Mol Neurobiol DOI 10.1007/s12035-015-9318-8

Minocycline Protects Against NLRP3 Inflammasome-Induced Inflammation and P53-Associated Apoptosis in Early Brain Injury After Subarachnoid Hemorrhage Jianru Li 1 & Jingsen Chen 1 & Hangbo Mo 1 & Jingyin Chen 1 & Cong Qian 1 & Feng Yan 1 & Chi Gu 1 & Qiang Hu 2 & Lin Wang 1 & Gao Chen 1

Received: 16 December 2014 / Accepted: 24 June 2015 # Springer Science+Business Media New York 2015

Abstract Minocycline has beneficial effects in early brain injury (EBI) following subarachnoid hemorrhage (SAH); however, the molecular mechanisms underlying these effects have not been clearly identified. This study was undertaken to determine the influence of minocycline on inflammation and neural apoptosis and the possible mechanisms of these effects in early brain injury following subarachnoid hemorrhage. SAH was induced by the filament perforation model of SAH in male Sprague–Dawley rats. Minocycline or vehicle was given via an intraperitoneal injection 1 h after SAH induction. Minocycline treatment markedly attenuated brain edema secondary to blood-brain barrier (BBB) dysfunction by inhibiting NLRP3 inflammasome activation, which controls the maturation and release of pro-inflammatory cytokines, especially interleukin-1β (IL-1β). Minocycline treatment also markedly reduced the number of terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive cells. To further identify the potential mechanisms, we demonstrated that minocycline increased Bcl2 expression and reduced the protein expression of P53, Bax, and cleaved caspase-3. In addition, minocycline reduced the cortical levels of reactive oxygen species (ROS), which are closely related to both NLRP3 inflammasome and P53 expression. Minocycline protects against NLRP3 inflammasomeinduced inflammation and P53-associated apoptosis in early Jianru Li, Jingsen Chen and Hangbo Mo contributed equally to this work. * Gao Chen [email protected] 1

Department of Neurosurgery, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China

2

Department of Neurosurgery, Hangzhou First People’s Hospital, Nanjing Medical University, Hangzhou, China

brain injury following SAH. Minocycline’s anti-inflammatory and anti-apoptotic effect may involve the reduction of ROS. Minocycline treatment may exhibit important clinical potentials in the management of SAH. Keywords Early brain injury . Minocycline . NLRP3 . Apoptosis . P53 . Subarachnoid hemorrhage

Introduction Subarachnoid hemorrhage(SAH) is a severe disease that accounts for approximately 6 to 8 % of all strokes and 22 to 25 % of cerebrovascular deaths [1]. Previous studies on SAH have been focused on vasospasm; however, anti-vasospastic drugs fail to improve outcome in clinical trials [2]. In recent years, growing evidence has indicated that early brain injury play an important role in SAH [2, 3]. The possible mechanisms include increased intracranial pressure, a reduction in cerebral blood flow, oxidative stress, inflammation, and apoptosis [4]. Thus, a novel, safe, and effective drug for both inflammation and apoptosis may be promising for patients with SAH. Inflammation is hypothesized to mediate brain injury and cerebral vasospasm after SAH [5]. An increase in proinflammatory cytokines, including TNF-nflammatory cy-1β (IL-1β) and IL-6, is observed acutely after SAH [6]. The potential mechanisms underlying the upregulation of proinflammatory cytokines involve the activation of nuclear factor-κB (NF-κB) and the activation of the mitogenactivated protein kinase pathway and toll-like receptor 4 (TLR4) signaling pathway [6–8]. Recently, a study focused on the inflammasome axis, which controls the maturation and release of pro-inflammatory cytokines, demonstrated neuroprotective effects in early brain injury after SAH [9].

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Apoptosis is reported to be a mechanism of early brain injury (EBI) after SAH and has been investigated in a number of studies. Apoptotic pathways and cascades within cortical, subcortical, or hippocampal neurons and vascular cells have been demonstrated following the onset of SAH [10]. The pathways involved in apoptosis after SAH include intrinsic (caspase-independent and mitochondrial) and extrinsic (cell-death receptor) pathways [11]. The tumor suppressor P53 is a transcription factor that may play a central role in the organization and orchestration of apoptosis. P53 has previously been shown to play an important role in endothelial apoptosis and cerebral vasospasm after SAH [12, 13]. P53 is thought to play a major role in the organization of caspase-dependent and caspase-independent pathways as well as the mitochondrial cascades after SAH [14]. Reactive oxygen species (ROS) are highly reactive free radicals that include superoxide anion (O2·), hydroxyl radical (OH·), and hydrogen peroxide (H2O2). ROS regulates several important physiological responses and plays an important role in the pathological processes of SAH [15]. ROS, especially mitochondrial ROS, induces NLRP3 inflammasome activation, and excessive ROS induces DNA damage, which activates P53 and increases P53 expression [16, 17]. Minocycline is a second-generation, semi-synthetic tetracycline that can easily pass though the blood-brain barrier [18] In recent years, minocycline has been shown to exhibit promising neuroprotective properties in various CNS disease models, such as ischemia, traumatic brain injury, and neurodegenerative diseases [19–21]. Recent studies have also demonstrated that minocycline improves outcomes and protects against early brain injury through MMP-9 inhibition after SAH [22]. Furthermore, early minocycline treatment after SAH provides long-term benefits with respect to cognitive function and improved histopathology [23]. However, the anti-inflammatory and anti-apoptotic effects of minocycline and the potential mechanisms underlying these effects have not been evaluated in EBI after SAH. In the present study, we aimed to investigate the following hypotheses: (1) Minocycline ameliorates functional deficits and brain edema as well as the destruction of the blood-brain barrier. (2) Minocycline treatment inhibits NLRP3 inflammasome activation, reduces levels of IL-1β, and thereby decreases neuroinflammation in EBI after SAH. (3) Minocycline decreases neural apoptosis induced by P53-associated apoptotic pathway. (4) The anti-inflammatory and anti-apoptotic effects of minocycline may involve the reduction of ROS.

cycle under controlled temperature and humidity conditions. All procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University and in accordance with the guidelines of National Institutes of Health Guide for the Care and Use of Laboratory Animals. Study Design One hundred and thirty adult rats were randomly assigned to three groups: the sham group (n=36), the SAH + vehicle group (n=48), and the SAH + minocycline group (n=46). All end points in this study were investigated at 24 h after SAH. Six animals in each group were used for Western blot analysis. Eight rats in each group were required for brain water content analysis and Evans blue dye extravasation as well as ROS assay. Six rats in each group were used for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and immunohistological staining. Drug Administration Minocycline was purchased from Sigma-Aldrich (St. Louis, MO, USA) and was dissolved in phosphate-buffered saline (PBS; 0.1 mol/L, pH 7.4). The treatment groups received 135 mg/kg of minocycline intraperitoneally 1 h after SAH induction. The dose of minocycline and the time point were chosen based on a previous study [23], in which beneficial effects were observed on improving functional outcomes and reducing memory deficits. The sham group and vehicle group received the same volume of PBS intraperitoneally 1 h after SAH induction. SAH Rat Model The endovascular perforation SAH rat model was produced as previously described with modifications [24]. Briefly, after the rats were anesthetized with an intraperitoneal injection of pentobarbital (40 mg/kg), the left carotid artery and its branches were exposed. A blunted 4-0 monofilament nylon suture was placed in the external carotid artery and advanced through the internal carotid artery until resistance was felt. The bifurcation of the anterior and middle cerebral artery was then punctured by inserting the suture an additional 3 mm. Sham rats underwent the same procedures with the exception of vessel puncture. Neurological Score and SAH Grade

Materials and Methods Animals Adult male Sprague–Dawley rats weighing 300–320 g were obtained from Slac Laboratory Animal Co., Ltd. (Shanghai, China). The animals were maintained on a 12-h light/dark

The neurological scores were evaluated 24 h after SAH using the previously described scoring system of Garcia et al. [25]. Briefly, the evaluation consists of six tests that can be scored 0–3 or 1–3 and include the following: spontaneous activity, symmetry in the movement of four limbs, forepaw outstretching, climbing, body proprioception, and the

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response to vibrissae touch. Possible scores ranged from 3 to 18. All of the tests were evaluated by an observer who was blind to the treatment conditions. A lower score represents serious neurological deficits. The severity of the SAH was quantified according to a previously described grading scale [26]. The scale was based on the amount of subarachnoid blood in six segments of the basal cistern A total score ranging from 0 to 18 was obtained by adding the scores. Brain Water Content Brains were removed 24 h after SAH and were separated into the left hemisphere, right hemisphere, and cerebellum. Left hemispheres were weighed immediately to obtain the wet weight and were then dried at 105 °C for 24 h to obtain the dry weight. The percentage of water content was calculated as follows: [(wet weight−dry weight)/wet weight]×100 % [27]. Evans Blue Dye Extravasation The permeability of the blood-brain barrier (BBB) was evaluated on the basis of Evans blue extravasation, as described previously [28]. Evans blue dye (2 %, 5 mL/kg) was administered into the left femoral vein after 24 h of SAH and allowed to circulate for 1 h. Subsequently, the rats were sacrificed under deep anesthesia by intracardial perfusion with PBS. Subsequently, the brain was removed and the left hemisphere was separated immediately. Brain samples were weighed and homogenized in 3 mL of 50 % trichloroacetic acid and then centrifuged at 15,000×g for 30 min. The supernatant (1 mL) was mixed with an equal volume of trichloroacetic acid with ethanol (1:3). After overnight incubation at 4 °C, the samples were centrifuged at 15,000×g for 30 min, and the supernatant was measured by spectrofluorophotomery at an excitation wavelength of 620 nm and an emission wavelength of 680 nm. ROS Assay The left basal cortical sample, which faced the blood clot was collected at 24 h after SAH. The total ROS levels in the brains were measured with a ROS assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), following the manufacturer’s instruction. Briefly, the fresh tissue were weighed (approximately 70 μg) and homogenized in PBS (approximately 1.5 mL), followed by centrifugation at 1000×g for 10 min at 4 °C. The protein content of the supernatant was measured using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). The supernatant (190 μL) was added to 96-well plates and mixed with 1 mmol/L DCFH-DA (10 μL). The supernatant in the control well was mixed with PBS (10 μL). The samples were incubated at 37 °C for 30 min. The mixture was measured by spectrofluorophotomery at an

excitation wavelength of 480 nm and an emission wavelength of 520 nm. The ROS levels in the fresh tissue were expressed as fluorescence intensity/gram protein. TUNEL and Immunohistological Staining After 24 h of SAH, the rats were sacrificed and perfused intracardially with PBS (pH 7.4) and 4 % paraformaldehyde (pH 7.4). Brains were collected and immersed in 4 % paraformaldehyde at 4 °C for 12 h and followed by immersion in 30 % sucrose solution until the tissue sank (3 days). Subsequently, the brains were frozen in tissue-freezing media and cut in 7 μm sections. The primary antibodies used were mouse monoclonal anti-Iba-1 antibody (1:300, Millipore). Afterward, the sections were washed with PBS several times and were then incubated with fluorescein isothiocyanate-labeled goat anti-mouse antibody (1:200, Jackson ImmunoResearch) for 2 h at room temperature in the dark. The sections were rinsed and stained with DAPI and then rinsed again and mounted with glycerol. TUNEL staining was performed according to the manufacturer’s protocol (Roche Inc, Basel, Switzerland) and examined under a laser scanning confocal microscope (LSM-710; Zeiss). To identify the location of NLRP3, double fluorescence labeling was performed on brain sections. The primary antibodies used included rabbit polyclonal anti-NLRP3 antibody (1:200, Abcam) and mouse monoclonal anti-Iba-1 antibody (1:300, Millipore). The secondary antibodies included rhodamine-conjugated goat antirabbit antibody (1:200, Jackson ImmunoResearch) and fluorescein isothiocyanate-labeled goat anti-mouse antibody (1:200, Jackson ImmunoResearch). Western Blot Western blot analysis was performed as previously described [24]. The cytoplasmic and nuclear protein extracts were prepared with the NE-PER nuclear and cytoplasmic extraction reagents (Thermo, Rockford, IL, USA) according to the manufacturer’s instructions. Briefly, the left hemispheres (perforation side) were homogenized and centrifuged at 1000×g for 10 min at 4 °C. The protein content was measured using the DC protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein samples (60 μg) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with a nonfat dry milk buffer for 2 h, followed by incubation overnight at 4 °C with the primary antibodies: rabbit polyclonal anti-cleaved IL-1β antibody (1:300, Santa Cruz), rabbit polyclonal anti-cleaved caspase-1 (1:300, Santa Cruz), rabbit polyclonal antiNLRP3 antibody (1:800, Abcam), rabbit polyclonal anti-NF-κB (1:1500, Abcam), rabbit polyclonal anti-P53 antibody (1:300, Santa Cruz), rabbit polyclonal anti-Bax

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antibody (1:500, Santa Cruz). Histone (H3) and β-actin were used as the positive control for nuclear extracts and cytoplasmic extracts, respectively. The membranes were processed with horseradish peroxidase-conjugated secondary antibodies for 1 h at 21 °C. Blot bands were detected by X-ray film and quantified using Image J software (NIH). Statistical Analysis To facilitate comparisons between the four groups, band density values were normalized to the mean value for the control group. Data are expressed as the mean±SD. Statistical significance was analyzed by a one-way analysis of variance (ANOVA) followed by Tukey test for multiple comparisons. Neurological scores and SAH grade were expressed in median values (range) and analyzed with a one-way ANOVA on ranks. The Mann–Whitney U test was used for comparisons between groups. Statistical significance was defined as P<0.05.

Results Mortality and SAH Grade A mortality rate of 25.0 % (n=12/48) was recorded in vehicle group, whereas 21.7 % (n=10/46) mortality was observed in the SAH + Mino group. However, our study was not sufficiently powered to detect the significant differences in the mortality between the SAH + vehicle group and SAH + Mino group. No mortality was recorded in the sham-operated rats. Similarly, the SAH grades did not differ significantly between the SAH + vehicle group and SAH + Mino group (Fig. 1b). Neurological Scores The neurological scores in the SAH + vehicle group were significantly lower than those in the sham group (P<0.05, Fig. 1c); however, the SAH rats that received minocycline treatment exhibited significantly improved neurological performances 24 h after SAH induction (P<0.05 compared with vehicle, Fig. 1c).

after surgery (P<0.05, Fig. 1d). Minocycline treatment significantly reduced Evans blue dye extravasation in the left hemisphere relative to the SAH + vehicle group (P<0.05, Fig. 1d).

NLRP3 Was Mainly Located in Microglia To date, NLRP3 location has not been identified in SAH. Therefore, we used double fluorescence labeling to identify the location of NLRP3 in SAH. Our results demonstrated that NLRP3 was mainly located in the microglia (Fig. 2).

Minocycline Reduced Cleaved IL-1β Expression and Inhibited NLRP3 Inflammasome Activation in the Left Hemisphere 24 h After SAH Induction Western blot results revealed that the pro-IL-1β levels were significantly increased 24 h after SAH, when compared with the sham group (P<0.05, Fig. 3a). However, there was no significant difference in the protein levels of pro-IL-1β between the SAH + vehicle group and the SAH + Mino group (P<0.05, Fig. 3a). Cleaved IL-1β was significantly increased 24 h after SAH (P < 0.05, Fig. 3b). Cleaved IL-1β expression was markedly decreased by minocycline treatment 24 h after SAH (P < 0.05, Fig. 3b). To explore the potential role of minocycline in NLRP3 inflammasome, Western blot results was used to demonstrate that NLRP3 and cleaved caspase-1 protein levels were significantly increased 24 h after SAH (P < 0.05, Fig. 3c, d) and were reduced by minocycline injection when relative to vehicle rats (P < 0.05, Fig. 3c, d). We also measured the effect of minocycline on NF-κB p65 level in cytoplasmic and nuclear fraction. Our results demonstrated that the level of p65 in nuclear was significantly increased in the SAH + vehicle group compared with the sham-operated group (P<0.05, Fig. 4), while minocycline did not affect the p65 level in nuclear (P>0.05, Fig. 4).

Brain Water Content and BBB Permeability

Minocycline Reduce Neural Apoptosis in the Cortex 24 h After SAH Induction

The brain water content in the SAH + vehicle group was significantly higher than that in the sham group (P<0.05, Fig. 1e); however, the minocycline-treated rats exhibited significantly reduced brain water content relative to the SAH + vehicle group (P<0.05, Fig. 1e). The Evans blue level significantly increased in the left hemisphere in the vehicle group compared with the sham group at 24 h

In sham rats, no TUNEL-positive cells were detected. Numerous TUNEL-positive cells were observed in the cortex of the SAH + vehicle rats, and the apoptotic index was significantly increased compared with the sham-operated group (P < 0.05, Fig. 5). Minocycline treatment markedly reduced the number of TUNELpositive cells (P<0.05, Fig. 5).

Mol Neurobiol Fig. 1 Representative pictures of brains from each group and SAH grade, neurological scores, brain water content, and Evans blue dye extravasation at 24 h after SAH. a Typical brains from sham, SAH + vehicle, and SAH + Mino. b Quantification of SAH severity. The bars represent the mean±SD. n=36. c The quantification of neurological scores. The bars represent the mean±SD. n=36. *P<0.05 vs sham, #P<0.05 vs SAH. Brain water content, and Evans blue dye extravasation at 24 h after SAH. d The quantification of Evans blue dye extravasation. The bars represent the mean±SD. n=8. *P<0.05 vs sham, #P<0.05 vs SAH. e Quantification of brain water content. The bars represent the mean±SD. n=8. *P<0.05 vs sham, #P<0.05 vs SAH

Minocycline Reduced the Protein Expression of P53 Associated Apoptotic Proteins and Increased Protein Expression of Bcl2 in the Left Hemisphere 24 h After SAH Induction Upregulation of P53 was observed 24 h after SAH in vehicle group when compared to sham (P<0.01, Fig. 6a). Accordingly, the protein expression levels of Bax and caspase-3 was significantly increased 24 h following SAH in the SAH + vehicle group relative to sham rats (P < 0.05, Fig. 6b, d). Minocycline treatment significantly reduced P53 expression relative to the SAH + vehicle group (P<0.05, Fig. 6a). Western Fig. 2 Double fluorescence labeling of NLRP3/Iba-1 in the ipsilateral basal cortex at 24 h after SAH (scale bar = 20 μm)

blot results revealed that Bax and caspase-3 protein levels were markedly reduced by minocycline injection compared with vehicle rats (P<0.05, Fig. 6b, d). In addition, the protein levels of Bcl2 were significantly decreased in SAH + vehicle group (P<0.05, Fig. 6c), whereas minocycline treatment increased the protein expression of Bcl2 (P<0.05, Fig. 6c). Minocycline Inhibited Activation of Microglia and Reduced ROS Level We studied the effects of minocycline on expression of Iba-1 in the cortex after SAH. Our results indicated that the number of

Mol Neurobiol Fig. 3 Minocycline reduced cleaved IL-1β expression and inhibited NLRP3 inflammasome activation. a Western blot assay for the expression of pro-IL-1β and b cleaved IL-1β and c NLRP3 as well as d cleaved caspase-1 in the ipsilateral hemisphere in sham, SAH + vehicle, and minocycline groups at 24 h after SAH induction; n=6 rats per group. The bars represent the mean±SD. n=6. *P<0.05 vs sham, #P<0.05 vs SAH

Iba-1-positive cells is significantly increased in the SAH + vehicle group compared with the sham-operated group (Fig. 7a, b; P<0.05) which indicate robust microglia activation after SAH. Minocycline administration significantly reduces the number of Iba-1-positive cells in comparison with the SAH + vehicle group Fig. 4 Effect of treatment with minocycline on NF-κB p65 nuclear translocation. a Western blot assay for the expression of Nuc-p65. b Western blot assay for the expression of Cyto-p65. The bars represent the mean±SD. n= 6. *P<0.05 vs sham, #P<0.05 vs SAH

(Fig. 7a, b; P<0.05). The ROS assay revealed that ROS was significantly increased by approximately threefold 24 h after SAH, relative to the sham group (P<0.01, Fig. 7c). ROS levels were markedly reduced by minocycline treatment compared with vehicle rats (P<0.01, Fig. 7c).

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Fig. 5 Effect of minocycline treatment on cell apoptosis in the ipsilateral basal cortex at 24 h after SAH. a–i Representative TUNEL/DAPI photomicrographs of the ipsilateral cortex in the different groups (scale bar = 100 μm). Fluorescence colors: DAPI—blue, and TUNEL—green.

j Quantification of TUNEL-positive cells in the groups, expressed as percentage of total (DAPI+) cells. The bars represent the mean±SD. n=6. *P<0.05 vs sham, #P<0.05 vs SAH

Discussion

initial stage, massive brain injury observed during patient autopsies has confirmed its primary importance in SAH [2]. Much of the research in the SAH field related to minocycline has focused on minocycline’s ability to inhibit MMP9 without considering minocycline’s potential influence on

Early brain injury refers to the immediate injury to the brain as a whole, within the first 72 h of ictus, secondary to a SAH [15]. Although research on early brain injury remains in its Fig. 6 Effect of treatment with minocycline on the P53 associated apoptotic proteins and Bcl2. a Western blot assay for the expression of P53 and b Bax and c Bcl2 as well as d caspase-3 in the ipsilateral hemisphere in sham, SAH, and minocycline groups at 24 h after SAH induction; n=5 rats per group. The bars represent the mean±SD. n=6. *P<0.05 vs sham, #P<0.05 vs SAH

Mol Neurobiol Fig. 7 Effect of minocycline treatment on microglia activation and reactive oxygen species. a Representative Iba-1/DAPI photomicrographs of the ipsilateral cortex in the different groups (scale bar = 50 μm). Fluorescence colors: DAPI— blue, and Iba-1—red. b Quantification of Iba-1-positive cells in the groups, expressed as percentage of total (DAPI+) cells. The bars represent the mean±SD. n=6. *P<0.05 vs sham, #P<0.05 vs SAH. c Quantification of ROS. The bars represent the mean±SD. n=8. *P<0.05 vs sham, #P<0.05 vs SAH

inflammation and neural apoptosis. In present study, we made the following observation: treatment with minocycline attenuated brain edema secondary to BBB destruction by inhibiting NLRP3 inflammasome activation in early brain injury after SAH. Furthermore, administration of minocycline attenuated P53 protein expression, Bax protein expression, and cleaved caspase-3 protein expression, which is related to neural apoptosis in early brain injury after SAH. The cortical levels of ROS, which are intimately related to NLRP3 inflammasome activation and P53 protein expression, were increased in early brain injury 24 h after SAH and were repressed by minocycline treatment. Brain edema is one of the common and important features of both clinical SAH and experimental SAH [29, 30]. Brain edema after SAH reflects disruption of the BBB, which is primarily induced by endothelial cells apoptosis, degradation of the basal-lamina by proteases, and diffuse inflammatory reaction [31–33]. Recent studies demonstrate that minocycline improves outcomes and protects against early brain injury after SAH [22]. Furthermore, early treatment with minocycline provides long-term benefits with respect to

cognitive function and improved histopathology [23]. These studies focused on minocycline’s inhibition of MMP9, which changes the permeability of the BBB by disrupting tight junction complexes containing occludin and claudin-5 [34, 35]. However, in addition to the proteases MMP9, inflammation and cytokines may participate in the pathology of BBB disruption and brain edema. A variety of inflammatory cytokines, including IL-1β, IL-6, and TNF-α, are strongly associated with brain injury in rats [6]. Activated NF-κB in neurons plays an important role in regulating inflammatory gene expression in the brain after SAH [7]. Early activation of the mitogenactivated protein kinase (MEK)-extracellular signal-regulated kinase 1/2 (ERK 1/2) also increased the protein expression of IL-1β, IL-6, and MMP-9 after SAH [6]. Recently, a study focused on the inflammasome axis, which controls the maturation and release of pro-inflammatory cytokines, demonstrated neuroprotective effects in early brain injury after SAH [9]. Additionally, pharmacologic inhibition of IL-1β has been shown to attenuate EBI and improve blood-brain barrier function after SAH [36]. Minocycline selectively inhibits microglia polarization to a proinflammatory state [37]. Therefore, we

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attempted to determine whether the left cerebral hemispheres levels of IL-1β were repressed by minocycline. A striking observation in the present study is that minocycline reduced the protein expression of IL-1β, rather than pro-IL-1β. Minocycline appears to inhibit the activation of IL-1β, rather than generation of IL-1β. To further analyze the potential mechanisms of this phenomenon, we measured the NLRP3 inflammasome, which controls the maturation and release of pro-inflammatory cytokines, especially IL-1β [38]. The NLRP3 inflammasome is an important player in the innate immune system and is comprised of NLRP3, the ASC adaptor and caspase-1. This multiprotein complex serves as a platform for caspase-1 activation and caspase-1-dependent maturation and secretion of IL-1β. In our study, we demonstrated that the protein expressions of NLRP3 and caspase-1 were increased in early brain injury, indicating that the NLRP3 inflammasome is activated in early brain injury. Minocycline treatment significantly reduced the protein expressions of NLRP3 and caspase-1. Together, these data indicate that minocycline decreases IL-1β by inhibiting the activation of NLRP3 inflammasome. In addition to its anti-inflammation effects, minocycline has been shown to be anti-apoptotic in various experimental models [20, 39, 40]. However, the anti-apoptotic properties of minocycline in early brain injury after SAH have never been reported. In our study, the results of TUNEL staining revealed that minocycline significantly reduced the number of TUNEL-positive cells, indicating that minocycline suppressed apoptosis in early brain injury after SAH. To further explore the possible mechanism of this phenomenon, we focused on P53 and associated apoptotic proteins. The tumor suppressor gene P53 is a transcription factor that possesses multiple functions in the orchestration of the cell cycle and apoptosis [41]. P53 is stabilized in the cytoplasm in response to SAH [42]. P53 play a major role in the organization of the caspasedependent and caspase-independent apoptotic pathway as well as the mitochondrial apoptosis cascade in SAH [14]. In our study, we examined the protein expressions of P53 in the left cerebral hemisphere 24 h after SAH. Consistent with previous studies, protein expressions of P53 was markedly increased 24 h after SAH and was repressed by minocycline treatment. Unlike previous studies, our research further explored the apoptotic proteins downstream of P53, including Bcl2, Bax, and cleaved caspase-3. P53 is able to influence the balance of pro- and anti-apoptotic members of the BCL-2 family, and thus can affect the subsequent formation of mitochondrial pores and, ultimately, the release of cytochrome C [43]. In the present study, our results demonstrated that Bax expression significantly increased and that Bcl2 expression was significantly decreased 24 h after SAH. Thus, P53 may induce Bax expression and inhibit the Bcl 2 expression. An interesting observation in our study is that minocycline treatment, which repressed the protein expression of P53,

increased Bcl 2 expression and reduced Bax expression. We also examined the protein expression of cleaved caspase-3, which triggers the cleavage of a number of proteins and ultimately leads to DNA fragmentation and apoptosis [44]. Our results demonstrated that minocycline also reduced cleaved caspase-3 expression, which increased 24 h after SAH. ROS are highly reactive free radicals that include superoxide anion (O2·), the hydroxyl radical (OH·), the hydrogen peroxide (H2O2). ROS regulates several important physiological responses and plays an important role in pathogenesis of SAH [15]. ROS, especially mitochondrial ROS, induces NLRP3 inflammasome activation [16]. In addition, excessive ROS induces DNA damage, which activates P53 and increases P53 protein expression [17]. In ischemic injury, minocycline has been demonstrated to reduce infarct size and neurological deficits in animal models of focal/global cerebral ischemia [45–47]. The potential mechanism underlying these neuroprotective effects of minocycline may include the following: inhibitory effect on cell apoptosis via the reduction expression of caspase-3 and poly polymerase-1, inhibitory effect on expression and activity of MMPs, and inhibitory effect on activation of microglia. In our study, our results demonstrated that minocycline effectively inhibited activation of microglia. Furthermore, activated microglia generates ROS [48]. Therefore, we wondered whether ROS is involved in the neuroprotection effect of minocycline. We examined the levels of ROS in the cortex 24 h after SAH. The cortical levels of ROS increased markedly at 2 h after SAH and were repressed by minocycline treatment. These observations suggest that the antiinflammatory and anti-apoptotic effect of minocycline may involve the reduction of ROS levels. There are several weaknesses in our study. First, many studies have proposed mechanisms involved in the neuroprotective effects of minocycline [18], including the following: inhibition of matrix metalloproteinases (MMPs) and phospholipase A2 (PLA2), inhibition of caspase-1 and caspase-3, and reduction of p38 mitogen-activated protein kinase (MAPK) phosphorylation. We cannot exclude the possibility that these effects also play a role in the neuroprotective effect of minocycline. In addition, our present study indicated that minocycline’s anti-inflammatory and anti-apoptotic effect may involve the reduction of ROS. Additional experiments with Mito-TEMPO (mitochondrial-specific ROS scavenger) or diphenyleneiodonium chloride (DPI, an NADPH oxidase inhibitor) are still needed in the future. In conclusion, our study extends the current understanding of the mechanisms of minocycline-related neuroprotective effects in early brain injury following SAH. Our results suggest that minocycline inhibits NLRP3 inflammasome activation, which may play an important role in inflammation and brain edema. Minocycline may repress neural apoptosis induced by P53-associated apoptotic proteins in early brain injury following SAH. In addition, our present study also indicates that the

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anti-inflammatory and anti-apoptotic effects of minocycline may involve the reduction of ROS. Minocycline treatment may have important clinical potentials for the management of SAH. Acknowledgments This study was supported by the Scientific and Technological Project of Zhejiang Province (2013C33138) and the Qianjiang rencai project (2013R10029).

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