Neuroprotection by arbutin against haloperidol-induced Tardive Dyskinesia in rats and reducing neurotoxicity in SHSY-5Y cells

doi:10.21203/rs.3.rs-2211641/v1

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Neuroprotection by arbutin against haloperidol-induced Tardive Dyskinesia in rats and reducing neurotoxicity in SHSY-5Y cells

https://doi.org/10.21203/rs.3.rs-2211641/v1

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Background: Schizophrenia is a psychological condition, and its primary treatment is an antipsychotic medication. However, long-term use of typical antipsychotics often causes irregular involuntary movements, targeting the orofacial region. Due to its complex pathophysiology, there is no appropriate cure for Tardive Dyskinesia (TD). Arbutin, is a natural polyphenol, which is well known for neuroprotection. Therefore, this study evaluated the neuroprotective potential of arbutin against haloperidol induced neurotoxicity and orofacial dysfunction in TD rats.

Methods & results: SH-SY5Y cells were treated with 1 mM concentration of haloperidol and arbutin (5, 10, 15, and 20 µM) for 48 hours, arbutin significantly reduced haloperidol-induced neurotoxicity. Other side, Wistar rats were treated with haloperidol (1 mg/kg/for 21 days) for induced TD like symptoms and treatment with arbutin (50 and 100 mg/kg i.p.) for 21 days show significant decrease in vacuous chewing movements, tongue protrusions, and facial jerking and improved locomotor activity and motor coordination in haloperidol-treated rats. Further, arbutin treatment causes a significant reduction in nitric oxide, MDA, TNF-α, IL-β, and increases SOD, GSH and catalase levels in the striatum region in contrast to haloperidol-treated rats.

Conclusion: In-vitro and In-vivo experimental outcomes suggest that arbutin has the neuroprotective potential that limit TD progression. Their results indicate that arbutin has anti-inflammatory and antioxidant properties that strengthen motor activity and could be explored for cellular and molecular pathways for possible use in the treatment of TD.

Tardive Dyskinesia

Haloperidol

Arbutin

Oxidative stress

Neuroinflammation

Tardive dyskinesia (TD) is a hyperkinetic movement disorder influenced by repetitive and involuntary movements, mostly affecting the orofacial part of the body, it can also hamper the trunk and limbs [1]. The extrapyramidal system regulates movement via "direct" and "indirect" pathways and dopamine is a key regulator of these two pathways that restrict and facilitate motor movements [2, 3]. In the schizophrenia condition, excessive release of dopamine is a major concern. To overcome this problem, dopamine receptor blocking agents (DRBAs) such as typical antipsychotics are used to treat schizophrenia. Their prolonged treatment (months or years) increases the supersensitivity of D2 receptors, which results from aberrant hyperkinetic/involuntary movements [4, 5]. The antipsychotics-induced pathogenic mechanisms are linked to the degeneration of striatal neurons, which is a key factor in TD progression [6]. The earlier study found that prolonged antipsychotic treatment enhances oxidative stress, neuroinflammation, and neuronal excitotoxicity results from hyperkinetic motor activity [7, 8]. Furthermore, anti-psychotic altered antioxidant enzyme activity leads to excessive oxidative insults that contribute to the pathophysiology of TD; several antipsychotic-induced preclinical animal models have been developed to investigate the neuroprotective effect of drugs against TD [8–10].

Haloperidol-induced neurotoxicity is a well-established model used to investigate the therapeutic effects of compounds against TD. Acute haloperidol administration enhances the neurotoxicity in rats and causes orofacial dyskinesia (OD), which is marked by increased vacuous chewing movement (VCM), facial jerking, and tongue protrusion (TP) [9]. An accumulative study indicates that haloperidol has the potential to induce TD-like pathogenic conditions in animals via modifying oxidative stress, mitochondrial functioning, and the apoptosis process [7, 11].

Arbutin is a bioactive hydroquinone glucoside that is produced mostly by the Ericaceae family plant Arctostaphyllosuvaursi, it is naturally present in many other plants, animals, and microbes [12]. According to the report, arbutin was found to possess antioxidant and anti-inflammatory properties and is traditionally used for the treatment of hypertension, and diabetes [13]. In contrast to that, it has been utilized as an anti-microbial, anti-tussive, and anti-infective agent in the treatment of several disease conditions [14, 15]. Additionally, arbutin shows neuroprotective effects on various diseases such as ischemia-reperfusion [16], Alzheimer's disease [17], Parkinson's disease [18], and epilepsy [19]. Moreover, arbutin can improve cognitive function [20].

Therefore, this study was designed to evaluate the neuroprotective effect of arbutin against haloperidol-induced behavioural, and biochemical alteration in TD rats and neurotoxicity in the SH-SY5Y neuronal cell line. Our findings suggest that arbutin could be a treatment option for TD patients.

2.1 Animals

Wistar rats (180–250 g) were procured from the Central Animal House of Panjab University in Chandigarh, (India). Four rats were housed together in polyacrylic cages under conventional standard conditions; 22 ± 2°C room temperature with 60% relative humidity and a 12-hour (light/dark) schedule was operated. The rats had free access to purified fresh water and food with ad libitum. All behavioural activity was performed between 9:00 and 17:00 hours, the current study includes similar-age rats to reduce variation across experiments, and this protocol was approved by Institutional Animal Ethics Committee (IAEC) with protocol no. CUPB/IAEC/2022/005.

3.2 Cell Culture

SH-SY5Y neuroblastoma cell lines were grown in standard media (Dulbecco’s Modified Eagle Medium (DMEM) including streptomycin (100 mg/ml), penicillin (100 U/ml), and 10% of Fetal Bovine Serum (FBS). All these items were bought from HiMedia Pvt. Ltd., Mumbai, India. Cells were routinely grown at 37°C under humidified incubator with 5% CO2 [21].

2.3 Drugs and treatment schedule

Haloperidol (Serenace® Inj.) was purchased from Searle India Pvt Ltd. and dissolved in distilled water before administration. Arbutin was buying from Sigma-Aldrich® Pvt. Ltd. India. Haloperidol (1 mg/kg) and arbutin (50 and 100 mg/kg) were given through the intraperitoneal route. All the required ELISA kits were obtained from Krishgen Biosciences, USA. Other high-quality analytical grade chemicals were used in the present study.

2.4 Experimental Protocol

The total protocol schedule was of 21st days. Haloperidol (1 mg/kg) was injected continuously for 21 days through the i.p. route. Arbutin (50 and 100mg/kg) was given intraperitoneally for 21 days after giving the haloperidol treatment. All experimental rats were divided into four groups (n = 6 animals in each group).

Group-1; Vehicle control

Group-2: Haloperidol (1 mg/kg, i.p.) for 21 days.

Group-3: Haloperidol (1 mg/kg, i.p.) + Arbutin (50 mg/kg, i.p.) for 21 days.

Group-4: Haloperidol (1 mg/kg, i.p.) + Arbutin (100 mg/kg, i.p.) for 21 days.

On the 21st day, various behavioural parameters were performed to examine the impact of arbutin on motor abnormalities. At the end of the protocol (22nd day), all experimental rats were sacrificed, and the striatum was removed, the homogenates was prepared for further biochemical analysis (Fig. 1).

2.5 Parameters

2.5.1 Cell viability

The cell viability was quantified using an MTT assay. SHSY-5Y cell lines were obtained from exponential phase cultures and were proliferated on 96 well plates at a density of 2X10⁴. The SH-SY5Y cells were then treated with a 1 mM concentration of haloperidol dissolved in a 5% DMSO solution and then treated with arbutin (5, 10, 15, and 20 µM) dissolved in distilled water. After a 48-hour incubation period, cells were cleaned with PBS, and a 100 µl MTT dye was loaded in all wells and retained for 3 hours at 37°C. After that DMSO was added to solubilize the crystal formed by converting the well color from yellow to blue. Absorbance was assessed using a Bio-Rad microplate reader at 570 nm. Graphs were generated by plotting the cell viability (%) against the drug concentration [7, 22].

2.5.2 Behavioural parameters:

2.5.2.1 Analysis of orofacial movements

The small Plexiglass chamber (30×20×30 cm) was utilized to analyze oral dyskinesia. The experimental rat was placed separately in an observation chamber to assess oral movement. Before behavioral analysis, the rat was given 10 minutes to explore the observation chamber. After 10 min, a hand-operated counter was started to record the no of scoring for VCMs, tongue protrusions, and facial jerking was analyzed for oral dyskinesia in rats. VCMs and facial jerking were not counted during the time of grooming. When the rat started grooming, counting was paused, and it was resumed when it finished. A total of 10 min was observed for the rat to analyze the oral dyskinesia. During the protocol, the scorer was unaware of the treatment given to the animals [8, 23].

2.5.2.2 Rotarod activity

The Rotarod instrument was utilized to assess motor coordination and balance abilities before starting the experimentation. Rats were allowed to explore the rotarod apparatus and during the trial, a 25 rpm speed was maintained. The average fall-off times were recorded after each rat completed three separate trials with a five-minute break [9].

2.5.2.3 Locomotor activity

A double-beam photo-actometer was used to record the locomotor activity. The animals were individually placed there to test their locomotion. The rats were allowed to move throughout the chamber for five minutes, and the number of beams crossed within that time was recorded [24].

2.5.2.4 Narrow Beam Walking

The narrow beam walking was conducted to assess gait abnormalities. It entails allowing animals to freely roam in a fixed, constrained wooden beam while keeping its equilibrium and stability. The rats were habituated to walking from one end (Start area) to another end (Target area) of the narrow beam. During the experiment, the time it took to cross the beam was recorded [25].

2.5.3 Biochemical analysis

2.5.3.1 Dissection and Homogenization

At the end of protocol day 22nd, each experimental animal was sacrificed via injecting thiopentone 100 mg/kg, i.p., and the animal's brain was then isolated, rinsed with cold isotonic saline, the striatum was separated, and homogenate was prepared using child 0.1 M phosphate buffer solution having a 7.4 PH and centrifuged at 10,000 g for 15 minutes. The supernatant was collected and stored for further biochemical assessment [26].

2.5.4 Biochemical Parameters

2.5.4.1 Quantification of Malondialdehyde

Malondialdehyde (MDA), a by-product of lipid peroxidation, was measured in the striatum using the standard method developed by Wills in 1996 [23].

2.5.4.2 Estimation of nitrite

The concentration of nitrite in the supernatant was measured using the Griess reagent as described previously, which serves as a marker for the formation of nitric oxide (NO). The nitrite standard curve value is expressed as mol/mg protein [24].

2.5.4.3 Estimation of Catalase

The catalase activity was estimated using a standard method developed by Luck et al. [27].

2.5.4.4 Estimation of Superoxide Dismutase (SOD)

The SOD concentration was assessed in the striatum supernatant by using the method described by Kono et al., 1978 [28].

2.5.4.5 Measurement of reduced glutathione (GSH)

The Ellman method was adopted to determine glutathione (GSH) concentration [25].

2.5.4.6 Protein estimation

The total protein composition of the rat striatum was assessed using the Lowry method [29].

2.5.4.7 Evaluation of TNF-α and IL-1β

The inflammatory cytokines (TNF-α and IL-1β) were quantified in the stratum tissue of rat brain by using an ELISA kit, followed by the standard guidelines of Krishgen Biosciences USA [30].

2.6 Statistical analysis

GraphPad Prism (8.0.1 V) was applied for data analysis. The behavioural data were analyzed by two-way (ANOVA) variance, applied by Bonferroni's post hoc test for multiple comparisons. However, all biochemical data were analyzed using one-way (ANOVA), followed by Tukey's post hoc test. All the observed results were obtained as Mean\(\pm\)S.D. all the results are reliable and statistically significant.

3.1 Effect of arbutin in haloperidol-induced neurotoxicity in SH-SY5Y Cell

The SH-SY5Y cell lines were utilized to confirm the neurotoxicity of arbutin. Treatment of SH-SY5Y cells with a concentration of 1 mM haloperidol showed a significant (P < 0.0001) decrease in cell survival up to 35% after 48 hours compared to the control. Arbutin treatment with different concentrations (5, 10, 15, and 20 µM) significantly (PP < 0.001)) increased cell survival rates after 48 hours when compared to 1 mM haloperidol treated cells. The results showed that arbutin treatment reduced cell death by 16%, also indicating that arbutin could protect SH-SY5Y cells against haloperidol-induced neurotoxicity (Fig. 2).

3.2 Effect of arbutin on haloperidol-induced VCMs, tongue protrusions, and facial jerking in rats

Acute haloperidol (1 mg/kg) injection for 21 days showed a significant (P < 0.0001) increase in VCMs, tongue protrusion, and facial jerking on the 21st day than the control group. Long-term treatment with arbutin (50 and 100 mg/kg) substantial declines the VCMs (P < 0.001), facial jerking(P < 0.001), and tongue protrusion(P < 0.05) on the 21st day in comparison with haloperidol group (Fig. 3, 4, 5).

3.3 Effect of arbutin on rotarod in haloperidol-induced neurotoxicity in rats

Acute haloperidol injection for 21 days dramatically (P < 0.0001) reduces motor coordination on the 21st day compare with the control group. Prolonged injection of arbutin (50 and 100 mg/kg) considerably (P < 0.0001) improved motor coordination compare to the haloperidol-treated group (Fig. 6).

3.4 Effect of arbutin on locomotor activity in haloperidol-induced neurotoxicity in rats

Acute treatment with haloperidol results in a substantial(P < 0.0001) reduction in locomotor activity on the 21st day in comparison to the control group. Arbutin at the dose of 50 and 100 mg/kg, substantially (P < 0.0001) improved locomotor activity in comparison to haloperidol-injected rats (Fig. 7)

3.5 Effect of arbutin on narrow beam walking activity in haloperidol-induced neurotoxicity in rats

Chronic treatment with haloperidol for 21 days shows a substantial (P < 0.0001) reduction in the transfer latency on the 21st day in contrast with a control group. Continuously administration of arbutin (50 and 100 mg/kg) showed significantly (P < 0.0001) improvement in transfer latency than the haloperidol injected group (Fig. 8).

3.6 Effect of arbutin on MDA and nitrite in haloperidol-induced neurotoxicity in rats

Acute haloperidol injection significantly (P < 0.0001) increased in MDA and nitrite levels in the striatum then the control group. Arbutin (50 and 100 mg/kg) treatment significantly (P < 0.001) decreased MDA (Fig. 9a), and nitrite (Fig. 9b) levels as compared to the haloperidol-treated group.

3.7 Effect of arbutin on antioxidants in haloperidol-induced neurotoxicity in rats

Haloperidol injection significantly (P < 0.0001) reduced in the SOD, GSH, and catalase levels in contrast with control group. Co-administration of arbutin (50 and 100 mg/kg) dramatically (P < 0.001) enhanced SOD (Fig. 10a), GSH (Fig. 10b), and catalase (Fig. 10c) levels as compared to the haloperidol-treated group.

3.8 Effect of arbutin on TNF-α and IL-1β levels in haloperidol-induced neurotoxicity in rats

Acute treatment with haloperidol showed a considerable (P < 0.0001) increase in the TNF-α and IL-1β levels in comparison to the control group. Long-term administration of arbutin (50 and 100 mg/kg) substantially (P < 0.001) decreased the TNF-α and IL-1β in the striatum in comparision to the haloperidol group (Fig. 11).

Arbutin is a bioactive hydroquinone glucoside obtained from Arctostaphyllosuvaursi, it is known for its antioxidant and anti-inflammatory properties, which provide neuroprotective action against brain disorders [17, 18]. The present study shows arbutin has the ability to protect SH-SY5Y cells from the neurotoxicity induced by haloperidol. The earlier study revealed that haloperidol treatment increases the reactive oxygen species that induce mitochondrial dysfunction, leading to the death of SH-SY5Y cells [7, 21]. In this investigation, SH-SY5Y cells treated with haloperidol (1mM) reduced cell viability, indicating cell death; however, arbutin (5,10,15, and 20 M) treated cells significantly increased cell viability and reduced cell death when compared to haloperidol treated cells. These findings indicate that arbutin has the potential to reduce neurotoxicity by providing neuroprotection against haloperidol-induced neuronal death. These findings indicate that arbutin has the potential to reduce neurotoxicity by providing neuroprotection against haloperidol-induced neuronal cell death.

The present study employed a haloperidol-induced experimental rat model to investigate behavioral and biochemical changes in rats presenting TD-like symptoms [10]. The haloperidol (1 mg/kg) treated group caused a significant increase in VCMs, tongue protrusion, and facial jerking in TD rats, similar to that observed in patients taking antipsychotic medication [31]. While arbutin (50 and 100 mg/kg) has the potential to reverse the orofacial dysfunctions in haloperidol-treated rats. Evidence from prior research suggests that antipsychotic treatment like haloperidol enhances the generation of free radicals, which is associated with hyperkinetic movement[32]. Furthermore, another study revealed that arbutin administration exerted a neuroprotective effect by improving motor function in MPTP-treated rats. Their results revealed that arbutin has the potential to reverse motor abnormalities [33]. In this experiment, arbutin showed a significant increase in muscular coordination observed by using rotarod, locomotor activity by using an actophotometer, and gait abnormality by using the narrow beam walk test as compared to haloperidol-treated rats.

Furthermore, several studies have found that haloperidol treatment causes dopamine receptor hypersensitivity by inhibiting the dopamine D2 receptors specifically located in the striatum. As a result, there is an increase in oxidative stress that leads to neurodegeneration [5, 34]. Another study reported higher levels of free radical formation in TD patients compared to those who do not have the illness [35]. According to the report, arbutin has antioxidant and anti-inflammatory properties which provide a neuroprotective effect against various neurodegenerative disorders [18, 19]. The present study found that arbutin treatment substantially reduced the MDA and nitrite levels in rats treated with haloperidol. The increase in MDA showed the formation of lipid peroxidation, which is associated with TD pathogenesis. Previous study outcomes suggested that arbutin treatment has the potential to decrease MDA and nitrite in rat brains treated with streptozotocin [36].

Additionally, prolonged treatment with antipsychotic drugs altered the antioxidant enzyme activity and imbalanced the oxidative stress that is responsible for the progression of TD [5]. Furthermore, another study found that arbutin has the potential to enhance the antioxidant enzyme activity by stimulating the Nrf2/HO-1 signaling pathway, which is responsible for the transcription of several antioxidant enzymes that provide neuroprotection against isolectin-induced multiple sclerosis in rats [37]. In the current study, the arbutin treatment showed a considerable increase in antioxidant enzymes such as SOD, GSH, and catalase levels in the striatum region of rats in comparison with haloperidol-treated rats. This result indicates that arbutin can halt the pathogenesis of TD. Moreover, the prior researcher observed that the long-term arbutin treatment inhibit the production of pro-inflammatory cytokines (TNF-α and IL-1β) showing neuroprotection against MPTP-treated PD rats [18]. Furthermore, other research evidence revealed that prolonged antipsychotic treatment impaired the production of inflammatory markers that lead to the degeneration of striatal neurons, which is linked to the pathogenesis of TD [38]. In this research, arbutin treatment significantly reduced the TNF-α and IL-1β levels in the striatum, which was found to be increased in haloperidol-treated rats. The study outcomes indicate that arbutin has a neuroprotective potential that reduces the behavioural and biochemical abnormalities in haloperidol induced TD rats. The present study indicates that arbutin has a neuroprotective effect; arbutin ameliorates the behaviour deficit in haloperidol-induced TD in rats. Besides that, arbutin administrations restored the antioxidant enzyme levels, alleviating oxidative stress and decreasing the pro-inflammatory cytokine levels in the rat’s brain and thereby providing a reason for arbutin could be used in the treatment of TD. The outcomes indicate that arbutin could be a better option to treat TD-like symptoms. Nevertheless, cellular and molecular studies are needed to explore and verify the potential neuroprotective mechanism of arbutin.

Acknowledgment

Author would like to acknowledge the facility provided by the Central University of Punjab (CUPB), Bathinda, India. The authors would like to acknowledge Indian Council of Medical Research, New Delhi, India for providing a Senior Research Fellowship to Mr. Shubham Upadhayay to pursue his research at the department of Pharmacology, CUPB.

Funding

This study had no funding

Author contribution statement

G.S: Reviewed the literature, and contribute to behavioural experiments and data acquisition S.U: Manuscript drafting, data collection, and statistical analysis U.N: Manuscript editing, data interpretation, P.K: Concept design, critically reviewed the manuscript. All authors read the final version of the manuscript and approved the authorship list.

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Conflict of interest

The authors report there are no competing interests to declare.

Data availability statement

The authors confirm that the data supporting the findings of this study are available in the article

Author contribution statement

Ethics approval

Ethical clearance for conducting this study was obtained from Institutional Animal Ethics Committee (IAEC) at the Central University of Punjab, Bhatinda, India with protocol no CUPB/IAEC/2022/005.

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Neuroprotection by arbutin against haloperidol-induced Tardive Dyskinesia in rats and reducing neurotoxicity in SHSY-5Y cells

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Abstract

Figures

1. Introduction

2. Experimental

2.1 Animals

3.2 Cell Culture

2.3 Drugs and treatment schedule

2.4 Experimental Protocol

2.5 Parameters

2.5.1 Cell viability

2.5.2 Behavioural parameters:

2.5.2.1 Analysis of orofacial movements

2.5.2.2 Rotarod activity

2.5.2.3 Locomotor activity

2.5.2.4 Narrow Beam Walking

2.5.3 Biochemical analysis

2.5.3.1 Dissection and Homogenization

2.5.4 Biochemical Parameters

2.5.4.1 Quantification of Malondialdehyde

2.5.4.2 Estimation of nitrite

2.5.4.3 Estimation of Catalase

2.5.4.4 Estimation of Superoxide Dismutase (SOD)

2.5.4.5 Measurement of reduced glutathione (GSH)

2.5.4.6 Protein estimation

2.5.4.7 Evaluation of TNF-α and IL-1β

2.6 Statistical analysis

3. Results

3.1 Effect of arbutin in haloperidol-induced neurotoxicity in SH-SY5Y Cell

3.2 Effect of arbutin on haloperidol-induced VCMs, tongue protrusions, and facial jerking in rats

3.3 Effect of arbutin on rotarod in haloperidol-induced neurotoxicity in rats

3.4 Effect of arbutin on locomotor activity in haloperidol-induced neurotoxicity in rats

3.5 Effect of arbutin on narrow beam walking activity in haloperidol-induced neurotoxicity in rats

3.6 Effect of arbutin on MDA and nitrite in haloperidol-induced neurotoxicity in rats

3.7 Effect of arbutin on antioxidants in haloperidol-induced neurotoxicity in rats

3.8 Effect of arbutin on TNF-α and IL-1β levels in haloperidol-induced neurotoxicity in rats

4. Discussion

Declarations

References

Supplementary Files

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