Neuroprotective Effect of Swertiamain on Cerebral Ischemia

Subscriber access provided by Macquarie University

Article

Neuroprotective Effect of Swertiamain on Cerebral Ischemia/ Reperfusion Injury by Inducing the Nrf2 Protective Pathway Hui Wang, Wei Wei, Xiaobing Lan, Ning Liu, Yuxiang Li, Hanxiang Ma, Tao Sun, Xiaodong Peng, Chunlin Zhuang, and Jian Qiang Yu ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00605 • Publication Date (Web): 12 Feb 2019 Downloaded from http://pubs.acs.org on February 13, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

Neuroprotective Effect of Swertiamain on Cerebral Ischemia/Reperfusion Injury by Inducing the Nrf2 Protective Pathway Hui Wanga,1, Wei Weia,1, Xiaobing Lana, Ning Liua,b, Yuxiang Lic, Hanxiang Mad, Tao Sune, Xiaodong Penga, *, Chunlin Zhuangb,f,* , Jianqiang Yua,b,e,* a.

Department of Pharmacology, College of Pharmacy, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region 750004, P.R. China

b.

Ningxia Hui Medicine Modern Engineering Research Center and Collaborative Innovation Center, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region 750004, P.R. China

c.

College of Nursing, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region 750004, P.R. China

d.

Department of Anesthesiology, General Hospital of Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region 750004, P.R. China

e.

Ningxia Key Laboratory of Craniocerebral Diseases of Ningxia Hui Autonomous Region, Ningxia Medical University, Yinchuan, Ningxia Hui Autonomous Region 750004, P.R. China

f.

School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, P.R. China

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 29

ABSTRACT Oxidative stress plays a vital role in the development of cerebral ischemic/reperfusion (I/R). Targeting oxidative stress is proposed to be an effective strategy to treat cerebral I/R injury. Gentiana macrophylla Pall is reported to have potential protective effect against stroke. Swertiamarin (Swe), an active secoiridoid glycoside compound isolated from Gentiana macrophylla Pall, has been reported to possess the antioxidative potential. This study is to explore whether Swe could prevent brain from I/R injury and the related mechanisms of oxidative stress is also elucidated using mice middle cerebral artery occlusion (MCAO) model and primary hippocampal neurons oxygen-glucose deprivation/reperfusion (OGD/R) model. Swe (25, 100, or 400 mg/kg) was pre-treated intraperitoneally for 7 days until establishment of the MCAO model, while hippocampal neurons were maintained in Swe (0.1, 1 or 10 μM) in the entire process of reoxygenation. The results indicated that Swe pre-treatment markedly decreased infarct volume, apoptotic neurons and oxidative damage and promoted neurologic recovery in vivo. It also decreased reactive oxygen species (ROS) and increased cell viability in vitro. Western blot analyses and immunofluorescence staining demonstrated that Swe pre-treatment promoted Nrf2 nuclear translocation from Keap1-Nrf2 complex and enhanced the expressions of NAD(P)H: quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO-1) both in vivo and in vitro, while the expressions could be reversed by a Nrf2 inhibitor. The binding mode of Keap1 with Swe was also proposed by covalent molecular docking. Collectively, Swe could be considered as a promising protective agent against cerebral I/R injury through suppressing oxidative stress by activation of the Nrf2 protective pathway.

KEYWORDS: Swertiamarin; Cerebral ischemic/reperfusion; Neuroprotection; Stroke; Oxidative stress; Nrf2


Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphical Table of Contents



Page 4 of 29

INTRODUCTION Stroke has become a major public health problem with high death and disability in the world.1, 2 About 87% of all strokes belongs to ischemic stroke.3,

4

Restoring the blood flow as soon as possible is the main treatment for this

condition. However, cerebral ischemia-reperfusion injury (CIRI) is a main risk of the treatment.5, 6 Therefore, it is of great importance to find an effective strategy to prevent ischemia/reperfusion (I/R) injury. Oxidative stress is recognized to be closely related to CIRI in stroke. During CIRI, reactive oxygen species (ROS) production is greatly increased. Then, the excessive ROS causes the inactivation of antioxidant enzymes, energy production disorder, mitochondrial dysfunction, and alterations in homeostasis, finally, leading to neuronal death.7 Thus, targeting oxidative stress has been investigated as a strategy to reduce the damage caused by the I/R injury.8 The Nrf2 pathway is an essential cellular system that maintains cellular redox homeostasis through the antioxidant response element (ARE).9 In normal conditions, Nrf2 binds to Kelch-like ECH-associated protein 1 (Keap1) in the cytoplasm and then is degraded by the proteasome.10 Under oxidative stress conditions, Nrf2 is released from Keap1, then translocates into the nucleus and binds to the ARE to induce the production of antioxidant enzymes and phase II detoxification enzymes, such as heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1).11-14 Moreover, the expression of downstream antioxidants including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GSH-PX) can be increased by the upregulation of Nrf2. They together play a critical part in oxidative stress by regulating and maintaining intracellular redox states.15, 16 The therapeutic potential of targeting the Nrf2 pathway to protect CIRI have been demonstrated.17, 18 In recent years, some natural compounds are demonstrated to have the neuroprotective effects against oxidative stress by activating Nrf2 and increasing expression of downstream antioxidants.19 Swertiamarin (Swe, Figure 1) is a secoiridoid glycoside and one of the main bioactive components in Gentiana macrophylla Pall (Gentianaceae), which is a traditional Chinese medicine for treatment of stroke. Nowadays, Swe has been studied to have neuritogenic activity and antioxidant capacity in varieties of diseases.20-24 Besides, our group reported previously that Swe protected neurons against pilocarpine-induced seizures injury in mice.20 Swe has been ACS Paragon Plus Environment

Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


demonstrated to have a significant antioxidant capacity as it can scavenge ROS and reduce oxidative stress in cisplatin-induced renal damage, and ameliorate the CCl4-induced liver injury via activating the Nrf2 pathway.23,

24

However, the neuroprotective effect of Swe against CIRI by modulating oxidative stress remains unclear. In the present study, we explored the neuroprotective effects of Swe both in vivo and in vitro ischemic paradigms, and tried to elucidate the underlying mechanism. O HO

O

O

HO H2C H

O OH

HO

HO

Figure 1. The chemical structure of Swe.

RESULTS AND DISCUSSION Swe protects brain against I/R injury. At 24 h after reperfusion, we first examined the neurologic scores to determine neurological deficits. As shown in Figure 2a, the neurological deficit score of the vehicle group were significantly increased (p < 0.01) compared with the sham group. In the pre-treated groups with Swe (100 and 400 mg/kg), the neurological deficits were significantly decreased. The infarct volume of the cerebral ischemic area was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining (Figure 2b and Figure 2c). The infarct volume in I/R group was remarkably higher than that in the sham group. Pre-treatment of Swe dose-dependently reduced infarct volume. At a dose of 400 mg/kg, it decreased the infarct volume from 46.85 ± 1.99% to 16.50 ± 3.62% (p < 0.01), comparable to that of the positive drug nimodipine. Furthermore, the protective effect of Swe against CIRI was further determined by HE staining (Figure 3) on sections from ischemic hippocampus and cortex at 24 h after reperfusion. Compared with the sham group, the number of cells in vehicle group was decreased and arranged irregularly, and karyopyknosis were observed. In contrast, Swe pre-treatment reversed the change of pathological to a certain extent, indicating that Swe protected brain from I/R ACS Paragon Plus Environment


Page 6 of 29

injury.

Figure 2. Swe protected against CIRI in MCAO mice. (a) Neurological deficit scores at 24 h after reperfusion, n =10 per group. (b) Representative TTC staining of the cerebral infarct in brain. (c) The percentage of infarct volume was detected for each group, n = 6 per group. ##p < 0.01 versus the sham group. *p < 0.05, **p < 0.01 versus the I/R group.


Page 7 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. HE staining performed on sections from ischemic hippocampus and cortex at 24 h after reperfusion. Ischemic cerebral cortex (magnification: ×400), ischemic cerebral hippocampus CA3 (magnification: ×200) and ischemic cerebral hippocampus CA1 (magnification: ×400), n = 6 per group.

Swe reduces I/R-induced apoptosis To evaluate the effect of Swe in apoptosis, TUNEL staining was used to determine cell death (Figure 4a). The number of dying cells was notably augmented in the cortex of the vehicle group compared with the sham group. Swe treatment markedly decreased the cell apoptosis from 45.8 ± 2.27% to 30.6 ± 2.47% in the infarct region (p < 0.01). In addition, we detected the expression of apoptotic related proteins including Bcl-2 and Bax (Figure 4b). Consistent with TUNEL staining, Swe reduced the decrease of Bcl-2, the increase of Bax induced by I/R and markedly increased the ratio of Bcl-2/Bax protein in I/R mice. These results indicated that Swe significantly inhibited I/R-induced neuronal apoptosis.



Page 8 of 29

Figure 4. Swe reduces I/R-induced apoptosis. (a) TUNEL staining performed on sections from ischemic cortex at 24 h

after reperfusion and quantification of TUNEL-positive cells was performed. Scale bar = 20 μm. (b) Representative Western blots of Bcl-2 and Bax and the quantitative analyses of Bcl-2/Bax ratio were shown. n = 6 per group. ##p < 0.01 versus the sham group. **p < 0.01 versus the I/R group.

Swe significantly reduces oxidative damage. Malondialdehyde (MDA) is generated from lipid peroxidation and usually acted as a biomarker of oxidative stress.25 The SOD, GSH-PX and CAT were endogenous scavenger enzymes. SOD is a free radical scavenger that converts superoxide to hydrogen peroxide and oxygen.26 GSH-PX is involved in scavenging free radicals, maintaining redox status, and electrophilic intermediates. CAT can further metabolize peroxide, and defend oxidative stress as a second line.27 GSH is an endogenous antioxidant.28 The activities of these enzymes and GSH content have been used for reflecting the state of oxidative stress.29 We assessed oxidative damage by measuring the production of SOD, GSH-PX, CAT, GSH and MDA contents (Figure 5). In I/R group, the activities of SOD (Figure 5a), GSH-PX (Figure 5b) and CAT (Figure 5c) in brain tissue were significantly lower than those in the sham group, as well as the content ACS Paragon Plus Environment

Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of GSH (Figure 5d). The content of MDA in brain tissue was notably increased in I/R group than in the sham group (Figure 5e). Pre-treatment with Swe dose-dependently increased the GSH-PX, SOD and CAT activities. In the groups with Swe (100 and 400 mg/kg), the content of GSH was markedly increased while the MDA levels were significantly diminished. No obvious difference was observed in the content of GSH treated with Swe (25 mg/kg), as well as the MDA level, consistent with no improvement of the neurologic scores at the dose of 25 mg/kg. These results suggested the severe oxidative stress during CIRI and pre-treatment with Swe could markedly decrease MDA level and significantly upregulate these main antioxidant enzymes in vivo.

Figure 5. Effect of Swe on I/R-induced oxidative stress in mice. (a) SOD. (b) GSH-PX. (c) CAT. (d) GSH. (e) MDA. n = 6 per group. ##p < 0.01 versus the sham group. *p < 0.05, **p < 0.01 versus the I/R group.

Swe activates the Nrf2 pathway and induces the expression of HO-1, NQO1 after I/R in mice.



Page 10 of 29

Figure 6. Effects of Swe (400 mg/kg) on the protein expression levels of Keap1, Nrf2, HO-1 and NQO1 and the binding affinity between Nrf2-ARE in ischemic brain tissue of MCAO mice. Representative Western blots and quantitative analyses show Keap1 (a), nuclear Nrf2 (b), cytosolic Nrf2 (c), HO-1 (d) and NQO1 (e) protein expression. An EMSA was performed (f). n = 6 per group. #p < 0.05, ##p < 0.01 versus the sham group. **p < 0.01 versus the I/R group.

Nrf2 is a transcription factor sensitive to redox, and it can regulate many detoxifying and antioxidant defense gene expressions, such as HO-1 and NQO-1, to protect from oxidative stress.30 It was reported that mice with Nrf2 deficiency were more vulnerable to oxidative stress.31 Furthermore, Nrf2 knockout and wild-type mice indicated that Nrf2 attenuated cerebral ischemic injury by preventing from oxidative stress.32 HO-1 acts a vital part in protecting against cerebral ischemic injury as mice with HO-1 knockout exhibit greater ischemic brain injury compared with that of wild-type mice.33, 34 NQO1 is a cytosolic flavoenzyme that exerts a chemopreventive function.35 The upregulation ACS Paragon Plus Environment

Page 11 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of NOQ1 by Nrf2 signaling pathway can exert protective effect against stroke injury.36 Small molecules, such as natural products, activating the Nrf2 pathway to reinforce redox homeostasis, may have preventive and therapeutic potentials as neuroprotective agents.36-39 In a CCl4-induced liver damage model, it can activate the Nrf2 protective pathway and induce phase II enzyme expression, including HO-1 and NQO1, and reduce oxidative stress injury, thereby exert a protective effect.24 Thus, to explore the mechanism underlying the neuroprotective effect of Swe, the expression of Keap1, Nrf2, HO-1 and NQO1 from ischemic brain tissue were analyzed at 24 h after MCAO. Swe treatment significantly decreased the level of Keap1 compared with sham group (Figure 6a). Compared with I/R group, the nucleus protein levels of Nrf2 were markedly increased in Swe treatment group (Figure 6b), while the cytoplasm protein levels of Nrf2 were decreased (Figure 6c). The downstream phase II enzymes HO-1 (Figure 6d) and NQO1 (Figure 6e) were then significantly elevated in the Swe + I/R group than the I/R group. In addition, an electrophoretic mobility shift assay (EMSA) was performed to assess the binding affinity between Nrf2-ARE. The results indicated that Swe treatment could significantly increase the binding affinity between Nrf2-ARE (Figure 6f).

Swe protects neurons against OGD/R-induced injury We further investigated the neuroprotective effect of Swe in hippocampal neurons using an OGD/R model. The MTT (thereduction 3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide) assay was performed to detect the cell viability after injury. OGD/R treatment resulted in 60 % cell death (Figure 7a). Swe dose-dependently blocked OGD/R induced cell death. Treatment with Swe (10 μM) induced the increase of cell viability by approximately 30% than the OGD/R group. The damaged cells were detected by the LDH assay. The LDH release was reduced in the groups treated with Swe (1 and 10 μM) compared with the OGD/R group (Figure 7b). The intracellular ROS level was assessed (Figure 7c). The level of ROS significantly decreased by approximately 32 % and 43 % in the groups treated with Swe (1 μM and 10 μM) compared with the OGD/R group, and no obvious difference with Swe (0.1 μM). On the basis of the MTT assay and LDH detection, treatment with Swe reduced neuronal cell death triggered by OGD. During CIRI, ROS was increased sharply, and oxidative stress injury was induced. Swe inhibited the ROS increase ACS Paragon Plus Environment


Page 12 of 29

and the antioxidative effect of Swe was shown.

Figure 7. Swe treatment decreased cell death and intracellular ROS level induced by OGD/R in primary cultured hippocampal neurons. (a) Cell viability assay. (b) Cytotoxicity determination using an LDH release assay. (c) Intracellular ROS level. n = 6 per group. ##p < 0.01 versus the control group. *p < 0.05, **p < 0.01 versus the OGD/R group.

Swe promotes the release of Nrf2 from Keap1-Nrf2 complex. The immunoprecipitation assay was performed to detect whether Swe promoted the release of Nrf2 from Keap1. As shown in Figure 8, the results showed that the level of Keap1-Nrf2 complex in Swe group was observably decreased compared with OGD/R group, indicating that Swe induced the release of Nrf2 from the Keap1-Nrf2 complex and prevented Nrf2 from the degradation.


Page 13 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. Keap1 in the cytoplasmic fraction was immunoprecipitated with an anti-Keap1 antibody and the immunoprecipitated proteins were analyzed using antibodies to Nrf2 and Keap1.

The neuronal protection of Swe is involved in the Nrf2 protective pathway. To confirm the function of Nrf2 pathway in Swe induced neuroprotection, a specific inhibitor of Nrf2 activation brusatol, was used to knockdown the Nrf2 expression.40-43 After OGD treatment, cells were co-treated with Swe (10 μM) and brusatol (40 nM) for 24 h, and then cell viability was analyzed and the level of ROS was measured. As shown in Figure 9a, cell viability, which was increased by Swe under OGD/R, was inhibited by brusatol. Consistently, ROS level was increased by brusatol (Figure 9b). Western blot analysis performed that the protein levels of Nrf2, HO-1 and NQO1 were remarkably increased by Swe compared with the OGD/R group, while brusatol attenuated the increases (Figures 9c-e). It was observed that Nrf2 was generally located in the cytoplasm in normal neuron by immunofluorescence staining (Figure 9f). After treatment with Swe (10 μM), Nrf2 was markedly increased in the nucleus and this nuclear translocation could also be attenuated by co-treatment with brusatol. Consistent with the results in vivo, the increased expression level of Nrf2 by Swe in primary hippocampal neurons was observed as well. More nuclear immunofluorescence was obviously visualized in Swe group, indicating that Swe promoted nuclear translocation of Nrf2 in neurons. These findings indicated Nrf2 pathway was involved in the neuroprotective effect of Swe. The effects of Swe on the nuclear translocation of Nrf2 and downstream enzymes were reversed when brusatol was added in hippocampal neurons. This suggested that Swe-induced up-regulation of HO-1 and NQO1 was Nrf2 ACS Paragon Plus Environment


Page 14 of 29

dependent. Brusatol reversed the effects of Swe on protecting neuron and decreasing ROS levels, indicating Swe attenuated oxidative damage associated with Nrf2 activation. These results provided strong evidence that the activation of the Nrf2 pathway was indeed related to Swe-induced neuroprotective effects.

Figure 9. Brusatol reversed the protective effects conferred by Swe in primary cultured hippocampal neurons and Swe (10 μM) upregulated HO-1 and NQO1 expression may be mediated by Nrf2 activation. (a) Cell viability. (b) ACS Paragon Plus Environment

Page 15 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Intracellular ROS level. Nrf2 (c), HO-1 (d) and NQO1 (e) Protein expression were determined by Western blots. Swe treatment (10 μM) induced Nrf2 (red fluorescence) translocation into the nucleus (blue) was determined by immunofluorescence (f). n = 6 per group. ##p < 0.05 versus the control group. *p < 0.05, **p < 0.01 versus the OGD/R group. △p < 0.05, △△p < 0.01 versus the Swe group.

Swe is proposed to covalently modify Keap1 protein, leading to Nrf2 release. Natural products with electrophiles in chemical structures, such as bardoxolonemethyl (CDDO-Me) and dimethyl fumarate (DMF), have high reactivity with the cysteine in Keap1 protein to release Nrf2 into nucleus and protect cells from the oxidative stress.19, 44 The structure of Swe contains an electrophile (Figure 10, highlighted in blue), which might be a reactive position for cysteine, having the potential to react with Keap1 and leading to the conformational change of Keap1. Molecular modeling was performed to predict the binding mode of Swe with Keap1 protein. The crystal structure of BTB domain of Keap1 (PDB: 4CXI), which is thought to contain the key cysteine residue responsible for interaction with electrophiles,45 was selected for docking. As shown in Figure 10, Swe could covalently modify Keap1 at Cys151 and two hydrogen bonds with Tyr85 and His129 residues were observed with a docking score of -5.672 KJ/mol. With this modification, the conformational changes of Keap1 occurred to release Nrf2 from the Keap1-Nrf2 complex. Experimentally, Swe significantly increased the level of Nrf2 in the nucleus and decreased Nrf2 in the cytoplasm simultaneously, indicating Swe induced Nrf2 nuclear translocation (Figure 6 and Figure 8). These findings supported our hypothesis that Swe with potential electrophile exhibited its protective effects against ischemic injury through activating Nrf2 pathway.

CONCLUSION To our knowledge, this study for the first time demonstrated that Swe protected against I/R-induced injury both in vivo and in vitro. This protection potentially relied on antioxidative stress capabilities that were mediated by activating the Nrf2 protective pathway. We found the preventive potential of Swe for ischemic brain damage. Additional ACS Paragon Plus Environment


Page 16 of 29

research in Nrf2 knockout mice to verify the present study is ongoing.

O

O

O

O S

HO

O

Keap1

O

HO H2C H

HO H2C H

O OH

O OH

HO

HO

Keap1-Cys151

HO

HO

HO

Figure 10. Predicted binding mode and Michael addition of Swe with Keap1 BTB domain (PDB: 4CXI). Keap1 protein is shown as surface mode. Hydrogen bonds are depicted as yellow dash line.

MATERIALS AND METHODS Drugs and reagents Swe and brusatol were purchased from Zhongke Quality Inspection Biotechnology Co., Ltd with a purity higher than 98%. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin liquid (100×), neurobasal medium, B-27 supplement, phosphate buffer saline (PBS) and trypsin (0.25%) were obtained from Gibco (Life Technologies, China). Earle，s balanced salt solution (EBSS) (in g/l: 6.802 NaCl, 0.403 KCl, 0.2 CaCl2, 0.4 MgSO4, 0.405 NaH2PO4·2H2O, 2.201 NaHCO3, 4.79 HEPES), and MTT were purchased from Solarbio (Beijing, China). Nimodipine (0.2 mg/ml) was purchased from the German Bayer Company. The 2,3,5-triphenyl tetrazolium chloride (TTC) was obtained from Sigma (St. Louis, MO). Focal cerebral ischemia model and treatment with Swe ACS Paragon Plus Environment

Page 17 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Healthy adult male Institute of Cancer Research (ICR) mice weighing 20.0-25.0 g were purchased from the Experimental Animal Center of Ningxia Medical University, Yinchuan, China (certificate no. SYXK Ningxia 2015-0001). All animals were housed in a room under a 12-h dark/light cycle where the temperature was controlled at 25-28°C, and they were fed with standard chow and tap water. All experiments involving animals were performed according to the protocols approved by the Institutional Animal Ethics Committee of Ningxia Medical University (ethical number: 2016-032) and the ARRIVE2009 Guidelines for Reporting Animal Research.46 Focal cerebral ischemia was produced by middle cerebral artery occlusion (MCAO). In brief, mice were anesthetized with 3.5% chloral hydrate (0.1 ml/10g, i.p.). A 4−0 monofilament nylon suture was inserted into the internal carotid artery (ICA), and then it was advanced to block the left middle cerebral artery (MCA). After 2 h of occlusion, the thread was dismantled carefully to achieve the reperfusion for 24 h. Sham-operated mice were exposed to the same surgical procedure, except for arteries occlusion. Animals were eliminated from the groups if the cerebral blood flow (CBF) did not decrease at least 70% or animals died after ischemia induction. All mice were divided randomly into six groups as follows using a random number table: sham operated (sham), I/R (vehicle), I/R + Swe (25, 100, and 400 mg/kg) and I/R + nimodipine (1.4 mg/kg) groups. After diluted with physiological saline, Swe was administered via intraperitoneal injection. Mice were administered with 25, 100, or 400 mg/kg Swe daily, for 7 days, respectively. One hour after administration, then mice were exposed to MCAO for 2 h after reperfusion for 24 h. The time-line diagram of animal experiments was shown in Figure 11.

Figure 11. The time-line diagram of animal experiments. Calcium channel blockers can reduce free radical stress to arrest the neural cell death and alleviate the damage after ischemic stroke, and nimodipine has been convinced as the first-line treatment for stroke.38, 47, 48 Thus, nimodipine was ACS Paragon Plus Environment


Page 18 of 29

used as a positive control. Neurological deficit score Neurological deficits were evaluated blindly after 24 h of reperfusion with a 5-point scale scoring system.49 The scoring system is as follows: 0 = no deficit; 1 = failure to stretch the contralateral forelimb fully; 2 = circling to the contralateral side; 3 = falling over to the contralateral side; 4 = no spontaneous locomotor activity. Infarct volume evaluation After 24 h of reperfusion, six animals of each group were cervical dislocation followed by decapitated then brains were used to measure infarct volume immediately. The animal brains were cut into 2 mm thick coronal sections and exposed to TTC staining after immersion in 4% paraformaldehyde overnight at 4 °C. Unstained areas were defined as infarcts and measured using microscope image-analysis software (Image-Pro Plus, USA). The percentage of the infarct volume was calculated by the following formula: Infarct volume (%) = (normal hemisphere volume − non-infarct volume of the infarct side) / normal hemisphere volume × 100. Hematoxylin-Eosin (HE) and TUNEL staining Mice were deeply anesthetized with 3.5 % choral hydrate after 24 h of reperfusion, perfused with physiological saline and 4 % paraformaldehyde and then decapitated. Brains were dehydrated and hembedded in paraffin, and cut into 5 μm coronal sections, followed by brain sections were deparaffinized, hydrated for HE and TUNEL staining. Neuronal apoptosis was detected with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) using a cell death detection kit (Roche, Germany) following the manufacturer's instructions. In three different fields, the number of TUNEL positive neurons in the cerebral cortex was counted blindly for each section, using a laser scanning confocal microscope (Olympus FV1000, Japan). The result was expressed by the percentage of TUNEL positive cells. Determination of oxidative stress After 24 h of reperfusion, the mice (n = 6 for each group) were decapitated. The ischemic hemispheres tissue was homogenized in phosphate buffer (PBS) then centrifugation for 10 min at 12,000g, and the supernatants were used for ACS Paragon Plus Environment

Page 19 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


detection. The SOD, GSH-PX, CAT, GSH and MDA contents were assessed by assay kits from Nanjing Jiancheng Bioengineering Institute following the manufacturer's instruction. The concentration of protein was detected using the BCA protein assay reagent kit (KeyGEN, China). The absorbance was determined with a microplate reader. Primary culture of hippocampal neurons Primary hippocampal cultures were gained from neonatal rats.50 Briefly, hippocampi were rapidly separated from one-day-old Sprague-Dawley rats, shredded into small pieces, and then incubated in 0.125% of trypsin for 20 minutes to get single cell suspensions. After that, cells were cultured in DMEM medium with 10% FBS, then placed into incubator with 5% CO2 at 37 °C for 2 h. Then, the DMEM medium was replaced with neurobasal medium supplemented with 2% B-27. Culture medium was replaced once every 2 days. On the seventh day, hippocampal neurons were used for following studies. The purity of neurons was evaluated by immunofluorescence staining. Oxygen-glucose deprivation/reperfusion (OGD/R) and Swe treatment Primary cultured hippocampal neurons were subjected to OGD in vitro to simulate ischemia-like conditions. Briefly, the culture medium was replaced by glucose-free EBSS, followed by cells were placed into an oxygen-deprived (95% air / 5% CO2) incubator for 2 h at 37 °C. After that, the EBSS was replaced with regular medium and putted back into an incubator at normal conditions to reperfusion for 24 h. Neurons were treated with Swe (0.1 μM, 1 μM and 10 μM, respectively.) for 24 h in the entire process of reperfusion. For inhibiting Nrf2 pathway in vitro, the brusatol was administered at a final concentration of 40 nM. Cell viability The viability of hippocampal neurons was assessed by MTT assay. In brief, the hippocampal neurons were seeded in 96-well culture plates for 7 days. The cells were exposed to OGD for 2 h after that returned to normal culture conditions with or without Swe for another 24 h. After treatment, MTT (5 mg/ml) was given to the cells, incubated at 37 °C for 4 h. Then, remove the supernatant carefully and add 150 μl DMSO to solubilize purple formazan. Absorbance was detected at a wavelength of 570 nm with a microplate reader (Thermo, USA). Lactate dehydrogenase (LDH) release assay ACS Paragon Plus Environment


Page 20 of 29

The release of LDH demonstrates the lack of integrity of cell membrane. The OGD/R induced cellular damage was evaluated by LDH activity detection in intracellular and supernatant.51 The experimental procedure followed instructions of LDH assay kit (Jiancheng, China). The results were expressed as percentages of the total LDH. Measurement of ROS production The level of intracellular ROS was evaluated by an oxidation-sensitive fluorescent probe (DCFH-DA) Kit (Jiancheng, China). After treatment, the cells were reacted with a 10 μM working solution of DCFH-DA at 37 °C for 30 min in the dark, followed by they were washed with PBS for 3 times. After that, the cells were collected and re-suspended in PBS for detection. Fluorescence intensity was measured under a fluorescence spectrophotometer at excitation wavelengths of 485 and emission of 535 nm. Immunofluorescence staining The nuclear distribution of Nrf2 was observed with specific antibody against Nrf2 using immunofluorescence staining.52 The cells were seeded on coverslips coated with poly-D-lysine hydrobromide, fixed in 4% paraformaldehyde for 30 min, then washed with PBS, and permeabilized in 0.1% (w/v) Triton X-100 for 30 min. The cells were blocked with 1% albumin from bovine serum for 1 h at room temperature and then incubation with primary anti-Nrf2 antibody (1:200, Abcam, Catalogue numbers: ab137550, RRID: AB_2687540) at 4 °C overnight. Cells were incubated with Rhodamine (TRITC)–conjugated secondary antibody (ZSGB-BIO, USA) in the dark for 1 h. Images were taken using a laser scanning confocal microscope. Immunoprecipitation assay The hippocampal neurons were collected and washed with PBS for 3 times, then lysed by immunoprecipitation (IP) lysis buffer (protease inhibitor) for 30 min on ice. The concentration of protein was detected using the BCA protein assay reagent kit (KeyGEN, China). Sufficient amount of Keap1 antibody or rabbit IgG negative control was added to 300 µg protein, and then rotational incubation at 4 °C overnight. The immunoprecipitation was captured using 50 µl protein. A sepharose bead slurry was rotated for 4 h at 4°C (Proteintech, USA). The mixture was washed for 4 times using washing buffer and collected by centrifugation, followed by western blotting with Nrf2 or Keap1 antibody. ACS Paragon Plus Environment

Page 21 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Electrophoretic mobility shift assay (EMSA) The nuclear protein was extracted using nuclear and cytoplasmic extraction kits (KeyGEN, China) according to the manufacturer's

instruction.

The

ARE

sequence

was

5'-ACTGAGGGTGACTCAGCAAAATC-3',

3'-TGACTCCCACTGAGTCGTTTTAG-5' and end-labeled with biotin. Binding reactions was carried out at room temperature for 30 min. The DNA-protein complexes were separated by 6.5% polyacrylamide gel electrophoresis. Finally, the signal was visualized using ECL reagents (Pierce, Rockford, IL, USA). Western blotting After 24 h of OGD/R and MCAO treatment, the mice were sacrificed and the ischemic hemispheres tissue were rapidly separated on ice, weighed, and fully homogenized. Nuclear proteins were extracted using the nuclear and cytoplasmic extraction kits following the manufacturer’s instructions (Beyotime). The cellular proteins of primary hippocampal neuron were collected with lysis buffer on the basis of the manufacturers’ instructions (KeyGEN, China). Protein concentrations were determined by a BCA protein assay reagent kit. The same amounts of proteins were resolved over sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocked for 1 h with 5% non-fat milk, the bands were probed by primary antibodies: anti-Nrf2 antibody (1:800, Abcam, Catalogue numbers: ab137550, RRID: AB_2687540), anti-HO-1 (1:1000, Proteintech, Catalogue numbers: 10701-1-AP, RRID:AB_2118685), anti-NQO1 (1:800, Abcam, Catalogue numbers: ab28947, RRID: AB_881738), anti-Histone-H3 (1:800, Proteintech, Catalogue numbers: 17168-1-AP, RRID: AB_2716755), anti-Keap1 antibody (1:1000, Proteintech, Catalogue numbers: 10503-2-AP, RRID: AB_2132625), anti-Bcl-2 antibody (1:2000, Abcam, Catalogue numbers: ab182858, RRID: AB_2715467), anti-Bax antibody (1:1000, Abcam, Catalogue numbers: ab32503, RRID: AB_725631), anti-β-actin (1:1000, Proteintech, Catalogue numbers: 20536-1-AP, RRID: AB_10700003) at 4 °C overnight. After washed with PBST the bands were probed with the secondary antibodies goat anti-rabbit IgG (SA00001-2, Proteintech, USA) or goat anti-mouse IgG (SA00001-1, Proteintech, USA) for 2 h. Protein signal was visualized using ECL reagents (Pierce, Rockford, IL, USA). ACS Paragon Plus Environment


Page 22 of 29

Molecular modeling The docking study was carried out using the Schrodinger Maestro 11.4. The covalent docking was performed by setting Cys151 of Keap1 as the reactive residue and Michael addition as the reaction type. The other parameters choose the default without any constraints. Statistical analyses The statistical analyses were performed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Shapiro-Wilk test was carried out to determine whether the data are normally distributed. Normally distributed data were shown as mean ± SD. Statistical differences was determined by One-Way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test. Neurobehavioral scores were presented as median (range) and analyzed by the Mann– Whitney U test. A value of p < 0.05 was considered statistically significant.

ABBREVIATIONS Swe: swertiamarin; MCAO: middle cerebral artery occlusion; OGD/R: oxygen–glucose-deprivation/reperfusion; Nrf2: nuclear factor erythroid-2-related factor 2; HO-1: heme oxygenase-1; NQO1: NAD(P)H: quinone oxidoreductase-1; Keap1: Kelch-like ECH-associated protein 1; CIRI: cerebral ischemia-reperfusion injury; LDH: lactate dehydrogenase; ARE: antioxidant response element; ROS: reactive oxygen species; MTT: thereduction 3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide); MDA: malondialdehyde; SOD: superoxide dismutase; GSH: glutathione; GSH-PX: glutathione peroxidase; CAT: catalase; CDDO-Me: bardoxolonemethyl; DMF: dimethyl fumarate; TTC: 2,3,5-triphenyltetrazolium chloride; EMSA: electrophoretic mobility shift assay.

AUTHOR INFORMATION Corresponding authors: E-mail: [email protected] (Jianqiang Yu); [email protected] (Chunlin Zhuang); [email protected] (Xiaodong Peng). ACS Paragon Plus Environment

Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Author Contributions 1These

authors contributed equally to this work.

Funding Sources The authors gratefully acknowledge the financial supported from the National Natural Science Foundation of China (Grant No. 81660261, 81872791), the Ningxia Hui Autonomous Region Science and Technology Support Program (Grant No. 2015BAK45B01), the Ningxia Hui Autonomous Region Key Research and Development Project (Grant No. 2017BY079), the Ningxia 13th Plan of 5-year major scientific program (Grant No. 2016BZ07) and the Key Research and Development Program of Ningxia (2018BFH02001). Conflict of Interest The authors declare no competing financial interest.

ACKNOWLEDGEMENT We thank the staff in the Animal Center and the Science and Technology Center, Ningxia Medical University.

REFERENCES 1. Feigin, V. L., Mensah, G. A., Norrving, B., Murray, C. J., Roth, G. A., and Group, G. B. D. S. P. E. (2015) Atlas of the Global Burden of Stroke (1990-2013): The GBD 2013 Study, Neuroepidemiology 45, 230-236. 2. Disease, G. B. D., Injury, I., and Prevalence, C. (2017) Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016, Lancet 390, 1211-1259. 3. Amarenco, P., Bogousslavsky, J., Caplan, L. R., Donnan, G. A., Wolf, M. E., and Hennerici, M. G. (2013) The ASCOD phenotyping of ischemic stroke (Updated ASCO Phenotyping), Cerebrovasc Dis 36, 1-5. 4. Ouyang, Y. B., Stary, C. M., Yang, G. Y., and Giffard, R. (2013) microRNAs: innovative targets for cerebral ischemia and stroke, Curr Drug Targets 14, 90-101. ACS Paragon Plus Environment


Page 24 of 29

5. Puyal, J., Ginet, V., and Clarke, P. G. (2013) Multiple interacting cell death mechanisms in the mediation of excitotoxicity and ischemic brain damage: a challenge for neuroprotection, Prog Neurobiol 105, 24-48. 6. Chen, H., Yoshioka, H., Kim, G. S., Jung, J. E., Okami, N., Sakata, H., Maier, C. M., Narasimhan, P., Goeders, C. E., and Chan, P. H. (2011) Oxidative stress in ischemic brain damage: mechanisms of cell death and potential molecular targets for neuroprotection, Antioxid Redox Signal 14, 1505-1517. 7. Sims, N. R., and Muyderman, H. (2010) Mitochondria, oxidative metabolism and cell death in stroke, Biochim Biophys Acta 1802, 80-91. 8. Xiao, A. J., He, L., Ouyang, X., Liu, J. M., and Chen, M. R. (2018) Comparison of the anti-apoptotic effects of 15and 35-minute suspended moxibustion after focal cerebral ischemia/reperfusion injury, Neural Regen Res 13, 257-264. 9. Kobayashi, M., and Yamamoto, M. (2006) Nrf2-Keap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species, Adv Enzyme Regul 46, 113-140. 10. Kang, M. I., Kobayashi, A., Wakabayashi, N., Kim, S. G., and Yamamoto, M. (2004) Scaffolding of Keap1 to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes, Proc Natl Acad Sci U S A 101, 2046-2051. 11. Deshmukh, P., Unni, S., Krishnappa, G., and Padmanabhan, B. (2017) The Keap1-Nrf2 pathway: promising therapeutic target to counteract ROS-mediated damage in cancers and neurodegenerative diseases, Biophys Rev 9, 41-56. 12. Kensler, T. W., Wakabayashi, N., and Biswal, S. (2007) Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway, Annu Rev Pharmacol Toxicol 47, 89-116. 13. Zhuang, C., Narayanapillai, S., Zhang, W., Sham, Y. Y., and Xing, C. (2014) Rapid identification of Keap1-Nrf2 small-molecule inhibitors through structure-based virtual screening and hit-based substructure search, J Med Chem 57, 1121-1126. 14. Meng, N., Tang, H., Zhang, H., Jiang, C., Su, L., Min, X., Zhang, W., Zhang, H., Miao, Z., Zhang, W., and ACS Paragon Plus Environment

Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Zhuang, C. (2018) Fragment-growing guided design of Keap1-Nrf2 protein-protein interaction inhibitors for targeting myocarditis, Free Radic Biol Med 117, 228-237. 15. Zhao, Z., Lu, C., Li, T., Wang, W., Ye, W., Zeng, R., Ni, L., Lai, Z., Wang, X., and Liu, C. (2018) The protective effect of melatonin on brain ischemia and reperfusion in rats and humans: In vivo assessment and a randomized controlled trial, J Pineal Res 65, e12521. 16. Ding, Y., Chen, M., Wang, M., Wang, M., Zhang, T., Park, J., Zhu, Y., Guo, C., Jia, Y., Li, Y., and Wen, A. (2014) Neuroprotection by acetyl-11-keto-beta-Boswellic acid, in ischemic brain injury involves the Nrf2/HO-1 defense pathway, Sci Rep 4, 7002. 17. Satoh, T., Okamoto, S. I., Cui, J., Watanabe, Y., Furuta, K., Suzuki, M., Tohyama, K., and Lipton, S. A. (2006) Activation of the Keap1/Nrf2 pathway for neuroprotection by electrophilic [correction of electrophillic] phase II inducers, Proc Natl Acad Sci U S A 103, 768-773. 18. Wu, G., Zhu, L., Yuan, X., Chen, H., Xiong, R., Zhang, S., Cheng, H., Shen, Y., An, H., Li, T., Li, H., and Zhang, W. (2017) Britanin Ameliorates Cerebral Ischemia-Reperfusion Injury by Inducing the Nrf2 Protective Pathway, Antioxid Redox Signal 27, 754-768. 19. Lu, M. C., Ji, J. A., Jiang, Z. Y., and You, Q. D. (2016) The Keap1-Nrf2-ARE Pathway As a Potential Preventive and Therapeutic Target: An Update, Med Res Rev 36, 924-963. 20. Deng, X. H., Zhang, X., Wang, J., Ma, P. S., Ma, L., Niu, Y., Sun, T., Zhou, R., and Yu, J. Q. (2017) Anticonvulsant Effect of Swertiamarin Against Pilocarpine-Induced Seizures in Adult Male Mice, Neurochem Res 42, 3103-3113. 21. Chiba, K., Yamazaki, M., Kikuchi, M., Kakuda, R., and Kikuchi, M. (2011) New physiological function of secoiridoids: neuritogenic activity in PC12h cells, J Nat Med 65, 186-190. 22. Bhattacharya, S. K., Reddy, P. K., Ghosal, S., Singh, A. K., and Sharma, P. V. (1976) Chemical constituents of Gentianaceae XIX: CNS-depressant effects of swertiamarin, J Pharm Sci 65, 1547-1549. 23. Sharma, S., Modi, A., Narayan, G., and Hemalatha, S. (2018) Protective Effect of Exacum lawii on ACS Paragon Plus Environment


Page 26 of 29

Cisplatin-induced Oxidative Renal Damage in Rats, Pharmacogn Mag 13, S807-S816. 24. Wu, T., Li, J., Li, Y., and Song, H. (2017) Antioxidant and Hepatoprotective Effect of Swertiamarin on Carbon Tetrachloride-Induced Hepatotoxicity via the Nrf2/HO-1 Pathway, Cell Physiol Biochem 41, 2242-2254. 25. Adibhatla, R. M., and Hatcher, J. F. (2010) Lipid oxidation and peroxidation in CNS health and disease: from molecular mechanisms to therapeutic opportunities, Antioxid Redox Signal 12, 125-169. 26. Kelly, P. J., Morrow, J. D., Ning, M., Koroshetz, W., Lo, E. H., Terry, E., Milne, G. L., Hubbard, J., Lee, H., Stevenson, E., Lederer, M., and Furie, K. L. (2008) Oxidative stress and matrix metalloproteinase-9 in acute ischemic stroke: the Biomarker Evaluation for Antioxidant Therapies in Stroke (BEAT-Stroke) study, Stroke 39, 100-104. 27. Li, Y., Bao, Y., Jiang, B., Wang, Z., Liu, Y., Zhang, C., and An, L. (2008) Catalpol protects primary cultured astrocytes from in vitro ischemia-induced damage, Int J Dev Neurosci 26, 309-317. 28. Kho, A. R., Choi, B. Y., Lee, S. H., Hong, D. K., Lee, S. H., Jeong, J. H., Park, K. H., Song, H. K., Choi, H. C., and Suh, S. W. (2018) Effects of Protocatechuic Acid (PCA) on Global Cerebral Ischemia-Induced Hippocampal Neuronal Death, Int J Mol Sci 19, E1420. 29. Yin, M. C., Lin, M. C., Mong, M. C., and Lin, C. Y. (2012) Bioavailability, distribution, and antioxidative effects of selected triterpenes in mice, J Agric Food Chem 60, 7697-7701. 30. Shah, Z. A., Li, R. C., Thimmulappa, R. K., Kensler, T. W., Yamamoto, M., Biswal, S., and Dore, S. (2007) Role of reactive oxygen species in modulation of Nrf2 following ischemic reperfusion injury, Neuroscience 147, 53-59. 31. Loboda, A., Damulewicz, M., Pyza, E., Jozkowicz, A., and Dulak, J. (2016) Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism, Cell Mol Life Sci 73, 3221-3247. 32. Narayanan, S. V., Dave, K. R., Saul, I., and Perez-Pinzon, M. A. (2015) Resveratrol Preconditioning Protects Against Cerebral Ischemic Injury via Nuclear Erythroid 2-Related Factor 2, Stroke 46, 1626-1632. 33. Magesh, S., Chen, Y., and Hu, L. (2012) Small molecule modulators of Keap1-Nrf2-ARE pathway as potential preventive and therapeutic agents, Med Res Rev 32, 687-726. ACS Paragon Plus Environment

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


34. Ryter, S. W., Alam, J., and Choi, A. M. (2006) Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications, Physiol Rev 86, 583-650. 35. Winski, S. L., Faig, M., Bianchet, M. A., Siegel, D., Swann, E., Fung, K., Duncan, M. W., Moody, C. J., Amzel, L. M., and Ross, D. (2001) Characterization of a mechanism-based inhibitor of NAD(P)H:quinone oxidoreductase 1 by biochemical, X-ray crystallographic, and mass spectrometric approaches, Biochemistry 40, 15135-15142. 36. Wu, J., Li, R., Li, W., Ren, M., Thangthaeng, N., Sumien, N., Liu, R., Yang, S., Simpkins, J. W., Forster, M. J., and Yan, L. J. (2017) Administration of 5-methoxyindole-2-carboxylic acid that potentially targets mitochondrial dihydrolipoamide dehydrogenase confers cerebral preconditioning against ischemic stroke injury, Free Radic Biol Med 113, 244-254. 37. Wen, Z., Hou, W., Wu, W., Zhao, Y., Dong, X., Bai, X., Peng, L., and Song, L. (2018) 6'-O-Galloylpaeoniflorin Attenuates Cerebral Ischemia Reperfusion-Induced Neuroinflammation and Oxidative Stress via PI3K/Akt/Nrf2 Activation, Oxid Med Cell Longev 2018, 8678267. 38. Shi, A., Xiang, J., He, F., Zhu, Y., Zhu, G., Lin, Y., and Zhou, N. (2018) The Phenolic Components of Gastrodia elata improve Prognosis in Rats after Cerebral Ischemia/Reperfusion by Enhancing the Endogenous Antioxidant Mechanisms, Oxid Med Cell Longev 2018, 7642158. 39. Yang, R. X., Lei, J., Wang, B. D., Feng, D. Y., Huang, L., Li, Y. Q., Li, T., Zhu, G., Li, C., Lu, F. F., Nie, T. J., Gao, G. D., and Gao, L. (2017) Pretreatment with Sodium Phenylbutyrate Alleviates Cerebral Ischemia/Reperfusion Injury by Upregulating DJ-1 Protein, Front Neurol 8, 256. 40. Chen, B., Cao, H., Chen, L., Yang, X., Tian, X., Li, R., and Cheng, O. (2016) Rifampicin Attenuated Global Cerebral Ischemia Injury via Activating the Nuclear Factor Erythroid 2-Related Factor Pathway, Front Cell Neurosci 10, 273. 41. Chao, X. J., Chen, Z. W., Liu, A. M., He, X. X., Wang, S. G., Wang, Y. T., Liu, P. Q., Ramassamy, C., Mak, S. H., Cui, W., Kong, A. N., Yu, Z. L., Han, Y. F., and Pi, R. B. (2014) Effect of tacrine-3-caffeic acid, a novel multifunctional anti-Alzheimer's dimer, against oxidative-stress-induced cell death in HT22 hippocampal neurons: ACS Paragon Plus Environment


Page 28 of 29

involvement of Nrf2/HO-1 pathway, CNS Neurosci Ther 20, 840-850. 42. Sun, Q., Wu, Y., Zhao, F., and Wang, J. (2017) Maresin 1 Ameliorates Lung Ischemia/Reperfusion Injury by Suppressing Oxidative Stress via Activation of the Nrf-2-Mediated HO-1 Signaling Pathway, Oxid Med Cell Longev 2017, 9634803. 43. Meng, Q. T., Cao, C., Wu, Y., Liu, H. M., Li, W., Sun, Q., Chen, R., Xiao, Y. G., Tang, L. H., Jiang, Y., Leng, Y., Lei, S. Q., Lee, C. C., Barry, D. M., Chen, X., and Xia, Z. Y. (2016) Ischemic post-conditioning attenuates acute lung injury induced by intestinal ischemia-reperfusion in mice: role of Nrf2, Lab Invest 96, 1087-1104. 44. Kansanen, E., Bonacci, G., Schopfer, F. J., Kuosmanen, S. M., Tong, K. I., Leinonen, H., Woodcock, S. R., Yamamoto, M., Carlberg, C., Yla-Herttuala, S., Freeman, B. A., and Levonen, A. L. (2011) Electrophilic nitro-fatty acids activate NRF2 by a KEAP1 cysteine 151-independent mechanism, J Biol Chem 286, 14019-14027. 45. Cleasby, A., Yon, J., Day, P. J., Richardson, C., Tickle, I. J., Williams, P. A., Callahan, J. F., Carr, R., Concha, N., Kerns, J. K., Qi, H., Sweitzer, T., Ward, P., and Davies, T. G. (2014) Structure of the BTB domain of Keap1 and its interaction with the triterpenoid antagonist CDDO, Plos One 9, e98896. 46. Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., and Altman, D. G. (2010) Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research, PLoS Biol 8, e1000412. 47. Monzani, D., Genovese, E., Pini, L. A., Di Berardino, F., Alicandri Ciufelli, M., Galeazzi, G. M., and Presutti, L. (2015) Nimodipine in otolaryngology: from past evidence to clinical perspectives, Acta Otorhinolaryngol Ital 35, 135-145. 48. Kim, S. Y., Kim, K. H., Cho, J. H., and Kim, Y. D. (2015) Clinical Variables Correlated with Numbers of Intra-arterial Nimodipine Infusion in Patients with Medically Refractory Cerebral Vasospasm, J Cerebrovasc Endovasc Neurosurg 17, 157-165. 49. Bederson, J. B., Pitts, L. H., Tsuji, M., Nishimura, M. C., Davis, R. L., and Bartkowski, H. (1986) Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination, Stroke 17, 472-476. 50. Ma, N. T., Zhou, R., Chang, R. Y., Hao, Y. J., Ma, L., Jin, S. J., Du, J., Zheng, J., Zhao, C. J., Niu, Y., Sun, T., Li, ACS Paragon Plus Environment

Page 29 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


W., Koike, K., Yu, J. Q., and Li, Y. X. (2015) Protective effects of aloperine on neonatal rat primary cultured hippocampal neurons injured by oxygen-glucose deprivation and reperfusion, J Nat Med 69, 575-583. 51. Lobner, D. (2000) Comparison of the LDH and MTT assays for quantifying cell death: validity for neuronal apoptosis?, J Neurosci Methods 96, 147-152. 52. Feng, R., Lu, Y., Bowman, L. L., Qian, Y., Castranova, V., and Ding, M. (2005) Inhibition of activator protein-1, NF-kappaB, and MAPKs and induction of phase 2 detoxifying enzyme activity by chlorogenic acid, J Biol Chem 280, 27888-27895.


Neuroprotective Effect of Swertiamain on Cerebral Ischemia

Recommend Documents