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Tanshinol protects hippocampus and attenuates vascular dementia development a
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Cui-Ge Shi , Yi-Shu Yang , Hui Li , Ying Zhang , Ning Wang , a
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Shang-Ming Wang , Jie-Dong Wang & Shu-Cheng Zhang
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Department of Cell Biology, National Research Institute of Family Planning, Beijing 100081, China b
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Department of Neurology in Medical Healthcare Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China c
Department of Anatomy, Capital Medical University, Beijing 100069, China d
WHO Collaborating Center for Research in Human Reproduction, National Research Institute of Family Planning, Beijing 100081, China Published online: 24 Jun 2014.
To cite this article: Cui-Ge Shi, Yi-Shu Yang, Hui Li, Ying Zhang, Ning Wang, Shang-Ming Wang, Jie-Dong Wang & Shu-Cheng Zhang (2014) Tanshinol protects hippocampus and attenuates vascular dementia development, Journal of Asian Natural Products Research, 16:6, 667-676, DOI: 10.1080/10286020.2014.930131 To link to this article: http://dx.doi.org/10.1080/10286020.2014.930131
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 6, 667–676, http://dx.doi.org/10.1080/10286020.2014.930131
Tanshinol protects hippocampus and attenuates vascular dementia development Cui-Ge Shia1, Yi-Shu Yangb1, Hui Lic, Ying Zhangd, Ning Wanga, Shang-Ming Wanga, Jie-Dong Wanga and Shu-Cheng Zhanga*
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a
Department of Cell Biology, National Research Institute of Family Planning, Beijing 100081, China; bDepartment of Neurology in Medical Healthcare Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China; cDepartment of Anatomy, Capital Medical University, Beijing 100069, China; dWHO Collaborating Center for Research in Human Reproduction, National Research Institute of Family Planning, Beijing 100081, China (Received 23 February 2014; final version received 28 May 2014) Tanshinol (3-(30 ,40 -dihydroxyphenyl)-(2R)-lactic acid, TSL) is widely used in traditional Chinese medicine for the treatment of cardiovascular and cerebrovascular diseases. Here, we assessed whether TSL protected hippocampus and attenuated vascular dementia (VD) development in rats. The behavioral analysis showed that TSL could decrease the distance and latency time, and increase the swim speed in water maze in rats subjected to VD. TSL remarkably increased acetylcholine level and decreased acetylcholinesterase activity in rats subjected to VD. Likewise, TSL remarkably decreased malondialdehyde and increased superoxide dismutase levels in rats subjected to VD. Furthermore, treatment with TSL reduced the level of dead neurons in dentate gyrus. In addition, TSL upregulated growth-associated protein 43 (GAP43) and vascular endothelial growth factor (VEGF) expression and downregulated phosphorylated Akt (p-AKt) and phosphorylated glycogen synthase kinase (p-GSK3b) expression in hippocampus in rats subjected to VD. These results suggest that TSL may be a potential compound in VD model. Keywords: tanshinol; vascular dementia; hippocampus; animal model
1. Introduction Vascular dementia (VD) is one of the most frequent causes of dementia in the elderly, causing a major burden on health care systems in aging societies. VD is the second-most frequent cause of dementia after Alzheimer’s disease. Approximately one-third of individuals above 85 years of age had a dementia syndrome and about one-sixth of them suffer from VD, resulting in a prevalence of about 4– 5%. Cognitive dysfunction frequently occurs after a clinically obvious ischemic or hemorrhagic stroke. Treatment of VD can be approached from two different angles: the control of classical vascular risk *Corresponding author. Email: [email protected] q 2014 Taylor & Francis
factors that obviously contribute to the development of vascular disease and dementia, as well as the use of drugs such as acetylcholine (ACh) esterase inhibitors [1]. Studies in humans have documented that damage to the hippocampus produces retrograde amnesia. Typically, the retrograde amnesia is temporally graded, such that information acquired long before hippocampal damage is remembered better than information acquired recently [2]. Temporally graded retrograde amnesia has been studied extensively in the experimental animal using prospective methods. When the hippocampus is damaged at
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Figure 1. Chemical structure of TSL.
different times after learning, the large majority of studies have found that remote memory is spared relative to more recent memory [3]. Tanshinol (3-(30 ,40 -dihydroxyphenyl)(2R)-lactic acid, TSL, Figure 1) is widely used in traditional Chinese medicine for the treatment of coronary heart disease, cerebrovascular disease, bone loss, hepatocirrhosis, and chronic renal failure [4,5]. It has been reported that TSL could protect the cardiac muscle by calcium antagonizing, reducing oxygen-free radical generation, inhibiting peroxidative damage, and preventing platelet aggregation [6]. The activities of TSL on other aspects, such as antitumor and nerve cells injury protection, have also been reported [7]. As a result, great research interests have been focused on the therapeutic potential of TSL. Therefore, the present study focused on evaluating the TSL-induced attenuation of VD development and its protective mechanisms on hippocampus with the goal of identifying therapeutic strategies against VD in the future. 2.
Results
2.1 The role of TSL in learning and memory impairment To determine whether the middle cerebral artery occlusion induced deficits in spatial working memory, the rats were trained in the water maze for 4 days. Figure 2 shows distance swam (a), latency to platform (b), and swim speed (c) for rats. For distance, VD group swam further to reach the platform than did the control group, wherein TSL group performed signifi-
cantly better than the VD group. There was also a main effect for test day (P , 0.01) in which performance of control group improved over the 4 days of testing. There was a similar pattern of results for latency to reach the platform. Swim speed results also showed that VD group swam more slowly than did the control group, and TSL group performed significantly better than did the VD group. These data suggest that the ability to integrate the platform position with existing memories of the spatial cues is disrupted by VD and this disruption could be improved partly by TSL treatment. 2.2
Nissl staining
The dentate gyrus granule cells of rat hippocampus after VD exhibited morphological alterations consistent with a degenerative process. In the control group, most of the granule neurons had a round or oval nucleus, located in the center of perikarion that is surrounded by pale cytoplasm. In comparison with the control animals, the rats submitted to VD showed slight morphological alterations in most of their neurons. The neuronal changes were triangular in shape mostly exhibiting a dark staining due to condensation of cytoplasm and karyoplasms. Treatment with TSL could improve the morphological alterations of the dentate gyrus granule cells after VD (Figure 3). 2.3 ACh level and acetylcholinesterase activity in hippocampus TSL remarkably increased Ach level and decreased acetylcholinesterase (AChE) activity in rats subjected to VD (Figure 4). The ACh level was 203.6 ^ 25.4 nmol/L in control group, which were much lower than those subjected to VD (93.2 ^ 12.1 nmol/ L). During the 2-week treatment period of TSL at the doses of 15 and 30 mg/kg, the ACh levels decreased to 149.5 ^ 19.3 mL and 160.6 ^ 18.3 nmol/L. The AChE
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Figure 2. Swim distance (a), latency to platform (b), and swim speed (c) in water maze. Rats were trained in the water maze for 4 days with a static platform position. **P , 0.01, *P , 0.05 versus control, #P , 0.05 versus VD. n ¼ 10.
activity was 0.36 ^ 0.04 in control group, which were much higher than those subjected to VD (1.54 ^ 0.22). After TSL
treatment at the doses of 15 and 30 mg/kg, the superoxide dismutase (SOD) levels increased to 0.9 ^ 0.12 and 0.7 ^ 0.1.
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C.-G. Shi et al. in rats subjected to VD (Figure 5). The MDA level was 1.2 ^ 0.18 nmol/mg protein in control group, which was much lower than those subjected to VD (3.8 ^ 0.4 nmol/mg protein). During the 2-week treatment period of TSL at the doses of 15 and 30 mg/kg, the MDA levels decreased to 2.9 ^ 0.4 mL and 2.48 ^ 0.24 nmol/mg protein. The SOD level was 174.2 ^ 25.2 nmol/mg protein in control group, which were much higher than those subjected to VD (93.2 ^ 13.4 nmol/mg protein). After TSL treatment at the doses of 15 and 30 mg/kg, the SOD levels increased to 135.5 ^ 19.3 mL and 149.6 ^ 18.6 nmol/mg protein.
2.5 TSL upregulates growth-associated protein 43 and vascular endothelial growth factor expression in hippocampus Figure 3. Different morphological profiles of apoptotic granule cells by Nissl-stained sections in the dentate gyrus of a control rat (a), a rat receiving VD (b), a rat receiving UCMS and TSL 15 mg/kg (c), a rat receiving UCMS and TSL 30 mg/kg (d). a1 – d1 are magnified views of regions from a to d. Scale bars: 100 mm. n ¼ 3.
2.4 Malondialdehyde and SOD levels in serum TSL remarkably decreased malondialdehyde (MDA) level and increased SOD level
Growth-associated protein 43 (GAP43) and vascular endothelial growth factor (VEGF) protein expressions in hippocampus were quantified using Western blotting (Figure 6). These results demonstrated that relative protein GAP43 and VEGF levels in the control group were 1.36 ^ 0.24 and 1.06 ^ 0.15, respectively. VD significantly downregulated relative protein GAP43 and VEGF levels to 0.54 ^ 0.07 and 0.44 ^ 0.06, respectively. TSL treatment upregulated GAP43 expression to 1.03 ^ 0.15 and 1.2 ^ 0.19 and VEGF expression to
Figure 4. ACh level (a) and AChE activity (b) in hippocampus. TSL remarkably increased ACh level and decreased AChE activity in hippocampus in rats subjected to VD. **P , 0.01, *P , 0.05 versus control, #P , 0.05 versus VD. n ¼ 10.
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Figure 5. Serum levels of MDA (a) and SOD (b). TSL remarkably decreased MDA and increased SOD levels in rats subjected to VD. **P , 0.01, *P , 0.05 versus control, #P , 0.05 versus VD. n ¼ 10.
0.7 ^ 0.12 and 1.1 ^ 0.16 at the doses of 15 and 30 mg/kg (P , 0.05 vs. VD) 2.6 TSL inhibits phosphorylated Akt and glycogen synthase kinase 3b expression in hippocampus Phosphorylated Akt (p-AKt) and glycogen synthase kinase 3b (GSK3b) protein
expressions in hippocampus were quantified using Western blotting (Figure 7). These results demonstrated that relative protein p-AKt and GSK3b levels in the control group were 0.56 ^ 0.06 and 0.06 ^ 0.04, respectively. VD significantly upregulated relative protein p-AKt and GSK3b levels to 1.24 ^ 0.2 and
Figure 6. TSL increased GAP43 and VEGF expressions in hippocampus in rats subjected to VD. GAP43 and VEGF expressions were determined by Western blot. *P , 0.05, **P , 0.01 versus control. #P , 0.05 versus VD. n ¼ 4.
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Figure 7. TSL attenuates the expression of p-AKt and p-GSK3b protein in hippocampus in rats subjected to VD. p-AKt and p-GSK3b expressions were determined by Western blot analysis. *P , 0.05, **P , 0.01 versus control. #P , 0.05 versus VD. n ¼ 3.
1.5 ^ 0.2, respectively. TSL treatment downregulated p-AKt expression to 0.9 ^ 0.12 and 0.7 ^ 0.16 and GSK3b expression to 1.2 ^ 0.14 and 0.6 ^ 0.09 at the doses of 15 and 30 mg/kg (P , 0.05 vs. VD) 3.
Discussion
Herein we showed that TSL could not only improve the spatial working memory, but also promote the consequences of rat subjected to VD. These studies evaluated the protective role of TSL on VD. TSL induces multiple changes in the animal model of VD: (1) a reduction in the distance and latency times, and a promotion in the swim speed in water maze; (2) an improvement on the morphological alterations of the dentate gyrus granule cells; (3) a promotion in the levels of ACh and SOD, and a depression in the levels of MDA and AChE; and (4) upregulation of
the expression of GAP43 and VEGF, and downregulation of the expression of p-AKt and phosphorylated GSK3b (p-GSK3b). Chronic cerebral hypoperfusion has been assumed as a pathogenic mechanism in patients with VD. Cognitive impairment has been demonstrated in animals with chronic cerebral hypoperfusion and modeling patients with VD [8]. Rats with untreated VD in the present study were found to have abnormal cognitive function, as demonstrated by their impaired learning ability in the water maze test [9]. The behavioral results are consistent with hippocampal dysfunction, as the hippocampus seems to be a critical component for forming relational representations and strategic flexibility in the Morris water maze. This learning ability was significantly better in rats with VD that were treated with TSL. It had been shown that a relationship exists between changes in
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Journal of Asian Natural Products Research cognitive function and hippocampus damage, and that TSL treatment reversed the impairment. Our study demonstrated that loss of VD definitely resulted in hippocampal granule cell degeneration [10 – 12]. This study showed that dentate gyrus granule cell degeneration following an ischemia was related to the vessel occlusion (VO). VD-induced granule cell apoptosis was blocked by the treatment with TSL. These results suggested that TSL in this study could protect granule cells continuously against VD-induced apoptosis. An important hypothesis in this study was that TSL would rescue the uninjured granule cells. It is noteworthy that the relationship between vascular function and cognition in our study was much stronger than the relationship between cognition and many other individual vascular-related variables that were analyzed [13,14]. Therefore, the vasorelaxation is obtained by assessing vascular response to ACh [15,16]. Ischemia resulted in a significant decrease in ACh level, but recirculation was associated with a rapid increase in ACh concentration. Impaired synthesis and/or increased release of ACh can be responsible for the decrease in ACh concentration during ischemia. Early postischemic elevation of ACh may be related to the large increase in brain choline brought about by ischemia. In this study, we found an increase in the levels of ACh and a decrease in the activity of AChE. The degradation product of MDA was considered to reflect the degree of lipid peroxidation. The past work showed that, during ischemia-reperfusion injury process, in memantine intervention group, the increase in expression level of caspase-3 in cortex neuron and MDA release in cortex homogenate were inhibited significantly at 12, 24, and 48 h time points, compared with that of rats in ischemia-reperfusion model group. As expected, VD was associated with oxidative stress as evidenced by elevated MDA levels in hippocampus. The SOD-mimetic effects
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of VD were confirmed by the findings of hippocampus. MDA were lowered by TSL treatment and by the fact that SOD activity was elevated in the TSL-treated group. Therefore, TSL should exert protection against hippocampus damage in VD. GAP43 is widely considered to be involved in neuronal mechanisms underlying axonal growth, axonal regeneration, and synaptic plasticity [17,18]. GAP43 is thought to contribute directly to the outgrowth of neuronal processes, while first recognized for its increased synthesis and axonal transport in neurons responding to peripheral nerve injury [19]. GAP43 and its mRNA are also detectable in some regions of the normal brain [20]. VEGF is reported to stimulate adult neurogenesis in vivo and proliferation of brain-derived neural stem/progenitor cells [21]. It is a target zone of several important afferent inputs to hippocampus. The results have functional implications because of the known effects of VEGF in hippocampus, such as stimulation of neurogenesis in the dentate gyrus in rats of VD. The main biological consequences of p-Akt activation that are relevant to cancer cell growth can be classified loosely into three categories: survival, proliferation (increased cell number), and growth (increased cell size). p-Akt activation is also associated with enhanced cell invasion. Gao et al. [22] showed that ischemic postconditioning enhanced phosphorylation of Akt. The inhibition of Akt activity partly abolished the protective effects of postconditioning. Many neuroprotectants, including propofol, humanin, estradiol, melatonin, isoflurane, atorvastatin, and tissue-type plasminogen activator, exert their protective effects through the Akt pathway. Akt enhances invasiveness of pancreatic carcinoma cells via upregulation of VEGF [23]. Gong reported that p-Akt were significantly decreased in the hippocampus at 1, 2, and 4 months following 2-VO injury [24]. On the contrary, Shu et al. reported that p-Akt
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were significantly increased in the hippocampus at 1, 4, and 8 weeks following 2-VO injury [25]. The expression level of p-Akt might be related to time following 2-VO injury. GSK3b activation is mediated through PP2A-regulated inactivation of the PI 3-kinase-Akt pathway. GSK3b acts downstream of PP2A and the PI3K-Akt pathway, and upstream of caspase-2 in ceramide induced mitochondrial apoptosis. Our analysis showed that after 2 weeks following VD, p-Akt and p-GSK3b protein expressions were significantly increased in VD groups as compared with the control group. TSL decreased p-AKt and p-GSK3b expression in hippocampus lesions in VD. It is suggested that TSL could affect PI3KAkt-GSK3b pathway. In conclusion, we demonstrated that TSL significantly protected VD damage. These results suggest that this may be a potential compound in VD model. 4. Experimental 4.1 Animals Adult male Sprague–Dawley rats with weights of 180 – 220 g were used. All animals were housed 5 per cage under standardized light/dark cycle condition (lights on at 7:00 am, lights off at 7:00 pm) at a room temperature of 24 ^ 18C and humidity of 60% ^ 10% with food and water ad libitum. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the National Research Institute of Family Planning. 4.2
Materials
TSL (purity . 99%) was purchased from Tong Ren Tang Company (Beijing, China). Following primary antibodies were used in Western blot: anti-VEGF, anti-bFGF, anti-EGF, anti-CREB, and
anti-p-CREB (PI3K, AKT) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Kits to measure SOD, MDA, ACh, and AChE were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 4.3
Study design
TSL and saline were administered by oral gavage daily for 14 days. The rats were randomly divided into the following four groups (n ¼ 10 for each group): (1) control; (2) VD group: VD (60 min) þ saline (2 mL/ kg, once per day for 14 days); (3) low-dose TSL groups: TSL (15 mg/kg, once per day) was administrated for 14 days after VD; (4) high-dose TSL groups: TSL (30 mg/kg, once per day) was administrated for 14 days after VD. In our study, TSL was administered at the doses of 15 and 30 mg/kg, which is modified by the human therapeutic dose in clinic. 4.4 Establishment of VD Rats were deeply anesthetized with mebumal sodium (50 mg/kg) by intraperitoneal injection, and a midline incision was made to expose the bilateral common carotid arteries. Briefly, ischemia was induced by intraluminal filament (18.5 ^ 0.5 mm internal diameter) occlusion of the middle cerebral artery for 60 min. Postoperative neurological function was scored on a fivepoint scale, where 0 indicated no neurological deficit, 1 (failure to extend left forepaw fully) indicated mild focal neurological deficit, 2 (circling to the left) indicated moderate focal neurological deficit, 3 (falling to the left) indicated severe focal deficit, and 4 (did not walk spontaneously) indicated a depressed level of consciousness. Rats that scored $ 3 points were selected and used for experiments. 4.5 Water maze Rats were trained in a 1.5-m diameter open-field water maze filled with water
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Journal of Asian Natural Products Research (268C) and made opaque with latex liquid. Prominent extra-maze visual cues around the room remained in fixed positions throughout the experiment. During behavioral testing, animals were required to locate a hidden submerged platform 10 cm in diameter (1.5 cm below the surface), which remained in the same position across trials for individual animals but was counterbalanced across animals. Four equally spaced points (north, south, east, and west) around the edge of the pool were used as starting positions. The animals were given four trials per day for 4 days. Trials began as the rat placed in the pool facing the side wall at a start position and ended once the animal had found the platform; if the rat had not found the platform within 120 s, it was guided there by hand. After a period of 30 s on the platform, the rat was immediately re-placed in the pool at a different start position for the next trial. The latency time, distance and swimming speed of the rats were monitored. A video camera mounted to the ceiling directly above the center of the maze was used in conjunction with the EthoVision animal-tracking system (Noldus Information Technologies, Wageningen, the Netherlands). 4.6
Nissl staining
Rats were deeply anesthetized with mebumal sodium and perfused transcardially with 0.9% NaCl for 5 min, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) for 20 min. Then, the brains were removed, postfixed in 4% PFA, and embedded in paraffin. Threemicrometer thick paraffin sections were cut from the hippocampus, and Nissl staining was successfully optimized on paraffin section.
4.8 Western blot analysis The isolated cortex and hippocampus were homogenized in lysis buffer (20 mM Tris pH 7.5, 30 mM NaCl, 1% nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor cocktail), centrifuged at 3000 g for 10 min. Protein concentration was determined by BioRad protein assay. Samples were electrophoresed in SDS/PAGE gels and transferred onto a PVDF membrane. The membrane was blocked with 5% nonfat milk in 1 £ TBS, 0.1% Tween-20 at 258C for 1 h, and subsequently incubated overnight at 48C with appropriate primary antibody. Anti-VEGF, anti-GAP43, antiVEGF, anti-p-AKt, and anti-p-GSK3b were purchased from Santa Cruz or Cell Signal Transduction, diluted in TBST [TBS, 0.1% (v/v) Tween-20, and 5% (w/v) BSA]. After incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h, the blots were developed with chemiluminescence reagent and exposed to X-ray film. 4.9 Statistical analyses The water Morris was performed with repeated measures and two-way ANOVA followed by a Bonferroni multiple-group comparison. For statistical analysis, a standard software package (SAS 10.0) was used. All data are presented as means ^ SEM. Statistical significance was set at P , 0.05. Acknowledgments The work was financially supported by the National Key Grant of Basic Research Project (2010CB530403) and Capital Medical University Key Laboratory Project (2013NZDJ01).
Note 1.
4.7 Measurement of serum Ach/AChE and MDA/SOD levels Serum Ach/AChE and MDA/SOD levels were measured by commercially available ELISA kits.
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Cui-Ge Shi and Yi-Shu Yang contributed equally to this work.
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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Tanshinol protects hippocampus and attenuates vascular dementia development a
b
c
d
a
Cui-Ge Shi , Yi-Shu Yang , Hui Li , Ying Zhang , Ning Wang , a
a
Shang-Ming Wang , Jie-Dong Wang & Shu-Cheng Zhang
a
a
Department of Cell Biology, National Research Institute of Family Planning, Beijing 100081, China b
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Department of Neurology in Medical Healthcare Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China c
Department of Anatomy, Capital Medical University, Beijing 100069, China d
WHO Collaborating Center for Research in Human Reproduction, National Research Institute of Family Planning, Beijing 100081, China Published online: 24 Jun 2014.
To cite this article: Cui-Ge Shi, Yi-Shu Yang, Hui Li, Ying Zhang, Ning Wang, Shang-Ming Wang, Jie-Dong Wang & Shu-Cheng Zhang (2014) Tanshinol protects hippocampus and attenuates vascular dementia development, Journal of Asian Natural Products Research, 16:6, 667-676, DOI: 10.1080/10286020.2014.930131 To link to this article: http://dx.doi.org/10.1080/10286020.2014.930131
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 6, 667–676, http://dx.doi.org/10.1080/10286020.2014.930131
Tanshinol protects hippocampus and attenuates vascular dementia development Cui-Ge Shia1, Yi-Shu Yangb1, Hui Lic, Ying Zhangd, Ning Wanga, Shang-Ming Wanga, Jie-Dong Wanga and Shu-Cheng Zhanga*
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a
Department of Cell Biology, National Research Institute of Family Planning, Beijing 100081, China; bDepartment of Neurology in Medical Healthcare Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China; cDepartment of Anatomy, Capital Medical University, Beijing 100069, China; dWHO Collaborating Center for Research in Human Reproduction, National Research Institute of Family Planning, Beijing 100081, China (Received 23 February 2014; final version received 28 May 2014) Tanshinol (3-(30 ,40 -dihydroxyphenyl)-(2R)-lactic acid, TSL) is widely used in traditional Chinese medicine for the treatment of cardiovascular and cerebrovascular diseases. Here, we assessed whether TSL protected hippocampus and attenuated vascular dementia (VD) development in rats. The behavioral analysis showed that TSL could decrease the distance and latency time, and increase the swim speed in water maze in rats subjected to VD. TSL remarkably increased acetylcholine level and decreased acetylcholinesterase activity in rats subjected to VD. Likewise, TSL remarkably decreased malondialdehyde and increased superoxide dismutase levels in rats subjected to VD. Furthermore, treatment with TSL reduced the level of dead neurons in dentate gyrus. In addition, TSL upregulated growth-associated protein 43 (GAP43) and vascular endothelial growth factor (VEGF) expression and downregulated phosphorylated Akt (p-AKt) and phosphorylated glycogen synthase kinase (p-GSK3b) expression in hippocampus in rats subjected to VD. These results suggest that TSL may be a potential compound in VD model. Keywords: tanshinol; vascular dementia; hippocampus; animal model
1. Introduction Vascular dementia (VD) is one of the most frequent causes of dementia in the elderly, causing a major burden on health care systems in aging societies. VD is the second-most frequent cause of dementia after Alzheimer’s disease. Approximately one-third of individuals above 85 years of age had a dementia syndrome and about one-sixth of them suffer from VD, resulting in a prevalence of about 4– 5%. Cognitive dysfunction frequently occurs after a clinically obvious ischemic or hemorrhagic stroke. Treatment of VD can be approached from two different angles: the control of classical vascular risk *Corresponding author. Email: [email protected] q 2014 Taylor & Francis
factors that obviously contribute to the development of vascular disease and dementia, as well as the use of drugs such as acetylcholine (ACh) esterase inhibitors [1]. Studies in humans have documented that damage to the hippocampus produces retrograde amnesia. Typically, the retrograde amnesia is temporally graded, such that information acquired long before hippocampal damage is remembered better than information acquired recently [2]. Temporally graded retrograde amnesia has been studied extensively in the experimental animal using prospective methods. When the hippocampus is damaged at
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Figure 1. Chemical structure of TSL.
different times after learning, the large majority of studies have found that remote memory is spared relative to more recent memory [3]. Tanshinol (3-(30 ,40 -dihydroxyphenyl)(2R)-lactic acid, TSL, Figure 1) is widely used in traditional Chinese medicine for the treatment of coronary heart disease, cerebrovascular disease, bone loss, hepatocirrhosis, and chronic renal failure [4,5]. It has been reported that TSL could protect the cardiac muscle by calcium antagonizing, reducing oxygen-free radical generation, inhibiting peroxidative damage, and preventing platelet aggregation [6]. The activities of TSL on other aspects, such as antitumor and nerve cells injury protection, have also been reported [7]. As a result, great research interests have been focused on the therapeutic potential of TSL. Therefore, the present study focused on evaluating the TSL-induced attenuation of VD development and its protective mechanisms on hippocampus with the goal of identifying therapeutic strategies against VD in the future. 2.
Results
2.1 The role of TSL in learning and memory impairment To determine whether the middle cerebral artery occlusion induced deficits in spatial working memory, the rats were trained in the water maze for 4 days. Figure 2 shows distance swam (a), latency to platform (b), and swim speed (c) for rats. For distance, VD group swam further to reach the platform than did the control group, wherein TSL group performed signifi-
cantly better than the VD group. There was also a main effect for test day (P , 0.01) in which performance of control group improved over the 4 days of testing. There was a similar pattern of results for latency to reach the platform. Swim speed results also showed that VD group swam more slowly than did the control group, and TSL group performed significantly better than did the VD group. These data suggest that the ability to integrate the platform position with existing memories of the spatial cues is disrupted by VD and this disruption could be improved partly by TSL treatment. 2.2
Nissl staining
The dentate gyrus granule cells of rat hippocampus after VD exhibited morphological alterations consistent with a degenerative process. In the control group, most of the granule neurons had a round or oval nucleus, located in the center of perikarion that is surrounded by pale cytoplasm. In comparison with the control animals, the rats submitted to VD showed slight morphological alterations in most of their neurons. The neuronal changes were triangular in shape mostly exhibiting a dark staining due to condensation of cytoplasm and karyoplasms. Treatment with TSL could improve the morphological alterations of the dentate gyrus granule cells after VD (Figure 3). 2.3 ACh level and acetylcholinesterase activity in hippocampus TSL remarkably increased Ach level and decreased acetylcholinesterase (AChE) activity in rats subjected to VD (Figure 4). The ACh level was 203.6 ^ 25.4 nmol/L in control group, which were much lower than those subjected to VD (93.2 ^ 12.1 nmol/ L). During the 2-week treatment period of TSL at the doses of 15 and 30 mg/kg, the ACh levels decreased to 149.5 ^ 19.3 mL and 160.6 ^ 18.3 nmol/L. The AChE
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Figure 2. Swim distance (a), latency to platform (b), and swim speed (c) in water maze. Rats were trained in the water maze for 4 days with a static platform position. **P , 0.01, *P , 0.05 versus control, #P , 0.05 versus VD. n ¼ 10.
activity was 0.36 ^ 0.04 in control group, which were much higher than those subjected to VD (1.54 ^ 0.22). After TSL
treatment at the doses of 15 and 30 mg/kg, the superoxide dismutase (SOD) levels increased to 0.9 ^ 0.12 and 0.7 ^ 0.1.
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C.-G. Shi et al. in rats subjected to VD (Figure 5). The MDA level was 1.2 ^ 0.18 nmol/mg protein in control group, which was much lower than those subjected to VD (3.8 ^ 0.4 nmol/mg protein). During the 2-week treatment period of TSL at the doses of 15 and 30 mg/kg, the MDA levels decreased to 2.9 ^ 0.4 mL and 2.48 ^ 0.24 nmol/mg protein. The SOD level was 174.2 ^ 25.2 nmol/mg protein in control group, which were much higher than those subjected to VD (93.2 ^ 13.4 nmol/mg protein). After TSL treatment at the doses of 15 and 30 mg/kg, the SOD levels increased to 135.5 ^ 19.3 mL and 149.6 ^ 18.6 nmol/mg protein.
2.5 TSL upregulates growth-associated protein 43 and vascular endothelial growth factor expression in hippocampus Figure 3. Different morphological profiles of apoptotic granule cells by Nissl-stained sections in the dentate gyrus of a control rat (a), a rat receiving VD (b), a rat receiving UCMS and TSL 15 mg/kg (c), a rat receiving UCMS and TSL 30 mg/kg (d). a1 – d1 are magnified views of regions from a to d. Scale bars: 100 mm. n ¼ 3.
2.4 Malondialdehyde and SOD levels in serum TSL remarkably decreased malondialdehyde (MDA) level and increased SOD level
Growth-associated protein 43 (GAP43) and vascular endothelial growth factor (VEGF) protein expressions in hippocampus were quantified using Western blotting (Figure 6). These results demonstrated that relative protein GAP43 and VEGF levels in the control group were 1.36 ^ 0.24 and 1.06 ^ 0.15, respectively. VD significantly downregulated relative protein GAP43 and VEGF levels to 0.54 ^ 0.07 and 0.44 ^ 0.06, respectively. TSL treatment upregulated GAP43 expression to 1.03 ^ 0.15 and 1.2 ^ 0.19 and VEGF expression to
Figure 4. ACh level (a) and AChE activity (b) in hippocampus. TSL remarkably increased ACh level and decreased AChE activity in hippocampus in rats subjected to VD. **P , 0.01, *P , 0.05 versus control, #P , 0.05 versus VD. n ¼ 10.
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Figure 5. Serum levels of MDA (a) and SOD (b). TSL remarkably decreased MDA and increased SOD levels in rats subjected to VD. **P , 0.01, *P , 0.05 versus control, #P , 0.05 versus VD. n ¼ 10.
0.7 ^ 0.12 and 1.1 ^ 0.16 at the doses of 15 and 30 mg/kg (P , 0.05 vs. VD) 2.6 TSL inhibits phosphorylated Akt and glycogen synthase kinase 3b expression in hippocampus Phosphorylated Akt (p-AKt) and glycogen synthase kinase 3b (GSK3b) protein
expressions in hippocampus were quantified using Western blotting (Figure 7). These results demonstrated that relative protein p-AKt and GSK3b levels in the control group were 0.56 ^ 0.06 and 0.06 ^ 0.04, respectively. VD significantly upregulated relative protein p-AKt and GSK3b levels to 1.24 ^ 0.2 and
Figure 6. TSL increased GAP43 and VEGF expressions in hippocampus in rats subjected to VD. GAP43 and VEGF expressions were determined by Western blot. *P , 0.05, **P , 0.01 versus control. #P , 0.05 versus VD. n ¼ 4.
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Figure 7. TSL attenuates the expression of p-AKt and p-GSK3b protein in hippocampus in rats subjected to VD. p-AKt and p-GSK3b expressions were determined by Western blot analysis. *P , 0.05, **P , 0.01 versus control. #P , 0.05 versus VD. n ¼ 3.
1.5 ^ 0.2, respectively. TSL treatment downregulated p-AKt expression to 0.9 ^ 0.12 and 0.7 ^ 0.16 and GSK3b expression to 1.2 ^ 0.14 and 0.6 ^ 0.09 at the doses of 15 and 30 mg/kg (P , 0.05 vs. VD) 3.
Discussion
Herein we showed that TSL could not only improve the spatial working memory, but also promote the consequences of rat subjected to VD. These studies evaluated the protective role of TSL on VD. TSL induces multiple changes in the animal model of VD: (1) a reduction in the distance and latency times, and a promotion in the swim speed in water maze; (2) an improvement on the morphological alterations of the dentate gyrus granule cells; (3) a promotion in the levels of ACh and SOD, and a depression in the levels of MDA and AChE; and (4) upregulation of
the expression of GAP43 and VEGF, and downregulation of the expression of p-AKt and phosphorylated GSK3b (p-GSK3b). Chronic cerebral hypoperfusion has been assumed as a pathogenic mechanism in patients with VD. Cognitive impairment has been demonstrated in animals with chronic cerebral hypoperfusion and modeling patients with VD [8]. Rats with untreated VD in the present study were found to have abnormal cognitive function, as demonstrated by their impaired learning ability in the water maze test [9]. The behavioral results are consistent with hippocampal dysfunction, as the hippocampus seems to be a critical component for forming relational representations and strategic flexibility in the Morris water maze. This learning ability was significantly better in rats with VD that were treated with TSL. It had been shown that a relationship exists between changes in
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Journal of Asian Natural Products Research cognitive function and hippocampus damage, and that TSL treatment reversed the impairment. Our study demonstrated that loss of VD definitely resulted in hippocampal granule cell degeneration [10 – 12]. This study showed that dentate gyrus granule cell degeneration following an ischemia was related to the vessel occlusion (VO). VD-induced granule cell apoptosis was blocked by the treatment with TSL. These results suggested that TSL in this study could protect granule cells continuously against VD-induced apoptosis. An important hypothesis in this study was that TSL would rescue the uninjured granule cells. It is noteworthy that the relationship between vascular function and cognition in our study was much stronger than the relationship between cognition and many other individual vascular-related variables that were analyzed [13,14]. Therefore, the vasorelaxation is obtained by assessing vascular response to ACh [15,16]. Ischemia resulted in a significant decrease in ACh level, but recirculation was associated with a rapid increase in ACh concentration. Impaired synthesis and/or increased release of ACh can be responsible for the decrease in ACh concentration during ischemia. Early postischemic elevation of ACh may be related to the large increase in brain choline brought about by ischemia. In this study, we found an increase in the levels of ACh and a decrease in the activity of AChE. The degradation product of MDA was considered to reflect the degree of lipid peroxidation. The past work showed that, during ischemia-reperfusion injury process, in memantine intervention group, the increase in expression level of caspase-3 in cortex neuron and MDA release in cortex homogenate were inhibited significantly at 12, 24, and 48 h time points, compared with that of rats in ischemia-reperfusion model group. As expected, VD was associated with oxidative stress as evidenced by elevated MDA levels in hippocampus. The SOD-mimetic effects
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of VD were confirmed by the findings of hippocampus. MDA were lowered by TSL treatment and by the fact that SOD activity was elevated in the TSL-treated group. Therefore, TSL should exert protection against hippocampus damage in VD. GAP43 is widely considered to be involved in neuronal mechanisms underlying axonal growth, axonal regeneration, and synaptic plasticity [17,18]. GAP43 is thought to contribute directly to the outgrowth of neuronal processes, while first recognized for its increased synthesis and axonal transport in neurons responding to peripheral nerve injury [19]. GAP43 and its mRNA are also detectable in some regions of the normal brain [20]. VEGF is reported to stimulate adult neurogenesis in vivo and proliferation of brain-derived neural stem/progenitor cells [21]. It is a target zone of several important afferent inputs to hippocampus. The results have functional implications because of the known effects of VEGF in hippocampus, such as stimulation of neurogenesis in the dentate gyrus in rats of VD. The main biological consequences of p-Akt activation that are relevant to cancer cell growth can be classified loosely into three categories: survival, proliferation (increased cell number), and growth (increased cell size). p-Akt activation is also associated with enhanced cell invasion. Gao et al. [22] showed that ischemic postconditioning enhanced phosphorylation of Akt. The inhibition of Akt activity partly abolished the protective effects of postconditioning. Many neuroprotectants, including propofol, humanin, estradiol, melatonin, isoflurane, atorvastatin, and tissue-type plasminogen activator, exert their protective effects through the Akt pathway. Akt enhances invasiveness of pancreatic carcinoma cells via upregulation of VEGF [23]. Gong reported that p-Akt were significantly decreased in the hippocampus at 1, 2, and 4 months following 2-VO injury [24]. On the contrary, Shu et al. reported that p-Akt
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were significantly increased in the hippocampus at 1, 4, and 8 weeks following 2-VO injury [25]. The expression level of p-Akt might be related to time following 2-VO injury. GSK3b activation is mediated through PP2A-regulated inactivation of the PI 3-kinase-Akt pathway. GSK3b acts downstream of PP2A and the PI3K-Akt pathway, and upstream of caspase-2 in ceramide induced mitochondrial apoptosis. Our analysis showed that after 2 weeks following VD, p-Akt and p-GSK3b protein expressions were significantly increased in VD groups as compared with the control group. TSL decreased p-AKt and p-GSK3b expression in hippocampus lesions in VD. It is suggested that TSL could affect PI3KAkt-GSK3b pathway. In conclusion, we demonstrated that TSL significantly protected VD damage. These results suggest that this may be a potential compound in VD model. 4. Experimental 4.1 Animals Adult male Sprague–Dawley rats with weights of 180 – 220 g were used. All animals were housed 5 per cage under standardized light/dark cycle condition (lights on at 7:00 am, lights off at 7:00 pm) at a room temperature of 24 ^ 18C and humidity of 60% ^ 10% with food and water ad libitum. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the National Research Institute of Family Planning. 4.2
Materials
TSL (purity . 99%) was purchased from Tong Ren Tang Company (Beijing, China). Following primary antibodies were used in Western blot: anti-VEGF, anti-bFGF, anti-EGF, anti-CREB, and
anti-p-CREB (PI3K, AKT) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Kits to measure SOD, MDA, ACh, and AChE were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 4.3
Study design
TSL and saline were administered by oral gavage daily for 14 days. The rats were randomly divided into the following four groups (n ¼ 10 for each group): (1) control; (2) VD group: VD (60 min) þ saline (2 mL/ kg, once per day for 14 days); (3) low-dose TSL groups: TSL (15 mg/kg, once per day) was administrated for 14 days after VD; (4) high-dose TSL groups: TSL (30 mg/kg, once per day) was administrated for 14 days after VD. In our study, TSL was administered at the doses of 15 and 30 mg/kg, which is modified by the human therapeutic dose in clinic. 4.4 Establishment of VD Rats were deeply anesthetized with mebumal sodium (50 mg/kg) by intraperitoneal injection, and a midline incision was made to expose the bilateral common carotid arteries. Briefly, ischemia was induced by intraluminal filament (18.5 ^ 0.5 mm internal diameter) occlusion of the middle cerebral artery for 60 min. Postoperative neurological function was scored on a fivepoint scale, where 0 indicated no neurological deficit, 1 (failure to extend left forepaw fully) indicated mild focal neurological deficit, 2 (circling to the left) indicated moderate focal neurological deficit, 3 (falling to the left) indicated severe focal deficit, and 4 (did not walk spontaneously) indicated a depressed level of consciousness. Rats that scored $ 3 points were selected and used for experiments. 4.5 Water maze Rats were trained in a 1.5-m diameter open-field water maze filled with water
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Journal of Asian Natural Products Research (268C) and made opaque with latex liquid. Prominent extra-maze visual cues around the room remained in fixed positions throughout the experiment. During behavioral testing, animals were required to locate a hidden submerged platform 10 cm in diameter (1.5 cm below the surface), which remained in the same position across trials for individual animals but was counterbalanced across animals. Four equally spaced points (north, south, east, and west) around the edge of the pool were used as starting positions. The animals were given four trials per day for 4 days. Trials began as the rat placed in the pool facing the side wall at a start position and ended once the animal had found the platform; if the rat had not found the platform within 120 s, it was guided there by hand. After a period of 30 s on the platform, the rat was immediately re-placed in the pool at a different start position for the next trial. The latency time, distance and swimming speed of the rats were monitored. A video camera mounted to the ceiling directly above the center of the maze was used in conjunction with the EthoVision animal-tracking system (Noldus Information Technologies, Wageningen, the Netherlands). 4.6
Nissl staining
Rats were deeply anesthetized with mebumal sodium and perfused transcardially with 0.9% NaCl for 5 min, followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4) for 20 min. Then, the brains were removed, postfixed in 4% PFA, and embedded in paraffin. Threemicrometer thick paraffin sections were cut from the hippocampus, and Nissl staining was successfully optimized on paraffin section.
4.8 Western blot analysis The isolated cortex and hippocampus were homogenized in lysis buffer (20 mM Tris pH 7.5, 30 mM NaCl, 1% nonidet P-40, 0.5% sodium deoxycholate, and protease inhibitor cocktail), centrifuged at 3000 g for 10 min. Protein concentration was determined by BioRad protein assay. Samples were electrophoresed in SDS/PAGE gels and transferred onto a PVDF membrane. The membrane was blocked with 5% nonfat milk in 1 £ TBS, 0.1% Tween-20 at 258C for 1 h, and subsequently incubated overnight at 48C with appropriate primary antibody. Anti-VEGF, anti-GAP43, antiVEGF, anti-p-AKt, and anti-p-GSK3b were purchased from Santa Cruz or Cell Signal Transduction, diluted in TBST [TBS, 0.1% (v/v) Tween-20, and 5% (w/v) BSA]. After incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h, the blots were developed with chemiluminescence reagent and exposed to X-ray film. 4.9 Statistical analyses The water Morris was performed with repeated measures and two-way ANOVA followed by a Bonferroni multiple-group comparison. For statistical analysis, a standard software package (SAS 10.0) was used. All data are presented as means ^ SEM. Statistical significance was set at P , 0.05. Acknowledgments The work was financially supported by the National Key Grant of Basic Research Project (2010CB530403) and Capital Medical University Key Laboratory Project (2013NZDJ01).
Note 1.
4.7 Measurement of serum Ach/AChE and MDA/SOD levels Serum Ach/AChE and MDA/SOD levels were measured by commercially available ELISA kits.
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Cui-Ge Shi and Yi-Shu Yang contributed equally to this work.
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