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memory impairment by regulating the levels of Arc
Pharmacology, Biochemistry and Behavior 117 (2014) 47–51
Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Berberine rescues D-galactose-induced synaptic/memory impairment by regulating the levels of Arc Zhan Pei-Yan ⁎,1, Peng Cai-Xia 1, Zhang Lin-Hong Department of Neurology, Central Hospital of Wuhan, Wuhan 430014, China
a r t i c l e
i n f o
Article history: Received 10 July 2013 Received in revised form 29 November 2013 Accepted 5 December 2013 Available online 14 December 2013 Keywords: Berberine Synapse Arc protein
a b s t r a c t Synaptic communication forms the basis of learning and memory. Disruptions of synaptic function and memory have been widely reported in many neurological diseases, such as dementia. Thus, restoration of impaired synaptic communication is a potential therapeutic approach for these diseases. In this study, we demonstrated that supplementation with berberine, a plant alkaloid with a long history of medicinal usage in Chinese medicine, effectively reverses the synaptic deficits induced by D -galactose. We also found that berberine rescued D-galactose-induced memory impairment and additionally rescued the mRNA and protein levels of Arc/Arg3.1, an important immediate early gene that is crucial for maintaining normal synaptic plasticity. Our study provides the first piece of evidence supporting the potential use of berberine in the treatment of neural diseases with synaptic/memory impairments. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Synaptic plasticity is the experience-dependent alteration in connectivity between neurons and is believed to be the neurobiological basis of learning and memory (Ho et al., 2011). Disruptions of synaptic plasticity have been found in multiple neurological disorders, including neurodevelopmental diseases and neurodegenerative diseases such as Down syndrome and Alzheimer's disease (Garner and Wetmore, 2012; Selkoe, 2002). A better understanding of the molecular mechanisms of synaptic plasticity would be helpful in developing appropriate treatments for these diseases. The maintenance of normal synaptic plasticity requires numerous genes and proteins, including immediate early genes (IEGs) (Okuno, 2011). Arc (activity-regulated cytoskeletonassociated protein), also known as Arg3.1, is a highly conserved IEG that is expressed only in vertebrates (Bramham et al., 2008). It has been reported that Arc is essential for the formation and consolidation of long-term memories (Korb and Finkbeiner, 2011). The mRNA for Arc is specifically located in the active synaptic loci for the locally synthesized Arc protein (Bramham, 2008). The tight association between Arc expression and the strength of excitatory synapses strongly suggests a critical role for Arc in synaptic plasticity (Korb and Finkbeiner, 2011). D-Galactose is a reducing sugar that can form advanced glycation end products (AGEs); consequently, the administration of D-galactose can induce cognitive deficits and disruptions in synaptic communication. ⁎ Corresponding author. Tel.: +86 2782211413; fax: +86 2782211409. E-mail addresses: [email protected] (P.-Y. Zhan), [email protected] (L.-H. Zhang). 1 The first two authors contributed equally to this paper. 0091-3057/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pbb.2013.12.006
Thus, D-galactose-treated rats have been widely studied as an animal model of synaptic disruption and memory impairment. Many drugs, including some traditional Chinese medicines, have been tested in efforts to reverse D-galactose-induced memory deficits. Berberine is a quaternary ammonium salt derived from the plants of the genus Berberis. Berberine (2000) reportedly helps the body fight infections, lowers elevated blood glucose (Yin et al., 2008), lowers elevated blood total cholesterol (Zhou et al., 2008), inhibits hepatic stellate cell proliferation (X. Sun et al., 2009), and has antineoplastic activity (Y. Sun et al., 2009; Tang et al., 2009). In addition, it has been suggested to exert potentially beneficial effects on central nervous system disorders via effects such as neuroprotection, anti-neuronal apoptosis, and improvement of cerebral microcirculation (Kulkarni and Dhir, 2010). As a traditional medicine, berberine has been shown to be an effective treatment for fungal, bacterial, and viral infections (Zhu et al., 2004). During the past few decades, many studies have suggested that berberine also has various beneficial effects on the nervous system as a neuroprotective agent (Kulkarni and Dhir, 2009, 2010). For example, berberine appears to produce its antiamnesic effects by augmenting the activity of the cholinergic neuronal system (Peng et al., 1997). Recently, derivatives of berberine have been designed to inhibit the activity of acetylcholinesterase (AChE) (Huang et al., 2010), mimicking an important mechanism of action for potential therapeutics for AD. In a transgenic mouse model of AD, a neuroprotective effect of berberine has also been well demonstrated (Durairajan et al., 2012). However, whether berberine can reverse deficits in synaptic function is still unclear. In the current study, we intended to study the possible protective effect of berberine on the D-galactose induced memory and synaptic disorder by using Morris water maze and in vivo LTP recording. We also
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P.-Y. Zhan et al. / Pharmacology, Biochemistry and Behavior 117 (2014) 47–51
2.2. Electrophysiology To detect the long-term potentiation (LTP), 6–8 rats from each group were anesthetized and fixed on a Narishige (Tokyo, Japan) stereotaxic instrument (SR-6 N) (Wang et al., 2012). The stimulating electrode and the recording electrode were placed in the perforant path and dentate gyrus (DG) region of rat hippocampus separately. The basal line recording was obtained by delivering a single pulse of stimulation once every 10 s. Before LTP induction, we recorded a stable baseline for at least 30 min. To induce LTP, high-frequency stimulation (HFS) consisting of four trains of 50 pulses were delivered at 200 Hz with a 2 s intertrain interval. The slope of EPSP was recorded for 90 min and calculated automatedly by a computerized program (RM6240BD; Chengdu, China). After LTP recording, the rats were sacrificed by using overdose of urethane. 2.3. Morris water maze
Fig. 1. Berberine rescues D-galactose-induced impairment in long-term potentiation (LTP). LTP recordings were conducted as described previously. The representative traces (A) of EPSP before (pre-HFS) and after (post-HFS), the normalized population spike (PS) amplitude (B) and normalized field excitatory postsynaptic potentiation (fEPSP) slope (C) were analyzed. The arrow at time 0 indicates the application of high-frequency stimulation to induce LTP. Con: vehicle treated group, D-gal: D-galactose-treated group, D-gal + Ber: D-galactose- and berberine-treated group. N = 6–8 rats for each group. The black bar indicates the time window for the statistical analysis.
explore the possible underlying mechanisms for the neuroprotection of berberine. We found that D-galactose administration induced deficits in long-term potentiation along with a reduction in the levels of Arc and supplementation with berberine could reverse these effects in D-galactose-treated rats.
2. Materials and methods 2.1. Animals and treatment Male Wistar rats (10–12 weeks, 180–220 g) were purchased from Center for Disease Control and Prevention of Hubei province of China. The rats were housed in an air-conditioned room (22 ± 2 °C, 12 h light and 12 h dark; lights on at 06:00 am) with food and water ad libitum. All animal experiments were performed according to the ‘National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).’ D-galactose (100 mg/kg) was obtained from Shanghai Bio Life Science & Technology Co. (Shanghai, China) and was administered intraperitoneally once a day for 7 weeks (Cui et al., 2006). Berberine was purchased from Beijing Ocean Pharmacy Co. Ltd. (Batch No. 090601) and was delivered by gavage once daily to the rats with the dose of 100 mg/kg per day for 7 weeks too (Bhutada et al., 2011). All the experimental protocols were approved the review committee of Wuhan Central Hospital.
The Morris water maze was performed according to a previous published paper (Yao et al., 2012). Briefly, another 8–10 rats per group were training for six continuous days to find a hidden platform in the water by using a stationary array of cues inside or outside the pool (the diameter is 180 cm). For each trial, the rat was placed at the middle of any no-target quadrants (northeast, southeast, northwest) and facing the wall of the pool. If the rat did not reach the platform within 60 s, the trial was terminated, and the rat was guided to the platform manually and placed on the platform for another 30 s. An upper camera tracking device was used to record the swimming pathway and escape latency. The learning curve during the first 6 days was used to analyze the learning ability. At the 7th day, the platform was removed and the percent of time in target quadrant and the numbers of platform quadrant crosses were recorded to analyze the memory retention. Then the rats were sacrificed after Morris water maze for the biochemical experiments. 2.4. Western blot Hippocampi were homogenized in the buffer that contains Tris × Cl (pH 7.6) 10 mmol/L, NaF 50 mmol/L, Na3VO4 1 mmol/L, edetic acid 1 mmol/L, benzamidine 1 mmol/L, and Phenylmethanesulfonyl fluoride (PMSF) 1 mmol/L mixture (2 mg/L each of aprotinin, leupeptin and pepstain A). The hippocampi were lysate by the buffer containing Tris × Cl (pH 7.6) 200 mmol/L, 8% sodium dodecyl sulfate (SDS), 40% glycerol, and boiled at water bath for 10 min. After centrifuged at 12 000 × g for 5 min, the samples are subjected for protein concentration measurement and dithiothreitol (DTT) was added to attain a final concentration 100 mmol/L. The proteins were then separated by SDSpolyacrylamide gel electrophoresis (10% gel) and transferred to nitrocellulose membrane. Immunoreactive materials were detected using Chemiluminescent Substrate kit (Pierce, Rockford, IL, USA) and exposed to CL-XPosure film (Wang et al., 2007). The blots were scanned and the protein bands were quantitatively analyzed by Image J (NIH, USA). 2.5. RT-PCR Total RNA was isolated using Trizol reagents according to the instructions (Tiangen Technologies, Beijing, China). Then total RNA (3 μg in 25 μl) was reversely transcribed by commercial kit (Takara, Dalian, China) and the produced cDNA (1 μl) was used to detect the transcripts. For Arc/Arg3.1, the following primers were used: 5′-GGGA GGTCTTCTACCGTCTG-3′ (forward primer), 5′-CTTCACCGAGCCCTGT TT-3′ (reverse primer); for β-actin, 5′-GTAAAGACCTCTATGCCAACA-3′ (forward primer), 5′-GGACTCATCGTACTCCTGCT-3′ (reverse primer). PCR amplification consists of 32 cycles, each cycle was run with the following program: denaturing at 94 °C, 60 s, annealing at 48 °C (at 50 °C for Arc/Arg3.1), 50 s; and chain extension at 72 °C, 1 min. The PCR
P.-Y. Zhan et al. / Pharmacology, Biochemistry and Behavior 117 (2014) 47–51
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Fig. 2. Berberine rescues D-galactose-induced memory impairment. The Morris water maze was used to examine memory. The learning curve (A) for the first 6 days and the representative traces in the probe test (B). The time spent on the target quadrant (C) and the crossing times (D) are also analyzed. **P b 0.01, compared with Con, ##P b 0.01, compared with the D-gal group. N = 8–10 rats for each group.
products were separated on 1.0% agarose gels and stained with GoldView and visualized under UV light. 2.6. Statistical analysis All data were expressed as means ± S.E, and analyzed by SPSS 14.0 statistical software (SPSS, Chicago, IL, USA) by one-way ANOVA followed by Student t (least significant difference) post hoc test.
longer latency to reach the hidden platform from the third day, while the rats co-administrated with D-galactose and berberine doesn't shown any difference with vehicle rats (Fig. 2A). Moreover, in the probe test, we found that D-galactose treatment significantly decreased the time spent in the target quadrant and the crossing time to the platform regions, while berberine supplement effectively restores those abnormalities (Fig. 2B–D). We also measured the swimming speed and the total swimming distance, and found there is no difference between each group.
3. Results 3.3. Berberine reversed the reduction of Arc/Arg3.1 induced by D-galactose 3.1. Berberine restores LTP deficits induced by D-galactose During the whole experimental procedures, we measured the temperature, body weight, respiratory rate and heart rate of all rats, and found that both D-galactose and berberine didn't alter those physiological parameters. We then performed an in vivo LTP recording experiment according to a well-known method. We found that neither berberine nor D-galactose altered basal synaptic transmission. After high-frequency stimulation, the amplitude of the population spike (PS) and the slope of the excitatory postsynaptic potential (EPSP) increased 1.6- to 1.8-fold in vehicle-treated rats, whereas the increments were much lower in D-galactose-treated rats. Interestingly, supplementation with berberine effectively reversed the D-galactose-induced inhibition of these LTP measures (Fig. 1). No difference in excitation was found in rats treated with berberine alone (data not shown). These results suggest that berberine supplementation can reverse the LTP deficits induced by D-galactose.
IEGs such as Arc/Arg3.1, c-fos, Zif268, and Homer1a are crucial for regulating synaptic plasticity. Western blot analyses revealed that the levels of Arc/Arg3.1 are significantly decreased in D-galactose-treated rats compared to vehicle-treated rats, whereas berberine administration clearly rescued the reduction of Arc/Arg3.1 observed in the hippocampus (Fig. 3A-B). No apparent alterations in the protein levels of other IEGs were found (Fig. 3C). These results indicate that among the IEGs that we studied, only Arc/Arg3.1 is important for berberine's beneficial effects on the synaptic disruptions induced by D-galactose. We subsequently examined Arc/Arg3.1 mRNA levels with semiquantitative PCR and found that D-galactose suppressed Arc/Arg3.1 mRNA to 40% of the levels detected in vehicle-treated rats; moreover, supplementation with berberine effectively restored the levels of Arc/ Arg3.1 mRNA (Fig. 4). These data further demonstrate the key role of Arc/Arg3.1 in the beneficial effects of berberine. 4. Discussions
3.2. Berberine recovers the memory impairment caused by D-galactose We then used the Morris water maze to evaluate the learning and memory ability. We found that rats treated with D-galactose exhibit a
The application of berberine in the treatment of many neurological disorders had been widely reported. For example, in ischemia animal models, it protects from hippocampal neuronal damage following
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Fig. 3. Berberine restores protein levels of Arc/Arg3.1 but not levels of other IEGs. Hippocampal homogenates were prepared as described, and western blotting was performed to evaluate the protein levels of Arc/Arg3.1 (A) and (C), c-fos, zif268, and Homer1a (B). Statistical analysis was performed on Arc/Arg3.1 protein levels. **P b 0.01, compared with Con, ##P b 0.01, compared with the D-gal group. N = 6–8 rats for each group.
transient global ischemia by reducing MMP-9 activity (Hong et al., 2012) and exerts its anti-apoptotic effect by enhancing PI3K p55γ promoter activity in cerebral ischemia-reperfusion (Hu et al., 2012). Additionally, it is reported that berberine reverses the memory deficits caused by scopolamine by decreasing the expression of proinflammatory cytokines such as interleukin-1β, tumor necrosis factor-α and
Fig. 4. Berberine rescues Arc/Arg3.1 mRNA expression. Total RNA samples from hippocampi were prepared, and semi-quantitative PCR was used to examine the mRNA levels of Arc/ Arg3.1 (A). Quantitative analysis was also performed (B). **P b 0.01, compared with Con, ##P b 0.01, compared with the D-gal group. N = 6–8 rats for each group.
cyclooxygenase-2 mRNA in the hippocampus (Lee et al., 2012). Here, we found the berberine supplement restores the learning and memory impairment induced by D-galactose, indicating that berberine may be useful as therapeutic agents for improving cognitive functioning for many neurodegenerative diseases. As reported previously, berberine was widely used for the treatment of many other diseases rather than neuroprotection (Zhu et al., 2004). In our study, to exclude the possible non-neuronal effects of berberine, we first examined the temperature, body weight, respiratory rate and heart rate and didn't find any difference, which suggests berberine treatment doesn't alter the basic physiological parameters. This further verifies the neuronal specific protection of berberine in our study. LTP is used to evaluate long-term synaptic plasticity in rodents and is widely recognized as a cellular basis for learning and memory (Bliss and Collingridge, 1993). Previous study has also shown that chronic berberine treatment significantly attenuates learning and memory deficits and restores PS amplitude and fEPSP slopes in the CA1 neurons of streptozocin (STZ)-treated diabetic rats (Kalalian-Moghaddam et al., 2012), suggesting a preservation effect of berberine in the synaptic plasticity, which is highly consistent with current study that berberine reverses the synaptic impairments induced by D-galactose. We also demonstrated that berberine rescues the mRNA and protein levels of Arc, an IEG that is important for the normal generation of LTP. The role of Arc in LTP is a dynamic one. Transient infusion of Arc antisense oligodeoxynucleotides at different experimental time points has revealed that an early presence of Arc is required for LTP induction (Messaoudi et al., 2007), whereas later on, Arc is necessary for LTP consolidation. Arc expression is also highly correlated with multiple behavioral activities such as spatial memory and contextual fear conditioning (Huff et al., 2006; Monti et al., 2006). Given that berberine effectively restored mRNA and protein levels of Arc in our study, we can speculate that berberine supplementation may stimulate the activity of MAPK (Alzamora et al., 2011), which plays an important role in activityinduced Arc/Arg3.1 expression in neurons (Waltereit et al., 2001). Further experimental studies will be necessary to elucidate the role of MAPK in these phenomena. In conclusion, we found in this study that berberine rescued memory deficits and synaptic impairments induced by D-galactose in rats, and that Arc/Arg3.1 expression may be an important mediator of the beneficial effects of berberine.
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P.-Y. Zhan et al. / Pharmacology, Biochemistry and Behavior 117 (2014) 47–51 Huang L, Shi A, He F, Li X. Synthesis, biological evaluation, and molecular modeling of berberine derivatives as potent acetylcholinesterase inhibitors. Bioorg Med Chem 2010;18:1244–51. Huff NC, Frank M, Wright-Hardesty K, Sprunger D, Matus-Amat P, Higgins E, et al. Amygdala regulation of immediate-early gene expression in the hippocampus induced by contextual fear conditioning. J Neurosci 2006;26:1616–23. Kalalian-Moghaddam H, Baluchnejadmojarad T, Roghani M, Goshadrou F, Ronaghi A. Hippocampal synaptic plasticity restoration and anti-apoptotic effect underlie berberine improvement of learning and memory in streptozotocin-diabetic rats. Eur J Pharmacol 2013;698(1–3):259–66. Korb E, Finkbeiner S. Arc in synaptic plasticity: from gene to behavior. Trends Neurosci 2011;34:591–8. Kulkarni SK, Dhir A. Current investigational drugs for major depression. Expert Opin Investig Drugs 2009;18:767–88. Kulkarni SK, Dhir A. Berberine: a plant alkaloid with therapeutic potential for central nervous system disorders. Phytother Res 2010;24:317–24. Lee B, Sur B, Shim I, Lee H, Hahm DH. Phellodendron amurense and its major alkaloid compound, berberine ameliorates scopolamine-induced neuronal impairment and memory dysfunction in rats. Korean J Physiol Pharmacol 2012;16: 79–89. Messaoudi E, Kanhema T, Soule J, Tiron A, Dagyte G, da Silva B, et al. Sustained Arc/Arg3.1 synthesis controls long-term potentiation consolidation through regulation of local actin polymerization in the dentate gyrus in vivo. J Neurosci 2007;27:10445–55. Monti B, Berteotti C, Contestabile A. Subchronic rolipram delivery activates hippocampal CREB and arc, enhances retention and slows down extinction of conditioned fear. Neuropsychopharmacology 2006;31:278–86. Okuno H. Regulation and function of immediate-early genes in the brain: beyond neuronal activity markers. Neurosci Res 2011;69:175–86.
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Peng WH, Hsieh MT, Wu CR. Effect of long-term administration of berberine on scopolamine-induced amnesia in rats. Jpn J Pharmacol 1997;74:261–6. Selkoe DJ. Alzheimer's disease is a synaptic failure. Science 2002;298:789–91. Sun X, Zhang X, Hu H, Lu Y, Chen J, Yasuda K, et al. Berberine inhibits hepatic stellate cell proliferation and prevents experimental liver fibrosis. Biol Pharm Bull 2009a;32:1533–7. Sun Y, Xun K, Wang Y, Chen X. A systematic review of the anticancer properties of berberine, a natural product from Chinese herbs. Anticancer Drugs 2009b;20:757–69. Tang J, Feng Y, Tsao S, Wang N, Curtain R, Wang Y. Berberine and Coptidis rhizoma as novel antineoplastic agents: a review of traditional use and biomedical investigations. J Ethnopharmacol 2009;126:5–17. Waltereit R, Dammermann B, Wulff P, Scafidi J, Staubli U, Kauselmann G, et al. Arg3.1/Arc mRNA induction by Ca2+ and cAMP requires protein kinase A and mitogen-activated protein kinase/extracellular regulated kinase activation. J Neurosci 2001;21:5484–93. Wang S, Zhu L, Shi H, Zheng H, Tian Q, Wang Q, et al. Inhibition of melatonin biosynthesis induces neurofilament hyperphosphorylation with activation of cyclin-dependent kinase 5. Neurochem Res 2007;32:1329–35. Wang SH, Liao XM, Liu D, Hu J, Yin YY, Wang JZ, et al. NGF promotes long-term memory formation by activating poly(ADP-ribose)polymerase-1. Neuropharmacology 2012;63:1085–92. Yao ZH, Zhang JJ, Xie XF. Enriched environment prevents cognitive impairment and tau hyperphosphorylation after chronic cerebral hypoperfusion. Curr Neurovasc Res 2012;9:176–84. Yin J, Xing H, Ye J. Efficacy of berberine in patients with type 2 diabetes mellitus. Metabolism 2008;57:712–7. Zhou JY, Zhou SW, Zhang KB, Tang JL, Guang LX, Ying Y, et al. Chronic effects of berberine on blood, liver glucolipid metabolism and liver PPARs expression in diabetic hyperlipidemic rats. Biol Pharm Bull 2008;31:1169–76. Zhu XZ, Li XY, Liu J. Recent pharmacological studies on natural products in China. Eur J Pharmacol 2004;500:221–30.
Contents lists available at ScienceDirect
Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh
Berberine rescues D-galactose-induced synaptic/memory impairment by regulating the levels of Arc Zhan Pei-Yan ⁎,1, Peng Cai-Xia 1, Zhang Lin-Hong Department of Neurology, Central Hospital of Wuhan, Wuhan 430014, China
a r t i c l e
i n f o
Article history: Received 10 July 2013 Received in revised form 29 November 2013 Accepted 5 December 2013 Available online 14 December 2013 Keywords: Berberine Synapse Arc protein
a b s t r a c t Synaptic communication forms the basis of learning and memory. Disruptions of synaptic function and memory have been widely reported in many neurological diseases, such as dementia. Thus, restoration of impaired synaptic communication is a potential therapeutic approach for these diseases. In this study, we demonstrated that supplementation with berberine, a plant alkaloid with a long history of medicinal usage in Chinese medicine, effectively reverses the synaptic deficits induced by D -galactose. We also found that berberine rescued D-galactose-induced memory impairment and additionally rescued the mRNA and protein levels of Arc/Arg3.1, an important immediate early gene that is crucial for maintaining normal synaptic plasticity. Our study provides the first piece of evidence supporting the potential use of berberine in the treatment of neural diseases with synaptic/memory impairments. © 2013 Elsevier Inc. All rights reserved.
1. Introduction Synaptic plasticity is the experience-dependent alteration in connectivity between neurons and is believed to be the neurobiological basis of learning and memory (Ho et al., 2011). Disruptions of synaptic plasticity have been found in multiple neurological disorders, including neurodevelopmental diseases and neurodegenerative diseases such as Down syndrome and Alzheimer's disease (Garner and Wetmore, 2012; Selkoe, 2002). A better understanding of the molecular mechanisms of synaptic plasticity would be helpful in developing appropriate treatments for these diseases. The maintenance of normal synaptic plasticity requires numerous genes and proteins, including immediate early genes (IEGs) (Okuno, 2011). Arc (activity-regulated cytoskeletonassociated protein), also known as Arg3.1, is a highly conserved IEG that is expressed only in vertebrates (Bramham et al., 2008). It has been reported that Arc is essential for the formation and consolidation of long-term memories (Korb and Finkbeiner, 2011). The mRNA for Arc is specifically located in the active synaptic loci for the locally synthesized Arc protein (Bramham, 2008). The tight association between Arc expression and the strength of excitatory synapses strongly suggests a critical role for Arc in synaptic plasticity (Korb and Finkbeiner, 2011). D-Galactose is a reducing sugar that can form advanced glycation end products (AGEs); consequently, the administration of D-galactose can induce cognitive deficits and disruptions in synaptic communication. ⁎ Corresponding author. Tel.: +86 2782211413; fax: +86 2782211409. E-mail addresses: [email protected] (P.-Y. Zhan), [email protected] (L.-H. Zhang). 1 The first two authors contributed equally to this paper. 0091-3057/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pbb.2013.12.006
Thus, D-galactose-treated rats have been widely studied as an animal model of synaptic disruption and memory impairment. Many drugs, including some traditional Chinese medicines, have been tested in efforts to reverse D-galactose-induced memory deficits. Berberine is a quaternary ammonium salt derived from the plants of the genus Berberis. Berberine (2000) reportedly helps the body fight infections, lowers elevated blood glucose (Yin et al., 2008), lowers elevated blood total cholesterol (Zhou et al., 2008), inhibits hepatic stellate cell proliferation (X. Sun et al., 2009), and has antineoplastic activity (Y. Sun et al., 2009; Tang et al., 2009). In addition, it has been suggested to exert potentially beneficial effects on central nervous system disorders via effects such as neuroprotection, anti-neuronal apoptosis, and improvement of cerebral microcirculation (Kulkarni and Dhir, 2010). As a traditional medicine, berberine has been shown to be an effective treatment for fungal, bacterial, and viral infections (Zhu et al., 2004). During the past few decades, many studies have suggested that berberine also has various beneficial effects on the nervous system as a neuroprotective agent (Kulkarni and Dhir, 2009, 2010). For example, berberine appears to produce its antiamnesic effects by augmenting the activity of the cholinergic neuronal system (Peng et al., 1997). Recently, derivatives of berberine have been designed to inhibit the activity of acetylcholinesterase (AChE) (Huang et al., 2010), mimicking an important mechanism of action for potential therapeutics for AD. In a transgenic mouse model of AD, a neuroprotective effect of berberine has also been well demonstrated (Durairajan et al., 2012). However, whether berberine can reverse deficits in synaptic function is still unclear. In the current study, we intended to study the possible protective effect of berberine on the D-galactose induced memory and synaptic disorder by using Morris water maze and in vivo LTP recording. We also
48
P.-Y. Zhan et al. / Pharmacology, Biochemistry and Behavior 117 (2014) 47–51
2.2. Electrophysiology To detect the long-term potentiation (LTP), 6–8 rats from each group were anesthetized and fixed on a Narishige (Tokyo, Japan) stereotaxic instrument (SR-6 N) (Wang et al., 2012). The stimulating electrode and the recording electrode were placed in the perforant path and dentate gyrus (DG) region of rat hippocampus separately. The basal line recording was obtained by delivering a single pulse of stimulation once every 10 s. Before LTP induction, we recorded a stable baseline for at least 30 min. To induce LTP, high-frequency stimulation (HFS) consisting of four trains of 50 pulses were delivered at 200 Hz with a 2 s intertrain interval. The slope of EPSP was recorded for 90 min and calculated automatedly by a computerized program (RM6240BD; Chengdu, China). After LTP recording, the rats were sacrificed by using overdose of urethane. 2.3. Morris water maze
Fig. 1. Berberine rescues D-galactose-induced impairment in long-term potentiation (LTP). LTP recordings were conducted as described previously. The representative traces (A) of EPSP before (pre-HFS) and after (post-HFS), the normalized population spike (PS) amplitude (B) and normalized field excitatory postsynaptic potentiation (fEPSP) slope (C) were analyzed. The arrow at time 0 indicates the application of high-frequency stimulation to induce LTP. Con: vehicle treated group, D-gal: D-galactose-treated group, D-gal + Ber: D-galactose- and berberine-treated group. N = 6–8 rats for each group. The black bar indicates the time window for the statistical analysis.
explore the possible underlying mechanisms for the neuroprotection of berberine. We found that D-galactose administration induced deficits in long-term potentiation along with a reduction in the levels of Arc and supplementation with berberine could reverse these effects in D-galactose-treated rats.
2. Materials and methods 2.1. Animals and treatment Male Wistar rats (10–12 weeks, 180–220 g) were purchased from Center for Disease Control and Prevention of Hubei province of China. The rats were housed in an air-conditioned room (22 ± 2 °C, 12 h light and 12 h dark; lights on at 06:00 am) with food and water ad libitum. All animal experiments were performed according to the ‘National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).’ D-galactose (100 mg/kg) was obtained from Shanghai Bio Life Science & Technology Co. (Shanghai, China) and was administered intraperitoneally once a day for 7 weeks (Cui et al., 2006). Berberine was purchased from Beijing Ocean Pharmacy Co. Ltd. (Batch No. 090601) and was delivered by gavage once daily to the rats with the dose of 100 mg/kg per day for 7 weeks too (Bhutada et al., 2011). All the experimental protocols were approved the review committee of Wuhan Central Hospital.
The Morris water maze was performed according to a previous published paper (Yao et al., 2012). Briefly, another 8–10 rats per group were training for six continuous days to find a hidden platform in the water by using a stationary array of cues inside or outside the pool (the diameter is 180 cm). For each trial, the rat was placed at the middle of any no-target quadrants (northeast, southeast, northwest) and facing the wall of the pool. If the rat did not reach the platform within 60 s, the trial was terminated, and the rat was guided to the platform manually and placed on the platform for another 30 s. An upper camera tracking device was used to record the swimming pathway and escape latency. The learning curve during the first 6 days was used to analyze the learning ability. At the 7th day, the platform was removed and the percent of time in target quadrant and the numbers of platform quadrant crosses were recorded to analyze the memory retention. Then the rats were sacrificed after Morris water maze for the biochemical experiments. 2.4. Western blot Hippocampi were homogenized in the buffer that contains Tris × Cl (pH 7.6) 10 mmol/L, NaF 50 mmol/L, Na3VO4 1 mmol/L, edetic acid 1 mmol/L, benzamidine 1 mmol/L, and Phenylmethanesulfonyl fluoride (PMSF) 1 mmol/L mixture (2 mg/L each of aprotinin, leupeptin and pepstain A). The hippocampi were lysate by the buffer containing Tris × Cl (pH 7.6) 200 mmol/L, 8% sodium dodecyl sulfate (SDS), 40% glycerol, and boiled at water bath for 10 min. After centrifuged at 12 000 × g for 5 min, the samples are subjected for protein concentration measurement and dithiothreitol (DTT) was added to attain a final concentration 100 mmol/L. The proteins were then separated by SDSpolyacrylamide gel electrophoresis (10% gel) and transferred to nitrocellulose membrane. Immunoreactive materials were detected using Chemiluminescent Substrate kit (Pierce, Rockford, IL, USA) and exposed to CL-XPosure film (Wang et al., 2007). The blots were scanned and the protein bands were quantitatively analyzed by Image J (NIH, USA). 2.5. RT-PCR Total RNA was isolated using Trizol reagents according to the instructions (Tiangen Technologies, Beijing, China). Then total RNA (3 μg in 25 μl) was reversely transcribed by commercial kit (Takara, Dalian, China) and the produced cDNA (1 μl) was used to detect the transcripts. For Arc/Arg3.1, the following primers were used: 5′-GGGA GGTCTTCTACCGTCTG-3′ (forward primer), 5′-CTTCACCGAGCCCTGT TT-3′ (reverse primer); for β-actin, 5′-GTAAAGACCTCTATGCCAACA-3′ (forward primer), 5′-GGACTCATCGTACTCCTGCT-3′ (reverse primer). PCR amplification consists of 32 cycles, each cycle was run with the following program: denaturing at 94 °C, 60 s, annealing at 48 °C (at 50 °C for Arc/Arg3.1), 50 s; and chain extension at 72 °C, 1 min. The PCR
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Fig. 2. Berberine rescues D-galactose-induced memory impairment. The Morris water maze was used to examine memory. The learning curve (A) for the first 6 days and the representative traces in the probe test (B). The time spent on the target quadrant (C) and the crossing times (D) are also analyzed. **P b 0.01, compared with Con, ##P b 0.01, compared with the D-gal group. N = 8–10 rats for each group.
products were separated on 1.0% agarose gels and stained with GoldView and visualized under UV light. 2.6. Statistical analysis All data were expressed as means ± S.E, and analyzed by SPSS 14.0 statistical software (SPSS, Chicago, IL, USA) by one-way ANOVA followed by Student t (least significant difference) post hoc test.
longer latency to reach the hidden platform from the third day, while the rats co-administrated with D-galactose and berberine doesn't shown any difference with vehicle rats (Fig. 2A). Moreover, in the probe test, we found that D-galactose treatment significantly decreased the time spent in the target quadrant and the crossing time to the platform regions, while berberine supplement effectively restores those abnormalities (Fig. 2B–D). We also measured the swimming speed and the total swimming distance, and found there is no difference between each group.
3. Results 3.3. Berberine reversed the reduction of Arc/Arg3.1 induced by D-galactose 3.1. Berberine restores LTP deficits induced by D-galactose During the whole experimental procedures, we measured the temperature, body weight, respiratory rate and heart rate of all rats, and found that both D-galactose and berberine didn't alter those physiological parameters. We then performed an in vivo LTP recording experiment according to a well-known method. We found that neither berberine nor D-galactose altered basal synaptic transmission. After high-frequency stimulation, the amplitude of the population spike (PS) and the slope of the excitatory postsynaptic potential (EPSP) increased 1.6- to 1.8-fold in vehicle-treated rats, whereas the increments were much lower in D-galactose-treated rats. Interestingly, supplementation with berberine effectively reversed the D-galactose-induced inhibition of these LTP measures (Fig. 1). No difference in excitation was found in rats treated with berberine alone (data not shown). These results suggest that berberine supplementation can reverse the LTP deficits induced by D-galactose.
IEGs such as Arc/Arg3.1, c-fos, Zif268, and Homer1a are crucial for regulating synaptic plasticity. Western blot analyses revealed that the levels of Arc/Arg3.1 are significantly decreased in D-galactose-treated rats compared to vehicle-treated rats, whereas berberine administration clearly rescued the reduction of Arc/Arg3.1 observed in the hippocampus (Fig. 3A-B). No apparent alterations in the protein levels of other IEGs were found (Fig. 3C). These results indicate that among the IEGs that we studied, only Arc/Arg3.1 is important for berberine's beneficial effects on the synaptic disruptions induced by D-galactose. We subsequently examined Arc/Arg3.1 mRNA levels with semiquantitative PCR and found that D-galactose suppressed Arc/Arg3.1 mRNA to 40% of the levels detected in vehicle-treated rats; moreover, supplementation with berberine effectively restored the levels of Arc/ Arg3.1 mRNA (Fig. 4). These data further demonstrate the key role of Arc/Arg3.1 in the beneficial effects of berberine. 4. Discussions
3.2. Berberine recovers the memory impairment caused by D-galactose We then used the Morris water maze to evaluate the learning and memory ability. We found that rats treated with D-galactose exhibit a
The application of berberine in the treatment of many neurological disorders had been widely reported. For example, in ischemia animal models, it protects from hippocampal neuronal damage following
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Fig. 3. Berberine restores protein levels of Arc/Arg3.1 but not levels of other IEGs. Hippocampal homogenates were prepared as described, and western blotting was performed to evaluate the protein levels of Arc/Arg3.1 (A) and (C), c-fos, zif268, and Homer1a (B). Statistical analysis was performed on Arc/Arg3.1 protein levels. **P b 0.01, compared with Con, ##P b 0.01, compared with the D-gal group. N = 6–8 rats for each group.
transient global ischemia by reducing MMP-9 activity (Hong et al., 2012) and exerts its anti-apoptotic effect by enhancing PI3K p55γ promoter activity in cerebral ischemia-reperfusion (Hu et al., 2012). Additionally, it is reported that berberine reverses the memory deficits caused by scopolamine by decreasing the expression of proinflammatory cytokines such as interleukin-1β, tumor necrosis factor-α and
Fig. 4. Berberine rescues Arc/Arg3.1 mRNA expression. Total RNA samples from hippocampi were prepared, and semi-quantitative PCR was used to examine the mRNA levels of Arc/ Arg3.1 (A). Quantitative analysis was also performed (B). **P b 0.01, compared with Con, ##P b 0.01, compared with the D-gal group. N = 6–8 rats for each group.
cyclooxygenase-2 mRNA in the hippocampus (Lee et al., 2012). Here, we found the berberine supplement restores the learning and memory impairment induced by D-galactose, indicating that berberine may be useful as therapeutic agents for improving cognitive functioning for many neurodegenerative diseases. As reported previously, berberine was widely used for the treatment of many other diseases rather than neuroprotection (Zhu et al., 2004). In our study, to exclude the possible non-neuronal effects of berberine, we first examined the temperature, body weight, respiratory rate and heart rate and didn't find any difference, which suggests berberine treatment doesn't alter the basic physiological parameters. This further verifies the neuronal specific protection of berberine in our study. LTP is used to evaluate long-term synaptic plasticity in rodents and is widely recognized as a cellular basis for learning and memory (Bliss and Collingridge, 1993). Previous study has also shown that chronic berberine treatment significantly attenuates learning and memory deficits and restores PS amplitude and fEPSP slopes in the CA1 neurons of streptozocin (STZ)-treated diabetic rats (Kalalian-Moghaddam et al., 2012), suggesting a preservation effect of berberine in the synaptic plasticity, which is highly consistent with current study that berberine reverses the synaptic impairments induced by D-galactose. We also demonstrated that berberine rescues the mRNA and protein levels of Arc, an IEG that is important for the normal generation of LTP. The role of Arc in LTP is a dynamic one. Transient infusion of Arc antisense oligodeoxynucleotides at different experimental time points has revealed that an early presence of Arc is required for LTP induction (Messaoudi et al., 2007), whereas later on, Arc is necessary for LTP consolidation. Arc expression is also highly correlated with multiple behavioral activities such as spatial memory and contextual fear conditioning (Huff et al., 2006; Monti et al., 2006). Given that berberine effectively restored mRNA and protein levels of Arc in our study, we can speculate that berberine supplementation may stimulate the activity of MAPK (Alzamora et al., 2011), which plays an important role in activityinduced Arc/Arg3.1 expression in neurons (Waltereit et al., 2001). Further experimental studies will be necessary to elucidate the role of MAPK in these phenomena. In conclusion, we found in this study that berberine rescued memory deficits and synaptic impairments induced by D-galactose in rats, and that Arc/Arg3.1 expression may be an important mediator of the beneficial effects of berberine.
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