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Leonurine ameliorates cognitive dysfunction via antagonizing excitotoxic glutamate insults and inhibiting autophagy
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Leonurine ameliorates cognitive dysfunction via antagonizing excitotoxic glutamate insults and inhibiting autophagy Chunhua Liu , Hongqiang Yin , Jing Gao , Xiaxia Xu , Tao Zhang , Zhuo Yang PII: DOI: Reference:
S0944-7113(16)30180-5 10.1016/j.phymed.2016.10.005 PHYMED 52088
To appear in:
Phytomedicine
Received date: Revised date: Accepted date:
13 May 2016 28 September 2016 2 October 2016
Please cite this article as: Chunhua Liu , Hongqiang Yin , Jing Gao , Xiaxia Xu , Tao Zhang , Zhuo Yang , Leonurine ameliorates cognitive dysfunction via antagonizing excitotoxic glutamate insults and inhibiting autophagy, Phytomedicine (2016), doi: 10.1016/j.phymed.2016.10.005
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Graphical abstract
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Leonurine ameliorates cognitive dysfunction via antagonizing excitotoxic glutamate insults and inhibiting autophagy
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Chunhua Liua, Hongqiang Yina, Jing Gaoa, Xiaxia Xub, Tao Zhangb, Zhuo Yanga,*
School of Medicine, State Key Laboratory of Medicinal Chemical Biology,
Key Laboratory of Bioactive Materials Ministry of Education, Nankai University, Tianjin 300071, China; College of Life Sciences, Nankai University, Tianjin 300071, China
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*Corresponding author.
23504364; fax: +86 22 23502554
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School of Medicine, Nankai University, 94 Weijin Road, Tianjin 300071, China. Tel.: +86 22
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E-mail address: [email protected] (Z. Yang)
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ABSTRACT Background: Chronic cerebral hypoperfusion is related with cognitive deficits in different types of dementia. Purpose: In this study, we aimed to investigate the effect and potential mechanisms of leonurine on
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chronic cerebral hypoperfusion both in vitro and in vivo. Study Design: Chronic cerebral hypoperfusion was duplicated by oxygen-glucose deprivation (OGD) in vitro and by ligation of bilateral common carotid arteries (2-VO) in vivo.
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Methods: In in vitro study, there were control group, OGD group, OGD + 100 μM leonurin group, and OGD+10 μM donepezil group. The spontaneous excitatory postsynaptic current amplitude and frequency were recorded. In in vivo study, the chronic cerebral hypoperfusion model was induced by
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ligated bilateral common carotid arteries. Rats were randomly divided into Sham group, 2-VO group, 2-VO + 60 mg/kg/day leonurine group, and 2-VO + 4 mg/kg/day donepezil group. After three weeks,
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the Morris water maze and Long-term depression recording were observed. Then N-methyl-D-aspartate
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receptor-associated proteins and autophagy-associated proteins were detected by Western blot assay. Results: In in vitro experiment, results showed that leonurine could obviously attenuate the
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spontaneous excitatory postsynaptic current amplitude and frequency on pyramidal neurons. In in vivo
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experiment, leonurine significantly decreased levels of glutamate and hydrogen peroxide, improved both the cognitive flexibility and the spatial learning and memory abilities. Moreover, leonurine obviously enhanced long-term depression, elevated the ratio of N-methyl-D-aspartate receptor 2A/2B, and decreased the expression of postsynaptic density protein-95. Interestingly, the ratio of LC3II/LC3I and beclin-1 expression were markedly down-regulated by leonurine. Conclusion: These findings suggest that leonurine ameliorates cognitive dysfunction at least partly via
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antagonizing excitotoxic glutamate insults and inhibiting autophagy. Furthermore, it might become a potential drug candidate of chronic cerebral hyperfusion in future.
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Keywords: Chronic cerebral hypofusion; Long-term depression; Leonurine; Hippocampus; Autophagy
Abbreviations
Chronic cerebral hypoperfusion CCH; oxygen–glucose deprivation OGD; artificial cerebral spinal fluid
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aCSF; bilateral common carotid arteries 2-VO; N-methyl-D-aspartate receptors NMDARs; long-term depression LTD; Morris water maze MWM; initial training IT; initial probe trials IPT; reversal training
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RT; reversal probe trials RPT.
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1. Introduction Chronic cerebral hypoperfusion (CCH) has been identified to be related with the cognitive deficits in different types of dementia, such as Alzheimer‟s disease, vascular dementia and frontotemporal dementia (Osawa et al., 2004; Shu et al., 2013). CCH leads to the inadequate blood supply in different
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regions of brain, which is accompanied with the deficiency of oxygen and nutrients. As known, the hippocampus formation is the most sensitive brain area to the cerebral ischemia and plays a key role in spatial learning and memory (Li et al., 2011). Accumulating evidences demonstrate that there is a
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causal relationship between CCH and dementia (Osawa et al., 2004).
Brain ischemia and hypoxia could lead to the depolarization of neurons, and then a large sum of glutamate will release into the synaptic cleft to excessively agitate the N-methyl-D-aspartate receptors (NMDARs or NRs), which will cause lots of Ca2+ flux into neurons and result in cell death by this
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excitotoxic glutamate insults. Autophagy is necessary for cell degrading cellular “garbage” and for
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maintaining cell homeostasis by lysosome-mediated catabolic machinery. It plays an important role not
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only at physiological status, such as cell survival, development and differentiation, but also at pathological condition, such as Alzheimer‟s disease, Parkinson‟s disease and brain ischemia disease
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(Caberlotto and Nguyen, 2014). Interestingly, studies demonstrated that brain hypoxia and ischemia
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obviously enhanced the neuronal autophagy, and the inhibition of the autophagy was of benefit to hippocampus (Koike et al., 2008). On the contrary, there were also some other results demonstrated that autophagy played neuroprotective effects on brain ischemia (Balduini et al., 2001; Carloni et al., 2008). Therefore, the role of autophagy in brain ischemia and hypoxia still needs to be further investigated. Then, how about the role of autophagy in CCH and what the underlying mechanism is still not completely understand.
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Leonurus cardiaca is an herbaceous perennial plant in the mint family and has a long history in traditional medicine to treat a variety of diseases in China, Japan, Korea and European countries. Leonurine (LEO), an active alkaloid extracts from Leonurus cardiaca, has recently been demonstrated to be effective on the treatment of cardiovascular disease and nervous system diseases (Liu et al., 2013;
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Qi et al., 2010; Wojtyniak et al., 2013). Studies have demonstrated that LEO has neuroprotective effects on middle cerebral artery occlusion rats via anti-oxidantion and anti-apoptosis (Qi et al., 2010). However, the effect and underlying mechanism of LEO on CCH are still poorly understood. In this
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study, the CCH model was duplicated by oxygen-glucose deprivation (OGD) in vitro and by ligation of bilateral common carotid arteries (2-VO) in vivo. We aimed to investigate whether or not LEO could alleviate the cognitive impairment of CCH.
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2. Materials and methods
The brain slices were removed from 3-4 week Wistar rats and there were control group (CON)
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incubated in oxygenated artificial cerebrospinal fluid (aCSF); OGD group incubated with aCSF
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containing 95% N2 and 5% CO2 (OGD); OGD group treated with aCSF containing 95% N2 and 5% CO2 and different concentrations of LEO (OGD+25μM LEO, OGD+50μM LEO and OGD+100μM
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LEO groups). The cell viability of brain slices was determined by PI staining, and the patch-clamp
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recording was performed in CON, OGD and OGD+100μM LEO, and OGD+10μM donepezil
hydrochloride (OGD+DON) groups. Adult male Wistar rats (250-280g) were randomly divided into four groups, i.e. sham operation group with vehicle treatment (Sham); 2-VO group with vehicle treatment (2-VO); 2-VO group treated with 60 mg/kg/day of LEO (2-VO+LEO); and 2-VO group treated with 4 mg/kg/day of DON (2-VO+DON). The CCH model was induced by 2-VO surgery. After three weeks, spatial learning and memory performances of rats were evaluated by Morris water
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maze (MWM). Then the long-term depression (LTD) recording was performed immediately after MWM test. In order to investigate the neuroprotective mechanism of LEO, the levels of glutamate and H2O2 of hippocampus were measured. Moreover, levels of NR2A, NR2B, PSD-95 (1:2000, abcam, UK), LC3I/II (1:1000, Medical & biological laboratories Co. LTD., JPN), beclin-1 (1:2000, Cell
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Signaling Technology, USA) and -actin (1:4000, abcam, UK) were tested by Western blot assay. All data were analyzed by SPSS 16.0 software. Escape latencies and swimming speeds in MWM experiment were analyzed by two-way repeated ANOVA followed by the Bonferroni multiple group
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comparison. Other data were analyzed by one-way ANOVA followed by a post Turkey test. Data were presented as means ± S.E.M and defined differences at p < 0.05 as statistically significant. All pictures were processed with Photoshop software (sources and detailed methods in Supporting Text).
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3. Results
3.1 LEO attenuated the excitotoxic insults induced by OGD
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As shown in Fig. 1A and 1B, OGD significantly increased brain cell death. However, LEO
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alleviated the OGD-induced damage in a dose-dependent manner (p < 0.05). In the OGD+100 M LEO group, LEO could significantly decrease the brain cell death to 130.36 ± 9.06% (fold of CON
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group), so we selected the 100 LEO for next experiments. OGD induced the increase of the
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amplitude and expressed a markedly right shift in the cumulative probability curve of spontaneous excitatory postsynaptic current (sEPSC) amplitude (p < 0.01, Fig. 1C, 1D, 1F, 1G, 1I and 1J). However, LEO and DON obviously attenuated the amplitude of pyramidal neurons (p < 0.05), and presented a markedly left shift in the cumulative probability curve of sEPSC amplitude (Fig. 1C, 1D, 1F, 1G, 1I and 1J). As to the sEPSCs frequency, OGD produced obviously higher time-dependent frequency of pyramidal neurons, which was also ameliorated by LEO and DON (p < 0.01, Fig. 1C, 1E, 1F, 1H, 1I
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and 1K). 3.2 LEO alleviated the impaired spatial learning and memory in 2-VO model rats Rats were subjected to MWM test to investigate the spatial learning and memory ability. The average escape latency was obviously decreased in all four groups during initial training (IT) without
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affecting swimming speeds (Fig. 2A and 2B). However, the escape latency was much longer in 2-VO group from day 2 to day 4 (p < 0.05, Fig. 2A). Interestingly, rats in 2-VO+LEO group and 2-VO+DON group located the platform much more faster on day 3 and day 4 (p < 0.05, Fig. 2A). In initial probe
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trials (IPT), the platform crossings (1.1 ± 0.31) and the quadrant dwell time (29.22 ± 2.44%) were obviously decreased in 2-VO group compared with that of Sham group (2.5 ± 0.54; 50.44 ± 3.06%, p < 0.01, Fig. 2C and 2D). Although there was no significant difference in platform crossings between
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2-VO group and 2-VO+LEO group or 2-VO+DON group, LEO and DON remarkably improved the quadrant dwell time compared with that of 2-VO group (p < 0.05, Fig. 2C and 2D).
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The average escape latency was markedly longer in 2-VO group than that of Sham group on both
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day 6 and day 7 during reversal training (RT) (p < 0.05, Fig. 2A). Rats of 2-VO+LEO group and 2-VO+DON group spent less time to find the platform than that of 2-VO group without affecting
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swimming speeds (p < 0.05, Fig. 2A and 2B). There was no significant difference in platform crossings
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between 2-VO group and 2-VO+LEO group or 2-VO+DON group (p >0.05, Fig. 2E). However, the quadrant dwell time in reversal probe trials (RPT) were obviously increased in 2-VO+LEO group and 2-VO+DON group compared with those in 2-VO group (p < 0.05, Fig. 2F). In addition, we analyzed the temporal distribution of the quadrants. Rats of 2-VO group spent much more time in zone 1 (p < 0.01, Fig. 3A) on day 7. In contrast, they spent less time in zone 4 (p < 0.05, Fig. 3D). There was no significant difference between 2-VO group and 2-VO+LEO group or
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2-VO+DON group in the time spent in both zone 2 and zone 3 (p >0.05, Fig. 3B and 3C). However, rats administrated with LEO or DON spent less time in zone 1 and more time in zone 4 on day 7 (p < 0.05, Fig. 3A and 3D). Moreover, the swim traces of all rats in RT stage were showed in Fig. 3E. In Sham group, 2-VO+LEO group and 2-VO+DON group, the trajectories of reversal learning stage were
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shorten, whereas the trajectory was significantly longer in 2-VO group (Fig. 3E). Also, rats of 2-VO group showed much more swimming trajectories in zone 1. In contrast, rats treated with LEO or DON showed much more swimming trajectories in zone 4 rather than other zones (Fig. 3E).
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3.3 LEO ameliorated the impaired LTD in hippocampus of 2-VO model rats
As shown in Fig. 4, field excitatory postsynaptic potentials (fEPSPs) slope was obviously decreased after the LFS and maintained lower than the baseline in Sham group. In contrast, the fEPSPs
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slope was higher in 2-VO group, which indicated that LTD was suppressed in 2-VO group (p < 0.01, Fig. 4). Interestingly, LEO and DON could significantly reduce the fEPSPs slope and maintained lower,
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which suggested that LEO and DON alleviated the impaired synaptic plasticity induced by 2-VO (p <
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0.01, Fig. 4).
3.4 LEO decreased the concentrations of glutamate and hydrogen peroxide (H2O2) in
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hippocampus of 2-VO model rats
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Data showed that both levels of glutamate and H2O2 were significantly increased in hippocampus
of 2-VO group compared with those of Sham group (p < 0.01, Fig. 5). After administration with LEO and DON, the concentration of glutamate and H2O2 were obviously decreased (p < 0.05, Fig. 5). 3.5 LEO improved cognitive function by modulating the NMDARs-associated proteins As shown in Fig. 6A-C, there was no change in NR2B expression in hippocampus of 2-VO group (p > 0.05), but NR2A expression was markedly decreased to 0.68-fold compared with that of Sham
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group, then the ratio of NR2A/2B was decreased to 0.62 (p < 0.01, Fig. 6C). Interestingly, LEO and DON were able to increase the expression of NR2A so as to improve the ratio of NR2A/2B (p < 0.01, Fig. 6A-C). As to the expression of PSD-95, there was a significantly difference between Sham group and 2-VO group (p < 0.01, Fig. 6D). After administration with LEO and DON, the PSD-95 expression
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were obviously down-regulated (p < 0.05, Fig. 6D). 3.6 LEO protected rats from 2-VO induced damage by inhibiting autophagy
Data showed that the CCH markedly up-regulated the beclin-1 expression (p < 0.01, Fig. 6E),
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which suggested that autophagy was activated. LEO and DON were able to attenuate the level of beclin-1 (p < 0.05, Fig. 6E). As shown in Fig. 6F, the CCH increased the ratio of LC3II/I compared with that of Sham group (p < 0.01). Interestingly, LEO and DON significantly reduced the ratio of
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LC3II/I (p < 0.05), which suggested that LEO could inhibit the excessive activation of autophagy. 4. Discussion
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Cerebral hypoxia could lead to the depolarization of neurons, and then the induced massive glutamate
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will release into the synaptic cleft. When inhibiting the sEPSC events, the excitotoxicity was alleviated and the neuronal death was reduced (Yang et al., 2014). Recently, studies reported that LEO has
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neuroprotective effects on middle cerebral artery occlusion rats via anti-oxidation and anti-apoptosis
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(Qi et al., 2010). Moreover, it can also attenuate cognitive impairments of AD via JNK and NF-кB pathways (Hong et al., 2015). As known, the synaptic plasticity is closely related with cognitive function, so we speculated that whether or not the neuroprotective effect of LEO on CCH was correlated with the protection of synaptic plasticity. Furthermore, the pathology of CCH was related with cerebral ischemia. Therefore, we employed the OGD to partly duplicate the CCH for in vitro experiment according to previous studies (Le et al., 2015; Yang et al., 2014).
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From in vitro experiments, data showed that hippocampal slices treated by OGD displayed excessive events of sEPSCs, which suggested that OGD led to the depolarization of CA1 pyramidal neurons and induced neuronal excitotoxicity. When administrated with LEO, it could obviously alleviate the depolarization of neurons by reducing the sEPSCs amplitude and frequency. These results
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suggested that LEO protected synaptic plasticity from OGD-induced damage. Considering the in vitro experimental results, we next preliminarily observed the potential neuroprotection of LEO on CCH in in vivo experiment. We duplicated the rat model of CCH by 2-VO
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(Li et al., 2011). Our data showed that there was obvious learning and memory deficiency in 2-VO group. However, LEO could effectively alleviate cognitive deficiency to some extent in IT stage, it suggested that the acquisition and retention capacities were improved in 2-VO+LEO group. Then, rats
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were subjected to spatial reversal learning. Our data suggested that the information storage was partly damaged by 2-VO. However, rats of 2-VO+LEO group could successfully find the platform much
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faster than that of 2-VO group. Moreover, the time spent in the novel quadrant was longer and the time
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spent in the original quadrant was shorter in 2-VO+LEO group compared with those of 2-VO group. It implied that rats of 2-VO+LEO group established a new strategy to explore the platform in the novel
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environment. Our results indicated that LEO not only ameliorated the spatial learning and memory, but
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also alleviated the deficits in cognitive flexibility in 2-VO rats. As known, memory is achieved by experience-dependent changes in synaptic strength. These
changes can take the form of LTP or LTD of synaptic transmission. Studies have reported that LTD underlies storage of memory and the acquisition of novel information, especially in the acquisition of object-place configuration (Kemp and Manahan-Vaughan, 2004; Manahan-Vaughan and Braunewell, 1999). When rats were explored to a novel task, LTD was helpful to weaken previous memory traces,
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which led to the decreased synaptic efficacy. Then, LTD contributed to increase the „signal to noise ratio‟ and prevent previous memory from interfering with newly encoded information (Nicholls et al., 2008). In this study, our results showed that CCH led to the increase of fEPSPs slope of 2-VO group, which demonstrated that synaptic plasticity was impaired. However, the LTD was ameliorated via
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administrating LEO. So our data suggested that LEO was able to protect rats from the impairment of synaptic plasticity induced by 2-VO.
LEO prevented the neuron death from the injury induced by 2-VO. The neuroprotection of LEO
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was associated with the lower levels of glutamate and H2O2 in hippocampus, so as to decrease the damage from excitatory toxicity. It suggested that LEO could partly modulate the damage of glutamate system. However, the neuroprotective mechanism of LEO still needs to be further investigated.
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Recently studies reported that NMDARs related proteins and autophagic related proteins were involved in modulating synaptic plasticity (Fu et al., 2016; Yu et al., 2016; Zhang et al., 2016). So we
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examined these proteins mentioned above, in order to preliminarily explore the neuroprotective
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mechanism of LEO. As known, NMDARs are involved in hippocampal synaptic plasticity and in mediating learning and memory processes. Preferential inhibition of NR2A prevented the induction of
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LTP without affecting LTD production (Ge et al., 2010; Liu et al., 2004). By contrast, other studies
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found that the inhibition of NR2A impaired both LTD and LTP (Bartlett et al., 2007; Liu et al., 2004). Thus there is so far some controversy about NMDAR subunits dependence of LTD, which makes it hard to draw an unifying conclusion. In our study, the NR2A expression was significantly decreased, as well as the LTD was severely impaired in 2-VO group. In view of our results, we considered that NR2A played a key role in LTD. In addition, the activation of NR2B resulted in neuronal death (Hardingham et al., 2002; Liu et al., 2007). However, the inhibited NR2B ameliorated the impairment
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of ischemia/reperfusion (Meng and Zhang, 2002). Besides, inhibiting the PSD-95 expression selectively
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excitotoxicity,
because
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activated nitricoxide production by NMDARs selectively (Sattler et al., 1999). Interestingly,
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activation of NR2A contributed to neuronal survival via NMDA receptor-mediated and non-NMDA
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receptor-mediated neuronal damage (Hardingham et al., 2002). In this experiment, the NR2A expression was significantly decreased accompanied with the increase of PSD-95 in 2-VO group, it implied that serious excitotoxicity was induced and the capability of neuronal survival was markedly
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weakened in 2-VO group. Fortunately, LEO was able to dramatically up-regulated NR2A expression and down-regulated PSD-95 expression, which was beneficial to protect rats from the damage induced by 2-VO.
A large sum of glutamate will rush into the synaptic cleft to excessively agitate NMDARs in
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cerebral ischemia, which will cause lots of Ca2+ flux into neurons and result in cell death. Moreover,
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Ca2+ fluxing into neurons will also induce excessive H2O2, which could improve the level of autophagy
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(Ashabi et al., 2013). The inhibition of autophagy could help neuron survive from ischemia (Zheng et al., 2014). On the contrary, some studies reported that the improvement of autophagy could antagonize
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neuronal apoptosis after cerebral ischemia (Su et al., 2014; Yang et al., 2015). Therefore, the effect of
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autophagy on the cerebral ischemia is still controversial. In our study, rats presented higher levels of glutamate and H2O2 in hippocampus of 2-VO group. In addition, the ratio of NR2A/NR2B was significantly reduced and PSD-95 expression was obviously increased by 2-VO. We inferred that the higher level of glutamate triggered the excitotoxicity, and then led to an excess of H2O2, which could improve the level of autophagy (Ashabi et al., 2013). So we observed the expression of autophagic related proteins. Data showed that the ratio of LC3II/I and the expression of beclin-1 were significantly
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improved in 2-VO group, which could be partly reversed after treatment with LEO. These results demonstrated that LEO prevented the cognitive impairment from 2-VO partly via inhibiting autophagy. 5. Conclusion In conclusion, we reported that LEO obviously attenuated the amplitude and frequency of
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pyramidal neurons in vitro. Moreover, LEO not only improved the spatial learning and memory, but also alleviated the impairment of LTD. It increased the NR2A protein expression in hippocampus in order to improve the ratio of NR2A/2B, and attenuated the over-expression of PSD-95, which was
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beneficial for the synaptic plasticity and neuronal survival. Interestingly, LEO reduced the expression of LC3II and beclin-1 in order to inhibit the activation of autophagy. Thus, we conclude that LEO protects synaptic plasticity from cognitive impairment partly via antagonizing excitotoxic glutamate
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insults and inhibiting autophagy. Our results will shed new light on the neuroprotective mechanism of
Acknowledgments
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LEO, which might be used as a potential drug candidate of CCH in the future.
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This work was partly supported by grants from the National Natural Science Foundation of China (31400986, 11232005), the Applied Basic Research Programs of Science and Technology Commission
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Foundation of Tianjin (14JCQNJC11800, 14JCZDJC35000). We thank Prof. Yizhun Zhu (School of
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Pharmacy, Fudan University, Shanghai, China) for kindly providing LEO. Conflict of interest There is no conflict of interest.
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Wojtyniak, K., Szymanski, M., Matlawska, I., 2013. Leonurus cardiaca L. (Motherwort): A Review of its Phytochemistry and Pharmacology. Phytother Res 27, 1115-1120. Yang, J.J., Yao, Y., Chen, T., Zhang, T., 2014. VEGF Ameliorates Cognitive Impairment in In Vivo
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and In Vitro Ischemia via Improving Neuronal Viability and Function. Neuromolecular medicine 16, 376-388.
Yang, Y., Gao, K., Hu, Z., Li, W., Davies, H., Ling, S., Rudd, J.A., Fang, M., 2015. Autophagy upregulation and apoptosis downregulation in DAHP and triptolide treated cerebral ischemia. Mediators of inflammation 2015, 120198. Yu, M., Zhang, Y., Chen, X., Zhang, T., 2016. Antidepressant-like effects and possible mechanisms of amantadine on cognitive and synaptic deficits in a rat model of chronic stress. Stress 19, 104-113. Zhang, H., Wang, H., Xiao, X., Zhang, T., 2016. Melamine Alters Glutamatergic Synaptic Transmission of CA3-CA1 Synapses Presynaptically Through Autophagy Activation in the Rat Hippocampus. Neurotoxicity research 29, 135-142. Zheng, Y., Hou, J., Liu, J., Yao, M., Li, L., Zhang, B., Zhu, H., Wang, Z., 2014. Inhibition of 16
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in rats. Journal of pharmacological sciences 124, 354-364.
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Figure legends
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Fig. 1. LEO attenuated brain cell death and the excitotoxic insults induced by OGD. (A) Representative pictures of PI staining in CON, OGD, OGD+25 LEO, OGD+50 LEO and OGD+100 LEO
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groups. (B) Quantification of PI staining. (C) and (F) Representative pictures of sEPSCs of LEO. (D) and (G) Alterations in cumulative probability of sEPSCs amplitude. (E) and (H) Alterations in cumulative probability of sEPSCs frequency. (I) Scheme of patch-clamp recording. (J) Changes in slope of sEPSCs amplitude. (K) Changes in slope of sEPSCs frequency. Data are expressed as mean ± S.E.M. #p < 0.05,
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p < 0.01 compared with CON group. *p < 0.05, *p < 0.01 compared with OGD
group. n =8~10 for per group. 18
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Fig. 2. Measurement of spatial learning and reversal learning. (A) Escape latency calculated for each
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day in Sham, 2-VO, 2-VO+LEO and 2-VO+DON groups on each training day in IT and RT stages. (B)
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Swimming speed on each training day in IT and RT stages. (C) Platform area crossings in IPT stage. (D) Percentage of time in target quadrant in IPT stage. (E) Platform area crossings in RPT stage. (F) Percentage of time in target quadrant in RPT stage. Data are expressed as mean ± S.E.M. # p < 0.05, ## p < 0.01 comparison between Sham group v.s. 2-VO group. *p < 0.05 comparison between 2-VO+LEO group v.s. 2-VO group. $ p < 0.05, $$ p < 0.01 comparison between 2-VO group v.s. 2-VO+DON group. n =8 for per group.
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Fig. 3. Comparison of temporal distribution in each quadrant of RT stage. (A) Time spent in zone 1. (B) Time spent in zone 2. (C) Time spent in zone 3. (D) Time spent in zone 4. Data are expressed as mean ± S.E.M. (E) Representative swim traces of rats for each day in Sham, 2-VO, 2-VO+LEO and 2-VO+DON groups.
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p < 0.01 compared with Sham group. * p < 0.05 compared with 2-VO+LEO
group. $ p< 0.05 compared with 2-VO+DON group. n =8 for per group.
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Fig. 4. Protective effect of LEO on LTD from CA3 to CA1 region of hippocampus. (A) Alterations in
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fEPSPs slopes after LFS in Sham, 2-VO, 2-VO+LEO and 2-VO+DON groups. (B) Changes in fEPSPs slope in Sham, 2-VO, 2-VO+LEO and 2-VO+DON groups. Data are expressed as mean ± S.E.M. ## p <
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0.01 compared with Sham group. * p < 0.05 compared with 2-VO group. n = 8 for per group.
Fig. 5. The contents of glutamate and H2O2 in hippocampus of Sham, 2-VO and 2-VO+LEO. (A) The content of glutamate. (B) The content of H2O2. Data are expressed as mean ± S.E.M. ## p < 0.01 compared with Sham group. *p < 0.05 compared with 2-VO group. n =8 for per group.
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Fig. 6. The synapse-associated proteins were detected by Western blot assay. (A) Quantitative analysis of protein expression of NR2A. (B) Quantitative analysis of protein expression of NR2B. (C) The ratio of NR2A/NR2B. (D) Quantitative analysis of protein expression of PSD-95. (E) The ratio of LC3II/I. (F) Quantitative analysis of protein expression of beclin-1. Data are expressed as mean ± S.E.M. 0.01 compared with Sham group. *p < 0.05 compared with 2-VO group. n = 4~6 for per group.
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Leonurine ameliorates cognitive dysfunction via antagonizing excitotoxic glutamate insults and inhibiting autophagy Chunhua Liu , Hongqiang Yin , Jing Gao , Xiaxia Xu , Tao Zhang , Zhuo Yang PII: DOI: Reference:
S0944-7113(16)30180-5 10.1016/j.phymed.2016.10.005 PHYMED 52088
To appear in:
Phytomedicine
Received date: Revised date: Accepted date:
13 May 2016 28 September 2016 2 October 2016
Please cite this article as: Chunhua Liu , Hongqiang Yin , Jing Gao , Xiaxia Xu , Tao Zhang , Zhuo Yang , Leonurine ameliorates cognitive dysfunction via antagonizing excitotoxic glutamate insults and inhibiting autophagy, Phytomedicine (2016), doi: 10.1016/j.phymed.2016.10.005
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Graphical abstract
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Leonurine ameliorates cognitive dysfunction via antagonizing excitotoxic glutamate insults and inhibiting autophagy
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Chunhua Liua, Hongqiang Yina, Jing Gaoa, Xiaxia Xub, Tao Zhangb, Zhuo Yanga,*
School of Medicine, State Key Laboratory of Medicinal Chemical Biology,
Key Laboratory of Bioactive Materials Ministry of Education, Nankai University, Tianjin 300071, China; College of Life Sciences, Nankai University, Tianjin 300071, China
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*Corresponding author.
23504364; fax: +86 22 23502554
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School of Medicine, Nankai University, 94 Weijin Road, Tianjin 300071, China. Tel.: +86 22
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E-mail address: [email protected] (Z. Yang)
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ABSTRACT Background: Chronic cerebral hypoperfusion is related with cognitive deficits in different types of dementia. Purpose: In this study, we aimed to investigate the effect and potential mechanisms of leonurine on
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chronic cerebral hypoperfusion both in vitro and in vivo. Study Design: Chronic cerebral hypoperfusion was duplicated by oxygen-glucose deprivation (OGD) in vitro and by ligation of bilateral common carotid arteries (2-VO) in vivo.
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Methods: In in vitro study, there were control group, OGD group, OGD + 100 μM leonurin group, and OGD+10 μM donepezil group. The spontaneous excitatory postsynaptic current amplitude and frequency were recorded. In in vivo study, the chronic cerebral hypoperfusion model was induced by
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ligated bilateral common carotid arteries. Rats were randomly divided into Sham group, 2-VO group, 2-VO + 60 mg/kg/day leonurine group, and 2-VO + 4 mg/kg/day donepezil group. After three weeks,
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the Morris water maze and Long-term depression recording were observed. Then N-methyl-D-aspartate
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receptor-associated proteins and autophagy-associated proteins were detected by Western blot assay. Results: In in vitro experiment, results showed that leonurine could obviously attenuate the
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spontaneous excitatory postsynaptic current amplitude and frequency on pyramidal neurons. In in vivo
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experiment, leonurine significantly decreased levels of glutamate and hydrogen peroxide, improved both the cognitive flexibility and the spatial learning and memory abilities. Moreover, leonurine obviously enhanced long-term depression, elevated the ratio of N-methyl-D-aspartate receptor 2A/2B, and decreased the expression of postsynaptic density protein-95. Interestingly, the ratio of LC3II/LC3I and beclin-1 expression were markedly down-regulated by leonurine. Conclusion: These findings suggest that leonurine ameliorates cognitive dysfunction at least partly via
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antagonizing excitotoxic glutamate insults and inhibiting autophagy. Furthermore, it might become a potential drug candidate of chronic cerebral hyperfusion in future.
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Keywords: Chronic cerebral hypofusion; Long-term depression; Leonurine; Hippocampus; Autophagy
Abbreviations
Chronic cerebral hypoperfusion CCH; oxygen–glucose deprivation OGD; artificial cerebral spinal fluid
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aCSF; bilateral common carotid arteries 2-VO; N-methyl-D-aspartate receptors NMDARs; long-term depression LTD; Morris water maze MWM; initial training IT; initial probe trials IPT; reversal training
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RT; reversal probe trials RPT.
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1. Introduction Chronic cerebral hypoperfusion (CCH) has been identified to be related with the cognitive deficits in different types of dementia, such as Alzheimer‟s disease, vascular dementia and frontotemporal dementia (Osawa et al., 2004; Shu et al., 2013). CCH leads to the inadequate blood supply in different
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regions of brain, which is accompanied with the deficiency of oxygen and nutrients. As known, the hippocampus formation is the most sensitive brain area to the cerebral ischemia and plays a key role in spatial learning and memory (Li et al., 2011). Accumulating evidences demonstrate that there is a
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causal relationship between CCH and dementia (Osawa et al., 2004).
Brain ischemia and hypoxia could lead to the depolarization of neurons, and then a large sum of glutamate will release into the synaptic cleft to excessively agitate the N-methyl-D-aspartate receptors (NMDARs or NRs), which will cause lots of Ca2+ flux into neurons and result in cell death by this
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excitotoxic glutamate insults. Autophagy is necessary for cell degrading cellular “garbage” and for
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maintaining cell homeostasis by lysosome-mediated catabolic machinery. It plays an important role not
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only at physiological status, such as cell survival, development and differentiation, but also at pathological condition, such as Alzheimer‟s disease, Parkinson‟s disease and brain ischemia disease
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(Caberlotto and Nguyen, 2014). Interestingly, studies demonstrated that brain hypoxia and ischemia
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obviously enhanced the neuronal autophagy, and the inhibition of the autophagy was of benefit to hippocampus (Koike et al., 2008). On the contrary, there were also some other results demonstrated that autophagy played neuroprotective effects on brain ischemia (Balduini et al., 2001; Carloni et al., 2008). Therefore, the role of autophagy in brain ischemia and hypoxia still needs to be further investigated. Then, how about the role of autophagy in CCH and what the underlying mechanism is still not completely understand.
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Leonurus cardiaca is an herbaceous perennial plant in the mint family and has a long history in traditional medicine to treat a variety of diseases in China, Japan, Korea and European countries. Leonurine (LEO), an active alkaloid extracts from Leonurus cardiaca, has recently been demonstrated to be effective on the treatment of cardiovascular disease and nervous system diseases (Liu et al., 2013;
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Qi et al., 2010; Wojtyniak et al., 2013). Studies have demonstrated that LEO has neuroprotective effects on middle cerebral artery occlusion rats via anti-oxidantion and anti-apoptosis (Qi et al., 2010). However, the effect and underlying mechanism of LEO on CCH are still poorly understood. In this
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study, the CCH model was duplicated by oxygen-glucose deprivation (OGD) in vitro and by ligation of bilateral common carotid arteries (2-VO) in vivo. We aimed to investigate whether or not LEO could alleviate the cognitive impairment of CCH.
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2. Materials and methods
The brain slices were removed from 3-4 week Wistar rats and there were control group (CON)
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incubated in oxygenated artificial cerebrospinal fluid (aCSF); OGD group incubated with aCSF
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containing 95% N2 and 5% CO2 (OGD); OGD group treated with aCSF containing 95% N2 and 5% CO2 and different concentrations of LEO (OGD+25μM LEO, OGD+50μM LEO and OGD+100μM
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LEO groups). The cell viability of brain slices was determined by PI staining, and the patch-clamp
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recording was performed in CON, OGD and OGD+100μM LEO, and OGD+10μM donepezil
hydrochloride (OGD+DON) groups. Adult male Wistar rats (250-280g) were randomly divided into four groups, i.e. sham operation group with vehicle treatment (Sham); 2-VO group with vehicle treatment (2-VO); 2-VO group treated with 60 mg/kg/day of LEO (2-VO+LEO); and 2-VO group treated with 4 mg/kg/day of DON (2-VO+DON). The CCH model was induced by 2-VO surgery. After three weeks, spatial learning and memory performances of rats were evaluated by Morris water
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maze (MWM). Then the long-term depression (LTD) recording was performed immediately after MWM test. In order to investigate the neuroprotective mechanism of LEO, the levels of glutamate and H2O2 of hippocampus were measured. Moreover, levels of NR2A, NR2B, PSD-95 (1:2000, abcam, UK), LC3I/II (1:1000, Medical & biological laboratories Co. LTD., JPN), beclin-1 (1:2000, Cell
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Signaling Technology, USA) and -actin (1:4000, abcam, UK) were tested by Western blot assay. All data were analyzed by SPSS 16.0 software. Escape latencies and swimming speeds in MWM experiment were analyzed by two-way repeated ANOVA followed by the Bonferroni multiple group
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comparison. Other data were analyzed by one-way ANOVA followed by a post Turkey test. Data were presented as means ± S.E.M and defined differences at p < 0.05 as statistically significant. All pictures were processed with Photoshop software (sources and detailed methods in Supporting Text).
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3. Results
3.1 LEO attenuated the excitotoxic insults induced by OGD
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As shown in Fig. 1A and 1B, OGD significantly increased brain cell death. However, LEO
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alleviated the OGD-induced damage in a dose-dependent manner (p < 0.05). In the OGD+100 M LEO group, LEO could significantly decrease the brain cell death to 130.36 ± 9.06% (fold of CON
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group), so we selected the 100 LEO for next experiments. OGD induced the increase of the
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amplitude and expressed a markedly right shift in the cumulative probability curve of spontaneous excitatory postsynaptic current (sEPSC) amplitude (p < 0.01, Fig. 1C, 1D, 1F, 1G, 1I and 1J). However, LEO and DON obviously attenuated the amplitude of pyramidal neurons (p < 0.05), and presented a markedly left shift in the cumulative probability curve of sEPSC amplitude (Fig. 1C, 1D, 1F, 1G, 1I and 1J). As to the sEPSCs frequency, OGD produced obviously higher time-dependent frequency of pyramidal neurons, which was also ameliorated by LEO and DON (p < 0.01, Fig. 1C, 1E, 1F, 1H, 1I
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and 1K). 3.2 LEO alleviated the impaired spatial learning and memory in 2-VO model rats Rats were subjected to MWM test to investigate the spatial learning and memory ability. The average escape latency was obviously decreased in all four groups during initial training (IT) without
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affecting swimming speeds (Fig. 2A and 2B). However, the escape latency was much longer in 2-VO group from day 2 to day 4 (p < 0.05, Fig. 2A). Interestingly, rats in 2-VO+LEO group and 2-VO+DON group located the platform much more faster on day 3 and day 4 (p < 0.05, Fig. 2A). In initial probe
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trials (IPT), the platform crossings (1.1 ± 0.31) and the quadrant dwell time (29.22 ± 2.44%) were obviously decreased in 2-VO group compared with that of Sham group (2.5 ± 0.54; 50.44 ± 3.06%, p < 0.01, Fig. 2C and 2D). Although there was no significant difference in platform crossings between
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2-VO group and 2-VO+LEO group or 2-VO+DON group, LEO and DON remarkably improved the quadrant dwell time compared with that of 2-VO group (p < 0.05, Fig. 2C and 2D).
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The average escape latency was markedly longer in 2-VO group than that of Sham group on both
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day 6 and day 7 during reversal training (RT) (p < 0.05, Fig. 2A). Rats of 2-VO+LEO group and 2-VO+DON group spent less time to find the platform than that of 2-VO group without affecting
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swimming speeds (p < 0.05, Fig. 2A and 2B). There was no significant difference in platform crossings
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between 2-VO group and 2-VO+LEO group or 2-VO+DON group (p >0.05, Fig. 2E). However, the quadrant dwell time in reversal probe trials (RPT) were obviously increased in 2-VO+LEO group and 2-VO+DON group compared with those in 2-VO group (p < 0.05, Fig. 2F). In addition, we analyzed the temporal distribution of the quadrants. Rats of 2-VO group spent much more time in zone 1 (p < 0.01, Fig. 3A) on day 7. In contrast, they spent less time in zone 4 (p < 0.05, Fig. 3D). There was no significant difference between 2-VO group and 2-VO+LEO group or
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2-VO+DON group in the time spent in both zone 2 and zone 3 (p >0.05, Fig. 3B and 3C). However, rats administrated with LEO or DON spent less time in zone 1 and more time in zone 4 on day 7 (p < 0.05, Fig. 3A and 3D). Moreover, the swim traces of all rats in RT stage were showed in Fig. 3E. In Sham group, 2-VO+LEO group and 2-VO+DON group, the trajectories of reversal learning stage were
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shorten, whereas the trajectory was significantly longer in 2-VO group (Fig. 3E). Also, rats of 2-VO group showed much more swimming trajectories in zone 1. In contrast, rats treated with LEO or DON showed much more swimming trajectories in zone 4 rather than other zones (Fig. 3E).
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3.3 LEO ameliorated the impaired LTD in hippocampus of 2-VO model rats
As shown in Fig. 4, field excitatory postsynaptic potentials (fEPSPs) slope was obviously decreased after the LFS and maintained lower than the baseline in Sham group. In contrast, the fEPSPs
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slope was higher in 2-VO group, which indicated that LTD was suppressed in 2-VO group (p < 0.01, Fig. 4). Interestingly, LEO and DON could significantly reduce the fEPSPs slope and maintained lower,
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which suggested that LEO and DON alleviated the impaired synaptic plasticity induced by 2-VO (p <
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0.01, Fig. 4).
3.4 LEO decreased the concentrations of glutamate and hydrogen peroxide (H2O2) in
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hippocampus of 2-VO model rats
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Data showed that both levels of glutamate and H2O2 were significantly increased in hippocampus
of 2-VO group compared with those of Sham group (p < 0.01, Fig. 5). After administration with LEO and DON, the concentration of glutamate and H2O2 were obviously decreased (p < 0.05, Fig. 5). 3.5 LEO improved cognitive function by modulating the NMDARs-associated proteins As shown in Fig. 6A-C, there was no change in NR2B expression in hippocampus of 2-VO group (p > 0.05), but NR2A expression was markedly decreased to 0.68-fold compared with that of Sham
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group, then the ratio of NR2A/2B was decreased to 0.62 (p < 0.01, Fig. 6C). Interestingly, LEO and DON were able to increase the expression of NR2A so as to improve the ratio of NR2A/2B (p < 0.01, Fig. 6A-C). As to the expression of PSD-95, there was a significantly difference between Sham group and 2-VO group (p < 0.01, Fig. 6D). After administration with LEO and DON, the PSD-95 expression
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were obviously down-regulated (p < 0.05, Fig. 6D). 3.6 LEO protected rats from 2-VO induced damage by inhibiting autophagy
Data showed that the CCH markedly up-regulated the beclin-1 expression (p < 0.01, Fig. 6E),
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which suggested that autophagy was activated. LEO and DON were able to attenuate the level of beclin-1 (p < 0.05, Fig. 6E). As shown in Fig. 6F, the CCH increased the ratio of LC3II/I compared with that of Sham group (p < 0.01). Interestingly, LEO and DON significantly reduced the ratio of
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LC3II/I (p < 0.05), which suggested that LEO could inhibit the excessive activation of autophagy. 4. Discussion
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Cerebral hypoxia could lead to the depolarization of neurons, and then the induced massive glutamate
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will release into the synaptic cleft. When inhibiting the sEPSC events, the excitotoxicity was alleviated and the neuronal death was reduced (Yang et al., 2014). Recently, studies reported that LEO has
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neuroprotective effects on middle cerebral artery occlusion rats via anti-oxidation and anti-apoptosis
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(Qi et al., 2010). Moreover, it can also attenuate cognitive impairments of AD via JNK and NF-кB pathways (Hong et al., 2015). As known, the synaptic plasticity is closely related with cognitive function, so we speculated that whether or not the neuroprotective effect of LEO on CCH was correlated with the protection of synaptic plasticity. Furthermore, the pathology of CCH was related with cerebral ischemia. Therefore, we employed the OGD to partly duplicate the CCH for in vitro experiment according to previous studies (Le et al., 2015; Yang et al., 2014).
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From in vitro experiments, data showed that hippocampal slices treated by OGD displayed excessive events of sEPSCs, which suggested that OGD led to the depolarization of CA1 pyramidal neurons and induced neuronal excitotoxicity. When administrated with LEO, it could obviously alleviate the depolarization of neurons by reducing the sEPSCs amplitude and frequency. These results
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suggested that LEO protected synaptic plasticity from OGD-induced damage. Considering the in vitro experimental results, we next preliminarily observed the potential neuroprotection of LEO on CCH in in vivo experiment. We duplicated the rat model of CCH by 2-VO
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(Li et al., 2011). Our data showed that there was obvious learning and memory deficiency in 2-VO group. However, LEO could effectively alleviate cognitive deficiency to some extent in IT stage, it suggested that the acquisition and retention capacities were improved in 2-VO+LEO group. Then, rats
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were subjected to spatial reversal learning. Our data suggested that the information storage was partly damaged by 2-VO. However, rats of 2-VO+LEO group could successfully find the platform much
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faster than that of 2-VO group. Moreover, the time spent in the novel quadrant was longer and the time
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spent in the original quadrant was shorter in 2-VO+LEO group compared with those of 2-VO group. It implied that rats of 2-VO+LEO group established a new strategy to explore the platform in the novel
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environment. Our results indicated that LEO not only ameliorated the spatial learning and memory, but
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also alleviated the deficits in cognitive flexibility in 2-VO rats. As known, memory is achieved by experience-dependent changes in synaptic strength. These
changes can take the form of LTP or LTD of synaptic transmission. Studies have reported that LTD underlies storage of memory and the acquisition of novel information, especially in the acquisition of object-place configuration (Kemp and Manahan-Vaughan, 2004; Manahan-Vaughan and Braunewell, 1999). When rats were explored to a novel task, LTD was helpful to weaken previous memory traces,
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which led to the decreased synaptic efficacy. Then, LTD contributed to increase the „signal to noise ratio‟ and prevent previous memory from interfering with newly encoded information (Nicholls et al., 2008). In this study, our results showed that CCH led to the increase of fEPSPs slope of 2-VO group, which demonstrated that synaptic plasticity was impaired. However, the LTD was ameliorated via
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administrating LEO. So our data suggested that LEO was able to protect rats from the impairment of synaptic plasticity induced by 2-VO.
LEO prevented the neuron death from the injury induced by 2-VO. The neuroprotection of LEO
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was associated with the lower levels of glutamate and H2O2 in hippocampus, so as to decrease the damage from excitatory toxicity. It suggested that LEO could partly modulate the damage of glutamate system. However, the neuroprotective mechanism of LEO still needs to be further investigated.
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Recently studies reported that NMDARs related proteins and autophagic related proteins were involved in modulating synaptic plasticity (Fu et al., 2016; Yu et al., 2016; Zhang et al., 2016). So we
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examined these proteins mentioned above, in order to preliminarily explore the neuroprotective
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mechanism of LEO. As known, NMDARs are involved in hippocampal synaptic plasticity and in mediating learning and memory processes. Preferential inhibition of NR2A prevented the induction of
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LTP without affecting LTD production (Ge et al., 2010; Liu et al., 2004). By contrast, other studies
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found that the inhibition of NR2A impaired both LTD and LTP (Bartlett et al., 2007; Liu et al., 2004). Thus there is so far some controversy about NMDAR subunits dependence of LTD, which makes it hard to draw an unifying conclusion. In our study, the NR2A expression was significantly decreased, as well as the LTD was severely impaired in 2-VO group. In view of our results, we considered that NR2A played a key role in LTD. In addition, the activation of NR2B resulted in neuronal death (Hardingham et al., 2002; Liu et al., 2007). However, the inhibited NR2B ameliorated the impairment
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of ischemia/reperfusion (Meng and Zhang, 2002). Besides, inhibiting the PSD-95 expression selectively
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receptor-mediated neuronal damage (Hardingham et al., 2002). In this experiment, the NR2A expression was significantly decreased accompanied with the increase of PSD-95 in 2-VO group, it implied that serious excitotoxicity was induced and the capability of neuronal survival was markedly
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weakened in 2-VO group. Fortunately, LEO was able to dramatically up-regulated NR2A expression and down-regulated PSD-95 expression, which was beneficial to protect rats from the damage induced by 2-VO.
A large sum of glutamate will rush into the synaptic cleft to excessively agitate NMDARs in
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cerebral ischemia, which will cause lots of Ca2+ flux into neurons and result in cell death. Moreover,
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Ca2+ fluxing into neurons will also induce excessive H2O2, which could improve the level of autophagy
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(Ashabi et al., 2013). The inhibition of autophagy could help neuron survive from ischemia (Zheng et al., 2014). On the contrary, some studies reported that the improvement of autophagy could antagonize
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neuronal apoptosis after cerebral ischemia (Su et al., 2014; Yang et al., 2015). Therefore, the effect of
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autophagy on the cerebral ischemia is still controversial. In our study, rats presented higher levels of glutamate and H2O2 in hippocampus of 2-VO group. In addition, the ratio of NR2A/NR2B was significantly reduced and PSD-95 expression was obviously increased by 2-VO. We inferred that the higher level of glutamate triggered the excitotoxicity, and then led to an excess of H2O2, which could improve the level of autophagy (Ashabi et al., 2013). So we observed the expression of autophagic related proteins. Data showed that the ratio of LC3II/I and the expression of beclin-1 were significantly
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improved in 2-VO group, which could be partly reversed after treatment with LEO. These results demonstrated that LEO prevented the cognitive impairment from 2-VO partly via inhibiting autophagy. 5. Conclusion In conclusion, we reported that LEO obviously attenuated the amplitude and frequency of
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pyramidal neurons in vitro. Moreover, LEO not only improved the spatial learning and memory, but also alleviated the impairment of LTD. It increased the NR2A protein expression in hippocampus in order to improve the ratio of NR2A/2B, and attenuated the over-expression of PSD-95, which was
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beneficial for the synaptic plasticity and neuronal survival. Interestingly, LEO reduced the expression of LC3II and beclin-1 in order to inhibit the activation of autophagy. Thus, we conclude that LEO protects synaptic plasticity from cognitive impairment partly via antagonizing excitotoxic glutamate
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insults and inhibiting autophagy. Our results will shed new light on the neuroprotective mechanism of
Acknowledgments
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LEO, which might be used as a potential drug candidate of CCH in the future.
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This work was partly supported by grants from the National Natural Science Foundation of China (31400986, 11232005), the Applied Basic Research Programs of Science and Technology Commission
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Foundation of Tianjin (14JCQNJC11800, 14JCZDJC35000). We thank Prof. Yizhun Zhu (School of
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Pharmacy, Fudan University, Shanghai, China) for kindly providing LEO. Conflict of interest There is no conflict of interest.
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autophagy contributes to melatonin-mediated neuroprotection against transient focal cerebral ischemia
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Figure legends
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Fig. 1. LEO attenuated brain cell death and the excitotoxic insults induced by OGD. (A) Representative pictures of PI staining in CON, OGD, OGD+25 LEO, OGD+50 LEO and OGD+100 LEO
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groups. (B) Quantification of PI staining. (C) and (F) Representative pictures of sEPSCs of LEO. (D) and (G) Alterations in cumulative probability of sEPSCs amplitude. (E) and (H) Alterations in cumulative probability of sEPSCs frequency. (I) Scheme of patch-clamp recording. (J) Changes in slope of sEPSCs amplitude. (K) Changes in slope of sEPSCs frequency. Data are expressed as mean ± S.E.M. #p < 0.05,
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p < 0.01 compared with CON group. *p < 0.05, *p < 0.01 compared with OGD
group. n =8~10 for per group. 18
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Fig. 2. Measurement of spatial learning and reversal learning. (A) Escape latency calculated for each
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day in Sham, 2-VO, 2-VO+LEO and 2-VO+DON groups on each training day in IT and RT stages. (B)
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Swimming speed on each training day in IT and RT stages. (C) Platform area crossings in IPT stage. (D) Percentage of time in target quadrant in IPT stage. (E) Platform area crossings in RPT stage. (F) Percentage of time in target quadrant in RPT stage. Data are expressed as mean ± S.E.M. # p < 0.05, ## p < 0.01 comparison between Sham group v.s. 2-VO group. *p < 0.05 comparison between 2-VO+LEO group v.s. 2-VO group. $ p < 0.05, $$ p < 0.01 comparison between 2-VO group v.s. 2-VO+DON group. n =8 for per group.
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Fig. 3. Comparison of temporal distribution in each quadrant of RT stage. (A) Time spent in zone 1. (B) Time spent in zone 2. (C) Time spent in zone 3. (D) Time spent in zone 4. Data are expressed as mean ± S.E.M. (E) Representative swim traces of rats for each day in Sham, 2-VO, 2-VO+LEO and 2-VO+DON groups.
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p < 0.01 compared with Sham group. * p < 0.05 compared with 2-VO+LEO
group. $ p< 0.05 compared with 2-VO+DON group. n =8 for per group.
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Fig. 4. Protective effect of LEO on LTD from CA3 to CA1 region of hippocampus. (A) Alterations in
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fEPSPs slopes after LFS in Sham, 2-VO, 2-VO+LEO and 2-VO+DON groups. (B) Changes in fEPSPs slope in Sham, 2-VO, 2-VO+LEO and 2-VO+DON groups. Data are expressed as mean ± S.E.M. ## p <
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0.01 compared with Sham group. * p < 0.05 compared with 2-VO group. n = 8 for per group.
Fig. 5. The contents of glutamate and H2O2 in hippocampus of Sham, 2-VO and 2-VO+LEO. (A) The content of glutamate. (B) The content of H2O2. Data are expressed as mean ± S.E.M. ## p < 0.01 compared with Sham group. *p < 0.05 compared with 2-VO group. n =8 for per group.
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Fig. 6. The synapse-associated proteins were detected by Western blot assay. (A) Quantitative analysis of protein expression of NR2A. (B) Quantitative analysis of protein expression of NR2B. (C) The ratio of NR2A/NR2B. (D) Quantitative analysis of protein expression of PSD-95. (E) The ratio of LC3II/I. (F) Quantitative analysis of protein expression of beclin-1. Data are expressed as mean ± S.E.M. 0.01 compared with Sham group. *p < 0.05 compared with 2-VO group. n = 4~6 for per group.
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p<