The Neuroprotective Effect of Clove Essential Oil Against 6-Ohda-Induced Cell Death in Sh-Sy5y and A Rat Model of Parkinson’s Disease

    

    

    Figure 5: The figure represents the TH immunostaining in sham (A), 6-OHDA-lesioned (B), and CEO-treated (C) animals. For cell count, data are displayed as mean ± SEM and statistical analysis was done using two-way ANOVA with additional THSD tests to handle post-hoc multiple comparisons. In the sham, TH immunostaining was localized in numerous neuronal cell bodies within bilateral SNc (arrows). A differential TH labeling was observed between the ipsilateral and the contralateral of the SNc in both 6-OHDA-lesioned rat (B) and CEO-treated (C). A very intense TH immunolabeling was present within the contralateral when compared to the ipsilateral SNc. The number of TH positive cell was counted in ipsilateral (red) and contralateral (blue) SNc (D). Between the three groups, there was no significant TH cell count difference in the contralateral SNc. Comparing ipsilateral and contralateral SNc in the 6-OHDA-lesioned and CEO-treated groups revealed a significant (p<0.0001) difference in TH cell count. When compared to the 6-OHDA-lesioned SNc, the CEO-treated ipsilateral SNc exhibit a greater number of TH-positive cells (p<0.0001). The bar represents 200μm.
    

Discussion

The potential neuroprotective efficacy of CEO was evaluated against cell toxicity caused by 6-OHDA both in vitro in SH-SY5Y and in vivo in a rat PD model. The current study is the first to demonstrate that CEO, as well as its most abundant component EG, protects SH-SY5Y cells from 6-OHDA-induced cell death in vitro. In addition, CEO treatment improves motor performance in 6-OHDA-lesioned rats, which could be linked to the neuroprotective effect of CEO on dopaminergic neurons of the SNc. This is corroborated by a higher number of TH cells present in CEO-treated compared to 6-OHDA-lesioned rats.

All used 6-OHDA concentrations (20, 40, 60, 80, and 100μM) showed a significant SH-SY5Y cell viability reduction compared to the control cell culture. The highest dose of 100μM caused the most cell damage (around 58.58±3.4% cell death). This result is in accord with previous reports demonstrating the use of 6-OHDA as a successful tool for inducing neurodegeneration in cell culture [12,13]. Therefore, the neuroprotective effects of CEO and EG against the 6-OHDA cytotoxicity were investigated using the dose of 100 μM.

SH-SY5Y were also treated with each of CEO or EG to determine their potent cytotoxic doses. At concentrations below 20 μg/ml, neither EG nor CEO had cytotoxic effects on SH-SY5Y cells. There was no significant difference in viable cell number between CEO or EG-treated and the control cultures at these concentrations. Only at the highest doses, 40μg/ml for the CEO and 80μg/ml for EG, a significant cytotoxic effect was observed. There are several inconsistencies in the literature when it comes to EG's cytotoxic (apoptosis) and anti-proliferative actions [45]. These disparities could be caused by differences in concentrations, EG purity, and cell line types. The CEO's toxicity has been linked to the compounds EG and phenolic terpene. The cytotoxic doses of EG range from 100 to 300μM, depending on the cell line culture [3,4,16]. CEO and EG toxicity have been linked to their effects on ion fluxes [6,23,36] or the intracellular synthesis of metabolic change of EG to a quinone methide [19,43].

In the present study, the neuroprotective effects of CEO and EG against 6-OHDA have been investigated in vitro at concentrations lesser than 20μg/ml. Both CEO and EG showed a significant neuroprotective effect on 6-OHDA-treated SH-SY5Y cells at doses ranging from 2.5 to 20μg/ml, with the highest effect obtained at 5μg/ml. To our knowledge, this is the first study demonstrating a protective effect of CEO and EG against 6-OHDA-induced cell death on SH-SY5Y cells. Comparative analysis between CEO and EG revealed no significant difference in cell viability. This suggests that the CEO neuroprotective effect is probably due to its major compound EG. The current study's in vitro findings on CEO and EG effects matched those of a previous study on different cell lines [37]. EG has been shown to protect PC12 cells against amyloid-induced cytotoxicity through its ability to prevent calcium intake induced by amyloid [18]. EG also protects cortical cell culture from NMDA-induced cytotoxicity through the modulation of NMDA-receptors [44].

Further research into the protective impact of CEO in rat models of PD was examined in light of the promising in vitro neuroprotectivity evidence. CEO's neuroprotective effect was tested in vivo against 6-OHDA-induced neuronal degeneration in the SNc. To investigate the impact of 6-OHDA on SNc neuronal death, a locomotor function was first assessed in 6-OHDA-lesioned and CEO-treated rats and compared to the sham group. Motor performance constitutes an important tool that could predict the degree of neuronal cell loss [27]. CEO treatment significantly enhanced rat motor performance, as indicated by both cylinder and actimetry tests one and two weeks after the 6-OHDA lesion. This is most likely due to CEO treatment's impact against 6-OHDA-induced cell death in the SNc. In addition, this is the first study demonstrating a neuroprotective effect of CEO on a rat model of PD. The findings backed up a prior study that used EG, which represents the major CEO compound in a rat model of PD [28,34]. EG has been demonstrated to improve motor performance using different motor tests [7,28,34,35]. The motor effect of EG was attributed to its ability to reduce oxidative stress and the modulation of genes related to antioxidant activity caused by 6-OHDA [28,34]. One study, however, reported that ingestion of EG after brain injury or after the onset of Parkinson's disease may increase oxidative stress and exacerbate the injury [21]. This discrepancy may be due to the EG doses, the way of administration, and the animal model used.

The CEO effect on motor performance is also supported by the presence of a high number of TH-labeled cells in the ipsilateral SNc of CEO-treated rats when compared to 6-OHDA-lesioned. TH cell count demonstrated the impact of the neurotoxin 6-OHDA on the SNc dopaminergic neurons. It is well established that 6-OHDA neurotoxic injection within the striatum causes dopaminergic neurons in the SNc to degenerate retrogradely [20]. Ipsilateral SNc in both CEO-treated and 6-OHDA-lesion rats had a significantly high TH cell number when compared to the contralateral. Indeed, within the ipsilateral SNc of the CEO-treated, the TH staining demonstrated a higher cell number than that of the 6-OHDA-lesion rats. The number of cells in the contralateral SNc side (non-lesioned side) did not differ significantly across groups, indicating that CEO had no cytotoxic effect on neurons in the non-lesioned SNc side. Overall, the findings showed that the CEO had neuroprotective effects on SNc neurons damaged by 6-OHDA.

In conclusion, the current study demonstrated that CEO protects against 6-OHDA cytotoxicity both in vitro and in vivo. Clove buds may constitute a potent compound that could be used as a food supplement to aid those at risk of Parkinson's disease.

Materials and Methods

Chemicals and Reagents

Dimethylsulfoxide (DMSO) (D8418, Sigma-Aldrich, France)

Tween 20 (P1379, Sigma-Aldrich, France)

SH-SY5Y cells (Sigma-Aldrich, France)

DMEM1X medium (31885-023, Gibco, France)

Vitamin 100X (11120-037, Gibco, France)

Non-essential amino acid 100X (11140-035, Gibco, France)

Heat inactivated FBS (F9665, Sigma-Aldrich, France)

Penicillin-streptomycin solution (P0781, Sigma-Aldrich, France)

Trypsin-EDTA (0.05%) (59417C, Sigma-Aldrich, France)

Trypan blue (0.4%) (T8154, Sigma-Aldrich, France)

Cell counting slide (C10283, Invitrogen, France)

MTT (tetrazolium salt) (M5655, Sigma-Aldrich, France)

Eugenol (E51791, Sigma-Aldrich, France)

Xylazine (ROMPUN 2%, Bayer Pharma)

Ketamine (Imalgene 1000, Merial)

Pentobarbital (Ceva Santé Animale, France)

Primary antibody (mouse anti-TH, sigma-Aldrich, France)

Secondary antibodies (anti-mouseDylight549, Vector, France)

Slides (Superfrost^® plus, Thermo scientific)

Fluorescence microscope (Nikon Eclipse Ni)

Coverslip in mounting medium (Dako Fluorescence Mounting Medium, Dako)

Extraction and Preparation of Clove Essential Oil (CEO)

CEO was hydrodistilled using Clevenger type equipment as described previously [25]. The condensed vapor transforms into an organic phase (essential oil). The CEO was extracted from the hydrolate, stripped of any leftover water, and kept in dark glass pill containers at 4°C until analysis. For the in vitro pretreatment, the CEO was dissolved in 0.1% DMSO. Intraperitoneal injectable solutions of 10mg/kg CEO [28] were made in 0.9 % saline solution diluted in 3% Tween 20 (Sigma, France) for the rats' treatment.

SH-SY5Y Cell Line Culture

SH-SY5Y cell line was purchased from Sigma-Aldrich, France. The cells were cultured in DMEM medium (31885-023, Gibco) supplemented with 1% vitamin 100X (11120-037, Gibco), 1% non-essential amino acid 100X (11140-035, Gibco), 10% heat-inactivated FBS (F9665, Sigma) and 100X penicillin-streptomycin solution (P0781, Sigma). The cells were incubated at 37°C in a humidified atmosphere with 5% CO2.

Cell Viability Test

For this analysis, 3.104 cells were seeded in 96-well plates in a complete medium and incubated at 37°C and 5% CO2. For 6-OHDA toxicity, SH-SY5Y cells were treated with 20, 40, 60, 80, and 100μM of 6-OHDA for 24 hours. Potent cytotoxicity of CEO and its major component eugenol (Sigma-Aldrich, France) was determined by treating SH-SY5Y for 24 hours with different concentrations (5, 10, 20, 40, 80μg/ml) of each drug diluted in complete media containing 0.1% DMSO (D8418, Sigma). In addition, a 0.1% DMSO-containing vehicle group was used [9,39]. To test the neuroprotective effects of CEO and EG, against 6-OHDA-induced cell death, SH-SY5Y were pretreated for one hour with each aromatic compound at varying concentrations (2.5, 5, 10, and 20μg/mL), and subsequently, cells were cultured in the presence of 6-OHDA (100μM) for an additional 24 hours. The 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (M5655, Sigma) test was employed at a dose of 0.5mg/mL to assess cell viability. At the end of treatment, the medium was replaced by 110μL of fresh complete medium containing 10μL of MTT solution (tetrazolium salt) and then incubated at 37°C. Two hours later, the medium was removed and 100μL of DMSO was added. The optical density was measured at 540 and 630nm using Biotech Epoch spectrophotometer. Viable cells were counted (percentage) by the formula [(Optic Density treated cells/Optic density control cells) x100] [33].

Animals

Charles River (L'Arbresle, France) Sprague-Dawley male rats (n=18: 176-200g) were used. The animals were kept in a controlled environment with standard feed and water accessible at all times (lighting from 7a.m. to 7p.m., 22°C). The testing followed the ethical guidelines of the International Association for the Study of Pain, as well as directions from the European Community Council and the Animal Ethics Committee at the University of Clermont Auvergne. A variety of measures was taken to minimize the number of animals and their suffering.

Stereotaxic Surgery and Treatment

The animals were random divided into three groups (n=6 per group) (Figure 1). Group 1 (Sham) the animals received aunilateral intrastriatal injection of 0.9% saline solution containing 0.02% of L-ascorbic acid into the rat striatum. In group 2 (6-OHDA-lesioned) and 3 (CEO-treated), the lesion was carried out by unilateral injection of 21μg of 6-OHDA solution dissolved in a 0.9% saline solution containing 0.02% of L-ascorbic acid into the rat striatum. The treatments were performed 3 days as pretreatment and 7 days after 6-OHDA-lesion by intraperitoneal injection (i.p). The group 2 were i.p injected with a saline solution containing Tween 20 (3%). Rats in group 3 received 10mg/kg of CEO diluted in a saline solution containing Tween 20 (3%) [24,28]. Stereotaxic surgery Moreira Vasconcelos et al., 2020 is performed 3-day after a daily i.p injection of CEO (10mg/Kg) or saline solution containing Tween 20 (3%). One hour after the third i.p injection, the rats were anesthetized with 10mg/Kg of Xylazine (ROMPUN 2%, Bayer Pharma) and 60mg/ kg of Ketamine (Imalgene 1000, Merial) administered intraperitoneally. The stereotaxic coordinates were determined according to the Paxinos and Watson, 2004 atlas at the following coordinates: + 0.8mm Anteroposterior (AP) from the bregma, ± 2.7mm laterally from the Midline (ML), -4.5mm in dorso ventral (DV), tooth bar at + 3.4mm and + 0.8mm (AP), ± 2.7mm (ML), -5.2mm (DV), tooth bar at + 3.4mm [46]. The injections were carried out at a speed of 0.5μl / min with a 10μL Hamilton syringe connected to a microinjector. At the end of the injection, the needle remains in place for 5 min then was raised by 1 mm/min [32].

Motor Performance

Actimetry test: Locomotor activity was performed in rats in weeks one and two after a stereotaxic surgery. A 15-minute test, using an infrared probe, records the rat's total movements in the CPP apparatus. The data were analyzed with the IMétronic software (Bordeaux, France). The sum of all movements was made to obtain the overall data on the locomotor activity of the rat [32].

Cylinder test: The cylinder test [40] was used to assess the movement behavior of the lesioned rat after CEO treatment and in control. A beaker (30cm high and 20cm in diameter) was used to conduct the cylinder test. At one and two weeks following the 6-OHDA lesion, the rat was video recorded for 5 minutes. Videotapes were evaluated to measure the time the animal spends on its two hind paws throughout the course of a five-minute session. The ability of the rat to stand and retain balance on its hind paws was determined by this test.

Immunohistochemistry

At the end of the behavioral tests, the animals were anesthetized with a lethal dose of pentobarbital (80mg/kg), then infused with 0.5% heparinized saline via the cardiac route, followed by a 4% paraformaldehyde solution made in a Tris Phosphate-buffered Saline (TPS) solution (0.2M). The brains were extracted and put in Phosphate-Buffered Saline (PBS) solution (0.1M). Thirty μm vibratome sections were collected in PBS. The sections were saturated in a TBS, 0.3% X100 triton, and 1% Bovine Serum Albumin (BSA) for 1 hour before being incubated overnight in a solution of primary antibody (1: 5000 mouse anti-TH, Sigma-Aldrich) at room temperature. The sections were then washed several times in PBS before being treated with secondary antibody (1: 500 anti-mouse Dylight 549, Vector) for 2 hours at room temperature. TBS containing 1% BSA and 0.3% Triton X-100 was used to dilute all antibodies. The sections were then rinsed several times in PBS before being placed on slides (Thermo Scientific's Superfrost^® plus), dried, and then mounted between slides and coverslipped in mounting medium (Dako Fluorescence Mounting Medium, Dako). Slides were examined using a fluorescent microscope (Nikon Eclipse Ni) and the TH-positive cells were counted using ImageJ software. The number of brain sections (each SNc side) used for TH count was n=29 for for sham, n=35 for 6-OHDA-lesioned, and n=39 for the CEO-treated group.

Figure 1 presents the experiment's time course.

Statistical Snalysis

Cell viability was subjected to One-way ANOVA, followed by Tukey Honestly Significant Difference (THSD) tests. The interaction plot of cell viability between CEO and EG was done using two-way ANOVA followed by THSD (results are shown in figures 1 to 3). For behavioral characteristics (actimetry and cylinder test) and immunohistochemistry (cell count), which were all considered quantitative criteria and displayed as mean ± SE (standard error mean), to test for both effects of CEO treatment and time (week 1 and 2) (including their interaction), two-way ANOVA was performed with additional THSD test to handle post-hoc multiple comparisons. These results are shown in figures 4 and 5. All statistical analysis were performed using SAS v9.4 (SAS Institute Inc., Cary, NC, USA) with a type I error set at 0.05.

Author Statements

Author Contributions

All authors contributed substantially to this study: conception and design of the study (DH, AH, OO, CM); acquisition and interpretation of data (DH, AH, OO); drafting of the article (DH, AH), revising the article critically for important intellectual content (DH, AH, CM); and final approval of the version to be submitted (DH, AH, OO, CM, HOR).

Competing Interests

The present study was funded by University Clermont Auvergne. The testing followed the ethical guidelines of the International Association for the Study of Pain, as well as directions from the European Community Council and the Animal Ethics Committee at the University of Clermont Auvergne (D6311316). A variety of measures was taken to minimize the number of animals and their suffering. The authors have no conflict of interest to declare in this work.

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