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Berberine attenuates cognitive impairment and ameliorates tau hyperphosphorylation by limiting the self-perpetuating pathogenic cycle between NF-κB signaling, oxidative stress and neuroinflammation
Accepted Manuscript Title: Berberine attenuates cognitive impairment and ameliorates tau hyperphosphorylation by limiting the self-perpetuating pathogenic cycle between NF-B signaling, oxidative stress and neuro-inflammation Authors: Wenbo He, Chuanlin Wang, Yi Chen, Yongli He, Zhiyou Cai PII: DOI: Reference:
S1734-1140(17)30257-8 http://dx.doi.org/doi:10.1016/j.pharep.2017.06.006 PHAREP 745
To appear in: Received date: Revised date: Accepted date:
7-4-2017 25-5-2017 13-6-2017
Please cite this article as: Wenbo He, Chuanlin Wang, Yi Chen, Yongli He, Zhiyou Cai, Berberine attenuates cognitive impairment and ameliorates tau hyperphosphorylation by limiting the self-perpetuating pathogenic cycle between NF-B signaling, oxidative stress and neuro-inflammation (2010), http://dx.doi.org/10.1016/j.pharep.2017.06.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Berberine attenuates cognitive impairment and ameliorates tau hyperphosphorylation by limiting the self-perpetuating pathogenic cycle between NF-κB signaling, oxidative stress and neuro-inflammation Wenbo He1, Chuanlin Wang1, Yi Chen1, Yongli He2, Zhiyou Cai3 1
Department of Neurology, Renmin Hospital, Hubei University of Medicine, Shiyan Renmin Hospital, Shiyan, 442000,
Hubei Province, China 2
Department of Internal Medicine, Chongqing Prevention and Treatment Center for Occupational Diseases, Chongqing,
400060, Chongqing, China 3
Department of Neurology, Chongqing General Hospital, Chongqing, 400013, Chongqing, China Correspondence to Yongli He and Zhiyou Cai Yongli He Department of Internal Medicine, Chongqing Prevention and Treatment Center for Occupational Diseases, No. 312 Nancheng Road, Nan-an District, Chongqing, People’s Republic of China. 400060, Phone: +86-23-61929106, Fax: +86-23-61929503 Email: [email protected] Zhiyou Cai Department of Neurology, Chongqing General Hospital, No. 312 Zhongshan First Road, Yuzhong District, Chongqing, People’s Republic of China. 400013, Phone: +86-23-63515796, Fax: +86-23-63515796 Email: [email protected]
Running title：Berberine ameliorates tau hyperphosphorylation by limiting NF-κB signaling Conflict of interest: None declared.
Abstract Background: Berberine (BBR) plays an important role in the prevention and treatment of Alzheimer’s disease (AD). The present work was to explore whether BBR ameliorates cognitive deficits in AD and to investigate whether its underlying mechanism involves inhibiting hyperphosphorylated tau protein. Methods: The cognitive function was measured by the Morris water maze (MWM) test. The levels of hyperphosphorylated tau proteins were determined by Western blot. The biomarkers of NF-κB signaling pathway and oxidative stress were detected by Western blot and biochemical assays. The biomarkers of neuroinflammation were determined by Western blot and immunohistochemistry. Results: BBR improved learning and memory in APP/PS1 mice. BBR decreased the hyperphosphorylated tau protein in the hippocampus of APP/PS1 mice. BBR lowered the activity of NF-κB signaling in the hippocampus of AD mice. BBR-administration promoted the activity of glutathione (GSH) and inhibited lipid peroxidation in the hippocampus of AD mice. Conclusion: BBR attenuated cognitive deficits and limited hyperphosphorylation of tau via inhibiting the activation of NF-κB signaling pathway, and by retarding oxidative stress and neuro-inflammation. Keywords: tau hyperphosphorylation; cognitive dysfunction; oxidative stress; inflammation; berberine
Introduction Alzheimer's disease (AD), the most prevalent form of dementia in the elderly, is an irreversible neurodegenerative disorder which is clinically featured with progressive cognitive deficits and dementia. AD is pathologically characterized by amyloid plaques, neurofibrillary tangles (NFTs) [1, 2]. To date, a variety of medications are used to prevent the progress of AD. Some of them may slow down memory loss, and improve communication skills, and help with certain behavioral problems. However, all the drugs had limited effects, or no effects with more sideeffects [3]. Until now, drugs that can be successfully used for AD treatment have not been developed. Berberine (BBR) is a yellow-colored alkaloid compound that can be extracted from several different plants, including European barberry, goldenseal, goldthread, Oregon grape phellodendron, and tree turmeric. It has been reported that BBR has therapeutic effects on metabolic disorders [4, 5], cardiovascular diseases [6], congestive heart failure [7], and neurodegenerative diseases [8]. Increasing evidence has also indicated its beneficial effect on stroke and various neurodegenerative and neuropsychiatric disorders [9, 10]. Substantial studies have demonstrated that BBR limits the pathogenesis of extracellular amyloid plaques and intracellular NFTs [11, 12]. BBR could potentially be developed as an effective therapeutic strategy for AD patients [13, 14]. However, the mechanism remains obscure. The aim of this study was to investigate the effects of BBR on tau hyperphosphorylation. As numerous results have supported the role of NF-κB signaling pathway, oxidative stress and neuro-inflammation in the pathogenesis of AD [11, 15], we analyzed the change of NF-κB signaling pathway, oxidative stress and neuro-inflammation in the hippocampus of APP/PS1 transgenic mice after BBR administration. Our results indicated that it is via inhibiting the activation of NF-κB signaling pathway, and by retarding oxidative stress and neuro-inflammation that BBR effectively attenuated cognitive impairment and limited tau hyperphosphorylation in an AD mouse model.
Materials and methods
Animals and drugs Thirty 120-day male APP/PS1 transgenic mice were used in the present study. The APP/PS1 transgenic mice were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences & Peking Union Medical College. They were housed in individual cages at a constant temperature (25°C) under a 12-h light-dark cycle and given free access to standard laboratory food and water. All mice were acclimated for at least 5 days before the experiments. The mice were randomly assigned into three groups: control group (n=10), 50mg/kg-BBR (n=10) and 100mg/kg-BBR (n=10). BBR (100 mg/tablet, Hangzhou Sanofi Minsheng Health Pharmaceutical Co. Ltd., China) was diluted to 0.5 mg/mL by normal saline. For the 50mg/kg-BBR group (50 mg/kg/d), mouse was given BBR through intragastric administration (ig) by 50 mg/kg/d for 14 days; for the 100mg/kg-BBR group (100 mg/kg/d), mouse was given 100 mg/kg/d also for 14 days. The BBR dosage was determined based on previous studies [16, 17]. Control group was administrated with the same volume saline. All the experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animal experiments followed the Research Ethics standards.
Morris water maze (MWM) Test The MWM test was performed according to the instrument instructions. The MWM test was implemented in the Basic Research Center of Hubei University of Medicine, including place navigation and spatial probe. The experimental procedures of the MWM test were recorded with Morris Image System (Shanghai DOiT Industrial Co., Ltd.). First, the mice were given three days to familiarize themselves familiar with the pool environment in a steady room temperature (20°C -24°C) and humidity (60–80%). This was followed by the MWM test with 4 trials per day. Then the acquisition trial for the mice was the last training trials to find the hidden platform at a target Quadrant A. The escape latency was recorded by the finding-
platform time. At Day 8, the mice were subjected to the probe trial to assess spatial memory. The time in the target quadrant and the times crossing the former platform area were recorded for testing spatial memory.
Lipid peroxidation assay The levels of malonaldehyde (MDA) and lipid peroxide (LPO) were measured with biochemical assay according to the manufacturer's instructions provided in the MDA and LPO assay kits (Nanjing Jiancheng Bioengineering Institute, China). Fifty microliter of sample or standard was placed into the microtiter plate wells and then mixed with 250 µL aqueous working reagent. Then samples were incubated for 30 min on a shaker and the absorbance was taken at 540 nm. The content of MDA and LPO was determined by comparison with a predetermined standard curve.
Western blot After the Morris water maze test, mice were anesthetized with pentobarbital, and then the brain was quickly removed, and the hippocampus tissues were dissected and homogenized on ice in T-PER buffer in the presence of protease inhibitors. After the samples were centrifuged at 5000g for 10 minutes at 4°C, the supernatant was collected and used for Western blot using Ciphergen (Biosource) protein chip arrays. Western blot was carried out according to the manufacturer’s instructions and previous studies [18, 19]. The p-S199, p-S202, p-T205, pT231, p-S396, p-S404, GPx-1/2, GSS, GGT1, GR, GFAP, CD45, IL-1β and TNF-α antibodies were purchased from Abcam, Cambridge, MA, USA. NF-κB elements (p50, p52, p65, c-Rel and RelB) and IκB proteins (IκBα and IκBβ) were obtained from Cell Signaling Technology, Inc. USA. Goat anti-mouse IgG labeled with biotin was from Santa Cruz Biotechnology, Inc. USA. The optical densities of the specific bands were scanned and measured by image analysis software (HPIAS 2000, Tongji Qianping Company, Wuhan, China). Rabbit anti-mouse β-actin (Santa Cruz Biotechnology, Inc. USA) was used as an internal control.
Immunohistochemical staining
Immunohistochemical staining was conducted as described in the previous study [20]. After deparaffination and rehydration of brain sections, antigen retrieval was performed by treatment with proteinase K for GFAP and CD45 staining. After the sections were blocked for nonspecific binding, the samples were incubated with monoclonal anti-GFAP and anti-CD45 antibody overnight. Primary antibody was measured with peroxidase-conjugated secondary antibody and detected with a stable diaminobenzidine solution (Vector Laboratories, Burlingame, CA, USA). Images were captured by using Olympus microscope connected to a digital microscope camera. All slides were tested in duplicate. Samples with a positive score over 10 or frequency over 5% were considered as positive. The percentage of area occupied by positive expression was calculated using the Image-Pro Plus imaging software from Media Cybernetics, Bethesda, MD, USA. Mean values for each parameter were recorded from six equidistant sections through the hippocampal region per mouse in each group.
Statistical analysis Quantitative data were expressed as the mean ± standard error of the mean (SEM). All statistical analyses were performed using the GraphPad Prism 6.0 software for Windows (GraphPad Software, La Jolla, CA, USA). For statistical evaluation of intergroup differences, paired t test was used, and one-way analyses of variance (ANOVA) were employed. Significant difference was considered at p<0.05.
Results BBR mitigated cognitive impairment of AD mice The latency to find the platform in the acquisition trials of the MWM task was shown in Figure 1A, B and C. The BBR-treated mice exhibited lower escape latency on days 3, 4, 5, 6 and 7 during training trials (p<0.05). However, there is no difference between BBR-treated groups (50mg and 100mg). In the probe trial, the mice were placed into the non-target quadrants respectively and allowed to swim freely for 60 seconds. The BBR-treated mice demonstrated a significant increase in the number of times crossing the target Quadrant A (Figure 1D, p<0.05)
the time staying in the target Quadrant A (Figure 1E, p<0.05). These results indicated that BBR improved the learning and memory capability of AD mice.
BBR inhibited phosphorylation of tau Hippocampal samples were obtained from AD mice to measure tau hyperphosphorylation in the hippocampus. We selected 6 phosphorylated tau monoclonal antibodies (phosphorylated at S199, S202, T205, T231, S396 and S404: p-S199, p-S202, p-T205, p-T231, p-S396 and pS404) to evaluate changes in tau protein. Western blot revealed a substantial decrease in the Tau-6 levels (p-S199, p-S202, p-T205, p-T231, p-S396 and p-S404) in the hippocampus of BBRtreated AD mice, compared to the control groups (p<0.001) (Figure 2).
BBR inhibits the activation of NF-κB signaling pathway The NF-κB family mainly includes five related transcription factors: p50, p52, RelA (p65), c-Rel and RelB. The activation of NF-κB is inhibited by IκB proteins in the cytoplasm. Figure 3 showed that BBR lowered the activity of p50, p52, p65, c-Rel and RelB in the hippocampus of AD mice. BBR improved the activity of IκB proteins (IκBα and IκBβ). These results implicated that BBR suppressed the activation of NF-κB signaling pathway in the brain of AD mice.
BBR enhanced the activity of glutathione BBR promoted the activity of glutathione (GSH) in the hippocampus of AD mice (Figure 4). BBR-administration up-regulated the level of GPx-1/2, GSS and GR in the hippocampus of AD mice, and reduced the activity of γ-glutamyltranspeptidase (GGT), which acts as a glutathionase and catalyzes glutathione into L-cysteinylglycine and L-glutamatethe, compared to control groups (Figure 4).
BBR limited lipid peroxidation Lipid peroxidation produces a wide variety of oxidation products such as malondialdehyde (MDA) and other lipid peroxide (LPO). Figure 5 showed that BBR decreased the activity of
MDA and LPO in the hippocampus of AD mice. The result implicated that BBR inhibited lipid peroxidation in the pathogenesis of AD.
BBR inhibited neuro-inflammation Gliocytes play an important role in the inflammatory process of AD. Increasing studies have reported that activated astrocytes and microglia are associated with extracellular amyloid peptide deposition, intracellular accumulation of hyperphosphorylated tau protein (formation of NFTs), neuro-inflammation and oxidative stress. To investigate whether BBR treatment has anti-inflammatory effects in the hippocampus of AD mice, we measured the biomarkers of astrocyte and microglia reactivity with an anti-GFAP antibody for astrocytes and an anti-CD45 antibody for microglia. Results indicate BBR significantly decreased both GFAP and CD45 immunoreactivity in the hippocampus of AD mice when compared with those in the control mice (Figure 6A, B). In order to clarify the anti-inflammatory role of BBR in AD, we detected the levels of IL-1β and TNF-α, the classic inflammatory biomarkers. Results demonstrate that BBR down-regulated the levels of IL-1β and TNF-α in the hippocampus of AD mice, when compared to the control groups (Figure 6A).
Discussion NFTs are aggregates of hyperphosphorylated tau protein that are most commonly known as a primary hallmark of AD. In the present study, we demonstrated that BBR-treatment strongly decreased tau hyperphosphorylation in the hippocampus of APP/PS1 mice, which resulted in improvement of cognitive deficits. Importantly, we found that BBR markedly inhibited the activation of NF-κB signaling pathway and counteracted the progress of oxidative stress and ameliorated tau hyperphosphorylation, while BBR enhanced the activity of GSH, and limited lipid peroxidation. Furthermore, this study demonstrates that BBR retarded neuroinflammation by inhibiting the activation of astrocytes and microglia and down-regulating the levels of IL-1β and TNF-α (the classic inflammatory biomarkers) in the hippocampus of AD mice. All of these parameters indicated that it is via inhibiting the activation of NF-κB signaling pathway, and by retarding its related oxidative stress and neuro-inflammation that BBR
effectively attenuated cognitive impairment and limited tau hyperphosphorylation in an AD mice model (APP/PS1 transgenic mice). NF-κB signaling pathway plays an exceptionally important role due to its pleiotropic effects, its unique regulatory mechanisms, and large number of activating signaling pathways and number of genes that it controls. Activation of NF-κB is association with many diseases, such as cardiovascular disease, diabetes, and neurodegenerative diseases [21, 22]. Several studies have highlighted the progress on the role of NF-κB signaling pathway in AD [22, 23]. It has been found that activated NF-κB contributed to Cdk5/p25-induced phosphorylated tau [24]. NF-κB family mainly includes five related transcription factors: p50, p52, RelA (p65), c-Rel and RelB. We found that BBR increased the levels of IκB proteins (IκBα and IκBβ) which inhibit the activation of NF-κB, whereas BBR decreased the levels of p50, p52, p65, c-Rel and RelB, respectively, in the hippocampus of APP/PS1 mice. Oxidative stress and neuro-inflammation are major pathogenesis of AD leading to neurological sequelae [25]. Oxidative stress is essentially an imbalance between the production of reactive oxygen species (ROS) and the ability of the body to counteract their harmful effects through neutralization by antioxidants. Glutathione (GSH), an important antioxidant, prevents tissue damage from oxidative stress induced by ROS including free radicals, peroxides and lipid peroxides [26]. Glutathione peroxidase (GPx), a free radical scavenger, plays an important role in the antioxidant defense of central nervous system in response to oxidative stress [27, 28]. BBR-administration increased the level of GPx-1/2 in the hippocampus of AD mice. Glutathione synthetase (GSS) induces an increase in glutathione levels in brain [29]. There was also a markedly increase in GSS in the hippocampus of AD mice after the intervention of BBR. Glutathione reductase (GR) converts oxidized GSH to the reduced form and hold high levels of reduced glutathione in the cytosol of neural cells [30, 31]. BBR-treatment dramatically promotes the biological activity of GR in the hippocampus of AD mice. Additionally, BBR lowered the activity of GGT which acts as a glutathionase and catalyzes glutathione into Lcysteinylglycine and L-glutamatethe. Anti-oxidative enzymes, such as GSH, GPx, catalase, and superoxide dismutase (SOD), are essential to prevent cells from oxidative damage [32, 33]. An
antioxidant activity of BBR has been assumed in our studies in which neuro-protection was demonstrated in AD mice. Our results found that BBR improved the biological activity of GPx1/2, GSS and GR, while reduced the GGT1 activity in the hippocampus of AD mice. Therefore, the anti-oxidative property of BBR in the process of AD pathogenesis has an association with its modifying GSH enzymes and enhancing the activity of GSH. We found a highly significant anti-oxidative property of BBR via modifying antioxidant enzymes and enhancing the activity of antioxidants. Lipid peroxidation is considered as a process of the oxidative degradation of lipids containing any number of carbon-carbon double bonds [34]. Lipid peroxidation generates multiple oxidation products, including MDA and LPO. Numerous studies have evidenced a crucial role of lipid peroxidation in the process of AD pathogenesis [35, 36]. Our study showed that BBR limited the deleterious activity of MDA and LPO in the hippocampus of AD mice. Therefore, such an anti-oxidative property of BBR is reflected by its ability to limit lipid peroxidation. Neuro-inflammation, a common feature of neurodegenerative diseases, plays an important role in the progression of AD and may be responsible for degeneration in vulnerable regions such as the hippocampus [37, 38]. Activated microglia and astrocytes contribute to extracellular amyloid peptide deposition, intracellular accumulation of hyperphosphorylated tau protein (formation of NFTs), neuro-inflammation and oxidative stress [39-41]. We measured the GFAP biomarker for astrocyte and CD45 for microglia. BBR strongly reduced the levels of both GFAP and CD45 in the hippocampus of AD mice compared with the control mice. Moreover, we also investigated the levels of IL-1β and TNF-α (the classic inflammatory biomarkers). The result demonstrated that BBR lowered the levels of IL-1β and TNF-α in the hippocampus of AD mice. The self-perpetuating cycle of chronic neuro-inflammation and oxidative stress may contribute to irreversible neuronal dysfunction, acting as an important feature of AD pathogenesis [42, 43]. It has been reported that interaction between oxidative stress and neuro-inflammation is strongly associated with NF-κB signaling pathway while NF-κB signaling interacts with both oxidative stress and neuro-inflammation [44-47]. NF-κB signaling, oxidative stress and inflammation are composed of an important pathogenic cascade of neuro-degeneration in AD,
suggesting that therapeutic efforts aimed at the pathogenic cascade may be beneficial for AD treatment and prevention. The present study demonstrated that BBR was conducive to the decrease in the biological activity of NF-κB signaling, oxidative stress and inflammation in the hippocampus of AD mice. Taken together, attenuating cognitive impairment and inhibiting tau hyperphosphorylation by BBR are related to its ability in limiting the pathogenic cascade and the self-perpetuating cycle between NF-κB signaling, oxidative stress and neuro-inflammation (Figure 7). In summary, the current study shows the neuro-protective property of BBR against AD lies with its ability to inhibit NF-κB signaling, oxidative stress and inflammation, to lower tau hyperphosphorylation, and to attenuate cognitive deficits. From clinical point of view, the ability of BBR to modulate oxidative stress and inflammation and to regulate NF-κB signaling pathway, may be of great importance in neuro-protection in AD treatment and prevention. For the clinical practice, the strategy to use BBR for brain protection would provide great advantages for AD patients. However, it would be important to precisely define the effects of BBR on AD brains. Hence, further studies elucidating BBR neuroprotective mechanisms may provide insights into novel strategies for AD treatments.
Acknowledgments: This work was supported by grants from Chongqing General Hospital (CGHK201701), the Natural Science Foundation of Hubei Province (2015CFB260), and the Hubei Province Health and Family Planning Scientific Research Project (WJ2015MB219) and the Shiyan Natural Science Foundation (15K70).
References 1. Carreiras MC, Mendes E, Perry MJ, Francisco AP, Marco-Contelles J: The multifactorial nature of Alzheimer's disease for developing potential therapeutics. Curr Top Med Chem 2013, 13(15):1745-1770.
2. Galimberti D, Scarpini E: Alzheimer's disease: from pathogenesis to disease-modifying approaches. CNS Neurol Disord Drug Targets 2011, 10(2):163-174. 3. Mancuso C, Siciliano R, Barone E, Butterfield DA, Preziosi P: Pharmacologists and Alzheimer disease therapy: to boldly go where no scientist has gone before. Expert Opin Investig Drugs 2011, 20(9):1243-1261. 4. Caliceti C, Franco P, Spinozzi S, Roda A, Cicero AF: Berberine: New Insights from Pharmacological Aspects to Clinical Evidences in the Management of Metabolic Disorders. Curr Med Chem 2016, 23(14):1460-1476. 5. Pirillo A, Catapano AL: Berberine, a plant alkaloid with lipid- and glucose-lowering properties: From in vitro evidence to clinical studies. Atherosclerosis 2015, 243(2):449-461. 6. Jin Y, Khadka DB, Cho WJ: Pharmacological effects of berberine and its derivatives: a patent update. Expert Opin Ther Pat 2016, 26(2):229-243. 7. Chang W, Li K, Guan F, Yao F, Yu Y, Zhang M, et al. Berberine Pretreatment Confers Cardioprotection Against Ischemia-Reperfusion Injury in a Rat Model of Type 2 Diabetes. J Cardiovasc Pharmacol Ther. 2016;21(5):486494. 8. Jiang W, Li S, Li X: Therapeutic potential of berberine against neurodegenerative diseases. Sci China Life Sci 2015, 58(6):564-569. 9. Friedemann T, Schumacher U, Tao Y, Leung AK, Schroder S: Neuroprotective Activity of Coptisine from Coptis chinensis (Franch). Evid Based Complement Alternat Med 2015, 2015:827308. 10. Simoes Pires EN, Frozza RL, Hoppe JB, Menezes Bde M, Salbego CG: Berberine was neuroprotective against an in vitro model of brain ischemia: survival and apoptosis pathways involved. Brain Res 2014, 1557:26-33. 11. Cai Z, Wang C, Yang W: Role of berberine in Alzheimer's disease. Neuropsychiatr Dis Treat 2016, 12:25092520. 12. de Oliveira JS, Abdalla FH, Dornelles GL, Adefegha SA, Palma TV, Signor C, et al: Berberine protects against memory impairment and anxiogenic-like behavior in rats submitted to sporadic Alzheimer's-like dementia: Involvement of acetylcholinesterase and cell death. Neurotoxicology 2016, 57:241-250. 13. Mak S, Luk WW, Cui W, Hu S, Tsim KW, Han Y: Synergistic inhibition on acetylcholinesterase by the combination of berberine and palmatine originally isolated from Chinese medicinal herbs. J Mol Neurosci 2014, 53(3):511-516. 14. Kim MH, Kim SH, Yang WM: Mechanisms of action of phytochemicals from medicinal herbs in the treatment of Alzheimer's disease. Planta Med 2014, 80(15):1249-1258. 15. Kaur U, Banerjee P, Bir A, Sinha M, Biswas A, Chakrabarti S: Reactive oxygen species, redox signaling and neuroinflammation in Alzheimer's disease: the NF-kappaB connection. Curr Top Med Chem 2015, 15(5):446457.
16. Zhu F, Qian C: Berberine chloride can ameliorate the spatial memory impairment and increase the expression of interleukin-1beta and inducible nitric oxide synthase in the rat model of Alzheimer's disease. BMC Neurosci 2006, 7:78. 17. Patil S, Tawari S, Mundhada D, Nadeem S: Protective effect of berberine, an isoquinoline alkaloid ameliorates ethanol-induced oxidative stress and memory dysfunction in rats. Pharmacol Biochem Behav 2015, 136:1320. 18. Meng F, Wang J, Ding F, Xie Y, Zhang Y, Zhu J: Neuroprotective effect of matrine on MPTP-induced Parkinson's disease and on Nrf2 expression. Oncol Lett 2017, 13(1):296-300. 19. Cai Z, Yan Y, Wang Y: Minocycline alleviates beta-amyloid protein and tau pathology via restraining neuroinflammation induced by diabetic metabolic disorder. Clin Interv Aging 2013, 8:1089-1095. 20. Zhao Y, Xiao M, He W, Cai Z: Minocycline upregulates cyclic AMP response element binding protein and brainderived neurotrophic factor in the hippocampus of cerebral ischemia rats and improves behavioral deficits. Neuropsychiatr Dis Treat 2015, 11:507-516. 21. Vicente Miranda H, Outeiro TF: The sour side of neurodegenerative disorders: the effects of protein glycation. J Pathol 2010, 221(1):13-25. 22. Salminen A, Kaarniranta K, Haapasalo A, Hiltunen M, Soininen H, Alafuzoff I: Emerging role of p62/sequestosome-1 in the pathogenesis of Alzheimer's disease. Prog Neurobiol 2012, 96(1):87-95. 23. Kitazawa M, Cheng D, Tsukamoto MR, Koike MA, Wes PD, Vasilevko V, et al: Blocking IL-1 signaling rescues cognition, attenuates tau pathology, and restores neuronal beta-catenin pathway function in an Alzheimer's disease model. J Immunol 2011, 187(12):6539-6549. 24. Sun LH, Ban T, Liu CD, Chen QX, Wang X, Yan ML, et al: Activation of Cdk5/p25 and tau phosphorylation following chronic brain hypoperfusion in rats involves microRNA-195 down-regulation. J Neurochem 2015, 134(6):1139-1151. 25. Chen SD, Yin JH, Hwang CS, Tang CM, Yang DI: Anti-apoptotic and anti-oxidative mechanisms of minocycline against sphingomyelinase/ceramide neurotoxicity: implication in Alzheimer's disease and cerebral ischemia. Free Radic Res 2012, 46(8):940-950. 26. Rodriguez VM, Del Razo LM, Limon-Pacheco JH, Giordano M, Sanchez-Pena LC, Uribe-Querol E, et al.: Glutathione reductase inhibition and methylated arsenic distribution in Cd1 mice brain and liver. Toxicol Sci 2005, 84(1):157-166. 27. Comparsi B, Meinerz DF, Franco JL, Posser T, Prestes Ade S, Stefanello ST, et al: Diphenyl ditelluride targets brain selenoproteins in vivo: inhibition of cerebral thioredoxin reductase and glutathione peroxidase in mice after acute exposure. Mol Cell Biochem 2012, 370(1-2):173-182. 28. Dudek H, Farbiszewski R, Michno T, Kozlowski A: [Activity of glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R) and superoxide dismutase (SOD-1) in single brain metastasis]. Wiad Lek 2002, 55(56):252-256.
29. Kang Y, Viswanath V, Jha N, Qiao X, Mo JQ, Andersen JK: Brain gamma-glutamyl cysteine synthetase (GCS) mRNA expression patterns correlate with regional-specific enzyme activities and glutathione levels. J Neurosci Res 1999, 58(3):436-441. 30. Singh R, Pathak DN: Lipid peroxidation and glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase activities in FeCl3-induced epileptogenic foci in the rat brain. Epilepsia 1990, 31(1):15-26. 31. Uchoa MF, de Souza LF, Dos Santos DB, Peres TV, Mello DF, Leal RB, et al: Modulation of Brain Glutathione Reductase and Peroxiredoxin 2 by alpha-Tocopheryl Phosphate. Cell Mol Neurobiol 2016, 36(6):1015-1022. 32. Kumar R, Kaur R, Singh AP, Arora S: Diminution of hepatic response to 7, 12-dimethylbenz(alpha)anthracene by ethyl acetate fraction of Acacia catechu willd. through modulation of xenobiotic and anti-oxidative enzymes in rats. PLoS One 2014, 9(2):e90083. 33. Nikolic-Kokic A, Stevic Z, Blagojevic D, Davidovic B, Jones DR, Spasic MB: Alterations in anti-oxidative defence enzymes in erythrocytes from sporadic amyotrophic lateral sclerosis (SALS) and familial ALS patients. Clin Chem Lab Med 2006, 44(5):589-593. 34. Ayala A, Munoz MF, Arguelles S: Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxid Med Cell Longev 2014, 2014:360438. 35. Caliskan M, Mogulkoc R, Baltaci AK, Menevse E: The Effect of 3',4'-Dihydroxyflavonol on Lipid Peroxidation in Rats with Cerebral Ischemia Reperfusion Injury. Neurochem Res 2016, 41(7):1732-1740. 36. Muralikrishna Adibhatla R, Hatcher JF: Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ischemia. Free Radic Biol Med 2006, 40(3):376-387. 37. Ardestani PM, Evans AK, Yi B, Nguyen T, Coutellier L, Shamloo M: Modulation of neuroinflammation and pathology in the 5XFAD mouse model of Alzheimer's disease using a biased and selective beta-1 adrenergic receptor partial agonist. Neuropharmacology 2017, 116:371-386. 38. Popp J, Oikonomidi A, Tautvydaite D, Dayon L, Bacher M, Migliavacca E, et al: Markers of neuroinflammation associated with Alzheimer's disease pathology in older adults. Brain Behav Immun 2017, 62:203-211. 39. Goetzl EJ, Mustapic M, Kapogiannis D, Eitan E, Lobach IV, Goetzl L, et al: Cargo proteins of plasma astrocytederived exosomes in Alzheimer's disease. FASEB J 2016, 30(11):3853-3859. 40. Peters O, Schipke CG, Philipps A, Haas B, Pannasch U, Wang LP, et al: Astrocyte function is modified by Alzheimer's disease-like pathology in aged mice. J Alzheimers Dis 2009, 18(1):177-189. 41. Baker C, Nielsen HM, Minthon L, Wright HT, Chappell S, Okyere J, et al: Effects of Alzheimer's peptide and alpha1-antichymotrypsin on astrocyte gene expression. Neurobiol Aging 2007, 28(1):51-61. 42. Vaziri ND, Rodriguez-Iturbe B: Mechanisms of disease: oxidative stress and inflammation in the pathogenesis of hypertension. Nat Clin Pract Nephrol 2006, 2(10):582-593.
43. Ortiz GG, Pacheco-Moises FP, Bitzer-Quintero OK, Ramirez-Anguiano AC, Flores-Alvarado LJ, Ramirez-Ramirez V, et al: Immunology and oxidative stress in multiple sclerosis: clinical and basic approach. Clin Dev Immunol 2013, 2013:708659. 44. Raish M: Momordica charantia polysaccharides ameliorate oxidative stress, hyperlipidemia, inflammation, and apoptosis during myocardial infarction by inhibiting the NF-kappaB signaling pathway. Int J Biol Macromol 2017, 97:544-551. 45. Zhong W, Qian K, Xiong J, Ma K, Wang A, Zou Y: Curcumin alleviates lipopolysaccharide induced sepsis and liver failure by suppression of oxidative stress-related inflammation via PI3K/AKT and NF-kappaB related signaling. Biomed Pharmacother 2016, 83:302-313. 46. Wang YR, Tian FL, Yan MX, Fan JH, Wang LY, Kuang RG, et al: Sulfasalazine inhibits inflammation and fibrogenesis in pancreas via NF-kappaB signaling pathway in rats with oxidative stress-induced pancreatic injury. Drug Des Devel Ther 2016, 10:1743-1751. 47. Wang YL, Sun GY, Zhang Y, He JJ, Zheng S, Lin JN: Tormentic acid inhibits H2O2-induced oxidative stress and inflammation in rat vascular smooth muscle cells via inhibition of the NF-kappaB signaling pathway. Mol Med Rep 2016, 14(4):3559-3564.
Figure 1. The Morris water maze (MWM). After treatment with BBR (50 or 100mg/kg/d, ig) for 14 days, mice were allowed for the MWM task. The latency to find the platform during 7 days in the acquisition trials was recorded in the navigation training (Quadrant A). The BBR-treated mice exhibited lower escape latency on days 3, 4, 5, 6 and 7 during training trials (**p<0.05) (A, B and C). The number of times crossing the target quadrant (removed platform) and the time keeping in the target quadrant was analyzed (D and E). Values represented as mean±SEM (n=10). **p<0.05, compared with APP/PS1 transgenic control mice. The swimming speed had no difference between the three groups (F). The swimming tracks of mice searching for the underwater platform at the 7 training days were investigated (G). Figure 2 BBR inhibited phosphorylation of tau in the hippocampus of AD mice. BBR downregulated the levels of different phosphorylated tau (p-S199, p-S202, p-T205, p-T231, p-S396 and pS404) (**p<0.001 vs. the control group) in the hippocampus of AD mice. BBR: berberine; OD: optical density. Figure 3 BBR suppressed the activation of NF-κB signaling pathway in the hippocampus of AD mice. BBR decreased the expression of NF-κB elements (p50, p52, p65, c-Rel and RelB) (**p<0.001 vs. the control group) and increased the activity of IκB proteins (IκBα and IκBβ) (**p<0.001 vs. the control group) in the hippocampus of AD mice. BBR: berberine; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; OD: optical density. Figure 4 BBR enhances the activity of glutathione (GSH). BBR increased the biological activity of GPx-1/2, GSS and GR (**p<0.001 vs. the control group), while inhibited the GGT1 activity in the hippocampus of AD mice (**p<0.001 vs. the control group). BBR: berberine; GPx: Glutathione peroxidase; GR: Glutathione reductase; GSS: glutathione synthetase; GGT: γ-glutamyltranspeptidase; OD: optical density. Figure 5 BBR limited lipid peroxidation. BBR decreased the biological activity of MDA and LPO in the hippocampus of AD mice (**p<0.001 vs. the control group). BBR:berberine; LPO: lipid peroxide; MDA: malondialdehyde.
Figure 6 BBR inhibited neuro-inflammation. BBR decreased the biomarker levels of astrocyte and microglia (an anti-GFAP antibody for astrocytes and an anti-CD45 antibody for microglia: Western blot and immunohistochemistry) (**p<0.001 vs. the control group), and the levels of IL-1β and TNF-α (**p<0.001 vs. the control group) in the hippocampus of AD mice. BBR: berberine; GFAP: glial fibrillary acidic protein; IL-1β: interleukin 1 beta; TNF-α: tumor necrosis factor alpha; OD: optical density.
Figure 7 A possible mechanism for the role of BBR in inhibiting tau hyperphosphorylation. BBR inhibited the pathogenic cascade and the self-perpetuating cycle between NF-κB signaling, oxidative stress and neuroinflammation, improved cognitive impairment and limited tau hyperphosphorylation.
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S1734-1140(17)30257-8 http://dx.doi.org/doi:10.1016/j.pharep.2017.06.006 PHAREP 745
To appear in: Received date: Revised date: Accepted date:
7-4-2017 25-5-2017 13-6-2017
Please cite this article as: Wenbo He, Chuanlin Wang, Yi Chen, Yongli He, Zhiyou Cai, Berberine attenuates cognitive impairment and ameliorates tau hyperphosphorylation by limiting the self-perpetuating pathogenic cycle between NF-B signaling, oxidative stress and neuro-inflammation (2010), http://dx.doi.org/10.1016/j.pharep.2017.06.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Berberine attenuates cognitive impairment and ameliorates tau hyperphosphorylation by limiting the self-perpetuating pathogenic cycle between NF-κB signaling, oxidative stress and neuro-inflammation Wenbo He1, Chuanlin Wang1, Yi Chen1, Yongli He2, Zhiyou Cai3 1
Department of Neurology, Renmin Hospital, Hubei University of Medicine, Shiyan Renmin Hospital, Shiyan, 442000,
Hubei Province, China 2
Department of Internal Medicine, Chongqing Prevention and Treatment Center for Occupational Diseases, Chongqing,
400060, Chongqing, China 3
Department of Neurology, Chongqing General Hospital, Chongqing, 400013, Chongqing, China Correspondence to Yongli He and Zhiyou Cai Yongli He Department of Internal Medicine, Chongqing Prevention and Treatment Center for Occupational Diseases, No. 312 Nancheng Road, Nan-an District, Chongqing, People’s Republic of China. 400060, Phone: +86-23-61929106, Fax: +86-23-61929503 Email: [email protected] Zhiyou Cai Department of Neurology, Chongqing General Hospital, No. 312 Zhongshan First Road, Yuzhong District, Chongqing, People’s Republic of China. 400013, Phone: +86-23-63515796, Fax: +86-23-63515796 Email: [email protected]
Running title：Berberine ameliorates tau hyperphosphorylation by limiting NF-κB signaling Conflict of interest: None declared.
Abstract Background: Berberine (BBR) plays an important role in the prevention and treatment of Alzheimer’s disease (AD). The present work was to explore whether BBR ameliorates cognitive deficits in AD and to investigate whether its underlying mechanism involves inhibiting hyperphosphorylated tau protein. Methods: The cognitive function was measured by the Morris water maze (MWM) test. The levels of hyperphosphorylated tau proteins were determined by Western blot. The biomarkers of NF-κB signaling pathway and oxidative stress were detected by Western blot and biochemical assays. The biomarkers of neuroinflammation were determined by Western blot and immunohistochemistry. Results: BBR improved learning and memory in APP/PS1 mice. BBR decreased the hyperphosphorylated tau protein in the hippocampus of APP/PS1 mice. BBR lowered the activity of NF-κB signaling in the hippocampus of AD mice. BBR-administration promoted the activity of glutathione (GSH) and inhibited lipid peroxidation in the hippocampus of AD mice. Conclusion: BBR attenuated cognitive deficits and limited hyperphosphorylation of tau via inhibiting the activation of NF-κB signaling pathway, and by retarding oxidative stress and neuro-inflammation. Keywords: tau hyperphosphorylation; cognitive dysfunction; oxidative stress; inflammation; berberine
Introduction Alzheimer's disease (AD), the most prevalent form of dementia in the elderly, is an irreversible neurodegenerative disorder which is clinically featured with progressive cognitive deficits and dementia. AD is pathologically characterized by amyloid plaques, neurofibrillary tangles (NFTs) [1, 2]. To date, a variety of medications are used to prevent the progress of AD. Some of them may slow down memory loss, and improve communication skills, and help with certain behavioral problems. However, all the drugs had limited effects, or no effects with more sideeffects [3]. Until now, drugs that can be successfully used for AD treatment have not been developed. Berberine (BBR) is a yellow-colored alkaloid compound that can be extracted from several different plants, including European barberry, goldenseal, goldthread, Oregon grape phellodendron, and tree turmeric. It has been reported that BBR has therapeutic effects on metabolic disorders [4, 5], cardiovascular diseases [6], congestive heart failure [7], and neurodegenerative diseases [8]. Increasing evidence has also indicated its beneficial effect on stroke and various neurodegenerative and neuropsychiatric disorders [9, 10]. Substantial studies have demonstrated that BBR limits the pathogenesis of extracellular amyloid plaques and intracellular NFTs [11, 12]. BBR could potentially be developed as an effective therapeutic strategy for AD patients [13, 14]. However, the mechanism remains obscure. The aim of this study was to investigate the effects of BBR on tau hyperphosphorylation. As numerous results have supported the role of NF-κB signaling pathway, oxidative stress and neuro-inflammation in the pathogenesis of AD [11, 15], we analyzed the change of NF-κB signaling pathway, oxidative stress and neuro-inflammation in the hippocampus of APP/PS1 transgenic mice after BBR administration. Our results indicated that it is via inhibiting the activation of NF-κB signaling pathway, and by retarding oxidative stress and neuro-inflammation that BBR effectively attenuated cognitive impairment and limited tau hyperphosphorylation in an AD mouse model.
Materials and methods
Animals and drugs Thirty 120-day male APP/PS1 transgenic mice were used in the present study. The APP/PS1 transgenic mice were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences & Peking Union Medical College. They were housed in individual cages at a constant temperature (25°C) under a 12-h light-dark cycle and given free access to standard laboratory food and water. All mice were acclimated for at least 5 days before the experiments. The mice were randomly assigned into three groups: control group (n=10), 50mg/kg-BBR (n=10) and 100mg/kg-BBR (n=10). BBR (100 mg/tablet, Hangzhou Sanofi Minsheng Health Pharmaceutical Co. Ltd., China) was diluted to 0.5 mg/mL by normal saline. For the 50mg/kg-BBR group (50 mg/kg/d), mouse was given BBR through intragastric administration (ig) by 50 mg/kg/d for 14 days; for the 100mg/kg-BBR group (100 mg/kg/d), mouse was given 100 mg/kg/d also for 14 days. The BBR dosage was determined based on previous studies [16, 17]. Control group was administrated with the same volume saline. All the experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animal experiments followed the Research Ethics standards.
Morris water maze (MWM) Test The MWM test was performed according to the instrument instructions. The MWM test was implemented in the Basic Research Center of Hubei University of Medicine, including place navigation and spatial probe. The experimental procedures of the MWM test were recorded with Morris Image System (Shanghai DOiT Industrial Co., Ltd.). First, the mice were given three days to familiarize themselves familiar with the pool environment in a steady room temperature (20°C -24°C) and humidity (60–80%). This was followed by the MWM test with 4 trials per day. Then the acquisition trial for the mice was the last training trials to find the hidden platform at a target Quadrant A. The escape latency was recorded by the finding-
platform time. At Day 8, the mice were subjected to the probe trial to assess spatial memory. The time in the target quadrant and the times crossing the former platform area were recorded for testing spatial memory.
Lipid peroxidation assay The levels of malonaldehyde (MDA) and lipid peroxide (LPO) were measured with biochemical assay according to the manufacturer's instructions provided in the MDA and LPO assay kits (Nanjing Jiancheng Bioengineering Institute, China). Fifty microliter of sample or standard was placed into the microtiter plate wells and then mixed with 250 µL aqueous working reagent. Then samples were incubated for 30 min on a shaker and the absorbance was taken at 540 nm. The content of MDA and LPO was determined by comparison with a predetermined standard curve.
Western blot After the Morris water maze test, mice were anesthetized with pentobarbital, and then the brain was quickly removed, and the hippocampus tissues were dissected and homogenized on ice in T-PER buffer in the presence of protease inhibitors. After the samples were centrifuged at 5000g for 10 minutes at 4°C, the supernatant was collected and used for Western blot using Ciphergen (Biosource) protein chip arrays. Western blot was carried out according to the manufacturer’s instructions and previous studies [18, 19]. The p-S199, p-S202, p-T205, pT231, p-S396, p-S404, GPx-1/2, GSS, GGT1, GR, GFAP, CD45, IL-1β and TNF-α antibodies were purchased from Abcam, Cambridge, MA, USA. NF-κB elements (p50, p52, p65, c-Rel and RelB) and IκB proteins (IκBα and IκBβ) were obtained from Cell Signaling Technology, Inc. USA. Goat anti-mouse IgG labeled with biotin was from Santa Cruz Biotechnology, Inc. USA. The optical densities of the specific bands were scanned and measured by image analysis software (HPIAS 2000, Tongji Qianping Company, Wuhan, China). Rabbit anti-mouse β-actin (Santa Cruz Biotechnology, Inc. USA) was used as an internal control.
Immunohistochemical staining
Immunohistochemical staining was conducted as described in the previous study [20]. After deparaffination and rehydration of brain sections, antigen retrieval was performed by treatment with proteinase K for GFAP and CD45 staining. After the sections were blocked for nonspecific binding, the samples were incubated with monoclonal anti-GFAP and anti-CD45 antibody overnight. Primary antibody was measured with peroxidase-conjugated secondary antibody and detected with a stable diaminobenzidine solution (Vector Laboratories, Burlingame, CA, USA). Images were captured by using Olympus microscope connected to a digital microscope camera. All slides were tested in duplicate. Samples with a positive score over 10 or frequency over 5% were considered as positive. The percentage of area occupied by positive expression was calculated using the Image-Pro Plus imaging software from Media Cybernetics, Bethesda, MD, USA. Mean values for each parameter were recorded from six equidistant sections through the hippocampal region per mouse in each group.
Statistical analysis Quantitative data were expressed as the mean ± standard error of the mean (SEM). All statistical analyses were performed using the GraphPad Prism 6.0 software for Windows (GraphPad Software, La Jolla, CA, USA). For statistical evaluation of intergroup differences, paired t test was used, and one-way analyses of variance (ANOVA) were employed. Significant difference was considered at p<0.05.
Results BBR mitigated cognitive impairment of AD mice The latency to find the platform in the acquisition trials of the MWM task was shown in Figure 1A, B and C. The BBR-treated mice exhibited lower escape latency on days 3, 4, 5, 6 and 7 during training trials (p<0.05). However, there is no difference between BBR-treated groups (50mg and 100mg). In the probe trial, the mice were placed into the non-target quadrants respectively and allowed to swim freely for 60 seconds. The BBR-treated mice demonstrated a significant increase in the number of times crossing the target Quadrant A (Figure 1D, p<0.05)
the time staying in the target Quadrant A (Figure 1E, p<0.05). These results indicated that BBR improved the learning and memory capability of AD mice.
BBR inhibited phosphorylation of tau Hippocampal samples were obtained from AD mice to measure tau hyperphosphorylation in the hippocampus. We selected 6 phosphorylated tau monoclonal antibodies (phosphorylated at S199, S202, T205, T231, S396 and S404: p-S199, p-S202, p-T205, p-T231, p-S396 and pS404) to evaluate changes in tau protein. Western blot revealed a substantial decrease in the Tau-6 levels (p-S199, p-S202, p-T205, p-T231, p-S396 and p-S404) in the hippocampus of BBRtreated AD mice, compared to the control groups (p<0.001) (Figure 2).
BBR inhibits the activation of NF-κB signaling pathway The NF-κB family mainly includes five related transcription factors: p50, p52, RelA (p65), c-Rel and RelB. The activation of NF-κB is inhibited by IκB proteins in the cytoplasm. Figure 3 showed that BBR lowered the activity of p50, p52, p65, c-Rel and RelB in the hippocampus of AD mice. BBR improved the activity of IκB proteins (IκBα and IκBβ). These results implicated that BBR suppressed the activation of NF-κB signaling pathway in the brain of AD mice.
BBR enhanced the activity of glutathione BBR promoted the activity of glutathione (GSH) in the hippocampus of AD mice (Figure 4). BBR-administration up-regulated the level of GPx-1/2, GSS and GR in the hippocampus of AD mice, and reduced the activity of γ-glutamyltranspeptidase (GGT), which acts as a glutathionase and catalyzes glutathione into L-cysteinylglycine and L-glutamatethe, compared to control groups (Figure 4).
BBR limited lipid peroxidation Lipid peroxidation produces a wide variety of oxidation products such as malondialdehyde (MDA) and other lipid peroxide (LPO). Figure 5 showed that BBR decreased the activity of
MDA and LPO in the hippocampus of AD mice. The result implicated that BBR inhibited lipid peroxidation in the pathogenesis of AD.
BBR inhibited neuro-inflammation Gliocytes play an important role in the inflammatory process of AD. Increasing studies have reported that activated astrocytes and microglia are associated with extracellular amyloid peptide deposition, intracellular accumulation of hyperphosphorylated tau protein (formation of NFTs), neuro-inflammation and oxidative stress. To investigate whether BBR treatment has anti-inflammatory effects in the hippocampus of AD mice, we measured the biomarkers of astrocyte and microglia reactivity with an anti-GFAP antibody for astrocytes and an anti-CD45 antibody for microglia. Results indicate BBR significantly decreased both GFAP and CD45 immunoreactivity in the hippocampus of AD mice when compared with those in the control mice (Figure 6A, B). In order to clarify the anti-inflammatory role of BBR in AD, we detected the levels of IL-1β and TNF-α, the classic inflammatory biomarkers. Results demonstrate that BBR down-regulated the levels of IL-1β and TNF-α in the hippocampus of AD mice, when compared to the control groups (Figure 6A).
Discussion NFTs are aggregates of hyperphosphorylated tau protein that are most commonly known as a primary hallmark of AD. In the present study, we demonstrated that BBR-treatment strongly decreased tau hyperphosphorylation in the hippocampus of APP/PS1 mice, which resulted in improvement of cognitive deficits. Importantly, we found that BBR markedly inhibited the activation of NF-κB signaling pathway and counteracted the progress of oxidative stress and ameliorated tau hyperphosphorylation, while BBR enhanced the activity of GSH, and limited lipid peroxidation. Furthermore, this study demonstrates that BBR retarded neuroinflammation by inhibiting the activation of astrocytes and microglia and down-regulating the levels of IL-1β and TNF-α (the classic inflammatory biomarkers) in the hippocampus of AD mice. All of these parameters indicated that it is via inhibiting the activation of NF-κB signaling pathway, and by retarding its related oxidative stress and neuro-inflammation that BBR
effectively attenuated cognitive impairment and limited tau hyperphosphorylation in an AD mice model (APP/PS1 transgenic mice). NF-κB signaling pathway plays an exceptionally important role due to its pleiotropic effects, its unique regulatory mechanisms, and large number of activating signaling pathways and number of genes that it controls. Activation of NF-κB is association with many diseases, such as cardiovascular disease, diabetes, and neurodegenerative diseases [21, 22]. Several studies have highlighted the progress on the role of NF-κB signaling pathway in AD [22, 23]. It has been found that activated NF-κB contributed to Cdk5/p25-induced phosphorylated tau [24]. NF-κB family mainly includes five related transcription factors: p50, p52, RelA (p65), c-Rel and RelB. We found that BBR increased the levels of IκB proteins (IκBα and IκBβ) which inhibit the activation of NF-κB, whereas BBR decreased the levels of p50, p52, p65, c-Rel and RelB, respectively, in the hippocampus of APP/PS1 mice. Oxidative stress and neuro-inflammation are major pathogenesis of AD leading to neurological sequelae [25]. Oxidative stress is essentially an imbalance between the production of reactive oxygen species (ROS) and the ability of the body to counteract their harmful effects through neutralization by antioxidants. Glutathione (GSH), an important antioxidant, prevents tissue damage from oxidative stress induced by ROS including free radicals, peroxides and lipid peroxides [26]. Glutathione peroxidase (GPx), a free radical scavenger, plays an important role in the antioxidant defense of central nervous system in response to oxidative stress [27, 28]. BBR-administration increased the level of GPx-1/2 in the hippocampus of AD mice. Glutathione synthetase (GSS) induces an increase in glutathione levels in brain [29]. There was also a markedly increase in GSS in the hippocampus of AD mice after the intervention of BBR. Glutathione reductase (GR) converts oxidized GSH to the reduced form and hold high levels of reduced glutathione in the cytosol of neural cells [30, 31]. BBR-treatment dramatically promotes the biological activity of GR in the hippocampus of AD mice. Additionally, BBR lowered the activity of GGT which acts as a glutathionase and catalyzes glutathione into Lcysteinylglycine and L-glutamatethe. Anti-oxidative enzymes, such as GSH, GPx, catalase, and superoxide dismutase (SOD), are essential to prevent cells from oxidative damage [32, 33]. An
antioxidant activity of BBR has been assumed in our studies in which neuro-protection was demonstrated in AD mice. Our results found that BBR improved the biological activity of GPx1/2, GSS and GR, while reduced the GGT1 activity in the hippocampus of AD mice. Therefore, the anti-oxidative property of BBR in the process of AD pathogenesis has an association with its modifying GSH enzymes and enhancing the activity of GSH. We found a highly significant anti-oxidative property of BBR via modifying antioxidant enzymes and enhancing the activity of antioxidants. Lipid peroxidation is considered as a process of the oxidative degradation of lipids containing any number of carbon-carbon double bonds [34]. Lipid peroxidation generates multiple oxidation products, including MDA and LPO. Numerous studies have evidenced a crucial role of lipid peroxidation in the process of AD pathogenesis [35, 36]. Our study showed that BBR limited the deleterious activity of MDA and LPO in the hippocampus of AD mice. Therefore, such an anti-oxidative property of BBR is reflected by its ability to limit lipid peroxidation. Neuro-inflammation, a common feature of neurodegenerative diseases, plays an important role in the progression of AD and may be responsible for degeneration in vulnerable regions such as the hippocampus [37, 38]. Activated microglia and astrocytes contribute to extracellular amyloid peptide deposition, intracellular accumulation of hyperphosphorylated tau protein (formation of NFTs), neuro-inflammation and oxidative stress [39-41]. We measured the GFAP biomarker for astrocyte and CD45 for microglia. BBR strongly reduced the levels of both GFAP and CD45 in the hippocampus of AD mice compared with the control mice. Moreover, we also investigated the levels of IL-1β and TNF-α (the classic inflammatory biomarkers). The result demonstrated that BBR lowered the levels of IL-1β and TNF-α in the hippocampus of AD mice. The self-perpetuating cycle of chronic neuro-inflammation and oxidative stress may contribute to irreversible neuronal dysfunction, acting as an important feature of AD pathogenesis [42, 43]. It has been reported that interaction between oxidative stress and neuro-inflammation is strongly associated with NF-κB signaling pathway while NF-κB signaling interacts with both oxidative stress and neuro-inflammation [44-47]. NF-κB signaling, oxidative stress and inflammation are composed of an important pathogenic cascade of neuro-degeneration in AD,
suggesting that therapeutic efforts aimed at the pathogenic cascade may be beneficial for AD treatment and prevention. The present study demonstrated that BBR was conducive to the decrease in the biological activity of NF-κB signaling, oxidative stress and inflammation in the hippocampus of AD mice. Taken together, attenuating cognitive impairment and inhibiting tau hyperphosphorylation by BBR are related to its ability in limiting the pathogenic cascade and the self-perpetuating cycle between NF-κB signaling, oxidative stress and neuro-inflammation (Figure 7). In summary, the current study shows the neuro-protective property of BBR against AD lies with its ability to inhibit NF-κB signaling, oxidative stress and inflammation, to lower tau hyperphosphorylation, and to attenuate cognitive deficits. From clinical point of view, the ability of BBR to modulate oxidative stress and inflammation and to regulate NF-κB signaling pathway, may be of great importance in neuro-protection in AD treatment and prevention. For the clinical practice, the strategy to use BBR for brain protection would provide great advantages for AD patients. However, it would be important to precisely define the effects of BBR on AD brains. Hence, further studies elucidating BBR neuroprotective mechanisms may provide insights into novel strategies for AD treatments.
Acknowledgments: This work was supported by grants from Chongqing General Hospital (CGHK201701), the Natural Science Foundation of Hubei Province (2015CFB260), and the Hubei Province Health and Family Planning Scientific Research Project (WJ2015MB219) and the Shiyan Natural Science Foundation (15K70).
References 1. Carreiras MC, Mendes E, Perry MJ, Francisco AP, Marco-Contelles J: The multifactorial nature of Alzheimer's disease for developing potential therapeutics. Curr Top Med Chem 2013, 13(15):1745-1770.
2. Galimberti D, Scarpini E: Alzheimer's disease: from pathogenesis to disease-modifying approaches. CNS Neurol Disord Drug Targets 2011, 10(2):163-174. 3. Mancuso C, Siciliano R, Barone E, Butterfield DA, Preziosi P: Pharmacologists and Alzheimer disease therapy: to boldly go where no scientist has gone before. Expert Opin Investig Drugs 2011, 20(9):1243-1261. 4. Caliceti C, Franco P, Spinozzi S, Roda A, Cicero AF: Berberine: New Insights from Pharmacological Aspects to Clinical Evidences in the Management of Metabolic Disorders. Curr Med Chem 2016, 23(14):1460-1476. 5. Pirillo A, Catapano AL: Berberine, a plant alkaloid with lipid- and glucose-lowering properties: From in vitro evidence to clinical studies. Atherosclerosis 2015, 243(2):449-461. 6. Jin Y, Khadka DB, Cho WJ: Pharmacological effects of berberine and its derivatives: a patent update. Expert Opin Ther Pat 2016, 26(2):229-243. 7. Chang W, Li K, Guan F, Yao F, Yu Y, Zhang M, et al. Berberine Pretreatment Confers Cardioprotection Against Ischemia-Reperfusion Injury in a Rat Model of Type 2 Diabetes. J Cardiovasc Pharmacol Ther. 2016;21(5):486494. 8. Jiang W, Li S, Li X: Therapeutic potential of berberine against neurodegenerative diseases. Sci China Life Sci 2015, 58(6):564-569. 9. Friedemann T, Schumacher U, Tao Y, Leung AK, Schroder S: Neuroprotective Activity of Coptisine from Coptis chinensis (Franch). Evid Based Complement Alternat Med 2015, 2015:827308. 10. Simoes Pires EN, Frozza RL, Hoppe JB, Menezes Bde M, Salbego CG: Berberine was neuroprotective against an in vitro model of brain ischemia: survival and apoptosis pathways involved. Brain Res 2014, 1557:26-33. 11. Cai Z, Wang C, Yang W: Role of berberine in Alzheimer's disease. Neuropsychiatr Dis Treat 2016, 12:25092520. 12. de Oliveira JS, Abdalla FH, Dornelles GL, Adefegha SA, Palma TV, Signor C, et al: Berberine protects against memory impairment and anxiogenic-like behavior in rats submitted to sporadic Alzheimer's-like dementia: Involvement of acetylcholinesterase and cell death. Neurotoxicology 2016, 57:241-250. 13. Mak S, Luk WW, Cui W, Hu S, Tsim KW, Han Y: Synergistic inhibition on acetylcholinesterase by the combination of berberine and palmatine originally isolated from Chinese medicinal herbs. J Mol Neurosci 2014, 53(3):511-516. 14. Kim MH, Kim SH, Yang WM: Mechanisms of action of phytochemicals from medicinal herbs in the treatment of Alzheimer's disease. Planta Med 2014, 80(15):1249-1258. 15. Kaur U, Banerjee P, Bir A, Sinha M, Biswas A, Chakrabarti S: Reactive oxygen species, redox signaling and neuroinflammation in Alzheimer's disease: the NF-kappaB connection. Curr Top Med Chem 2015, 15(5):446457.
16. Zhu F, Qian C: Berberine chloride can ameliorate the spatial memory impairment and increase the expression of interleukin-1beta and inducible nitric oxide synthase in the rat model of Alzheimer's disease. BMC Neurosci 2006, 7:78. 17. Patil S, Tawari S, Mundhada D, Nadeem S: Protective effect of berberine, an isoquinoline alkaloid ameliorates ethanol-induced oxidative stress and memory dysfunction in rats. Pharmacol Biochem Behav 2015, 136:1320. 18. Meng F, Wang J, Ding F, Xie Y, Zhang Y, Zhu J: Neuroprotective effect of matrine on MPTP-induced Parkinson's disease and on Nrf2 expression. Oncol Lett 2017, 13(1):296-300. 19. Cai Z, Yan Y, Wang Y: Minocycline alleviates beta-amyloid protein and tau pathology via restraining neuroinflammation induced by diabetic metabolic disorder. Clin Interv Aging 2013, 8:1089-1095. 20. Zhao Y, Xiao M, He W, Cai Z: Minocycline upregulates cyclic AMP response element binding protein and brainderived neurotrophic factor in the hippocampus of cerebral ischemia rats and improves behavioral deficits. Neuropsychiatr Dis Treat 2015, 11:507-516. 21. Vicente Miranda H, Outeiro TF: The sour side of neurodegenerative disorders: the effects of protein glycation. J Pathol 2010, 221(1):13-25. 22. Salminen A, Kaarniranta K, Haapasalo A, Hiltunen M, Soininen H, Alafuzoff I: Emerging role of p62/sequestosome-1 in the pathogenesis of Alzheimer's disease. Prog Neurobiol 2012, 96(1):87-95. 23. Kitazawa M, Cheng D, Tsukamoto MR, Koike MA, Wes PD, Vasilevko V, et al: Blocking IL-1 signaling rescues cognition, attenuates tau pathology, and restores neuronal beta-catenin pathway function in an Alzheimer's disease model. J Immunol 2011, 187(12):6539-6549. 24. Sun LH, Ban T, Liu CD, Chen QX, Wang X, Yan ML, et al: Activation of Cdk5/p25 and tau phosphorylation following chronic brain hypoperfusion in rats involves microRNA-195 down-regulation. J Neurochem 2015, 134(6):1139-1151. 25. Chen SD, Yin JH, Hwang CS, Tang CM, Yang DI: Anti-apoptotic and anti-oxidative mechanisms of minocycline against sphingomyelinase/ceramide neurotoxicity: implication in Alzheimer's disease and cerebral ischemia. Free Radic Res 2012, 46(8):940-950. 26. Rodriguez VM, Del Razo LM, Limon-Pacheco JH, Giordano M, Sanchez-Pena LC, Uribe-Querol E, et al.: Glutathione reductase inhibition and methylated arsenic distribution in Cd1 mice brain and liver. Toxicol Sci 2005, 84(1):157-166. 27. Comparsi B, Meinerz DF, Franco JL, Posser T, Prestes Ade S, Stefanello ST, et al: Diphenyl ditelluride targets brain selenoproteins in vivo: inhibition of cerebral thioredoxin reductase and glutathione peroxidase in mice after acute exposure. Mol Cell Biochem 2012, 370(1-2):173-182. 28. Dudek H, Farbiszewski R, Michno T, Kozlowski A: [Activity of glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R) and superoxide dismutase (SOD-1) in single brain metastasis]. Wiad Lek 2002, 55(56):252-256.
29. Kang Y, Viswanath V, Jha N, Qiao X, Mo JQ, Andersen JK: Brain gamma-glutamyl cysteine synthetase (GCS) mRNA expression patterns correlate with regional-specific enzyme activities and glutathione levels. J Neurosci Res 1999, 58(3):436-441. 30. Singh R, Pathak DN: Lipid peroxidation and glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase activities in FeCl3-induced epileptogenic foci in the rat brain. Epilepsia 1990, 31(1):15-26. 31. Uchoa MF, de Souza LF, Dos Santos DB, Peres TV, Mello DF, Leal RB, et al: Modulation of Brain Glutathione Reductase and Peroxiredoxin 2 by alpha-Tocopheryl Phosphate. Cell Mol Neurobiol 2016, 36(6):1015-1022. 32. Kumar R, Kaur R, Singh AP, Arora S: Diminution of hepatic response to 7, 12-dimethylbenz(alpha)anthracene by ethyl acetate fraction of Acacia catechu willd. through modulation of xenobiotic and anti-oxidative enzymes in rats. PLoS One 2014, 9(2):e90083. 33. Nikolic-Kokic A, Stevic Z, Blagojevic D, Davidovic B, Jones DR, Spasic MB: Alterations in anti-oxidative defence enzymes in erythrocytes from sporadic amyotrophic lateral sclerosis (SALS) and familial ALS patients. Clin Chem Lab Med 2006, 44(5):589-593. 34. Ayala A, Munoz MF, Arguelles S: Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxid Med Cell Longev 2014, 2014:360438. 35. Caliskan M, Mogulkoc R, Baltaci AK, Menevse E: The Effect of 3',4'-Dihydroxyflavonol on Lipid Peroxidation in Rats with Cerebral Ischemia Reperfusion Injury. Neurochem Res 2016, 41(7):1732-1740. 36. Muralikrishna Adibhatla R, Hatcher JF: Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ischemia. Free Radic Biol Med 2006, 40(3):376-387. 37. Ardestani PM, Evans AK, Yi B, Nguyen T, Coutellier L, Shamloo M: Modulation of neuroinflammation and pathology in the 5XFAD mouse model of Alzheimer's disease using a biased and selective beta-1 adrenergic receptor partial agonist. Neuropharmacology 2017, 116:371-386. 38. Popp J, Oikonomidi A, Tautvydaite D, Dayon L, Bacher M, Migliavacca E, et al: Markers of neuroinflammation associated with Alzheimer's disease pathology in older adults. Brain Behav Immun 2017, 62:203-211. 39. Goetzl EJ, Mustapic M, Kapogiannis D, Eitan E, Lobach IV, Goetzl L, et al: Cargo proteins of plasma astrocytederived exosomes in Alzheimer's disease. FASEB J 2016, 30(11):3853-3859. 40. Peters O, Schipke CG, Philipps A, Haas B, Pannasch U, Wang LP, et al: Astrocyte function is modified by Alzheimer's disease-like pathology in aged mice. J Alzheimers Dis 2009, 18(1):177-189. 41. Baker C, Nielsen HM, Minthon L, Wright HT, Chappell S, Okyere J, et al: Effects of Alzheimer's peptide and alpha1-antichymotrypsin on astrocyte gene expression. Neurobiol Aging 2007, 28(1):51-61. 42. Vaziri ND, Rodriguez-Iturbe B: Mechanisms of disease: oxidative stress and inflammation in the pathogenesis of hypertension. Nat Clin Pract Nephrol 2006, 2(10):582-593.
43. Ortiz GG, Pacheco-Moises FP, Bitzer-Quintero OK, Ramirez-Anguiano AC, Flores-Alvarado LJ, Ramirez-Ramirez V, et al: Immunology and oxidative stress in multiple sclerosis: clinical and basic approach. Clin Dev Immunol 2013, 2013:708659. 44. Raish M: Momordica charantia polysaccharides ameliorate oxidative stress, hyperlipidemia, inflammation, and apoptosis during myocardial infarction by inhibiting the NF-kappaB signaling pathway. Int J Biol Macromol 2017, 97:544-551. 45. Zhong W, Qian K, Xiong J, Ma K, Wang A, Zou Y: Curcumin alleviates lipopolysaccharide induced sepsis and liver failure by suppression of oxidative stress-related inflammation via PI3K/AKT and NF-kappaB related signaling. Biomed Pharmacother 2016, 83:302-313. 46. Wang YR, Tian FL, Yan MX, Fan JH, Wang LY, Kuang RG, et al: Sulfasalazine inhibits inflammation and fibrogenesis in pancreas via NF-kappaB signaling pathway in rats with oxidative stress-induced pancreatic injury. Drug Des Devel Ther 2016, 10:1743-1751. 47. Wang YL, Sun GY, Zhang Y, He JJ, Zheng S, Lin JN: Tormentic acid inhibits H2O2-induced oxidative stress and inflammation in rat vascular smooth muscle cells via inhibition of the NF-kappaB signaling pathway. Mol Med Rep 2016, 14(4):3559-3564.
Figure 1. The Morris water maze (MWM). After treatment with BBR (50 or 100mg/kg/d, ig) for 14 days, mice were allowed for the MWM task. The latency to find the platform during 7 days in the acquisition trials was recorded in the navigation training (Quadrant A). The BBR-treated mice exhibited lower escape latency on days 3, 4, 5, 6 and 7 during training trials (**p<0.05) (A, B and C). The number of times crossing the target quadrant (removed platform) and the time keeping in the target quadrant was analyzed (D and E). Values represented as mean±SEM (n=10). **p<0.05, compared with APP/PS1 transgenic control mice. The swimming speed had no difference between the three groups (F). The swimming tracks of mice searching for the underwater platform at the 7 training days were investigated (G). Figure 2 BBR inhibited phosphorylation of tau in the hippocampus of AD mice. BBR downregulated the levels of different phosphorylated tau (p-S199, p-S202, p-T205, p-T231, p-S396 and pS404) (**p<0.001 vs. the control group) in the hippocampus of AD mice. BBR: berberine; OD: optical density. Figure 3 BBR suppressed the activation of NF-κB signaling pathway in the hippocampus of AD mice. BBR decreased the expression of NF-κB elements (p50, p52, p65, c-Rel and RelB) (**p<0.001 vs. the control group) and increased the activity of IκB proteins (IκBα and IκBβ) (**p<0.001 vs. the control group) in the hippocampus of AD mice. BBR: berberine; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; OD: optical density. Figure 4 BBR enhances the activity of glutathione (GSH). BBR increased the biological activity of GPx-1/2, GSS and GR (**p<0.001 vs. the control group), while inhibited the GGT1 activity in the hippocampus of AD mice (**p<0.001 vs. the control group). BBR: berberine; GPx: Glutathione peroxidase; GR: Glutathione reductase; GSS: glutathione synthetase; GGT: γ-glutamyltranspeptidase; OD: optical density. Figure 5 BBR limited lipid peroxidation. BBR decreased the biological activity of MDA and LPO in the hippocampus of AD mice (**p<0.001 vs. the control group). BBR:berberine; LPO: lipid peroxide; MDA: malondialdehyde.
Figure 6 BBR inhibited neuro-inflammation. BBR decreased the biomarker levels of astrocyte and microglia (an anti-GFAP antibody for astrocytes and an anti-CD45 antibody for microglia: Western blot and immunohistochemistry) (**p<0.001 vs. the control group), and the levels of IL-1β and TNF-α (**p<0.001 vs. the control group) in the hippocampus of AD mice. BBR: berberine; GFAP: glial fibrillary acidic protein; IL-1β: interleukin 1 beta; TNF-α: tumor necrosis factor alpha; OD: optical density.
Figure 7 A possible mechanism for the role of BBR in inhibiting tau hyperphosphorylation. BBR inhibited the pathogenic cascade and the self-perpetuating cycle between NF-κB signaling, oxidative stress and neuroinflammation, improved cognitive impairment and limited tau hyperphosphorylation.
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