J Nat Med DOI 10.1007/s11418-015-0901-0
ORIGINAL PAPER
(2R,3S)-Pinobanksin-3-cinnamate improves cognition and reduces oxidative stress in rats with vascular dementia Hong Liu1 • Min Zhao1 • Shen Yang2 • Dian-Rong Gong1 • De-Zhe Chen1 De-Yong Du3
•
Received: 3 December 2014 / Accepted: 10 March 2015 Ó The Japanese Society of Pharmacognosy and Springer Japan 2015
Abstract This study investigated the neuroprotective effects of (2R,3S)-pinobanksin-3-cinnamate (PNC) in rats with occlusion-damaged bilateral common carotid arteries. Administration with PNC (5 and 10 mg/kg/day) for 5 weeks significantly improved the behavioral performance of rats with vascular dementia, as showed in the Morris water maze test by shortening the escape latency and latency of crossing, completing more platform crossings, as well as spending more time in the target zone. Further evaluations found that PNC could markedly decrease malondialdehyde levels, enhance superoxide dismutase activity and glutathione levels, and decrease the release of cytochrome c as well as the activities of caspases. Moreover, PNC increased Nrf2 and anti-apoptotic bcl-2 protein expression, while Nox1 and pro-apopotic bax protein expression was decreased. PNC may exert its neuroprotective effects through counteracting oxidative stress and has the potential to treat vascular dementia. Keywords (2R,3S)-Pinobanksin-3-cinnamate Neuroprotective effects Oxidative stress Morris water maze
H. Liu and M. Zhao contributed equally. & Min Zhao [email protected] 1
Department of Neurology, Liaocheng Hospital, Liaocheng 252000, China
2
Department of Neurology, Taian Central Hospital, Taian 271000, China
3
Department of Neurosurgery, The Affiliated Hospital of Binzhou Medical University, Binzhou 256600, China
Introduction Dementia is a syndrome due to brain disease where there is disturbance of multiple higher cortical and neuropsychological functions including memory, orientation, thinking, calculation, learning capacity and language [1]. Vascular dementia (VD) is a mental disorder caused by brain damage due to cerebrovascular disease and has gained increasing attention in recent years for being the second most common type of dementia following Alzheimer’s disease [2, 3]. Together with the increasing age of the population and improved survival rates from cardiovascular diseases, VD may affect more individuals in the future [4]. About 1–4 % of people over 65 years old suffer from VD and the morbidity rate after 65 years of age doubles every 5–10 years [5]. Moreover, VD in turn increases the risk of recurrent stroke, dependent living and death [6]. There is substantial evidence that conventional risk factors such as hypertension, hyperhomocysteinemia and dyslipidemia play an important role in the development of VD [7–9]. Etiopathogenic mechanisms leading to VD include oxidative stress, cytotoxicity of reactive oxygen species (ROS), mitochondrial dysfunction and apoptosis [10, 11]. Supplementation of exogenous anti-oxidants can effectively curtail the oxidative damage to cellular macromolecules. One category of such anti-oxidants is flavonone, with diversified biological activities including anti-oxidative, radical scavenging and anti-inflammatory effects, most interestingly in attenuating the redox imbalance and improving neuro-cognitive performance [12, 13]. (2R,3S)Pinobanksin-3-cinnamate (PNC, Fig. 1) is a new flavonone, isolated from the seed of Alpinia galanga Willd., which has shown a neuroprotective effect on H2O2-damaged PC12 cells [14].
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model group received vehicle (0.5 % methylcellulose) during the same period. All animal experiments were performed in compliance with the Chinese legislation on the use and care of laboratory animals and were approved by the Ethical Committee on Animal Care and Use. Bilateral common carotid artery occlusion
Fig. 1 Structure of (2R,3S)-pinobanksin-3-cinnamate (PNC)
When the blood supplied to the brain is reduced, VD occurs and leads to a progressive decline in memory and cognitive function [15]. VD can be successfully induced by bilateral common carotid artery occlusion in rats, resulting in significant white matter lesions, learning and memory impairment, and hippocampal neuronal damage [16, 17]. In this study, we will investigate the protective effect of (2R,3S)-pinobanksin-3-cinnamate on rats with VD.
After being anesthetized with 10 % chloral hydrate, the skin of the rat was incised along the midline of the cervical region to expose the bilateral common carotid arteries without nerve damage; they were carefully separated from the surrounding tissues, approximately 1 cm inferior to the origin of the external carotid artery [16]. Both common carotid arteries were tied twice with silk sutures. The skin incision was sutured and the animals were allowed to recover from the anesthesia. Control rats were subjected to the same surgical procedure without occlusion of arteries. Animals that exhibited an abnormal post-operative condition were excluded from the study. Morris water maze test
Materials and methods Chemicals and reagents Malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) assay kits were purchased from Jiancheng Institute of Biological Engineering (Nanjing, China). Cytochrome c enzyme-linked immunosorbent assay kit was from R&D Systems (Minneapolis, MN, USA). Caspase-3 and caspase-9 activity assay kits were purchased from Kaiji Institute of Biological Engineering (Nanjing, China). In Situ Cell Death Detection Kit was purchased from Roche (Mannheim, Germany). (2R,3S)-Pinobanksin3-cinnamate (purity [ 98 %) was isolated and purified by Department of Natural Product, Institute of Materia Medica, Shandong Academy of Medical Sciences. Experimental groups and drug treatment Forty-eight healthy male Wistar rats (280–300 g) were obtained from the Animal Center of Shandong Lukang Pharmaceutical Group Co., Ltd. (Jinan, China). Rats were kept under conditions of constant temperature (25 ± 2 °C) and humidity (55 ± 5 %), 12-h light/12-h dark photoperiod, and free access to food and water. After acclimatization to the laboratory environment, rats were divided randomly into four groups: control group, VD model group and PNC groups (low and high dose). Rats in the PNC groups received a daily oral dose of 5 or 10 mg/kg, respectively, from the eighth week to the twelfth week after the operation. Rats in the control group and
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The rat behavioral test was performed by the Morris water maze (MWM) test [18]. After treatment, rats were trained four times per day for four consecutive days with an interval between sessions of 30–40 min. The rat was placed in the water facing the wall at one of three random starting positions (in three different quadrants that did not contain the platform). Each trial had a ceiling time of 60 s and a trial interval of 60 s. After climbing onto the platform, rats stayed there for 30 s before the next trial. If the rat failed to reach the platform within 60 s, it was gently placed on the platform and remained there for 30 s. Latency to escape from the water maze was calculated for each trial. On the fifth day, a probe test was carried out by removing the platform and each rat was allowed to swim freely for 120 s. The latency of first crossing the platform, the time which a rat spent in the target quadrant and the number of crossings of the non-exits were recorded for each trial. All data were recorded and analyzed by a video tracking system. Sample preparation and biochemical evaluation Rats were killed for biochemical evaluation after MWM tests. Brains were quickly removed and the cerebral cortex and hippocampus were separated on ice. Some tissues were weighed and homogenated in phosphate-buffered saline (PBS; pH 7.4) and centrifuged at 10,000 rpm at 4 °C for 15 min to remove cellular debris. Supernatants were kept at -80 °C and used for further tests. Other tissues were fixed with freshly prepared 4 % paraformaldehyde in PBS (pH 7.4) overnight, dehydrated by gradient sucrose, and
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transferred into 30 % sucrose solution for cryoprotection. Sections of 15 lm thickness were made using a freezing microtome. Measurement of MDA and GSH levels and SOD activity MDA, SOD and GSH assays were conducted by colorimetric kits according to the manufacturers’ instructions. The levels of MDA and GSH and activity of SOD were expressed as per gram tissue. Assessment of cytochrome c release The concentration of cytochrome c was measured according to the manufacturer’s instructions. After reaction with reagents, the optical density was measured by a microplate reader at 490 nm. The concentration was calculated according to the standard curve. Measurement of caspase activity The caspase activity was measured according to the manufacturer’s instructions. Briefly, aliquots of supernatants containing 25 mg protein were added to a reaction buffer supplemented with 0.1 % CHAPS, 5 mM DTT and 100 mM PMSF. The reactions were initiated after adding the following fluorescent substrates (50 mM at the final concentration): Ac-DEVD-Amc for caspase-3 activity and Ac-LEDH-Afc for caspase-9 activity. After incubation for 2 h at 37 °C, the cleavage of the substrates was measured (Amc: 390/475 nm; Afc: 400/505 nm) by a microplate reader. The protein content was determined by the bicinchoninic acid method. The activity of caspase was expressed as per min per mg protein. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay
subjected to electrophoresis on a 12 % (v/v) SDS–polyacrylamide gel. After proteins were electroblotted to a PVDF membrane, the membrane was blocked with PBST containing 5 % dried non-fat milk at room temperature, washed three times and incubated with indicated primary antibodies at 4 °C overnight, followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h. After incubation, the membrane was washed three times, and the antigen–antibody complexes were visualized by the enhanced chemiluminescence system (PerkinElmer). Relative protein expression was compared to the control group, which was set at 1. Statistical analysis Values are presented as mean ± SD. Results were statistically analyzed by one-way analysis of variance (ANOVA) followed by multiple comparison tests using Sigma Stat statistical software (SPSS Inc., Chicago, IL, USA). Group differences in the escape latency in the MWM training task were analyzed with two-way ANOVA with repeated measures, the factors being treatment and training day. Differences were considered significant at P \ 0.05.
Results Measurement of MDA and GSH levels and SOD activity Oxidative stress was assessed by MDA and GSH levels and SOD activity. Figure 2 shows that MDA level was significantly increased in the model group, while GSH level and SOD activity were decreased. Treatment with PNC (5 and 10 mg/kg) significantly reversed the oxidative stress in rats, compared with the model group (P \ 0.05).
Apoptotic cells in the hippocampus were detected by TUNEL staining using an In Situ Cell Death Detection Kit, according to the manufacturer’s instructions. Sections were rinsed and visualized with DAB, and then mounted with coverslips. The number of TUNEL-positive cells was counted under the microscope and compared in each group.
Measurement of cytochrome c
Western blot analysis
Activities of caspase-3 and caspase-9
Total cellular and nuclear protein was quantified using the protein assay kit (Biyotime, China). Homogenate in 59 SDS sample buffer was boiled for 5 min, and then equal amounts of protein (50 lg) from each sample were
As shown in Fig. 4, activities of caspase-3 and caspase-9 in the model group increased significantly. After treatment with PNC, caspase activities were significantly decreased compared to the model group (P \ 0.01).
Figure 3 shows that the cytochrome c released into the cytoplasm was significantly increased in the model group. However, treatment with PNC (5 and 10 mg/kg) significantly decreased cytochrome c levels in rats (P \ 0.05).
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Fig. 3 Effect of PNC on cytochrome c release in the cerebral cortex and hippocampus of VD rats. Values are expressed as mean ± SD (n = 8). ##P \ 0.01 vs control group; *P \ 0.05, **P \ 0.01 vs model group
Fig. 4 Effect of PNC on caspase activity in the cerebral cortex and hippocampus of VD rats. Values are expressed as mean ± SD (n = 8). ##P \ 0.01 vs control group; **P \ 0.01 vs model group
Apoptosis detection by TUNEL staining Fig. 2 Effect of PNC on oxidative stress in VD rats. Oxidative stress was evaluated by examining MDA content (a), SOD activity (b), and GSH content (c). Values are expressed as mean ± SD (n = 8). ## P \ 0.01 vs control group; *P \ 0.05, **P \ 0.01 vs model group
Protein expression Western blotting results confirmed that nuclear factor erythroid 2-related factor 2 (Nrf2) and anti-apopotic bcl2 protein expression was significantly increased, while Nox1 and pro-apoptotic bax protein expression was significantly decreased after treatment with PNC (Fig. 5).
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Figure 6 shows that apoptotic cells were significantly increased in the model group. However, after treatment with PNC (5 and 10 mg/kg), apoptosis was significantly decreased in the hippocampus (P \ 0.05). PNC improved the behavioral performance of rats with VD Figure 7a shows that after training for 4 days, the performance of rats in all groups was improved with shortened escape latencies. During the MWM test, the tested rats displayed no difference in motor activity. However, rats in the model group had longer escape latencies than rats in the
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Figure 7b–d shows that in the probe trial, rats in the model group had longer latency of crossing (P \ 0.01), completed fewer platform crossings (P \ 0.01) and spent less time in the target zone (P \ 0.01) than rats in the control group. Significant improvement of these indexes was observed in the PNC group.
Discussion
Fig. 5 Effect of PNC on apoptosis-related and oxidative stressrelated protein expression. Lane 1: control group; lane 2: model group; lane 3: PNC, 5 mg/kg; lane 4: PNC, 10 mg/kg. Values are expressed as mean ± SD (n = 4). #P \ 0.05 vs control group; **P \ 0.01 vs model group
control group, showing that VD caused cognitive impairment in the model group. Conversely, 5-week administration of PNC significantly reduced the prolonged escape latency in the model group on all 4 days.
VD comprises dementias resulting from all types of vascular pathologies, with increasing prevalence in the elderly population [19]. The activity of anti-oxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and heme oxygenase/biliverdin reductase, is decreased in patients with VD [20, 21]. Oxidative stress is considered to be a major contributing factor in the pathogenesis of VD [10, 11]. Increased oxidative stress in the brain parenchyma, as manifested by lipid peroxidation, protein oxidation, and DNA oxidative damage, is a main characteristic feature of VD [22]. Oxidative stress occurs as the results of a shift in balance that favors the generation of oxygen-derived free radicals or ROS over anti-oxidant defense mechanisms [23]. MDA is a by-product of lipid peroxidation produced under oxidative stress and is well-known as a widely used marker for oxidative damage of plasma membrane, which is proportional to lipid peroxidation and oxidative stress [24]. In vascular diseases, ROS have direct effects on vascular tone and also impair vasomotor responses to other stimuli [25]. Cerebral blood vessels have the capacity to generate high levels of superoxide and are particularly sensitive to the effects of ROS [26]. Therefore, the imbalance between the intracellular oxidative and anti-oxidative defense systems, including SOD and GSH, requires the supplement of
Fig. 6 Effect of PNC on apoptosis in the hippocampus. Values are expressed as mean ± SD (n = 4). ## P \ 0.01 vs control group; *P \ 0.05, **P \ 0.01 vs model group
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the MWM test. a Mean escape latency in the hidden platform test during four consecutive training days; b latency of first crossing the platform in the probe trial; c number of crossings over the exact former location of the platform in the probe trial; d comparison of time spent in the target quadrant in the probe trial. Values are expressed as mean ± SD (n = 8). ##P \ 0.01 vs control group; *P \ 0.05, **P \ 0.01 vs model group
external anti-oxidants to eliminate ROS as a potential therapeutics in some central nervous diseases. It is well known that flavonones have potent anti-oxidative and free radical-scavenging activities [27–30]. In this study, we demonstrated that PNC dramatically improved the cognitive performance of rats with VD. The protective effect of PNC might be attributed to its powerful anti-oxidant action, as evidenced by the markedly reduced MDA level as well as the enhanced SOD activity and GSH level. PNC possibly stabilizes the mitochondrial redox balance by scavenging ROS directly or decreasing the ROS formation through protecting the electron transfer chain. Disturbance in mitochondrial membrane potential (MMP) is followed by generation of superoxide anion radical. The later steps of the mitochondrial-dependent cell death pathway involve the translocation of cytochrome c from the mitochondria to the nucleus [31]. Cytochrome c facilitates the formation of apoptosome complexes, which leads to chromatin condensation and DNA cleavage. Caspases play an important role in the mitochondria-dependent apoptotic process [32]. Caspase-3 acts as an apoptotic executor and activates DNA fragmentation factor, which in turn activates endonucleases to cleave nuclear DNA and ultimately leads to cell death. So inhibition of caspase-3 has a promising effect in attenuating apoptosis. Moreover, caspase-9 was generally activated following the disruption of the outer mitochondrial membrane. A previous study showed that the cytochrome c released from mitochondria combined with caspase-9 precursor and led to activation of caspase-9 activity [33]. Our results showed that the increased cytochrome c level and caspase activities could be recovered, and as a result the apoptosis was significantly decreased after treatment with PNC. Moreover, behavioral performance was tested by the Morris water maze, suggesting that long-term administration of PNC improved spatial learning and memory in rats with VD. Increasing evidence suggests that nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxs), the enzyme complexes that transport electrons across the membrane and generate superoxide, play an important role in generating ROS in various types of tissue [34]. In the central nervous system, although Nox-mediated ROS are required for normal cellular functions, such as long-term potentiation and cardiovascular homeostasis, excess ROS
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generation may contribute to pathological conditions [35, 36]. Studies have shown that ROS derived from Nox1 in microglia, an immune component in the brain, cause oxidative stress in several brain diseases [37]. Recent studies, however, have indicated that Nox1 expression is not limited to microglia, but is involved in various pathological conditions in neuronal cells [38]. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor necessary for the induction of antioxidant enzymes, is a master regulator for the expression of many anti-oxidative genes, such as HO-1 and nicotinamide adenine dinucleotide phosphate quinone oxidoreductase-1 [39, 40]. Activated Nrf2 is released from its cytosolic repressor Keap1 and then translocates into the nucleus, where it binds to anti-oxidant response elements in the promoter regions of target anti-oxidative genes [41]. Expression of Nrf2-mediated target genes promotes cell survival in oxidizing environments via regulation of proteasome function, enhancement of free radical metabolism, and maintenance of glutathione homeostasis [42]. The results of this study clearly indicated that PNC increased the expression of Nrf2 and decreased the expression of Nox1. In summary, this study showed that treatment with PNC could markedly attenuate the cognitive impairment of rats with VD, and that the protective effects may be mediated through its anti-oxidant activities. Therefore, PNC may be a potential agent for the treatment of VD. Conflict of interest The authors declare that no actual or potential conflict of interest exists in relation to this article.
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ORIGINAL PAPER
(2R,3S)-Pinobanksin-3-cinnamate improves cognition and reduces oxidative stress in rats with vascular dementia Hong Liu1 • Min Zhao1 • Shen Yang2 • Dian-Rong Gong1 • De-Zhe Chen1 De-Yong Du3
•
Received: 3 December 2014 / Accepted: 10 March 2015 Ó The Japanese Society of Pharmacognosy and Springer Japan 2015
Abstract This study investigated the neuroprotective effects of (2R,3S)-pinobanksin-3-cinnamate (PNC) in rats with occlusion-damaged bilateral common carotid arteries. Administration with PNC (5 and 10 mg/kg/day) for 5 weeks significantly improved the behavioral performance of rats with vascular dementia, as showed in the Morris water maze test by shortening the escape latency and latency of crossing, completing more platform crossings, as well as spending more time in the target zone. Further evaluations found that PNC could markedly decrease malondialdehyde levels, enhance superoxide dismutase activity and glutathione levels, and decrease the release of cytochrome c as well as the activities of caspases. Moreover, PNC increased Nrf2 and anti-apoptotic bcl-2 protein expression, while Nox1 and pro-apopotic bax protein expression was decreased. PNC may exert its neuroprotective effects through counteracting oxidative stress and has the potential to treat vascular dementia. Keywords (2R,3S)-Pinobanksin-3-cinnamate Neuroprotective effects Oxidative stress Morris water maze
H. Liu and M. Zhao contributed equally. & Min Zhao [email protected] 1
Department of Neurology, Liaocheng Hospital, Liaocheng 252000, China
2
Department of Neurology, Taian Central Hospital, Taian 271000, China
3
Department of Neurosurgery, The Affiliated Hospital of Binzhou Medical University, Binzhou 256600, China
Introduction Dementia is a syndrome due to brain disease where there is disturbance of multiple higher cortical and neuropsychological functions including memory, orientation, thinking, calculation, learning capacity and language [1]. Vascular dementia (VD) is a mental disorder caused by brain damage due to cerebrovascular disease and has gained increasing attention in recent years for being the second most common type of dementia following Alzheimer’s disease [2, 3]. Together with the increasing age of the population and improved survival rates from cardiovascular diseases, VD may affect more individuals in the future [4]. About 1–4 % of people over 65 years old suffer from VD and the morbidity rate after 65 years of age doubles every 5–10 years [5]. Moreover, VD in turn increases the risk of recurrent stroke, dependent living and death [6]. There is substantial evidence that conventional risk factors such as hypertension, hyperhomocysteinemia and dyslipidemia play an important role in the development of VD [7–9]. Etiopathogenic mechanisms leading to VD include oxidative stress, cytotoxicity of reactive oxygen species (ROS), mitochondrial dysfunction and apoptosis [10, 11]. Supplementation of exogenous anti-oxidants can effectively curtail the oxidative damage to cellular macromolecules. One category of such anti-oxidants is flavonone, with diversified biological activities including anti-oxidative, radical scavenging and anti-inflammatory effects, most interestingly in attenuating the redox imbalance and improving neuro-cognitive performance [12, 13]. (2R,3S)Pinobanksin-3-cinnamate (PNC, Fig. 1) is a new flavonone, isolated from the seed of Alpinia galanga Willd., which has shown a neuroprotective effect on H2O2-damaged PC12 cells [14].
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model group received vehicle (0.5 % methylcellulose) during the same period. All animal experiments were performed in compliance with the Chinese legislation on the use and care of laboratory animals and were approved by the Ethical Committee on Animal Care and Use. Bilateral common carotid artery occlusion
Fig. 1 Structure of (2R,3S)-pinobanksin-3-cinnamate (PNC)
When the blood supplied to the brain is reduced, VD occurs and leads to a progressive decline in memory and cognitive function [15]. VD can be successfully induced by bilateral common carotid artery occlusion in rats, resulting in significant white matter lesions, learning and memory impairment, and hippocampal neuronal damage [16, 17]. In this study, we will investigate the protective effect of (2R,3S)-pinobanksin-3-cinnamate on rats with VD.
After being anesthetized with 10 % chloral hydrate, the skin of the rat was incised along the midline of the cervical region to expose the bilateral common carotid arteries without nerve damage; they were carefully separated from the surrounding tissues, approximately 1 cm inferior to the origin of the external carotid artery [16]. Both common carotid arteries were tied twice with silk sutures. The skin incision was sutured and the animals were allowed to recover from the anesthesia. Control rats were subjected to the same surgical procedure without occlusion of arteries. Animals that exhibited an abnormal post-operative condition were excluded from the study. Morris water maze test
Materials and methods Chemicals and reagents Malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) assay kits were purchased from Jiancheng Institute of Biological Engineering (Nanjing, China). Cytochrome c enzyme-linked immunosorbent assay kit was from R&D Systems (Minneapolis, MN, USA). Caspase-3 and caspase-9 activity assay kits were purchased from Kaiji Institute of Biological Engineering (Nanjing, China). In Situ Cell Death Detection Kit was purchased from Roche (Mannheim, Germany). (2R,3S)-Pinobanksin3-cinnamate (purity [ 98 %) was isolated and purified by Department of Natural Product, Institute of Materia Medica, Shandong Academy of Medical Sciences. Experimental groups and drug treatment Forty-eight healthy male Wistar rats (280–300 g) were obtained from the Animal Center of Shandong Lukang Pharmaceutical Group Co., Ltd. (Jinan, China). Rats were kept under conditions of constant temperature (25 ± 2 °C) and humidity (55 ± 5 %), 12-h light/12-h dark photoperiod, and free access to food and water. After acclimatization to the laboratory environment, rats were divided randomly into four groups: control group, VD model group and PNC groups (low and high dose). Rats in the PNC groups received a daily oral dose of 5 or 10 mg/kg, respectively, from the eighth week to the twelfth week after the operation. Rats in the control group and
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The rat behavioral test was performed by the Morris water maze (MWM) test [18]. After treatment, rats were trained four times per day for four consecutive days with an interval between sessions of 30–40 min. The rat was placed in the water facing the wall at one of three random starting positions (in three different quadrants that did not contain the platform). Each trial had a ceiling time of 60 s and a trial interval of 60 s. After climbing onto the platform, rats stayed there for 30 s before the next trial. If the rat failed to reach the platform within 60 s, it was gently placed on the platform and remained there for 30 s. Latency to escape from the water maze was calculated for each trial. On the fifth day, a probe test was carried out by removing the platform and each rat was allowed to swim freely for 120 s. The latency of first crossing the platform, the time which a rat spent in the target quadrant and the number of crossings of the non-exits were recorded for each trial. All data were recorded and analyzed by a video tracking system. Sample preparation and biochemical evaluation Rats were killed for biochemical evaluation after MWM tests. Brains were quickly removed and the cerebral cortex and hippocampus were separated on ice. Some tissues were weighed and homogenated in phosphate-buffered saline (PBS; pH 7.4) and centrifuged at 10,000 rpm at 4 °C for 15 min to remove cellular debris. Supernatants were kept at -80 °C and used for further tests. Other tissues were fixed with freshly prepared 4 % paraformaldehyde in PBS (pH 7.4) overnight, dehydrated by gradient sucrose, and
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transferred into 30 % sucrose solution for cryoprotection. Sections of 15 lm thickness were made using a freezing microtome. Measurement of MDA and GSH levels and SOD activity MDA, SOD and GSH assays were conducted by colorimetric kits according to the manufacturers’ instructions. The levels of MDA and GSH and activity of SOD were expressed as per gram tissue. Assessment of cytochrome c release The concentration of cytochrome c was measured according to the manufacturer’s instructions. After reaction with reagents, the optical density was measured by a microplate reader at 490 nm. The concentration was calculated according to the standard curve. Measurement of caspase activity The caspase activity was measured according to the manufacturer’s instructions. Briefly, aliquots of supernatants containing 25 mg protein were added to a reaction buffer supplemented with 0.1 % CHAPS, 5 mM DTT and 100 mM PMSF. The reactions were initiated after adding the following fluorescent substrates (50 mM at the final concentration): Ac-DEVD-Amc for caspase-3 activity and Ac-LEDH-Afc for caspase-9 activity. After incubation for 2 h at 37 °C, the cleavage of the substrates was measured (Amc: 390/475 nm; Afc: 400/505 nm) by a microplate reader. The protein content was determined by the bicinchoninic acid method. The activity of caspase was expressed as per min per mg protein. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay
subjected to electrophoresis on a 12 % (v/v) SDS–polyacrylamide gel. After proteins were electroblotted to a PVDF membrane, the membrane was blocked with PBST containing 5 % dried non-fat milk at room temperature, washed three times and incubated with indicated primary antibodies at 4 °C overnight, followed by incubation with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h. After incubation, the membrane was washed three times, and the antigen–antibody complexes were visualized by the enhanced chemiluminescence system (PerkinElmer). Relative protein expression was compared to the control group, which was set at 1. Statistical analysis Values are presented as mean ± SD. Results were statistically analyzed by one-way analysis of variance (ANOVA) followed by multiple comparison tests using Sigma Stat statistical software (SPSS Inc., Chicago, IL, USA). Group differences in the escape latency in the MWM training task were analyzed with two-way ANOVA with repeated measures, the factors being treatment and training day. Differences were considered significant at P \ 0.05.
Results Measurement of MDA and GSH levels and SOD activity Oxidative stress was assessed by MDA and GSH levels and SOD activity. Figure 2 shows that MDA level was significantly increased in the model group, while GSH level and SOD activity were decreased. Treatment with PNC (5 and 10 mg/kg) significantly reversed the oxidative stress in rats, compared with the model group (P \ 0.05).
Apoptotic cells in the hippocampus were detected by TUNEL staining using an In Situ Cell Death Detection Kit, according to the manufacturer’s instructions. Sections were rinsed and visualized with DAB, and then mounted with coverslips. The number of TUNEL-positive cells was counted under the microscope and compared in each group.
Measurement of cytochrome c
Western blot analysis
Activities of caspase-3 and caspase-9
Total cellular and nuclear protein was quantified using the protein assay kit (Biyotime, China). Homogenate in 59 SDS sample buffer was boiled for 5 min, and then equal amounts of protein (50 lg) from each sample were
As shown in Fig. 4, activities of caspase-3 and caspase-9 in the model group increased significantly. After treatment with PNC, caspase activities were significantly decreased compared to the model group (P \ 0.01).
Figure 3 shows that the cytochrome c released into the cytoplasm was significantly increased in the model group. However, treatment with PNC (5 and 10 mg/kg) significantly decreased cytochrome c levels in rats (P \ 0.05).
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Fig. 3 Effect of PNC on cytochrome c release in the cerebral cortex and hippocampus of VD rats. Values are expressed as mean ± SD (n = 8). ##P \ 0.01 vs control group; *P \ 0.05, **P \ 0.01 vs model group
Fig. 4 Effect of PNC on caspase activity in the cerebral cortex and hippocampus of VD rats. Values are expressed as mean ± SD (n = 8). ##P \ 0.01 vs control group; **P \ 0.01 vs model group
Apoptosis detection by TUNEL staining Fig. 2 Effect of PNC on oxidative stress in VD rats. Oxidative stress was evaluated by examining MDA content (a), SOD activity (b), and GSH content (c). Values are expressed as mean ± SD (n = 8). ## P \ 0.01 vs control group; *P \ 0.05, **P \ 0.01 vs model group
Protein expression Western blotting results confirmed that nuclear factor erythroid 2-related factor 2 (Nrf2) and anti-apopotic bcl2 protein expression was significantly increased, while Nox1 and pro-apoptotic bax protein expression was significantly decreased after treatment with PNC (Fig. 5).
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Figure 6 shows that apoptotic cells were significantly increased in the model group. However, after treatment with PNC (5 and 10 mg/kg), apoptosis was significantly decreased in the hippocampus (P \ 0.05). PNC improved the behavioral performance of rats with VD Figure 7a shows that after training for 4 days, the performance of rats in all groups was improved with shortened escape latencies. During the MWM test, the tested rats displayed no difference in motor activity. However, rats in the model group had longer escape latencies than rats in the
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Figure 7b–d shows that in the probe trial, rats in the model group had longer latency of crossing (P \ 0.01), completed fewer platform crossings (P \ 0.01) and spent less time in the target zone (P \ 0.01) than rats in the control group. Significant improvement of these indexes was observed in the PNC group.
Discussion
Fig. 5 Effect of PNC on apoptosis-related and oxidative stressrelated protein expression. Lane 1: control group; lane 2: model group; lane 3: PNC, 5 mg/kg; lane 4: PNC, 10 mg/kg. Values are expressed as mean ± SD (n = 4). #P \ 0.05 vs control group; **P \ 0.01 vs model group
control group, showing that VD caused cognitive impairment in the model group. Conversely, 5-week administration of PNC significantly reduced the prolonged escape latency in the model group on all 4 days.
VD comprises dementias resulting from all types of vascular pathologies, with increasing prevalence in the elderly population [19]. The activity of anti-oxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and heme oxygenase/biliverdin reductase, is decreased in patients with VD [20, 21]. Oxidative stress is considered to be a major contributing factor in the pathogenesis of VD [10, 11]. Increased oxidative stress in the brain parenchyma, as manifested by lipid peroxidation, protein oxidation, and DNA oxidative damage, is a main characteristic feature of VD [22]. Oxidative stress occurs as the results of a shift in balance that favors the generation of oxygen-derived free radicals or ROS over anti-oxidant defense mechanisms [23]. MDA is a by-product of lipid peroxidation produced under oxidative stress and is well-known as a widely used marker for oxidative damage of plasma membrane, which is proportional to lipid peroxidation and oxidative stress [24]. In vascular diseases, ROS have direct effects on vascular tone and also impair vasomotor responses to other stimuli [25]. Cerebral blood vessels have the capacity to generate high levels of superoxide and are particularly sensitive to the effects of ROS [26]. Therefore, the imbalance between the intracellular oxidative and anti-oxidative defense systems, including SOD and GSH, requires the supplement of
Fig. 6 Effect of PNC on apoptosis in the hippocampus. Values are expressed as mean ± SD (n = 4). ## P \ 0.01 vs control group; *P \ 0.05, **P \ 0.01 vs model group
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the MWM test. a Mean escape latency in the hidden platform test during four consecutive training days; b latency of first crossing the platform in the probe trial; c number of crossings over the exact former location of the platform in the probe trial; d comparison of time spent in the target quadrant in the probe trial. Values are expressed as mean ± SD (n = 8). ##P \ 0.01 vs control group; *P \ 0.05, **P \ 0.01 vs model group
external anti-oxidants to eliminate ROS as a potential therapeutics in some central nervous diseases. It is well known that flavonones have potent anti-oxidative and free radical-scavenging activities [27–30]. In this study, we demonstrated that PNC dramatically improved the cognitive performance of rats with VD. The protective effect of PNC might be attributed to its powerful anti-oxidant action, as evidenced by the markedly reduced MDA level as well as the enhanced SOD activity and GSH level. PNC possibly stabilizes the mitochondrial redox balance by scavenging ROS directly or decreasing the ROS formation through protecting the electron transfer chain. Disturbance in mitochondrial membrane potential (MMP) is followed by generation of superoxide anion radical. The later steps of the mitochondrial-dependent cell death pathway involve the translocation of cytochrome c from the mitochondria to the nucleus [31]. Cytochrome c facilitates the formation of apoptosome complexes, which leads to chromatin condensation and DNA cleavage. Caspases play an important role in the mitochondria-dependent apoptotic process [32]. Caspase-3 acts as an apoptotic executor and activates DNA fragmentation factor, which in turn activates endonucleases to cleave nuclear DNA and ultimately leads to cell death. So inhibition of caspase-3 has a promising effect in attenuating apoptosis. Moreover, caspase-9 was generally activated following the disruption of the outer mitochondrial membrane. A previous study showed that the cytochrome c released from mitochondria combined with caspase-9 precursor and led to activation of caspase-9 activity [33]. Our results showed that the increased cytochrome c level and caspase activities could be recovered, and as a result the apoptosis was significantly decreased after treatment with PNC. Moreover, behavioral performance was tested by the Morris water maze, suggesting that long-term administration of PNC improved spatial learning and memory in rats with VD. Increasing evidence suggests that nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxs), the enzyme complexes that transport electrons across the membrane and generate superoxide, play an important role in generating ROS in various types of tissue [34]. In the central nervous system, although Nox-mediated ROS are required for normal cellular functions, such as long-term potentiation and cardiovascular homeostasis, excess ROS
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generation may contribute to pathological conditions [35, 36]. Studies have shown that ROS derived from Nox1 in microglia, an immune component in the brain, cause oxidative stress in several brain diseases [37]. Recent studies, however, have indicated that Nox1 expression is not limited to microglia, but is involved in various pathological conditions in neuronal cells [38]. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor necessary for the induction of antioxidant enzymes, is a master regulator for the expression of many anti-oxidative genes, such as HO-1 and nicotinamide adenine dinucleotide phosphate quinone oxidoreductase-1 [39, 40]. Activated Nrf2 is released from its cytosolic repressor Keap1 and then translocates into the nucleus, where it binds to anti-oxidant response elements in the promoter regions of target anti-oxidative genes [41]. Expression of Nrf2-mediated target genes promotes cell survival in oxidizing environments via regulation of proteasome function, enhancement of free radical metabolism, and maintenance of glutathione homeostasis [42]. The results of this study clearly indicated that PNC increased the expression of Nrf2 and decreased the expression of Nox1. In summary, this study showed that treatment with PNC could markedly attenuate the cognitive impairment of rats with VD, and that the protective effects may be mediated through its anti-oxidant activities. Therefore, PNC may be a potential agent for the treatment of VD. Conflict of interest The authors declare that no actual or potential conflict of interest exists in relation to this article.
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