Neuroprotective potential of erucic acid via inhibition of N2a cell lines and rotenone induced Parkinson’s disease rat model

Abstract

OBJECTIVE:

The objective of this study was to investigate the potential for erucic acid (EA), an omega-9 monounsaturated fatty acid, to act as a neuroprotective agent.

MATERIALS AND METHODS:

In this study, EA was investigated against N2a cell lines and a rotenone (ROT)-induced model of Parkinson’s disease for its neuroprotective potential. The N2a cell line was incubated with fetal bovine serum, penicillin, and streptomycin supplemented with Dulbecco’s Modified Eagle’s Medium, and the following assays were carried out: (i) MTT, (ii) biocompatibility, (iii) DCFDA, and (iv) diphenylamine. A cell morphology study was also performed. Further, ROT 1 mg/kg s.c. and EA 3 and 10 mg/kg p.o. were given to rats on a daily basis for 21 days, and the following parameters were assessed: (i) neurobehavioral studies, (ii) oxidative stress markers, (iii) neuroinflammatory markers, (iv) neurotransmitters, and (v) histopathological study.

RESULTS:

The cell viability assay revealed that EA showed protection against ROT-induced toxicity in N2a cells, which was confirmed by a cell morphology study. EA decreased oxidative stress and % DNA fragmentation significantly. EA also prevented ROT-induced motor impairment and altered levels of oxidative stress markers, neurotransmitters, and neuroinflammatory markers significantly. When compared to the ROT group, a histological investigation of the EA group showed partial neuronal loss with the existence of intact neurons in between the vacuolated gaps.

CONCLUSION:

This study revealed that EA possesses profound neuroprotective properties in in vitro and in vivo studies. Additional research can be carried out to study the mechanism of EA with respect to its neuroprotective potential.

Introduction

Neurodegenerative diseases (NDs) are a wide range of neurological conditions characterized by the deterioration of neurons, glial cells, synapses, and other networks.[1] They lead to protein buildup within neurons, which alters physicochemical properties, especially in the brain and peripheral organs.

Among the various NDs, Parkinson’s disease (PD) is linked with the degeneration of dopamine (DA)-producing cells in the substantia nigra along with the decline of voluntary motor skills over time. Some of the signs and symptoms associated with PD are bad posture, tremors when at rest, akinesia, postural instability, bradykinesia, rigidity, and hypokinesia.

A large number of monocarboxylic acids with carbon atoms between C12 and C26 are found in the human brain. Fatty acids having just one double bond are known as monounsaturated fatty acids. Examples are Erucic acid (EA), oleic acid, palmitoleic acid, and nervonic acid. However, polyunsaturated fatty acids refer to fatty acids comprising several double bonds, for example – α-linoleic acid and γ-linoleic acid. Docosahexaenoic acid was reported to improve mitochondrial function in a number of aging-related animal models, PD, AD, HD, etc.[2]

Similarly, EA (with the formula CH₃(CH₂)₇CH = CH(CH₂)₁₁COOH; 22:1 ω9) is a monounsaturated omega-9 fatty acid that was recently reported to have many neuroprotective effects in the studies conducted earlier, for example: (i) enhanced memory effects in a scopolamine-induced rat model,[3] (ii) positive cognitive effects with erucamide,[4] (iii) management of X-linked adrenoleukodystrophy along with oleic acid,[5] (iv) peroxisome proliferator-activated receptor-delta interaction,[6] and (v) thrombin inhibitory and elastase inhibitory actions that have anti-neuroinflammatory properties.[7] These observed results lead to the conclusion that EA might be helpful in the treatment of NDs. Therefore, an in vitro study using an N2a cell line was executed to explore the neuroprotective effect of EA. Further, neurobehavioral study, oxidative stress markers, neurotransmitters, and neuroinflammatory markers were evaluated using a rat model of PD triggered by rotenone (ROT). The effect of EA was further confirmed by a histopathological study of the rat’s striatum.

Materials and Methods

In vitro study

Maintenance of cell line

10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin added to Dulbecco’s Modified Eagle’s Medium were used to cultivate N2a cells at 37°C in an incubator with 5% carbon dioxide. The media got changed twice a week, and 0.25% trypsin was used to pass the cells once they had reached confluency (70%–80%).

Cytotoxicity assay

In order to determine the IC₅₀ value and a safer dose, the cytotoxicity of ROT at concentrations of 1, 2, 4, 8, 16, 32, 64, and 128 μM and EA at concentrations of 2.5, 5, 10, 20, 40, 80, and 160 μM on N2a cell line were assessed. 96-well microtiter plates containing 5 × 10³ cells/well were seeded with 10% FBS, and the plates were then kept incubating for 24 h at 37°C and carbon dioxide 5%. Each well received an MTT solution (20 μL), followed by incubation for 4 h. The optical density of the formazan crystals was measured after they had been solubilized in 100% dimethyl sulfoxide (DMSO). Percentage Cytotoxicity was determined using the outlined formula:[8]

A_test – Absorbance of the sample; A_control – Absorbance of the control sample:

Biocompatibility assay

After the detection of cytotoxicity of ROT, the toxicity of ROT was tried to minimize using a safer dose of EA. 5 × 10³ N2a cells/well and 10% FBS were added to a well plate, and incubated for 24 h. Fixed quantities of ROT (8 μM, 12 μM, and 16 μM) were given to the cells and cultured for 48 h after 4 h of incubation with 5 μM EA. Cells were incubated with MTT dye and optical density was obtained at 570 nm wavelength using an ELISA plate reader. Observed optical density is directly proportional to cell viability.[9]

Further, the combined effect of selected doses of ROT (8 μM, 12 μM, and 16 μM) with a safer dose of EA (5 μM) was calculated using the same method, and the results are presented in Table 1.

Table 1.

Effect of erucic acid on percentage cell viability, fluorescent intensity, and percentage DNA fragmentation

Parameters	Groups
	Control	EA5	ROT8	EA5 + ROT8	ROT12	EA5 + ROT12	ROT16	EA5 + ROT16
Percentage cell viability	100±0.59	116.45±1.35	56.47±0.78	57.09±0.05*	51.36±0.24	56.92±0.03**	44.83±1.3	59.31±0.07***
Fluorescent intensity	179±2.08	157.33±1.45	432.67±2.60	289.33±2.18***	594.33±1.85	274.33±2.40***	664.67±1.85	460±1.52***
Percentage DNA fragmentation	27±1.52	30.33±1.20	48.33±1.20	36.33±2.33**	57±1.15	49±2.08***	59.66±0.33	50.66±0.88***

*P<0.05, **P<0.01, ***P<0.001 when ROT8, ROT12 and ROT16 compared to EA5 + ROT8, EA5 + ROT12 and EA5 + ROT16 respectively. All values are mean±SEM (n=3). EA=Erucic acid, ROT=Rotenone, SEM=Standard error of mean

Cellular morphology

In a 6-well plate, 1 × 10⁵ N2a cells per well were seeded. EA (5 μM) for 4 h followed by ROT (8 μM, 12 μM, and 16 μM) was added in the cells. The images of the cells incubated with EA and ROT were captured through the inverted microscope.[10]

DCFDA assay

A 6-well plate with 2 × 10⁵ N2a cells in each well was treated with EA (5 μM) for 4 h, followed by the addition of ROT (8 μM, 12 μM, and 16 μM), and the cells were incubated for 48 h. Fluorescent dye DCFDA solution (1 μM) was applied to the cells at 37°C in the dark for a period of ½ h. Following incubation, collected cells were redispersed in phosphate-buffered saline (PBS). A spectrofluorometer with a wavelength for excitation (488 nm) and for emission (522 nm) was used to measure fluorescence intensity.[11] Reactive oxygen species (ROS) was presented by arbitrary unit (a.u.).

Diphenylamine assay

2 × 10⁵ cells were subsequently seeded in plates followed by treatment with EA (5 μM) for 4 h, and ROT (8 μM, 12 μM, and 16 μM) for 48 h. Cells were harvested in 10 mM PBS and ice-cold lysis buffer (Triton X-100, Tris-HCl, and EDTA). The supernatant from centrifuging the lysate was put in a new tube. Centrifugation was performed again after the addition of 10% trichloroacetic acid (TCA) and incubation for 10 min. The leftover pellet was redispersed in 5% TCA. One milliliter of diphenylamine (DPA) reagent and 0.5 ml of the supernatant were combined and stored at 30°C overnight. An ELISA reader was used to measure the absorbance at 600 nm. % DNA fragmentation was determined using the following outlined formula:[12]

In vivo study

Acute toxicity study

All animals were assessed for changes in the skin, mucous membrane, reactivity to stimuli, body weight, etc., for a period of 14 days (OECD Guideline No. 425) for choosing the dose of EA for experiment.[3]

Experimental design and drug treatment

The study was carried out at Trans-Genica Services Pvt. Ltd., Maharashtra (IAEC Approval Number – IAEC-TRS/PT/021/005).

By using simple randomization, the following nine groups (each with n = 6) of male Wistar rats (150–200 g) were created: Group I: vehicle control (VC) group treated with 0.5% carboxy methyl cellulose (CMC), Group II: ROT (1 mg/kg s.c.), Group III: EA (3 mg/kg p.o.), Group IV: EA (3 mg/kg p.o.) + ROT, Group V: EA (10 mg/kg p.o.), Group VI: EA (10 mg/kg p.o.) + ROT, Group VII: LA (3 mg/kg p.o.) + ROT, Group VIII: LA (10 mg/kg p.o.) + ROT, and Group IX: RIL (8 mg/kg p.o.) + ROT.

In 0.5% CMC, all of the test compounds were dispersed, and ROT was dissolved in a 1:1 mixture of DMSO and polyethylene glycol before being further diluted to a final 0.1% DMSO concentration in sterile saline. All the treatments were given for 21 days, once a day. The EA administration began 60 min before the ROT. Rats were given injections of xylazine (10 mg/kg) and ketamine (75 mg/kg) as anesthetics.

Neurobehavioral studies

Open field test

Rats’ voluntary movement activity was measured using an open field test equipment made of wood and measuring 100 cm × 100 cm × 40 cm. The number of squares passed by the rats during the previous 10 min was tabled.[13]

Catalepsy

The rats were placed in the standing position for the bar test, and the catalepsy behavior was calculated by adding the times it took them over three efforts to withdraw one of their forelimbs from a 10-cm-long rod (cutoff time, 60 s).[14]

Oxidative stress markers

The striatum was isolated from the animal’s brains after 21 days of treatment with EA. After being centrifuged, the supernatant was removed and used for additional calculations.

Glutathione level

Ellman’s reagent (1.0 mL) was combined with the tissue homogenate and glutathione (GSH), which was quantified as 10% TCA. At a wavelength of 412 nm, the completed product’s absorbance was measured.[15] The GSH/mg of tissue was calculated using the formula:

Catalase level

In the catalase (CAT) activity assay, a 3 mL reaction mixture (H₂O₂ phosphate buffer) and supernatant (0.05 mL) were used. From the H₂O₂ standard graph, activity units were derived. The data were shown in mg/protein units of CAT activity.[14]

CAT activity = Change in absorbance per minute/extinction coefficient of hydrogen peroxide × volume of sample × milligram of protein

Superoxide dismutase level

Carbonate buffer and EDTA were mixed with the supernatant (0.05 mL) and epinephrine (0.5 mL). The variation in OD was measured at a wavelength of 480 nm. The results showed superoxide dismutase (SOD) activity units (mg per protein).[15]

Malondialdehyde level

The malondialdehyde (MDA) quantity was observed to determine lipid peroxidation in the tissue homogenate of the treated animal.[16] A test tube containing supernatant (1 mL) and TBA (3 mL) was mixed. At 532 nm, the upper layer’s absorbance was measured at 600 nm.

MDA concentration = Absorbance at 532 nm × 100 × total volume of the mixture /1.56 ×10⁵ × weight of the dissected brain × aliquot volume

Neuro-inflammatory markers and neurotransmitters

Neuro-inflammatory markers

An immunoassay kit was used to quantify these markers. At a wavelength of 450 nm, the optical density value was measured. Standard curves were used to measure the amounts of tumor necrosis factor (TNF-α), interleukin (IL)-1β, and IL-6, which were then measured in pg/mL protein.[17]

Neurotransmitters

Using high-performance liquid chromatography, an electrochemical detector was used to assess the catecholamine levels of DA, 5-hydroxyindoleacetic acid (5-HIAA), 5-hydroxytryptamine (5-HT3), and norepinephrine (NE). Frozen brain samples were defrosted and 0.2 M perchloric acid was used to homogenize the mixture. After that, centrifugation was done and the supernatant was passed through 0.22 mm nylon filters. 20 ml samples were added and the concentrations of neurotransmitters and their metabolites were estimated from the standard curve created by using standards in the concentration range of 10–100 ng/mL.[15]

Histopathological study

The striatum tissue samples were embedded in paraffin blocks after being serially dehydrated in alcohol and cleaned in xylene. The microsections (4–5 μ wide) were sliced, smudged with dyes – hematoxylin and eosin, and evaluated for histological alterations using a routine procedure.[18]

Statistical analysis

As shown Table 2 all observations are presented as mean ± standard error of the mean (n = 3/group for in vitro study and n = 6/group for in vivo study). Data were investigated using Graph Pad Prism Software version 5 for Mac (GraphPad Software, San Diego, CA, USA). Analysis of variance was used to calculate all parameters followed by Tukey’s post hoc test. The threshold for significance level was fixed at P < 0.05 for all reported P values.

Table 2.

Percentage cell viability by MTT assay at different concentrations of rotenone and erucic acid

Concentration of ROT (µM)	Percentage cell viability	Concentration of EA (µM)	Percentage cell viability
8	57.09	1.25	100.43
12	51.36	2.5	109.03
16	44.83	5	116.45

EA=Erucic acid, ROT=Rotenone

Results

In vitro study

% cytotoxicity

The ROT had a direct cytotoxic impact on the N2a cell line in the MTT assay with respect to concentration (R² = 0.89) [Figure 1A]. The ROT shows 50% cytotoxicity in the range of 8 μM–16 μM for 48 h, and the ROT’s IC₅₀ value for the N2a cell line was discovered to be 15.76 μM. Similarly, EA showed low cytotoxicity (10%) at a dose of 5 μM [Figure 1A].

Figure 1.

(A) % cytotoxicity of rotenone (ROT) and EA at diverse concentrations in MTT assay, (B) Morphological assessment of the N2a cells by bright field microscopy: (a) Control; (b-d) reflect ROT (8 μM, 12 μM, and 16 μM) induced morphological aberration in N2a cells; (e) high density of cells treated with erucic acid (5 μM); (f-h) indicated cell morphology to normal or regular, after prior treatment with erucic acid (5 μM) along with ROT in different doses (8 μM, 12 μM, and 16 μM). ROT: Rotenone, EA: Erucic acid

% cell viability in MTT assay

The % cytotoxicity of ROT was found to be 57.09% ±1.35%, 51.36% ±0.41%, and 44.83% ±2.26%, respectively, at concentrations 8 μM, 12 μM, and 16 μM, and for EA (1.25 μM, 2.5 μM, and 5 μM), % cell viability was found to be 100.43% ±1.55%; 109.03% ±9.77%, and 116.45% ±2.34%, respectively [Table 2]. Therefore, a 5 μM dose of EA was selected for further studies.

% cell viability, fluorescent intensity, and % DNA fragmentation

Four hours pretreatment of EA (5 μM) followed by ROT (8 μM, 12 μM, and 16 μM) exposure for 48 h indicated that EA cells increased viability by 0.67%, 5.55%, and 14.48%, respectively [Table 1].

On pretreatment with EA, ROS generation was found to be decreased, i.e., 289.33 ± 2.18, 274.33 ± 2.40, and 460 ± 1.52, respectively, when compared to enhance intracellular ROS generation, i.e. 432.67 ± 2.60, 594.33 ± 1.85, and 664.67 ± 1.85 in the ROT8, ROT12, and ROT16 groups [Table 1]. Similarly, pretreatment with EA minimized % DNA fragmentation (36.33% ±2.33%, 49% ±2.08%, and 50.66% ±0.88%, respectively) as compared to ROT8, ROT12, and ROT16 per se.

Cellular morphology

Pretreatment with EA resulted in their morphology returning to normal, with proliferating cells that were less prone to cell death when compared to rounded and detached cells with ROT [Figure 1B].

In vivo study

Acute toxicity studies

We selected 3 and 10 mg/kg p.o. doses of EA for the study since no death or clinical hallmarks were seen with such doses of EA.

Neurobehavioral studies

Open field test

Rats receiving ROT injections had significantly lower overall rat activity levels (P < 0.001) against the VC group. However, EA (both doses) significantly improved (P < 0.001) the overall activity when compared to the total activity for ROT-treated rats. The results obtained for the LA- and RIL-treated groups were in line with those of EA in comparison to the ROT-treated group [Table 3].

Table 3.

Effect of erucic acid on neurobehavioral studies and oxidative stress markers

Parameter	Groups
	VC	ROT	EA3	EA10	EA3 + ROT	EA10 + ROT	LA3 + ROT	LA10 + ROT	RIL8 + ROT
Catalepsy time (s)	5.31±0.41	17.92±0.73###	4.31±0.25	4.28±0.22	11.05±0.69***	8.81±0.42***	7.70±0.42***	7.29±0.42***	6.76±0.45***
Open field test (total activity)	189.85±6.04	53.08±4.39###	251.61±3.10	251.15±0.93	180.9±5.08***	199.56±7.35***	199.95±4.44***	216±6.42***	211.81±5.16***
GSH (nmol/mg)	13.13±0.60	8.36±0.43###	12.84±0.37	12.41±0.29	11.76±0.53**	12.88±0.64***	13.72±0.59***	14.27±0.83***	13.42±0.54***
SOD (U/mg)	3.58±0.11	2.53±0.14##	3.14±0.21	2.94±0.22	3.59±0.09**	3.7±0.10***	3.81±0.09***	3.76±0.23***	4.08±0.20***
CAT (U/mg)	9.72±0.30	4.96±0.15###	7.27±0.14	7.12±0.08	6.5±0.49*	6.52±0.30*	7.34±0.27***	7.49±0.37***	7.46±0.24***
MDA (nmol/mg)	2.88±0.31	5.47±0.38###	3.22±0.13	3.18±0.19	3.65±0.24***	3.66±0.14***	3.26±0.15***	3.24±0.14***	2.82±0.20***

*P<0.05 when compared to ROT, **P<0.01 when compared to ROT, ***P<0.001 when compared to ROT, ^##P<0.01 compared to VC, ^###P<0.001 compared to VC. All values are mean±SEM (n=6). VC=Vehicle control, EA=Erucic acid, ROT=Rotenone, GSH=Glutathione, SOD=Superoxide dismutase, CAT=Catalase, MDA=Malondialdehyde, SEM=Standard error of mean, LA=Linoleic Acid

Catalepsy test

When compared to the VC-administered rats, catalepsy time was seen to be considerably significantly larger (P < 0.001) in rats which were given ROT. The EA-administered animals at both doses significantly decreased (P < 0.001) the catalepsy time in comparison to rats which were given ROT. A similar pattern was observed in the LA- and RIL-treated groups [Table 3].

Oxidative stress markers

When compared to rats that received a VC treatment, animals administered with ROT had lowered GSH, SOD, and CAT levels (P < 0.001, P < 0.01, and P < 0.001, respectively) significantly and increased MDA levels (P < 0.001) significantly. However, comparing rats administered with ROT to those given EA (3 mg/kg), the levels of GSH, CAT, and SOD were increased significantly (P < 0.01, P < 0.01, and P < 0.05 respectively), while the MDA levels were dramatically decreased (P < 0.001). On the contrary, when compared to ROT-treated rats, after EA (10 mg/kg) administration, the levels of GSH, SOD, and CAT were significantly higher (P < 0.001, P < 0.001, and P < 0.05) and the levels of MDA were significantly lower (P < 0.001). In addition, when combined with ROT-treated rats, significant elevation in GSH and CAT levels (P < 0.001) and reduction in MDA levels (P < 0.001) were seen in the LA- and RIL-treated groups [Table 3].

Neuro-inflammatory markers and neurotransmitters

In comparison to the VC group, TNF-α, IL-1β, and IL-6 levels in the ROT group were observed to be raised significantly (P < 0.001). When EA (3 mg/kg) was administered, the elevated levels of neuro-inflammatory markers in the ROT group were reduced significantly with respect to P values of P < 0.001, P < 0.01, and P < 0.001, respectively. However, when EA (10 mg/kg) was administered, the elevated levels of pro-inflammatory cytokines were significantly lowered with P < 0.001. A similar pattern of results (P < 0.001) was observed in the LA- and RIL-treated groups when collated with ROT-treated rats [Figure 2].

Figure 2.

Effect of erucic acid on neuro-inflammatory markers and neurotransmitters. All values are mean ± standard error of the mean, (n = 6); ^###P < 0.001 compared to VC; ^####P < 0.0001 compared to VC; **P < 0.01 when compared to ROT; ***P < 0.001 when compared to ROT; ****P < 0.0001 when compared to ROT. DA: Dopamine, HIAA: 5-hydroxyindoleacetic acid, 5-HT3: 5-hydroxytryptamine, EA: Erucic acid, VC: Vehicle control, ROT: Rotenone, TNF: Tumor necrosis factor, IL: Interleukin

The level of all neurotransmitters was observed to be markedly reduced (P < 0.001) in ROT-treated rats in comparison to VC-administered rats. However, when compared to the ROT group, the EA (3 mg/kg and 10 mg/kg)-treated rats had enhanced levels of DA, NE, 5-HT3, and 5-HIAA significantly (P < 0.001, P < 0.001, P < 0.001, and P < 0.001, respectively). Furthermore, the results obtained in the LA- and RIL (P < 0.001)-treated groups were in a similar pattern with EA-treated rats when collated with ROT-treated rats [Figure 2].

Histopathological study

Sections from the VC group displayed healthy cells without any pathologic alterations; the neurons and astrocytes had prominent nuclei and showed multipolarity in comparison with the section from the ROT group, which revealed neuron degeneration, apoptosis, and darkly colored nuclei, which are signs of injury to neurons. However, the comparable space in the parts from EA + ROT and LA + ROT exhibited some neuronal death, but among the vacuolated areas, there were still some intact neurons. The treatment groups RIL + ROT retained normal neuronal structure and normal cellular formations [Figure 3].

Figure 3.

Effect of erucic acid on histopathological study. EA: Erucic acid, ROT: Rotenone

Discussion

Fatty acids are found to be present to a rich extent in various natural products including fruits, vegetables, dairy products, edible oils, and nuts. According to reports, fatty acids have a variety of biological roles in regulating gene expression, intracellular signaling pathway, transcription factor activity, inflammatory cascade; and reducing oxidative stress, and thereby exhibiting neuroprotection and cancer protection.[16] An increased level of oxidative stress and apoptosis rate is reported to be directly related to neurological disorders.[19]

According to reports, complex I of the mitochondrial electron transport chain is severely inhibited by ROT in rats.[20,21] The cell viability assay and cell morphology results in this study are consistent with the earlier research.[22] ROT impaired the mitochondrial system, which led to increased production of ROS and death of cells in a dominant dopaminergic area of cells.[23] ROT-induced ROS generation was reduced in the N2a cells pretreated with EA. One of the most noticeable biological characteristics of apoptosis is DNA nucleosomal fragmentation.[24] DPA is a simple and quantitative method for biochemical confirmation and calculating the fragmentation proportion during apoptosis. In this study, EA reduced DNA fragmentation, thereby reducing apoptosis caused by ROT.

ROT 1 mg/kg s.c. for 21 days was reported to not only cause progressive neurodegeneration of nigrostriatal DA neurons but also cause severe olfactory bulb deficits[25] and thereby increase oxidative stress, neuro-inflammation, and a decrease in neurotransmitters. All these features observed in rats bear a resemblance to the main characteristics of human brain disease.

In our study, repeated ROT administration significantly disturbed the locomotor activities, which is in line with the earlier studies.[19] However, treatment with EA increased the movement of the animals and muscle activity in a dose-dependent manner. PD is also found to be associated with cognitive disorders apart from motor complications. EA was also revealed to improve cognitive performance in rats with harm in memory caused by scopolamine.[4] In our study, a considerable rise in MDA levels and a significant fall in CAT, SOD, and GSH were observed in ROT-treated rats when correlated to the VC group which was in line with the study conducted earlier.[26] EA was earlier reported to have direct inhibitory effects on lipid peroxidation and induction of an antioxidant peroxisomal enzyme – CAT, as observed in this study.[11] The oxidation inflammatory cascade reaction is further triggered, resulting in apoptosis, necrosis, and finally neuronal degeneration. It was also reported that in patients with PD and in experimental animal models of PD, TNF-α, IL-1β, and IFN-γ were found in raised amounts.[27] However, EA dramatically reduced the expression of TNF-α, IL-1β, and IL-6 levels as well. EA was earlier reported to regulate neuroinflammation in AD by acting as an elastase inhibitor (IC₅₀ – 0.45 μM) and as a thrombin inhibitor (IC₅₀ – 5 μM).[7] The results of our investigation also indicated that loss of dopaminergic, cholinergic, and serotonergic neuronal degeneration may occur after 21 days after s.c. injection of ROT. Similar results were observed in the study conducted earlier.[15]

Conclusion

This is the first report indicating the role of EA as a neuroprotective and antioxidant agent using an N2a cell line and against a ROT-induced PD model in experimental rats. However, future studies can be conducted to explore the mechanisms of EA.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.