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NF-κB pathway
Behavioural Brain Research 235 (2012) 200–209
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Research report
Akebia Saponin D attenuates amyloid -induced cognitive deficits and inflammatory response in rats: Involvement of Akt/NF-B pathway Xing Yu a,b , Lin-na Wang a,b , Qian-ming Du a,b , Lin Ma a,b , Li Chen a,b , Ran You a,b , Ling Liu a,b , Jing-jing Ling a,b , Zhong-lin Yang c , Hui Ji a,b,∗ a
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, 210009 Nanjing, PR China Department of Pharmacology, China Pharmaceutical University, 24 Tong Jia Xiang, 210009 Nanjing, PR China c Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, 210009 Nanjing, PR China b
h i g h l i g h t s Akebia Saponin D is a typical saponin component from Dipsacus asper Wall. ASD improved memory impairment in the A1–42-induced dementia model. ASD attenuates inflammatory response induced by A1–42.
a r t i c l e
i n f o
Article history: Received 3 May 2012 Received in revised form 27 July 2012 Accepted 31 July 2012 Available online 8 August 2012 Keywords: Alzheimer’s disease Akebia Saponin D (ASD) Amyloid  (A)1–42 Cognitive impairment Inflammatory responses Akt/NF-B
a b s t r a c t Neuroinflammatory responses caused by amyloid (A) play an important role in the pathogenesis of Alzheimer’s disease (AD). A is known to be directly responsible for the activation of glial cells and induction of apoptosis. Akebia Saponin D (ASD) is extracted from a traditional herbal medicine Dipsacus asper Wall, which has been shown to protect against ibotenic acid-induced cognitive deficits and cell death in rats. In this study, we investigated the in vivo protective effect of ASD on learning and memory impairment induced by bilateral intracerebroventricular injections of A1–42 using Morris water and Y-maze task. Furthermore, the anti-inflammatory activity and neuroprotective effect of ASD was examined with methods of histochemistry and biochemistry. These data showed that oral gavage with ASD at doses of 30, 90 and 270 mg/kg for 4 weeks exerted an improved effect on cognitive impairment. Subsequently, the ASD inhibited the activation of glial cells and the expression of tumor necrosis factor (TNF)-␣, interleukin-1 beta (IL-1) and cyclooxygenase-2 (COX-2) in rat brain. Moreover, ASD afforded beneficial actions on inhibitions of Akt and IB kinase (IKK) phosphorylations, as well as nuclear factor B (NF-B) activation induced by A1–42. These results suggest that ASD may be a potential agent for suppressing both Alzheimer’s disease-related neuroinflammation and memory system dysfunction. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is a multifaceted neurodegenerative disorder characterized by progressive cognitive dysfunction and neuronal cell death, which correlates with brain deposition of senile plaques and accumulation of neurofibrillatory tangles [1]. Accumulation of A is hypothesized to initiate a pathogenic cascade that eventually results in AD [2]. The mechanism underlying A-induced neurotoxicity is complex, involving several pathways as signaling events [3]. It is now well documented that fibrillar
∗ Corresponding author at: State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, 210009 Nanjing, PR China. Tel.: +86 258 602 1369. E-mail address: [email protected] (H. Ji). 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2012.07.045
forms of amyloid  may serve as an inflammatory stimulus for neuronal cells, and the underlying signaling mechanisms have been identified by several research groups [4–6]. Thus, in addition to direct neurotoxic effects, A assemblies have the ability to stimulate the proliferation of glial cells, and microglial-mediated inflammatory responses in the AD brain are well-documented [7,8]. Furthermore, increased numbers of morphologically reactive microglia is a common histological observation from AD brains as compared to non-demented control brains [9]. Neuroinflammation in AD is considered a downstream consequence of A aggregation in the brain, which results in microglial activation initiating a pro-inflammatory cascade and release of cytokines, chemokines, reactive oxygen and nitrogen species, and proteolytic enzymes, all of which contribute to neuronal degeneration [10–12]. There may also be other as yet unknown actions.
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according to the stereotaxic atlas of Paxinos and Watson [20]. Animals were injected with 5.0 l sterile bidistiled water (vehicle-treated, 10 rats) or 5.0 l aggregated A1–42 solution (50 rats) into each cerebral lateral ventricle at a rate of 1 l/min by pump.
2.4. Drug administration and experimental design
Fig. 1. Chemical structure of ASD.
At present, there is no definitive treatment or cure for AD. A large segment of the public finds solace in herbs, in part believing that herbs are natural and hence safer than synthetic drugs, and that a complex mixture of herbs can effectively treat complex disease. In addition, the metabolite profile of herbal medicines is important for screening its active constituents, thus providing a valuable contribution to the drug discovery process and elucidation of the underlying mechanism of action. Dipsacus asper Wall (DAW), belonging to Dipsacaceae, is a kind of perennial herb growing in moist fields and mountain [13]. The roots of DAW have been used as a tonic, an analgesic and anti-inflammatory agent in China for the therapy of low back pain, rheumatic arthritis, traumatic hematoma, threaten abortion and bone fractures. Akebia Saponin D (ASD), is a typical bioactive triterpenoid saponin isolated from the rhizome of DAW. Pharmacological study has demonstrated that ASD can promote human osteoblast proliferation and differentiation [14], anti-oxidation, anti-nociceptive and neuroprotective activities [15]. In this study, we aimed to observe the protective effects of ASD on cognitive deficits in a rat model of AD induced by A1–42. Furthermore, we addressed the mechanisms that contribute to the neuroprotective effects of ASD and whether it occurs via anti-inflammatory pathway.
The surgery took place on Day 0. ASD at the dose of 30 mg/kg, 90 mg/kg and 270 mg/kg, or donepezil at the dose of 0.5 mg/kg was intragastrically administered once per day for consecutive 28 days starting on Day 1. Control and model groups received the same volume of vehicle (0.5% sodium carboxyl methyl cellulose, CMCNa) as ASD. The dosage of donepezil was similar to those commonly used in clinical practice and in previous reports [21–23]. Morris water maze task took place from Day 22 to Day 26. The next day after the water-maze probe trial test (i.e. 27 days after A1–42 infusions), the same rats were tested for memory using the Y-maze task. All the treatment was given 1 h before the maze training. The rats were decapitated after the Y maze for biochemical and histological examination.
2.5. Morris water maze task The Morris water maze task was carried out from Day 22 to Day 26 after A1–42injection as described. The water maze [24] was a black circular pool (120 cm in diameter, 50 cm deep) divided into 4 equal imaginary quadrants for data analysis. The water temperature was maintained between 21 and 23 ◦ C. One centimeter beneath the surface of the water and hidden from rats view was a black circular platform 10 cm in diameter. The swimming patterns of the rats were recorded with a video camera mounted above the center of the pool and analyzed with a videotracking system set at 10 samples/s. The water maze was located in a room with several visual stimuli hanging on the walls to provide spatial cues. The acquisition phase was carried out during 4 consecutive days (from Day 22 to Day 25). On each training day/session, the rats received 4 consecutive training trials with the hidden platform kept in a constant position. A different starting location was used for each trial, which consisted of swimming followed by remaining on the platform for at least 10 s. Rats not finding the platform within 90 s were guided to it by the experimenter. The time latency to reach the platform, the distance traveled and the swimming speed were recorded. On Day 26, to assess spatial retention, a 90 s probe trial with the platform removed from the pool was given one day after the last hidden platform trial. The time spent swimming in the target quadrant (where the platform was located during hidden platform training) was measured. In probe trials the time latency to reach the target quadrant and the time each rat spent in it were calculated. At the end of the probe test a highly visible platform was placed in the center of the pool. The visible platform subtest was performed to determine whether the experimental manipulation affects visual acuity or motivation.
2. Materials and methods
2.6. Y-maze task
2.1. Animals and housing
The Y-maze test was performed as previously described [25,26] on Day 27 and Day 28. The Y-maze [27] was constructed of black plastic walls (10 cm high). It consisted of three compartments (10 cm × 10 cm) connected with 4 cm × 5 cm passages, floors were constructed of 3.175 mm stainless steel rods set 8 mm apart. The test was conducted for 2 consecutive days at the same time each day. On the first day (learning trial) each rat was placed in one of the compartments and allowed to move freely for 5 min. Then turning on the power, two of the compartments have electric shock (10 V) through the stainless steel grid floor, another one has no electric shock, but its light was turned on. If the rat enters the compartment which has no electric shock, we record it right. Otherwise, the rat was gently navigated to the compartment which has no electric shock for 30 s. The times of electric shock with 9 positive in 10 continuous trials were recorded. On the second day (testing trial), the rats were tested 10 times, the number of corrects were recorded manually. ASD (30 mg/kg, 90 mg/kg and 270 mg/kg, i.g.) was administered 1 h before the trial.
60 adult male 3-months-old Sprague–Dawley rats (B&K Laboratory Animal Corp. Ltd., Shanghai) weighing 250–300 g were maintained under standardized conditions (22 ± 1 ◦ C, 60 ± 10% humidity, 12 h light/dark cycle), and provided free access to food and water ad libitum. The rats were randomly divided into 6 groups of 10 rats each: Control, Model (A1–42), Donepezil (A1–42 + 0.5 mg/kg donepezil) as a positive control, ASD-L (A1–42 + 30 mg/kg ASD), ASD-M (A1–42 + 90 mg/kg ASD) and ASD-H (A1–42 + 270 mg/kg ASD) groups. All experiments were approved and performed in accordance with National Institutes of Health Guide for Care and Use of Laboratory Animals (publication no. 85-23, revised 1985). 2.2. Drugs ASD (Fig. 1) was prepared by Professor Zhong-Lin Yang. It was isolated from Dipsacus asperoides Wall at the Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University [16]. Commercially available A1–42 (Invitrogen, USA) aggregated according to previous study [17]. Briefly, the peptide was dissolved in sterile bidistiled water at concentration of 2 g/l, aliquoted into tubes and stored at −20 ◦ C. It was “aged” by incubation at 37 ◦ C for 4 days before the surgery [18,19]. Light microscopic observation demonstrated the existence of both birefringent fibril-like structures and globular aggregates.
2.7. Preparation of cytosolic and nuclear extracts After Y-maze, the animals were decapitated (Day 28). Brains were immediately taken out and washed in ice-cold PBS solution. Hippocampus tissues were isolated and frozen in liquid nitrogen before analysis. Protein extraction of cytosolic and nuclear fractions was performed using a cytosolic/nuclei isolation kit (KGP150, NanJing KeyGEN Biotechnology Co., Ltd., Nanjing, China), according to a multiple centrifugation method. Protein concentration was determined using an enhanced BCA protein assay (Beyotime Institute of Biotechnology Co., Ltd. Haimen, China).
2.3. Surgery Rats were anaesthetized with intraperitoneal (i.p.) administration of sodium pentobarbital (30 mg/kg) before they placed in a stereotaxic apparatus (RWD Life Science Co., Ltd. Shenzhen, China). Guide cannulae (22-gauge, 6 mm; Ka Science, Germany) were implanted into bilateral lateral ventricles using the following coordinates: AP-3.3 mm from Bregma; ML 2.0 mm from the midline; DV-2.5 mm from dura
2.8. Measurement of TNF-˛ and IL-1ˇ level The TNF-␣ and IL-1 levels were assayed using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Jianglai Bioengineering Institute Co., Ltd., Shanghai, China) according to the manufacturer’s protocol.
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2.9. Immunohistochemistry Free-floating brain slices (6-m) were washed in PBS to remove cryopreservative. The tissues were incubated for 10 min at room temperature in 3% H2 O2 solution to reduce endogenous peroxidase activity, then washed in PBS again. The samples were placed in rabbit serum for 1 h at room temperature (SP immunohistochemical staining kit, Maxim biotechnology Co., Ltd, Fuzhou, China). Incubation overnight at 4 ◦ C was performed with anti-macrophage antibody [MAC387] (1:500, Abcam,
Cambridge, UK), anti-GFAP antibody (1:600, Millipore Corporation, MA, USA). After that, the slices were washed in PBS and incubated with biotinylated rabbit anti-rat IgG for 1 h at room temperature, again washed, and then incubated in avidin–biotin horseradish peroxidase macromolecular complex (Maxim biotechnology Co., Ltd., Fuzhou, China) for 1 h at room temperature. To develop color, slices were incubated briefly in DAB substrate kit (Maxim biotechnology Co., Ltd., Fuzhou, China). After a final set of washed in PBS, the slices were dehydrated, cleared, and coverslipped with mounting medium.
Fig. 2. Effects of ASD (30, 90 and 270 mg/kg, 4 weeks) on A1–42-induced spatial cognitive deficits in the Morris water-maze test in rats. (A) The latency to escape onto the submerged platform and (B) distance traveled during the 4-days acquisition trials. (C) The times passed through the former platform location and (D) distance swum in the target quadrant in the probe trial test performed 24 h after the last acquisition trial. (E) Representative searching swimming paths by rats with different treatments in the probe trial test. The data were expressed as mean ± SEM, n = 10 per group, *p < 0.05, **p < 0.01, ***p < 0.001 vs model group.
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2.10. Western blot analysis For western blotting assay, the remaining 7 rats of each group were euthanized by rapid decapitation on Day 28, and 3 brains rapidly frozen at −80 ◦ C each group. The hippocampus were cut into small pieces, and lysed in ice-cold RIPA buffer (Beyotime Institute of Biotechnology Co., Ltd. Haimen, China). The lysate was centrifuged at 12,000 × g for 5 min at 4 ◦ C and the supernatant was collected. Equal amounts of protein were separated on 15% SDS-polyacrylamide gels. Proteins on the gel were blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature. Subsequently, the membranes were incubated at 4 ◦ C overnight with the rabbit anti-COX-2 antibody(1:500, Proteintech Group Inc., USA), rabbit anti-TNF-␣ antibody(1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-IL-1 antibody(1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-bcl-2 antibody(1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-Bax(1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-NF-B p65 antibody(1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-IKK antibody (1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-IKK␣/ antibody (1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-Akt antibody(1:1000, Cell Signaling Technology Co., Ltd., MN, USA), rabbit anti-phosphor-Akt antibody (1:1000, Cell Signaling Technology Co., Ltd, MN, USA). Following three washes with TBST, the blots were incubated with the secondary horseradish peroxidase-conjugated antibody (goat anti-rabbit IgG, 1:10,000, Bioworld Technology Co., Ltd., MN, USA) at the room temperature for 2 h. The antibody-reactive bands were visualized by using the enhanced chemiluminescence detection reagents (Keygen Biotechnology Co., Ltd., Nanjing, China) by a gel imaging system (ChemiScope 2850, Clinx Science Instruments Co., Ltd., Shanghai, China). 2.11. Statistical analysis The density of positive cells in histochemistry analysis was calculated by ImagePro Plus image analysis software. The band intensity in western blot analysis was calculated by AlphaEaseFC software. Data were analyzed statistically using one-way analysis of variance followed by Tukey’s post hoc test. Summarized data were expressed as mean ± SEM. Statistical significance was accepted at p < 0.05. All statistical analysis was undertaken using SPSS vs 11.5.
3. Results 3.1. ASD reversed memory impairment induced by Aˇ1–42 in Morris water-maze and Y-maze tasks To establish memory deficits produced by A1–42, we examined memory performance in rats micro-infused with aged A1–42 at 10 g/side into lateral ventricles using the Morris water-maze and Y-maze tests. During the acquisition trials in the water-maze test, all the rats, regardless of the different treatments, displayed progressive decrease in escape latency to reach the hidden platform. Results indicated that A1–42, administered 23 days prior to the beginning of acquisition training, increased escaped latencies on sessions 2, 3, 4 compared to the corresponding vehicle controls (p < 0.01 or 0.001, Fig. 2A), these were reversed by ASD at different doses especially at 270 mg/kg for session 2–4 and 90 mg/kg for session 3 (p < 0.05 or 0.01, Fig. 2A). In addition, the rats also displayed slight, progressive decrease in distance traveled over time. The groups treated with ASD showed a significant decrease in distance traveled compared to the model group for session 3 (p < 0.01 or 0.001, Fig. 2B). 24 h after the last acquisition trial, rats were tested for spatial memory in the probe trial test, during which the platform was removed. Drug treatment altered entries and distance swum in the target quadrant. Results indicated that A1–42 decreased entries (p < 0.05) and distance (p < 0.01) compared to the respective vehicle controls, these were reversed by chronic treatment with ASD (p < 0.05 or p < 0.01, Fig. 2C and D). Consistent with this, the behavioral tracking results revealed that both vehicle - and A/ASD-treated rats displayed more exploration in the target quadrant than the rats treated with A1–42 alone (Fig. 2E). This result is consistent with ASD’s reversal of A1–42-induced decreases in escape latency and length of swimming path. The next day after the water-maze probe trial test (i.e. 26 days after A1–42 infusions), the same rats were tested for short-term
Fig. 3. Effect of ASD on the performance in the presence of A1–42 in the Y-maze test in rats. (A) Changes in the arm entries on learning training; (B) Changes in the arm entries on memory training. The data were expressed as mean ± SEM, n = 10 per group, *p < 0.05, **p < 0.01, ***p < 0.001 vs model group.
memory using the Y-maze task, with daily ASD treatment continued. As shown in Fig. 3A, Compared to the saline injected control group, the spontaneous alternations of arm entries were lowered in A1–42 injected model group (***p < 0.001). The alternation memory reduction observed in the A1–42 injected model was significantly improved by the administration of 90 and 270 mg/kg ASD (*p < 0.05, **p < 0.01, Fig. 3B). 3.2. ASD suppress the activation of microglia and astroglia in the brain following a Aˇ1–42 insult To assess the involvement of glial cells following A1–42 infusion, immunohistochemical analysis of macrophage, a marker protein for microglia (Fig. 4A) and GFAP, a marker for astroglia (Fig. 4B) were carried out in the brain. Quantitative analysis showed that infusion of soluble A1–42 produced a significant increase of GFAP-positive astroglial cells and reactive microglia in the brain compared with the vehicle control (Fig. 4C and D). Treatment with ASD significantly suppressed the number of GFAP-positive astrocytes (90 and 270 mg/kg, p < 0.05) in the brain. 3.3. ASD inhibits Aˇ1–42-induced TNF-˛, IL-1ˇ and COX-2 production and expression To examine the potential mechanisms of ASD to inhibit inflammation induced by A1–42, the levels of TNF-␣ and IL-1 were measured by ELISA. The result (Fig. 5) showed that brain concentration of cytokines increased in rats treated with A1–42 compared with the control group. However, TNF-␣ and IL-1 decreased in rats treated with ASD for 4 weeks. We further examined the expressions
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Fig. 4. Effect of ASD on the activation of microglia and astroglia in the brain following a A1–42 insult. (A) Represents photomicrographs of macrophage immunohistochemistry in brain (magnification 400×). Scale bar = 50 m. (B) Represents photomicrographs of GFAP immunohistochemistry in brain (magnification 400×). Scale bar = 50 m. (C) The number of macrophage immunopositive cells. (D) The number GFAP immunopositive cells. The data were expressed as mean ± SEM, n = 3 per group, *p < 0.05, **p < 0.01, ***p < 0.001 vs model group. The result indicated at least three independent experiments in each animal.
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Fig. 5. Effect of ASD treatment on A1–42 induced enhancement of TNF-␣ and IL-1 expression in brain of rats by ELISA. (A) TNF-␣ level in brain; (B) IL-1 level in brain. The data were expressed as mean ± SEM, n = 7 per group, *p < 0.05, **p < 0.01, ***p < 0.001 vs model group.
of TNF-␣, IL-1 and COX-2 protein by western blot. Consistent with the result of ELISA, as shown in Fig. 6 expressions of TNF-␣, IL-1 and COX-2 were markedly increased in response to A. However, after treatment with ASD at the doses of 90 and 270 mg/kg, the TNF␣, IL-1 and COX-2 protein expressions significantly decreased compared with model group (p < 0.01, Fig. 6). All these results
Fig. 6. Effect of ASD treatment on A1–42 induced COX-2, TNF-␣ and IL-1 expression changes in brain of rats using western blotting analysis. (A) Assessment of COX-2, TNF-␣ and IL-1 protein levels in brain by western blotting. (B) The ratio of COX-2 was calculated to the actin. (C) The ratio of TNF-␣ was calculated to the actin. (D) The ratio of IL-1 was calculated to the actin. The data were expressed as mean ± SEM of three independent experiments, *p < 0.05 vs model group.
indicated that ASD could inhibit the generation of inflammatory cytokines induced by A1–42. 3.4. ASD reverses Aˇ1–42-induced apoptotic effects Since A1–42 decreases neuronal cell viability via increased apoptotic, we examined expression of Bax, a pro-apoptotic protein that induces rapid human neuronal cell death, and Bcl-2, another cell death-associated protein, to determine whether apoptotic responses were involved in the effect of ASD on A1–42-induced toxicity. As shown in Fig. 7B and C, in treatment of ASD at the dosage of 90, 270 mg/kg., proapoptic Bax was down-regulated (p < 0.01) and antiapoptic Bcl-2 was up-regulated (p < 0.01).
Fig. 7. Effect of ASD on A1–42-induced changes in Bcl-2 and Bax in the brain in rats. (A) Assessment of Bax and Bcl-2 protein levels in brain by western blotting. (B) ASD reversed A1–42-induced increases in expression of the apoptotic mediator Bax. (C) ASD reversed A1–42-induced increases in expression of anti-apoptotic mediator Bcl-2. The data were expressed as mean ± SEM of three independent experiments, * p < 0.05 vs model group.
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Fig. 9. Effect of ASD on Akt phosphorylation was detected by western blotting analysis. (A) Assessment of Akt protein levels in brain by western blotting. (B) The ratio of p-Akt/Akt was calculated by the mean of p-Akt divided the mean of Akt.
4. Discussion
Fig. 8. Effect of ASD treatment on A1–42 induced NF-B expression changes and IKK phosphorylation in brain of rats using western blotting analysis. (A) Assessment of NF-B and IKK protein levels in brain by western blotting. (B) The ratio of NF-B expression in nucleus was calculated to the actin. (C) The ratio of p-IKK/IKK was calculated by the mean of p-IKK divided the mean of IKK.
3.5. ASD attenuates Aˇ1–42-induced IKK phosphorylation and decreases NF-B p65 nuclear translocation To further explore the mechanisms underlying the protective effect of ASD, we analyzed nuclear levels of NF-B, since A1–42 induced damage provokes the activation of inflammatory pathway. We found that in A-injected rats, the levels of NF-B were significantly increased in the nucleus after 4 weeks by western blot. The increase was reduced by treating with ASD significantly, compared to A-infused animals. As shown in Fig. 8, western blot analysis of phosphor-IKK (p-IKK) and total IKK demonstrates an increase in phosphor-IKK with A1–42 stimulation that is significantly attenuated with the treatment of ASD at the dosage of 90 and 270 mg/kg.
3.6. ASD decreases Akt phosphorylation As Akt are major contributors to neuroprotection, we determined the levels of active Akt in rats injected with A1–42. Increased posphorylation of Akt reflects increased activity of Akt. Quantification of western blot revealed that the amount of Akt has no significant alteration in rats injected A < beta > 1–42 in brain when compared with vehicle control rats. However, ASD treatment at the dose of 30, 90 and 270 mg/kg significantly decreased phosphor-Akt in rats injected with A1–42 (Fig. 9).
A peptides have been shown to impair memory [28,29], produce inflammatory responses [30,31], and activate the apoptotic pathway [32,33]. Cognitive impairment is a major feature of Alzheimer’s disease and is accompanied by A deposition [34]. Microinfusions of A1–42 into the hippocampus or lateral ventricle in the brain have been shown to impair memory [28,35,36]. Consistent with these reports, we observed that A1–42-injected rats showed spatial memory deterioration in the Morris water maze and Y-maze task, and this impairment was significantly attenuated by ASD administrations. The protective effects of ASD on learning and memory impairment are in line with our previous study showing that ASD rescues ibotenic acid-associated deficits in spatial memory and learning in rats [21]. A peptides have been shown to produce inflammatory responses [30,31]. Inflammation responses mediated by glial cells play a critical role in many pathological situations related to neurodegeneration such as Alzheimer’s disease. A deposits or senile plaques are frequently associated with reactive microglia and astrocytes [37,38]. Activation of microglia and astrocytes has beneficial effects on neurodegenerative disease, including Alzheimer’s disease (AD), Parkinson’s disease, stroke and so on [39–41]. It is now well established that most types of injury or pathological processes within the CNS lead to activation of those cells from their resting state [42,43]. Microglial and astrocyte activation have been previously demonstrated in the brains of AD patients [11]. In present study, we observed that ASD also functions as a microglia and astrocyte-deactivating factor in our A1–42 injection model of AD by IHC. Many studies have shown that A neurotoxicity induces cytokine production and release of TNF-␣ and IL-1 [11,44]. COX-2 is an important mediator that is involved in the inflammatory cascade in AD [45,46]. This inflammatory process has also largely been described in brain [47,48] and in the periphery in plasma, serum or mononuclear cells of patients with AD [49,50]. To further investigate the protective effects of ASD on inflammatory responses, the levels of TNF-␣ and IL-1 were detected by Elisa, and the protein expressions of TNF-␣, IL-1 and COX-2 were determined by western blot, respectively. Our results revealed that ASD may inhibit the productions and protein expressions of TNF-␣, IL-1 and COX2, which suggested that inhibitions of TNF-␣, IL-1 and COX-2 are involved in protective effects of ASD on A-induced inflammatory responses.
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NF-B plays a critical role in many cellular events, such as expression of cytokine genes that affect inflammatory process. Concerning AD, NF-B has been shown to be upregulated and responsible for the induction of TNF-␣ and IL-1 [51–54], particularly in glial and astrocyte cells. In resting cells, NF-B exists primarily in the cytoplasm as an inactive form bound to its distinct inhibitory factor IB. Upon activation, IB is rapidly phosphorylated by IKK␣/, subsequently ubiquitinated and degraded by 26S proteasome complex. This permits the liberated NF-B dimers to translocate into the nucleus, where it binds to the promoter of target genes and activates transcription [55,56]. In the present study, our results indicated that ASD significantly inhibited NF-B transcriptional activity by attenuating A1–42-induced IKK␣/ and IB␣ phosphorylation and subsequent p65 nuclear translocation. NF-B-dependent upregulation of TNF-␣, IL-1 and COX-2 were also strongly suppressed. Since NF-B signaling enhances apoptosis [57] and inhibition of NF-B not only protracts inflammatory responses but also prevents apoptosis [58]. The bcl-2 family members are the most important molecules in regulating apoptosis [59,60]. They can be divided into anti-apoptotic members, such as Bcl-2, Bcl-xl, and Bcl-w, and pro-apoptotic members, such as Bax and Bak [61]. It has been considered that the ratio of Bcl-2/Bax is crucial in the regulation of apoptosis [61,62]. Microinfusions of A1–42 decreased Bcl-2 and increased Bax, leading to reduction of the Bcl-2/Bax ratio and increases in apoptotic responses. In the present study, our results indicated that ASD reversed the A-induced decrease in the Bcl2/Bax ratio (i.e. decreases in Bcl-2 and increases in Bax). On the basis of our results that ASD inhibited A1–42-induced NF-B activation, we further investigated the effects of ASD on upstream signaling pathways. The signal transduction pathways involved in A-dependent neurotoxicity have become a major focus in AD research. Though the activation of Akt pathway has been demonstrated to protect neurons against apoptosis [63] as phosphorylated Akt acts both to stimulated anti-apoptotic factors and to inhibit proapoptotic factors [64]. There is a general consensus that A induces Akt abnormalities as shown in the brain of AD patients [65] and animal models [66,67]. An activation of Akt was reported in hippocampal slices after treatment with soluble A1–42 [68], and its inhibition could attenuate cellular injury and neuronal death, indicating a pro-death signaling role of Akt [69,70]. Previous articles had demonstrated that Akt promotes IKK␣/ phosphorylation in a reciprocal activation mode and IKK␣/ is required for Aktmediated degradation of IB [71,72]. Further studies reported Akt stimulates the transactivation potential of the p65 subunit of NFB through IKK␣/ [73]. There suggested that Akt pathway is important not only for the canonical NF-B but also for the regulation of trans-activation potential of p65. In accordance with the above studies, our findings indicate that an obvious activation of Akt induced by A1–42 infusion. However, treatments with ASD notably decreased an elevation of phosphorylated Akt. Thus, it is concluded that inhibition of Akt activation is implicated in protective effects of ASD on A1–42-induced cognitive deficits and neuronal ultrastructure. Donepezil, an acetylcholinestrase inhibitor, is currently used to treat Alzheimer’s disease patients [74–76]. Treatment with donepezil significantly improved cognitive dysfunction in the animal model of A injury [77,78]. Therefore, donepezil was chosen as a positive control to evaluate the therapeutic efficacy of ASD. Donepezil treatment significantly improved cognitive dysfunction in rats, as assessed by the Morris water maze task in this study (Fig. 2). Meanwhile, although had no statistic significancy, donepezil did exert mild protection against astrogila and microglia activation and inflammatory reactions, attributing to decrease of Akt activation. However, unlike ASD, donepezil failed to completely
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block the NF-B pathway, which probably can explain the limited the anti-inflammatory activity. In addition, it is the improvement of ChAT activity and muscarinic receptor (M receptor) density, instead of anti-inflammatory effects,that is appreciated as playing the essencial roles in the protection of donepezil against neurodegenerative changes, as demonstrated by lots of researches from various laboratories [79–81]. In summary, the present study showed that ASD, a typical compound of Dipsacus asper Wall, may inhibit cognitive impairment and inflammatory responses induced by A1–42 for the first time. ASD afforded a beneficial action on inhibitions of Akt, as well as IKK degradation and NF-B activation produced by A. These findings suggested that ASD might be a potential agent for treatment of AD. Acknowledgments This work was supported by a National Natural Sciences Foundation of China grant (contract grant number: 30730113) and “Scientific and Technological Major Special Project-Significant Creation of New Drugs (contract grant number: 2009ZX09102-118) in the Eleventh Five-Year”. References [1] Selkoe DJ. Translating cell biology into therapeutic advances in Alzheimer’s disease. Nature 1999;399:A23–31. [2] Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002;297:353–6. [3] Verdile G, Fuller S, Atwood CS, Laws SM, Gandy SE, Martins RN. The role of beta amyloid in Alzheimer’s disease: still a cause of everything or the only one who got caught? Pharmaceutical Research 2004;50:397–409. [4] Giulian D, Haverkamp LJ, Li J, Karshin WL, Yu J, Tom D, et al. Senile plaques stimulate microglia to release a neurotoxin found in Alzheimer brain. Neurochemistry International 1995;27:119–37. [5] Klegeris A, Walker DG, McGeer PL. Interaction of Alzheimer beta-amyloid peptide with the human monocytic cell line THP-1 results in a protein kinase C-dependent secretion of tumor necrosis factor-alpha. Brain Research 1997;747:114–21. [6] Combs CK, Johnson DE, Karlo JC, Cannady SB, Landreth GE. Inflammatory mechanisms in Alzheimer’s disease: inhibition of beta-amyloid-stimulated proinflammatory responses and neurotoxicity by PPAR gamma agonists. Journal of Neuroscience 2000;20:558–67. [7] Combs CK. Inflammation and microglia actions in Alzheimer’s disease. Journal of NeuroImmune Pharmacology 2009;4:380–8. [8] Cameron B, Landreth GE. Inflammation, microglia, and Alzheimer’s disease. Neurobiology of Disease 2010;37:503–9. [9] Lue LF, Rydel R, Brigham EF, Yang LB, Hampel H, Murphy Jr GM, et al. Inflammatory repertoire of Alzheimer’s disease and nondemented elderly microglia in vitro. Glia 2001;35:72–9. [10] Kalaria RN. Microglia and Alzheimer’s disease. Current Opinion in Hematology 1999;6:15–24. [11] Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, et al. Inflammation and Alzheimer’s disease. Neurobiology of Aging 2000;21:383–421. [12] Eikelenboom P, Veerhuis R, Scheper W, Rozemuller AJ, van Gool WA, Hoozemans JJ. The significance of neuroinflammation in understanding Alzheimer’s disease. Journal of Neural Transmission 2006;113:1685–95. [13] Hung TM, Na M, Thuong PT, Su ND, Sok D, Song KS, et al. Antioxidant activity of caffeoyl quinic acid derivatives from the roots of Dipsacus asper Wall. Journal of Eyhnopharmacology 2006;108:188–92. [14] Ginty DD, Bonni A, Greenberg ME. Nerve growth factor activates a Ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB. Cell 1994;77:713–25. [15] Suh H, Song D, Huh S, Son K, Kim Y. Antinociceptive mechanisms of Dipsacus saponin C administered intrathecally in mice. Journal of Ethnopharmacology 2000;71:211–8. [16] Zhou YQ, Yang ZL, Xu L, Li P, Hu YZ, Akebia Saponin D. a saponin component from Dipsacus asper Wall, protects PC 12 cells against amyloid-beta induced cytotoxicity. Cell Biology International 2009;33:1102–10. [17] Wei W, Wang X, Kusiak JW. Signaling events in amyloid beta-peptide-induced neuronal death and insulin-like growth factor I protection. Journal of Biological Chemistry 2002;277:17649–56. [18] Delobette S, Privat A, Maurice T. In vitro aggregation facilities beta-amyloid peptide-(25–35)-induced amnesia in the rat. European Journal of Pharmacology 1997;319:1–4. [19] Pike CJ, Burdick D, Walencewicz AJ, Glabe CG, Cotman CW. Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state. Journal of Neuroscience 1993;13:1676–87. [20] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 6th ed. Amsterdam, Boston: Academic Press/Elsevier; 2007.
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Research report
Akebia Saponin D attenuates amyloid -induced cognitive deficits and inflammatory response in rats: Involvement of Akt/NF-B pathway Xing Yu a,b , Lin-na Wang a,b , Qian-ming Du a,b , Lin Ma a,b , Li Chen a,b , Ran You a,b , Ling Liu a,b , Jing-jing Ling a,b , Zhong-lin Yang c , Hui Ji a,b,∗ a
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, 210009 Nanjing, PR China Department of Pharmacology, China Pharmaceutical University, 24 Tong Jia Xiang, 210009 Nanjing, PR China c Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, 210009 Nanjing, PR China b
h i g h l i g h t s Akebia Saponin D is a typical saponin component from Dipsacus asper Wall. ASD improved memory impairment in the A1–42-induced dementia model. ASD attenuates inflammatory response induced by A1–42.
a r t i c l e
i n f o
Article history: Received 3 May 2012 Received in revised form 27 July 2012 Accepted 31 July 2012 Available online 8 August 2012 Keywords: Alzheimer’s disease Akebia Saponin D (ASD) Amyloid  (A)1–42 Cognitive impairment Inflammatory responses Akt/NF-B
a b s t r a c t Neuroinflammatory responses caused by amyloid (A) play an important role in the pathogenesis of Alzheimer’s disease (AD). A is known to be directly responsible for the activation of glial cells and induction of apoptosis. Akebia Saponin D (ASD) is extracted from a traditional herbal medicine Dipsacus asper Wall, which has been shown to protect against ibotenic acid-induced cognitive deficits and cell death in rats. In this study, we investigated the in vivo protective effect of ASD on learning and memory impairment induced by bilateral intracerebroventricular injections of A1–42 using Morris water and Y-maze task. Furthermore, the anti-inflammatory activity and neuroprotective effect of ASD was examined with methods of histochemistry and biochemistry. These data showed that oral gavage with ASD at doses of 30, 90 and 270 mg/kg for 4 weeks exerted an improved effect on cognitive impairment. Subsequently, the ASD inhibited the activation of glial cells and the expression of tumor necrosis factor (TNF)-␣, interleukin-1 beta (IL-1) and cyclooxygenase-2 (COX-2) in rat brain. Moreover, ASD afforded beneficial actions on inhibitions of Akt and IB kinase (IKK) phosphorylations, as well as nuclear factor B (NF-B) activation induced by A1–42. These results suggest that ASD may be a potential agent for suppressing both Alzheimer’s disease-related neuroinflammation and memory system dysfunction. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is a multifaceted neurodegenerative disorder characterized by progressive cognitive dysfunction and neuronal cell death, which correlates with brain deposition of senile plaques and accumulation of neurofibrillatory tangles [1]. Accumulation of A is hypothesized to initiate a pathogenic cascade that eventually results in AD [2]. The mechanism underlying A-induced neurotoxicity is complex, involving several pathways as signaling events [3]. It is now well documented that fibrillar
∗ Corresponding author at: State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia Xiang, 210009 Nanjing, PR China. Tel.: +86 258 602 1369. E-mail address: [email protected] (H. Ji). 0166-4328/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2012.07.045
forms of amyloid  may serve as an inflammatory stimulus for neuronal cells, and the underlying signaling mechanisms have been identified by several research groups [4–6]. Thus, in addition to direct neurotoxic effects, A assemblies have the ability to stimulate the proliferation of glial cells, and microglial-mediated inflammatory responses in the AD brain are well-documented [7,8]. Furthermore, increased numbers of morphologically reactive microglia is a common histological observation from AD brains as compared to non-demented control brains [9]. Neuroinflammation in AD is considered a downstream consequence of A aggregation in the brain, which results in microglial activation initiating a pro-inflammatory cascade and release of cytokines, chemokines, reactive oxygen and nitrogen species, and proteolytic enzymes, all of which contribute to neuronal degeneration [10–12]. There may also be other as yet unknown actions.
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according to the stereotaxic atlas of Paxinos and Watson [20]. Animals were injected with 5.0 l sterile bidistiled water (vehicle-treated, 10 rats) or 5.0 l aggregated A1–42 solution (50 rats) into each cerebral lateral ventricle at a rate of 1 l/min by pump.
2.4. Drug administration and experimental design
Fig. 1. Chemical structure of ASD.
At present, there is no definitive treatment or cure for AD. A large segment of the public finds solace in herbs, in part believing that herbs are natural and hence safer than synthetic drugs, and that a complex mixture of herbs can effectively treat complex disease. In addition, the metabolite profile of herbal medicines is important for screening its active constituents, thus providing a valuable contribution to the drug discovery process and elucidation of the underlying mechanism of action. Dipsacus asper Wall (DAW), belonging to Dipsacaceae, is a kind of perennial herb growing in moist fields and mountain [13]. The roots of DAW have been used as a tonic, an analgesic and anti-inflammatory agent in China for the therapy of low back pain, rheumatic arthritis, traumatic hematoma, threaten abortion and bone fractures. Akebia Saponin D (ASD), is a typical bioactive triterpenoid saponin isolated from the rhizome of DAW. Pharmacological study has demonstrated that ASD can promote human osteoblast proliferation and differentiation [14], anti-oxidation, anti-nociceptive and neuroprotective activities [15]. In this study, we aimed to observe the protective effects of ASD on cognitive deficits in a rat model of AD induced by A1–42. Furthermore, we addressed the mechanisms that contribute to the neuroprotective effects of ASD and whether it occurs via anti-inflammatory pathway.
The surgery took place on Day 0. ASD at the dose of 30 mg/kg, 90 mg/kg and 270 mg/kg, or donepezil at the dose of 0.5 mg/kg was intragastrically administered once per day for consecutive 28 days starting on Day 1. Control and model groups received the same volume of vehicle (0.5% sodium carboxyl methyl cellulose, CMCNa) as ASD. The dosage of donepezil was similar to those commonly used in clinical practice and in previous reports [21–23]. Morris water maze task took place from Day 22 to Day 26. The next day after the water-maze probe trial test (i.e. 27 days after A1–42 infusions), the same rats were tested for memory using the Y-maze task. All the treatment was given 1 h before the maze training. The rats were decapitated after the Y maze for biochemical and histological examination.
2.5. Morris water maze task The Morris water maze task was carried out from Day 22 to Day 26 after A1–42injection as described. The water maze [24] was a black circular pool (120 cm in diameter, 50 cm deep) divided into 4 equal imaginary quadrants for data analysis. The water temperature was maintained between 21 and 23 ◦ C. One centimeter beneath the surface of the water and hidden from rats view was a black circular platform 10 cm in diameter. The swimming patterns of the rats were recorded with a video camera mounted above the center of the pool and analyzed with a videotracking system set at 10 samples/s. The water maze was located in a room with several visual stimuli hanging on the walls to provide spatial cues. The acquisition phase was carried out during 4 consecutive days (from Day 22 to Day 25). On each training day/session, the rats received 4 consecutive training trials with the hidden platform kept in a constant position. A different starting location was used for each trial, which consisted of swimming followed by remaining on the platform for at least 10 s. Rats not finding the platform within 90 s were guided to it by the experimenter. The time latency to reach the platform, the distance traveled and the swimming speed were recorded. On Day 26, to assess spatial retention, a 90 s probe trial with the platform removed from the pool was given one day after the last hidden platform trial. The time spent swimming in the target quadrant (where the platform was located during hidden platform training) was measured. In probe trials the time latency to reach the target quadrant and the time each rat spent in it were calculated. At the end of the probe test a highly visible platform was placed in the center of the pool. The visible platform subtest was performed to determine whether the experimental manipulation affects visual acuity or motivation.
2. Materials and methods
2.6. Y-maze task
2.1. Animals and housing
The Y-maze test was performed as previously described [25,26] on Day 27 and Day 28. The Y-maze [27] was constructed of black plastic walls (10 cm high). It consisted of three compartments (10 cm × 10 cm) connected with 4 cm × 5 cm passages, floors were constructed of 3.175 mm stainless steel rods set 8 mm apart. The test was conducted for 2 consecutive days at the same time each day. On the first day (learning trial) each rat was placed in one of the compartments and allowed to move freely for 5 min. Then turning on the power, two of the compartments have electric shock (10 V) through the stainless steel grid floor, another one has no electric shock, but its light was turned on. If the rat enters the compartment which has no electric shock, we record it right. Otherwise, the rat was gently navigated to the compartment which has no electric shock for 30 s. The times of electric shock with 9 positive in 10 continuous trials were recorded. On the second day (testing trial), the rats were tested 10 times, the number of corrects were recorded manually. ASD (30 mg/kg, 90 mg/kg and 270 mg/kg, i.g.) was administered 1 h before the trial.
60 adult male 3-months-old Sprague–Dawley rats (B&K Laboratory Animal Corp. Ltd., Shanghai) weighing 250–300 g were maintained under standardized conditions (22 ± 1 ◦ C, 60 ± 10% humidity, 12 h light/dark cycle), and provided free access to food and water ad libitum. The rats were randomly divided into 6 groups of 10 rats each: Control, Model (A1–42), Donepezil (A1–42 + 0.5 mg/kg donepezil) as a positive control, ASD-L (A1–42 + 30 mg/kg ASD), ASD-M (A1–42 + 90 mg/kg ASD) and ASD-H (A1–42 + 270 mg/kg ASD) groups. All experiments were approved and performed in accordance with National Institutes of Health Guide for Care and Use of Laboratory Animals (publication no. 85-23, revised 1985). 2.2. Drugs ASD (Fig. 1) was prepared by Professor Zhong-Lin Yang. It was isolated from Dipsacus asperoides Wall at the Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University [16]. Commercially available A1–42 (Invitrogen, USA) aggregated according to previous study [17]. Briefly, the peptide was dissolved in sterile bidistiled water at concentration of 2 g/l, aliquoted into tubes and stored at −20 ◦ C. It was “aged” by incubation at 37 ◦ C for 4 days before the surgery [18,19]. Light microscopic observation demonstrated the existence of both birefringent fibril-like structures and globular aggregates.
2.7. Preparation of cytosolic and nuclear extracts After Y-maze, the animals were decapitated (Day 28). Brains were immediately taken out and washed in ice-cold PBS solution. Hippocampus tissues were isolated and frozen in liquid nitrogen before analysis. Protein extraction of cytosolic and nuclear fractions was performed using a cytosolic/nuclei isolation kit (KGP150, NanJing KeyGEN Biotechnology Co., Ltd., Nanjing, China), according to a multiple centrifugation method. Protein concentration was determined using an enhanced BCA protein assay (Beyotime Institute of Biotechnology Co., Ltd. Haimen, China).
2.3. Surgery Rats were anaesthetized with intraperitoneal (i.p.) administration of sodium pentobarbital (30 mg/kg) before they placed in a stereotaxic apparatus (RWD Life Science Co., Ltd. Shenzhen, China). Guide cannulae (22-gauge, 6 mm; Ka Science, Germany) were implanted into bilateral lateral ventricles using the following coordinates: AP-3.3 mm from Bregma; ML 2.0 mm from the midline; DV-2.5 mm from dura
2.8. Measurement of TNF-˛ and IL-1ˇ level The TNF-␣ and IL-1 levels were assayed using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Jianglai Bioengineering Institute Co., Ltd., Shanghai, China) according to the manufacturer’s protocol.
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2.9. Immunohistochemistry Free-floating brain slices (6-m) were washed in PBS to remove cryopreservative. The tissues were incubated for 10 min at room temperature in 3% H2 O2 solution to reduce endogenous peroxidase activity, then washed in PBS again. The samples were placed in rabbit serum for 1 h at room temperature (SP immunohistochemical staining kit, Maxim biotechnology Co., Ltd, Fuzhou, China). Incubation overnight at 4 ◦ C was performed with anti-macrophage antibody [MAC387] (1:500, Abcam,
Cambridge, UK), anti-GFAP antibody (1:600, Millipore Corporation, MA, USA). After that, the slices were washed in PBS and incubated with biotinylated rabbit anti-rat IgG for 1 h at room temperature, again washed, and then incubated in avidin–biotin horseradish peroxidase macromolecular complex (Maxim biotechnology Co., Ltd., Fuzhou, China) for 1 h at room temperature. To develop color, slices were incubated briefly in DAB substrate kit (Maxim biotechnology Co., Ltd., Fuzhou, China). After a final set of washed in PBS, the slices were dehydrated, cleared, and coverslipped with mounting medium.
Fig. 2. Effects of ASD (30, 90 and 270 mg/kg, 4 weeks) on A1–42-induced spatial cognitive deficits in the Morris water-maze test in rats. (A) The latency to escape onto the submerged platform and (B) distance traveled during the 4-days acquisition trials. (C) The times passed through the former platform location and (D) distance swum in the target quadrant in the probe trial test performed 24 h after the last acquisition trial. (E) Representative searching swimming paths by rats with different treatments in the probe trial test. The data were expressed as mean ± SEM, n = 10 per group, *p < 0.05, **p < 0.01, ***p < 0.001 vs model group.
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2.10. Western blot analysis For western blotting assay, the remaining 7 rats of each group were euthanized by rapid decapitation on Day 28, and 3 brains rapidly frozen at −80 ◦ C each group. The hippocampus were cut into small pieces, and lysed in ice-cold RIPA buffer (Beyotime Institute of Biotechnology Co., Ltd. Haimen, China). The lysate was centrifuged at 12,000 × g for 5 min at 4 ◦ C and the supernatant was collected. Equal amounts of protein were separated on 15% SDS-polyacrylamide gels. Proteins on the gel were blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature. Subsequently, the membranes were incubated at 4 ◦ C overnight with the rabbit anti-COX-2 antibody(1:500, Proteintech Group Inc., USA), rabbit anti-TNF-␣ antibody(1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-IL-1 antibody(1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-bcl-2 antibody(1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-Bax(1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-NF-B p65 antibody(1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-IKK antibody (1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-IKK␣/ antibody (1:500, Bioworld Technology Co., Ltd., MN, USA), rabbit anti-Akt antibody(1:1000, Cell Signaling Technology Co., Ltd., MN, USA), rabbit anti-phosphor-Akt antibody (1:1000, Cell Signaling Technology Co., Ltd, MN, USA). Following three washes with TBST, the blots were incubated with the secondary horseradish peroxidase-conjugated antibody (goat anti-rabbit IgG, 1:10,000, Bioworld Technology Co., Ltd., MN, USA) at the room temperature for 2 h. The antibody-reactive bands were visualized by using the enhanced chemiluminescence detection reagents (Keygen Biotechnology Co., Ltd., Nanjing, China) by a gel imaging system (ChemiScope 2850, Clinx Science Instruments Co., Ltd., Shanghai, China). 2.11. Statistical analysis The density of positive cells in histochemistry analysis was calculated by ImagePro Plus image analysis software. The band intensity in western blot analysis was calculated by AlphaEaseFC software. Data were analyzed statistically using one-way analysis of variance followed by Tukey’s post hoc test. Summarized data were expressed as mean ± SEM. Statistical significance was accepted at p < 0.05. All statistical analysis was undertaken using SPSS vs 11.5.
3. Results 3.1. ASD reversed memory impairment induced by Aˇ1–42 in Morris water-maze and Y-maze tasks To establish memory deficits produced by A1–42, we examined memory performance in rats micro-infused with aged A1–42 at 10 g/side into lateral ventricles using the Morris water-maze and Y-maze tests. During the acquisition trials in the water-maze test, all the rats, regardless of the different treatments, displayed progressive decrease in escape latency to reach the hidden platform. Results indicated that A1–42, administered 23 days prior to the beginning of acquisition training, increased escaped latencies on sessions 2, 3, 4 compared to the corresponding vehicle controls (p < 0.01 or 0.001, Fig. 2A), these were reversed by ASD at different doses especially at 270 mg/kg for session 2–4 and 90 mg/kg for session 3 (p < 0.05 or 0.01, Fig. 2A). In addition, the rats also displayed slight, progressive decrease in distance traveled over time. The groups treated with ASD showed a significant decrease in distance traveled compared to the model group for session 3 (p < 0.01 or 0.001, Fig. 2B). 24 h after the last acquisition trial, rats were tested for spatial memory in the probe trial test, during which the platform was removed. Drug treatment altered entries and distance swum in the target quadrant. Results indicated that A1–42 decreased entries (p < 0.05) and distance (p < 0.01) compared to the respective vehicle controls, these were reversed by chronic treatment with ASD (p < 0.05 or p < 0.01, Fig. 2C and D). Consistent with this, the behavioral tracking results revealed that both vehicle - and A/ASD-treated rats displayed more exploration in the target quadrant than the rats treated with A1–42 alone (Fig. 2E). This result is consistent with ASD’s reversal of A1–42-induced decreases in escape latency and length of swimming path. The next day after the water-maze probe trial test (i.e. 26 days after A1–42 infusions), the same rats were tested for short-term
Fig. 3. Effect of ASD on the performance in the presence of A1–42 in the Y-maze test in rats. (A) Changes in the arm entries on learning training; (B) Changes in the arm entries on memory training. The data were expressed as mean ± SEM, n = 10 per group, *p < 0.05, **p < 0.01, ***p < 0.001 vs model group.
memory using the Y-maze task, with daily ASD treatment continued. As shown in Fig. 3A, Compared to the saline injected control group, the spontaneous alternations of arm entries were lowered in A1–42 injected model group (***p < 0.001). The alternation memory reduction observed in the A1–42 injected model was significantly improved by the administration of 90 and 270 mg/kg ASD (*p < 0.05, **p < 0.01, Fig. 3B). 3.2. ASD suppress the activation of microglia and astroglia in the brain following a Aˇ1–42 insult To assess the involvement of glial cells following A1–42 infusion, immunohistochemical analysis of macrophage, a marker protein for microglia (Fig. 4A) and GFAP, a marker for astroglia (Fig. 4B) were carried out in the brain. Quantitative analysis showed that infusion of soluble A1–42 produced a significant increase of GFAP-positive astroglial cells and reactive microglia in the brain compared with the vehicle control (Fig. 4C and D). Treatment with ASD significantly suppressed the number of GFAP-positive astrocytes (90 and 270 mg/kg, p < 0.05) in the brain. 3.3. ASD inhibits Aˇ1–42-induced TNF-˛, IL-1ˇ and COX-2 production and expression To examine the potential mechanisms of ASD to inhibit inflammation induced by A1–42, the levels of TNF-␣ and IL-1 were measured by ELISA. The result (Fig. 5) showed that brain concentration of cytokines increased in rats treated with A1–42 compared with the control group. However, TNF-␣ and IL-1 decreased in rats treated with ASD for 4 weeks. We further examined the expressions
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Fig. 4. Effect of ASD on the activation of microglia and astroglia in the brain following a A1–42 insult. (A) Represents photomicrographs of macrophage immunohistochemistry in brain (magnification 400×). Scale bar = 50 m. (B) Represents photomicrographs of GFAP immunohistochemistry in brain (magnification 400×). Scale bar = 50 m. (C) The number of macrophage immunopositive cells. (D) The number GFAP immunopositive cells. The data were expressed as mean ± SEM, n = 3 per group, *p < 0.05, **p < 0.01, ***p < 0.001 vs model group. The result indicated at least three independent experiments in each animal.
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Fig. 5. Effect of ASD treatment on A1–42 induced enhancement of TNF-␣ and IL-1 expression in brain of rats by ELISA. (A) TNF-␣ level in brain; (B) IL-1 level in brain. The data were expressed as mean ± SEM, n = 7 per group, *p < 0.05, **p < 0.01, ***p < 0.001 vs model group.
of TNF-␣, IL-1 and COX-2 protein by western blot. Consistent with the result of ELISA, as shown in Fig. 6 expressions of TNF-␣, IL-1 and COX-2 were markedly increased in response to A. However, after treatment with ASD at the doses of 90 and 270 mg/kg, the TNF␣, IL-1 and COX-2 protein expressions significantly decreased compared with model group (p < 0.01, Fig. 6). All these results
Fig. 6. Effect of ASD treatment on A1–42 induced COX-2, TNF-␣ and IL-1 expression changes in brain of rats using western blotting analysis. (A) Assessment of COX-2, TNF-␣ and IL-1 protein levels in brain by western blotting. (B) The ratio of COX-2 was calculated to the actin. (C) The ratio of TNF-␣ was calculated to the actin. (D) The ratio of IL-1 was calculated to the actin. The data were expressed as mean ± SEM of three independent experiments, *p < 0.05 vs model group.
indicated that ASD could inhibit the generation of inflammatory cytokines induced by A1–42. 3.4. ASD reverses Aˇ1–42-induced apoptotic effects Since A1–42 decreases neuronal cell viability via increased apoptotic, we examined expression of Bax, a pro-apoptotic protein that induces rapid human neuronal cell death, and Bcl-2, another cell death-associated protein, to determine whether apoptotic responses were involved in the effect of ASD on A1–42-induced toxicity. As shown in Fig. 7B and C, in treatment of ASD at the dosage of 90, 270 mg/kg., proapoptic Bax was down-regulated (p < 0.01) and antiapoptic Bcl-2 was up-regulated (p < 0.01).
Fig. 7. Effect of ASD on A1–42-induced changes in Bcl-2 and Bax in the brain in rats. (A) Assessment of Bax and Bcl-2 protein levels in brain by western blotting. (B) ASD reversed A1–42-induced increases in expression of the apoptotic mediator Bax. (C) ASD reversed A1–42-induced increases in expression of anti-apoptotic mediator Bcl-2. The data were expressed as mean ± SEM of three independent experiments, * p < 0.05 vs model group.
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Fig. 9. Effect of ASD on Akt phosphorylation was detected by western blotting analysis. (A) Assessment of Akt protein levels in brain by western blotting. (B) The ratio of p-Akt/Akt was calculated by the mean of p-Akt divided the mean of Akt.
4. Discussion
Fig. 8. Effect of ASD treatment on A1–42 induced NF-B expression changes and IKK phosphorylation in brain of rats using western blotting analysis. (A) Assessment of NF-B and IKK protein levels in brain by western blotting. (B) The ratio of NF-B expression in nucleus was calculated to the actin. (C) The ratio of p-IKK/IKK was calculated by the mean of p-IKK divided the mean of IKK.
3.5. ASD attenuates Aˇ1–42-induced IKK phosphorylation and decreases NF-B p65 nuclear translocation To further explore the mechanisms underlying the protective effect of ASD, we analyzed nuclear levels of NF-B, since A1–42 induced damage provokes the activation of inflammatory pathway. We found that in A-injected rats, the levels of NF-B were significantly increased in the nucleus after 4 weeks by western blot. The increase was reduced by treating with ASD significantly, compared to A-infused animals. As shown in Fig. 8, western blot analysis of phosphor-IKK (p-IKK) and total IKK demonstrates an increase in phosphor-IKK with A1–42 stimulation that is significantly attenuated with the treatment of ASD at the dosage of 90 and 270 mg/kg.
3.6. ASD decreases Akt phosphorylation As Akt are major contributors to neuroprotection, we determined the levels of active Akt in rats injected with A1–42. Increased posphorylation of Akt reflects increased activity of Akt. Quantification of western blot revealed that the amount of Akt has no significant alteration in rats injected A < beta > 1–42 in brain when compared with vehicle control rats. However, ASD treatment at the dose of 30, 90 and 270 mg/kg significantly decreased phosphor-Akt in rats injected with A1–42 (Fig. 9).
A peptides have been shown to impair memory [28,29], produce inflammatory responses [30,31], and activate the apoptotic pathway [32,33]. Cognitive impairment is a major feature of Alzheimer’s disease and is accompanied by A deposition [34]. Microinfusions of A1–42 into the hippocampus or lateral ventricle in the brain have been shown to impair memory [28,35,36]. Consistent with these reports, we observed that A1–42-injected rats showed spatial memory deterioration in the Morris water maze and Y-maze task, and this impairment was significantly attenuated by ASD administrations. The protective effects of ASD on learning and memory impairment are in line with our previous study showing that ASD rescues ibotenic acid-associated deficits in spatial memory and learning in rats [21]. A peptides have been shown to produce inflammatory responses [30,31]. Inflammation responses mediated by glial cells play a critical role in many pathological situations related to neurodegeneration such as Alzheimer’s disease. A deposits or senile plaques are frequently associated with reactive microglia and astrocytes [37,38]. Activation of microglia and astrocytes has beneficial effects on neurodegenerative disease, including Alzheimer’s disease (AD), Parkinson’s disease, stroke and so on [39–41]. It is now well established that most types of injury or pathological processes within the CNS lead to activation of those cells from their resting state [42,43]. Microglial and astrocyte activation have been previously demonstrated in the brains of AD patients [11]. In present study, we observed that ASD also functions as a microglia and astrocyte-deactivating factor in our A1–42 injection model of AD by IHC. Many studies have shown that A neurotoxicity induces cytokine production and release of TNF-␣ and IL-1 [11,44]. COX-2 is an important mediator that is involved in the inflammatory cascade in AD [45,46]. This inflammatory process has also largely been described in brain [47,48] and in the periphery in plasma, serum or mononuclear cells of patients with AD [49,50]. To further investigate the protective effects of ASD on inflammatory responses, the levels of TNF-␣ and IL-1 were detected by Elisa, and the protein expressions of TNF-␣, IL-1 and COX-2 were determined by western blot, respectively. Our results revealed that ASD may inhibit the productions and protein expressions of TNF-␣, IL-1 and COX2, which suggested that inhibitions of TNF-␣, IL-1 and COX-2 are involved in protective effects of ASD on A-induced inflammatory responses.
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NF-B plays a critical role in many cellular events, such as expression of cytokine genes that affect inflammatory process. Concerning AD, NF-B has been shown to be upregulated and responsible for the induction of TNF-␣ and IL-1 [51–54], particularly in glial and astrocyte cells. In resting cells, NF-B exists primarily in the cytoplasm as an inactive form bound to its distinct inhibitory factor IB. Upon activation, IB is rapidly phosphorylated by IKK␣/, subsequently ubiquitinated and degraded by 26S proteasome complex. This permits the liberated NF-B dimers to translocate into the nucleus, where it binds to the promoter of target genes and activates transcription [55,56]. In the present study, our results indicated that ASD significantly inhibited NF-B transcriptional activity by attenuating A1–42-induced IKK␣/ and IB␣ phosphorylation and subsequent p65 nuclear translocation. NF-B-dependent upregulation of TNF-␣, IL-1 and COX-2 were also strongly suppressed. Since NF-B signaling enhances apoptosis [57] and inhibition of NF-B not only protracts inflammatory responses but also prevents apoptosis [58]. The bcl-2 family members are the most important molecules in regulating apoptosis [59,60]. They can be divided into anti-apoptotic members, such as Bcl-2, Bcl-xl, and Bcl-w, and pro-apoptotic members, such as Bax and Bak [61]. It has been considered that the ratio of Bcl-2/Bax is crucial in the regulation of apoptosis [61,62]. Microinfusions of A1–42 decreased Bcl-2 and increased Bax, leading to reduction of the Bcl-2/Bax ratio and increases in apoptotic responses. In the present study, our results indicated that ASD reversed the A-induced decrease in the Bcl2/Bax ratio (i.e. decreases in Bcl-2 and increases in Bax). On the basis of our results that ASD inhibited A1–42-induced NF-B activation, we further investigated the effects of ASD on upstream signaling pathways. The signal transduction pathways involved in A-dependent neurotoxicity have become a major focus in AD research. Though the activation of Akt pathway has been demonstrated to protect neurons against apoptosis [63] as phosphorylated Akt acts both to stimulated anti-apoptotic factors and to inhibit proapoptotic factors [64]. There is a general consensus that A induces Akt abnormalities as shown in the brain of AD patients [65] and animal models [66,67]. An activation of Akt was reported in hippocampal slices after treatment with soluble A1–42 [68], and its inhibition could attenuate cellular injury and neuronal death, indicating a pro-death signaling role of Akt [69,70]. Previous articles had demonstrated that Akt promotes IKK␣/ phosphorylation in a reciprocal activation mode and IKK␣/ is required for Aktmediated degradation of IB [71,72]. Further studies reported Akt stimulates the transactivation potential of the p65 subunit of NFB through IKK␣/ [73]. There suggested that Akt pathway is important not only for the canonical NF-B but also for the regulation of trans-activation potential of p65. In accordance with the above studies, our findings indicate that an obvious activation of Akt induced by A1–42 infusion. However, treatments with ASD notably decreased an elevation of phosphorylated Akt. Thus, it is concluded that inhibition of Akt activation is implicated in protective effects of ASD on A1–42-induced cognitive deficits and neuronal ultrastructure. Donepezil, an acetylcholinestrase inhibitor, is currently used to treat Alzheimer’s disease patients [74–76]. Treatment with donepezil significantly improved cognitive dysfunction in the animal model of A injury [77,78]. Therefore, donepezil was chosen as a positive control to evaluate the therapeutic efficacy of ASD. Donepezil treatment significantly improved cognitive dysfunction in rats, as assessed by the Morris water maze task in this study (Fig. 2). Meanwhile, although had no statistic significancy, donepezil did exert mild protection against astrogila and microglia activation and inflammatory reactions, attributing to decrease of Akt activation. However, unlike ASD, donepezil failed to completely
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block the NF-B pathway, which probably can explain the limited the anti-inflammatory activity. In addition, it is the improvement of ChAT activity and muscarinic receptor (M receptor) density, instead of anti-inflammatory effects,that is appreciated as playing the essencial roles in the protection of donepezil against neurodegenerative changes, as demonstrated by lots of researches from various laboratories [79–81]. In summary, the present study showed that ASD, a typical compound of Dipsacus asper Wall, may inhibit cognitive impairment and inflammatory responses induced by A1–42 for the first time. ASD afforded a beneficial action on inhibitions of Akt, as well as IKK degradation and NF-B activation produced by A. These findings suggested that ASD might be a potential agent for treatment of AD. Acknowledgments This work was supported by a National Natural Sciences Foundation of China grant (contract grant number: 30730113) and “Scientific and Technological Major Special Project-Significant Creation of New Drugs (contract grant number: 2009ZX09102-118) in the Eleventh Five-Year”. References [1] Selkoe DJ. Translating cell biology into therapeutic advances in Alzheimer’s disease. Nature 1999;399:A23–31. [2] Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002;297:353–6. [3] Verdile G, Fuller S, Atwood CS, Laws SM, Gandy SE, Martins RN. The role of beta amyloid in Alzheimer’s disease: still a cause of everything or the only one who got caught? Pharmaceutical Research 2004;50:397–409. [4] Giulian D, Haverkamp LJ, Li J, Karshin WL, Yu J, Tom D, et al. Senile plaques stimulate microglia to release a neurotoxin found in Alzheimer brain. Neurochemistry International 1995;27:119–37. [5] Klegeris A, Walker DG, McGeer PL. Interaction of Alzheimer beta-amyloid peptide with the human monocytic cell line THP-1 results in a protein kinase C-dependent secretion of tumor necrosis factor-alpha. Brain Research 1997;747:114–21. [6] Combs CK, Johnson DE, Karlo JC, Cannady SB, Landreth GE. Inflammatory mechanisms in Alzheimer’s disease: inhibition of beta-amyloid-stimulated proinflammatory responses and neurotoxicity by PPAR gamma agonists. Journal of Neuroscience 2000;20:558–67. [7] Combs CK. Inflammation and microglia actions in Alzheimer’s disease. Journal of NeuroImmune Pharmacology 2009;4:380–8. [8] Cameron B, Landreth GE. Inflammation, microglia, and Alzheimer’s disease. Neurobiology of Disease 2010;37:503–9. [9] Lue LF, Rydel R, Brigham EF, Yang LB, Hampel H, Murphy Jr GM, et al. Inflammatory repertoire of Alzheimer’s disease and nondemented elderly microglia in vitro. Glia 2001;35:72–9. [10] Kalaria RN. Microglia and Alzheimer’s disease. Current Opinion in Hematology 1999;6:15–24. [11] Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, et al. Inflammation and Alzheimer’s disease. Neurobiology of Aging 2000;21:383–421. [12] Eikelenboom P, Veerhuis R, Scheper W, Rozemuller AJ, van Gool WA, Hoozemans JJ. The significance of neuroinflammation in understanding Alzheimer’s disease. Journal of Neural Transmission 2006;113:1685–95. [13] Hung TM, Na M, Thuong PT, Su ND, Sok D, Song KS, et al. Antioxidant activity of caffeoyl quinic acid derivatives from the roots of Dipsacus asper Wall. Journal of Eyhnopharmacology 2006;108:188–92. [14] Ginty DD, Bonni A, Greenberg ME. Nerve growth factor activates a Ras-dependent protein kinase that stimulates c-fos transcription via phosphorylation of CREB. Cell 1994;77:713–25. [15] Suh H, Song D, Huh S, Son K, Kim Y. Antinociceptive mechanisms of Dipsacus saponin C administered intrathecally in mice. Journal of Ethnopharmacology 2000;71:211–8. [16] Zhou YQ, Yang ZL, Xu L, Li P, Hu YZ, Akebia Saponin D. a saponin component from Dipsacus asper Wall, protects PC 12 cells against amyloid-beta induced cytotoxicity. Cell Biology International 2009;33:1102–10. [17] Wei W, Wang X, Kusiak JW. Signaling events in amyloid beta-peptide-induced neuronal death and insulin-like growth factor I protection. Journal of Biological Chemistry 2002;277:17649–56. [18] Delobette S, Privat A, Maurice T. In vitro aggregation facilities beta-amyloid peptide-(25–35)-induced amnesia in the rat. European Journal of Pharmacology 1997;319:1–4. [19] Pike CJ, Burdick D, Walencewicz AJ, Glabe CG, Cotman CW. Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state. Journal of Neuroscience 1993;13:1676–87. [20] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 6th ed. Amsterdam, Boston: Academic Press/Elsevier; 2007.
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