Parkinson's disease (PD) is an age-related complex neurodegenerative disease that affects ≤80% of dopaminergic neurons in the substantia nigra pars compacta (SNpc). It has previously been suggested that mitochondrial dysfunction, oxidative stress and oxidative damage underlie the pathogenesis of PD. Curcumin, which is a major active polyphenol component extracted from the rhizomes of
Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder worldwide, which is well characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The diagnosis of PD is associated with various symptoms of motor dysfunction, including progressive resting tremor, rigidity, bradykinesia and postural instability (
Rotenone (ROT) is an active herbicide that can be used to induce dopaminergic neuronal oxidative damage in experimental animal models of PD (
Previous studies have demonstrated that Akt phosphorylation facilitates nuclear factor erythroid 2-related factor 2 (Nrf2) translocation into the nucleus, which has an important role in the defense against oxidative stress by regulating the expression levels of antioxidant enzymes, including heme oxygenase (HO)-1 and NAD(P)H:quinone oxidoreductase 1 (NQO1) (
A total of 50 male Lewis rats (age, 7–8 weeks; weight, 260–280 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The rats were housed in a room maintained at 22°C with 40–70% humidity, under a 12-h light/dark cycle with
Human embryonic kidney (HEK)293T cells and SK-N-SH human neuroblastoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 4.5 g/l high glucose, 2 mM L-glutamine, 1 mM sodium pyruvate (Thermo Fisher Scientific, Inc.) and 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in an atmosphere containing 5% CO2 and 95% air.
Rotenone (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sunflower seed oil at 1.5 mg/ml. The rats were divided randomly into four groups (n=6/group): (i) The CON group, in which rats were injected with sunflower seed oil (0.5 ml/kg/day, twice daily) as a vehicle for 50 days; (ii) the ROT + curcumin treatment group (Sigma-Aldrich), in which rats were intragastrically administered curcumin (100 mg/kg) twice a day for 50 days, and ROT was subcutaneously administered twice a day at 1 ml/kg/day on day 4 of curcumin treatment; (iii) the ROT treatment group, in which the rats received the same as the ROT + curcumin group beginning on day 1, except that gum arabica (Sigma-Aldrich) was administered instead of curcumin; (iv) the curcumin treatment group, in which the rats received the same as the ROT + curcumin group, except that sunflower seed oil was administered instead of ROT. During the experimental period, the food intake was monitored daily and body weight was measured regularly. Behavioral assessments were carried out among rats of all groups. Subsequently, the rats were sacrificed by decapitation, and tissues were removed from the brain on a Petri dish placed on ice under a microscope (Olympus BX50, Tokyo, Japan). The SNpc regions were stored at −80°C, and the cytosolic fraction was subsequently extracted and subjected to various biochemical investigations and western blot analyses.
A total of 24 h after the final injection, the rotational behavior of the rats was determined following intraperitoneal injection with freshly prepared apomorphine hydrochloride (1 mg/kg, s.c.; Sigma-Aldrich) for 5–10 min. Subsequently, full 360° unilateral turns were recorded in a 10 min period. The rats that turned unilaterally >7 cycles/min, were considered successful models of PD.
In the catalepsy test, rats were hung by the paws on a vertical grid (25×45 cm, 1 cm space between each wire). The time taken for the rats to move their paws or to perform any sort of initial movement was noted. The rats were then placed with both forepaws on a bar 10 cm above, parallel from the base in a half-rearing position. The paw removal latency was noted, and the maximum descent latency was fixed at 180 sec for both tests.
Behavioral tests were carried out in an open field using a spontaneous activity instrument (Shanghai Jiliang Software Technology Co., Ltd., Shanghai, China), including four boxes (40×40×50 cm). Each box housed one rat at a time. An infrared camera was attached to the top of the box to record the movement time, movement distance and movement speed for 1 h. Subsequently, the video images were sent to a computer and analyzed with the JLBehv-LAR-4 software (Shanghai Jiliang Software Technology Co., Shanghai, China).
The obtained SNpc regions were immediately homogenized using an ultrasonic cell disruptor (Model 150 V/T; BioLogics, Manassas, VA, USA), and then subjected to centrifugation at 14,000 × g for 15 min at 4°C. The supernatant was used for the determination of the activity of various enzyme activities. GSH content in the SNpc regions was measured as described previously (
HEK293T cells were co-transfected with envelope plasmid, packaging plasmid and expression plasmid (pLKO.1; Sigma-Aldrich) containing the shRNA using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and the Lentiviral Packaging Mix (Sigma-Aldrich), according to the manufacturers' protocols. The short hairpin (sh)RNA sequence targeting coding regions of the rat Nrf2 gene (shNrf2) and the control shScramble plasmid (GenePharma Co., Ltd., Shanghai, China) were used. The cell supernatant containing the lentivirus was collected, and filtered through membrane filters (0.45-
In the shNrf2 lentivirus experiment, nine groups (n=6/group) were designed: CON group; shScramble (control) group; shScramble + ROT group; shScramble + curcumin group; shScramble + ROT + curcumin group; shNrf2 group; shNrf2 + ROT group; shNrf2 + curcumin group; shNrf2 + ROT + curcumin group. Lentiviruses containing shScramble or shNrf2 (2×107 RIU; 10
In the LY294002 experiment, five groups (n=6/group) were designed: CON group; LY294002 (control) group; LY294002 + ROT group; LY294002 + curcumin group; LY294002 + ROT + curcumin group. ALZET® osmotic mini-pumps (0.5
Tissues were homogenized on ice using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) and were centrifuged at 14,000 × g for 15 min at 4°C. Protein concentration was determined using the Pierce Bicinchoninic Acid Protein Assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Equal amounts of protein (25
Data are presented as the mean ± standard deviation representative of three individual experiments performed in triplicate. Statistical analyses were conducted using one-way analysis of variance followed by the Student Newman-Keuls test. Statistical analyses were performed using GraphPad Prism software 5.0 (GraphPad Software Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.
Almost all ROT-treated rats displayed significant weight loss during the first 10 days of ROT treatment, which was reversed after curcumin pretreatment. In the CON and curcumin-treated rats, body weight increased slowly and no significant difference was observed (
ROT-treated rats developed typical disordered behavioral features, including catalepsy, unsteady movement and hunched posture to varying degrees. ROT-treated rats exhibited prolonged descent latency in the grid and bar test (
The present study aimed to determine the protective effects of curcumin on the neurochemical features of dopaminergic neurons in ROT-treated rats. Western blot analysis revealed that curcumin pretreatment restored the ROT-induced loss of TH protein, which is considered a marker of dopaminergic neurons, in the SNpc (
The protective effects of Akt/Nrf2 signaling against oxidative stress have previously been reported in PD (
Transfection with a shNrf2 lentivirus or sublethal chronic exposure to LY294002 prior to treatment with ROT/curcumin, resulted in a failure of curcumin to prevent ROT-induced reductions in TH and GSH expression (
PD is regarded as a neurodegenerative disease, which is characterized by the age-associated accumulation of oxidative damage in human brain tissue. ROT, which is a commonly used natural substance obtained from the roots of certain tropical plants including
To better understand the therapeutic effects of drugs specific to certain disease processes, animal models are often used. Rats have been reported to develop motor and postural deficits characteristic of PD following ROT minipump administration for 35 days (
A previous study demonstrated that the levels of endogenous oxidative markers (ROS and MDA), redox status (GSH), and response of antioxidant enzymes were associated with the state of oxidative stress in rat brain tissues (
It is well documented that Nrf2 is a basic leucine zipper transcription factor that protects various tissues and cells from ROS via the antioxidant response element (ARE)-mediated induction of diverse antioxidant proteins and phase II detoxifying enzymes, including HO-1 and NQO1 (
In conclusion, the present study established a rat model of PD using ROT, and examined the behavioral performance, endogenous markers of oxidative stress, the activities of antioxidant enzymes, and the activation of the related signaling pathways in the SNpc following curcumin pretreatment. The results indicated that the protective effects of curcumin against ROT-induced dopaminergic neuronal oxidative damage were mediated by activation of the Akt/Nrf2 signaling pathway. These results demonstrated that curcumin exerts potential neuroprotective therapeutic effects, and may be considered a novel therapeutic agent for the treatment of PD.
Parkinson's disease
substantia nigra pars compacta
nuclear factor erythroid 2-related factor 2
phosphoinositide 3-kinase
NAD(P)H:quinone oxidoreductase 1
lipid peroxidation
malondialdehyde
reactive oxygen species
heme oxygenase-1
rotenone
tyrosine hydroxylase
glutathione
short hairpin RNA
Effects of curcumin (CUR) and rotenone (ROT) on parkinsonian symptoms in rats. (A) Body weight of the rats was recorded daily and no mortality occurred. (B) Rotational behavior of all rats was recorded after 50 days of treatment with ROT and CUR. (C) Catalepsy of the rats was measured on a vertical grid and horizontal bar. (D) Locomotor activity assay of the rats recorded the movement time (min), movement distance (m) and movement speed (mm/s). Data are presented as the mean ± standard deviation. *P<0.05 vs. the CON group. #P<0.05 vs. the ROT group (n=6).
Effects of curcumin (CUR) and rotenone (ROT) on the state of dopaminergic neurons, and the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and glutathione (GSH) in the substantia nigra pars compacta (SNpc) of rats. (A) SNpc sections were lysed and subjected to western blotting with anti-tyrosine hydroxylase (TH) antibody. Quantification analysis was performed using Gel-Pro Analyzer 4.0 and normalized to β-actin. Markers of oxidative status: (B) ROS, (C) MDA and (D) GSH, were examined using commercial kits. Data are presented as the mean ± standard deviation. *P<0.05 vs. the CON group. #P<0.05 vs. the ROT group (n=6). DCF, dichlorodihydrofluorescein.
Effects of curcumin (CUR) and rotenone (ROT) on the Akt/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling axis in the substantia nigra pars compacta of rats. The protein expression levels of (A) heme-oxygenase (HO)-1, NAD(P)H:quinone oxidoreductase 1 (NQO1) and β-actin, and (B) Nrf2, phosphorylated (p)-Nrf2, Akt and p-Akt were detected by western blotting. Quantification analysis was performed using Gel-Pro Analyzer 4.0 and normalized to β-actin in (A) and normalized to total Akt or total Nrf2 in (B). Data are presented as the mean ± standard deviation. *P<0.05 vs. the CON group. #P<0.05 vs. the ROT group (n=6).
Effects of short hairpin RNA nuclear factor erythroid 2-related factor 2 (shNrf2) lentivirus and LY294002 treatment on the neuroprotective effects of curcumin (CUR) on rotenone (ROT)-induced substantia nigra pars compacta dopaminergic neuronal oxidative damage. (A) Protein expression levels of tyrosine hydroxylase (TH) and β-actin, as determined by western blotting. (B) Glutathione (GSH) levels were examined using a commercial kit. (C) Protein expression levels of phosphorylated (p)-Nrf2 and Nrf2, as detected by western blotting. Quantification analysis of western blotting was performed using Gel-Pro Analyzer 4.0 and normalized to β-actin in (A) and total Nrf2 in (C). CON group was untreated. Control group was treated with shScramble, shNrf2, or LY294002. Data are presented as the mean ± standard deviation. *P<0.05 vs. the CON group. #P<0.05 vs. the ROT group (n=6). Control group was regarded as the group treated with shScramble, shNrf2, or LY294002.