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Neuroprotective Effects of BHDPC, a Novel Neuroprotectant, on Experimental Stroke by Modulating Microglia Polarization Chuwen Li, Yaqi Bian, Yu Feng, Fan Tang, Liang Wang, Maggie Pui Man Hoi, Dan Ma, Chao Zhao, and Simon Ming-Yuen Lee ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00713 • Publication Date (Web): 06 Mar 2019 Downloaded from http://pubs.acs.org on March 7, 2019
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ACS Chemical Neuroscience
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Title Page
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Neuroprotective Effects of BHDPC, a Novel Neuroprotectant, on Experimental
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Stroke by Modulating Microglia Polarization
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Chuwen Li,†,§ Yaqi Bian,§ Yu Feng,§ Fan Tang,§ Liang Wang,§ Maggie Pui Man Hoi,§
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Dan Ma,‡ Chao Zhao,‡ and Simon Ming Yuen Lee*§
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† Key Laboratory of Molecular Target & Clinical Pharmacology, School of
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Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou, China
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§ State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese
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Medical Sciences, University of Macau, Macau, China
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‡ Department of Clinical Neurosciences, Wellcome Trust-MRC Cambridge Stem Cell
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Institute, University of Cambridge, Cambridge CB2 0AH, UK
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* To whom correspondence should be addressed:
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Simon Ming-Yuen Lee, Ph.D., Professor (Full)
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Institute of Chinese Medical Sciences, University of Macau
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Tel: (+853) 8822-4695; Fax: (+853) 8822-1358
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E-mail:
[email protected] 1 ACS Paragon Plus Environment
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ABSTRACT
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This study mainly aimed to investigate the therapeutic effects of BHDPC on ischemic
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stroke and its underlying mechanisms. In vivo, the transient middle cerebral artery
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occlusion (MCAO) was used to induce ischemic model. In vitro, oxygen and glucose
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deprivation/reperfusion (OGD/R)-induced ischemic stroke in BV-2 microglia, primary
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neurons, and b.End3 mouse cerebral microvascular endothelial cells (ECs) were also
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performed. Firstly, we found that BHDPC exerts considerable neuroprotection against
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MCAO-induced ischemic injury mice, via alleviating neurological deficits and brain
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infarcts, inhibiting neuronal cells loss and apoptosis, and attenuating blood-brain barrier
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(BBB) disruption and tight junction proteins changes. Next, we observed that BHDPC
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could significantly reduce microglial M1 activation but enhances M2 polarization in
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MCAO-induced ischemic brain. Further experiments in vitro indicated that BHDPC
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suppressed microglial activation but promotes M2 microglial polarization in OGD/R-
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induced BV-2 microglia. In addition, CM experiments showed that CM from BHDPC-
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treated BV-2 microglia provided protectons against OGD/R-induced ischemic damage
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in primary neurons and bEnd.3 ECs. Moreover, we found that BHDPC actions on
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microglial inflammation were associated with the inactivation of NF- κ B signaling.
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Interestingly, we also found that BHDPC enhanced phosphorylation of PKA and CREB.
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The pharmacological inhibition or gene knockdown of PKA/CREB signaling
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diminished BHDPC-promoted microglial M2 polarization. In summary, BHDPC
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conferred neuroprotection against ischemic injury in experimental stroke models. 2 ACS Paragon Plus Environment
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Modulating microglial activation and polarization contributes to BHDPC-mediated
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neuroprotective actions, which, in part were mediated by NF-κB and PKA/CREB
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signaling pathway.
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KEYWORDS
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BHDPC; Ischemic Stroke; Microglia; NF-κB; CREB; PKA
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INTRODUCTION
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Cerebral ischemic stroke, contributing to the most population of total stroke incidence,
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is a major cause of death and disability worldwide
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effective agents for ischemic stroke treatments in the clinic. Ischemic stroke lesions
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result in neuronal function losses, multiple tissues damages, and inflammatory
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responses. Currently, modulating inflammatory processes has been considered as one
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of the promising therapeutic approaches for ischemic stroke 1-2. Accumulating evidence
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has well established activated microglia as major cellular elements of inflammatory
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processes, which execute specific immune functions to maintain physiological
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homeostasis 3-5. Once activated, microglia can be phenotypically polarized into either a
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classical (pro-inflammatory; M1) or an alternative (anti-inflammatory; M2) phenotype
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6-10.
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that exacerbate neuronal injury and impede cellular repair in ischemic brains, whereas
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M2 phenotype produces anti-inflammatory cytokines and neurotrophic factors that
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confer neuroprotection and promote recovery and remodeling after ischemic lesions 6-
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12.
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microglia is a promising target for ischemic stroke treatments 10-14.
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The transcription factor nuclear factor kappa B (NF-κB) is a crucial modulator of
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various kinds of pro-inflammatory mediators and cytokines, which is closely involved
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in microglia-mediated inflammatory processes 12, 15-16. Suppression of NF-κB activation
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is an action of many well-known anti-inflammatory agents and is also a well-reported
1-2.
However, there is still no
Generally, M1 state microglia produce pro-inflammatory cytokines and mediators
Therefore, the combination of inhibiting the M1 phenotype and promoting the M2
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target for ischemic stroke treatments
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protein (CREB) is one of the best understood phosphorylation-dependent cellular
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transcription factors
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survival, neural differentiation, and neurite outgrowth
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mainly modulated by the cAMP-dependent protein kinase A (PKA) promote via
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promoting CREB the phosphorylation of CREB at its transcription-activating site 11, 22-
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24.
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factors in activated microglia, which are typically involved in microglia de-activation
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and microglia polarization into the M2 phenotype
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signaling has also been considered as an attractive target for treatments of inflammation
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in ischemic stroke 10-14.
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In our previous studies, we discovered a pyrimidine derivative, benzyl 7-(4-hydroxy-3-
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methoxyphenyl)-5-methyl-4,7-dihydrotetrazolo
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(BHDPC, ChemBridge ID: 7989205; the chemical structure is shown in Figure 1A) that
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can prevent chemical-induced cerebral hemorrhage injury using docking-based virtual
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screening and zebrafish models 11, 26-27. In addition, we found that BHDPC could confer
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neuroprotective actions in neurotoxin-induced Parkinson’s disease (PD) models, which
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was closely associated with BHDPC-mediated phosphorylation of PKA/CREB
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signaling
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considerable anti-neuroinflammatory effects in lipopolysaccharide (LPS)-stimulated
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BV-2 microglia
10, 19-21.
12, 17-18.
The cAMP-response element-binding
CREB mediates genes closely associated with neuronal 10, 19-21.
The CREB activity is
The PKA/CREB signaling regulates expressions of anti-inflammatory cytokines and
28.
2, 24-25.
[1,5–a]
Therefore, the PKA/CREB
pyrimidine-6-carboxylate
Moreover, our previous study also confirmed that BHDPC provided
11.
Considering its potential effects in the nervous system, further 5 ACS Paragon Plus Environment
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explorations are necessary for BHDPC in ischemic stroke and related disorders.
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Therefore, in the current study, we aimed to investigate the therapeutic effects of
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BHDPC on ischemic stroke models in vitro and in vivo. Moreover, the protective effects
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of BHDPC in ischemic stroke-induced neuroinflammatory responses were further
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investigated. Finally, the role of BHDPC in NF-κB and PKA/CREB signaling was also
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explored.
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RESULTS AND DISCUSSION
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Our previous studies have indicated a potential relationship between BHDPC and
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cerebral ischemic injury neurotoxicity
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performed experiments in vivo and in vitro to investigate BHDPC actions on ischemic
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stroke and to explore underlying mechanisms. In vivo, the transient middle cerebral
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artery occlusion (MCAO) was used to induce ischemic model. In vitro, oxygen and
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glucose deprivation/reperfusion (OGD/R)-induced ischemic stroke in BV-2 microglia,
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primary neurons, and b.End3 mouse cerebral microvascular endothelial cells (ECs)
11, 26-28.
Therefore, in the current study, we
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were also performed.
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In summary, the key findings in this study included (1) BHDPC could confer potent
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therapeutic effects on ischemic stroke in mice by inhibiting neuronal death,
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ameliorating BBB impairment and promoting microglial M1-M2 phenotype
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polarization; (2) HHDPC inhibited M1 microglial inflammation and modulating M2
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microglial polarization in OGD/R-induced BV-2 microglia, which in part contributed 6 ACS Paragon Plus Environment
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to its protective effects on OGD/R neurons and endothelial cells; (3) BHDPC
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modulated microglial polarization by inhibiting NF-κB activation but promoting
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PKA/CREB phosphorylation, which is most likely to be attributable to its
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neuroprotection. These findings suggest that BHDPC may constitute a new agent for
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treating ischemic stroke.
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BHDPC exerts neuroprotective effects against MCAO-induced neurological
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deficits and brain infarcts.
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MCAO-induced rodents are well-known cerebral ischemia models to evaluate the
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progression of ischemic stroke and develop novel neuroprotectant for stroke treatments
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10-14.
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MCAO with the postischemic treatment of BHDPC. Firstly, we observed that that
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BHDPC (2.5, 10, and 40 mg/kg) did not affect cerebral blood flow (CBF) and relevant
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physiological parameters in MCAO mice in current experimental conditions
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(Supporting Information, Table S1). Ischemic insults lead to neurological deficits in
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MCAO animals 10-14. Thus, behavior function tests, including mNSS testing, foot-fault
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test, adhesion-removal test, and inclined plane test were performed at 24 h after MCAO
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surgery, to evaluate the effect of BHDPC on neurological functions. As shown in Figure
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2A, mNSS testing demonstrated that treatment with BHDPC at doses of 10 and 40
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mg/kg could significantly ameliorate total neurological deficit scores and improve
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neurological functions when compared to the vehicle groups, respectively (both p
