Alpha-Pinene Alleviates Motor Activity in Animal Model of Huntington’s Disease via Enhancing Antioxidant Capacity

Huntington’s disease (HD) is a progressive, neurodegenerative, and inherited disease. Antioxidants have been shown to be effective in slowing disease progression in animal models of HD and are under investigation in human clinical trials. α-pinene, a member of the monoterpene class, has been shown to exert antioxidant activity. Therefore, this study aimed to investigate the impact of α-pinene on animal model of HD. Thirty-two male Wistar rats received 3-Nitropropionic acid (3-NP) for induction of the disease model or treated with α-pinene + 3-NP in different groups. Motor skill, and biochemical evaluations to detect oxidant/antioxidant markers in rat cortex and striatum were performed in all groups. We found that α-pinene significantly improved 3-NP-induced changes in the body weight, rotarod activity, time taken to cross the narrow beam, and locomotor activity. Biochemical analysis revealed that α-pinene significantly decreased the 3NP-induced elevation in oxidant markers, nitrite, and malondialdehyde in both cortex and striatum. In addition, α-pinene counteracted the 3-NP-induced fall in antioxidant enzymes, including superoxide dismutase, catalase, and glutathione in the cortex and striatum. In conclusion, we found that α-pinene prevented the motor dysfunction induced by 3-NP in the animal model of Huntington’s disease. Oxidants-antioxidant balance might be involved in the protective effect of α-pinene.


Introduction
Huntington's disease (HD) is a progressive, fatal disease associated with neurodegeneration and is characterized by spontaneous choreiform movements, cognitive disorders, and weight loss [1]. Its prevalence is about 5 to 20 cases per 100,000, and there is evidence of an increase in prevalence of between 15 and 20% per decade in studies from Australia, North America, and Western Europe [2,3]. Despite the several hypotheses in disease pathology, including mitochondrial malfunction, abnormal repetition of the poly-glutamine sequences in the Huntingtin protein, apoptosis, neurochemical imbalance, oxidative stress, and neuroinflammation, the molecular mechanism involved in neurodegeneration of HD has not yet been precisely uncovered [4].
Several genetic and toxin-induced animal models have been developed to mimic HD pathophysiology and test treatment strategies [5,6]. Among the toxin-induced models, the 3-Nitropropionic acid (3-NP) model can simulate HD pathophysiology, and with a pattern similar to neuronal death in the striatum of patients with HD, causes GABAergic medium spiny neurons degeneration [7][8][9]. Indeed, HD mainly affects the striatum and cerebral cortex, as is evident from the cell loss in the medium-sized spiny neurons of the striatum and the large glutamatergic pyramidal neurons of the cortex [8].
Elevated oxidative stress and imbalanced oxidant/antioxidant signaling have been known as hallmarks of HD. Dysfunction of antioxidant molecules and enzymes participate in scavenging or withdrawing oxidative damage and are associated with HD's pathophysiology. In this regard, markers of damage like protein oxidation, lipid peroxidation, and DNA damage have been elevated in HD [10]. Recently the beneficial effects of α-pinene (a member of the monoterpene class) on the nervous system have been reported [11]. α-pinene is found in various plants of the coniferous genus, including the Pistacia Atlantica subsp. Kurdica tree. α-pinene exerts anti-inflammatory [12] and antioxidant effects. The protective effects of α-pinene have also been demonstrated in PC12 cell lines. Porres-Martínez and colleagues reported that treating these cells with α-pinene increased the cell viability and markedly improved the expression of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and heme-oxygenase 1 (HO-1) [13].
Antioxidant and anti-inflammatory activity of α-pinene and similar monoterpenes have been shown to be involved in their protective effect on central nervous system disorders [13,14]. It has been shown that α-pinene improves motor activity, avoidance memory, and lipid peroxidation in an animal model of Parkinson's disease [15]. Also, it was effective in scopolamine-induced memory and learning disorders and exerted beneficial effects in the management of dementia, memory impairment, and learning. Lee and colleagues showed that α-pinene augmented the protein levels of antioxidant enzymes such as manganese superoxide dismutase in the hippocampus of animals with memory impairment induced by scopolamine [16].
According to the antioxidant and neuroprotective effects of α-pinene, this study aimed to investigate the effect of α-pinene on motor functions and oxidant/antioxidant parameters in 3-NP-induced Huntington's animal model. In order to respond to the research question, first, animals were rendered and treated with or without α-pinene, then the motor/ behavioral skills and biochemical assays related to oxidative stress and inflammation were evaluated.

Experimental Animals and Grouping
All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85 -23, revised 1985) and approved by the Research Ethics Committee of Kurdistan University of Medical Sciences (IR. MUK.REC.1398.251). Thirty-two male Wistar rats weighing 250-300 g were rendered and randomly divided into four groups (n = 8). Sham group: received saline 1 ml/kg, and α-pinene vehicle (10% DMSO in saline) 1 ml/kg. Control group: received 3-NP (10 mg/kg, ip) and α-pinene vehicle (1 ml/kg). Treatment groups: received 3-NP (10 mg/kg, ip) [17,18] and α-pinene at 1 and 5 mg/kg intraperitoneally [16,19]. All groups were subjected to the same injections and behavioral tests, and the person conducting the behavioral tests was not aware of the nature of the treatment.
3-NP was dissolved in normal saline and injected for 21 days to induce the HD model. Also, the treatments were done every day before 3-NP injection. All injections were given intraperitoneally, and solutions were prepared freshly every day just before injections [20]. 3-NP was purchased from Sigma Aldrich and α-pinene was provided by Saghez Sazi Kurdistan Manufacturing Co. (Van).

Measurement of Body Weight
Bodyweight loss is a typical sign of HD disease induced by 3-NP injection [21]. Therefore, animal body weight was recorded on the first and the last day of experiments, and the change in the body weight was calculated and reported as a percentage [20].

Motor Skill Assessments
In each group, locomotor activity, rotarod, and Narrow beam walking tests were performed at 4-time points: before the first 3-NP injection (day 0), on the seventh day (day 7), fourteenth day (day 14), and the twenty-first day (day 21) 4 h after 3-NP injection.
An open field test was performed for the assessment of locomotor activity. The apparatus consists of a metal, rectangular, black colored square (60 × 60 × 40 cm), and the floor was divided into nine small squares (20 cm). In order to adapt, the animals were given 5 min to move freely. Each rat was then placed in the center of the device, and then the number of grooming, rearing, and squares crossed were recorded for 10 min. Then the sum of all three above indices was considered as the total locomotor activity [17]. The total distance that animal travels in the open field apparatus was measured as another parameter of locomotor activity.
A narrow beam walk apparatus measured gait abnormalities. First, the animals were trained to walk across a narrow wooden beam. The beam consisted of two platforms (eight cm in diameter) connected by a wooden beam (5 mm in thickness, 2.0 cm in width, and 120 cm in length). The beam was elevated 50 cm above the ground. A box full of sawdust was placed under the beam to act as a protector for falling the rats. Rats were allowed to move on the beam for 5 min before training to adapt to it. Then a trial was begun by placing a rat on one end of the platform, and the time required to cross the beam in each trial was recorded [18].
A rotarod (moving rod with a diameter of 7 cm) test was done to determine coordination, balance, and muscle tone in animals. In order to achieve a similar level of coordination, the animals underwent a 5-day training program before the first injection to achieve stable performance, and the animals that were not well balanced were replaced. After the training program, the fall-off time was recorded with the cut-off time of 180 s for each animal [18,21].

Sample Preparation
After behavioral assessments, rats were anesthetized (using a cocktail of 50 mg/kg ketamine and 4 mg/kg xylazine) and immediately (< 1 min) sacrificed. Subsequently, the skull was opened, the brain removed, the cerebral cortex and striatum were dissected on ice, frozen in liquid nitrogen, and stored at − 70 °C until biochemical analysis. Thirty mg of the striatum or cortex were homogenized (10% w/v) in PBS (0.1 M, pH 7.4) or homogenization buffer by a homogenizer and were centrifuged at 10,000 g for 15 min at 4 °C. The supernatant was collected and used for the measurement of the total protein content and oxidant-antioxidant parameters [17].

Biochemical Assays
Samples of the cerebral cortex and striatum were used to analyze oxidative stress parameters such as nitrite (Kiazist, Iran cat#KNIT96) and malondialdehyde (MDA, Kiazist, Iran cat#KMDA96), as well as antioxidant parameters such as glutathione (GSH, Kiazist, Iran cat#KTHI96), superoxide dismutase (SOD, Kiazist, Iran cat#KSOD96) and catalase (CAT, Kiazist, Iran cat#KCAT96). The total protein content was measured in all cerebral cortex and striatum samples by the Bradford method (Kiazist, Iran cat#KBRD96), and data were normalized with tissue protein content. Using commercial kits, these parameters were determined by colorimetric methods.

Statistical Analysis
Behavioral data were presented as the mean ± SEM of eight animals in each group. One-way Analyses of Variance (ANOVA) followed by tukey's post hoc test was used to analyze the statistical significance in multiple comparisons. Graphs were assembled and analyzed using Graph Pad Prism 8 (Graph Pad Software). A p value < 0.05 was considered statistically significant.

α-Pinene Effectively Prevented Weight Loss in the 3-NP Induced HD Model
One-way analysis of variance revealed, as depicted in Fig. 1, that there were significant variations between the groups' percentages of animal weight change at the end of the study [F (3, 28) = 7.525, P = 0.0008]. A 3-NP injection significantly decreased the weight of the animals in the control group compared to the sham group, according to post-hoc analyses [q (28) = 6.026, p = 0.0011]. The weight loss caused by 3-NP was considerably reduced when 1 and 5 mg/kg/ day of α-pinene were administered compared to the control group [q (28) = 5.508, p = 0.003 and 4.445, p = 0.019, respectively].

α-Pinene Ameliorated Locomotor Activity Deficit Induced by 3-NP
Decreased locomotor activity is a prominent feature of the HD in animal model induced by 3-NP injection. To evaluate the locomotor activity, the animals' movement performance, including grooming, rearing, and squares crossed the lines of the chamber surface within 10 min, were counted and compared. Statistical analysis showed that above mentioned parameters in different groups had significant differences [F (15, 112) = 55.31, P < 0.0001]. Post-hoc comparisons revealed that no significant difference was observed in the locomotor activity between different experimental groups on the baseline (Day 0). 3-NP injection significantly reduced locomotor activity in the control group on different time points [day 7: q (112) = 15.86, p < 0.0001, day 14: q (112) = 16.54, p < 0.0001, and day 21: q (112) = 16.72, Fig. 1 Effect of α-pinene on body weight change in different groups. Results are expressed as mean ± SEM (n = 8). P values of less than 0.05 were considered as significant. One-way ANOVA followed by Tukey's test was used to identify differences. 3-NP 3-nitropropionic acid, α-p alpha pinene, Ctrl control p < 0.0001] compared to the sham group, indicating a motor deficit in the control group (Fig. 2a). While, there was a significantly increased locomotor activity in the group that received α-pinene 5 compared to the control group [day 7: q (112) = 5.822, p = 0.007, day 14: q (112) = 5.719, p = 0.009, and day 21: q (112) = 6.545, p = 0.001]. In addition, α-pinene 1 showed a significant increase compared to the control on day 21 [(q (112) = 6.304, p = 0.002)].

α-Pinene Improved the Latency to Fall in the rotarod Test Performed in the 3-NP-Induced Rat Model of HD
Since the fall time of animals from the rotarod apparatus is one of the features of 3-NP treatment, the effect of α-pinene on balance was investigated by the rotarod test [15].
As shown in Fig. 3b,   open field test. a the total count of movements, b the total distance. Animals were treated with 3-NP or 3-NP + α-pinene. Data are expressed as mean ± SEM (n = 8). P values of less than 0.05 were considered as significant. One-way ANOVA followed by Tukey's test was used to identify differences. 3-NP 3-nitropropionic acid, α-p alpha pinene, Ctrl control

Effect of α-Pinene on biochemical Parameters in the Brain Cortex and Striatum of Animals Received 3-NP
Our findings showed that MDA level had significant differences in different experimental groups [F (7, 56) = 35.96, P < 0.0001]. Post-hoc comparisons indicated that administration of 3-NP significantly increased MDA levels in both cortex [q (56) = 8.986, p < 0.0001], and striatum [q (56) = 11.14, p < 0.0001], of the control group received 3-NP compared with the sham group (Fig. 4a).

Discussion
We found that injection of 3-NP to animals induced a significant motor dysfunction and weight loss compared to the healthy sham group. In addition, biochemical evaluations in Fig. 3 Effect of α-pinene on balance and coordination. a time taken by the animals to traverse from the Narrow beam. b Latency time to fall on the rotarod apparatus. Results are expressed as mean ± SEM (n=8). P values of less than 0.05 were considered as significant. One-way ANOVA followed by Tukey's test was used to identify differences. 3-NP 3-nitropropionic acid, α-p alpha pinene, Ctrl control Fig. 4 Biochemical analysis in different groups. Effect of α-pinene on malondialdehyde (a), nitrite (b), glutathione level (c), superoxide dismutase (d), and catalase activity (e) in animal's brain (cortex and striatum). These parameters were determined by colorimetric commercial kits. Results are expressed as mean ± SEM (n=8). P values of less than 0.05 were considered as significant. One-way ANOVA followed by Tukey's test was used to identify differences. 3-NP 3-nitropropionic acid,α-p alpha pinene, Ctrl control, MDAMalondialdehyde, GSH Glutathione, SOD Superoxide dismutase, CAT Catalase the cortex and striatum revealed that the antioxidant capacity (glutathione level, catalase, and, superoxide dismutase activity) was significantly decreased and the indicators of oxidative stress, MDA, and nitrite, were markedly increased in the control animals. Altogether, these data indicate that our model was successfully induced in rats that received 3-NP for 21 days.
Previously, we obtained a similar pattern in locomotor activity, body weight, and biochemical parameters following 3-NP injection in rats [17]. 3-Nitropropionic acid is gaining attention as a valuable tool to mimic behavioral, biochemical, and morphologic changes similar to those occurring in HD. Indeed, it affects the striatum, causes degeneration of GABAergic medium spiny neurons, and resembles those pathologies involved in HD [22].
Our results also showed that α-pinene in both doses used in this study alleviated the motor defect induced by 3-NP. These results were in accordance with those of Goudarzi et al., who investigated the effect of α-pinene in an animal model of Parkinson's disease. They found that administration of α-pinene significantly improved memory function and the latency time to fall on the rotarod device [15]. In another study, Kasuya and colleagues examined the effect of Chamaecyparis obtuse essential oil, which contains a high percentage of α-pinene, on emotional behavior and locomotor activity. They found that α-pinene is distributed in different parts of the brain, and it exerts anxiolytic, or excitatory-like effects dose-dependently. They suggested that besides the striatum and hippocampus, other areas in the brain involve in increasing locomotor activity induced by high concentrations of α-pinene [23]. In line with our results indicating the beneficial effect of α-pinene on locomotor activity, Abbasi Maleki et al. found a reduction in the immobility time and increased the swimming time and climbing the walls in animals received Origanum majorana oil, contains α-pinene, in the forced swimming test indicating an increase in locomotor activity by the oil [24].
Exploring the mechanisms involved in the effect of α-pinene, we found that it was able to decrease the 3-NPinduced elevation of MDA in both striatum and cortex. Confirming these results, Saeedipour et al. showed that α-pinene significantly reduced anxiety behaviors and MDA levels compared to the control group [25]. Also, Zamyad and colleagues reported that α-pinene decreased the MDA level and increased catalase activity compared to the control group [26].
In addition, our findings showed that α-pinene prevented the 3-NP-induced reduction of GSH levels in the cortex and striatum significantly, indicating its antioxidant activity. In fact, glutathione prevents the free radicals, peroxides, lipid peroxides, and heavy metals-induced damage to the vital cell components. In addition, glutathione peroxidase activity has provided neuroprotection in Huntington's disease models [27]. These findings were also in accordance with those of Saeedipoor et al., who reported α-pinene was able to increase the glutathione levels in rats [25].
Being catalase reduced in patients with HD, we explored the effect of α-pinene on catalase in animals [28]. Interestingly, we found that α-pinene prevented the 3-NPinduced reduction of catalase and SOD activity, indicating the possible mechanism behind the beneficial effect of α-pinene on Huntington's animal model.
In addition, in an animal model of local ischemic stroke, it has been shown that α-pinene improved the neurobehavioral function and increased the antioxidant capacity via restoring the SOD, catalase, and glutathione peroxidase to the normal condition. It also reduced the concentrations of MDA, nitric oxide, and IL-6 in the hippocampus, cortex and striatum [29].
Other mechanisms should be taken into account for the beneficial effect of α-pinene. For instance, it has been shown that its beneficial effect on the motor activity was in association with increase in the brain-derived neurotrophic factor (BDNF) and tyrosine hydroxylase expression in the brain of mice [30].
Recent research [31] examined the effects of α-pinene and trans-anethole on 3-NP-induced motor skill and oxidative stress in the striatum. Oral administration of α-pinene may explain why a larger dose of the compound was administered in this study. Motor activities were assessed in our study using the open field, narrow beam, and rotarod apparatus, which are the most motor skill test for animal models of Huntington's disease [18]. Their results for grip strength supported our rotarod test results, and their impact on weight is comparable with our data. Their biochemical findings, such as levels of MDA, SOD, GSH, and catalase activity, mainly supported our conclusions.
In summary, our findings demonstrate the beneficial effects of α-pinene on motor symptoms in the animal model of HD. These data shed light on the mechanisms involved in the protective effect of α-pinene through enhancing the antioxidant capacity. However, further studies are needed to determine the other pathways that participate in the protective effect of α-pinene in HD.
Data Availability Enquiries about data availability should be directed to the authors.