Free Radical Biology and Medicine 71 (2014) 186–195

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Original Contribution

Luteolin provides neuroprotection in models of traumatic brain injury via the Nrf2–ARE pathway Jianguo Xu a, Handong Wang a,n, Ke Ding a, Li Zhang a, Chunxi Wang a, Tao Li a, Wuting Wei b, Xinyu Lu a a

Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu Province, People's Republic of China Department of Neurosurgery, Jinling Hospital, School of Medicine, Southern Medical University (Guangzhou), Nanjing, Jiangsu Province, People's Republic of China

b

art ic l e i nf o

a b s t r a c t

Article history: Received 30 December 2013 Received in revised form 16 February 2014 Accepted 7 March 2014 Available online 15 March 2014

Luteolin has recently been proven to exert neuroprotection in a variety of neurological diseases; however, its roles and the underlying mechanisms in traumatic brain injury are not fully understood. The present study was aimed to investigate the neuroprotective effects of luteolin in models of traumatic brain injury (TBI) and the possible role of the Nrf2–ARE pathway in the putative neuroprotection. A modified Marmarou's weight-drop model in mice and the scratch model in mice primary cultured neurons were used to induce TBI. We determined that luteolin significantly ameliorated secondary brain injury induced by TBI, including neurological deficits, brain water content, and neuronal apoptosis. Furthermore, the level of malondialdehyde (MDA) and the activity of glutathione peroxidase (GPx) were restored in the group with luteolin treatment. in vitro studies showed that luteolin administration lowered the intracellular reactive oxygen species (ROS) level and increased the neuron survival. Moreover, luteolin enhanced the translocation of Nrf2 to the nucleus both in vivo and in vitro, which was proved by the results of Western blot, immunohistochemistry, and electrophoretic mobility shift assay (EMSA). Subsequently upregulation of the expression of the downstream factors such as heme oxygenase 1 (HO1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) was also examined. However, luteolin treatment failed to provide neuroprotection after TBI in Nrf2-/- mice. Taken together, these in vivo and in vitro data demonstrated that luteolin provided neuroprotective effects in the models of TBI, possibly through the activation of the Nrf2–ARE pathway. & 2014 Elsevier Inc. All rights reserved.

Keywords: Luteolin Neuroprotection Traumatic brain injury Nrf2

Introduction Traumatic brain injury (TBI) is a major public health problem in modern society, consuming a lot of medical resources and ending up with poor prognosis and a high mobility of long-term disability [1]. Although the severity of primary insult is the major factor determining the outcomes, the secondary insult caused by pathological processes, including oxidative stress, excitotoxicity, inflammation, and increased vascular permeability, aggravates the damage of TBI [2]. In the past decades, great efforts were concentrated on seeking effective ways to alleviate the secondary insult, thereby

Abbreviations: EMSA, electrophoretic mobility shift assay; GPx, glutathione peroxidase; HO1, heme oxygenase 1; Keap1, Kelch-like ECH-associated protein 1; MDA, malondialdehyde; NQO1, NAD(P)H:quinone oxidoreductase 1; Nrf2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species; RT-PCR, real-time quantitative polymerase chain reaction; TBI, traumatic brain injury; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling n Corresponding author. Fax: þ86 25 51805396. E-mail address: [email protected] (H. Wang). http://dx.doi.org/10.1016/j.freeradbiomed.2014.03.009 0891-5849/& 2014 Elsevier Inc. All rights reserved.

improving the outcome of TBI. To date, however, most approaches to the treatment of TBI which target a single injury mechanism have failed in clinical trials [3]. Oxidative stress resulting from the production of reactive oxygen species (ROS) causes severe damage to the brain tissue after TBI [4]. It is not only because of the excessive production of ROS due to excitotoxicity and exhaustion of the endogenous antioxidant system [2], but also because there is a high content of polyunsaturated fatty acids in the brain tissue, which makes it vulnerable to free radical attacks and lipid peroxidation [5]. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a known transcription factor as the cell's main defense mechanism against a variety of harmful stresses. The main function of Nrf2 is to activate the antioxidant response and induce transcription of a wide range of genes that are able to counteract the harmful effects of oxidative stress, thus restoring intracellular homeostasis [6]. The most wellknown downstream genes of Nrf2 include heme oxygenase 1 (HO1) and NADPH:quinine oxidoreductase 1 (NQO1), both of which are able to regulate intracellular redox balancing [7].

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Under basal conditions, Nrf2 is located in cytoplasm and is bound to its inhibitor, Kelch-like ECH-associated protein 1 (Keap1), which associates with the actin cytoskeleton and prevents Nrf2 from entering the nucleus. In addition, Keap1 facilitates the Cul3mediated polyubiquitination of Nrf2, leading to its proteasomal degradation [8]. Nrf2–ARE is proved to be activated in many neurological diseases [9,10], including TBI [11], which is considered as an endogenous compensatory adaptation against TBI. Exogenous administration of the Nrf2 stabilizer molecule tertbutylhydroquinone also conferred neuroprotective effects in brain injury [12,13]. Luteolin belongs to the flavonoid family and is abundant in fruits and vegetables. It exhibits a wide variety of pharmacological properties including antioxidant free radical scavenging and antiinflammatory effects [14–16]. The present study was aimed to investigate the neuroprotection of luteolin after traumatic brain injury in vivo and in vitro. The possible involvement of the Nrf2– ARE pathway and the regulation of redox state are the main focus.

Materials and methods Animals Male ICR mice (Experiment Animal Centre of Nanjing Medical University, Jiangsu, China) and Nrf2-deficient (Nrf2-/-) mice (with permission from Dr. Thomas W. Kensler, Johns Hopkins University, Baltimore, MD, USA) weighing 28–32 g were used in this study. Mice were housed on a 12 h light/dark cycle with free access to food and water and were acclimatized for at least 4 days before any experiment. Experiment protocols were approved by the Animal Care and Use Committee of Nanjing University and conformed to the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. Primary culture of mouse cortical neurons Cortical neuronal cells were isolated from embryos of timemated pregnant mice and cultured onto poly-D-lysine-coated 6-well dishes at a density of 1  106 cells/well in neurobasal medium (Life Technologies, Carlsbad, CA, USA) supplemented with 2% B27 (Life Technologies) and 1 mM glutamate (Sigma Aldrich, Shanghai, China) as described previously [17,18]. Cells were maintained in growth medium at 37 1C in 5% CO2/95% air. Half of the culture medium was replaced with fresh medium every 3 days. All experiments were performed after 10–12 days in vitro. Models of TBI The in vivo model of TBI used in this study was based on Marmarou's weight-drop model previously described by Flierl et al. with some modifications [19,20]. Briefly, mice were anesthetized with an intraperitoneal injection of chloral hydrate and then placed onto the platform directly under the weight of the weightdrop device. A 1.5-cm midline longitudinal scalp incision was made and the skull exposed. After locating the left anterior frontal area as the impact area, a 200-g weight was released onto the skull. The scalp wound was then closed with standard suture material, and the mice were returned to cages, where they had free access to water and food. Sham-injured animals underwent the same procedures but did not undergo the weight drop. A transection model was employed in the present study as described previously [17]. Briefly, each well of 6-well plates was manually scratched with a sterile plastic needle following a 9  9 square grid (with 4-mm spacing between the lines). Cell cultures were then placed back in an incubator at 37 1C for another 24 h.

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Cultures were incubated without change of medium. Uninjured cultures were used as controls. Because scratch injury activates neurons first at the wound edge and later expands to the entire neuron monolayer, the entire culture on each dish was used for all experiments.

Groups and drug administration For in vivo experiments, animals were randomly divided into 6 groups: sham, TBI, TBI þ vehicle, TBI þ luteolin (3 subgroups: 10, 30, and 50 mg/kg). Luteolin (Shanghai Yuanye Bio-Technology Co., Ltd, Shanghai, China) with purity of more than 98% was dissolved in saline containing 1% DMSO. Different doses of luteolin or equal volumes of 1% DMSO were injected intraperitoneally 30 min after onset of TBI. The doses used in this study were based on a study of neuroprotection of luteolin in a middle cerebral artery occlusion model [21]. For in vitro experiments, cells were divided into 6 groups: sham, TBI, TBI þ vehicle, TBI þ luteolin (3 subgroups: 5, 10, and 25 mM). Luteolin was first dissolved in DMSO as a stock solution and then added directly to cultured media to achieve different final concentrations [22].

Neurological deficit and brain water content Neurological deficit was evaluated by the grip test which was developed on the basis of the test of gross vestibulomotor function as described elsewhere [19,23]. Briefly, mice were placed on a thin, horizontal, metal wire (45 cm long) that was suspended between two vertical poles 45 cm above a foam pad. A score of zero was given if the mouse was unable to remain on the wire for less than 30 s; one point was given if the mouse failed to hold on to the wire with both forepaws and hind paws together; two points were given if the mice held on to the wire with both forepaws and hind paws but not the tail; three points were given if the mouse used its tail along with both forepaws and both hind paws; four points were given if the mouse moved along the wire on all four paws plus tail; and five points were given if mice that scored four points also ambulated down one of the posts used to support the wire. The grip test was performed in triplicate, and a total value was calculated for each mouse. The test was carried out by an investigator who was blinded to the experimental groups. Brain water content was performed according to a previous study [19]. In brief, mouse brain was removed and placed on a cooled brain matrix 24 h after TBI. The brain stem and cerebellum were taken away, and ipsilateral tissue was weighed immediately following removal to obtain wet weight (ww). Then the hemisphere was dried at 80 1C for 72 h and weighed to obtain dry weight (dw). We calculated water content as a percentage using the following formula: (ww – dw)/ww  100%.

Tissue processing For biochemical measurements, Western blot analysis, realtime quantitative polymerase chain reaction (RT-PCR), and electrophoresis mobility shift assay (EMSA), animals were rapidly killed 24 h post-TBI, and the ipsilateral cortex was collected. The tissue was positioned directly over the center of the injury site and included both contused and penumbra. Samples were immediately frozen in liquid nitrogen, and then stored at  80 1C in a freezer until use. For immunohistochemistry and TUNEL analysis, the whole brain was removed 24 h after TBI and immersed in 4% paraformaldehyde overnight.

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Determination of malondialdehyde (MDA) content and glutathione peroxidase (GPx) activity Tissue samples were homogenized in 2 ml phosphate-buffer saline (PBS, pH 7.4) and centrifuged at 12,000 rpm for 15 min/4 1C to remove cell debris. MDA content and GPx activity were measured using a spectrophotometer according to the manufacturer's instructions (Nanjing Jiancheng Biochemistry Co., Nanjing, China). Total protein concentrations were determined by the Bradford method. The content of MDA was expressed as nanomole per milligram protein and the activity of GPx was expressed as units per milligram protein, respectively. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis The formalin-fixed tissues were embedded in paraffin and sectioned at 4 mm thickness with a microtome. The apoptosis cells were determined using a TUNEL detection kit (Roche, Indianapolis, IN, USA). The procedures were according to the protocol of the kit and a previous study [24]. Briefly, the sections underwent deparaffinization and rehydration first, and then were washed with PBS followed by digesting for 15 min in proteinase K. After being washed with PBS (2  5 min), sections were incubated at 37 1C with labeling solution containing TUNEL reaction fluid for 60 min. After that, sections were washed with PBS (3  10 min) and then blocked with 10% goat serum in 0.1 M Tris for 15 min. DNA was visualized by treating the tissue with a 1:40 dilution of streptavidin peroxidase (HRP) and staining with DAB as chromogen. The apoptotic cells showed cell shrinkage with condensed nuclei stained brown. For negative control purposes, some slides were incubated with label solution not containing TdT. The distinctive morphological features of apoptosis were used to recognize apoptotic cells. The positive cells were identified, counted, and analyzed under a light microscope by an investigator blinded to the grouping. The extent of brain damage was evaluated by apoptotic index, defined as the average percentage of TUNELpositive cells in each section counted in 10 cortical microscopic fields (at  400 magnification). For the quantification of apoptotic cells, a total of eight coronal sections from the identical brain region of each animal was selected (from approximately bregma -1.0 mm to bregma -3.0 mm, at 200-mm intervals). The final average percentage of TUNEL-positive cells of the eight sections was regarded as the data for each sample. Immunohistochemistry and Western blot For immunohistochemistry, consecutive coronal sections were cut at 4-mm intervals from approximately bregma – 1.0 mm to bregma – 3.0 mm to collect the lesioned cortex. After routine deparaffinization, endogenous peroxidase was blocked with 3% H2O2/methanol. Nonspecific antibody binding was blocked by incubating the sections in blocking buffer (10% normal goat serum in PBS) for 30 min. Primary antibodies against Nrf2 (1:100; Abcam, Cambridge, MA, USA) were applied overnight at 4 1C. After being washed three times in PBS for 5 min each, the sections were incubated with horseradish peroxidase-conjugated IgG (1:500; Santa Cruz Biotechnology) for 60 min. 3,3-Diaminobenzidine (DAB)/H2O2 solution was used to visualize Nrf2. Before the sections were mounted, cell nuclei were counterstained with hematoxylin. For Western blot, as described in our previous study [19], equal amounts of protein were separated by SDS-PAGE on 8–12% Bis-Tris gels, transferred to PVDF membranes, and incubated overnight at 4 1C with corresponding primary antibodies. The antibodies used were Nrf2 (1:1000; Abcam), HO1 (1:2000; Abcam), NQO1 (1:1000; Abcam), H3 (1:1000; Cell Signaling Technology, Danvers, MA,

USA), and β-actin (1:5000; Bioworld Technology, Minneapolis, MN, USA). Subsequently, the membranes were incubated for 2 h with corresponding secondary antibodies. Nuclear extraction and electrophoresis mobility shift assay Nuclear extraction was according to the manufacturer's instructions of the nuclear and cytoplasmic protein extraction kit (Beyotime Biotech Inc., Nantong, China). EMSA was performed using a kit (Pierce Biotechnology, Rockford, IL, USA) to assay Nrf2 DNAbinding activity according to the manufacturer's instructions and a previous study [25]. Consensus oligonucleotide probes of Nrf2 (50 -TGGGGAACCTGTGCTGAGTCACTGGAG-30 ) were end-labeled with biotin. Binding reactions were carried out for 20 min at room temperature in the presence of 50 ng/μl poly(dI-dC), 0.05% Nonidet P-40, 5 mM MgCl2, 10 mM EDTA, and 2.5% glycerol in 1X binding buffer using 20 fmol of biotin-end-labeled target DNA and 10 μg of nuclear extract. Assays were loaded onto native 4% polyacrylamide gels preelectrophoresed for 60 min in 0.5X Trisborate-EDTA (TBE) and electrophoresed at 100 V before being transferred onto a positively charged nylon membrane in 0.5X TBE at 100 V for 30 min. Transferred DNAs were cross-linked to the membrane and detected using horseradish peroxidaseconjugated streptavidin. Real-time quantitative polymerase chain reaction Total RNA was extracted from ipsilateral cortex samples with RNAiso Plus (TaKaRa Bio., Dalian, China) and immediately reversetranscribed to cDNA with the PrimeScript RT reagent kit (TaKaRa Bio.). The primers were designed according to PubMed GenBank and synthesized by Invitrogen Life Technologies (Shanghai, China). The primer sequences were as follows: NQO1: F, 50 -CATTCTGAAAGGCTGGTTTGA-30 ; R, 50 -CTAGCTTTGATCTGGTTGTCAG-30 ; HO-1: F, 50 -ATCGTGCTCGCATGAACACT-30 ; R, 50 -CCAACACTGCATTTACATGGC-30 ; β-actin: F, 50 -AGTGTGACGTTGACATCCGTA-30 ; R, 50 GCCAGAGCAGTAATCTCCTTCT-30 . Quantitative real-time PCR analysis was performed by using the Mx3000P System (Stratagene, San Diego, CA, USA), applying real-time SYBR Green PCR technology. All samples were analyzed in triplicate. β-Actin was used as an endogenous reference “housekeeping” gene. Cell viability analysis Primary cultured neuron viability was quantified by measuring the release of the cytosolic enzyme, lactate dehydrogenase (LDH), as previous described [26]. The enzyme activity was determined by using an assay kit according to the manufacturer's instructions (Beyotime Biotech Inc.). Briefly, cells were treated with LDH release agent (served as the maximum), and the media which contained detached cells were collected and centrifuged. The supernatant was used for the assay of LDH activity. A spectrophotometer was used to measure the OD value at 490 nm. Percentage of damaged cells was calculated according to the equation damaged cells (%) ¼ (OD490sample – OD490media)/ (OD490maximum – OD490media)  100%, where OD490media ¼ only media without any cells, and OD490maximum ¼ cells treated with LDH release agent. Furthermore, the trypan blue staining assay was performed to confirm LDH assay results. After each treatment, cells were stained with 0.4% TB (Beyotime Biotech Inc.). Unstained cells were regarded as viable, and stained cells were regarded as dead. Total cell number and the number of TBA-positive cells were counted via a light microscope in a blinded way. Survival value was calculated by using the formula: number of stained cells/number of total cells  100%.

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Intracellular ROS measurement Cellular levels of ROS were measured using a reactive oxygen species assay kit (Beyotime Biotech Inc.) according to the method described elsewhere [27]. Briefly, cells were preincubated with DCFH-DA at a concentration of 10 μM. After 20 min, DCFH fluorescence was measured at an excitation wavelength of 488 nm and an emission wavelength of 515–540 nm using a an Olympus IX71 inverted microscope system; to prevent the photo-oxidation of DCFH, the fluorescence images were collected using a single rapid scan. After collection of the fluorescence images, the cells were imaged by digital interference contrast, and the mean relative fluorescence intensity for each group of cells was then measured using the Image-Pro Plus system (version 6.0). Statistical analysis Each experiment was repeated at least three times, and the data were reported as the mean 7 SEM. Statistical analysis among groups was performed with one-way ANOVA followed by Tukey's

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test. SPSS 17.0 (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. A P value o 0.05 was considered statistically significant.

Results Luteolin improves the recovery of motor performance after TBI and reduces posttraumatic cerebral edema To test whether luteolin provides neuroprotection after TBI, we set six groups as follows: sham, TBI, TBI þ vehicle, and TBI þ luteolin (three different dose groups: 10, 30, and 50 mg/kg). First, we used the grip test to evaluate the motor performance after TBI at Day 1. All the mice were trained on the task 1 day before the onset of TBI. The sham group showed no difference between different time points and there is no difference between the TBI group and the vehicle-treated group (data no shown). As showed in Fig. 1A, all groups exhibited an improved motor performance over time after TBI. Within 3 days after TBI, the performance of the

Fig. 1. Administration of luteolin protects mice against secondary brain injury after TBI. (A, B) Mice were subjected to TBI and then received 10, 30, and 50 mg/kg of luteolin or vehicle 30 min after TBI. Grip test score was evaluated at 1, 3, and 7 days after TBI while brain water content was examined at 1 day after TBI. (A) Both 10 and 30 mg/kg groups had an improved motor performance within 3 days; however, there was no significant difference between the 50 mg/kg group and the vehicle group. This effect was no longer significant 7 days after TBI between all groups treated with different doses of luteolin and the vehicle-treated group. n ¼ 6 per group. (B) Mice subjected to TBI or treated with vehicle had an increased brain water content as compared with the sham group. Brain water content was significantly lower in the groups with administration of luteolin (10, 30, 50 mg/kg) than the vehicle-treated group. n ¼ 5 each group. (C) Apoptotic index was determined using TUNEL assays 1 day after TBI and the most effective dose of luteolin (30 mg/kg) was chosen in this experiment. The apoptotic index was significantly higher after TBI compared to the sham group. Luteolin treatment significantly decreased the percentage of apoptotic cells after TBI. Arrows show the typical apoptotic cells on the left panel. n ¼ 6 each group. Data are presented as mean 7 SEM; nnnP o 0.001 versus sham group; #P o 0.05, ##P o 0.01 versus TBI þ vehicle group. Scale bar: 50 mm.

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luteolin-treated group was significantly better than the vehicletreated group. Larger doses such as 50 mg/kg, however, did not exhibit a better neuroprotection (P 4 0.05; Fig. 1A). We also examined brain water content to confirm the neuroprotection of luteolin. The results showed that all of the three luteolin-treated groups significantly decreased the brain water content after TBI as compared with the vehicle-treated group (P o 0.05, P o 0.01, and P o 0.05, resp.; Fig. 1B). Consistent with the grip test, a dose of 30 mg/kg had the best effect in alleviating the brain edema induced by TBI, although there is no significant difference between them (P 4 0.05; Fig. 1B). Therefore, our data confirmed that luteolin is neuroprotective against TBI, and suggest that 30 mg/kg exhibits the best effect, which we used in the following experiments.

Luteolin inhibits neuronal apoptosis To determine whether the neuroprotective effects of luteolin can be detected on a histopathology level, we employed TUNEL to examine the apoptotic cells in the brain tissues. The observation showed that few TUNEL-positive cells were found in the sham group mice brain, while TBI induced an apoptotic index at approximate 33.8% (Fig. 1C). There is no difference between the TBI group and the vehicle-treated group (P 4 0.05); however, luteolin significantly decreased the apoptotic index to 23.5% (P o 0.01; Fig. 1C). This result indicated that luteolin administration following TBI could lead to less cell death in the brain surrounding the cortical contusion and had the potential to ameliorate the secondary brain insult following TBI.

Luteolin reduces oxidative stress in injured brains Next, to elucidate how luteolin affects the outcomes after TBI, we examined the oxidative stress level in brain tissue. MDA reflects the level of lipid peroxidation while GPx catalyzes the reactions of reduced glutathione. Our observations showed that elevated MDA was detected in the TBI group and the TBI þ vehicle group (P o 0.001; Fig. 2A). Administration of luteolin significantly reduced the generation of MDA (P o 0.01 versus TBI þ vehicle; Fig. 2A). GPx, in contrast, was decreased after TBI (P o 0.01; Fig. 2B), while luteolin could upregulate the activity of GPx significantly (P o 0.05 versus TBI þ vehicle; Fig. 2B).

Luteolin promotes Nrf2 nuclear translocation and enhances Nrf2-ARE binding Nrf2 has a potential role in the regulation of oxidative stress. Our results provided evidence that luteolin reversed the oxidative stress induced by TBI (Fig. 2). Also, a prior study showed that luteolin promoted Nrf2 translocation to the nucleus, protecting PC12 and C6 cells from MPP þ -induced toxicity, and this effect can be blocked by inhibition of ERK1/2 [28]. Therefore, it was reasonable to hypothesize that luteolin might activate Nrf2. The results of Western blot showed that compared with the sham group, both TBI and administration of luteolin were inductors of Nrf2 nuclear translocation (Fig. 3A). In addition, compared with the vehicletreated group, the luteolin-treated group significantly increased the nuclear location of Nrf2 and reduced the cytoplasm location of Nrf2 (P o 0.01 and P o 0.001, resp.; Fig. 3A), which indicated that luteolin promoted Nrf2 nuclear translocation. This effect was also confirmed by immunohistochemistry. As seen in Fig. 3B, TBI enhanced the expression of Nrf2 in the nucleus, and luteolin enhanced Nrf2 concentration in the nucleus. Consistent with these observations, EMSA results showed that luteolin enhanced nuclear protein Nrf2 binding to AREs in the HO1 enhancer (Fig. 3C). These abundant evidences indicated that luteolin can promote Nrf2 translocation from cytoplasm to nucleus, thereby obtaining elevated binding ability to the downstream genes.

Luteolin upregulates expression of Nrf2 downstream proteins The observations showed that luteolin was able to activate Nrf2 and provide neuroprotection against TBI, so we hypothesized that luteolin might regulate the Nrf2 downstream pathway. Therefore, we examined the effect of luteolin on expression of antioxidative proteins downstream of Nrf2. RT-PCR results indicated that both HO1 and NQO1 mRNA were upregulated after TBI (P o 0.001 and P o 0.05, resp.; Fig. 4A and B). Additionally, administration of luteolin further enhanced their expression of mRNA as compared with the vehicle-treated group (P o 0.001 and P o 0.05, resp.; Fig. 4A and B). On the protein level, consistent with changes of mRNA, luteolin enhanced the expression of HO1 and NQO1 as compared with TBI þ vehicle groups (P o 0.05, Fig. 4C). These results indicated that luteolin induced expression of Nrf2 downstream proteins on both mRNA and protein levels through activation of Nrf2.

Fig. 2. Luteolin reduces oxidative stress in brain tissue after TBI. (A, B) oxidative stress is represented by the level of MDA and the activity of GPx. Mice were subjected to TBI and then administrated luteolin or vehicle 30 min after TBI. Ipsilateral cortex was collected 1 day after TBI and underwent biochemistry detection. (A) MDA was significantly increased after TBI compared to the sham group, while treatment with luteolin significantly restored this change. (B) Inversely, TBI significantly lowered the activity of GPx; however, the activity of GPx was raised after treatment with luteolin. Data are presented as mean 7 SEM, n ¼ 6 per group; nnP o 0.01, nnnP o 0.001 versus sham group; # P o 0.05, ##P o 0.01 versus TBI þ vehicle group.

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Fig. 3. Luteolin promotes translocation of Nrf2 from cytoplasm to nucleus and enhances Nrf2–ARE binding. (A) Mice brain tissues were collected 1 day after TBI in different groups and the Nrf2 levels in both cytoplasm and nucleus were measured by Western blot. Luteolin significantly increased the level of Nrf2 in nucleus and decreased the level of Nrf2 in their counterpart cytoplasm. β-Actin and H3 were used as protein loading control. (B) Representative photomicrographs showing Nrf2 immunohistochemistry of tissue from different groups 1 day after TBI. As compared with sham group, the TBI group presented a morphology with Nrf2 concentrated in the nucleus, and after treatment with luteolin, this concentration morphology was more apparent. (C) Based on EMSA, Nrf2–ARE-binding activity was enhanced after TBI and substantially increased after treatment with luteolin. Data are presented as mean 7 SEM, n ¼ 6 per group; nP o 0.05, nnP o 0.01, and nnnP o 0.001 versus sham group; ##P o 0.01, ### P o 0.001 versus TBI þ vehicle group. Scale bar: 20 mm.

Protective effect of luteolin in primary cultured neuron

Luteolin fails to protect brain injury in Nrf2-deficient mice

Prior studies also showed that luteolin could regulate the inflammatory process, and inflammation could in turn regulate the activity of Nrf2. In order to exclude this effect, we chose to confirm the neuroprotection of luteolin in primary cultured neurons. We first performed LDH release assay and trypan blue staining on cultured neurons with different concentrations of luteolin which underwent scratch. In LDH release assay, luteolin reduced the percentage of damaged cells in a dose-dependent manner, although there is no significant difference between 5 mM luteolin and DMSO-treated groups (Fig. 5A). Meanwhile, similar results were observed in trypan staining (Fig. 5B). Next, we examined the effect of luteolin on intracellular ROS production. As seen in Fig. 5C, luteolin reduced intracellular ROS production induced by scratch in a dose-depended manner, with 10 and 25 mM luteolin significantly depressing the level of ROS as compared with DMSO-treated group (P o 0.01 and P o 0.001, resp.).

As proof of our hypothesis, that is, neuroprotection of luteolin is attenuated in the absence of Nrf2, we evaluated the neuron apoptotic index, brain water content, and motor function in Nrf2-/- mice subjected to TBI. The mice were treated with vehicle or 30 mg/kg luteolin 30 min after the onset of TBI. The results showed that there was no significant difference of apoptotic index between the vehicle-treated group and the luteolin-treated group (P 4 0.05; Fig. 7A). Similarly, there were no significant differences in brain water content (P 4 0.05) and the grip test (P 4 0.05) (Fig. 7B and C), suggesting that luteolin loses its neuroprotection in Nrf2-/- mice and that the Nrf2–ARE pathway is a mechanism involved in luteolin neuroprotection.

Luteolin activates the Nrf2–ARE pathway in vitro The Western blot results showed that nuclear Nrf2 increased after TBI, and luteolin enhanced this concentration. The cytoplasmic Nrf2 as a counterpart of the nuclear Nrf2 was decreased. The downstream proteins HO1 and NQO1 were increased after luteolin treatment, and both of them were in a dose-dependent manner (Fig. 6).

Discussion In the present study, we investigated the neuroprotection of luteolin on TBI in wild-type mice, primary cultured neurons, and Nrf2-deficient mice. The main findings of this study are as follows. (1) Luteolin provides neuroprotection in wild-type mice and primary cultured neurons; specifically, it improves neurobehavioral performance, alleviates cerebral edema, and suppresses neuronal apoptosis. (2) Administration of luteolin reverses the oxidative stress state induced by TBI, which is represented by the level of MDA, the activity of GPx, and the production of intracellular ROS. (3) Nrf2 undergoes a translocation from cytoplasm to nucleus in the presence of luteolin, subsequently increasing the

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Fig. 4. Luteolin upregulates the expression of Nrf2 downstream in mice on both mRNA and protein levels. Mice were subjected to TBI and treated with 30 mg/kg of luteolin or vehicle 30 min after TBI. Ipsilateral brain tissues were collected 1 day after TBI. mRNA and protein expressions were examined by RT-PCR and Western blot, respectively. (A) HO1 mRNA was elevated after TBI and was further increased with administration of luteolin. (B) Similarly, NQO1 mRNA was elevated after TBI and was further increased with administration of luteolin. (C) Both HO1 and NQO1proteins were upregulated after TBI; additionally, luteolin further increased their expression in brain tissue. Actin was used as a loading control. Data are presented as mean 7 SEM, n ¼ 6 per group; nP o 0.05, nnP o 0.01, and nnnP o 0.001 versus sham group; #P o 0.05, ###P o 0.001 versus TBI þ vehicle group.

expression of downstream factors on mRNA and protein levels due to the elevated binding ability of Nrf2 to AREs enhancers. (4) Luteolin fails to provide similar protective effects in Nrf2-/mice as in wild-type mice. The secondary injury after TBI represents consecutive pathological processes which consist of excitotoxicity, inflammatory response, oxidative stress, and vascular abnormalities, etc. [2]. Several oxidants and their derivatives are generated after TBI, including superoxide anions and hydroxyl radicals [29]. This enhanced production of reactive oxygen species along with exhausted antioxidant defense enzymes, such as superoxide dismutase, catalase, and GPx, causes oxidative stress [30]. Substantial evidence indicates that oxidative stress is a major contributor to the pathophysiology of TBI. Specifically, it leads to damage in lipids, proteins, and nucleic acids [31,32]. Current antioxidant strategies are based on the following mechanisms: (1) removal of ROS, (2) inhibition of ROS formation, and (3) binding metal ions needed for catalysis of ROS generation [29]. Lipid peroxidation which refers to oxidative degradation of lipid increases the permeability of membranes, leading to cell damage [29]. MDA formation in the brain tissue is widely used as the index of lipid peroxidation. Hou and his colleagues reported that MDA increased as early as 1 min after TBI and maximized at 2 h post-TBI, persisting at 24 and 48 h after injury [33]. GPx is a well-known intracellular antioxidant enzyme, catalyzing the reactions of reduced glutathione. Its activity underwent a descent and minimum at 24 h after TBI, showing a 30% decrease with respect to controls [34]. Nrf2 as an important translation factor in the maintenance of cellular homeostasis has been widely studied in past decades. Much evidence indicated that Nrf2 and phase II enzymes such as NQO1 and HO1 were activated after TBI [11,35]. Besides, Nrf2-/mice exhibited poorer outcomes than the wild-type mice, while

administration of tBHQ or histone deacetylase inhibitors could protect against TBI by activation of Nrf2 [13,18]. These evidences demonstrate that activation of the Nrf2–ARE pathway is beneficial for TBI. It is now general accepted that the Nrf2–ARE pathway can be regulated in many different ways. Among all sorts of mechanisms reviewed by Bryan et al. [8], Keap1 degradation via the autophagy pathway is of great interest. The substrate adaptor Sequestosome1 (also known as p62) is a scaffold protein that can associate with both the autophagosome localizing protein LC3 and ubiquitinated proteins, thereby facilitating the degradation of these proteins in the autophagosome [36,37]. However, recently a physical and functional relationship between Keap1 and p62 has been elucidated [38,39]. Evidence suggested that p62 played a role in regulating Keap1 degradation via autophagy [40], and maybe related with the phosphorylation of p62 [41]. To investigate the effects of luteolin against secondary brain injury after TBI, mice were subjected to intraperitoneal injections of luteolin 30 min post-TBI. A subsequent question to this, namely, whether luteolin can across the blood brain barrier (BBB) by peripherally administration, is inevitable. Previous studies on different neurological diseases showed that peripherally administered luteolin had corresponding effects in brain tissue [21,42]. Thus, it is reasonable to predict that luteolin can penetrate the BBB and exhibit its property in brain tissue. More direct evidence on this question comes from a recent study [43], in which the brain tissue of total luteolin was analyzed by HPLC when mice were given systemic administration by gavage. All these data indicate that luteolin can freely penetrate the BBB and enter the brain. The neuroprotective effects of luteolin may involve multiple mechanisms. Previous studies suggested that luteolin could prevent monocyte migration across the brain endothelium by modulating the activity of Rho GTPases [44], decrease the elevated

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Fig. 5. Luteolin protects primary cultured neurons from TBI. (A, B) Primary cortical neurons were subjected to scratch injury and then treated with 5, 10, or 25 mM luteolin or DMSO for 1 day. The LDH release assay and TB staining were used to evaluate cell viability. The percentage of damaged cells significantly increased after TBI compared to the control group. Luteolin treatment (10 or 25 mM) significantly decreased damaged cells after TBI. (C) Luteolin repressed the production of ROS in primary cultured cells after TBI. Cells were subjected to scratch injury and subsequently treated with 5, 10, or 25 mM luteolin or DMSO for 1 day. Then cells were incubated with DCFH-DA, and subjected to fluorescent microscopy analysis. The intracellular ROS was significantly increased after TBI compared to the sham group, and administration of luteolin (10 mM or 25 mM) significantly repressed ROS production as compared to the TBI þ DMSO group. Data are presented as mean 7 SEM, n ¼ 6 per group; nP o 0.05, nnP o 0.01, and nnn P o 0.001. Scale bar: 50 mm.

Fig. 6. Luteolin promotes Nrf2 translocation from cytoplasm to nucleus and actives the proteins downstream in primary cultured neurons. Luteolin significantly increased the level of Nrf2 in the nucleus, and consequently reduced the level of Nrf2 in the cytoplasm. Elevated levels of HO1 and NQO1 which were downstream of Nrf2 were examined after TBI, and luteolin treatment significantly enhanced their expression as compared to DMSO treated. β-Actin and H3 were used as protein loading control. Data are presented as mean 7 SEM, n ¼ 6 per group; nP o 0.05, nnnP o 0.001 versus sham group; #P o 0.05, ##P o 0.01, and ###P o 0.001 versus TBI þ vehicle group.

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Fig. 7. Luteolin fails to provide neuroprotection against TBI in Nrf2-/- mice. Apoptotic index, brain water content, and the grip test score were used to evaluate the effects of luteolin on TBI in Nrf2-/- mice. Luteolin treatment (30 mg/kg) had no effect on reducing apoptotic cells (A), ameliorating brain edema (B), and improving motor performance (C) in Nrf2-/- mice compared to the vehicle-treated group. n ¼ 6 per group. Scale bar: 50 mm.

levels of proinflammatory cytokines IL-6, IL-8, and MCP-1 induced by IL-1β [45], and inhibit the activity of AP-1 and NF-κb [46,47] in different cerebral diseases, thereby mitigating neuroinflammation. Besides, luteolin was proven to play important roles in regulating redox balance. Its properties of scavenging free radicals [48] and modulating enzyme activities such as xanthine oxidase and nitric oxide synthase [14] are widely accepted as certain, while more recently, a variety of studies showed that luteolin confers neuroprotection by regulation of the Nrf2–ARE pathway [28,49,50]. Our observations in this study are consistent with prior reports, indicating that luteolin could regulate this antioxidant pathway by promoting Nrf2 translocation from cytoplasm to nucleus. More interesting, a recent study on the effects of luteolin on lung carcinoma cells indicated that the autophagy pathway could be induced by luteolin with evidence of accumulation of microtubuleassociated protein light chain-3 (LC3) II protein and the increase of LC3 puncta as well as an enhanced autophagy flux [51]. Therefore, we speculate that luteolin induces autophagy and enhances the degradation of Keap1 via interaction of Keap1 and p62. As a consequence, the Nrf2–Keap1 complex is dissociated ending up with Nrf2 nuclear translocation and activation of downstream AREs. Whether this is true, however, needs further study. Our in vivo study showed that luteolin provided neuroprotection within 3 days but did not last to 7 days which did seem satisfactory. Besides, a 50 mg/kg dose of luteolin did not show better effects than 30 mg/kg. Therefore, multiple administration of luteolin will be considered to improve efficacy in our future studies. In addition, more data about the effects of luteolin on normal animals should be established for the sake of clinical application.

Conclusion In conclusion, our data showed that luteolin exerted neuroprotection against TBI by combating oxidative stress, at least partly via

translocation of Nrf2 from cytoplasm to nucleus and activation of downstream proteins. However, further study is still needed to elucidate the underlying mechanism of Nrf2 translocation, especially where autophagy may be involved.

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Luteolin provides neuroprotection in models of traumatic brain injury via the Nrf2-ARE pathway.

Luteolin has recently been proven to exert neuroprotection in a variety of neurological diseases; however, its roles and the underlying mechanisms in ...
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