Neuroprotective Role of Ranolazine: ESR1 and NMDA Receptor Agonist in Traumatic Brain Injury in Drosophila melanogaster, In Silico and In Vivo Correlation

Abstract

Objective

In this study, a high-impact trauma (HIT) device was used for inducing moderate traumatic brain injury (TBI) in Drosophila melanogaster. Mechanical injuries in flies caused by rapid acceleration and assertion produce symptoms characteristics of TBI in humans.

Materials and Methods

Docking studies were carried out to check the binding affinity of the drug toward the receptors. Various oxidative stress parameters, catalase level, glutathione level, superoxide dismutase (SOD) level, malondialdehyde (MDA), and nitric oxide levels, were measured. The mortality index and neuroprotective potential were carried out in TBI in D. melanogaster models.

Results

In the current study, there was an increase in oxidative stress following TBI as evidenced by a significant decrease in the catalase, glutathione, and SOD levels and increase in the level of MDA and nitric oxide after 24 hours. Antioxidant enzymes, catalase and glutathione peroxidase, have a dominant role in TBI. Docking studies were carried out on estrogen receptor 1 (pdb: 1TVO and 1UOM) and NDMA receptor (pdb: 3QEL) as agonist showing the binding affinity of the drug toward the receptors. In comparison to the vehicle-treated group, there was a dose-dependent significant increase in the SOD level and percentage climbing along with a decrease in the MDA level and total protein content. The mortality index was also observed at three concentrations of ranolazine (1, 2, and 4 mg/mL) in D. melanogaster homogenate. These findings suggest that ranolazine has a good neuroprotective potential in the treatment of TBI in the D. melanogaster model.

Conclusion

Present study concluded the scientific evaluation of neuroprotective potential of ranolazine in the treatment of TBI in the D. melanogaster model.

 

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Keywords

traumatic - ranolazine - Drosophila melanogaster - docking - TBI

Introduction

Traumatic brain injury (TBI) remains a major reason of injury, disability, death, and socioeconomic fatalities in the world. It counts for about 30% of total death in cases of injury.[1] [2] TBI is a serious neuronal disorder commonly caused by car accidents, sports-related events, or cases of violence.[3] [4] TBI may occur due to external force with mechanical disruption of brain parts along with delayed pathogenic events leading to neurodegeneration.[5] [6]

Ranolazine possesses anti-ischemic, anti-inflammatory, anti-oxidant effect, etc. It decreases inflammatory mediators interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) and increases anti-inflammatory peroxisome proliferator-activated receptor gamma (PPARy) as well as the level of other antioxidants. Ranolazine decreases cell excitability of the dorsal root ganglion in neurons. Ranolazine prevents reverse mode sodium calcium exchanger.[7] [8] Drosophila melanogaster could be employed as a screening model for target finding, small-molecule selection, and validation of probable drug candidates from lead compounds like other traditional animal models used for of drug screening.[9] [10] [11]

Estrogen receptor 1 (ESR1) works as transcription factor and also plays crucial role in neuro-regulation. Data of some preclinical studies showed that ESR1 agonistic drugs may modify neurological function in TBI. On the basis of the results of reported studies, the potential neuroprotective action of ESR1 agonistic drugs could be further explored for TBI.[12] [13] Opening of the ionophore is facilitated by binding to a specific site on N-methyl-D-aspartate (NMDA) receptor.[14] The regional distribution of NMDA is directly related to excite toxicity in a specific region of the brain following TBI. So, docking studies were carried out to study the binding affinity of ranolazine toward these receptors. Two estrogens receptor Protein Data Bank (PDB) (1TVO and 1UOM) and one NMDA receptor PDB (3QEL) were selected for an in silico study from the literature.[15]

The current study focused on the applications of drug repurposing with integrated method in drug discovery of core targets along with the mechanisms of ranolazine impact in TBI drosophila model. The current study also postulates the treatment on the basis of network pharmacology and molecular docking.

 

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Materials and Methods

Drug and Chemicals

Ranolazine gift sample were collected from Belco Pharma, Bahadurgarh, India. The entire chemical used for the study were of analytical grades. Drosophila melanogaster of Oregon R⁺ strains having either sex were procured from Drosophila stock center situated in Shimla, India. The flies were raised on a defined food medium with corn meal, yeast, agar, and sugar with propanoic acid, and maintained in glass bottles at a temperature of 25°C with alternate light and dark cycle.

 

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In Silico Studies

In silico docking studies were accomplished by using Glide module of the Schrodinger suite 2016-1. The PDB were selected based on the literature, crystal structures of protein–ligand complexes with ranolazine, and the TBI mechanism (PDB code: 1TVO, 1UOM, and 3QEL). The protein was equipped with protein preparation wizard in Schrodinger Maestro. Ranolazine and the PDB ligand were docked into the catalytic domain of protein by using grid-based ligand docking. The Dock score, Glide score, and Glide energy were recorded for additional studies.[16] [17] [18] [19] [20]

 

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Organization of High-Impact Trauma Device

The high-impact trauma (HIT) device reproducibly inflicts closed head TBI in fruit flies. The device uses a metal spring clamped at one end to a wooden board and the other end located over the polyurethane cushion. We facilitated three springs with different impact forces (τ) of 3.675 Nm by using τ = force of spring × radius of semicircle × sin θ. We have performed our experiment using a moderate spring strength of 12.25N at different hits and recorded the mortality index at 24 and 48 hours. The obtained data from the moderate spring strength at 24 and 48 hours did not show significant differences, so we proceeded with our study by estimating the mortality index parameter at 24 hours (MI₂₄). The range of mortality index was 65% at 1 to 10 hits. On the basis of the mortality index, we chose 10 hits with a spring strength of 12.25 N.[21]

 

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Induction of Traumatic Brain Injury by HIT Device

TBI in fruit flies was induced in a standard plastic vial. All the flies used were unanesthetized and restricted to the bottom quarter of the vial with a stationary cotton ball attached to the spring. Then the spring was redirected and release from a 90-degree angle, causing the vial to quickly hit a polyurethane pad and delivering a mechanical force to the flies. The flies were subjected to 1 to 10 hits at an interval of 5 minutes between each hit. Then, the flies were examined for injury after 2 hours of hitting. Following this, the flies were observed for the evaluation of different parameters at different time points.[22] [23]

 

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Experimental Plan

The experimental study of flies was consisted in three parts (A, B, and C) for determination of different parameters at different time intervals. Each part consisted of five groups with 50 flies processed in each group. All the results were reported as mean along with ± standard error of the mean SEM. The data were checked by one-way analysis of variance (ANOVA), followed by Tukey's test.[24]

Part A involved the estimation of the mortality index of flies at 2 to 6 hours; part B involved the estimation of climbing assay at 2, 4, and 6 hours; part C involved estimating biochemical parameters such as oxidative stress at 6 hours.[25] [26]

 

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Treatment Schedule

Group 1, the control group, did not receive any drug and injury and group 2 received TBI with treatment. Group 3 (TBI + RA: ranolazine 1 mg/mL), group 4 (TBI + RA: ranolazine 2 mg/mL), and group 5 (TBI + RA: ranolazine 4 mg/mL) received TBI followed by treatment with ranolazine at the specified doses. First of all, head injury was induced using the HIT device in all the flies and then the flies were kept in a dry vial for some time. Before 24 hours of injury, ranolazine at doses of 1, 2, and 4 mg/mL was administered in groups 3, 4 and 5, respectively, by placing the flies in a food medium containing mentioned concentrations of ranolazine. The flies were starved overnight before drug administration.

First, the unanesthetized flies were taken in a plastic vial and then TBI was induced using the HIT device.

• Step 1: At the above-mentioned time points of 2, 4, and 6 hours, the number of incapacitated flies was observed under a microscope after injury and the mortality data were recorded.

• Step 2: Locomotor activities were checked by using a negative geotaxis assay as described by Bland et al[27] with slight modifications. To assess locomotor activity on vertical climbing, a single fly was placed in an empty glass vial without medium for an hour. The bottom of the vial was gently tapped to stimulate a negative geotactic climbing response and the time required for the fly to climb up 8 cm of the vial wall was recorded. Each fly was tested 4 times at 1-minute intervals. For each experiment, the mean climbing time was calculated. The climbing assay was performed at 2, 4, and 6 hours after brain injury.

• Step 3: At 6 hours, the flies were anaesthetized; the legs and wings were separated from the body with a sharp blade. Then, the flies were homogenized in 0.1-M sodium phosphate buffer with a pH of 8.0 and was centrifuged at 2,500 g for 10 minutes at low temperature (4°C). The supernatant was filtered through a nylon net and was used for measuring the biochemical parameters discussed in the following sections.

 

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Estimation of Biochemical Parameters

Total Protein Content

The principle behind the Lowry method of determining protein concentrations in aromatic acids was used.[28] [29] The phenolic group present in tyrosine and tryptophan residues of protein produced a blue purple color complex, having the highest absorption at 750 nm. The concentration of protein in the test sample was determined by using the following formula:

The protein concentration was expressed in milligram per milliliter.

 

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Superoxide Dismutase Estimation

Superoxide dismutase (SOD) activity was assayed as per the method of Kono et al.[30] [31] All the results were reported as unit/min/mg protein. The inhibition of Nitro Blue Tetrazonium (NBT) reduction by SOD present in the homogenate was then estimated by measuring the absorbance of mixture at 560 nm. The results were expressed as units/mg protein, where 1 unit of enzyme was defined as the amount of enzyme inhibiting the rate of reaction by 50%.

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Catalase Estimation

Dichromate in acetic acid reduced to chromic acetate when heated in the presence of hydrogen peroxide.[32] The green colored mixture was cooled and read at 570 nm against blank. An internal blank solution set was run under identical conditions without the addition of H₂O₂ (2–10 μmol/mL).

 

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Malondialdehyde

The malondialdehyde (MDA) content, a measure of lipid peroxidation, was assayed by the method of Ohkawa et al[33] and modified by Gupta et al.[34] A pink-colored organic layer was produced. This pink-colored layer was separated, and its absorbance was measured at 532 nm using a double-beam UV-visible spectrophotometer. The MDA concentration was calculated with the help of a standard curve prepared with tetra methoxy propane in (nmol/mg) protein.

 

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Results

Docking Result

The docking results were interpreted by using three pdb structures: two estrogen receptors in complex (pdb: 1UOM and 1TVO) and one amino terminal domains of the NMDA receptor subunit GluN1 and GluN2B in complex pdb: 3QEL and shows the interaction of the drug with the receptor. Ranolazine showed good docking scores compared to the PDB ligands, as shown in [Table 1], and based on the Glide score, Glide emodel, and Glide energy.

Table 1 Docking results

PDB	Ligand	Docking score	Glide score	Glide emodel	Glide energy
1TVO	1TVO ligand	−8.96	−8.96	−87.336	−56.386
	Ranolazine	−6.624	−6.624	−66.892	−49.77
1UOM	1UOM ligand	−7.41	−7.41	−47.991	−27.265
	Ranolazine	−5.911	−5.911	−55.91	−46.08
3QEL	3QEL ligand	−4.254	−4.254	−35.934	−29.403
	Ranolazine	−3.626	−3.626	−48.79	−39.35

 

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Effect of Ranolazine on Locomotor Activity in Flies

 

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Climbing Assay

After injury, most of the flies took more time to climb, so the percentage of climbing in 60 seconds was higher than that in 30 and 10 seconds. In the vehicle group, fewer flies climbed within 10 seconds compared to the control group. At 2 hours ([Fig. 1A]), RA concentrations of 1 mg/mL (16.67 ± 4; p < 0.001), 2 mg/mL (22.33 ± 4; p < 0.001), and 4 mg/mL (24.67 ± 3) were found to significantly increase the percentage of flies climbing within 10 seconds compared to the vehicle group. The same results were observed in 30-second climbing assay. At 4 hours ([Fig. 1B]), RA concentrations of 1 mg/mL (21 ± 3.786; p < 0.1), 2 mg/mL (23.67 ± 3.480; p < 0.01), 4 mg/mL (28.33 ± 3.333) showed a significant effect. At 6 hours ([Fig. 1C]), RA concentrations of 1 mg/mL (24 ± 4; p < 0.01), 2 mg/mL (26 ± 4.096; p < 0.01), and 4 mg/mL (31.33 ± 14.38) were found to significantly increase the percentage of flies climbing within 10 seconds, and the same result was observed in the percentage of flies climbing within 30 seconds as compared to the vehicle group (p < 0.05).

Fig. 1 Effect of ranolazine on climbing response of flies in 10, 30, and 60 seconds at (A) 2 hours, (B) 4 hours, and (C) 6 hours after traumatic brain injury (n = 40). At 4 hours, the F-value was found to be (3, 8) = 21.019 in 10 seconds, (3, 8) = 11.075 in 30 seconds, and (3, 8) = 11.075 in 60 seconds. At 6 hours, the F-value was found to be (4, 10) = 13.585 in 10 seconds, (3, 8) = 5.343 in 30 seconds, and (3, 8) = 10.856 in 60 seconds.

 

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Effect of Ranolazine on Percentage Mortality

Mortality Index

After injury, the mortality of the flies was observed at 2 to 6 hours. In the vehicle group, 44.67% mortality was found to occur at 2 to 6 hours after injury. The percentage of mortality reduced at 4 to 6 hours with respect to different drug dose concentrations. Maximum mortality was observed at 2 hours after injury, which was 50.67% in the vehicle group. Ranolazine concentration of 4 mg/mL (30.59 ± 0.8819; p < 0.001) showed a significant reduction in the percentage of mortality at 2 hours. [Fig. 2] shows the effect of acamprosate on mortality response of flies at 2 and 4 to 6 hours.

Fig. 2 Effect of acamprosate on (A) the mortality response of flies at 2 hours. (B) Mortality response of flies at 4 to 6 hours (n = 50). a, p < 0.001 vs. control; b, p > 0.05 vs. vehicle; and c, p < 0.5 vs. vehicle. The F-value is 209.38. The effect of ranolazine on the level of total protein content was significant.

 

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Effect of Ranolazine on the Level of Total Protein Content

In TBI, total protein content level decreases. In the present study, the protein level (0.761 ± 0.04470) was found to be significantly decreased (p < 0.001) in the vehicle group as compared to the control group (3.043 ± 0.04139). Ranolazine showed a significant effect on the total protein content level ([Fig. 3]).

Fig. 3 Effect of ranolazine on the total protein content at 6 hours. The effect of ranolazine on the total protein content in flies' homogenate (n = 50). a, p < 0.001 vs. control; b, p > 0.05 vs. vehicle; and c, p < 0.5 vs. vehicle. The F-value is 209.38. The effect of ranolazine on the level of the total protein content was significant.

 

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Effect of Ranolazine on Activity of Superoxide Dismutase

SOD is a family of metalloproteins that catalyze the dismutation of two molecules of O₂ (super oxides) to form hydrogen peroxide. After injury, the SOD level was significantly (p < 0.0001) decreased in the vehicle group (12.09 ± 0.7950 units/min/mg protein) compared to the control group (24.89 ± 0.9078). Ranolazine at dose 1 mg/mL (4.219 0.4148 units/min/mg protein; p > 0.05) showed non significant difference while at 2 mg/mL (7.8180.6056 units/min/mg protein; p < 0.01) showed a significant reduction in SOD level as compared to the vehicle group and ranolazine at 4 mg/mL (12.2701.291 units/min/mg of protein; p < 0.001). A significant effect was observed between ranolazine-treated groups ([Fig. 4]).

Fig. 4 Effect of ranolazine on superoxide dismutase (SOD) level at 6 hours (n = 50). The effect of ranolazine on the activity of the SOD level is as follows: a, p < 0.001 vs. control group; b, p > 0.05 vs. vehicle treated group; c, p < 0.01 vs. vehicle group. The F-value was found to be F (4, 10) = 36.205.

 

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Effect of Ranolazine on the Level of Catalase

A standard curve for catalase was plotted using the external standard H₂O₂ (2–10 µmol/mL). The concentration of catalase was determined by the linear standard curve (y = 0.157x – 0.051). In TBI, the catalase enzyme level was found to decrease. The catalase level was found to be significantly (p < 0.001) decreased in the vehicle group (1.92 ± 0.015 µmol/mg protein) compared to the control group (2.22788 ± 0.02563). Ranolazine at 1 mg/mL (2.06 ± 0.015 µmol/mg protein; p < 0.01) and 2 mg/mL (2.07 ± 0.012 µmol/mg protein; p < 0.01) showed a significant increase in the catalase level compared to the vehicle group (1.92 ± 0.015 µmol/mg protein). [Fig. 5] shows the effect of ranolazine on catalase at the 6-hour homogenate.

Fig. 5 Effect of ranolazine on catalase at 6 hours in flies' homogenate, (n = 50). The effect of ranolazine on the level of catalase is as follows: a, p < 0.001 vs. control group; b, p < 0.05 vs. vehicle-treated group; and c, p < 0.01. The F-value was found to be F (4, 10) = 32.973.

 

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Effect of Ranolazine on Level of Malondialdehyde

A standard curve for MDA was plotted using an external standard, that is, tetramethoxypropane (2–10 nmol/mL). The concentration of MDA was determined by the linear standard curve (y = 0.019x + 0.058). After injury, the MDA level was found to be increased in the vehicle group (1.23 ± 0.076 nmol/mg protein) as compared to the control group (0.1077 ± 0.02563). Changes in the MDA level in flies and its modulation by ranolazine was recorded. Ranolazine at 1 mg/mL (774 ± 0.076 nmol/mg protein; p < 0.05), 2 mg/mL (0.64 ± 0.10 nmol/mg protein; p < 0.05), and 4 mg/mL showed a significant reduction compared to the vehicle group, and when compared to each other, a significant effect was observed between the ranolazine-treated groups ([Fig. 6]).

Fig. 6 Effect of ranolazine on malondialdehyde (MDA) at 6 hours. The effect of ranolazine on MDA level in flies' homogenate, (n = 50). The effect of ranolazine on the level of MDA is as follows: a, p < 0.001 vs. control group; b, p > 0.05 vs. vehicle-treated group; c, p < 0.05 vs. vehicle-treated group; and d, p < 0.01 vs. vehicle-treated group. The F-value was found to be F (4, 10) = 15.577.

 

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Discussion

Docking results revealed the possible binding pattern and showed that ranolazine's binding affinity toward the NMDA receptor showed that the enzyme's catalytic cavity tightly interacted by pi–pi stacking, hydrophobic interaction, and hydrogen bonding. Glide energy results revealed the free binding energy in the interaction.

At different concentrations of the drug, the percentage of flies climbing improved. Injured flies take more time to climb, while at different drug concentrations more flies climbed within 10 and 30 seconds and the number of flies that climbed within 60 seconds decreased. At 6 hours after injury, the percentage of flies climbing in the vehicle group improved compared to 4 hours after injury, but the percentage of climbing decreased as compared to the control group.

The percentage mortality in flies was reduced at 4 to 6 hours at different doses of the drug. Ranolazine at a dose of 4 mg/mL showed a significant reduction in the percentage mortality at 2 hours. The effect of acamprosate on the mortality response of the flies was found to be effective. These results also strengthen the effectiveness of the drug in the treatment of TBI.

Ranolazine demonstrated its antioxidant potential by decreasing the level of MDA and increasing the level of SOD and catalase. Ranolazine also improves the total protein content. Thus, ranolazine acts as a free radical scavenger by decreasing or increasing the level of auto-destructive enzymes by reducing the overloading of calcium inside in the cells through NMDA receptor and voltage-gated calcium channels. It also inhibits nitric oxide production via nitric oxide synthase inhibition and reduces the release of inflammatory mediators such as TNF-α, and estrogen receptor alpha gene 1 prevents the invasion of proinflammatory cytokines to the damaged tissue following TBI. In the behavioral paradigms, ranolazine demonstrated effective results by reducing the mortality index at 2, 4, and 6 hours after injury and improved percentage of climbing flies at different concentrations of the drug.

 

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Conclusion

TBI is a serious clinical and socioeconomic problem associated with high morbidity and mortality rates. It has been occurring due to direct or indirect outside mechanical impact causing immediate distortion of brain tissue. The Drosophila model has potential as a useful model for studying human neurodegenerative disorders. In the present study, ranolazine was selected due to its pharmacological properties, such as ant-ischemic and anti-inflammatory activities. Docking studies were carried out on estrogen 1 (pdb: 1TVO and 1UOM) and NDMA receptor (pdb: 3QEL) as agonist showing the binding affinity of the drug toward the receptors. In vivo studies suggest that ranolazine provides protection against oxidative stress, possibly by reducing the lipid peroxidation and increasing the levels of SOD and catalase in the brain to combat oxidative stress induced by TBI. The agonistic effect of ranolazine on estrogen1 and NMDA receptors that attenuated the elevated calcium levels in neuronal cells. Ranolazine also directly activates voltage-gated calcium channels after TBI and leads to a reduction in excitotoxicity. The attenuation of excitotoxicity was also responsible for the decrease in the level of oxidative stress. The findings of the current study clearly show that ranolazine can be used for further evaluation in TBI.

 

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Conflict of Interest

None declared.

Acknowledgment

The authors would like to thank the Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak, for all its support.

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Publication History

Article published online:
25 November 2024