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Chronic administration of troxerutin protects mouse brain against d -galactose-induced impairment of cholinergic system
Neurobiology of Learning and Memory 93 (2010) 157–164
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Chronic administration of troxerutin protects mouse brain against D-galactose-induced impairment of cholinergic system Jun Lu a,1, Dong-mei Wu a,b,1, Bin Hu a,1, Wei Cheng b, Yuan-lin Zheng a,*, Zi-feng Zhang a, Qin Ye a, Shao-hua Fan a, Qun Shan a, Yong-jian Wang a a b
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Xuzhou Normal University, Xuzhou 221116, Jiangsu Province, PR China School of Environment and Spatial Informatics, China University of Mining and Technology, Xuzhou 221008, Jiangsu Province, PR China
a r t i c l e
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Article history: Received 3 August 2009 Revised 9 September 2009 Accepted 12 September 2009 Available online 17 September 2009 Keywords: Troxerutin D-Galactose ROS Cholinergic system AchE nAchRa7
a b s t r a c t Previous evidence showed that administration of D-galactose (D-gal) increased ROS production and resulted in impairment of cholinergic system. Troxerutin, a natural bioflavonoid, has been reported to have many benefits and medicinal properties. In this study, we evaluated the protective effect of troxerutin against D-gal-induced impairment of cholinergic system, and explored the potential mechanism of its action. Our results displayed that troxerutin administration significantly improved behavioral performance of D-gal-treated mice in step-through test and morris water maze task. One of the potential mechanisms of this action was decreased AGEs, ROS and protein carbonyl levels in the basal forebrain, hippocampus and front cortex of D-gal-treated mice. Furthermore, our results also showed that troxerutin significantly inhibited cholinesterase (AchE) activity, increased the expression of nicotinic acetylcholine receptor alpha 7 (nAchRa7) and enhanced interactions between nAchRa7 and either postsynaptic density protein 95 (PSD95) or N-methyl-D-aspartate receptors subunit 1 (NMDAR1) in the basal forebrain, hippocampus and front cortex of D-gal-treated mice, which could help restore impairment of brain function. Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved.
1. Introduction Troxerutin is a trihydroxyethylated derivative of the natural bioflavonoid rutin. Sufficient evidence has shown that it has many biologic effects, such as anti-oxidative, anti-inflammatory, antierythrocytic, anti-fibrinolyti, anti-thrombotic, antineoplastic, and anti-c-radiation effects (Blasig, Loewe, & Ebert, 1987, 1988; Boisseau et al., 1995; Fan et al., 2009; Gandhi et al., 2004; Kessler, Ubeaud, Walter, Sturm, & Jung, 2002; Wenisch & Biffignandi, 2001). Recently, reports from our laboratory also confirmed a renal protective effect of it. D-galactose (D-gal) is a reducing sugar and can be metabolized at normal concentration. However, at high levels, it reacts readily with the free amines of amino acids in proteins and peptides in vivo to form advanced glycation end products (AGEs) (Lu et al., 2007; Tian et al., 2005). A substantial amount of evidence reveals that AGEs induce ROS production and increase protein carbonyl levels, which will exacerbate and accelerate aging process and trigger the early phases of age-related disease such as diabetes, arteriosclerosis, nephropathy, infection, and Alzheimer’s disease (Cai, He, Zhu, Lu, & Vlassara, 2006; Srikanth et al., 2009; Tian * Corresponding author. Fax: +86 516 83500348. E-mail addresses: [email protected], [email protected] (Y.-l. Zheng). 1 These authors contributed equally to this work.
et al., 2005). Recent findings have further demonstrated that continuous subcutaneous injection of D-gal in rodent induced production of free radicals, subsequently leading to the decreased expression of memory-related protein and deterioration of learning and memory function which might be associated with impairment of the basal forebrain cholinergic system (Cui et al., 2006; Hua et al., 2007; Lei et al., 2008; Long et al., 2007; Lu et al., 2006; Zhang, Li, Cui, & Zuo, 2005). The basal forebrain cholinergic system comprised of closely associated nuclei containing cholinergic neurons that project to both hippocampus and cortical areas plays a key role in modulating learning and memory processes (Niewiadomska, Baksalerska-Pazera, & Riedel, 2009). Interest in the function of the basal forebrain cholinergic system greatly increased with the neuropathological demonstration that cholinergic markers in the hippocampus and cerebral cortex are changed in Alzheimer’s disease (AD) (Greig et al., 2005; Niewiadomska et al., 2009) and that these changes have been linked to its pathology and the degree of cognitive impairment (Greig et al., 2005). Furthermore, cholinergic and memory deficits (Decker, 1987; Kubanis & Zornetzer, 1981) are also found in normal aging, although these dysfunctions differ from those reported in AD. Therefore, much of the research on cognitive decline has focused on the cholinergic system (Bacciottini, Passani, Mannaioni, & Blandina, 2001; Everitt & Robbins, 1997), and related treatment strategies have traditionally aimed at restoring the cholinergic neurotransmission.
1074-7427/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.nlm.2009.09.006
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However, therapies with cholinesterase inhibitors or muscarinic agonists are limited by unacceptable side-effects. Now, no work has been done to study whether troxerutin has an effect against D-gal-induced impairment of cholinergic system in mouse model. In the present study, we addressed this issue and investigated the potential mechanism underlying its action. 2. Materials and methods 2.1. Animals and administration Thirty-two 10-week-old male Kunming strain mice were purchased from the branch of national breeder center of rodents (Shanghai). Prior to experiments mice had free access to food and water and were kept under constant conditions of temperature (23 ± 1 °C) and humidity (60%). Eight mice were housed per cage on a 12-h light/dark schedule (lights on 08:30–20:30). After a week of adaptation, mice were divided randomly into four groups. Group 1 and group 4 served as vehicle control with injection of saline (0.9%), and the other two groups of mice (groups 2 and 3) received daily subcutaneous injection of D-gal (Sigma– Aldrich, MO, USA) at dose of 500 mg/(kg day) for 8 weeks, respectively. At the same time, mice in groups 3 and groups 4 received daily troxerutin (troxerutin; Purity > 99%; Baoji Fangsheng Biotechnology Co., Ltd., Baoji, China) of 150 mg/(kg day) in distilled water by oral gavage for 8 weeks, and the mice of groups 1 and 2 were given distilled water orally at the same dose. All experiments were performed in compliance with the Chinese legislation on the use and care of laboratory animals and were approved by the respective university committees for animal experiments. After the behavioral testing, mice were sacrificed and brain tissues were immediately collected for experiments or stored at 70 °C for later use. 2.2. Behavioral tests 2.2.1. Step-through test The step-through passive avoidance apparatus consisted of an illuminated chamber (11.5 cm 9.5 cm 11 cm) attached to a darkened chamber (23.5 cm 9.5 cm 11 cm) containing a metal floor that could deliver footshocks. The two compartments were separated by a guillotine door. The illuminated chamber was lit with a 25 W lamp. The step-through test was performed as described previously (Lu et al., 2006). 2.2.2. Morris water maze test The Morris water maze (MWM) test was performed as described previously (Lu et al., 2006; Wu et al., 2008). The experimental apparatus consisted of a circular water tank (100 cm in diameter, 35 cm in height), containing water (23 ± 1 °C) to a depth of 15.5 cm, which was rendered opaque by adding ink. A platform (4.5 cm in diameter, 14.5 cm in height) was submerged 1 cm below the water surface and placed at the midpoint of one quadrant. The pool was located in a test room, which contained various prominent visual cues. The test was performed as described previously. Each mouse received four training periods per day for four consecutive days. Latency to escape from the water maze (finding the submerged escape platform) was calculated for each trial. On day five, the probe test was carried out by removing platform and allowing each mouse to swim freely for 60 s. The time that mice spent swimming in the target quadrant (where the platform was located during hidden platform training) was measured. For the probe trials, the number of times crossing over the platform site of each mouse was also measured and calculated. All data were recorded with a computerized video system.
2.3. Preparation of tissue samples 2.3.1. Tissue homogenates For biochemical studies performed as described previously (Lu et al., 2006, 2007), animals were deeply anesthetized and sacrificed. Brains were removed and dissected on ice to obtain the basal forebrain, hippocampus and front cortex. Tissues were homogenized in 1/5 (w/v) 50 mM (pH 7.4) ice-cold Tris buffer saline solution (TBS) containing a protease inhibitor cocktail (Sigma–Aldrich, MO, USA) with 10 strokes at 1200 rpm in a Potter homogenizer. Homogenates were centrifuged at 12,000g for 10 min to obtain the supernatant. Supernatant aliquots were used to determine brain ROS, cholinesterase (AchE) activity, protein carbonyl levels in the basal forebrain, hippocampus and front cortex. For Western blot analysis performed as described previously (Lu et al., 2006; Wu et al., 2008), the basal forebrain, hippocampus and front cortex were homogenized in 1/3 (w/v) ice cold RIPA lysis buffer (1 TBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium azide) combining 30 ll of 10 mg/ml PMSF solution, 30 ll of Na3VO4, 30 ll of NaF and 30 ll of protease inhibitors cocktail per gram of tissue. The homogenates were sonicated four times for 30 s with 20 s intervals using a sonicator, centrifuged at 15,000g for 10 min at 4 °C, and the supernatant was collected and stored at 70 °C for Western blot studies. Protein levels in the supernatants were determined using the BCA assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). 2.4. Biochemical analysis 2.4.1. Assay of ROS ROS was measured as described previously, based on the oxidation of 20 70 -dichlorodihydrofluorescein diacetate to 20 70 -dichlorofluorescein (Shinomol, 2007). Briefly, the homogenate was diluted 1:20 times with ice-cold Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.0 mM CaCl2, 10 mM D-glucose, and 5 mM HEPES, pH 7.4) to obtain a concentration of 5 mg tissue/ml. The reaction mixture (1 ml) containing Locke’s buffer (pH 7.4), 0.2 ml homogenate and 10 ml of DCFH-DA (5 mM) was incubated for 15 min at room temperature to allow the DCFH-DA to be incorporated into any membrane-bound vesicles and the diacetate group cleaved by esterases. After 30 min of further incubation, the conversion of DCFH-DA to the fluorescent product DCF was measured using a spectrofluorimeter with excitation at 484 nm and emission at 530 nm. Background fluorescence (conversion of DCFH-DA in the absence of homogenate) was corrected by the inclusion of parallel blanks. ROS formation was quantified from a DCF-standard curve and data are expressed as pmol DCF formed/ min/mg protein. 2.4.2. Oxidative stress marker: protein carbonyls Protein carbonyl content was measured as a marker of oxidative damage to proteins. They were determined the hydrazone derivatives between 360 and 390 nm (e = 21.0 mM1 cm1) according to the method described earlier (Shinomol, 2007). Data are expressed as nmol carbonyls/mg protein. 2.4.3. Assay of AchE activity AchE activity was determined according to the method of Shinomol et al. ( 2007). To a reaction mixture containing 2.85 ml phosphate buffer (0.1 M, pH 8.0), 50 ll of DTNB (10 mM), 50 ll sample and 20 ll acetylthiocholine iodide (78 mM) were added and the increase in absorbance was monitored at 412 nm for 5 min in a spectrophotometer and the enzyme activity is expressed as nmol of substrate hydrolyzed/min/mg protein.
J. Lu et al. / Neurobiology of Learning and Memory 93 (2010) 157–164
2.5. AGE-enzyme-linked immunosorbent assay Quantitative measurement of AGEs was performed by AGE-ELISA as described previously (Song, Bao, Li, & Li, 1999). Briefly, 96-well plates were coated with 100 ll/well of 3 lg/ml AGE-BSA in coating buffer overnight at 4 °C. Wells were washed three times with 150 ll washing buffer (PBS, 0.05% Tween-20, 1 mM NaN3), then blocked with 1% normal goat serum in 100 ll PBS for 2 h at 37 °C. After washing, 50 ll of 1:10 diluted samples in dilution buffer (PBS, 0.02% Tween-20, 1 mM NaN3, 1% normal goat serum) and 50 ll anti-AGE polyclonal antiserum in dilution buffer (1:2000) were added. Plates were incubated at room temperature for 2 h with gentle agitation on a horizontal rotary shaker. Wells were washed, 100 ll alkaline phosphate conjugated 2nd antibody in dilution buffer (1:2000) was added, and the plates were incubated at 37 °C for 1 h. They were then washed six times as above, 100 ll p-nitrophenyl phosphate substrate was added to each well. After 60 min optical density (OD) at 405 was determined by a microplate fluorometer Flex station II (Molecular Devices, San Francisco, CA, USA). The AGE-BSA was used as a competing antigen to generate an AGE standard curve. Sample AGE values were calculated from the standard curve. 2.6. Immunoprecipitation assay and Western blot analysis Immunoprecipitation assay was performed as described previously (Farias et al., 2007). The brain was homogenized in 1/3 (w/v) ice cold lysis buffer (25 mM HEPES, pH 7.4, 125 mM NaCl, 25 mM NaF, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1 mM Na3VO4, and the protease inhibitor mixture). Equal amounts of protein (500 lg) were precleared using protein A-Sepharose for 1 h at 4 °C and then incubated with 3 lg of antibody against the nAchRa7. The immune complexes were affinity precipitated with protein A-Sepharose beads and washed six times with 25 mM HEPES buffer, pH 7.4, 10 mM MgCl2, 1 mM NaF, 1% NP-40, and 1 mM Na3VO4. The immune complexes were then submitted to SDS–PAGE and analyzed by Western blot with mouse anti-post-synaptic density-95 (anti-PSD95) antibody (1:1000, Abcam, Cambridge, UK) and rabbit polyclonal antiNMDAR1 (1:1000, Cell Signaling Technology, Inc., Beverly, MA, USA). Samples (80 lg protein) were separated by denaturing SDS– PAGE and transferred to a PVDF membrane (Roche Diagnostics Corporation, Indianapolis, IN, USA) by electrophoretic transfer. The membrane was blocked with 5% non-fat milk and 0.1% Tween-20 in TBS, incubated overnight with rabbit anti-nicotinic acetylcholine receptor alpha 7 (anti-nAchRa7) (1:500, Abcam, Cambridge, UK). Quantitation of detected bands was performed with the scion image analysis software (Scion Corp., Frederick, MD, USA). The data were normalized using b-actin as an internal control and standardized with the vehicle control as 1.0. 2.7. Statistic analysis All statistical analyses were performed using the SPSS software, version 11.5. Group differences in the escape latency in the MWM training task were analyzed using two-way analysis of variance (ANOVA) with repeated measures, the factors being treatment and training day. The other data were analyzed with one-way ANOVA followed by Newman–Keuls or Tukey’s HSD post hoc test. Data were expressed as means ± SEM. Statistical significance was set at p < 0.05.
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the initial latencies did not differ significantly among the four groups [F(3, 28) = 0.259, p > 0.05] (Fig. 1). One-way ANOVA of passive avoidance task data revealed that D-gal significantly reduced the step-through latencies in the 24 h-retention trial [F(3, 28) = 4.888, p < 0.05]. This analysis also showed that the latencies in D-gal-treated mice received daily 150 mg/(kg day) troxerutin for 8 weeks were markedly lengthened as compared with D-gal-treated mice [F(3, 28) = 4.888, p < 0.05]. The shortened latencies induced by D-gal were restored to normal level after oral administration of troxerutin. There was no significant difference in step-through latencies between the control group and the troxerutin group. The result suggested that no obvious neural toxicity in troxerutin-treated mice was found. 3.1.2. Morris water maze test The Morris water maze is a useful tool for assessing spatial cognitive function. During the training session, all groups of mice improved their performance as indicated by the shortened escape latencies across successive days (Fig. 2A). Mice showed significant difference in mean latencies between training days [F(3, 112) = 25.698, p < 0.001] (Fig. 2A) and between treatments [F(3, 112) = 10.578, p < 0.001], but no interaction between the factors day and treatment [F(9, 112) = 1.207, p > 0.05]. Two-way ANOVA showed that the escape latencies were significantly higher in D-gal-treated mice than in control mice (p < 0.001). This result indicated that the spatial learning and memory ability in D-gal-treated mice was impaired. A comparison between the D-gal group and the troxerutin/D-gal group showed that the troxerutin could shorten escape latencies of D-gal-treated mice (p < 0.001). These data showed that troxerutin could improve cognitive performance in the D-gal-treated mice, and there was no significant difference in spatial learning and memory ability between the control group and the troxerutin/D-gal group. No significant difference between the control group and the troxerutin group was found. Note that mice of every treatment started at the same level of performance (no significant individual effect was observed for the first four trials of day 1). The withdrawal of the platform induced a general tendency to swim, preferentially to other equivalent zones, in the quadrant where the platform was previously located and in the platform zone. The control, the troxerutin and the troxerutin/D-gal group spent more time swimming in the target quadrant (where the platform was located) (no significant difference was observed among the three treated groups), while the D-gal tended to spend their time more averagely in each quadrant [F(3, 28) = 5.297, p < 0.05 vs. control group] (Fig. 2C). Furthermore, similar results were obtained from the former platform crossings experiments. The D-gal-treated mice crossed over the platform less frequently than the controls [F(3, 28) = 6.222, p < 0.01] (Fig. 2B). No significant difference between the control and the troxerutin/D-gal group was found, as well as the difference between the control group and the troxerutin group.
3. Results 3.1. Troxerutin counteracts cognitive impairment of D-gal-treated mice 3.1.1. Step-through passive avoidance task None of the mice tested had obvious health problems (e.g., weight loss, cataracts, or toxicity reaction). In the acquisition trial,
Fig. 1. Step-through passive avoidance task (n = 8). All values are expressed as mean ± SEM. *P < 0.05 vs. control group; #P < 0.05 vs. D-gal group.
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Fig. 2. Morris water maze test (n = 8). All values are expressed as mean ± SEM. (A) Mean latency in the hidden platform test. (B) The number of crossings over the exact location of the former platform. **P < 0.01 vs. control group; #P < 0.05, ##P < 0.01 vs. D-gal group. (C) Comparison of the time spent in target quadrant on day 5 (where the platform was located during hidden platform training). *P < 0.05 vs. control group; #P < 0.05 vs. D-gal group.
3.2. Troxerutin decreases AGEs, ROS and protein carbonyl levels in the basal forebrain, hippocampus and front cortex of D-gal-treated mice
There was no significant difference among the control group, the troxerutin group and the troxerutin/D-gal group.
Evidence reveals that D-gal can react with protein non-enzymatically forming Schiff bases and are converted into AGEs, which induces ROS and protein carbonyl production (Cai et al., 2006; Lu et al., 2007; Tian et al., 2005). The basal forebrain cholinergic system is involved in multiple aspects of learning and memory or processes of neuronal plasticity (Niewiadomska et al., 2009) in target structures such as neocortex and hippocampus. But it seems to be susceptible to oxidative stress induced by various neurotoxic agents such as D-gal (Lei et al., 2008) and b-amyloid (Olesen, Dag, & Mikkelsen, 1998). As evident from Fig. 3, D-gal administration significantly increased AGEs, ROS and protein carbonyl levels in the basal forebrain [FAGEs(3, 8) = 37.096, p < 0.001; FROS(3, 8) = 2!9.165, p < 0.001; Fprotein-carbonyl(3, 8) = 38.193, p < 0.001], hippocampus [FAGEs(3, 8) = 20.613, p < 0.01; FROS(3, 8) = 7.148, p < 0.05; Fprotein-carbonyl(3, 8) = 18.492, p < 0.01] and front cortex [FAGEs(3, 8) = 9.766, p < 0.01; FROS(3, 8) = 10.67, p < 0.01; Fprotein-carbonyl(3, 8) = 26.904, p < 0.001] as compared to the control group. The data indicated that oxidative stress in vivo was elevated in the brain of D-gal-treated mice. Interestingly, troxerutin could decrease AGEs, ROS and protein carbonyl levels in the basal forebrain [FAGEs(3, 8) = 37.096, p < 0.001; FROS(3, 8) = 29.165, p < 0.001; Fprotein-carbonyl(3, 8) = 38.193, p < 0.001], hippocampus [FAGEs(3, 8) = 20.613, p < 0.01; FROS(3, 8) = 7.148, p < 0.05; Fprotein-carbonyl(3, 8) = 18.492, p < 0.01] and front cortex [FAGEs(3, 8) = 9.766, p < 0.05; FROS(3, 8) = 10.67, p < 0.01; Fprotein-carbonyl(3, 8) = 26.904, p < 0.01].
3.3. Troxerutin decreases AchE activity and enhances nAchRa7 expression in the basal forebrain, hippocampus and front cortex of D-gal-treated mice Cholinergic system is of predominant importance in learning and memory processes (Muthuraju et al., 2009). Evidence shows that learning and memory deficits in a number of neurodegenerative disorders correlate with degeneration of cholinergic neurons (Muthuraju et al., 2009). In the present study, cholinergic markers like AchE levels and nAchRa7 expression in the basal forebrain, hippocampus and front cortex were studied (Fig. 4). Results displayed that D-gal administration induced a significant increase in AchE activity and a significant decrease in the nAchRa7 expression in mouse basal forebrain [FAchE(3, 8) = 15.032, p < 0.01 FnAchRa7(3, 8) = 7.087, p < 0.05; D-gal group vs. control group], hippocampus [FAchE(3, 8) = 15.366, p < 0.01; FnAchRa7(3, 8) = 19.283, p < 0.01; D-gal group vs. control group] and front cortex [FAchE(3, 8) = 34.124, p < 0.001; FnAchRa7(3, 8) = 6.768, p < 0.05; D-gal group vs. control group], which would impair learning and memory ablities of mice. However, troxerutin could significantly reverse the increased AchE activity and the decreased nAchRa7 expression in mouse basal forebrain [FAchE(3, 8) = 15.032, p < 0.01; FnAchRa7(3, 8) = 7.087, p < 0.05 vs. D-gal group], hippocampus [FAchE(3, 8) = 15.366, p < 0.01; FnAchRa7(3, 8) = 19.283, p < 0.01 vs. D-gal group] and front cortex [FAchE(3, 8) = 34.124, p < 0.001; FnAchRa7(3, 8) = 6.768, p < 0.05 vs.
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Fig. 3. Troxerutin decreases AGEs, ROS and protein carbonyl levels in the basal forebrain, hippocampus and front cortex of D-gal-treated mice. Values are averages from three independent experiments. All values are expressed as mean ± SEM. (A) Comparison of ROS levels in mouse basal forebrain, hippocampus and front cortex. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. D-gal group. (B) Comparison of protein carbonyl levels in mouse basal forebrain, hippocampus and front cortex. **P < 0.01, ***P < 0.001 vs. control group; ##P < 0.01, ###P < 0.001 vs. D-gal group. (C) Comparison of AGEs levels in mouse basal forebrain, hippocampus and front cortex. **P < 0.01, ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. D-gal group. D-gal
group] of D-gal treated mice. No significant difference among the control, the troxerutin and the troxerutin/D-gal group was found.
3.4. Troxerutin enhances interactions between nAchRa7 and the memory-related protein either PSD95 or NMDAR1 in the basal forebrain, hippocampus and front cortex of D-gal-treated mice nAchRa7 has been identified at postsynaptic and presynaptic sites, where it participates in numerous important processes including modulation of release of several neurotransmitters, mediation of postsynaptic excitatory responses, long-term potentiation (LTP), cognitive function and protection of neuron against toxicity (Fabian-Fine et al., 2001; Farias et al., 2007; Gahring, Meyer, & Rogers, 2003). We immunoprecipitated nAchRa7 and used Western blot analysis to assess nAchRa7 interactions with the memory-related protein either PSD95 or NMDAR1 in the basal forebrain, hippocampus and front cortex (Fig. 5). Our results showed that D-gal administration significantly reduced interactions between nAchRa7 and the memory-related protein PSD95 and NMDAR1 in the basal forebrain [FPSD95(3, 8) = 7.033, p < 0.05 FNMDAR1(3, 8) = 68.842, p < 0.001; D-gal group vs. control group], hippocampus [FPSD95(3, 8) = 13.863, p < 0.01 FNMDAR1(3, 8) = 46.852, p < 0.001; D-gal group vs. control group] and front cortex [FPSD95(3, 8) = 17.372, p < 0.01 FNMDAR1(3, 8) = 11.535, p < 0.01; D-gal group vs. control group] of mice. This could be one of the reasons why D-gal administration impaired learning and memory abilities of mice. On the contrary, troxerutin by oral gavage administration enhances interactions between nAchRa7 and the memory-related protein either PSD95 or NMDAR1 in the basal forebrain [FPSD95(3, 8) = 7.033, p < 0.05; FNMDAR1(3, 8) = 68.842, p < 0.001 vs. D-gal group], hippocampus [FPSD95(3, 8) = 13.863, p < 0.05; FNMDAR1(3, 8) = 46.852, p < 0.001 vs. D-gal group p] and front cortex [FPSD95(3, 8) = 17.372, p < 0.01; FNMDAR1(3, 8) = 11.535,
p < 0.01 vs. D-gal group] of D-gal-treated mice. No significant difference among the control, the troxerutin and the troxerutin + D-gal group was found. 4. Discussion D-gal is a reducing sugar and reacts readily with the free amines of amino acids in proteins and peptides both in vivo and in vitro to form AGEs. Evidence shows that AGEs is a harmless post-translational protein modification and it can accelerate the aging process (Srikanth et al., 2009). One of the potential mechanisms of AGE-induced damage is reactive oxygen species (ROS) production, especially superoxide and hydrogen peroxide release stimulated by AGEs (Srikanth et al., 2009). Our previous studies have demonstrated long-term injection of D-gal in mouse brain induces overproduction of ROS and leads to neuronal oxidative damage (Fan et al., 2009; Lu et al., 2006, 2007; Wu et al., 2008). Increasing evidence suggests that oxidative damage is an essential source of neurodegenerative diseases such as Parkinson’s disease (PD) and AD (Simonian & Coyle, 1996). In the present study, we confirmed that D-gal administration significantly increased AGEs, ROS and protein carbonyl levels in the basal forebrain, hippocampus and front cortex, which contributed to impairment of passive avoidant learning and memory function and spatial learning and memory ability. However, troxerutin by oral gavage administration effectively improved the impaired learning and memory performance, which could be associated with decreased AGEs, ROS and protein carbonyl levels in the basal forebrain, hippocampus and front cortex of D-gal-treated mice. Troxerutin, a rutoside derivative, is well known as antioxidant agent (Fan et al., 2009). Potential mechanisms underlying the neuroprotective effect of troxerutin in the D-gal-treated mice might be ascribed at least in part to its ability to scavenge and prevent free radical generation.
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Fig. 4. Troxerutin decreases AchE activity and enhances nAchRa7 expression in the basal forebrain, hippocampus and front cortex of D-gal-treated mice. Values are averages from three independent experiments. All values are expressed as mean ± SEM. (A) Representative immublot for nAchRa7 in mouse basal forebrain, hippocampus and front cortex. (B) Relative density analysis of nAchRa7 protein bands. The relative density is expressed as the ratio nAchRa7/b-actin and the vehicle control is set as 1.0. *P < 0.05, **P < 0.01 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. D-gal group. (C) Comparison of AchE activity in mouse basal forebrain, hippocampus and front cortex. **P < 0.01, ***P < 0.001 vs. control group; ##P < 0.01, ###P < 0.001 vs. D-gal group.
The basal forebrain cholinergic system, one of the most important modulatory neurotransmitter systems in the brain, provides a substantial source of cholinergic projections to the cortex and hippocampus and plays a pivotal role in attention, learning and memory (Bacciottini et al., 2001; von Engelhardt, Eliava, Meyer, Rozov, & Monyer, 2007). Its disruption produces a deficit in cognitive function, and its neurons are particularly vulnerable to oxidative stress (Mattson & Pedersen, 1998). Sufficient evidence suggests that oxidative stress and degeneration of basal forebrain cholinergic neurons play central roles in memory impairment in AD and PD (Niewiadomska et al., 2009). AchE is a cholinergic marker which is used to assess histopathologic changes of the cholinergic system. Previous studies showed D-gal administration significantly increased brain AchE activity in animal and resulted in dysfunction of the cholinergic system (Zhong, Ge, Qu, Li, & Ma, 2009). Our present data confirmed those findings. At the same time, the oral administration of troxerutin (150 mg/(kg day)) to D-gal-treated mice restored AchE activity to normal levels. Our experimental data also revealed that the basal forebrain was more sensitive to ROS than the hippocampus or the front cortex. nAchRa7, another cholinergic marker belongs to a superfamily of ionotropic receptors and participates in numerous important processes, including neuronal signaling, synaptic transmission and plasticity (Dehkordi, Kc, Balan, & Haxhiu, 2006). The present results showed that D-gal administration significantly decreased nAchRa7 expression in mouse basal forebrain, hippocampus and front cortex, which reflected the cholinergic impairment. How-
ever, the oral administration of troxerutin increased nAchRa7 expression in these brain regions and reversed the cholinergic impairment. On the other hand, recent evidence indicates that nAchRa7 can be expected to mediate modulation of synaptic transmission and plasticity by interacting with the memory-related protein PSD95 and NMDAR1 (Fabian-Fine et al., 2001; Farias et al., 2007). Furthermore, immunoprecipitation of nAchRa7 and Western immunodetection of PSD-95 and NMDAR1 demonstrated interactions between nAchRa7 and these two proteins in the basal forebrain, hippocampus and front cortex. D-gal administration significantly impaired the above-mentioned interactions. But the troxerutin administration reversed these changes in the brain of D-gal-treated mice. Taken together, impairment of the cholinergic system induced by D-gal injection might be associated with increased AchE activity, decreased nAchRa7 expression and reduced interactions between nAchRa7 and the protein of PSD95 or NMDAR1 in the basal forebrain, hippocampus and front cortex, which ultimately impaired learning and memory abilities of mice. Conversely the oral administration of troxerutin could reverse these changes and restore learning and memory abilities of mice. In conclusion, potential mechanisms underlying the neuroprotective effect of troxerutin in the D-gal-treated mice might be: (i) decreasing AGEs, ROS and protein carbonyl levels in the basal forebrain, hippocampus and front cortex and (ii) decreasing AchE activity, increasing nAchRa7 expression and enhancing interactions between nAchRa7 and the memory-related protein either
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cation Institutions of China (07KJA36029), Grants from Key Laboratory of Jiangsu Province, Grants from Qing Lan Project of Jiangsu Province, PR China and Grants from Natural Science Foundation by Xuzhou Normal University (08XLR09). References
Fig. 5. Troxerutin enhances interactions between nAchRa7 and the memoryrelated protein PSD95 and NMDAR1 in the basal forebrain, hippocampus and front cortex of D-gal-treated mice. Values are averages from three independent experiments. All values are expressed as mean ± SEM. (A) Representative Immunoprecipitation of nAchRa7 and Western immunodetection of PSD95 and NMDAR1 in mouse basal forebrain, hippocampus and front cortex. (B) Relative density analysis of PSD95 protein bands. The relative density is expressed as the ratio PSD95/b-actin and the vehicle control is set as 1.0. *P < 0.05, **P < 0.01 vs. control group; #P < 0.05, ## P < 0.01, ###P < 0.001 vs. D-gal group. (C) Relative density analysis of NMDAR1 protein bands. The relative density is expressed as the ratio NMDAR1/b-actin and the vehicle control is set as 1.0. **P < 0.01, ***P < 0.001 vs. control group; #P < 0.05, ## P < 0.01, ###P < 0.001 vs. D-gal group.
PSD95 or NMDAR1 in the basal forebrain, hippocampus and front cortex. Acknowledgments This work is supported by the Major Fundamental Research Program of Natural Science Foundation of the Jiangsu Higher Edu-
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Contents lists available at ScienceDirect
Neurobiology of Learning and Memory journal homepage: www.elsevier.com/locate/ynlme
Chronic administration of troxerutin protects mouse brain against D-galactose-induced impairment of cholinergic system Jun Lu a,1, Dong-mei Wu a,b,1, Bin Hu a,1, Wei Cheng b, Yuan-lin Zheng a,*, Zi-feng Zhang a, Qin Ye a, Shao-hua Fan a, Qun Shan a, Yong-jian Wang a a b
Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Xuzhou Normal University, Xuzhou 221116, Jiangsu Province, PR China School of Environment and Spatial Informatics, China University of Mining and Technology, Xuzhou 221008, Jiangsu Province, PR China
a r t i c l e
i n f o
Article history: Received 3 August 2009 Revised 9 September 2009 Accepted 12 September 2009 Available online 17 September 2009 Keywords: Troxerutin D-Galactose ROS Cholinergic system AchE nAchRa7
a b s t r a c t Previous evidence showed that administration of D-galactose (D-gal) increased ROS production and resulted in impairment of cholinergic system. Troxerutin, a natural bioflavonoid, has been reported to have many benefits and medicinal properties. In this study, we evaluated the protective effect of troxerutin against D-gal-induced impairment of cholinergic system, and explored the potential mechanism of its action. Our results displayed that troxerutin administration significantly improved behavioral performance of D-gal-treated mice in step-through test and morris water maze task. One of the potential mechanisms of this action was decreased AGEs, ROS and protein carbonyl levels in the basal forebrain, hippocampus and front cortex of D-gal-treated mice. Furthermore, our results also showed that troxerutin significantly inhibited cholinesterase (AchE) activity, increased the expression of nicotinic acetylcholine receptor alpha 7 (nAchRa7) and enhanced interactions between nAchRa7 and either postsynaptic density protein 95 (PSD95) or N-methyl-D-aspartate receptors subunit 1 (NMDAR1) in the basal forebrain, hippocampus and front cortex of D-gal-treated mice, which could help restore impairment of brain function. Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved.
1. Introduction Troxerutin is a trihydroxyethylated derivative of the natural bioflavonoid rutin. Sufficient evidence has shown that it has many biologic effects, such as anti-oxidative, anti-inflammatory, antierythrocytic, anti-fibrinolyti, anti-thrombotic, antineoplastic, and anti-c-radiation effects (Blasig, Loewe, & Ebert, 1987, 1988; Boisseau et al., 1995; Fan et al., 2009; Gandhi et al., 2004; Kessler, Ubeaud, Walter, Sturm, & Jung, 2002; Wenisch & Biffignandi, 2001). Recently, reports from our laboratory also confirmed a renal protective effect of it. D-galactose (D-gal) is a reducing sugar and can be metabolized at normal concentration. However, at high levels, it reacts readily with the free amines of amino acids in proteins and peptides in vivo to form advanced glycation end products (AGEs) (Lu et al., 2007; Tian et al., 2005). A substantial amount of evidence reveals that AGEs induce ROS production and increase protein carbonyl levels, which will exacerbate and accelerate aging process and trigger the early phases of age-related disease such as diabetes, arteriosclerosis, nephropathy, infection, and Alzheimer’s disease (Cai, He, Zhu, Lu, & Vlassara, 2006; Srikanth et al., 2009; Tian * Corresponding author. Fax: +86 516 83500348. E-mail addresses: [email protected], [email protected] (Y.-l. Zheng). 1 These authors contributed equally to this work.
et al., 2005). Recent findings have further demonstrated that continuous subcutaneous injection of D-gal in rodent induced production of free radicals, subsequently leading to the decreased expression of memory-related protein and deterioration of learning and memory function which might be associated with impairment of the basal forebrain cholinergic system (Cui et al., 2006; Hua et al., 2007; Lei et al., 2008; Long et al., 2007; Lu et al., 2006; Zhang, Li, Cui, & Zuo, 2005). The basal forebrain cholinergic system comprised of closely associated nuclei containing cholinergic neurons that project to both hippocampus and cortical areas plays a key role in modulating learning and memory processes (Niewiadomska, Baksalerska-Pazera, & Riedel, 2009). Interest in the function of the basal forebrain cholinergic system greatly increased with the neuropathological demonstration that cholinergic markers in the hippocampus and cerebral cortex are changed in Alzheimer’s disease (AD) (Greig et al., 2005; Niewiadomska et al., 2009) and that these changes have been linked to its pathology and the degree of cognitive impairment (Greig et al., 2005). Furthermore, cholinergic and memory deficits (Decker, 1987; Kubanis & Zornetzer, 1981) are also found in normal aging, although these dysfunctions differ from those reported in AD. Therefore, much of the research on cognitive decline has focused on the cholinergic system (Bacciottini, Passani, Mannaioni, & Blandina, 2001; Everitt & Robbins, 1997), and related treatment strategies have traditionally aimed at restoring the cholinergic neurotransmission.
1074-7427/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.nlm.2009.09.006
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However, therapies with cholinesterase inhibitors or muscarinic agonists are limited by unacceptable side-effects. Now, no work has been done to study whether troxerutin has an effect against D-gal-induced impairment of cholinergic system in mouse model. In the present study, we addressed this issue and investigated the potential mechanism underlying its action. 2. Materials and methods 2.1. Animals and administration Thirty-two 10-week-old male Kunming strain mice were purchased from the branch of national breeder center of rodents (Shanghai). Prior to experiments mice had free access to food and water and were kept under constant conditions of temperature (23 ± 1 °C) and humidity (60%). Eight mice were housed per cage on a 12-h light/dark schedule (lights on 08:30–20:30). After a week of adaptation, mice were divided randomly into four groups. Group 1 and group 4 served as vehicle control with injection of saline (0.9%), and the other two groups of mice (groups 2 and 3) received daily subcutaneous injection of D-gal (Sigma– Aldrich, MO, USA) at dose of 500 mg/(kg day) for 8 weeks, respectively. At the same time, mice in groups 3 and groups 4 received daily troxerutin (troxerutin; Purity > 99%; Baoji Fangsheng Biotechnology Co., Ltd., Baoji, China) of 150 mg/(kg day) in distilled water by oral gavage for 8 weeks, and the mice of groups 1 and 2 were given distilled water orally at the same dose. All experiments were performed in compliance with the Chinese legislation on the use and care of laboratory animals and were approved by the respective university committees for animal experiments. After the behavioral testing, mice were sacrificed and brain tissues were immediately collected for experiments or stored at 70 °C for later use. 2.2. Behavioral tests 2.2.1. Step-through test The step-through passive avoidance apparatus consisted of an illuminated chamber (11.5 cm 9.5 cm 11 cm) attached to a darkened chamber (23.5 cm 9.5 cm 11 cm) containing a metal floor that could deliver footshocks. The two compartments were separated by a guillotine door. The illuminated chamber was lit with a 25 W lamp. The step-through test was performed as described previously (Lu et al., 2006). 2.2.2. Morris water maze test The Morris water maze (MWM) test was performed as described previously (Lu et al., 2006; Wu et al., 2008). The experimental apparatus consisted of a circular water tank (100 cm in diameter, 35 cm in height), containing water (23 ± 1 °C) to a depth of 15.5 cm, which was rendered opaque by adding ink. A platform (4.5 cm in diameter, 14.5 cm in height) was submerged 1 cm below the water surface and placed at the midpoint of one quadrant. The pool was located in a test room, which contained various prominent visual cues. The test was performed as described previously. Each mouse received four training periods per day for four consecutive days. Latency to escape from the water maze (finding the submerged escape platform) was calculated for each trial. On day five, the probe test was carried out by removing platform and allowing each mouse to swim freely for 60 s. The time that mice spent swimming in the target quadrant (where the platform was located during hidden platform training) was measured. For the probe trials, the number of times crossing over the platform site of each mouse was also measured and calculated. All data were recorded with a computerized video system.
2.3. Preparation of tissue samples 2.3.1. Tissue homogenates For biochemical studies performed as described previously (Lu et al., 2006, 2007), animals were deeply anesthetized and sacrificed. Brains were removed and dissected on ice to obtain the basal forebrain, hippocampus and front cortex. Tissues were homogenized in 1/5 (w/v) 50 mM (pH 7.4) ice-cold Tris buffer saline solution (TBS) containing a protease inhibitor cocktail (Sigma–Aldrich, MO, USA) with 10 strokes at 1200 rpm in a Potter homogenizer. Homogenates were centrifuged at 12,000g for 10 min to obtain the supernatant. Supernatant aliquots were used to determine brain ROS, cholinesterase (AchE) activity, protein carbonyl levels in the basal forebrain, hippocampus and front cortex. For Western blot analysis performed as described previously (Lu et al., 2006; Wu et al., 2008), the basal forebrain, hippocampus and front cortex were homogenized in 1/3 (w/v) ice cold RIPA lysis buffer (1 TBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium azide) combining 30 ll of 10 mg/ml PMSF solution, 30 ll of Na3VO4, 30 ll of NaF and 30 ll of protease inhibitors cocktail per gram of tissue. The homogenates were sonicated four times for 30 s with 20 s intervals using a sonicator, centrifuged at 15,000g for 10 min at 4 °C, and the supernatant was collected and stored at 70 °C for Western blot studies. Protein levels in the supernatants were determined using the BCA assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). 2.4. Biochemical analysis 2.4.1. Assay of ROS ROS was measured as described previously, based on the oxidation of 20 70 -dichlorodihydrofluorescein diacetate to 20 70 -dichlorofluorescein (Shinomol, 2007). Briefly, the homogenate was diluted 1:20 times with ice-cold Locke’s buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.0 mM CaCl2, 10 mM D-glucose, and 5 mM HEPES, pH 7.4) to obtain a concentration of 5 mg tissue/ml. The reaction mixture (1 ml) containing Locke’s buffer (pH 7.4), 0.2 ml homogenate and 10 ml of DCFH-DA (5 mM) was incubated for 15 min at room temperature to allow the DCFH-DA to be incorporated into any membrane-bound vesicles and the diacetate group cleaved by esterases. After 30 min of further incubation, the conversion of DCFH-DA to the fluorescent product DCF was measured using a spectrofluorimeter with excitation at 484 nm and emission at 530 nm. Background fluorescence (conversion of DCFH-DA in the absence of homogenate) was corrected by the inclusion of parallel blanks. ROS formation was quantified from a DCF-standard curve and data are expressed as pmol DCF formed/ min/mg protein. 2.4.2. Oxidative stress marker: protein carbonyls Protein carbonyl content was measured as a marker of oxidative damage to proteins. They were determined the hydrazone derivatives between 360 and 390 nm (e = 21.0 mM1 cm1) according to the method described earlier (Shinomol, 2007). Data are expressed as nmol carbonyls/mg protein. 2.4.3. Assay of AchE activity AchE activity was determined according to the method of Shinomol et al. ( 2007). To a reaction mixture containing 2.85 ml phosphate buffer (0.1 M, pH 8.0), 50 ll of DTNB (10 mM), 50 ll sample and 20 ll acetylthiocholine iodide (78 mM) were added and the increase in absorbance was monitored at 412 nm for 5 min in a spectrophotometer and the enzyme activity is expressed as nmol of substrate hydrolyzed/min/mg protein.
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2.5. AGE-enzyme-linked immunosorbent assay Quantitative measurement of AGEs was performed by AGE-ELISA as described previously (Song, Bao, Li, & Li, 1999). Briefly, 96-well plates were coated with 100 ll/well of 3 lg/ml AGE-BSA in coating buffer overnight at 4 °C. Wells were washed three times with 150 ll washing buffer (PBS, 0.05% Tween-20, 1 mM NaN3), then blocked with 1% normal goat serum in 100 ll PBS for 2 h at 37 °C. After washing, 50 ll of 1:10 diluted samples in dilution buffer (PBS, 0.02% Tween-20, 1 mM NaN3, 1% normal goat serum) and 50 ll anti-AGE polyclonal antiserum in dilution buffer (1:2000) were added. Plates were incubated at room temperature for 2 h with gentle agitation on a horizontal rotary shaker. Wells were washed, 100 ll alkaline phosphate conjugated 2nd antibody in dilution buffer (1:2000) was added, and the plates were incubated at 37 °C for 1 h. They were then washed six times as above, 100 ll p-nitrophenyl phosphate substrate was added to each well. After 60 min optical density (OD) at 405 was determined by a microplate fluorometer Flex station II (Molecular Devices, San Francisco, CA, USA). The AGE-BSA was used as a competing antigen to generate an AGE standard curve. Sample AGE values were calculated from the standard curve. 2.6. Immunoprecipitation assay and Western blot analysis Immunoprecipitation assay was performed as described previously (Farias et al., 2007). The brain was homogenized in 1/3 (w/v) ice cold lysis buffer (25 mM HEPES, pH 7.4, 125 mM NaCl, 25 mM NaF, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1 mM Na3VO4, and the protease inhibitor mixture). Equal amounts of protein (500 lg) were precleared using protein A-Sepharose for 1 h at 4 °C and then incubated with 3 lg of antibody against the nAchRa7. The immune complexes were affinity precipitated with protein A-Sepharose beads and washed six times with 25 mM HEPES buffer, pH 7.4, 10 mM MgCl2, 1 mM NaF, 1% NP-40, and 1 mM Na3VO4. The immune complexes were then submitted to SDS–PAGE and analyzed by Western blot with mouse anti-post-synaptic density-95 (anti-PSD95) antibody (1:1000, Abcam, Cambridge, UK) and rabbit polyclonal antiNMDAR1 (1:1000, Cell Signaling Technology, Inc., Beverly, MA, USA). Samples (80 lg protein) were separated by denaturing SDS– PAGE and transferred to a PVDF membrane (Roche Diagnostics Corporation, Indianapolis, IN, USA) by electrophoretic transfer. The membrane was blocked with 5% non-fat milk and 0.1% Tween-20 in TBS, incubated overnight with rabbit anti-nicotinic acetylcholine receptor alpha 7 (anti-nAchRa7) (1:500, Abcam, Cambridge, UK). Quantitation of detected bands was performed with the scion image analysis software (Scion Corp., Frederick, MD, USA). The data were normalized using b-actin as an internal control and standardized with the vehicle control as 1.0. 2.7. Statistic analysis All statistical analyses were performed using the SPSS software, version 11.5. Group differences in the escape latency in the MWM training task were analyzed using two-way analysis of variance (ANOVA) with repeated measures, the factors being treatment and training day. The other data were analyzed with one-way ANOVA followed by Newman–Keuls or Tukey’s HSD post hoc test. Data were expressed as means ± SEM. Statistical significance was set at p < 0.05.
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the initial latencies did not differ significantly among the four groups [F(3, 28) = 0.259, p > 0.05] (Fig. 1). One-way ANOVA of passive avoidance task data revealed that D-gal significantly reduced the step-through latencies in the 24 h-retention trial [F(3, 28) = 4.888, p < 0.05]. This analysis also showed that the latencies in D-gal-treated mice received daily 150 mg/(kg day) troxerutin for 8 weeks were markedly lengthened as compared with D-gal-treated mice [F(3, 28) = 4.888, p < 0.05]. The shortened latencies induced by D-gal were restored to normal level after oral administration of troxerutin. There was no significant difference in step-through latencies between the control group and the troxerutin group. The result suggested that no obvious neural toxicity in troxerutin-treated mice was found. 3.1.2. Morris water maze test The Morris water maze is a useful tool for assessing spatial cognitive function. During the training session, all groups of mice improved their performance as indicated by the shortened escape latencies across successive days (Fig. 2A). Mice showed significant difference in mean latencies between training days [F(3, 112) = 25.698, p < 0.001] (Fig. 2A) and between treatments [F(3, 112) = 10.578, p < 0.001], but no interaction between the factors day and treatment [F(9, 112) = 1.207, p > 0.05]. Two-way ANOVA showed that the escape latencies were significantly higher in D-gal-treated mice than in control mice (p < 0.001). This result indicated that the spatial learning and memory ability in D-gal-treated mice was impaired. A comparison between the D-gal group and the troxerutin/D-gal group showed that the troxerutin could shorten escape latencies of D-gal-treated mice (p < 0.001). These data showed that troxerutin could improve cognitive performance in the D-gal-treated mice, and there was no significant difference in spatial learning and memory ability between the control group and the troxerutin/D-gal group. No significant difference between the control group and the troxerutin group was found. Note that mice of every treatment started at the same level of performance (no significant individual effect was observed for the first four trials of day 1). The withdrawal of the platform induced a general tendency to swim, preferentially to other equivalent zones, in the quadrant where the platform was previously located and in the platform zone. The control, the troxerutin and the troxerutin/D-gal group spent more time swimming in the target quadrant (where the platform was located) (no significant difference was observed among the three treated groups), while the D-gal tended to spend their time more averagely in each quadrant [F(3, 28) = 5.297, p < 0.05 vs. control group] (Fig. 2C). Furthermore, similar results were obtained from the former platform crossings experiments. The D-gal-treated mice crossed over the platform less frequently than the controls [F(3, 28) = 6.222, p < 0.01] (Fig. 2B). No significant difference between the control and the troxerutin/D-gal group was found, as well as the difference between the control group and the troxerutin group.
3. Results 3.1. Troxerutin counteracts cognitive impairment of D-gal-treated mice 3.1.1. Step-through passive avoidance task None of the mice tested had obvious health problems (e.g., weight loss, cataracts, or toxicity reaction). In the acquisition trial,
Fig. 1. Step-through passive avoidance task (n = 8). All values are expressed as mean ± SEM. *P < 0.05 vs. control group; #P < 0.05 vs. D-gal group.
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Fig. 2. Morris water maze test (n = 8). All values are expressed as mean ± SEM. (A) Mean latency in the hidden platform test. (B) The number of crossings over the exact location of the former platform. **P < 0.01 vs. control group; #P < 0.05, ##P < 0.01 vs. D-gal group. (C) Comparison of the time spent in target quadrant on day 5 (where the platform was located during hidden platform training). *P < 0.05 vs. control group; #P < 0.05 vs. D-gal group.
3.2. Troxerutin decreases AGEs, ROS and protein carbonyl levels in the basal forebrain, hippocampus and front cortex of D-gal-treated mice
There was no significant difference among the control group, the troxerutin group and the troxerutin/D-gal group.
Evidence reveals that D-gal can react with protein non-enzymatically forming Schiff bases and are converted into AGEs, which induces ROS and protein carbonyl production (Cai et al., 2006; Lu et al., 2007; Tian et al., 2005). The basal forebrain cholinergic system is involved in multiple aspects of learning and memory or processes of neuronal plasticity (Niewiadomska et al., 2009) in target structures such as neocortex and hippocampus. But it seems to be susceptible to oxidative stress induced by various neurotoxic agents such as D-gal (Lei et al., 2008) and b-amyloid (Olesen, Dag, & Mikkelsen, 1998). As evident from Fig. 3, D-gal administration significantly increased AGEs, ROS and protein carbonyl levels in the basal forebrain [FAGEs(3, 8) = 37.096, p < 0.001; FROS(3, 8) = 2!9.165, p < 0.001; Fprotein-carbonyl(3, 8) = 38.193, p < 0.001], hippocampus [FAGEs(3, 8) = 20.613, p < 0.01; FROS(3, 8) = 7.148, p < 0.05; Fprotein-carbonyl(3, 8) = 18.492, p < 0.01] and front cortex [FAGEs(3, 8) = 9.766, p < 0.01; FROS(3, 8) = 10.67, p < 0.01; Fprotein-carbonyl(3, 8) = 26.904, p < 0.001] as compared to the control group. The data indicated that oxidative stress in vivo was elevated in the brain of D-gal-treated mice. Interestingly, troxerutin could decrease AGEs, ROS and protein carbonyl levels in the basal forebrain [FAGEs(3, 8) = 37.096, p < 0.001; FROS(3, 8) = 29.165, p < 0.001; Fprotein-carbonyl(3, 8) = 38.193, p < 0.001], hippocampus [FAGEs(3, 8) = 20.613, p < 0.01; FROS(3, 8) = 7.148, p < 0.05; Fprotein-carbonyl(3, 8) = 18.492, p < 0.01] and front cortex [FAGEs(3, 8) = 9.766, p < 0.05; FROS(3, 8) = 10.67, p < 0.01; Fprotein-carbonyl(3, 8) = 26.904, p < 0.01].
3.3. Troxerutin decreases AchE activity and enhances nAchRa7 expression in the basal forebrain, hippocampus and front cortex of D-gal-treated mice Cholinergic system is of predominant importance in learning and memory processes (Muthuraju et al., 2009). Evidence shows that learning and memory deficits in a number of neurodegenerative disorders correlate with degeneration of cholinergic neurons (Muthuraju et al., 2009). In the present study, cholinergic markers like AchE levels and nAchRa7 expression in the basal forebrain, hippocampus and front cortex were studied (Fig. 4). Results displayed that D-gal administration induced a significant increase in AchE activity and a significant decrease in the nAchRa7 expression in mouse basal forebrain [FAchE(3, 8) = 15.032, p < 0.01 FnAchRa7(3, 8) = 7.087, p < 0.05; D-gal group vs. control group], hippocampus [FAchE(3, 8) = 15.366, p < 0.01; FnAchRa7(3, 8) = 19.283, p < 0.01; D-gal group vs. control group] and front cortex [FAchE(3, 8) = 34.124, p < 0.001; FnAchRa7(3, 8) = 6.768, p < 0.05; D-gal group vs. control group], which would impair learning and memory ablities of mice. However, troxerutin could significantly reverse the increased AchE activity and the decreased nAchRa7 expression in mouse basal forebrain [FAchE(3, 8) = 15.032, p < 0.01; FnAchRa7(3, 8) = 7.087, p < 0.05 vs. D-gal group], hippocampus [FAchE(3, 8) = 15.366, p < 0.01; FnAchRa7(3, 8) = 19.283, p < 0.01 vs. D-gal group] and front cortex [FAchE(3, 8) = 34.124, p < 0.001; FnAchRa7(3, 8) = 6.768, p < 0.05 vs.
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Fig. 3. Troxerutin decreases AGEs, ROS and protein carbonyl levels in the basal forebrain, hippocampus and front cortex of D-gal-treated mice. Values are averages from three independent experiments. All values are expressed as mean ± SEM. (A) Comparison of ROS levels in mouse basal forebrain, hippocampus and front cortex. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. D-gal group. (B) Comparison of protein carbonyl levels in mouse basal forebrain, hippocampus and front cortex. **P < 0.01, ***P < 0.001 vs. control group; ##P < 0.01, ###P < 0.001 vs. D-gal group. (C) Comparison of AGEs levels in mouse basal forebrain, hippocampus and front cortex. **P < 0.01, ***P < 0.001 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. D-gal group. D-gal
group] of D-gal treated mice. No significant difference among the control, the troxerutin and the troxerutin/D-gal group was found.
3.4. Troxerutin enhances interactions between nAchRa7 and the memory-related protein either PSD95 or NMDAR1 in the basal forebrain, hippocampus and front cortex of D-gal-treated mice nAchRa7 has been identified at postsynaptic and presynaptic sites, where it participates in numerous important processes including modulation of release of several neurotransmitters, mediation of postsynaptic excitatory responses, long-term potentiation (LTP), cognitive function and protection of neuron against toxicity (Fabian-Fine et al., 2001; Farias et al., 2007; Gahring, Meyer, & Rogers, 2003). We immunoprecipitated nAchRa7 and used Western blot analysis to assess nAchRa7 interactions with the memory-related protein either PSD95 or NMDAR1 in the basal forebrain, hippocampus and front cortex (Fig. 5). Our results showed that D-gal administration significantly reduced interactions between nAchRa7 and the memory-related protein PSD95 and NMDAR1 in the basal forebrain [FPSD95(3, 8) = 7.033, p < 0.05 FNMDAR1(3, 8) = 68.842, p < 0.001; D-gal group vs. control group], hippocampus [FPSD95(3, 8) = 13.863, p < 0.01 FNMDAR1(3, 8) = 46.852, p < 0.001; D-gal group vs. control group] and front cortex [FPSD95(3, 8) = 17.372, p < 0.01 FNMDAR1(3, 8) = 11.535, p < 0.01; D-gal group vs. control group] of mice. This could be one of the reasons why D-gal administration impaired learning and memory abilities of mice. On the contrary, troxerutin by oral gavage administration enhances interactions between nAchRa7 and the memory-related protein either PSD95 or NMDAR1 in the basal forebrain [FPSD95(3, 8) = 7.033, p < 0.05; FNMDAR1(3, 8) = 68.842, p < 0.001 vs. D-gal group], hippocampus [FPSD95(3, 8) = 13.863, p < 0.05; FNMDAR1(3, 8) = 46.852, p < 0.001 vs. D-gal group p] and front cortex [FPSD95(3, 8) = 17.372, p < 0.01; FNMDAR1(3, 8) = 11.535,
p < 0.01 vs. D-gal group] of D-gal-treated mice. No significant difference among the control, the troxerutin and the troxerutin + D-gal group was found. 4. Discussion D-gal is a reducing sugar and reacts readily with the free amines of amino acids in proteins and peptides both in vivo and in vitro to form AGEs. Evidence shows that AGEs is a harmless post-translational protein modification and it can accelerate the aging process (Srikanth et al., 2009). One of the potential mechanisms of AGE-induced damage is reactive oxygen species (ROS) production, especially superoxide and hydrogen peroxide release stimulated by AGEs (Srikanth et al., 2009). Our previous studies have demonstrated long-term injection of D-gal in mouse brain induces overproduction of ROS and leads to neuronal oxidative damage (Fan et al., 2009; Lu et al., 2006, 2007; Wu et al., 2008). Increasing evidence suggests that oxidative damage is an essential source of neurodegenerative diseases such as Parkinson’s disease (PD) and AD (Simonian & Coyle, 1996). In the present study, we confirmed that D-gal administration significantly increased AGEs, ROS and protein carbonyl levels in the basal forebrain, hippocampus and front cortex, which contributed to impairment of passive avoidant learning and memory function and spatial learning and memory ability. However, troxerutin by oral gavage administration effectively improved the impaired learning and memory performance, which could be associated with decreased AGEs, ROS and protein carbonyl levels in the basal forebrain, hippocampus and front cortex of D-gal-treated mice. Troxerutin, a rutoside derivative, is well known as antioxidant agent (Fan et al., 2009). Potential mechanisms underlying the neuroprotective effect of troxerutin in the D-gal-treated mice might be ascribed at least in part to its ability to scavenge and prevent free radical generation.
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Fig. 4. Troxerutin decreases AchE activity and enhances nAchRa7 expression in the basal forebrain, hippocampus and front cortex of D-gal-treated mice. Values are averages from three independent experiments. All values are expressed as mean ± SEM. (A) Representative immublot for nAchRa7 in mouse basal forebrain, hippocampus and front cortex. (B) Relative density analysis of nAchRa7 protein bands. The relative density is expressed as the ratio nAchRa7/b-actin and the vehicle control is set as 1.0. *P < 0.05, **P < 0.01 vs. control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. D-gal group. (C) Comparison of AchE activity in mouse basal forebrain, hippocampus and front cortex. **P < 0.01, ***P < 0.001 vs. control group; ##P < 0.01, ###P < 0.001 vs. D-gal group.
The basal forebrain cholinergic system, one of the most important modulatory neurotransmitter systems in the brain, provides a substantial source of cholinergic projections to the cortex and hippocampus and plays a pivotal role in attention, learning and memory (Bacciottini et al., 2001; von Engelhardt, Eliava, Meyer, Rozov, & Monyer, 2007). Its disruption produces a deficit in cognitive function, and its neurons are particularly vulnerable to oxidative stress (Mattson & Pedersen, 1998). Sufficient evidence suggests that oxidative stress and degeneration of basal forebrain cholinergic neurons play central roles in memory impairment in AD and PD (Niewiadomska et al., 2009). AchE is a cholinergic marker which is used to assess histopathologic changes of the cholinergic system. Previous studies showed D-gal administration significantly increased brain AchE activity in animal and resulted in dysfunction of the cholinergic system (Zhong, Ge, Qu, Li, & Ma, 2009). Our present data confirmed those findings. At the same time, the oral administration of troxerutin (150 mg/(kg day)) to D-gal-treated mice restored AchE activity to normal levels. Our experimental data also revealed that the basal forebrain was more sensitive to ROS than the hippocampus or the front cortex. nAchRa7, another cholinergic marker belongs to a superfamily of ionotropic receptors and participates in numerous important processes, including neuronal signaling, synaptic transmission and plasticity (Dehkordi, Kc, Balan, & Haxhiu, 2006). The present results showed that D-gal administration significantly decreased nAchRa7 expression in mouse basal forebrain, hippocampus and front cortex, which reflected the cholinergic impairment. How-
ever, the oral administration of troxerutin increased nAchRa7 expression in these brain regions and reversed the cholinergic impairment. On the other hand, recent evidence indicates that nAchRa7 can be expected to mediate modulation of synaptic transmission and plasticity by interacting with the memory-related protein PSD95 and NMDAR1 (Fabian-Fine et al., 2001; Farias et al., 2007). Furthermore, immunoprecipitation of nAchRa7 and Western immunodetection of PSD-95 and NMDAR1 demonstrated interactions between nAchRa7 and these two proteins in the basal forebrain, hippocampus and front cortex. D-gal administration significantly impaired the above-mentioned interactions. But the troxerutin administration reversed these changes in the brain of D-gal-treated mice. Taken together, impairment of the cholinergic system induced by D-gal injection might be associated with increased AchE activity, decreased nAchRa7 expression and reduced interactions between nAchRa7 and the protein of PSD95 or NMDAR1 in the basal forebrain, hippocampus and front cortex, which ultimately impaired learning and memory abilities of mice. Conversely the oral administration of troxerutin could reverse these changes and restore learning and memory abilities of mice. In conclusion, potential mechanisms underlying the neuroprotective effect of troxerutin in the D-gal-treated mice might be: (i) decreasing AGEs, ROS and protein carbonyl levels in the basal forebrain, hippocampus and front cortex and (ii) decreasing AchE activity, increasing nAchRa7 expression and enhancing interactions between nAchRa7 and the memory-related protein either
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cation Institutions of China (07KJA36029), Grants from Key Laboratory of Jiangsu Province, Grants from Qing Lan Project of Jiangsu Province, PR China and Grants from Natural Science Foundation by Xuzhou Normal University (08XLR09). References
Fig. 5. Troxerutin enhances interactions between nAchRa7 and the memoryrelated protein PSD95 and NMDAR1 in the basal forebrain, hippocampus and front cortex of D-gal-treated mice. Values are averages from three independent experiments. All values are expressed as mean ± SEM. (A) Representative Immunoprecipitation of nAchRa7 and Western immunodetection of PSD95 and NMDAR1 in mouse basal forebrain, hippocampus and front cortex. (B) Relative density analysis of PSD95 protein bands. The relative density is expressed as the ratio PSD95/b-actin and the vehicle control is set as 1.0. *P < 0.05, **P < 0.01 vs. control group; #P < 0.05, ## P < 0.01, ###P < 0.001 vs. D-gal group. (C) Relative density analysis of NMDAR1 protein bands. The relative density is expressed as the ratio NMDAR1/b-actin and the vehicle control is set as 1.0. **P < 0.01, ***P < 0.001 vs. control group; #P < 0.05, ## P < 0.01, ###P < 0.001 vs. D-gal group.
PSD95 or NMDAR1 in the basal forebrain, hippocampus and front cortex. Acknowledgments This work is supported by the Major Fundamental Research Program of Natural Science Foundation of the Jiangsu Higher Edu-
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