p<0.01) levels (Fig. 11A). Rotenone injected rats exhibited a significant increase in MDA levels (p<0.01) and significant decrease in GSH levels (p<0.01) in brain as compared to that of controls. Whereas, pre-administration of curcumin significantly decreased MDA levels (p<0.01) and increased GSH levels in Cur+Rot group when compared to that of rotenone group. To further test the relevance of rotenone-