Curcumin counteracts the aluminium-induced ageing-related alterations in oxidative stress, Na+, K+ ATPase and protein kinase C in adult and old rat brain regions
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- Sharma, D., Sethi, P., Hussain, E. et al. Biogerontology (2009) 10: 489. doi:10.1007/s10522-008-9195-x
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This study investigated the effect of curcumin on aluminium-induced alterations in ageing-related parameters: lipid peroxidation, superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-s-transferase (GST), protein kinase C (PKC), Na+, K+-adenosine triphosphatase (Na+, K+-ATPase) and acetylcholinesterase (AChE) in the cerebral cortex and hippocampus of the brain of 10- and 24-month-old rats. Measurements taken from aluminium-fed rats were compared with those from rats in which curcumin and aluminium were co-administered. In aluminium-treated rats the levels of lipid peroxidation, PKC and AChE were enhanced while the activities of SOD, GPx, GST and Na+, K+-ATPase were significantly decreased in both the brain regions of both age-groups. In animals co-administered with curcumin and aluminium, the levels of lipid peroxidation, activities of PKC and AChE were significantly lowered while the activities of SOD, GPx, GST and Na+, K+-ATPase were significantly enhanced in the two brain regions studied indicating curcumin’s protective effects against aluminium toxicity. Though the magnitudes of curcumin-induced alterations varied in young and old animals, the results of the present study also demonstrated that curcumin exerts a protective effect against aluminium-induced elevation of ageing-related changes by modulating the extent of oxidative stress (by upregulating the activities of antioxidant enzymes) and by regulating the activities of Na+, K+ ATPase, PKC and AChE. Therefore, it is suggested that curcumin counters aluminium-induced enhancement in ageing-related processes.
KeywordsAnti-ageing effectsAntioxidant enzymesCurcuminNa+ K+-ATPaseProtein kinase CAluminium-induced neurotoxicityGlutathione-s-transferaseSuperoxide dismutaseGlutathione peroxidaseLipid peroxidationOxidative stress
Curcumin (diferuloylmethane), a yellow pigment extracted from the rhizome of the plant Curcuma longa (turmeric) has hydrogen-donating antioxidant activity due to the presence of phenolic groups (Jovanovic et al. 2001; Barcley et al. 2000; Sun et al. 2002) in its chemical structure. Curcumin is widely used as a food additive, and also as an herbal medicine throughout Asia. In India it is an extensively consumed spice in food. As an Ayurvedic medicine it is of interest for its anticholesteremic (Patil and Srinivasan 1971), antidiabetic (Srinivasan 1972), anti-inflammatory (Srimal and Dhawan 1973; Mukhopadhyay et al. 1982), and antirheumatic (Deodhar et al. 1980) actions. It has antioxidant property (Reddy and Lokesh 1994, 1996), and may act directly as a chemical oxidant or as a modulator of cellular defenses (Awasthi et al. 2000; Chakravarty and Yasmin 2008). Several studies (Piper et al. 1998; Surh 1999; Watanabe and Fukui 2000) have thus reported that curcumin inhibits lipid peroxidation, and augments the activities of antioxidants: superoxide dismutase (SOD), glutathione (GSH), glutathione-s-transferase (GST) and glutathione peroxidase (GPx) in a variety of cancers of different body organs. Curcumin crosses the blood-brain barrier (Yang et al. 2006), and has beneficial antioxidant effects on the brain (Rajakumar and Rao 1994; Rajkrishnan et al. (1999). Curcumin exerts modulatory effects on Na+, K+-ATPase activity in brain microsomes (Kaul and Krishnakanth 1994). Furthermore, Frautschy et al. (2001) and Calabrese et al. (2003) reported curcumin’s ability to protect against the amyloid beta-protein-induced brain damage in old rats, and suggested its clinical application in prevention of Alzheimer’s disease (AD). Curcumin’s major metabolite tetrahydrocurcumin was shown to: scavenge the reactive oxygen species (ROS) formed during hyperglycemia; inhibit the antioxidant enzymes including detoxification enzymes such as GST; and elevate GSH concentration in the cultured rat lens (Osawa and Kato 2005). Goel et al. (2008) has reviewed the overall interactions of curcumin with target molecules directly or indirectly involved in the various metabolic functions and enlisted the clinical trials with curcumin in patients with different diseases.
Aluminium (Al) occurs in the earth’s crust and is released into the environment by both natural and anthropogenic sources, and has been shown to progressively accumulate in the brain with normal ageing (Gomez et al. 1997; Mc Dermott et al. 1979; Markesbery et al. 1984; Deloncle et al. 1990, 2001; Xu et al. 1992; Anane et al. 1995). Al crosses the blood-brain barrier and forms deposits in brain regions such as the striatum, hippocampus and occipital cortex (Lal et al. 1993; Deloncle et al. 1995). Recently, Walton (2006) has demonstrated a progressive intraneuronal accumulation of Al in the nuclei and cytoplasm of hippocampal neurons of the aged AD-affected human brain. Walton (2007) also showed that rats ingesting aluminium equivalent to the high end of the human dietary aluminium range developed cognitive damage and some features of AD. Al is also regarded as an etiological agent in the pathogenesis of several free radical-mediated degenerative disorders associated with the impairment of cognitive functions including long-term memory (Jacqmin et al. 1994; Bolla et al. 1992). Prolonged intake of even a very low concentration of aluminium in drinking water, and from other sources may lead to ageing-related neurological dysfunctions (Brichall and Chappell 1988; Varner et al. 1998). Furthermore, aluminium seems to have a role in the acceleration of the normal ageing process (Deloncle et al. 2001; Kaur et al. 2003a).
It would be of great interest to find out whether food supplements endowed with antioxidative potential could prevent/reverse or reduce the Al-induced neurological alterations. Experimentally, aluminium-induced neurotoxicity model is often used for pathological and pharmacological investigations (Sarin et al. 1997; Kaur et al. 2003a, Gong et al. 2005; Nehru and Bhalla 2006; Sethi et al. 2008). Curcumin consumed as a food additive (Reddy and Aggarwal 1994) was found to be more potent in counteracting free radical-induced damage in the brain than vitamin E (Martin-Aragno et al. 1997). It may be hypothesized that the amount of curcumin that is ingested routinely in diet may counteract Al-induced neurotoxicity that may result from the aluminium ingested in the normal course. The present study was thus designed to evaluate the protective effect of curcumin on aluminium-induced neurotoxicity changes in ageing-related parameters in brain regions. Furthermore, in order to determine whether there is any age-related difference in the effect of curcumin, we studied the effect on animals of two age groups. Data described in this paper were obtained from rats fed aluminium chloride as well as curcumin to young and old age rats. The following biochemical parameters were studied: lipid peroxidation, Na+, K+-ATPase, protein kinase C (PKC) and cellular antioxidant enzymes: SOD, GPx, GST and the acetylcholine-degrading enzyme acetylcholinesterase (AChE) in the cerebral cortex and hippocampus.
Materials and methods
Animals and curcumin treatment
Male Wistar rats of 4 and 18 months of age (at the beginning of experiments) were used in the present study. Animals were obtained from the Central Animal Facility, Jawaharlal Nehru University, New Delhi, and housed in polypropylene cages on a 12L: 12D (light from 0600 to 1800 hours) cycle and fed ad libitum on commercial laboratory food pellets. The health status of each rat was checked as in our previous work (Sharma et al. 1993). All experimental protocols were approved by the Institutional Animal Ethics Committee of the University in accordance with the Government of India’s Committee for the Purpose of Supervision and Control of Experiments on Animals.
Curcumin and other chemicals were obtained from Sigma Chemicals Co. USA. P32 was obtained from The Board of Radiation and Isotope Technology (BRIT), Department of Atomic Energy Government of India, Hyderabad, India.
Curcumin was fed orally at a dose of 30 mg ml−1 kg−1 body weight dissolved in corn oil daily as used earlier (Reddy and Lokesh 1996), while aluminium was given at a dose of 50 mg kg−1 day−1 in a drinking water. Curcumin and aluminium were co-administered for 6 months to 4- and 18-month-old rats. These rats constituted experimental groups (n = 5 for each group). Water consumption was recorded to calculate the amount of Al ingested rat−1 day−1 (Lal et al. 1993; Rodella et al. 2001). In our experiments, daily aluminium intake calculated on the basis of water consumption was 12.51 ± 0.52 mg rat−1 day−1. The water consumption was found to remain similar in all the groups throughout the experimental period. Body weight of the animals was recorded daily. Rats (n = 5) receiving only AlCl3 in drinking water (50 mg kg−1 day−1) daily for 6 months were used as treated controls for animals that received both curcumin as well as aluminium. Rats (n = 5) receiving normal drinking water served as untreated controls for Al-treated rats. The food and water intake by experimental animals did not differ significantly from that of untreated control. The maximum lifespan of animals in our housing conditions is 28–30 months. Therefore, 24-month-old animals used in the present study had completed ~80% of their lifespan.
At the end of the 6 months of treatment period, the animals had attained the age of 10 and 24 months. Animals were killed by decapitation. Brains were immediately taken out and frozen in a deep freezer. The parietal cortex and hippocampus were rapidly dissected out on an ice-cold plate. The tissue specimens dissected from the left and right sides of the brain were pooled to make one sample and each assay was performed on a minimum of five animals (n = 5). The tissue samples were homogenized in 0.1 M-phosphate buffer (pH 7.0). Lipid peroxidation was measured in this homogenate. Crude synaptosomal and cytosol fractions were prepared by further differentially centrifuging the homogenate according to the method of Flohe and Gunzler (1984).
Lipid peroxidation (thiobarbituric acid reactive substance; TBARS) was measured by the method of Okhawa et al. (1979) as per the protocol given in Kaizer et al. (2005). Brain tissue was weighed, homogenized at a proportion of 1 g of tissue to 10 ml of buffer Tris—HCl 10 mM pH 7.4 plus 10% of sodium dodecylsulphate (SDS). The reaction mixture contained 200 μl of brain homogenate or standard (MDA-malondialdehyde 0.03 mM), 200 μl of 8.1% sodium dodecylsulphate (SDS), 750 μl of acetic acid solution (2.5 M HCl, pH 3.5) and 750 μl of 0.8% TBA. The mixtures were heated at 95°C for 90 min. After centrifugation at 1,700g for 5 min, the absorbance was measured at 532 nm. TBARS tissue levels were expressed as nmol MDA mg−1 protein.
SOD activity was measured according to the method of Marklund and Marklund (1974). The supernatant was added to a mixture (1 ml) containing 0.05 M sodium phosphate buffer (pH 8), 0.1 mM EDTA, and 0.02 mM pyrogallol. The assay was based on the ability of the enzyme to inhibit auto-oxidation of pyrogallol. The stock solution of pyrogallol was made in 10 mM HCl. One unit of the enzyme was defined as the amount of SOD required for producing a half maximal inhibition of autooxidation. Absorbance was measured for 5 min at 420 nm spectrophotometrically, and expressed as units min−1 mg−1 protein.
GPx activity was assayed according to the method of Flohe and Gunzler (1984). The final concentration in 1 ml reaction volume were 50 mM sodium phosphate buffer (pH 7.0) having 0.5 mM EDTA, 0.7 U/ml glutathione reductase, 0.3 mM reduced GSH. 1.5 mM NADPH in 0.1% sodium bicarbonate, 1.5 mM hydrogen peroxide and an appropriate amount of the homogenate. The decrease in the absorbance was read at 340 nm using a spectrophotometer (Shimadzu UV-260A). The non-enzymatic reaction rate was assessed by replacing the enzyme sample with buffer. The specific activity was expressed as μmoles of NADPH oxidized min−1 mg−1 protein.
GST activity was assayed as described by Habig et al. (1974). The reaction mixture (1 ml) contained a final concentration of 0.1 M phosphate buffer (pH 6.5), 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) in ethanol, 1 mM GSH and was incubated at 37°C for 5 min. The reaction was initiated by addition of enzyme sample and the enzyme activity was followed for 5 min at 340 nm. The specific activity was calculated using extinction coefficient 9.6 nm−1 cm−1 at 340 nm and expressed in terms of μmoles of CDNB-GSH conjugate formed min−1 mg−1 protein.
Na+, K+-ATPase activity was measured in the crude synaptosomal fraction according to the method described by Tanaka and Ando (1990). The synaptosomal fraction was prepared according to the method of de Roberties et al. (1963) in 0.32 M sucrose, 12.5 mM Tris and 1 mM EDTA (pH 7.4). The reaction mixture contained 100 mM NaCl, 20 mM KCl, 5 mM MgCl2, 3 mM ATP, 100 mM ouabain, 50 mM Tris—HCl buffer (pH 7.4), enzyme preparation and the desired reagent in 1.0 ml. After preincubation for 5 min at 37°C, the reaction was initiated by adding ATP, and was followed by incubation for 20 min at the same temperature. The reaction was terminated by adding 1.0 ml of ice-cold trichloroacetic acid (TCA). After centrifugation at 1,000g for 10 min, the inorganic phosphate produced in the supernatant was determined as per the method described by Lowry et al. (1951). The Na+, K+-ATPase activity was calculated from the difference in the amount of inorganic phosphate released in the absence and presence of ouabain. The enzyme activity was expressed as mM Pi released h−1 mg−1 protein−1 h−1.
Ca2+-dependent PKC activity was estimated according to the method of Hetherington and Trewavas (1982) and Paulraj and Behari (2004). After subcellular fractionation, the homogenate was suspended in the incubation medium containing 100 mM Hepes, 120 mM NaCl, 1.2 mM MgSO4, 2.5 mM KCl, 15 mM NaHCO3, 10 mM Glucose, 1 mM EDTA and protein concentration was measured according to the method described by Lowry et al. (1951). PKC activity was assayed in a total volume of 0.5 ml of the incubation medium [50 mM Hepes (pH 7), 10 mM MgCl2, 0.5 mM CaCl2, and 0.2 mM EDTA (free calcium level of 0.1 mM)]. After addition of 100 μg protein, the reaction was initiated by addition of γP32 labelled ATP (specific activity, 3,000 Ci mmol−1 ATP). Incubation was carried out at 25°C. Samples of 50 μl were taken out at appropriate intervals (30–60 s) and pipetted onto 3 mm filter discs which had been pretreated with 10% TCA, 20 mM sodium pyrophosphate, and 10 mM EDTA. These filter discs were dropped into 500 ml of the TCA mixture and left overnight at 0°C. Filters were washed once in 5% TCA, heated to 90°C for 15 min in 10% TCA and extracted in hot ethanol/ether (3:1 v/v) before drying. Radioactivity was measured by a Beckman β counter (LS 1800). Results were expressed as β-counts mg−1 protein min−1.
Acetylcholinesterase activity was measured according to the method described by Ellman et al. (1961) using acetylthiolcholine iodide as a substrate. The assay mixture (1 ml final volume) contained the following: 84 mM sodium phosphate buffer (pH 8.0), 0.01 M DTNB (dinitrobenzoic acid), 0.48 mM acetylcholiniodide as substrate and 150 μg enzyme per assay. The enzyme activity was measured spectrophotometrically at 412 nm at an interval of 30 s for 3 min. The specific activity of the enzyme was calculated as micromoles of thiocholine produced mg−1 protein−1 min−1 at room temperature.
Results were evaluated by Student’s t-test. Data derived from aluminium-treated animals of both age groups were compared with those from curcumin plus aluminium treated animals to assess curcumin’s effect on aluminium-induced changes.
Effect of Aluminium
In 10-month-old aluminium-treated rats, levels of lipid peroxides, and the activities of PKC and AChE were significantly (P < 0.001) increased in the cortex (Fig. 1a) as well as in the hippocampus (Fig. 2b, c) compared to the age-matched controls. In terms of percent changes: lipid peroxidation was increased by 129.16% in the cortex and by 85.45% in the hippocampus; whereas for PKC and AChE, the increases were 39.90 and 61.20% respectively, in the cortex; and 33.29 and 43.33% respectively in the hippocampus. The data would thus show that aluminium treatment elevates lipid peroxidation as well as the activities of PKC and AChE. In aluminium-treated rats the activities of SOD, GPx and GST declined in both the brain regions compared to their age-matched controls (Fig. 1b–d, respectively). Percentage decrease for SOD, GPx and GST in the cortex was 43.74, 42.56 and 40.56% respectively, whereas in the hippocampus the decrease was 52.96, 40.00 and 23.08% respectively, showing thereby that aluminium treatment depressed the activities of SOD, GPx and GST. In aluminium-treated rats the activity of Na+, K+-ATPase declined significantly by 51.99% in the cortex and 43.34% in the hippocampus showing thereby that aluminium treatment inhibited Na+, K+ ATPase activity.
In 24-month-old aluminium-treated rats also, the levels of lipid peroxidation and the activities of PKC and AChE were significantly (P < 0.001) increased in both the brain regions compared to the age-matched controls (Figs. 1a, 2b, c, respectively). Lipid peroxidation in the cortex and hippocampus increased by 170.83 and 150.90% respectively, whereas the activities of PKC and AChE increased by 21.89 and 38% respectively in the cortex and by 39.36 and 20% respectively in the hippocampus, showing thereby that aluminium treatment caused elevation of lipid peroxidation and PKC and AChE activities. The activities of SOD, GPx and GST significantly decreased in comparison with age-matched controls. Similarly, unlike 10-month-old animals, the percent decreases in the activities of SOD, GPx and GST in 24-month-old animals were significantly (P < 0.001) higher (Fig. 1b–d, respectively). In the cortex the activities of SOD, GPx and GST declined by 40.93, 66.14 and 59.56% respectively, whereas in the hippocampus the activities declined by 49.6, 55.94 and 64.02% respectively, which were significantly higher (P < 0.001) than those of 10-month-old rats. The activity of Na+, K+-ATPase also showed a decrease in the cortex and hippocampus. The percentage decrease was 64.48 and 53.6% respectively.
Effect of co-administration of curcumin and aluminium
Measurement of parameters taken from the brain regions of animals of both age groups which were co-administered with curcumin and aluminium when compared with those from aluminium-treated ones showed that the levels of the various parameters remained near to the pre-aluminium treatment levels showing thereby that curcumin administration countered the toxic effects of aluminium (Figs. 1, 2). For example, in curcumin + aluminium-treated young animals, lipid peroxidation levels were elevated only by 33% in the cortex, and by 9% in the hippocampus when compared with those in aluminium-treated animals in which they were elevated by 129% in the cortex, and by 85% in the hippocampus. The levels thus became similar to those of untreated controls (Fig. 1a). This indicates that curcumin prevented aluminium-induced rise in lipid peroxidation in brain regions of both age groups. However, in old animals the elevation was a little higher than that of young animals. Similarly, in curcumin + aluminium-treated young animals, the activities of SOD, GPx, GST and Na+, K+-ATPase were decreased by 6.84, 2.13, 19.4 and 11.91% respectively, in the cortex and by 6.46, 2.0, 10.25 and 33.74% respectively in the hippocampus when compared with those in aluminium-treated age-matched animals in which they were decreased by 43.74, 42.56, 40.56 and 51.99% respectively in the cortex and by 52.96, 40, 23.08 and 53.6% respectively in the hippocampus. This indicated that curcumin prevented aluminium-induced inhibition in the activities of SOD, GPx, GST and Na+, K+ ATPase in the brain regions. However, in the old curcumin + aluminium-treated rats the decline was less when compared to the young curcumin + aluminium-treated animals. Thus the levels of various parameters remained similar to those of untreated controls (Figs. 1b–d, 2a). The activities of PKC and AChE were elevated by 2.55 and 12.0% respectively in the cortex and by 9.07 and 6.66% in the hippocampus of young curcumin + aluminium-treated young animals when compared with those of aluminium-treated age-matched animals in which they were elevated by 39.90 and 61.2%, respectively in the cortex, and by 33.29 and 43.33%, respectively in the hippocampus. The activities thus became similar to those of untreated controls (Fig. 2b, c). This indicated that curcumin prevented the aluminium-induced elevation of these enzyme activities.
Our data from the two age groups (10 and 24 months) revealed significant age-related alterations in the parameters studied. For example, there was a significant age-related increase in lipid peroxidation which is in agreement with previous observations on ageing-related changes in oxidative stress (Sharma et al. 1993; Kaur et al. 2001, 2003b). The results showed an age-related decline in PKC activity in 24-month-old animals. This is in agreement with previous reports (Friedman and Wang 1989; Battaini and Pascale 2005; Igwe and Filla 1995; Van der Zee et al. 2004). Recently, Pascale et al. (2007) have confirmed the deregulation of PKC with age. PKC, a proteolytically activated protein kinase, is said to modulate: ion conductance by phosphorylating membrane proteins (Nishizuka 1986), extrusion of Ca2+ immediately after its mobilization into the cytosol (Byus et al. 1984), neurite outgrowth and regrowth rate (Kabir et al. 2001) and synaptic plasticity (Sossin 2007). Spatial learning (Fordycee and Wehner 1993), and active maintenance of information required for the working memory (Runyan et al. 2005) involve transient changes in the balance of PKC activity. There are reports indicating involvement of the soluble PKC in the extrusion of Ca2+ immediately after its mobilization into the cytosol (Byus et al. 1984), and in Al-induced neurotoxicity (Katsuyama et al. 1989). Earlier reports have indicated: the translocation of PKC’s activity from cytosol to the membrane after lead treatment (Laterra et al. 1992; Markovac and Goldstein 1988; Zhao et al. 1998), and age-related loss in the activity of membrane-associated Ca2+/phospholipid dependent PKC isozymes leading to the loss in signal transduction mechanism (Igwe and Filla 1995) in membranes. Therefore, the aluminium-induced increase in PKC activity observed in our study could be a result of the movement of PKC from the membrane to the cytosol. The movement of PKC from the plasma membrane could be due to the loss of fluidity of the plasma membrane.
Our data showing a fall in the AChE activity from 10 to 24 months of age in the hippocampus is in agreement with our previous report (Sharma and Singh 1995). Nakamura and Ishihara (1989) and Sirvio et al. (1989) also showed a decline in AChE activity with advancing age. Our data also showed the age-related decline in the activities of SOD, GPx, GST and Na+, K+ ATPase in 24-month-old rats compared to 10-month-old rats. This is in agreement with our previous report (Kaur et al. 2001).
In aluminium-treated animals we have not measured the accumulation of Al in the brain regions studied. Earlier studies have already shown that ingested Al leads to the elevation of the brain content of Al in various brain regions (Deloncle et al. 2001; Kaur et al. 2003b, Lal et al. 1993; Rodella et al. 2001). In humans also an increased accumulation of Al in the brain due to industrial exposure to Al dust and other sources has been reported (Mclaughlin et al. 1962; Rifat et al. 1990). That aluminium accumulates in neurons overtime and causes oxidative stress both on its own (Zatta et al. 1993), as well as in association with that initiated by iron and copper (Roskans and Cinnor 1990; Redhead et al. 1992) in the brain has been reported in many studies (Florrence et al. 1994, 1995; Van Rensberg et al. 1995, 1997). Although the mechanism by which Al interacts with the nervous system is not well understood, results of Julka and Gill (1996) suggested that aluminium-induced neurotoxicity resulted from aluminium-induced modification of the intracellular calcium messenger system with detrimental consequences on neuronal functioning. In vitro, and electron microscopic studies have shown astrocytes in the brain as the principal target of Al’s toxic action leading to neuronal cell loss and degeneration (Suarez-Fernandez et al. 1999). There is evidence indicating that Al induces lipid peroxidation (Chainy et al. 1993; Lal et al. 1993; Kaur et al. 2003a), and inhibits the activity of Na+, K+ ATPase (Ure and Perassolo 2000), GPx and GST (Kaur et al. 2001, 2003a) in the brain which lends support to the present data. The results of the present study clearly showed that chronic aluminium treatment elevated the levels of lipid peroxidation and depressed the activities of SOD, GPx, GST in the brain cortex and hippocampus of 10-month-old as well as 24-month-old rats. This reflects aluminium-induced oxidative stress. The present data also showed that aluminium inhibited the enzyme Na+, K+ ATPase which is involved in maintaining Na+, K+ ion gradients across the cell membrane. Since this enzyme is susceptible to the cell membrane’s lipid environment, aluminium inhibition of this enzyme may largely be a consequence of aluminium-induced oxidative stress (lipid peroxidation). This has also been reported in many studies.
Our results showed that both AChE and PKC were activated by aluminium treatment. It is known that aluminium acts as a cholinotoxin and may cause cholinergic system dysfunction that may contribute to learning and memory deficits similar to those observed in Alzheimer’s dementia (Gulya et al. 1990). It is of interest to note here that cholinesterase inhibitors have some therapeutic significance in AD (Disterhoft and Oh 2006). Thus aluminium’s action on cholinesterase may be related to aluminium’s involvement in the aetiology of AD pathological process (Zatta et al. 1994). However, the effects of aluminium on AChE remain controversial as both aluminium-induced inhibition and activation have been reported (Kaizer et al. 2005).
In vitro studies of the effect of aluminium on PKC from mouse brain and rat hippocampal slices have indicated that aluminium inhibits PKC (Katsuyama et al. 1989; Proven and Yokel 1992). Some in vivo studies also indicate that aluminium inhibits PKC (Julka and Gill 1996). However, effects of aluminium on PKC in vivo have not been extensively studied. The PKC pathway is involved in diverse physiological, cognitive and pathological conditions including age-related pathologies that are responsible for learning and memory disturbance (Pascale et al. 2007). Aluminium is known to produce deficits in learning and memory (Proven and Yokel 1992) and PKC is also known to be involved in learning and memory (Fordycee and Wehner 1993), Our present in vivo study, however, showed that chronic aluminium treatment caused the activation of PKC both in the cerebral cortex and hippocampus. It will also be of interest to note that exposure to lead (Pb) also causes activation of cytosolic PKC activity (Zhao et al. 1998). There are studies which show that PKC activation increases basal neural activity (increased unit activity firing rate) (Kubo and Hagiwara 2005). We have also previously observed that chronic aluminium treatment results in enhanced multiple unit action potentials and produce epileptiform hyperactivity in brain regions (Kaur et al. 2003a). Therefore, the aluminium-induced enhanced activity of PKC observed in our study may have some relationship with increased electrical activity resulting from aluminium’s action. PKC activation has also been suggested to be neuroprotective against neuronal damage (Pascale et al. 2007). Therefore, aluminium-induced activation of PKC may also be a defensive response.
Our data show that administration of curcumin to aluminium-fed rats reversed the changes induced by aluminium. In curcumin + Al-fed 10- and 24-month-old rats the Al-induced increase in lipid peroxidation was significantly reduced and the Al-induced inhibition in the activities of SOD, GST and GPx was significantly retarded. This shows that curcumin has potential antioxidative properties. Curcumin is known for its antioxidant and antilipidperoxidative capability against a variety of oxidative stresses and thus can suppress oxidative damage (Aggarwal and Harikumar 2008). For example, the oxidative stress induced by ferrous ions (Rajakumar and Rao 1994), alcohol (Rajkrishnan et al. 1999), nicotine (Kalpana and Menon 2004) and the ageing process in the brain (Bala et al. 2006) was reversed by curcumin. Curcumin has been shown to inhibit the superoxide anion generation in xanthine–xanthine oxidase system as well as the hydroxyl (OH) ion generation to the extent of 40 and 76%, respectively (Reddy and Lokesh 1994, 1996). Thus, curcumin fed simultaneously with Al counters Al-induced free radical generation and to a great extent protects the brain against Al-induced peroxidative damage. In this respect, curcumin would appear to be similar to Ginko biloba (English-Kew Tree) which is a well known flavonoid glycosides-containing plant (Gong et al. 2005) and is currently under investigation for therapeutic use in Alzheimer disease (Khare 2007).
An earlier study has demonstrated that curcumin contains two electrophilic ά, β-unsaturated carbonyl groups, which can react with nucleophilic compounds such as GSH and form glutathionated products of curcumin (Awasthi et al. 2000). In previous studies, curcumin-induced reversal of lipid peroxidation and an enhancement of GSH in the brain of alcohol- intoxicated rats was reported (Rajkrishnan et al. 1999). Our studies show the curcumin inhibition of the Al-induced decline in SOD, GPx and GST activity. This shows the capability of curcumin to combat oxidative stress-related neurotoxicity. Oxidative stress is a prevalent condition in many clinical conditions including AD, traumatically injured brain, Parkinson’s disease etc. Thus curcumin is likely to be beneficial in such clinical conditions (Aggarwal and Harikumar 2008).
In Curcumin + Al-treated 10- and 24 month-old-rats, the Al-induced decline in Na+, K+ ATPase activity was significantly reduced, compared to age-matched Al-treated controls indicating that curcumin reversed aluminium-induced decline in Na+, K+ ATPase activity. Kaul and Krishnakanth (1994) showed a 148% increase in Na+, K+-ATPase activity in brain microsomes from curcumin-fed rats. Since an increase in Na+, K+-ATPase activity may be indicative of an increase in excitability, curcumin’s ability to increase the Na+, K+-ATPase activity in the ageing brain points to its potential in elevating the excitability of the aged neuronal tissue (Bala et al. 2006). The mechanism by which Na+, K+-ATPase activity may be increased by curcumin needs consideration. Phospholemman (PLM) is an accessory protein associated with Na+, K+-ATPase (Crambert et al. 2002). It has been shown to modulate the enzyme activity in the cardiac and skeletal muscle sarcolemma of Xenopus (Crambert et al. 2002) and rat. Recently, researchers have also shown the abundance of PLM in selected areas of the CNS. This protein has also been suggested to be an activator of Na+, K+-ATPase in the CNS (Feschenko et al. 2003). Curcumin activation of Na+, K+ ATPase may be a consequence of an activation of PLM. Since Na+, K+-ATPase activity is also known to be sensitive to lipid peroxidation (Mattson 1998), curcumin’s antilipidperoxidative activity may be responsible for the activation of Na+, K+-ATPase. Our data substantiate that curcumin, if fed simultaneously with Aluminium, tends to inhibit the Al-induced decrease in Na+, K+ ATPase. Curcumin’s influence on Na+, K+ ATPase would be indicative of its beneficial influence on neuronal membrane excitability since this enzyme is implicated in neuronal excitability process in ageing, epileptogenesis etc. (Kaur et al. 1998, 2003a, b).
In curcumin + aluminium-fed rats, the PKC activity was significantly reduced in comparison with that in aluminium-treated rats indicating that curcumin treatment reverses aluminium-induced enhancement in PKC activity. Recent findings indicate that polyphenoles and their in vivo metabolites do not act as conventional hydrogen-donating antioxidants but may exert their modulatory action in cells through a variety of protein kinases and signaling molecules. For example, Bastianetto et al. (2007) in their study on polyphenols derived from red wine and tea, suggested that polyphenoles are likely to modulate the plasma membrane and various intracellular effectors such as PKC and may directly interact with proteins that play a critical role in amyloiodogenesis. Similarly, curcumin has also been shown to significantly inhibit the activity of several protein kinases including phosphorylase kinase, PKC and protamine kinase (Aggarwal et al. 2006; Reddy and Aggarwal 1994; Shishodia et al. 2007). Therefore, curcumin by way of its antilipidperoxidative and cellular Ca2+-regulating capabilities (Logan-Smith et al. 2001) may help to keep the PKC activity significantly lower in aluminium-treated rats. Modulating PKC transduction pathways has been considered as an interesting novel approach for developing new therapeutic tool in physiologic and pathogenic conditions (Pascale et al. 2007). Thus, curcumin’s potential in regulating PKC pathway is of therapeutic significance.
Administration of curcumin reversed the increase in AChE activity induced by aluminium treatment. Since Al interacts with cholinergic system as a cholinotoxin (Gulya et al. 1990), it appears that curcumin given simultaneously with aluminium prevents aluminium-induced toxic effects on the cholinergic system and tends to optimize the AChE level in the brain. Aluminium, oxidative stress, and cholinergic dysfunction have all been implicated in AD (Kaizer et al. 2005). Oxidative stress can affect synaptic plasticity and cognition (Wu et al. 2006). Our data show that curcumin treatment significantly protects against aluminium-induced changes in oxidative stress and AChE dysfunctions. Curcumin’s chemoprotective effects in AD should thus be of clinical interest. Aluminium may be neurotoxic even at low doses (Kaizer et al. 2005; Walton 2007). In our current study, the dose of aluminium used is considerably high. Thus, our findings would indicate that curcumin is able to counter the effects of aluminium ingested even at a very high dose. It would thus be of interest to note that the curcumin that is consumed as spices in routine diet by humans would have protective effects against aluminium that may be ingested via routine diet. This shows the capacity of a dietary factor that can modulate a variety of alterations that may be caused in the brain function by adverse factors. For example supplementation of curcumin in the diet was found to normalize levels of BDNF (brain-derived nerve growth factor), synapsin I (involved in neurotransmitter release), and cyclic AMP-response element-binding protein (CREB—a transcription factor involved in learning and memory) which tend to be altered by the oxidative stress induced by traumatic brain injury (Wu et al. 2006).
In sum, the results of the present study show that curcumin exerts its protective influence against aluminium-induced toxicity and ageing-related acceleratory changes by modulating oxidative parameters and by augmenting and regulating antioxidant and other enzymatic systems such as PKC, Na+, K+ ATPase and cholinesterase. The present findings are of considerable clinical relevance as curcumin has been found to improve the clinical outcome of patients suffering from a great variety of diseases/disorders and extensive laboratory research has also produced data indicating its great relevance as a single therapeutic agent that can modulate multiple cellular targets (Goel et al. 2008; Aggarwal and Harikumar 2008).