Abstract
Epidemiological data imply links between the increasing incidences of Alzheimer’s disease (AD) and type 2 diabetes mellitus. In this study, an AD rat model was established by combining treatments with intracerebroventricular streptozotocin (icv-STZ) and subcutaneous D-galactose, and the effects of curcumin on depressing AD-like symptoms were investigated. In the AD model group, rats were treated with icv-STZ in each hippocampus with 3.0 mg/kg of bodyweight once and then were subcutaneously injected with D-galactose daily (125 mg/kg of bodyweight) for 7 weeks. In the curcumin-protective group, after icv-STZ treatment, rats were treated with D-galactose (the same as in the AD model group) and intraperitoneally injected with curcumin daily (10 mg/kg of bodyweight) for 7 weeks. Vehicle-treated rats were treated as control. Compared with the vehicle control, the amount of protein carbonylation and glutathione in liver, as well as malondialdehyde in serum, were upregulated but glutathione peroxidase activity in blood was downregulated in the AD model group. The shuttle index and locomotor activity of rats in the AD model group were decreased compared with the vehicle control group. Furthermore, AD model rats showed neuronal damage and neuron loss with formation of amyloid-like substances and neurofibrillary tangles, and the levels of both β-cleavage of AβPP and phosphorylation of tau (Ser396) were significantly increased compared with the vehicle control group. Notably, compared with the AD model group, oxidative stress was decreased and the abilities of active avoidance and locomotor activity were improved, as well as attenuated neurodegeneration, in the curcumin-protective group. These results imply the applications of this animal model for AD research and of curcumin in the treatment of AD.
INTRODUCTION
Alzheimer’s disease (AD) is one of common neurodegenerative disease. This disease is characterized in the clinic by the decline of cognitive abilities. Senile plaques and neurofibrillary tangles accompanied with loss of neurons are the major neuropathological features of AD. Senile plaques are mainly composed of amyloid-β (Aβ) while neurofibrillary tangles are formed by the paired helical filaments assembled from hyperphosphorylated tau. Aβ is one of the proteolytic products of amyloid-β protein precursor (AβPP), and Aβ peptides contain 39-43 amino acid residues. The amyloid cascade hypothesis suggests that Aβ may be the key factor leading to development of this disease, but the etiological mechanism of AD, especially of sporadic AD, is still not clear [1, 2]. This is particularly due to the fact that the biological function of AβPP is still not understood, and AD animal models are less available. So far, researchers have focused on the transgenic rats/mice models based on the genetics in the pathogenesis of familial AD, and these models require overexpression of human AβPP or other AD-related proteins [3–6]. These models have many advantages in the AD pathological research. For example, AβPP transgenic mice demonstrate Aβ deposition, which is seldom seen in aged mice in nature. However, there are still some limitations on the application of transgenic AD animals to understand all the pathological features of AD. For instance, human AβPP overexpressed in rats/mice is heterogeneous and therefore rats/mice might have a reduced ability to cleave human AβPP or its fragments, resulting in their deposition.
Epidemiological data imply the links between the increasing incidences of AD and type 2 diabetes mellitus (T2DM). T2DM shares similar pathological events with AD including insulin resistance, impaired glucose metabolism, and the presence of advanced glycation end products [7, 8]. A drastic increase in glucose transporter 2 (GLUT2), as well as a decrease in glucose transporter 1 (GLUT1) and glucose transporter 3 (GLUT3), has been reported in the AD brain [9], and abnormal expression of glucose transporters has also been suggested to link to hyperphosphorylation of tau and degeneration of neurofibrils [10]. The chemical streptozotocin (STZ), a selective GLUT2 substrate, can cause pancreatic β-cell destruction. Therefore, STZ is commonly used to induce diabetic models in mice and rats. Recently, the animals with intracerebroventricular injection of STZ (icv-STZ) have been applied as an AD model (about 100 times lower than that used to induce diabetes) [11, 12]. As a substrate of GLUT2, STZ administrated by icv injection results in regionally specific glucose hypometabolism [13] and damage to insulin/IGF signaling-related gene expression as well as the loss of oligodendrocytes and neurons [14], similar to that observed in the early stages of sporadic AD. However, icv-STZ animal model cannot simulate all of pathological features of AD, especially on the aging process with oxidative damage.
Chronic exposure to D-galactose (D-gal), which is commonly used to establish a model of accelerated animal aging, induces symptoms similar to natural aging in animals. Accumulated D-gal in vivo is metabolized by the D-gal oxidases accompanying by the generation of H2O2, resulting in the increase of oxidative damage. Mice injected subcutaneously with 125 mg/kg D-gal for 8 weeks show an increase of oxidative damage and mitochondrial dysfunction accompanied with deficiency in cognitive function and locomotor activity [15]. Further, D-gal administered (100 mg/kg intraperitoneally) for 60 days results in neuroinflammation, elevation of reactive oxygen species (ROS) and receptor for advanced glycation end products, and synaptic disorder [16].
However, negative reports suggested that the treatment with a 100 mg/kg dose for 6-weeks of D-gal in mice may not represent a suitable model for age-related neurobehavioral symptoms and serum lactate [17]. Therefore, the aged animal accelerated by D-gal on dose and treatment time is under argument on the neurobehavioral symptoms.
Curcumin, abundant in Curcuma longa, is a compound of polyphenolic compounds. In Asia, especially in India, curcumin is widely used as a food additive. Curcumin has been reported to show diverse biological functions including anticancer, anti-inflammation, and anti-oxidation. In recent years, curcumin has been paid great attention for the prevention and treatment for AD [18]. On one hand, among the population aged 60∼90 years old, people who often or occasionally eat curry have a better cognitive ability than those who never or rarely use curry by the performance analysis of the minimum-mean-square-error [19]. On the other hand, experiments on animals show that curcumin can improve the behavior of icv-Aβ rats [20]. Curcumin can not only decrease the deposition of Aβ in the brain of AD rats but also downregulate the generation of this peptide [21].
In this study, an AD rat model was established by combining treatments with a single injection of STZ in the bilateral hippocampus and a daily subcutaneous injection of D-gal. Furthermore, the effects of curcumin on behaviors and biochemical markers related to AD-like symptoms were investigated.
MATERIALS AND METHODS
Animals
Male SD rats (355±17 g) were obtained from the Chinese Academy of Military Medical Sciences, and rats were reared in accordance with the approved protocol from our institutional animal care and use committee. Rats were housed one animal per cage with access to food and water under a 12/12 h light/dark cycle.
Rat treatments
Before icv-STZ, the ability of active avoidance response (AAR) was assessed under light stimulation by shuttle box experiment. Rats were divided into three groups according to the assessment of AAR to make each group has almost the same ability on AAR. After anesthetization, rats were restrained on a stereotaxic apparatus and further injected with STZ. The bregma coordinates used for injection were as following: –1.5 mm lateral, –0.28 mm posterior, and –4.0 mm below. For vehicle control group, an amount of 10μL citrate buffer solution (pH4.5) was injected into hippocampus of each cerebral hemisphere. For the model group and cur-protective group, hippocampus of each cerebral hemisphere was injected with 3.0 mg/kg bodyweight STZ in 10μL citrate buffer solution (pH 4.5). After recovery for a week, rats of the vehicle control group were subcutaneously injected with 2.5 mL/kg bodyweight in 0.9% saline for 7 weeks; rats in the AD model group and cur-protective group were subcutaneously injected with 125 mg/kg bodyweight D-gal (50 mg/mL, 2.5 mL/kg bodyweight) in 0.9% saline daily for 7 weeks, and rats in the cur-protective group were further treated with 10 mg/kg curcumin by intraperitoneal (i.p.) injection (2.5 mg/mL, 4 mL/kg bodyweight) daily for 7 weeks.
Assessment of oxidative stress
Determination of protein carbonyl and GSH
Homogenate of 10% liver tissue was prepared using tissue lysis buffer with protease inhibitors. The supernatant was collected after centrifugation for 15 min at 3,000 g. The content of protein carbonyl in supernatant was determined based on the color reaction. Briefly in principle, amino acid residues tend to form carbonylate after being oxidation, and the formed carbonyl group can further react with 2,4-dinitrophenylhydrazine to form 2,4-dinitrophenylhydrazone depositions. After dissolved in guanidine hydrochloride solution, the OD370 nm represents the content of protein carbonyl. An amount of 450μL lysis was detected and the OD370 nm is recorded. The content of protein carbonyl was represented as the unit of nmol/g wet tissue.
The content of GSH in supernatant was determined based on the color reaction. Briefly in principle, GSH reacts with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) under the catalysis of GSH-Px to form 5-thioate 2-nitrobenzoic acid anion, which can be detected at the wavelength of 420 nm. The OD420 nm represents the content of GSH in the supernatant, and the data is represented as mg GSH per wet tissue.
GSH-Px activity and MDA content
The procedure is similar to the measure of GSH. In brief, an amount of 20μL blood was added into 2 mL ddH2O and mixed well. The mixture was further added 2.5 mL of 0.32 mol/L Na2HPO4 buffer and certain of DTNB. The color product was quantified under the 420 nm wavelength. One unit of GSH-Px activity was defined as that an amount of 4μL blood enzyme-catalyzed 1μM GSH to convert GSSG in 5 min at 37°C.
The content of MDA in serum was measured based on the principle that MDA reacts with 2-thiobarbituric acid (TBA) to form a fluorescent product 3,5,5-trimethyloxazolidine-2,4-dione. In brief, an amount of 100μL serum was reacted with 2 mL 0.6% TBA solution for 60 min. The fluorescence intensity of the product was measured under λex 532 nm and λex 553 nm with a fluorospectrophotometer. The content of MDA was calculated based on the MDA standard curve.
Behavioral tests
Shuttle box
The two compartments of shuttle-box (50 cm×16 cm×18 cm) were connected by a 4-cm-high open door. In the middle of the each compartment, there is a light and a linearly frequency-modulated tone, and the light or the sound was treated as a conditioned stimulus. As an unconditioned stimulus, electric footshock (0.5 mA) was applied through the grid floor. The training was carried out in weekly sessions consisting of 30 crossing trials. Rats were permitted to explore the compartments for 10 min before executing the testing procedure. There was an inter-trial interval of 30 s, and the response of crossing compartment on inter-trial interval was recorded. During a trial, rats were received a 15 s-stimulation of light or sound set in the compartment. If rats travelled to another compartment, a trial finished and a response of conditioned escape was recorded. The stimulation was going on 15 s if rats failed to cross to the door, and rats would receive a 15-s footshock as a punishment at the same time. If the rats travelled to another compartment during the footshock, the stimulation and the footshock were off and a response of unconditional escape was recorded. If rats failed to cross the channel during the 15-s footshock, a null response was recorded.
During the training course with 30-crossing trials, the latent time of conditioned escape was recorded, and the total number of conditioned escape (C) and the total number of inter-trial interval (N) of each rat were recorded and the ability of AAR was represented as the shuttle index (SI) in each group:
Where n is the number of rat in each group.
Morris water maze
The ability of spatial learning and memory was assessed by Morris water maze experiment and the trials were carried out once daily. A circular pool with 1.60-m diameter was filled with water (22∼25°C) with addition of black ink. Four markers with different shape were placed equidistantly on the margin of the pool, and the water surface was divided into 4 quadrants and further subdivided into 8 zones by 2-concentric circle with equal radius. A 12.0-cm-diameter platform was placed into one of the quadrants under the surface 1.0 cm. Rats were placed into the water at a sidewall of the pool. The latent time to find the platform was recorded. If the rat still did not find the platform within 2 min, it was guided onto the platform. The navigating trial was carried out for 3 day. On the fourth day, the platform was removed from the pool and rats were allowed to swim for 2 min to probe the place of former platform. The latent time of first crossing the former platform-placed zone and the times of crossing the quadrant where the former platform was located were recorded.
Tissue staining
Rat brains were fixed in 4% paraformaldehyde overnight. A section of brain including hippocampus from the fixed brain was dehydrated and embedded in paraffin according to the procedure: 70% ethanol overnight, 80% ethanol 2 h twice, and 95% ethanol 1 h twice, 100% ethanol 1 h twice, xylene 15 min twice. Then, 5μm-thin slices cut from embedded brain were stained with hematoxylin-eosin, Congo red, and silver ion after rehydration.
The rehydrated slices were also dyed by an immunocytochemical method to visualize the phosphorylation of tau. In brief, after antigen retrieval, slices were covered with antibody of phosphorylation tau at Ser396 residue (pSer396-Tau) (sc101815, Santa Cruz Biotech) overnight at 4°C. Then the slices were incubated with horseradish peroxidase-conjugated secondary antibody and visualized by the enzymatic detection to oxidize 3,3′-diaminobenzidine (DAB) to dark brow deposits.
Western blot
The hippocampal tissue was isolated and added into liquid nitrogen. Tissue was milled on a mortar and lysed with lysis buffer (1% Triton X-100, 1.0% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, and 50 mM Tris, pH 8.0) with inhibitors of protease and phosphatase. The lysates were centrifuged at 10,000 g in 4°C for 10 min and the supernatant was collected. After quantification with the bicinchoninic acid (BCA) assay, total protein was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene difluoride membrane (Millipore, USA). After blocking with skimmed milk, the membrane was incubated with primary antibodies overnight at 4°C. After incubation with the corresponding horseradish peroxidase-linked secondary antibodies, the optical density of bands were detected with an enhanced chemiluminescence reagent (ECL) using a gel detection system (GE Healthcare, USA). Quantification of the optical density was performed using Quantity one 4.62 software. The antibodies used for western blot analysis are as follows: anti-AβPP (A8717, Sigma-Aldrich), anti-BACE1 (ab2077, Abcam Inc.), and anti-PSEN1 (ab65293, Abcam Inc.), anti-pSer396-Tau (sc101815, Santa Cruz Biotech), and anti-β-actin (#8457 S, Cell Signaling Technology).
Content of hippocampal Aβx - 42
Total protein was extracted from hippocampal tissue using lysis buffer (1% Triton X-100, 1.0% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, and 50 mM Tris, pH 8.0) with inhibitors of protease phosphatase. The lysates were centrifuged at 10,000 g in 4°C for 10 min and the supernatant was collected. Total protein content in the supernatant was quantified with the BCA assay. The level of rat Aβx - 42 in hippocampus was detected by a sandwich ELISA kit (KMB3441, Life Technologies) according to the manual. The data were presented as ng/g hippocampal protein.
Statistical analysis
Data analysis was represent SE of the mean of each group and performed with Origin 8.0 software by one-way analysis of variance (ANOVA) followed by Tukey test for multiple comparisons. The minimum significance level was set at p < 0.05.
RESULTS
Curcumin attenuates oxidative damage induced by the combining treatments with STZ and D-gal
As shown in Table 1, both protein carbonyl and GSH are increased in liver tissue of AD model group, which were treated with icv-STZ and subcutaneous D-gal, compared to the vehicle control group. Serum MDA, a lipid oxidation product, is also higher but blood GSH-Px is lower in the AD model group than in the vehicle control group. Notably, the contents of protein carbonyl and GSH in liver tissue and serum MDA are decreased but blood GSH-Px activity is increased in the curcumin-protective group compared with the AD model group. These results indicated that the level of oxidative stress is elevated and rats might suffer from oxidative damage after treatment with an injection of icv-STZ and a daily subcutaneous injection of D-gal for 7 weeks. Curcumin attenuates oxidative stress induced by the combination of icv-STZ and subcutaneous D-gal and might decrease oxidative damage at an individual-animal-level.
Curcumin improves active behaviors in shutter box
As shown in Table 2, there is no significant difference on shuttle index among groups before STZ and D-gal administrations. The shuttle index either on light or sound stimulus tested at 5 and 7 weeks after D-gal treatment in the AD model groups was lower than in the vehicle control group, and the increment of shuttle index in the AD model group was also less than in the control group. However, rats in the curcumin-protective group had a higher shuttle index and an increment of shuttle index between the 5th and 7th week on light or sound stimulus than the AD model group (Tables 2 and 3). The latent time of conditioned response on both light and sound stimuli tested on week 5 and week 7 after D-gal treatment is decreased in the vehicle control, the AD model, and the curcumin-protective group, but the increment is less in the AD model group than in the vehicle control group; the increment is greater in the curcumin-protective group than in model group (Tables 4 and 5). These results indicated that curcumin depresses the decline in active avoidance response of rats induced by combining treatments of icv-STZ and subcutaneous D-gal.
Curcumin improves locomotor activity in the Morris water maze
As show in Table 6, the latent time for rats in the vehicle control and curcumin-protective groups to find the hidden platform was decreased but increased in the AD model group during the training course. The latent time in the AD model was significantly increased in the second and third training course compared with the vehicle control group. In the third training, notably, there is a significant decrease on latent time in the curcumin-protective group compared to the AD model group. That is to say, D-gal results in those rats spend more time to find the hidden platform under the water surface; however, rats in the curcumin-protective group have a better ability to search for the platform than in the AD model group.
In the aspect of orientation, the latent time to first enter the zone in which the former platform was placed was increased (Fig. 1A), and the times of crossing the zone were decreased in AD model group compared to the vehicle control group (Fig. 1B). In curcumin-protective group, however, the latent time of first entering the zone that the former platform placed was decreased and the times of crossing the zone were raised compared to the AD model group. These results indicated that curcumin attenuates the decline in locomotor activity of rats treated with icv-STZ and subcutaneous D-gal.
Curcumin attenuates neuronal damage and neuron loss
As shown in H&E-stained brain tissues (Fig. 2A-D), the nuclei of cells are eosinophilic and stained deeply violet. The brain tissues in the AD model group exhibit many pyknotic nuclei and even fragmentation of nuclei. On one hand, neurons in the cortex are shrunk and the soma is less visible; the density of neurons in the cortex are decreased in the AD model group compared to the vehicle control group. On the other hand, in the hippocampal CA1 and CA3 regions, neuron density is decreased and neuronal soma is shrunk in the AD model group; neuronal chromatin condensation in the AD model group is obvious, especially in the CA3 region. Notably, the nuclei and neuronal soma in the cortex and in the hippocampal CA regions are clearer in the curcumin-protective group compared to the AD model group. Furthermore, the hippocampus weight is significantly decreased in the AD model group compared to the vehicle control group, and treatment with curcumin (i.p) attenuates hippocampal loss induced by combining the treatments of icv-STZ and subcutaneous D-gal (Fig. 2E). The above results imply that curcumin decreases neuronal damage and neuron loss induced by combining treatments of icv-STZ and subcutaneous D-gal.
Curcumin decreases upregulation of AβPP β-cleavage and formation of amyloid-like substances
Aβ, a cleaving product of AβPP, is the main component of senile plaques. Aβ is considered to be an initiative pathological factor for the development of AD, resulting in tau hyperphosphorylation, synapse dysfunction, and neuron loss. As shown in Fig. 3A-C, the expressions of hippocampal AβPP and BACE1 in the AD model group are significantly higher than those in the vehicle control group. However, compared to the AD model group, the protein levels of hippocampal AβPP and BACE1 are decreased in the curcumin-preventive group.
The expression of protein PSEN1, a catalytic subunit of γ-secretase complex, in the AD model group is dramatically decreased compared to the vehicle control group, and the C-terminal fragment (20 kDa), a proteolytic product of PSEN1, is increased significantly in the AD model group (Fig. 3D). It is believed that PSEN1 undergoes proteolysis and the C-terminal fragment is further cleaved by caspase-3 to produce a 12-kd fragment [22]. The result of PSEN1 expression is correlative to a previous report that showed, in an AD brain, the level of the 20-kd C-terminal fragment is elevated [23].
Notably, the content of hippocampal Aβx - 42 is increased significantly in the AD model group compared to the vehicle control group; however, hippocampal Aβx - 42 is significantly decreased in the curcumin-protective group compared to the AD model group (Fig. 4). These results imply that curcumin depresses overexpression of AβPP and upregulation of AβPP β-cleavage in rats induced by the combined treatments of icv STZ and subcutaneous D-gal.
As shown in the tissues stained by Congo red (Fig. 5), red filaments and red precipitates can clearly be observed in the subcortical nuclei in the AD model group. However, compared to the AD model group, the Congo red-stained filaments and precipitates in the curcumin-protective group are decreased remarkably; similar to those in the vehicle control group, the filaments are stained slightly by Congo red in the curcumin-protective group. This result indicated that curcumin attenuates the formation of amyloid-like substances in rats induced by the combined treatments of icv-STZ and subcutaneous D-gal.
The above results indicated that curcumin depresses the upregulation of AβPP β-cleavage and the amyloid formation induced by the combined treatment of icv-STZ and subcutaneous D-gal.
Curcumin decreases tau phosphorylation and neurofibril formation
As shown in Fig. 6, the level of phosphorylated Tau protein at Ser396 (pSer396-Tau) in the AD model group is higher than in the vehicle control group. However, pSer396-Tau is decreased in the curcumin-protective group compared to the AD model group. In addition, stained with pSer396-Tau antibody (Fig. 7), neurons in the cortex and neurofilaments in the subcortex show more staining in the AD model group compared with the vehicle control group; however, both the neural processes in the cortex and the neurofilament in the subcortex are less stained in the curcumin-protective group compared with the AD model group.
Further, the tissues of the AD model group stained by silver ion showed more rough never fibers than those of the vehicle control group. The never fibers in the AD model group display a likelihood of neurofibrillary tangles. Notably, the tangle of never fibers in the curcumin-protective group is significantly decreased compared to the AD model group (Fig. 7).
These results imply that combining treatments of icv-STZ and subcutaneous D-gal induce tau phosphorylation and formation of neurofibrillary tangles.
DISCUSSION
AD is a degenerative disease of the central nervous system and is characterized by two defined pathological hallmarks: senile plaques and neurofibrillary tangles. Senile plaques are comprised of aggregates of Aβ as well as damaged neurons and reactive glia; neurofibrillary tangles are formed by paired helical filaments consisting of the hyperphosphorylated microtubule-associated protein tau. Senile plaques and neurofibrillary tangles are detected in cerebral cortex, hippocampus, and subcortical nuclei including the amygdala, basal nuclei, locus coeruleus, and hypothalamus [24]. The degree of senile plaques and neurofibrillary tangles are correlative to the process of AD. In AD brain, a neuronal damage like granulovacuolar degeneration of Simchowicz and Hirano body is intensive as well as neuron loss [25, 26]. In this study, brain tissues of rats treated with icv-STZ combining subcutaneous D-gal show an amyloid-like degeneration and formation of neurofibrillary tangles as well as neuronal pathological changes of neuronal soma and chromatin (Figs. 2, 3). It is interesting that Congo red is more prone to staining the subcortical nuclei rather than the cerebral cortex or hippocampus in our experiments, implying that the subcortical nuclei bear more amyloid-like substance than the cortex or the hippocampus. Furthermore, the combined treatments of icv-STZ and subcutaneous D-gal increase the β-cleavage of AβPP and tau phosphorylation (Figs. 4 and 5). Since accumulated Aβ is easy to aggregate with β-sheeted structure, Aβ might be one key compound of the amyloid-like substance. Neuronal damage is bound to affect the ability of learning and memory. In our experiment, the connecting door is open during trial and rats can cross the channel free to the other compartment (free crossing). Therefore, the recorded conditioned escape might not be the actual response to the light or sound stimulus but the desire for free crossing between the compartments. In this study, thus, the shuttle index was defined as the ratio of the number of conditioned escape and the number of inter-trial interval, and shuttle index, but not the number of conditioned escape, is employed to assess the ability on active avoidance directly. In AD model rats, the response to active avoidance and locomotor activity is desensitized (Tables 2-6- and Tables 2-6; Fig. 1), implying that the ability of learning and memory is decreased after rats were treated with icv-STZ combining subcutaneous D-gal.
Although the pathological hallmarks have been well identified for over a hundred years, the mechanism of AD pathology is still not understood completely. For familial AD, the genetic mutants including AβPP and presenilins contribute to the development of senile plaques and neurofibrillary tangles. For sporadic AD, the gene apolipoprotein E ɛ4 is believed to be related to AD pathological process and blocking the interaction between apolipoprotein E and Aβ in triple transgenic AD mice with PS1M146 V, AβPPswe, and tauP30IL transgenes reduces the amyloid burden and tau pathology [29]. Non-genetic factors, such as sustaining oxidative stress, traumatic brain injury, and metal ions, are increasingly considered to associate with the development of sporadic AD [30–33]. Oxidative damage to biological molecules have been found in AD brain, including lipid peroxidation, proteins carbonylation, and peroxidation of nucleic acids. Physiological metabolism generates oxygen free radicals and other ROS, which are needed for some enzymatic reactions and other physiological processes. There are antioxidant defense systems to terminate the excessive ROS, including superoxide dismutase (SOD), catalase (CAT), and GSH. Oxidative damage ensues when ROS are over-generated. The decreased activity of SOD was reported in late AD brain [34, 35]. Mice treated with D-gal show decreased activity of SOD and CAT [36]. Downregulation of SOD and CAT activity was also detected in mice treated with icv-STZ [37]. In this study, the results of physiological markers on oxidative stress indicated that protein carbonyl and GSH in liver as well as serum MDA are increased but blood GSH-Px activity is decreased in rats treated with icv-STZ combining subcutaneous D-gal (Table 1). Increase in protein carbonyl and MDA means the upregulation of oxidative stress and the occurrence of oxidative damage. The content of GSH, a physiological antioxidant, is promoted in liver; perhaps this is due to the feedback of increased ROS and decreased activities of SOD and CAT.
Curcumin is a yellow phenolic compound derived from the rhizome of Curcuma longa. Particular attention has been paid to curcumin because a regular diet of curcumin is a possible reason for the reduced risk of AD among the Indian populations. Curcumin has been found to attenuate inflammation and reduce oxidative damage in vivo. However, curcumin also results in cell damage at high dose [27], and there is a report that administration by intraperitoneal injection with more than 10 mg/kg bodyweight of curcumin might induce chromosomal aberrations in rats [28]. Therefore, in this study, we used s dose of 10 mg/kg bodyweight to investigate the inhibitory effects of curcumin on AD-like symptoms. The results indicated that curcumin attenuates the decline in learning and memory and AD-like pathological changes.
Conclusions
AD is the most common cause of dementia. Although we have learned much on the pathology and the treatment of AD, there are some factors that hamper the understanding of AD and drug development. For instance, the physiological function of AβPP and Aβ is not fully understood yet. Although transgenic animal models had been used widely and elucidate many AD features, some mechanisms like development of sporadic AD are still less understood. Epidemiological data imply the links between the increasing incidence of AD, especially sporadic AD, and T2DM. In this study, animals were treated with icv-STZ and subcutaneous D-gal to establish an AD model, and the attenuating effects of curcumin on AD-like symptoms were investigated. Rats in the AD model group showed an increase in oxidative stress, neurodegeneration, and decline in learning and memory; curcumin depressed the neurodegeneration and behavioral decline of rats resulting from the combined treatments of icv-STZ and subcutaneous D-gal. These results imply that rats treated with a single injection of STZ in the bilateral hippocampus combined with a sustaining subcutaneous injection of D-gal is a suitable animal model for AD research and that curcumin is a possible treatment for AD.
