The therapeutic potential of Berberine chloride hydrate against harmaline-induced motor impairments in a rat model of tremor

Neuroscience Letters 590 (2015) 84–90 Contents lists available at ScienceDirect Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet Research article The therapeutic potential of Berberine chloride hydrate against harmaline-induced motor impairments in a rat model of tremor Zohreh Vaziri a , Hassan Abbassian a , Vahid Sheibani a , Masoud Haghani b,c , Masoud Nazeri a , Iraj Aghaei a , Mohammad Shabani a,∗ a Neuroscience Research Center, Neuropharmacology Institute, Kerman University of Medical Sciences, Kerman, Iran Histomorphometry and Stereology Research Centre, Shiraz University of Medical Sciences, Shiraz, Iran c Department of Physiology, Shiraz University of Medical Sciences, Shiraz, Iran b h i g h l i g h t s • • • • • Harmaline significantly increased step width and tremor scores. Muscle strength and time on rod decreased in harmaline group. BBR partially reversed the effects of harmaline on motor and balance function. Berberine in a dose dependent manner improved harmaline – induced neurotoxicity. High dose of berberine had the same tremor score as compared to harmaline. a r t i c l e i n f o Article history: Received 14 December 2014 Received in revised form 24 January 2015 Accepted 29 January 2015 Available online 30 January 2015 Keywords: Tremor Berberine Harmaline Rat Motor function a b s t r a c t Essential tremor (ET) is a progressive neurological disorder with motor and non-motor symptoms. It has conclusively been shown that modulation of glutamate receptors could ameliorate ET. Recent studies have suggested that Berberine (BBR) has an inhibitory effect on glutamate receptors. Therefore, BBR may have therapeutic effects on ET. In this study, male Wistar rats (n = 10 in each group) weighing 40–60 g were divided into control, harmaline (30 mg/kg, i.p.) and berberine (10, 20 or 50 mg/kg, i.p, 15 min before harmaline injection) groups. Open field, rotarod, wire grip and foot print tests were used to evaluate motor performance. The results indicated that the administration of BBR (10 and 20 mg/kg) attenuated harmaline-induced tremor in rats, but the beneficial effects of BBR could not be identified at dose 50 mg/kg. In addition, BBR ameliorated gait disturbance in doses of 10 and 20 mg/kg. The high dose of BBR not only failed to recover step width but also showed an adverse effect on left and right step length. The results indicate that BBR only in dose of 20 mg/kg recovers mobility duration. The current study found a dose-dependent manner for the therapeutic effects of BBR in ET. Our study provides the initial evidence for the effects of BBR on motor function. Since BBR exerts its effects mainly through regulation of neurotransmitter release or blocke of NMDA receptors, thus, it is predicted that BBR ameliorate harmaline effect through blockade of NMDA receptors or glutamate release. This is an important issue for future research to evaluate the possible mechanisms involved. © 2015 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Tremor is defined as the involuntary, rhythmic and sinusoidal oscillation involving one or more body parts [1] seen in Essential tremor (ET) and Parkinson’s disease (PD) [2]. ET as a progressive neurological disorder has negative effects on the quality of ∗ Corresponding author at: Department of Neuroscience, Kerman Neuroscience Research Center, Kerman University of Medical Sciences, Kerman, Iran. Tel.: +98 9133978116. E-mail addresses: shabanimoh@yahoo.com, shabani@kmu.ac.ir (M. Shabani). http://dx.doi.org/10.1016/j.neulet.2015.01.078 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved. life in patients. By focusing on the concept of disability, its social and economical burden is estimated to be high [3,4]. The clinical manifestations consist of motor and non motor aspects, including tremor, ataxia and cognitive impairment [5]. In addition, postmortem studies have revealed that ET is a neurodegenerative disorder [6]. The pathophysiological mechanisms underlying ET are not clearly defined yet; however, the components that decreased neuronal excitability play an important role in the treatment of ET [7]. Therefore, there is an unambiguous relationship between ET and neuronal excitability. Z. Vaziri et al. / Neuroscience Letters 590 (2015) 84–90 Experimental studies using animal models of harmaline, a tremorgenic alkaloid that resembles centrally induced tremors in rodents and primates, have provided considerable insight into the pathogenesis and pharmacotherapy of ET [8]. Harmaline induces a nonspecific tremor by increasing glutamate discharge in climbing fibers, resulting in marked purkinje cell destruction and changes in the olivocerebellar pathway in the laboratory animals [9–11]. Considerable research has additionally been performed to investigate a large range of neuroprotective and neurotransmitter modulator agents using animal models which mimic such disorders [12,13]. Administration of harmaline causes excitotoxic damage of purkinje cells resulted by excessive release of glutamate from climbing fibers [14]. Hence, the modulation of glutamatergic system could preserve the normal levels of glutamate in the neuronal synapses, which positively affect motor disease. Previous studies have reported that activation of GABA by Gabapentin and inhibition of glutamatergic transmission by ethanol may be effective in ET [15,16], but questions have been raised about the safety of prolonged use of these drugs and medications. Thus, it is important to find new pharmacotherapies for inhibition of glutamatergic system and neuronal excitability as a therapeutic strategy for ET treatment. Berberine (BBR), an isoquinoline alkaloid is found in many plants such as Berberis, Coptidis rhizoma and Berberis integerrima or Zereshk and is widely used in traditional medicine [17,18]. BBR has been shown to exert neuroprotective, anti apoptotic, anti inflammatory and anti-oxidative properties in various animal models of CNS-related disorders such as Alzheimer’s disease, Parkinson, s disease, forebrain ischemia, mental depression and anxiety [17,20,21]. Previous studies have shown that BBR has an inhibitory effect on glutamate receptors and can alleviate neuronal injury by reducing glutamate, serotonin and norepinephrine levels [17]. Moreover, it has conclusively been shown that modulation of glutamate receptors could ameliorates ET and Parkinson disease (PD) [7,22]. More recently, Ji and Shen suggested that BBR can interact readily to three key enzymes AChE, butyrylcholinesterase (BChE), and monoamine oxidase (MAO) [23]. These findings have provided new insights for interaction of BBR with neurological disorders such as ET. In view of all that has been mentioned so far, one may suppose that BBR may have beneficial effects on ET. This study, therefore, set out to assess the neuroprotective effect of BBR on harmalineinduced termor and ataxia in rats. 85 20, 50 mg/kg, i.p). Berberine was administered 15 min before harmaline injection [17,20,24]. All behavioral assays were performed 30 min after BBR injection with suitable interval among each assay in the following order: observation, open field test, rotarod, wire grip test and footprint. 2.4. Observation The occurrence of tremors was rated by an observer who was blinded to the treatment groups. Fifteen minutes after harmaline administration, data was acquired at the same time of open field test. Balance disturbances were scored as the following scale: 0 = lack of disturbances, 1 = unsteady gait appearing occasionally, 2 = staggering while changing the body position and strongly unsteady gait, 3 = frequent episodes of losing a natural body posture and falling down and maintenance of lying position [25]. 2.5. Open field test (OFT) The open field test was used to evaluate the possible effects of BBR on locomotion and anxiety-like behaviors. The apparatus consisted of an arena made of opaque Plexiglas (90 × 90 × 45 [H] cm). The arena was divided into 16 small squares, so that the rats spent time in either central or peripheral squares. Rats were placed in the middle of the arena and their behavior was recorded and analyzed by an automated video tracking system (Ethovision, Noldus Technology, Netherlands) during a 5 min interval. Total time spent in the center or periphery, total distance moved (TDM), speed, and the number of grooming and rearing were recorded for each rat [26]. 2.6. Rotarod The accelerating rotarod was used in the current study. The rotarod experiment started at a speed of 10 revolutions per min (RPM) to the maximum speed of 60 RPM. Each rat underwent three trials (inter-trial interval = 5 min), with each trial lasting for a maximum of 300 s. Total duration that each rat spent on the rod maintaining its balance was recorded as a measure of balance [26]. 2.7. Wire grip test Harmaline 51330 and Berberine chloride hydrate were purchased from Sigma–Aldrich, India and dissolved in normal saline on the day of experiment. The wire grip test assays muscle strength and balance of the animals. Each rat was suspended on a horizontal steel wire hanging on both forepaws (80 cm long, 7 mm diameter). While the rat’s forepaws were put in contact with the steel wire, the rat was placed in a vertical posture and released whenever it grasped the wire. Latency to fall was recorded for each animal using a stop watch. Each rat underwent three trials with five minutes inter-trial interval [26]. 2.2. Animals 2.8. Footprint Male Wistar rats (40–60 g) were used for the current study. All the procedures in the current experiment were carried out according to the Kerman medical university guidelines for reporting research on animals (Ethics code: KNRC/93/46). Animals were kept under standard conditions (12/12 light-dark cycle) with access to food and water ad libitum. Footprint test was used to assess the walking pattern and gait kinematics of rats. Hindpaws of the animals were painted with nontoxic inks, and the rats were allowed to spontaneously transverse a clear plexiglass tunnel (100 cm × 10 cm × 10 cm) ending in a darkened cage. A sheet of white absorbent paper (100 cm × 10 cm) was placed at the bottom of the track. The resulting tracks provide the spatial relationship of consecutive footfalls from which the stride length and width were measured [27]. 2. Methods and materials 2.1. Drugs and chemicals 2.3. Drug preparation and administration On the day of experiments, animals were brought to the testing room and left for 1 h to acclimate. They were randomly assigned to five experimental groups: control group animals that received no treatment, harmaline (30 mg/kg, i.p), harmaline + Berberine (10, 2.9. Statistical analysis SPSS (V.16, IBM, USA) was used for the analysis of data. Standard error of the mean (Mean ± S.E.M) was used to describe the level of 86 Z. Vaziri et al. / Neuroscience Letters 590 (2015) 84–90 Fig. 1. Effect of different dose of BBR on tremor scores (A), step width (B), left (C) and right step length (D) after harmaline administration. Values show as means ± SE, significantly difference between harmaline versus control (**p < 0.01 and ***p < 0.001) and BBR versus harmaline (# p < 0.05 and ## p < 0.01) groups. behavioral parameters. ANOVA followed by Tukey’s post-hoc analysis was used to compare the differences between groups. Repeated measures ANOVA was used to analyze the data of rotarod task in the learning phase. p < 0.05 was considered statistically significant. 3. Result 3.1. The effect of BBR on balance disturbance The rats of BBR + harmaline and harmaline groups had increased tremor scale score compared to the control group (F (4, 35) = 24.8, p < 0.01, Fig. 1A), but as shown in Fig. 1A a clear decrement of tremor score is apparent by administration of BBR with dosage 10 and 20 mg/kg. These groups were shown a significant reduction in tremor score compared to harmaline group (p < 0.01, Fig. 1A). The most surprising aspect of the data is the treatment with high dose 50 mg/kg that tremor scale score of this group was reached to the level of harmaline group. Therefore, the beneficial effects of BBR in the prevention of tremor could not be identified in dose of 50 mg/kg. 3.2. The effect of BBR on gait disturbance The result of gait disturbance indicated that harmaline significantly increased step width as compared to the control group (F (4, 35) = 13.03, p < 0.01, ANOVA followed by Tukey’s test, Fig. 1B). This parameter was significantly decreased in harmaline + BBR (10 and 20 mg/kg) groups in comparison with the harmaline group (p < 0.05, Fig. 1), though there was no significant difference among harmaline + BBR (10 and 20 mg/kg) and control groups. However, no significant differences were found between high dose of BBR (50 mg/kg) and harmaline group. Overall, the high doses of BBR suppressed the improvement effects of BBR on step width in these measures. Moreover, the BBR (10,20 and 50 mg/kg) and harmaline groups had decreased left and right step length as compared to the control group (F (4, 35) = 15.4, p < 0.01, Fig. 1C and D). Interestingly, high dose of BBR has been found to have an adverse effects also on left and right step length; hence, this dose significantly decreased left (p < 0.01) and right (p < 0.05) step length even from the level of harmaline group (Fig. 1C and D). 3.3. The effect of BBR on explorative and anxiety-like behaviors Rats of the BBR (10, 20 and 50 mg/kg) and harmaline groups had a decrease in total distance moved as compared to the control group (F (4, 35) = 4.85, p < 0.05, Fig. 2A). There was no significant difference between BBR and harmaline groups (p > 0.05, Fig. 2A). Speed (F (4, 35) = 5.8, p < 0.01, Fig. 2B) and rearing numbers (F (4, 35) = 70.4, p < 0.001, Fig. 2C) were significantly decreased in BBR and harmaline treated rats as compared to the control group. No differences were observed among BBR and harmaline groups in speed and rearing number (p > 0.05). There was no significant difference between groups in grooming frequency, the time spent in perimeter and center in the open field test (Fig. 2D–F). Moreover, harmaline significantly deceased mobility duration compared to the control group (F (4, 35) = 5.1, p < 0.05, Fig. 2G). Z. Vaziri et al. / Neuroscience Letters 590 (2015) 84–90 87 Fig. 2. The effect of BBR on explorative behaviors changes induced by harmaline. Total distance moved (A), velocity(B) and rearing number (C) decreased in harmaline and berberine groups. There was no significant difference between groups in grooming frequency (D), time spent in perimeter (E) and center (F). Mobility duration (F) reversed by BBR only with dose 20 mg/kg. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to the control group; ## p < 0.01 as compared to the harmaline group. The results indicated that BBR only with dose 20 mg/kg recovered mobility duration to the control level but low (10 mg/kg) and high (50 mg/kg) doses of BBR did not affect harmaline inducedimpairment of mobility duration. 3.4. The effect of BBR on muscle strength, motor learning and balance function In three consecutive trials, rats of the BBR and harmaline groups had a decrease in falling time in comparison to the control group in muscle strength as measured in the wire grip test (F (4, 35) = 11.3, p < 0.01, Fig. 3A). Falling time increased in rats of the BBR (20 mg/kg) group in comparison to the harmaline group (p < 0.05, Fig. 3A). Balance function was evaluated by averaging three repeated trials of the time stay on rod, these results were similar to per trial alone (p < 0.01, Fig. 3B). Rats of the BBR and harmaline groups spent a shorter duration on the wire grip as compared to the control group (p < 0.05). BBR (20 mg/kg) counteracted with this effect of harmaline on balance function; BBR (20 mg/kg) + harmaline rats spent more time on the rod in comparison to the harmaline rats (p < 0.05) (Fig. 3C). 88 Z. Vaziri et al. / Neuroscience Letters 590 (2015) 84–90 Fig. 3. The effect of BBR on muscle strength, motor learning and balance function after harmaline administartion: (A): Falling time in three consecutive trials in rotarod test, (B): Mean of three repeated trials of staying time on the rod, (C): Mean latency to fall in wire grip test. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to the control group; # p < 0.05 as compare to the harmaline group. 4. Discussion This study set out with the aim of assessing the importance of BBR as an herbal neuroprotective on tremor induced by harmaline. Results revealed that BBR affected hypolocomotion, balance and gait disturbance induced by harmaline in a rat model of tremor in a dose dependent manner. Reduction of harmaline-induced tremors was evident from the decreasing severity of tremor scores in the rats treated with BBR. A number of studies have reported that harmaline produces generalized transient action tremor with kinetic and postural components that presents from minutes to hours [8]. It has been suggested that harmaline increased rebound T-type calcium pulses in inferior olive cells [28]. Based on both basic and clinical studies, T-type calcium channel blockers that transit into the CNS may have therapeutic potentials for tremor disorders [28]. Since BBR has blocking effects on T-type calcium channels in isolated guinea pig ventricular myocytes, therefore, we can assume that protective effect of on tremor can be probably resulted from entry of BBR to CNS and its blocking effect on T-type calcium channel. Harmaline may also induce tremor, at the cellular level, by acting on NMDA receptors but it seems, modulation of T-type calcium channels is the most likely mechanism for tremor [11]. In spite of this a generally accepted ET pathogenesis, very little was found in its therapy. The current study found that BBR had effects on motor and balance function in a dose-dependent manner. It has been shown that BBR has the ability to cross the blood brain barrier and transport to the neurons in a concentration-dependent and time-dependent manner; therefore, our results are justified through the fact that different doses of BBR might have diverse effects on motor and balance function based on the pharmacodynamics and bioavailiability of BBR in CNS [20]. The present findings seem to be consistent with other research which found a dose-dependent manner for the therapeutic effects of BBR in epilepsy [29]. Recent evidences suggest that neuroprotective activities of BBR mediated through interaction with NMDA [30], 5-HT receptors [31] and voltage gated Ca2+ channels [32]. Interestingly, modulations of these receptors or channels are reported to be effective on ET and PD. There is a large volume of published studies describing the role of BBR as a NMDA receptor antagonist [33,34]. It is reported that majority of NMDA receptor antagonists display beneficial effects on ET and PD [35]. Our results indicated that BBR has an alleviating effect on tremor scale in a specific dose, since BBR inhibits the release of glutamate in neurons, it might also be plausible that BBR exerts its effects through modulation of glutamate release. Consistent with these findings, Peng Z. Vaziri et al. / Neuroscience Letters 590 (2015) 84–90 et al. showed that BBR (20 mg/kg) increased NE and 5-HT levels in the hippocampus and frontal cortex but in higher doses (100 and 500 mg/kg) decreased NE, 5-HT and DA levels [31,36]. Altogether, there are several possible explanations for the beneficial effects of BBR in our result. However, the findings of the current study do not support the neuroprotective effects of high dose of BBR (50 mg/kg), this unexpected finding may be explained in several ways. The loss of significant protection with the highest doses of BER suggested the possibility that BER receptor subtypes or effect sites may have different functional and/or opposing effects. This result is in agreement with Peng et al. findings which showed BBR increased the level of some neurotransmitters in the frontal cortex but, it decreased those levels in higher doses [36]. However, other possible mechanisms can be explained in part by direct blockade of K+ channels by BBR [37]. It is well known that blockade of potassium channels increase neuronal excitability, hence the inhibition of NMDA receptors may compensate by blockade of K+ channels in high dose of BBR. The results of rotarod test revealed that BBR somewhat improved gait and balance disturbance in rats. Because harmaline disturbed movement coordination via NMDA-induced excitotoxicity, thus, it is predicted that BBR ameliorates harmaline effect through blocking of NMDA receptors or glutamate release. This hypothesis should be tested through administration of NMDA-R agonists and antagonists simultaneous with administration of BBR [8]. As a conclusion, it seems that BBR might have positive effects on tremor induced by harmaline. Though we did not evaluate the possible mechanisms involved, but it seems that BBR exerts its effects mainly through regulation of neurotransmitter release or block of NMDA receptors in different areas of the brain involved in motor and balance function. We suggest further studies are needed to evaluate the effect of harmaline and BBR on neurotransmitter release in the motor centers of the brain. Our results imply a promising effect of BBR consumption on tremors in a rat model of essential tremor. Conflict of interest The authors declare no conflict of interest. Acknowledgement Funding for this study was provided by Kerman University of Medical Sciences as a grant for the MSc thesis of Zohreh Vaziri. References [1] W.F. Abdo, D.J. van de Warrenburg, N.P. Burn, B.R. Bloem, The clinical approach to movement disorders, Nat. Rev. Neurol. 6 (2010) 29–37, http://dx.doi.org/10.1038/nrneurol.2009.196. [2] A.J. Hughes, S.E. Daniel, S. Blankson, A.J. 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