Effects of curcumin, quercetin, and their combinationson neurodegeneration and motor impairment in rotenone-induced Parkinson's disease in rats

doi:10.21203/rs.3.rs-2228676/v1

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Research Article

Effects of curcumin, quercetin, and their combinationson neurodegeneration and motor impairment in rotenone-induced Parkinson's disease in rats

https://doi.org/10.21203/rs.3.rs-2228676/v1

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Background

Parkinson’s disease (PD) is associated with motor and behavioral dysfunctions. Mitochondrial damage, increased oxidative stress, and the formation of Lewy bodies by misfolded proteins are major pathways for the development of PD. Using antioxidants could delay neurodegeneration in parkinsonism and improve the symptoms. We investigated the neuroprotective effects of quercetin, curcumin, and their combination on the rotenone-induced parkinsonism model.

Methods

PDwas induced by administration of rotenone 2mg/kg/day for 14 days in rats. Curcumin (100, 150, and 200 mg/kg), quercetin (30, 40, and 50 mg/kg),or their combination were given orally for 14 days concurrently with rotenone and for additional 14 days. On the 28th and 29th days, behavioral and histological studies were carried out.

Results

In the rotenone-induced parkinsonism model, curcumin and quercetin dose-dependently improved locomotor activity, motor coordination, and mobility. Also, they increased dopamine levels and mitigated the neural damage induced by rotenone.

Conclusion

Both curcumin and quercetin have neuroprotective effects against parkinsonism. The combination of curcumin and quercetin has more favorable effects than usingeither drug alone.

Parkinson’s disease

curcumin

quercetin

behavioral activity

motor activity

dopamine

Parkinson’s disease (PD), the second most prevalent neurologicaldisease[1],was first described in 1817[2].It affects 1% of people older than 65; however, it can occur before 40. The latter type is known as early-onset PD,representing about 5% of cases.The global burden of PD is increasing[3], with global estimates of 12 million cases in 2040[4].

Many etiologic factors contribute to the development of PD, including aging, exposure to heavy metals or pesticides, genetic factors, and, most recently, severe acute respiratory infection with coronavirus-2 (SARS-CoV-2) [5–8]. Increased oxidative stress and inflammatory mediators in SARS-CoV-2 infection induce neurodegeneration and initiate PD pathogenesis. Further, SARS-CoV-2 increases α-synuclein protein levels and thus accelerates the formation of Lewy bodies, a prominent pathological feature of PD[8, 9]. On the other hand, parkinsonism-mediated rigidity of respiratory muscles and loss of cough reflex increases the risk of hospitalization and mortality by COVID-19 respiratory infection [10, 11].

PDhighly affects the life of the patients; it is characterized by progressive motor disorders that range from bradykinesia, resting tremors,and musclerigidity to disability and postural instability[12]. Further, non-motor symptoms, including rapid eye movement disorders, anosmia, anxiety, depression, and autonomic dysfunctions such as constipation, urine retention, and orthostatic hypotension, may appear years earlier than the motor symptoms as features of the prodromal stage of PD[13, 14].

PD is characterized by dopaminergic neuronal cell damage in the region of substantia nigra pars compacta (SNpc). When dopamine levels in the basal ganglia decline by 70%, motor dysfunction appears [15]. However, the levels of other neurotransmitters such as noradrenaline, serotonin, and acetylcholine also change in PD, which may explain many of the manifestations associated with PD, including depression, cognitive impairment, and psychosis[16]. Based on the Braak hypothesis[17], the neuronal damage in PD follows an ascending pattern that starts in the brain medulla and the olfactory bulb. This stage occurs early in the pathology of PD with no motor symptoms. However, sleep disorders and loss of smell are the main manifestations. Then, the neuronal damage goeshigher and affects the SNpc and midbrain. In this stage, patients develop motor disorders. Finally, in advanced PD, the damage affects the cortical brain structures; thus, it represents the cognitive impairment stage[18].

The pathological factors involved in PD includedysfunction of mitochondrial complex 1, oxidative damage of neuronal cells, and the accumulation of Lewy bodies in the neuronal cell bodies. Lewy bodies are aggregates of misfolded α-synuclein and ubiquitin proteins, lipids, and other materials[19]. Other factors include neuronal inflammation, apoptosis, and accumulation of dysfunctional proteins due to defective clearance by the proteasome and autophagy systems [20–22].

Most drugs used to treat PD depend on substituting dopamine and other neurotransmitters to improve the symptoms. These drugs do not delay the neurodegenerative changes and have many adverse effects[23].However, using natural antioxidants and anti-inflammatory drugs may delay the progressive neurodegeneration in PD with the added safety benefit. Curcumin, a natural polyphenol extracted from turmeric roots, is widely used in cooking, cosmetics, coloring agents, and medicines. Accumulating research revealed curcumin's therapeutic potential in managing diabetes, cardiovascular disorders, bacterial infections, hyperlipidemia, and cancer[24, 25]. The high lipophilicity and the ability of curcumin to penetrate the blood-brain barrier allowits delivery tothe central nervous system. It has shownneuroprotective effects indifferent models, includingPD, Alzheimer's disease, and stroke[26–28]. Another antioxidant that possesses neuroprotective effects is quercetin,a flavonoid extracted from leaves, flowers, and fruits of several plants, which hasmultiple beneficial effects against cancer, diabetes, and inflammation[29]. Quercetin decreases neuronal cell degeneration by attenuatinginflammatory cytokine activation and reactive oxygen speciesproduction[30, 31].

In order to understand the disease pathogenesis, many neurotoxinsare used for the induction of parkinsonism,includingmethyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), and rotenone[32]. Rotenone, a natural isoflavone, is used as a pesticide.It is used for induction of an experimental parkinsonism model that mimics that in humans through the slow neurodegeneration and the presence of Lewy bodies inclusions in the brain[33].While the role of curcumin and quercetin against PD has been studied previously, in the present study, we studied the neuroprotective effects of different doses of either curcumin or quercetin their combinations against the rotenone-induced parkinsonism model. Also, we aimed to investigate the possible pathways mediating their neuroprotective effects.

2.1. Drugs and chemicals

Rotenone, amantadine, and quercetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pure salt of curcumin was obtainedas a gift sample from Ind-Swift Ltd (Chandigarh, India). All the chemicals employed in this research were of analytical grade.

2.2. Animals

Adult Sprague Dawley male rats (180–200 g) were left for seven days to acclimatize, housed under standard conditions of temperature (24–28°C) and relative humidity (60–70%) with a 12:12 light-dark cycle, and fed with standard pellet diet (Lipton IndiaLtd, Kolkata, India) and water ad libitum.All the experimental procedures were performed following the guidelines of the Institutional Animal Ethics Committee of M.M. College of Pharmacy, Mullana, Ambala, Haryana, India (MMCP/IAEC/65).

2.3. Preparation of drugs

Rotenone was dissolved in dimethyl sulfoxide and polyethylene glycol (1:1). Quercetin and amantadine were prepared in suspension form. Curcumin is highly hydrophobic; thus, to increase its bioavailability, curcumin was dissolved in milk (a food vehicle for hydrophobic substances) [34]. To prepare curcumin/milk emulsion, 32 g of curcumin was added to 250 ml of milk and then stirred for 5 min to obtain the final concentration of 128 mg/ml. Then the preparation was boiled and transferred to a glass bottle to cool[35].

2.4. Induction of PDand experimental design

Induction of PDwas done via using rotenone (2 mg/kg, i.p) for 14 days[36].On the 15th day, rats startedamantadine treatment, which continued for 14 days. Oral treatments with curcumin (100, 150, and 200 mg/kg), quercetin (30, 40, and 50 mg/kg) andtheircombinations, including: curcumin (100 mg/kg) + quercetin (30 mg/kg), curcumin (150 mg/kg) + quercetin (40 mg/kg) and curcumin (200 mg/kg) + quercetin (50 mg/kg),were given concurrently with rotenone for 14 days and continued for another 14 days after the discontinuation of rotenone. The whole period of treatment was 28 days.

2.5. Experimental groups

Animals were randomly divided into 12 groups (n = 6each).Group 1: Control; ratsreceived the vehicle of rotenone. Group 2: Rotenone (2 mg/kg, i.p for 14 days). Group 3: Rotenone (2 mg/kg, i.p for 14 days), then amantadine (10 mg/kg,p.o) 14 days. Group 4: Rotenone (2 mg/kg, i.p for 14 days) + curcumin (100 mg/kg,p.o) for 28 days. Group5: Rotenone (2 mg/kg, i.p for 14 days) + curcumin (150 mg/kg,p.o) for 28 days. Group 6: Rotenone (2 mg/kg, i.p for 14 days) + curcumin (200 mg/kg,p.o) for 28 days. Group 7: Rotenone (2 mg/kg, i.p for 14 days) + quercetin (30 mg/kg,p.o) for 28 days. Group 8: Rotenone (2 mg/kg, i.p for 14 days) + quercetin (40 mg/kg,p.o) for 28 days. Group 9: Rotenone (2 mg/kg, i.p for 14 days) + quercetin (50 mg/kg,p.o) for 28 days. Group10: Rotenone (2 mg/kg, i.p for 14 days) + combination of curcumin (100 mg/kg,p.o) + quercetin (30 mg/kg,p.o) for 28 days. Group 11: Rotenone (2 mg/kg, i.p for 14 days) + combination of curcumin (150 mg/kg p.o) + quercetin (40 mg/kg,p.o) for 28 days. Group12: Rotenone (2 mg/kg, i.p for 14 days) + combination of curcumin (200 mg/kg,p.o) + quercetin (50mg/kg,p.o) for 28 days.

2.6. Determination ofbody weight

Every rat's body weight was checked weekly, and the change during the research experiment was recorded.

2.7. Assessment of behavioral parameters

All behavioral analyses were performed by an observer blinded to theexperimental design. The following parameters were assessed on the 0th, 7th, 21st, and 28th day of the experiment.

2.7.1. Assessment of locomotor activity

Based on previous studies, total locomotor activity (ambulations and rearing) was determined using a computerized actophotometer. Rats were allowed to acclimatize for 2 min in the actophotometerchamber then motor activity was determined over 5min. Thenumber of crossings by the animal was checked and automatically recorded by the apparatus. Locomotion was expressed as the total photo beam counts per 5 min per animal.The activity chamber was swabbed with 10% alcohol every time to avoid interference due to animal odors[37].

2.7.2. Rota rod test (muscular coordination)

Rats were trained for this test for seven days beforestarting the experiment to reach a stable performance. This test aims to assess motor coordination and balance in animals. Ratswere mounted on a rod that rotated at different speeds. The rod has four compartments, so four rats simultaneously undergo this testing. The animals were placed in every compartment and rotated at a speed of 10 rpm. The time latency to fall from the rotating rod was recorded in seconds. The cut-off time for this test was 180 s. The test period was videotaped for all the animals [38].

2.7.3. Grid test

The grid test allows the evaluation of the grip or the muscle strength in rats. Rats were placed on the horizontal mesh, and locomotor behaviors like grasping strength and rearing events were visually observed. The cut-off time set for this test was 3 min[39].

2.7.4. Open-field test

This test assesses motor activity. Each rat was placed in the center of an open-field apparatus (a circular wooden box with a diameter of 72×72 cm with 36 cm height, with a floor divided into 16 regions). Rats were assessed individually for 5 min. Rearing was determined by the number of times the rat stood on their hind paws. The apparatus was cleared with 10% ethanol before behavioral testing to eliminate possible bias due to odors left by the previous rat[40].

2.7.5. Footprint test

The footprint test was carried out to demonstrate the change in the animal’s gait. Gait is the movement pattern of the limbs in rats and is changed in parkinsonism due to motor dysfunctions. The apparatus consists of a runway of 50–60 cm. The paws of each rat were painted with non-toxic paint then rats were allowed to walk on the narrow runway on which white paper was placed to take the footprints[41, 42]. The test period was videotaped. The apparatus was swiped with 10% ethanol before each experiment.

2.8.Estimation of dopamine levels

After the end of the experiment, dissected striata were weighed and homogenized (1:10, w/v) in 0.1 M perchloric acid with an ultrasonic disintegrator. The homogenates were centrifuged for 15 min at 15000g at 4°C, and supernatants were filtered and diluted (1:1) with 0.1 M perchloric acid. Separation and quantification of dopamine were done using a high-performance liquid chromatography (HPLC) system coupled with an electrochemical detector. Samples were separated on an ODS-C18 column using a flow rate of 1.0 ml/min. The column was maintained at 35°C. The mobile phase consisted of 0.1 M potassium phosphate (pH 4.0), 10% methanol, and 1.0 mMof 1-heptanesulfonic acid. The dopamine concentrationwas calculated using a standard curve generated by determining the ratio between known amounts of the amine and a constant amount of internal standard and represented as ng/mg of tissue[37, 43].

2.9. Histopathological analysis

After decapitation, brain tissues were rapidly removed, fixed in 10% buffered formalin,and then embedded in paraffin. Cutsof 4-µm thickness were taken at brain regions containing substantia nigra. Slides were stained using hematoxylin and eosin (H&E) for histopathological evaluation.

2.10. Statistical analysis

The data are expressed as mean ± standard deviation and statistically analyzed bythe SPSS program version 16.0 (SPSS Inc., Chicago, IL, USA).The statistical significances between means were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Multivariate analysis was done with the posthocBonferroni test. A p < 0.05 was considered statistically significant.

3.1. Effect of curcumin,quercetin, and their combination on rotenone-induced change in the body weight

The body weight was determined weekly, starting from the first day of the experiment till the end of the experiment (at 28 days). When compared with the control group, administration of rotenone (2 mg/kg) for 14 days resulted ina gradual decrease in the body weight throughout the experiment, but it was significantly (p < 0.01) decreasedon the 28th day of the experiment (Table 1). Treatment with amantadine (the standard anti-parkinsonism drug) abrogated this decline in body weight (Table 1).Treatment with curcumin, quercetin, and their combinations at different doses prevented the rotenone-induced decrease in body weight.The body weight at 28 days in thesetreated groups was significantly increased when compared to the untreated rotenone group.

Table 1

Effect of curcumin, quercetin, and their combination treatment on body weight (g) at different intervals in rotenone-induced Parkinson’s disease rat model.
Groups	0 day	7th day	21st day	28th day
Control	265 ± 16.4	266 ± 15.2	263 ± 9.81	261 ± 11.7
Rotenone	260 ± 1.50	253 ± 6.30	244 ± 18.4	235 ± 19.7#
Amantadine	263 ± 2.04	260 ± 7.70	263 ± 1.76	266 ± 6.63**
Curcumin (100 mg/kg)	271 ± 1.21	263 ± 14.4	261 ± 14.0	261 ± 13.5*
Curcumin (150 mg/kg)	263 ± 1.83	256 ± 12.0	261 ± 1.87	261 ± 3.86*
Curcumin (200 mg/kg)	263 ± 24.0	256 ± 29.0	261 ± 15.9	266 ± 17.8**
Quercetin (30 mg/kg)	265 ± 15.3	255 ± 26.2	261 ± 9.99	267 ± 6.05**
Quercetin (40 mg/kg)	265 ± 16.0	254 ± 12.8	252 ± 7.78	252 ± 8.13
Quercetin (50 mg/kg)	266 ± 0.81	260 ± 13.5	265 ± 1.47*	266 ± 2.13**
Curcumin (100 mg/kg) + Quercetin (30 mg/kg)	268 ± 1.21	257 ± 22.1	265 ± 2.25*	264 ± 3.44**
Curcumin (150 mg/kg) + Quercetin (40 mg/kg)	263 ± 24.2	257 ± 27.8	266 ± 6.81*	266 ± 16.5**
Curcumin (200 mg/kg) + Quercetin (50 mg/kg)	260 ± 3.54	252 ± 20.1	260 ± 5.77	268 ± 13.2***
Effect of curcumin (100, 150, and 200 mg/kg, p.o), quercetin (30, 40, and 50 mg/kg, p.o), and the combinations (curcumin 100 mg/kg + quercetin 30 mg/kg), (curcumin 150 mg/kg + quercetin 40 mg/kg), and (curcumin 200 mg/kg + quercetin 50 mg/kg) on the body weight of rats lesioned with 14 days injection with rotenone (2 mg/kg/day). Data are expressed as mean ± SD (n = 6); #: Significantly different from control group at p < 0.05; ,,**: Significantly different from disease control rotenone group at p < 0.05, p < 0.01, and p < 0.001, respectively.

3.2. Effect of curcumin,quercetin, and their combination on the rotenone-induced decrease in locomotor activity

Compared with control rats, administration of rotenone caused a significant (p < 0.001) progressive decline in the motor activity in rats starting from the 7th day of rotenone injection until the end of the experiment.The lowest motor activitywas observed on the 28th day of the experiment in the untreated rotenone group (Table 2). Amantadine treatment prevented the rotenone-induced decline in motor activity at different time intervals (Table 2).On the 28th day of the experiment, treatment with curcumin, quercetin, and their combinationsat different dosessignificantly increased locomotor activity. However, the groups treated with quercetin (50 mg/kg) and the combination of curcumin and quercetin at medium and higher doses showed noticeable improvements in motor activity than the other treated groups. The combination of the high-dose curcumin and quercetin significantly (p < 0.05) improved the locomotor activity when compared to the combination of curcumin + quercetin at the low dose, and significantly(p < 0.001) improved the motor activity in comparison with the group given the high-dose curcumin only. On 21th day of the experiment, compared to the untreated rotenone group, the groups treated with low doses of either curcumin (100 mg/kg) or quercetin (30 mg/kg) did not show a significant increase in locomotor activity (Table 2). The combination of high dose curcumin (200 mg/kg) + high dose quercetin (50 mg/kg), however, significantly (p < 0.001) improved the motor activity measured on days 7, 21, and 28 of the experiment when compared with the untreated rotenone group.

Table 2

Effect of curcumin, quercetin, and their combination treatment on locomotor activity monitored at different intervals in rotenone-induced Parkinson’s disease rat model.
Groups	0 day	7th day	21st day	28th day
Control	163 ± 14.3	163 ± 12.1	168 ± 18.8	166 ± 15.3
Rotenone	169 ± 19.8	120 ± 6.53#	105 ± 10.9#	93.7 ± 15.5#
Amantadine	167 ± 28.3	131 ± 10.7*	135 ± 14.9**	141 ± 4.62***
Curcumin (100 mg/kg)	161 ± 26.2	119 ± 14.8	126 ± 3.32	135 ± 17.5***
Curcumin (150 mg/kg)	164 ± 13.2	121 ± 24.5	134 ± 5.38*	137 ± 5.58***
Curcumin (200 mg/kg)	161 ± 8.80	128 ± 21.6	133 ± 9.39*	138 ± 6.17***$
Quercetin (30 mg/kg)	162 ± 15.5	117 ± 20.9	129 ± 11.4	134 ± 8.24***
Quercetin (40 mg/kg)	167 ± 95.5	125 ± 12.7	132 ± 16.4*	138 ± 6.08***
Quercetin (50 mg/kg)	163 ± 51.7	135 ± 12.7	142 ± 9.26***	153 ± 8.47***
Curcumin (100 mg/kg) + Quercetin (30 mg/kg)	161 ± 74.9	129 ± 5.34	132 ± 23.4*	133 ± 18.2***$
Curcumin (150 mg/kg) + Quercetin (40 mg/kg)	162 ± 56.2	132 ± 9.01	142 ± 8.08***	151 ± 15.1***
Curcumin (200 mg/kg) + Quercetin (50 mg/kg)	161 ± 31.6	151 ± 18.0***	151 ± 5.89***	166 ± 10.5***
Effect of curcumin (100, 150, and 200 mg/kg, p.o), quercetin (30, 40, and 50 mg/kg, p.o), and the combinations (curcumin 100 mg/kg + quercetin 30 mg/kg), (curcumin 150 mg/kg + quercetin 40 mg/kg), and (curcumin 200 mg/kg + quercetin 50 mg/kg) on the locomotor activity of rats lesioned with 14 days injection with rotenone (2 mg/kg/day). Data are expressed as mean ± SD (n = 6); #: Significantly different from control group at p < 0.001; ,,**: Significantly different from disease control rotenone group at p < 0.05, p < 0.01, and p < 0.001, respectively; $: Significantly different from curcumin 200 mg/kg + quercetin 50 mg/kg group.

3.3. Effect of curcumin,quercetin, and their combination on rotenone-induced change in motor coordination

The rota-rod test was used for the assessment of motor coordination. The latency time forfallingfroma rotating rodduring a 120-s trial was estimatedweekly.Administration of rotenone caused a progressive decline in the latency time for falling and thus a decrease in motor coordination and stability, which started onday 7 of the experiment. At the end of the experiment, motor coordination significantly (p < 0.001) declined in the rotenone group when compared with the control group (Table 3). Amantadine administration prevented the rotenone-induced motor coordination decline(Table 3).Treatment with curcumin and quercetin showed a dose-dependent improvement in motor coordination. At day 21 of the experiment, administration of curcumin (150 mg/kg), curcumin (200 mg/kg), quercetin (50 mg/kg),and the combination of curcumin 200 mg/kg + quercetin 50 mg/kg significantly increased the latency time of falling(Table 3). At the end of the experiment, treatment with the three different doses of curcumin, quercetin, or their combinationssignificantly abrogated the rotenone-induced motor dysfunction and increased the latency time for falling from the rotating rod(Table 3).

Table 3

Effect of treatment with curcumin, quercetin, and their combination on falling time (seconds) at different intervals in rotenone-induced Parkinson’s disease rat model.
Groups	0 day	7th day	21st day	28th day
Control	8.33 ± 1.03	8.5 ± 1.04	8.0 ± 0.89	8.50 ± 1.04
Rotenone	8.66 ± 1.03	5.33 ± 1.21	3.50 ± 1.04#	1.50 ± 0.83#
Amantadine	8.33 ± 0.81	6.5 ± 0.83	6.50 ± 1.04**	7.83 ± 0.98***
Curcumin (100 mg/kg)	8.0 ± 1.26	5.66 ± 1.21	5.50 ± 0.54	5.83 ± 0.75***
Curcumin (150 mg/kg)	9.5 ± 1.04	5.83 ± 1.94	6.0 ± 1.41*	6.33 ± 1.36***
Curcumin (200 mg/kg)	8.16 ± 1.16	6.0 ± 2.28	6.50 ± 0.83**	6.83 ± 1.16***
Quercetin (30 mg/kg)	9.5 ± 1.04	5.0 ± 1.26	4.16 ± 1.72	4.33 ± 0.81**
Quercetin (40 mg/kg)	8.33 ± 1.21	5.50 ± 1.51	5.83 ± 0.75	6.0 ± 1.09***
Quercetin (50 mg/kg)	9.16 ± 1.47	5.66 ± 1.50	6.16 ± 1.47*	6.66 ± 1.86***
Curcumin (100 mg/kg) + Quercetin (30 mg/kg)	8.5 ± 1.04	5.50 ± 1.04	5.16 ± 0.75	5.0 ± 0.89***
Curcumin (150 mg/kg) + Quercetin (40 mg/kg)	8.66 ± 0.81	5.66 ± 1.21	5.83 ± 1.32	6.50 ± 0.83***
Curcumin (200 mg/kg) + Quercetin (50 mg/kg)	8.83 ± 1.47	6.0 ± 1.09	6.66 ± 1.75**	7.66 ± 1.50***
Effect of curcumin (100, 150, and 200 mg/kg, p.o), quercetin (30, 40, and 50 mg/kg, p.o), and the combinations (curcumin 100 mg/kg + quercetin 30 mg/kg), (curcumin 150 mg/kg + quercetin 40 mg/kg), and (curcumin 200 mg/kg + quercetin 50 mg/kg) on falling time of the rods of rats lesioned with 14 days injection with rotenone (2 mg/kg/day). Data are expressed as mean ± SD (n = 6); #: Significantly different from control group at p < 0.001; ,,**: Significantly different from disease control rotenone group at p < 0.05, p < 0.01, and p < 0.001, respectively.

3.4. Effect of curcumin,quercetin, and their combination on rotenone-induced change in muscle strength

Compared with the control group, rotenonesignificantly decreased muscle strength starting from day seven of the experiment with a continuous decline till day 28 of the experiment (Table 4). Treatment with different doses of curcumin,quercetin, or their combination significantly abrogated the effects of rotenone observed on days 21 and 28 of the experiment in a dose-dependent manner.

Table 4

Effect of curcumin, quercetin, and their combination treatment on muscle strength monitored at different intervals in rotenone-induced Parkinson’s disease rat model.
Groups	0 day	7th day	21st day	28th day
Control	179 ± 1.04	176 ± 3.60	175 ± 2.80	178 ± 1.03
Rotenone	178 ± 0.75	71.2 ± 15.7#	29.5 ± 2.25#	15.3 ± 3.77#
Amantadine	178 ± 1.21	132 ± 13.8***	155 ± 13.0***	172 ± 2.33***
Curcumin (100 mg/kg)	179 ± 0.51	52.8 ± 1.94	58.0 ± 1.54**	62.3 ± 2.33***
Curcumin (150 mg/kg)	178 ± 1.36	81.3 ± 1.63	86.0 ± 1.54***	111 ± 17.1***
Curcumin (200 mg/kg)	178 ± 1.51	95.5 ± 21.6*	128 ± 5.70***	161 ± 1.75***
Quercetin (30 mg/kg)	178 ± 1.54	50.3 ± 4.13	52.8 ± 10.8**	61.2 ± 9.41***
Quercetin (40 mg/kg)	178 ± 0.98	74.5 ± 20.8	86.3 ± 22.1***	111 ± 16.2***
Quercetin (50 mg/kg)	178 ± 0.98	71.2 ± 47.5	95.5 ± 52.4***	126 ± 8.36***
Curcumin (100 mg/kg) + Quercetin (30 mg/kg)	178 ± 1.47	57.8 ± 8.10	66.5 ± 17.8***	91.7 ± 17.6***
Curcumin (150 mg/kg) + Quercetin (40 mg/kg)	177 ± 1.50	85.0 ± 27.0	99.3 ± 32.1***	124 ± 13.8***
Curcumin (200 mg/kg) + Quercetin (50 mg/kg)	179 ± 1.04	96.3 ± 27.7*	175 ± 2.80***	178 ± 1.03***
Effect of curcumin (100, 150, and 200 mg/kg, p.o), quercetin (30, 40, and 50 mg/kg, p.o), and the combinations (curcumin 100 mg/kg + quercetin 30 mg/kg), (curcumin 150 mg/kg + quercetin 40 mg/kg), and (curcumin 200 mg/kg + quercetin 50 mg/kg) on muscle strength of rats lesioned with 14 days injection with rotenone (2 mg/kg/day). Data are expressed as mean ± SD (n = 6); #: Significantly different from control group at p < 0.001; ,,**: Significantly different from disease control rotenone group at p < 0.05, p < 0.01, and p < 0.001, respectively.

3.5. Effect of curcumin,quercetin, and their combination on rotenone-induced change in rearing behavior

A significant (p < 0.001) decrease in the rearing behavior indicative of motor activity was observed in therotenone-treated group from day 7 to day 28 of the experiment. On days 21 and 28 of the experiment, the decrease in mobility was significantly abrogated by the treatment with amantadine, curcumin (100, 150, and 200 mg/kg), quercetin (30, 40, and 50 mg/kg), and its combination of curcumin at low, medium and high doses (Table 5). However, groups treated with curcumin (200 mg/kg), quercetin (40 and 50 mg/kg), and the combination of curcumin at low, medium, and high doses improved rat mobility significantly on day 7 of the experiment (Table 5).

Table 5

Effect of curcumin, quercetin, and their combination treatment on muscle mobility at different intervals in rotenone-induced Parkinson’s disease rat model.
Groups	0 day	7th day	21st day	28th day
Control	178 ± 1.16	176 ± 2.89	175 ± 4.35	177 ± 2.19
Rotenone	176 ± 1.75	56.7 ± 2.42#	29.7 ± 2.58#	15.3 ± 3.20#
Amantadine	176 ± 3.38	102 ± 46.1***	142 ± 14.9***	173 ± 1.16***
Curcumin (100 mg/kg)	177 ± 1.96	63.5 ± 2.16	72.7 ± 1.21***	80.3 ± 1.63***
Curcumin (150 mg/kg)	178 ± 1.86	85.3 ± 2.65	97.0 ± 2.96***	102 ± 1.03***
Curcumin (200 mg/kg)	177 ± 2.48	91.7 ± 22.1*	135 ± 24.6***	165 ± 4.80***$
Quercetin (30 mg/kg)	176 ± 1.47	70.7 ± 2.73	92.8 ± 0.75***	98.7 ± 1.03***
Quercetin (40 mg/kg)	177 ± 2.40	93.0 ± 1.41*	99.8 ± 1.47***	108 ± 1.16***
Quercetin (50 mg/kg)	176 ± 2.13	107 ± 13.9***	132 ± 18.7***	140 ± 13.6***$
Curcumin (100 mg/kg) + Quercetin (30 mg/kg)	179 ± 1.67	103 ± 2.63***	109 ± 1.36***	114 ± 2.31***
Curcumin (150 mg/kg) + Quercetin (40 mg/kg)	178 ± 1.36	1201 ± 1.47***	129 ± 2.0***	139 ± 1.22***$
Curcumin (200 mg/kg) + Quercetin (50 mg/kg)	178 ± 1.75	147 ± 17.3***	163 ± 12.5***	177 ± 1.16***$
Effect of curcumin (100, 150, and 200 mg/kg, p.o), quercetin (30, 40, and 50 mg/kg, p.o), and the combinations (curcumin 100 mg/kg + quercetin 30 mg/kg), (curcumin 150 mg/kg + quercetin 40 mg/kg), and (curcumin 200 mg/kg + quercetin 50 mg/kg) on mobility (rearing behavior) of rats lesioned with 14 days injection with rotenone (2 mg/kg/day). Data are expressed as mean ± SD (n = 6); #: Significantly different from control group at p < 0.001; ,**: Significantly different from disease control rotenone group at p < 0.05, and p < 0.001, respectively; $: Significantly different from curcumin 200 mg/kg + quercetin 50 mg/kg group.

3.6. Effect of curcumin,quercetin, and their combination on rotenone-induced change in the walking pattern

The footprint test determines the effect of rotenone on the gait or walking pattern of rats. On experiment day 21, compared with the control group, administration of rotenone impaired the walking pattern manifested as the inability of the rat to move more than two steps due to motor abnormalities (Fig. 1), which was even worse on day 28 of the experiment (Fig. 2). The use of amantadine abrogated this abnormality and improved the walking pattern (Figs. 1 and 2). The other treated groups showed improper double impressions of the fore and hind limbs (Fig. 1) on day 21. On day 28, however, the high doses of curcumin,quercetin, and their combination improved the walking pattern compared to the untreated rotenone group (Fig. 2).

3.7. Effect of curcumin,quercetin, and their combination on the rotenone-induced decrease in striatal dopamine levels

At the end of the experiment, levels of dopamine in striatal homogenates obtained from all groups were determined using HPLC-ECD. A significant (p < 0.01) decline in dopamine levels was observed in rotenone treated group (Table 6). Administration of amantadine significantly (p < 0.001) mitigated the rotenone-induced decline in dopamine levels (Table 6). On the other hand, treatment with curcumin and quercetin showed dose-dependent increases in dopamine levels;however, only the high doses of either curcumin (200 mg/kg) or quercetin (50 mg/kg) significantly(p < 0.05) increased the dopamine levels as compared with the rotenone-treated group (Table 6). Furthermore, the combination of 200 mg/kg of curcumin and 50 mg/kgof quercetin was the only combination that showed a significant (p < 0.001) elevation in dopamine levels when compared to the rotenone-treated group (Table 6).

Table 6

Effect of curcumin, quercetin, and their combination treatment on striatal dopamine levels in rotenone-induced Parkinson’s disease rat model.
Groups	Dopamine levels (ng/g)
Control	8.04 ± 0.91
Rotenone	4.25 ± 0.28#
Amantadine	7.10 ± 0.86***
Curcumin (100 mg/kg)	4.56 ± 0.27
Curcumin (150 mg/kg)	4.99 ± 0.44
Curcumin (200 mg/kg)	5.77 ± 0.62*
Quercetin (30 mg/kg)	4.41 ± 0.50
Quercetin (40 mg/kg)	5.21 ± 0.73
Quercetin (50 mg/kg)	5.90 ± 0.82*
Curcumin (100 mg/kg) + Quercetin (30 mg/kg)	4.34 ± 0.30
Curcumin (150 mg/kg) + Quercetin (40 mg/kg)	5.01 ± 0.29
Curcumin (200 mg/kg) + Quercetin (50 mg/kg)	6.78 ± 0.86***
Effect of curcumin (100, 150, and 200 mg/kg, p.o), quercetin (30, 40, and 50 mg/kg, p.o), and the combinations (curcumin 100 mg/kg + quercetin 30 mg/kg), (curcumin 150 mg/kg + quercetin 40 mg/kg), and (curcumin 200 mg/kg + quercetin 50 mg/kg) on striatal dopamine levels of rats lesioned with 14 days injection with rotenone (2 mg/kg/day). Data are expressed as mean ± SD (n = 6); #: Significantly different from control group at p < 0.01; ,**: Significantly different from disease control rotenone group at p < 0.05, and p < 0.001, respectively.

3.8. Effect of curcumin,quercetin, and their combination on rotenone-induced brain histopathological changes

The histopathological analysis of substantia nigrarevealed that treatment with rotenone showed degeneration of neurons, shrinkage, and changes in structure compared with the control group. On the other hand, curcumin (100 mg/kg), quercetin (30 mg/kg),and their combination showed mild restoration of neurons and mildimprovement of histopathological changes as compared with the rotenone group (Fig. 3). Curcumin (150 mg/kg and 200 mg/kg) and the combination of curcumin (200 mg/kg) + quercetin (50 mg/kg)showed marked improvement in the rotenone-induced histopathological changes (Fig. 3).

The chronic progressive decline in motor function in PD manifests when 70% of dopaminergic neurons in the substantia nigra are damaged. In the present study, using the natural antioxidants curcumin and quercetin protected against motor dysfunction and increased brain dopamine levels in the rotenone-induced rat model of parkinsonism. Rotenone administration is widely used to induce experimental parkinsonism[44, 45]. Chronic intraperitoneal injection of rotenone (2 mg/kg /day) in rats for 14 days reproduced many Parkinson-associated changes in the current study. Rotenone administration resulted in a gradual decline in body weight that was more prominent onthe 21st and 28th days of the experiment. Weight loss in parkinsonism is associated with gastrointestinal disorders, including early satiety, constipation, bloating, and dysphagia resulting from abnormalgastrointestinal motility. These symptoms frequently precede parkinsonism-induced motor dysfunction [46, 47]. Further, rotenone has been shown to induce neurodegeneration of the myenteric intestinal plexus leading to abnormal gastrointestinal motility and delayed gastric emptying, which induces weight loss [48]. Treatment with the standard antiparkinsonian drug, amantadine, prevented the decline in body weight. On the other hand, concurrent administration of curcumin,quercetin, or their combinations, along with rotenone, for 14 days and two weeks after its discontinuation prevented the rotenone-induced decline in body weight.

Rotenone induced behavioral and motor dysfunctions starting on the 7th day of the experiment and increased till the 28th day. Rotenone progressivelydecreased locomotion, motor coordination, muscle strength, and rearing in rats. Further, the results of the footprint test have shown Parkinson-like changes in the walking pattern. These motor dysfunctions are characteristics of parkinsonismthat were significantly abrogated by the standard antiparkinsonian drug, amantadine, which is in line with previous findings [49–51]. Rotenone isa highly lipid-soluble neurotoxin that passes rapidly through the blood-brain barrier and induces dopaminergic neuronal cell damage selectively in the nigrostriatum. The pathogenesis of parkinsonisminvolves mitochondrial dysfunction, particularly mitochondrial complex-1, leading to depletion of brain ATP molecules, increasedreactive oxygen speciesproduction, and celldeath[40, 52]. Also, rotenone increases oxidative stress due to glial cell activation[53, 54].Oxidative brain damage stimulates lipid peroxidation, protein destruction, DNA damage, and inflammatory and apoptotic pathways that eventually cause neuronal cell death, particularly since the brain has low antioxidant enzyme levels and is highly liable to oxidative damage[55].Further, the oxidation of α-synucleinprotein formsan insoluble form that aggregates and aids in the formation of Lewy bodies, the pathological hallmark of parkinsonism[56].

In the present study, in agreement with others,the use of polyphenolic antioxidants such as curcumin or quercetin showed promising improvementsin rotenone-induced behavioral and motor impairments[57]. Their neuroprotective effects were dose-dependent andwere significant over the last week (days 21 and 28) of the experiment. The footprint test results showed that the combination of curcumin and quercetin improved the walking pattern on the 28th day of the experiment. Importantly, the high dose combination of curcumin 200 mg/kg and quercetin 50 mg/kg has shown more beneficial effects; it almost retained the motor activity in rats lesioned with rotenone to the normal levels, likely due to its antioxidant activitymediating the neuroprotection against the rotenone-induced cellular damage.

Besides the protective antioxidant effects, curcumin showed inhibitory effects against the formation of α-synucleinaggregates, apoptosis, and inflammation, which enhances cell survival and abrogates neuroregenerationin different neuropathologies, including stroke and Alzheimer's disease [58, 59]. Additionally, the quercetin-mediated neuroprotective effects are attributed to its antioxidant effects andautophagy stimulation, representing a protein clearance system that helps remove misfolded proteins and inhibit their aggregation[60–62].

Increased dopamine levels are essential in improving parkinsonism's motor and behavioral deficits. Parallel to the improvement of motor functions, curcumin,quercetin, and their combination could preserve the neurons in the nigrostaiatum, which was evident by the improvement of histopathological changes induced by rotenone. Interestingly, the high doses of curcumin, quercetin, or their combination abrogated the rotenone-induced decrease in striatal dopamine levels [63, 64]. The increase in dopamine levels by curcumin or quercetin is likely attributed to neuronal cell protection and the preservation of dopaminergic neurons.

In conclusion, curcumin and quercetin have promising therapeutic potential by improving the motor and behavioral dysfunctions in the rotenone-induced parkinsonism model in a dose-based manner. Their antioxidant effects could protect against neuronal damage and preserve dopaminergic neurons, which may help delay the disease progression. The combination of curcumin and quercetin showed more beneficial neuroprotective effects than either drug alone.

Ethical Approbval

This study (MMCP/IAEC/65) was approved by Institutional Animal Ethics Committee

Competing interest

The authors declares that they have no conflict of interests.

Author’s contribution

ML, SD, MD, DM, RD, NG and SG carried out the experimental work in whole study. SG have been involved in revising it critically for important content. SG have made substantial contribution to concept, designing, and acquisition of data, analysis and interpretation of data and lastly drafting the manuscript. MAM, ABN, AIM, SG writing and editing of the manuscript. All authors approved the final manuscript for publication.

Funding

The authors have not received any funding from any sources.

Availiabity of data and materials

Not Applicable

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Effects of curcumin, quercetin, and their combinationson neurodegeneration and motor impairment in rotenone-induced Parkinson's disease in rats

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Abstract

Background

Methods

Results

Conclusion

Figures

1. Introduction

2. Material And Methods

2.1. Drugs and chemicals

2.2. Animals

2.3. Preparation of drugs

2.4. Induction of PDand experimental design

2.5. Experimental groups

2.6. Determination ofbody weight

2.7. Assessment of behavioral parameters

2.7.1. Assessment of locomotor activity

2.7.2. Rota rod test (muscular coordination)

2.7.3. Grid test

2.7.4. Open-field test

2.7.5. Footprint test

2.9. Histopathological analysis

2.10. Statistical analysis

3. Results

3.1. Effect of curcumin,quercetin, and their combination on rotenone-induced change in the body weight

3.2. Effect of curcumin,quercetin, and their combination on the rotenone-induced decrease in locomotor activity

3.3. Effect of curcumin,quercetin, and their combination on rotenone-induced change in motor coordination

3.4. Effect of curcumin,quercetin, and their combination on rotenone-induced change in muscle strength

3.5. Effect of curcumin,quercetin, and their combination on rotenone-induced change in rearing behavior

3.6. Effect of curcumin,quercetin, and their combination on rotenone-induced change in the walking pattern

3.7. Effect of curcumin,quercetin, and their combination on the rotenone-induced decrease in striatal dopamine levels

3.8. Effect of curcumin,quercetin, and their combination on rotenone-induced brain histopathological changes

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

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