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RIP1-RIP3-DRP1 pathway regulates NLRP3 inflammasome activation following subarachnoid hemorrhage
Experimental Neurology 295 (2017) 116–124
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Research Paper
RIP1-RIP3-DRP1 pathway regulates NLRP3 inflammasome activation following subarachnoid hemorrhage Keren Zhou a,b,c, Ligen Shi a,b,c, Zhen Wang a,b,c, Jingyi Zhou a,b,c, Anatol Manaenko d, Cesar Reis e, Sheng Chen a,b,c,⁎, Jianmin Zhang a,b,c,⁎ a
Department of Neurosurgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China Brain research institute, Zhejiang University, Hangzhou, Zhejiang, China Collaborative Innovation Center for Brain Science, Zhejiang University, Hangzhou, Zhejiang, China d Department of Neurology, University of Erlangen-Nuremberg, Erlangen, Germany e Department of Physiology and Pharmacology, Loma Linda University, Loma Linda, CA, USA b c
a r t i c l e
i n f o
Article history: Received 18 April 2017 Received in revised form 21 May 2017 Accepted 1 June 2017 Available online 2 June 2017 Keywords: RIP1 RIP3 DRP1 NLRP3 Early brain injury Subarachnoid hemorrhage
a b s t r a c t The NLRP3 inflammasome functions as a crucial component of the inflammatory response in early brain injury (EBI) after subarachnoid hemorrhage (SAH). However, the mechanisms underlying the activation of NLRP3 inflammasome has not been well elucidated. In this study, we hypothesized the RIP1-RIP3-DRP1 pathway was involved in the activation of the NLRP3 inflammasome following SAH. SAH was induced by endovascular perforation in rats. Necrostatin-1 (Nec-1) or mitochondrial division inhibitor (Mdivi-1) was administered 1 h after SAH by intraperitoneal injection. SAH grade, neurological function, brain water content, Western blot, ROS assay, immunofluorescence and transmission electron microscopy were performed. SAH led to the upregulation of RIP1, RIP3, phosphorylated DRP1 and NLRP3 inflammasome. Nec-1 treatment reduced RIP1, RIP3, phosphorylated DRP1 and NLRP3 inflammasome, subsequently alleviated brain edema and neurological deficits at 24 h following SAH. The treatment with Mdivi-1 inhibited the expression of DRP1 protein, attenuated mitochondria damage and the generation of ROS, inhibited NLRP3 inflammasome and ameliorated brain edema and neurological deficits at 24 h after SAH. The activation of the NLRP3 inflammasome in EBI after SAH was mediated by RIP1RIP3-DRP1 pathway. Nec-1 and Mdivi-1 can inhibit inflammation and improve neurological function after SAH. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Subarachnoid hemorrhage (SAH) is a severe cerebrovascular disease with high rate of mortality and disability (Etminan, 2015; van Gijn et al., 2007). The early brain injury (EBI), which occurs within first 72 h after SAH, has been considered a major cause of poor outcome in SAH patients (Caner et al., 2012; Sehba et al., 2012; Suzuki, 2015). The understanding of the molecular mechanisms underlying development of the EBI may improve the outcome of SAH patients. Recently, mounting evidence indicated that activation of NLRP3 inflammasome is a key component of post-SAH inflammatory response (Dong et al., 2016; Shao et al., 2016a). Previously, we demonstrated that P2X7R/NLRP3 inflammasome axis was involved in the pathogenesis of SAH. NLRP3 inflammasome expression significantly increased 24 h after SAH (Chen et al., 2013). However, the molecular mechanisms leading to the post-SAH NLRP3 inflammasome activation have not been well elucidated and the better understanding of them can lead to the development of new therapeutic approaches for SAH patients. ⁎ Corresponding authors at: 88 Jiefang Road, Hangzhou, 310009, Zhejiang, China. E-mail addresses: [email protected] (S. Chen), [email protected] (J. Zhang).
http://dx.doi.org/10.1016/j.expneurol.2017.06.003 0014-4886/© 2017 Elsevier Inc. All rights reserved.
The receptor-interacting protein (RIP) is a serine-threonine protein kinase family, consisting of four isoforms RIP 1–4 (Zhang et al., 2009). The active form of RIP1 and RIP3 can constitute a stable formation of RIP1-RIP3 complex, which has been demonstrated to regulate programmed necrosis (Cho et al., 2009). Furthermore, there are indications that the RIP1-RIP3 complex is involved in the activation of inflammasome (Kang et al., 2013; Yabal et al., 2014; X. Wang et al. 2014). Necrostatin-1 (Nec-1) is a small molecule capable of inhibiting RIP1 kinase activity by preventing RIP1-RIP3 interaction. It inhibits necroptosis in various central nervous system (CNS) diseases, such as: ischemia, traumatic brain injury, and intracerebral hemorrhage (Northington et al., 2011; Su et al., 2015; You et al., 2008). However, the role of RIP1-RIP3 complex in the activation of NLRP3 inflammasome and the neuroprotective role of Nec-1 in inflammation after SAH have not been studied. Reactive oxygen species (ROS), mainly associated with the malfunctioning mitochondria, played a significant role in the activation of NLRP3 inflammasome (Abais et al., 2015). Mitochondrial damage and fission are regulated by dynamin-related protein 1 (DRP1) (Tanaka and Youle, 2008). Mitochondrial division inhibitor (Mdivi-1), a selective inhibitor of DRP1, has been proved its neuroprotective effect in ischemia,
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traumatic brain injury and seizures (Wu et al., 2016; Xie et al., 2013; Zuo et al., 2014). In addition, infection with an RNA virus initiates assembly of the RIP1-RIP3 complex, which promotes activation of the GTPase DRP1 and its translocation to mitochondria to drive mitochondrial damage, production of ROS and eventually activation of the NLRP3 inflammasome (X. Wang et al. 2014). However, the anti-inflammatory effect of Mdivi-1 and the potential relation between RIP1-RIP3 and DRP1 have not been evaluated in EBI after SAH. In the present study, we hypothesized the RIP1-RIP3-DRP1 pathway played a key role in regulating the activation of NLRP3 inflammasome in EBI after SAH. We first applied Nec-1 to investigate the role of RIP1-RIP3 in the phosphorylation of DRP1 and the activation of NLRP3 inflammasome after SAH. Second, we investigated the effect of DRP1 in regulating mitochondria ROS and activating NLRP3 inflammasome after SAH. At last, we studied whether Nec-1 and Mdivi-1 can attenuate brain edema and improve neurological function after SAH. 2. Methods 2.1. Animals and SAH model Adult male Sprague–Dawley rats (300–340 g) were obtained from Animal Center of Zhejiang Chinese Medical University (Hangzhou, China). Rats were housed in a room with constant temperature (25 °C), humidity control and with a 12/12 h light/dark cycle, free access to food and water. All the experimental procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University. SAH model was conducted by modified endovascular perforation method (Sugawara et al., 2008). Briefly, rats were anesthetized with pentobarbital injection (intraperitoneally 40 mg/kg). The left external and internal carotid artery were exposed and a 4.0 monofilament nylon suture was inserted into the left internal carotid artery through the external carotid artery stump until feeling resistance, and then advanced 3 mm to perforate the bifurcation of the anterior and middle cerebral artery. Sham rats underwent the same procedures except for the perforation. The inflammasome level reached the peak around 24 h after SAH (Chen et al., 2013). So, all the parameters were investigated 24 h after SAH induction. 2.2. Experimental design (Fig. 1) In experiment 1, rats were randomly divided into 4 groups: the sham group (n = 16), SAH + vehicle group (n = 16), SAH + N(L) (low dose Nec-1, 3.5 mg/kg) group (n = 14), SAH + N(H) (high dose Nec-1, 10.5 mg/kg) group (n = 16). Postassessment included SAH grading, neurological score, brain water content, Western blot and immunofluorescence. In experiment 2, rats were randomly divided into 4 groups: sham group (n = 24), SAH + vehicle group (n = 24), SAH + M(L) (low
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dose Mdivi-1, 1.2 mg/kg) group (n = 14), SAH + M(H) (high dose Mdivi-1, 3.6 mg/kg) group (n = 24). Postassessment included SAH grading, neurological score, brain water content, Western blot immunofluorescence. High dose Mdivi-1 group was used to detect ROS assay and electron microscopy.
2.3. Drug administration Nec-1 was purchased from MedChem Express (Shanghai, CN) and was dissolved in DMSO (50 mg/ml) as stock. Before injection, it was diluted in PBS (Oerlemans et al., 2012). Nec-1 was administered in the SAH + N(L)(3.5 mg/kg) group and SAH + N(L)(10.5 mg/kg) group 1 h after surgery by intraperitoneal injection. The SAH + vehicle group received an equal volume of DMSO and PBS. Mdivi-1 was purchased from MedChem Express (Shanghai, CN) and was dissolved in DMSO (50 mg/ml) as stock. It was diluted in sterile saline before injection (Zhao et al., 2014). Animals of SAH + M(L) and SAH + M(H) group were respectively given an intraperitoneal injection of Mdivi-1(1.2 mg/kg) and Mdivi-1(3.6 mg/kg) 1 h after SAH induction. Animals of SAH + vehicle group received an equal volume of DMSO and sterile saline in the same way and at the same time point.
2.4. Assessment of neurological score The neurological status of all rats was evaluated at 24 h after SAH induction using the modified Garcia test (Sugawara et al., 2008). Briefly, the evaluation consists of six tests including: spontaneous activity (0– 3), spontaneous movements of all limbs (0–3), movements of forelimbs (0–3), climbing wall of wire cage (1–3), reaction to touch on both side of trunk (1–3), and response to vibrissae touch (1–3). Possible scores ranged from 3 to 18. A lower score represents serious neurological deficits. The assessment of neurological score was performed by a partner who was blind to the experiment.
2.5. Measurement of SAH grade The SAH grading score was used to estimate the degree of SAH as previously described (Sugawara et al., 2008). Briefly, the basal cistern was divided into six segments. Each segment was allotted a grade from 0 to 3 depending on the amount of subarachnoid blood clot: grade 0, no subarachnoid blood; grade 1, minimal subarachnoid clots; grade 2, moderate subarachnoid clots with recognizable arteries; and grade 3, blood clots covering all arteries. A total score ranging from 0 to 18 was obtained by adding the scores of six segments. The grading of SAH was performed by a partner who was blind to the experiment. Rats with the SAH grade lower than 9 were excluded from this study.
Fig. 1. Experimental designs and animal group classification.
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2.6. Evaluation of brain water content Rats were sacrificed at 24 h after SAH. The brain was removed and divided into four parts: the left hemisphere, right hemisphere, cerebellum, and brain stem. Each part was weighed immediately (wet weight) and then was dried in an oven at 105 °C for 24 h and weighed again (dry weight). The brain water content was calculated as [(wet weight − dry weight) / wet weight] × 100% (L. Li et al. 2015). 2.7. Western blot analysis Western blot analysis was performed as previously described (Shao et al., 2016b). Briefly, the left hemispheres (perforation side) were homogenized and centrifuged at 12,000 × g for 15 min at 4 °C. Supernatants were collected to measure the protein concentration by using a BCA kit (Beyotime, CN). Equal amounts of protein samples (60 μg) were subjected to a SDS-PAGE gel, electrophoresed, and transferred to a polyvinylidene fluoride membrane. After blocking with 5% nonfat dry milk in TBS for 1 h, membranes were incubated at 4 °C overnight with the primary antibodies: anti-RIP1 antibody (1:2000, Abcam, MA), anti-RIP3 antibody (1:3000, Abcam, MA), anti-NLRP3 antibody (1:1000, NOVUS, CO), anti-caspase-1 antibody (1:1000, Santa Cruz, CA), anti-DRP1 antibody (1:3000, Abcam, MA), anti-p-DRP1 antibody (1:2000, Abcam, MA). β-actin (1:2000, Santa Cruz, CA) was used as the positive control. The membranes were washed with TBST and incubated with horseradish-peroxidase conjugated secondary antibodies for 2 h at room temperature. Blot bands were detected by X-ray film and quantified by Image J software (NIH). 2.8. Immunofluorescence staining Rats were sacrificed at 24 h after SAH induction. A series of 8 μm slices were prepared. Double immunofluorescence staining was performed as previously described (Song et al., 2015). Briefly, after washing in PBS for 3 times and blocking in normal goat serum for 1 h, slices were incubated at 4 °C overnight with mouse monoclonal anti-Iba-1 antibody (1:200, Santa Cruz, CA) and rabbit monoclonal anti-RIP1 antibody (1:200, Abcam, MA) or rabbit polyclonal anti-RIP3 antibody (1:200, Abcam, MA) or rabbit monoclonal anti-DRP1 antibody (1:250, Abcam, MA). After washed with PBS, slices were incubated with a mixture of Alexa Flour 555-conjugated goat anti-mouse secondary antibody (1:250, CST, MA) or Alexa Flour 488-conjugated goat anti-rabbit secondary antibody (1:250, CST, MA) at 37 °C for 30 min. The fluorescent images were acquired in the left basal cortical regions with a fluorescent microscope (Olympus OX51). 2.9. ROS assay Rats were sacrificed at 24 h after SAH, and the left basal cortical samples were collected. The ROS levels were measured with a ROS assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the brain samples were weighed and homogenized in PBS (20 ml/g), followed by centrifugation at 1000 × g for 10 min at 4 °C. The protein content of the supernatant was measured using the Pierce BCA Protein Assays Kit (Thermo Scientific, Rockford, US). The supernatant (190 μl) was added to 96-well plates and mixed with 1 mmol/L DCFH-DA (10 μl). And the supernatant was mixed with PBS (10 μl) in anther well as control. The mixture was incubated at 37 °C for 30 min and then was measured by spectrofluorophotomery at an excitation wavelength of 500 nm and an emission wavelength of 525 nm. The ROS level in the brain sample was calculated as fluorescence intensity/g protein. 2.10. Transmission electron microscopy Rats were sacrificed 24 h after SAH and were perfused with 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 mol/l PBS. The brain
samples obtained from left cortex were cut into 1 mm3 pieces. Samples were then transferred into 2.5% glutaraldehyde and kept overnight at 4 °C. The samples were then rinsed several times with buffer and fixed with 1% osmium tetroxide for 1 h. After rinsing again with the distilled water several times, the samples were dehydrated with a series of graded ethanol. Next, infiltration was done with a solution of propylene oxide and resin (1:1). Samples were then embedded in resin. Sections (50 nm) were cut, and 4% uranyl acetate (20 min) and 0.5% lead citrate (5 min) were used to stain. A transmission electron microscope was used to examine the cortex ultrastructure. 2.11. Quantification and statistical analysis Data were presented as means ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare means of different groups followed by a Tukey multiple-comparisons test. Statistically significance was defined as p b 0.05. 3. Results 3.1. Severity and localization of SAH A total of 194 rats were used. 40 rats were sham group and 154 rats underwent SAH. At 24 h after SAH, the mortality of SAH rats is 20.8% (32 of 154) and no rats died in the sham group. 9.1% rats (14 of 154) were excluded because of low SAH grade. After SAH induction blood clots were mainly observed around the circle of Willis and ventral brain stem (Fig. 2A, Fig. 4A). No statistical differences in the average of SAH grades were observed between SAH groups (Fig. 2B, Fig. 4B). 3.2. Nec-1 improved neurobehavioral functions and attenuated brain edema after SAH At 24 h, SAH significant increased brain water content in the left hemisphere compared to sham operated animals (vehicle, 80.28 ± 0. 84 vs. sham, 79.10 ± 0.09, p b 0.01, Fig. 2C). Low dose of Nec-1 (SAH + N(L), 3.5 mg/kg) reduced brain water content, but there was no significant difference. The high dose of Nec-1 (SAH + N(H), 10.5 mg/kg) significantly attenuated brain edema 24 h after SAH (vehicle, 80.28 ± 0.84 vs. SAH + N(H), 79.56 ± 0.27, p b 0.05, Fig. 2C). There was no statistical difference of brain water content in the right hemisphere, cerebellum and brain stem among the groups. Compared to sham operated animals, SAH induced significant neurological deficits evaluated by the modified Garcia test (vehicle, 11.62 ± 2.34 vs. sham, 17.81 ± 0.40, p b 0.01, Fig. 2D). Both low (SAH + N(L), 3.5 mg/kg) and high (SAH + N(H), 10.5 mg/kg) dose of Nec-1 markedly improved neurobehavioral outcomes of SAH treated compared to the SAH + vehicle animals at 24 h after SAH (SAH + N(L), 14.29 ± 2.27 vs. vehicle, 11.62 ± 2.34, p b 0.01; SAH + N(H), 14.62 ± 1.75 vs. vehicle, 11.62 ± 2.34, p b 0.01, Fig. 2D). 3.3. Nec-1 inhibited RIP1-RIP3, p-DRP1 activation and reduced the expression of NLRP3 and cleaved caspase-1 In the Western blot test we demonstrated that SAH induced a 2.07 times increase of RIP1 expression 24 h after SAH. The high dose of Nec-1 significantly attenuated SAH-induced RIP1 overproduction (p b 0.01, Fig. 3B). Similarly, after SAH the expression of RIP3 protein was significantly increased (1.73 times) and the RIP3 expression was reduced by the high dose of Nec-1 (p b 0.05, Fig.3C). In addition, SAH elevated the expression of p-DRP1 at 24 h. This was associated with the increase of NLRP3 the cleaved caspase-1 (p b 0.01, Fig. 3D-F). The high dose of Nec-1 significantly reduced the SAH-induced overproduction of pDRP1, NLRP3 and cleaved caspase-1 at 24 h after SAH (p b 0.01, Fig. 3D-F).
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Fig. 2. Representative pictures of brains from each group, SAH grade, brain water content and neurological score at 24 h after SAH. (A) Typical pictures of rat brain from each group. (B) Similar SAH grades were observed in the SAH + vehicle group (n = 16), SAH + N(L) group (n = 14) and SAH + N(H) group (n = 16). (C) Nec-1 treatment decreased brain water content in the left hemisphere at 24 h after SAH (n = 8, **p b 0.01 vs. sham group; #p b 0.05 vs. vehicle group). (D) Nec-1 treatment significantly increased neurological scores (**p b 0.01 vs. sham group; ##p b 0.01 vs. vehicle group). Bars represent mean ± SD.
3.4. Mdivi-1 attenuated brain edema and improved neurologic function after SAH Compared to the sham group, the brain water content increased significantly in left hemisphere at 24 h after SAH (vehicle group, 80.23 ± 0.75 vs. sham, 79.07 ± 0.11, p b 0.01). High dose of Mdivi-1 significantly attenuated SAH-induced brain edema (SAH + M(H): 79.40 ± 0.14 vs.
vehicle, 80.23 ± 0.75, p b 0.05, Fig. 4C). There was no statistical difference of brain water content in the right hemisphere, cerebellum and brain stem among the groups. SAH resulted in significant neurological dysfunctions (vehicle, 11.92 ± 2.08 vs. sham, 17.83 ± 0.38, p b 0.01, Fig. 4D). Both low (SAH + M(L), 1.2 mg/kg) and high (SAH + M(H), 3.6 mg/kg) dose of Mdivi-1 significantly improved neurobehavioral outcome at 24 h after SAH (SAH
Fig. 3. Nec-1 treatment inhibited RIP1-RIP3, p-DRP1 activation and reduced the expression of NLRP3 and cleaved caspase-1 at 24 h after SAH. (A) Representative Western blots showing levels of RIP1, RIP3, p-DRP1, DRP1, NLRP3 and cleaved caspase-1. (B‐F) Quantification of protein levels of RIP1, RIP3, p-DRP1, NLRP3 and cleaved caspase-1. The high dose of Nec-1 significantly reduced the level of RIP1, RIP3, p-DRP1, NLRP3 and cleaved caspase-1 at 24 h after SAH (n = 6, *p b 0.05, **p b 0.01 vs. sham group; #p b 0.05, ##p b 0.01 vs. vehicle group). Bars represent mean ± SD.
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Fig. 4. Representative pictures of brains from each group, SAH grade, brain water content and neurological score at 24 h after SAH. (A) Typical pictures of rat brain from each group. (B) Similar SAH grades were observed in the SAH + vehicle group (n = 24), SAH + M(L) group (n = 14) and SAH + M(H) group (n = 24). (C) Mdivi-1 treatment decreased brain water content in the left hemisphere at 24 h after SAH (n = 8, **p b 0.01 vs. sham; #p b 0.05 vs. vehicle). (D) Mdivi-1 treatment significantly increased neurological scores (**p b 0.01 vs. sham; ##p b 0.01 vs. vehicle). Bars represent mean ± SD.
+ M(L), 14.29 ± 1.14 vs. vehicle, 11.92 ± 2.08, p b 0.01; SAH + M(H), 14.83 ± 1.47 vs. vehicle, 11.92 ± 2.08, p b 0.01, Fig. 4D).
3.5. Mdivi-1 inhibited DRP1 activation, alleviated ROS and reduced the expression of NLRP3 and cleaved caspase-1 Upregulation of DRP1 was observed by 1.70 times at 24 h after SAH in the vehicle group, when compared to the sham group (p b 0.01, Fig. 5B). The high dose of Mdivi-1 significantly attenuated SAH-induced upregulation of DRP1 (p b 0.05, Fig. 5B), NLRP3 and cleaved caspase-1 (p b 0.01, Fig. 5C, D). Moreover, the ROS assay showed that SAH significantly increased ROS production (3.60 times) compared to sham group at 24 h following SAH (p b 0.01, Fig. 5E). Compared to the SAH + vehicle group, high dose of Mdivi-1significantly reduced SAH-induced ROS release (p b 0.01, Fig .5E).
3.6. SAH induced mitochondrial damage can be alleviated by Mdivi-1 Using electron microscopy, we found that mitochondria in sham group were undamaged and had clear and complete double membrane and crista structures (Fig. 6A). Signs of severe mitochondrial damage including vacuolization in mitochondria and autophagosomes appeared in the vehicle group (Fig. 6B). In SAH + Mdivi-1 group, numerous mitochondria gathered and showed vague cristae and dense membrane (Fig. 6C).
3.7. RIP1, RIP3, DRP1 were mainly expressed with the activation of microglia cells To investigate the spatial expression of RIP1, RIP3 and DRP1 after SAH, we performed double-immunofluorescent staining. We demonstrated that proteins of interest are co-localized with marker of activated microglia, Iba-1, indicating that the proteins are expressed on microglia (Fig. 7A-C).
4. Discussion In the present study, we found that the administration of Nec-1 reduced the expression of RIP1 and RIP3, inhibited the phosphorylation of DRP1, downregulated the expression of NLRP3 inflammasome and subsequently alleviated brain edema and neurological deficits at 24 h following SAH. In addition, our study indicated that Mdivi-1 effectively inhibited the expression of DRP1, alleviated mitochondria damage, attenuated the generation of ROS, reduced the expression of NLRP3 inflammasome and ameliorated brain edema and neurological deficits at 24 h after SAH. Furthermore, we found that RIP1, RIP3, DRP1 were mainly expressed with the activation of microglia cells. The NLRP3 inflammasome, composed of the Nod-like receptor NLRP3, the adaptor protein ASC and effector enzyme caspase-1, is responsible for the maturation and secretion of proinflammatory cytokines (Chen et al., 2009). The NLRP3 inflammasome is activated by a wide range of signals that derive from both endogenous and exogenous stimuli (Jo et al., 2016). In previous studies, the role of NLRP3 inflammasome in the pathophysiology of SAH has been demonstrated. After SAH, the NLRP3 inflammasome activation can be inhibited with the reduction of mitochondrial ROS (Li et al. 2015a), through the NFκB pathway and NLRP3-associated apoptosis (Dong et al., 2016; Shao et al., 2016a). However, the potential mechanisms of NLRP3 inflammasome in EBI after SAH are still unclear. Here, we demonstrated a role for the RIP1-RIP3 complex in SAH induced activation of the NLRP3 inflammasome. We also identified DRP1 as a probable downstream of RIP1-RIP3 that promoted NLRP3 inflammasome activation. Following the stimulation of tumor necrosis factor(TNF), the RIP1RIP3 complex is formed and initiates the phosphorylation of mixed lineage kinase domain-like protein (MLKL) and phosphoglycerate mutase family member 5 (PGAM5), which eventually result in necroptosis (H. Wang et al. 2014; Wang et al., 2012). However, emerging evidence indicates that the RIP1 and RIP3 can also induce inflammatory cytokine production independent of necroptosis (Moriwaki and Chan, 2014). A study showed that the activity of the RIP1-RIP3 complex and its ability to trigger the NLRP3 inflammasome were mediated by two distinct pathways. One that involves the necrosis effector protein MLKL and
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Fig. 5. Mdivi-1 treatment inhibited the expression of DRP1, alleviated ROS and reduced the expression of NLRP3 and cleaved caspase-1 at 24 h after SAH. (A) Representative Western blots showing levels of DRP1. (B-D) Quantification of protein levels of DRP1, NLRP3 and cleaved caspase-1. Mdivi-1 significantly reduced SAH-induced upregulation of DRP1, NLRP3 and cleaved caspase-1. E: Mdivi-1 treatment decreased the expression of ROS. (n = 6, **p b 0.01 vs. sham group; #p b 0.05, ##p b 0.01 vs. vehicle group). Bars represent mean ± SD.
another that involves the mitochondrial fission protein DRP1 (X. Wang et al. 2014). In our study, we found the upregulation of RIP1 and RIP3 protein together with the increasing of NLRP3 inflammasome protein expression at 24 h after SAH. Moreover the administration of Nec-1 reduced the protein expression of RIP1, RIP3 and NLRP3 inflammasome simultaneously, suggesting an inhibitory effect of Nec-1 on RIP1-RIP3 activation and subsequent inflammatory response following SAH. It has been proved that TNF stimulates RIP1 kinase activity to drive formation of the RIP1-RIP3 complex, which acts as a scaffold for RIP3 kinase activation. The presence of Nec-1 abolishes the formation of the RIP1– RIP3 complex and affects the activation of RIP3 (Vandenabeele et al., 2010). This may explain why RIP1 and RIP3 were simultaneously inhibited by Nec-1, as a specific inhibitor of RIP1. However, how the RIP1-RIP3 complex is activated after SAH induction needs to be further studied. We investigated the role of DRP1 and mitochondrial ROS to study the mechanism of RIP1-RIP3 induced NLRP3 inflammasome activation. Emerging evidence suggests that mitochondrial dysfunction and excessive production of ROS may be associated with the activation of NLRP3 inflammasome (Heid et al., 2013). The regulation of mitochondrial dynamics is controlled by specific proteins such as mitofusions (Mfn1 and Mfn2), optic atrophy-1(OPA1) for fusion and DRP1 for fission (Liesa et al., 2009; Purnell and Fox, 2013). After translocated into mitochondrial outer-membrane from cytoplasm, DRP1 initiates mitochondrial fission and leads to mitochondrial dysfunction (Chan, 2006). However, the role of DRP1 and mitochondrial fission in inflammasome
activation is complex. It was reported that the activation of AMP-activated protein kinase (AMPK) inhibited mitochondrial fission via DRP1 and thus blocked mitochondrial ROS-associated endoplasmic reticulum stress and the NLRP3 inflammasome activation (A. Li et al. 2016; Li et al. 2015b). In addition, DRP1-knockdown cells containing elongated mitochondria induced NLRP3 activation in response to classical ATP or nigericin stimulation (Park et al., 2015). Our results showed that DRP1 were up-regulated after SAH induction, and thereby caused mitochondria damage, the release of ROS and the activation of NLRP3 inflammasome. Mdivi-1 reduced the expression of DRP1, alleviated mitochondria damage and attenuated the expression of ROS and NLRP3 inflammasome. We demonstrated a role of DRP1 in the modulation of mitochondrial dysfunction, accumulation of ROS and the activation of NLRP3 inflammasome. DRP1 activation is regulated by serine phosphorylation of DRP1 and it is reported that mitochondrial fission is induced by phosphorylation of DRP1 at serine 616 residue, leading to mitochondrial dysfunction (Y. Li et al. 2016). DRP1 has also been proposed to be downstream of PGAM5, which recruits DRP1 and activates its GTPase by dephosphorylating Ser637 of DRP1 to promote mitochondrial fission and necrosis (Wang et al., 2012). In our study, we found the upregulation of pDRP1 together with the increasing level of RIP1, RIP3 at 24 h after SAH, and the expression of p-DRP1 could be inhibited by Nec-1, the inhibitor of RIP1. It suggested a role of RIP1-RIP3 complex in the activation of DRP1. Our results were consistent with the recent study that revealed a specific role for the RIP1-RIP3-DRP1 pathway in RNA virus–induced
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Fig. 6. Effects of Mdivi-1 treatment on SAH induced mitochondria damage observed by transmission electron microscope. (A) Normal mitochondria (white arrows) were found in sham group. (B) Severe mitochondria damage appeared in the SAH + vehicle group. Black arrows indicate mitochondria vacuolization and white arrows indicate autophagosomes with mitochondria. (C) In SAH + Mdivi-1 (3.6 mg/kg) group, as white arrows show, mitochondria gathered and exhibited vague cristae and dense membrane (Scale bar = 1 μm).
Fig. 7. Double immunofluorescence staining for RIP1/Iba-1, RIP3/Iba-1 and DRP1/ Iba-1 in the left basal cortex at 24 h after SAH. (A) Colocalization of RIP1 and Iba-1. (B) Colocalization of RIP3 and Iba-1. (C) Colocalization of DRP1 and Iba-1 (Scale bar = 50 μm, Scale bar = 5 μm for smaller figure).
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activation of the NLRP3 inflammasome (X. Wang et al. 2014). However, the relation between RIP1-RIP3 complex and DRP1 after SAH remained to be further investigated. Our study also found that RIP1, RIP3 and DRP1 were mainly expressed with the activation of microglia cells. This is in accordance with the previous reports that indicated the role of microglia cells in innate inflammatory response in CNS (Hanamsagar et al., 2011) and the studies suggesting that NLRP3 was mainly expressed in microglia cells (Li et al. 2015a; Ma et al., 2014). This further confirmed the role of RIP1-RIP3-DRP1 in the activation of NLRP3 inflammasome following SAH. In addition, brain edema after SAH reflects disruption of the blood brain barrier, which can be resulted from microglia activation and inflammatory reaction (Li et al. 2015a). Besides inhibition of inflammation, previous studies have demonstrated that Nec-1 and mdivi-1 can prevent the endothelial dysfunction (Strilic et al., 2016; Tanner et al., 2017) and protect BBB permeability (King et al., 2014; Wu et al., 2017). In the present study, we found that Nec-1 and Mdivi-1 could attenuate brain edema through inhibiting inflammation after SAH. There are still some limitations in this study. We evaluated the neuroprotective functions of Nec-1 and Mdivi-1 at 24 h after SAH induction, while the long-term outcomes have not been investigated and need further study. In addition, the relation between RIP1-RIP3 complex and DRP1 has not been completely elucidated and the interaction between inflammation and necroptosis after SAH also need further study. In conclusion, the present study demonstrates the RIP1-RIP3-DRP1 pathway in regulating the activation of NLRP3 inflammasome in EBI after SAH. Nec-1 and Mdivi-1 can decrease brain edema and improve neurological function by suppressing inflammation after SAH. It provides a potential for the treatment of SAH. Acknowledgements This work was supported by National Natural Science Foundation of China (81500992, 81371433), and Natural Science Foundation of Zhejiang Province (LQ16H090002) and Medical and health key project of Zhejiang Province (2016RCA015). References Abais, J.M., Xia, M., Zhang, Y., Boini, K.M., Li, P.L., 2015. Redox regulation of NLRP3 inflammasomes: ROS as trigger or effector? Antioxid. Redox Signal. 22, 1111–1129. Caner, B., Hou, J., Altay, O., Fujii, M., Zhang, J.H., 2012. Transition of research focus from vasospasm to early brain injury after subarachnoid hemorrhage. J. Neurochem. 123 (Suppl. 2), 12–21. Chan, D.C., 2006. Mitochondria: dynamic organelles in disease, aging, and development. Cell 125, 1241–1252. Chen, G., Shaw, M.H., Kim, Y.G., Nunez, G., 2009. NOD-like receptors: role in innate immunity and inflammatory disease. Annu. Rev. Pathol. 4, 365–398. Chen, S., Ma, Q., Krafft, P.R., Hu, Q., Rolland 2nd, W., Sherchan, P., Zhang, J., Tang, J., Zhang, J.H., 2013. P2X7R/cryopyrin inflammasome axis inhibition reduces neuroinflammation after SAH. Neurobiol. Dis. 58, 296–307. Cho, Y.S., Challa, S., Moquin, D., Genga, R., Ray, T.D., Guildford, M., Chan, F.K., 2009. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137, 1112–1123. Dong, Y., Fan, C., Hu, W., Jiang, S., Ma, Z., Yan, X., Deng, C., Di, S., Xin, Z., Wu, G., Yang, Y., Reiter, R.J., Liang, G., 2016. Melatonin attenuated early brain injury induced by subarachnoid hemorrhage via regulating NLRP3 inflammasome and apoptosis signaling. J. Pineal Res. 60, 253–262. Etminan, N., 2015. Aneurysmal subarachnoid hemorrhage—status quo and perspective. Transl. Stroke Res. 6, 167–170. van Gijn, J., Kerr, R.S., Rinkel, G.J., 2007. Subarachnoid haemorrhage. Lancet 369, 306–318. Hanamsagar, R., Torres, V., Kielian, T., 2011. Inflammasome activation and IL-1beta/IL-18 processing are influenced by distinct pathways in microglia. J. Neurochem. 119, 736–748. Heid, M.E., Keyel, P.A., Kamga, C., Shiva, S., Watkins, S.C., Salter, R.D., 2013. Mitochondrial reactive oxygen species induces NLRP3-dependent lysosomal damage and inflammasome activation. J. Immunol. 191, 5230–5238. Jo, E.K., Kim, J.K., Shin, D.M., Sasakawa, C., 2016. Molecular mechanisms regulating NLRP3 inflammasome activation. Cell. Mol. Immunol. 13, 148–159. Kang, T.B., Yang, S.H., Toth, B., Kovalenko, A., Wallach, D., 2013. Caspase-8 blocks kinase RIPK3-mediated activation of the NLRP3 inflammasome. Immunity 38, 27–40. King, M.D., Whitaker-Lea, W.A., Campbell, J.M., Alleyne Jr., C.H., Dhandapani, K.M., 2014. Necrostatin-1 reduces neurovascular injury after intracerebral hemorrhage. Int J Cell Biol 2014, 495817.
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Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr
Research Paper
RIP1-RIP3-DRP1 pathway regulates NLRP3 inflammasome activation following subarachnoid hemorrhage Keren Zhou a,b,c, Ligen Shi a,b,c, Zhen Wang a,b,c, Jingyi Zhou a,b,c, Anatol Manaenko d, Cesar Reis e, Sheng Chen a,b,c,⁎, Jianmin Zhang a,b,c,⁎ a
Department of Neurosurgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China Brain research institute, Zhejiang University, Hangzhou, Zhejiang, China Collaborative Innovation Center for Brain Science, Zhejiang University, Hangzhou, Zhejiang, China d Department of Neurology, University of Erlangen-Nuremberg, Erlangen, Germany e Department of Physiology and Pharmacology, Loma Linda University, Loma Linda, CA, USA b c
a r t i c l e
i n f o
Article history: Received 18 April 2017 Received in revised form 21 May 2017 Accepted 1 June 2017 Available online 2 June 2017 Keywords: RIP1 RIP3 DRP1 NLRP3 Early brain injury Subarachnoid hemorrhage
a b s t r a c t The NLRP3 inflammasome functions as a crucial component of the inflammatory response in early brain injury (EBI) after subarachnoid hemorrhage (SAH). However, the mechanisms underlying the activation of NLRP3 inflammasome has not been well elucidated. In this study, we hypothesized the RIP1-RIP3-DRP1 pathway was involved in the activation of the NLRP3 inflammasome following SAH. SAH was induced by endovascular perforation in rats. Necrostatin-1 (Nec-1) or mitochondrial division inhibitor (Mdivi-1) was administered 1 h after SAH by intraperitoneal injection. SAH grade, neurological function, brain water content, Western blot, ROS assay, immunofluorescence and transmission electron microscopy were performed. SAH led to the upregulation of RIP1, RIP3, phosphorylated DRP1 and NLRP3 inflammasome. Nec-1 treatment reduced RIP1, RIP3, phosphorylated DRP1 and NLRP3 inflammasome, subsequently alleviated brain edema and neurological deficits at 24 h following SAH. The treatment with Mdivi-1 inhibited the expression of DRP1 protein, attenuated mitochondria damage and the generation of ROS, inhibited NLRP3 inflammasome and ameliorated brain edema and neurological deficits at 24 h after SAH. The activation of the NLRP3 inflammasome in EBI after SAH was mediated by RIP1RIP3-DRP1 pathway. Nec-1 and Mdivi-1 can inhibit inflammation and improve neurological function after SAH. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Subarachnoid hemorrhage (SAH) is a severe cerebrovascular disease with high rate of mortality and disability (Etminan, 2015; van Gijn et al., 2007). The early brain injury (EBI), which occurs within first 72 h after SAH, has been considered a major cause of poor outcome in SAH patients (Caner et al., 2012; Sehba et al., 2012; Suzuki, 2015). The understanding of the molecular mechanisms underlying development of the EBI may improve the outcome of SAH patients. Recently, mounting evidence indicated that activation of NLRP3 inflammasome is a key component of post-SAH inflammatory response (Dong et al., 2016; Shao et al., 2016a). Previously, we demonstrated that P2X7R/NLRP3 inflammasome axis was involved in the pathogenesis of SAH. NLRP3 inflammasome expression significantly increased 24 h after SAH (Chen et al., 2013). However, the molecular mechanisms leading to the post-SAH NLRP3 inflammasome activation have not been well elucidated and the better understanding of them can lead to the development of new therapeutic approaches for SAH patients. ⁎ Corresponding authors at: 88 Jiefang Road, Hangzhou, 310009, Zhejiang, China. E-mail addresses: [email protected] (S. Chen), [email protected] (J. Zhang).
http://dx.doi.org/10.1016/j.expneurol.2017.06.003 0014-4886/© 2017 Elsevier Inc. All rights reserved.
The receptor-interacting protein (RIP) is a serine-threonine protein kinase family, consisting of four isoforms RIP 1–4 (Zhang et al., 2009). The active form of RIP1 and RIP3 can constitute a stable formation of RIP1-RIP3 complex, which has been demonstrated to regulate programmed necrosis (Cho et al., 2009). Furthermore, there are indications that the RIP1-RIP3 complex is involved in the activation of inflammasome (Kang et al., 2013; Yabal et al., 2014; X. Wang et al. 2014). Necrostatin-1 (Nec-1) is a small molecule capable of inhibiting RIP1 kinase activity by preventing RIP1-RIP3 interaction. It inhibits necroptosis in various central nervous system (CNS) diseases, such as: ischemia, traumatic brain injury, and intracerebral hemorrhage (Northington et al., 2011; Su et al., 2015; You et al., 2008). However, the role of RIP1-RIP3 complex in the activation of NLRP3 inflammasome and the neuroprotective role of Nec-1 in inflammation after SAH have not been studied. Reactive oxygen species (ROS), mainly associated with the malfunctioning mitochondria, played a significant role in the activation of NLRP3 inflammasome (Abais et al., 2015). Mitochondrial damage and fission are regulated by dynamin-related protein 1 (DRP1) (Tanaka and Youle, 2008). Mitochondrial division inhibitor (Mdivi-1), a selective inhibitor of DRP1, has been proved its neuroprotective effect in ischemia,
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traumatic brain injury and seizures (Wu et al., 2016; Xie et al., 2013; Zuo et al., 2014). In addition, infection with an RNA virus initiates assembly of the RIP1-RIP3 complex, which promotes activation of the GTPase DRP1 and its translocation to mitochondria to drive mitochondrial damage, production of ROS and eventually activation of the NLRP3 inflammasome (X. Wang et al. 2014). However, the anti-inflammatory effect of Mdivi-1 and the potential relation between RIP1-RIP3 and DRP1 have not been evaluated in EBI after SAH. In the present study, we hypothesized the RIP1-RIP3-DRP1 pathway played a key role in regulating the activation of NLRP3 inflammasome in EBI after SAH. We first applied Nec-1 to investigate the role of RIP1-RIP3 in the phosphorylation of DRP1 and the activation of NLRP3 inflammasome after SAH. Second, we investigated the effect of DRP1 in regulating mitochondria ROS and activating NLRP3 inflammasome after SAH. At last, we studied whether Nec-1 and Mdivi-1 can attenuate brain edema and improve neurological function after SAH. 2. Methods 2.1. Animals and SAH model Adult male Sprague–Dawley rats (300–340 g) were obtained from Animal Center of Zhejiang Chinese Medical University (Hangzhou, China). Rats were housed in a room with constant temperature (25 °C), humidity control and with a 12/12 h light/dark cycle, free access to food and water. All the experimental procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University. SAH model was conducted by modified endovascular perforation method (Sugawara et al., 2008). Briefly, rats were anesthetized with pentobarbital injection (intraperitoneally 40 mg/kg). The left external and internal carotid artery were exposed and a 4.0 monofilament nylon suture was inserted into the left internal carotid artery through the external carotid artery stump until feeling resistance, and then advanced 3 mm to perforate the bifurcation of the anterior and middle cerebral artery. Sham rats underwent the same procedures except for the perforation. The inflammasome level reached the peak around 24 h after SAH (Chen et al., 2013). So, all the parameters were investigated 24 h after SAH induction. 2.2. Experimental design (Fig. 1) In experiment 1, rats were randomly divided into 4 groups: the sham group (n = 16), SAH + vehicle group (n = 16), SAH + N(L) (low dose Nec-1, 3.5 mg/kg) group (n = 14), SAH + N(H) (high dose Nec-1, 10.5 mg/kg) group (n = 16). Postassessment included SAH grading, neurological score, brain water content, Western blot and immunofluorescence. In experiment 2, rats were randomly divided into 4 groups: sham group (n = 24), SAH + vehicle group (n = 24), SAH + M(L) (low
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dose Mdivi-1, 1.2 mg/kg) group (n = 14), SAH + M(H) (high dose Mdivi-1, 3.6 mg/kg) group (n = 24). Postassessment included SAH grading, neurological score, brain water content, Western blot immunofluorescence. High dose Mdivi-1 group was used to detect ROS assay and electron microscopy.
2.3. Drug administration Nec-1 was purchased from MedChem Express (Shanghai, CN) and was dissolved in DMSO (50 mg/ml) as stock. Before injection, it was diluted in PBS (Oerlemans et al., 2012). Nec-1 was administered in the SAH + N(L)(3.5 mg/kg) group and SAH + N(L)(10.5 mg/kg) group 1 h after surgery by intraperitoneal injection. The SAH + vehicle group received an equal volume of DMSO and PBS. Mdivi-1 was purchased from MedChem Express (Shanghai, CN) and was dissolved in DMSO (50 mg/ml) as stock. It was diluted in sterile saline before injection (Zhao et al., 2014). Animals of SAH + M(L) and SAH + M(H) group were respectively given an intraperitoneal injection of Mdivi-1(1.2 mg/kg) and Mdivi-1(3.6 mg/kg) 1 h after SAH induction. Animals of SAH + vehicle group received an equal volume of DMSO and sterile saline in the same way and at the same time point.
2.4. Assessment of neurological score The neurological status of all rats was evaluated at 24 h after SAH induction using the modified Garcia test (Sugawara et al., 2008). Briefly, the evaluation consists of six tests including: spontaneous activity (0– 3), spontaneous movements of all limbs (0–3), movements of forelimbs (0–3), climbing wall of wire cage (1–3), reaction to touch on both side of trunk (1–3), and response to vibrissae touch (1–3). Possible scores ranged from 3 to 18. A lower score represents serious neurological deficits. The assessment of neurological score was performed by a partner who was blind to the experiment.
2.5. Measurement of SAH grade The SAH grading score was used to estimate the degree of SAH as previously described (Sugawara et al., 2008). Briefly, the basal cistern was divided into six segments. Each segment was allotted a grade from 0 to 3 depending on the amount of subarachnoid blood clot: grade 0, no subarachnoid blood; grade 1, minimal subarachnoid clots; grade 2, moderate subarachnoid clots with recognizable arteries; and grade 3, blood clots covering all arteries. A total score ranging from 0 to 18 was obtained by adding the scores of six segments. The grading of SAH was performed by a partner who was blind to the experiment. Rats with the SAH grade lower than 9 were excluded from this study.
Fig. 1. Experimental designs and animal group classification.
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2.6. Evaluation of brain water content Rats were sacrificed at 24 h after SAH. The brain was removed and divided into four parts: the left hemisphere, right hemisphere, cerebellum, and brain stem. Each part was weighed immediately (wet weight) and then was dried in an oven at 105 °C for 24 h and weighed again (dry weight). The brain water content was calculated as [(wet weight − dry weight) / wet weight] × 100% (L. Li et al. 2015). 2.7. Western blot analysis Western blot analysis was performed as previously described (Shao et al., 2016b). Briefly, the left hemispheres (perforation side) were homogenized and centrifuged at 12,000 × g for 15 min at 4 °C. Supernatants were collected to measure the protein concentration by using a BCA kit (Beyotime, CN). Equal amounts of protein samples (60 μg) were subjected to a SDS-PAGE gel, electrophoresed, and transferred to a polyvinylidene fluoride membrane. After blocking with 5% nonfat dry milk in TBS for 1 h, membranes were incubated at 4 °C overnight with the primary antibodies: anti-RIP1 antibody (1:2000, Abcam, MA), anti-RIP3 antibody (1:3000, Abcam, MA), anti-NLRP3 antibody (1:1000, NOVUS, CO), anti-caspase-1 antibody (1:1000, Santa Cruz, CA), anti-DRP1 antibody (1:3000, Abcam, MA), anti-p-DRP1 antibody (1:2000, Abcam, MA). β-actin (1:2000, Santa Cruz, CA) was used as the positive control. The membranes were washed with TBST and incubated with horseradish-peroxidase conjugated secondary antibodies for 2 h at room temperature. Blot bands were detected by X-ray film and quantified by Image J software (NIH). 2.8. Immunofluorescence staining Rats were sacrificed at 24 h after SAH induction. A series of 8 μm slices were prepared. Double immunofluorescence staining was performed as previously described (Song et al., 2015). Briefly, after washing in PBS for 3 times and blocking in normal goat serum for 1 h, slices were incubated at 4 °C overnight with mouse monoclonal anti-Iba-1 antibody (1:200, Santa Cruz, CA) and rabbit monoclonal anti-RIP1 antibody (1:200, Abcam, MA) or rabbit polyclonal anti-RIP3 antibody (1:200, Abcam, MA) or rabbit monoclonal anti-DRP1 antibody (1:250, Abcam, MA). After washed with PBS, slices were incubated with a mixture of Alexa Flour 555-conjugated goat anti-mouse secondary antibody (1:250, CST, MA) or Alexa Flour 488-conjugated goat anti-rabbit secondary antibody (1:250, CST, MA) at 37 °C for 30 min. The fluorescent images were acquired in the left basal cortical regions with a fluorescent microscope (Olympus OX51). 2.9. ROS assay Rats were sacrificed at 24 h after SAH, and the left basal cortical samples were collected. The ROS levels were measured with a ROS assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the brain samples were weighed and homogenized in PBS (20 ml/g), followed by centrifugation at 1000 × g for 10 min at 4 °C. The protein content of the supernatant was measured using the Pierce BCA Protein Assays Kit (Thermo Scientific, Rockford, US). The supernatant (190 μl) was added to 96-well plates and mixed with 1 mmol/L DCFH-DA (10 μl). And the supernatant was mixed with PBS (10 μl) in anther well as control. The mixture was incubated at 37 °C for 30 min and then was measured by spectrofluorophotomery at an excitation wavelength of 500 nm and an emission wavelength of 525 nm. The ROS level in the brain sample was calculated as fluorescence intensity/g protein. 2.10. Transmission electron microscopy Rats were sacrificed 24 h after SAH and were perfused with 0.5% glutaraldehyde and 4% paraformaldehyde in 0.1 mol/l PBS. The brain
samples obtained from left cortex were cut into 1 mm3 pieces. Samples were then transferred into 2.5% glutaraldehyde and kept overnight at 4 °C. The samples were then rinsed several times with buffer and fixed with 1% osmium tetroxide for 1 h. After rinsing again with the distilled water several times, the samples were dehydrated with a series of graded ethanol. Next, infiltration was done with a solution of propylene oxide and resin (1:1). Samples were then embedded in resin. Sections (50 nm) were cut, and 4% uranyl acetate (20 min) and 0.5% lead citrate (5 min) were used to stain. A transmission electron microscope was used to examine the cortex ultrastructure. 2.11. Quantification and statistical analysis Data were presented as means ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare means of different groups followed by a Tukey multiple-comparisons test. Statistically significance was defined as p b 0.05. 3. Results 3.1. Severity and localization of SAH A total of 194 rats were used. 40 rats were sham group and 154 rats underwent SAH. At 24 h after SAH, the mortality of SAH rats is 20.8% (32 of 154) and no rats died in the sham group. 9.1% rats (14 of 154) were excluded because of low SAH grade. After SAH induction blood clots were mainly observed around the circle of Willis and ventral brain stem (Fig. 2A, Fig. 4A). No statistical differences in the average of SAH grades were observed between SAH groups (Fig. 2B, Fig. 4B). 3.2. Nec-1 improved neurobehavioral functions and attenuated brain edema after SAH At 24 h, SAH significant increased brain water content in the left hemisphere compared to sham operated animals (vehicle, 80.28 ± 0. 84 vs. sham, 79.10 ± 0.09, p b 0.01, Fig. 2C). Low dose of Nec-1 (SAH + N(L), 3.5 mg/kg) reduced brain water content, but there was no significant difference. The high dose of Nec-1 (SAH + N(H), 10.5 mg/kg) significantly attenuated brain edema 24 h after SAH (vehicle, 80.28 ± 0.84 vs. SAH + N(H), 79.56 ± 0.27, p b 0.05, Fig. 2C). There was no statistical difference of brain water content in the right hemisphere, cerebellum and brain stem among the groups. Compared to sham operated animals, SAH induced significant neurological deficits evaluated by the modified Garcia test (vehicle, 11.62 ± 2.34 vs. sham, 17.81 ± 0.40, p b 0.01, Fig. 2D). Both low (SAH + N(L), 3.5 mg/kg) and high (SAH + N(H), 10.5 mg/kg) dose of Nec-1 markedly improved neurobehavioral outcomes of SAH treated compared to the SAH + vehicle animals at 24 h after SAH (SAH + N(L), 14.29 ± 2.27 vs. vehicle, 11.62 ± 2.34, p b 0.01; SAH + N(H), 14.62 ± 1.75 vs. vehicle, 11.62 ± 2.34, p b 0.01, Fig. 2D). 3.3. Nec-1 inhibited RIP1-RIP3, p-DRP1 activation and reduced the expression of NLRP3 and cleaved caspase-1 In the Western blot test we demonstrated that SAH induced a 2.07 times increase of RIP1 expression 24 h after SAH. The high dose of Nec-1 significantly attenuated SAH-induced RIP1 overproduction (p b 0.01, Fig. 3B). Similarly, after SAH the expression of RIP3 protein was significantly increased (1.73 times) and the RIP3 expression was reduced by the high dose of Nec-1 (p b 0.05, Fig.3C). In addition, SAH elevated the expression of p-DRP1 at 24 h. This was associated with the increase of NLRP3 the cleaved caspase-1 (p b 0.01, Fig. 3D-F). The high dose of Nec-1 significantly reduced the SAH-induced overproduction of pDRP1, NLRP3 and cleaved caspase-1 at 24 h after SAH (p b 0.01, Fig. 3D-F).
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Fig. 2. Representative pictures of brains from each group, SAH grade, brain water content and neurological score at 24 h after SAH. (A) Typical pictures of rat brain from each group. (B) Similar SAH grades were observed in the SAH + vehicle group (n = 16), SAH + N(L) group (n = 14) and SAH + N(H) group (n = 16). (C) Nec-1 treatment decreased brain water content in the left hemisphere at 24 h after SAH (n = 8, **p b 0.01 vs. sham group; #p b 0.05 vs. vehicle group). (D) Nec-1 treatment significantly increased neurological scores (**p b 0.01 vs. sham group; ##p b 0.01 vs. vehicle group). Bars represent mean ± SD.
3.4. Mdivi-1 attenuated brain edema and improved neurologic function after SAH Compared to the sham group, the brain water content increased significantly in left hemisphere at 24 h after SAH (vehicle group, 80.23 ± 0.75 vs. sham, 79.07 ± 0.11, p b 0.01). High dose of Mdivi-1 significantly attenuated SAH-induced brain edema (SAH + M(H): 79.40 ± 0.14 vs.
vehicle, 80.23 ± 0.75, p b 0.05, Fig. 4C). There was no statistical difference of brain water content in the right hemisphere, cerebellum and brain stem among the groups. SAH resulted in significant neurological dysfunctions (vehicle, 11.92 ± 2.08 vs. sham, 17.83 ± 0.38, p b 0.01, Fig. 4D). Both low (SAH + M(L), 1.2 mg/kg) and high (SAH + M(H), 3.6 mg/kg) dose of Mdivi-1 significantly improved neurobehavioral outcome at 24 h after SAH (SAH
Fig. 3. Nec-1 treatment inhibited RIP1-RIP3, p-DRP1 activation and reduced the expression of NLRP3 and cleaved caspase-1 at 24 h after SAH. (A) Representative Western blots showing levels of RIP1, RIP3, p-DRP1, DRP1, NLRP3 and cleaved caspase-1. (B‐F) Quantification of protein levels of RIP1, RIP3, p-DRP1, NLRP3 and cleaved caspase-1. The high dose of Nec-1 significantly reduced the level of RIP1, RIP3, p-DRP1, NLRP3 and cleaved caspase-1 at 24 h after SAH (n = 6, *p b 0.05, **p b 0.01 vs. sham group; #p b 0.05, ##p b 0.01 vs. vehicle group). Bars represent mean ± SD.
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Fig. 4. Representative pictures of brains from each group, SAH grade, brain water content and neurological score at 24 h after SAH. (A) Typical pictures of rat brain from each group. (B) Similar SAH grades were observed in the SAH + vehicle group (n = 24), SAH + M(L) group (n = 14) and SAH + M(H) group (n = 24). (C) Mdivi-1 treatment decreased brain water content in the left hemisphere at 24 h after SAH (n = 8, **p b 0.01 vs. sham; #p b 0.05 vs. vehicle). (D) Mdivi-1 treatment significantly increased neurological scores (**p b 0.01 vs. sham; ##p b 0.01 vs. vehicle). Bars represent mean ± SD.
+ M(L), 14.29 ± 1.14 vs. vehicle, 11.92 ± 2.08, p b 0.01; SAH + M(H), 14.83 ± 1.47 vs. vehicle, 11.92 ± 2.08, p b 0.01, Fig. 4D).
3.5. Mdivi-1 inhibited DRP1 activation, alleviated ROS and reduced the expression of NLRP3 and cleaved caspase-1 Upregulation of DRP1 was observed by 1.70 times at 24 h after SAH in the vehicle group, when compared to the sham group (p b 0.01, Fig. 5B). The high dose of Mdivi-1 significantly attenuated SAH-induced upregulation of DRP1 (p b 0.05, Fig. 5B), NLRP3 and cleaved caspase-1 (p b 0.01, Fig. 5C, D). Moreover, the ROS assay showed that SAH significantly increased ROS production (3.60 times) compared to sham group at 24 h following SAH (p b 0.01, Fig. 5E). Compared to the SAH + vehicle group, high dose of Mdivi-1significantly reduced SAH-induced ROS release (p b 0.01, Fig .5E).
3.6. SAH induced mitochondrial damage can be alleviated by Mdivi-1 Using electron microscopy, we found that mitochondria in sham group were undamaged and had clear and complete double membrane and crista structures (Fig. 6A). Signs of severe mitochondrial damage including vacuolization in mitochondria and autophagosomes appeared in the vehicle group (Fig. 6B). In SAH + Mdivi-1 group, numerous mitochondria gathered and showed vague cristae and dense membrane (Fig. 6C).
3.7. RIP1, RIP3, DRP1 were mainly expressed with the activation of microglia cells To investigate the spatial expression of RIP1, RIP3 and DRP1 after SAH, we performed double-immunofluorescent staining. We demonstrated that proteins of interest are co-localized with marker of activated microglia, Iba-1, indicating that the proteins are expressed on microglia (Fig. 7A-C).
4. Discussion In the present study, we found that the administration of Nec-1 reduced the expression of RIP1 and RIP3, inhibited the phosphorylation of DRP1, downregulated the expression of NLRP3 inflammasome and subsequently alleviated brain edema and neurological deficits at 24 h following SAH. In addition, our study indicated that Mdivi-1 effectively inhibited the expression of DRP1, alleviated mitochondria damage, attenuated the generation of ROS, reduced the expression of NLRP3 inflammasome and ameliorated brain edema and neurological deficits at 24 h after SAH. Furthermore, we found that RIP1, RIP3, DRP1 were mainly expressed with the activation of microglia cells. The NLRP3 inflammasome, composed of the Nod-like receptor NLRP3, the adaptor protein ASC and effector enzyme caspase-1, is responsible for the maturation and secretion of proinflammatory cytokines (Chen et al., 2009). The NLRP3 inflammasome is activated by a wide range of signals that derive from both endogenous and exogenous stimuli (Jo et al., 2016). In previous studies, the role of NLRP3 inflammasome in the pathophysiology of SAH has been demonstrated. After SAH, the NLRP3 inflammasome activation can be inhibited with the reduction of mitochondrial ROS (Li et al. 2015a), through the NFκB pathway and NLRP3-associated apoptosis (Dong et al., 2016; Shao et al., 2016a). However, the potential mechanisms of NLRP3 inflammasome in EBI after SAH are still unclear. Here, we demonstrated a role for the RIP1-RIP3 complex in SAH induced activation of the NLRP3 inflammasome. We also identified DRP1 as a probable downstream of RIP1-RIP3 that promoted NLRP3 inflammasome activation. Following the stimulation of tumor necrosis factor(TNF), the RIP1RIP3 complex is formed and initiates the phosphorylation of mixed lineage kinase domain-like protein (MLKL) and phosphoglycerate mutase family member 5 (PGAM5), which eventually result in necroptosis (H. Wang et al. 2014; Wang et al., 2012). However, emerging evidence indicates that the RIP1 and RIP3 can also induce inflammatory cytokine production independent of necroptosis (Moriwaki and Chan, 2014). A study showed that the activity of the RIP1-RIP3 complex and its ability to trigger the NLRP3 inflammasome were mediated by two distinct pathways. One that involves the necrosis effector protein MLKL and
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Fig. 5. Mdivi-1 treatment inhibited the expression of DRP1, alleviated ROS and reduced the expression of NLRP3 and cleaved caspase-1 at 24 h after SAH. (A) Representative Western blots showing levels of DRP1. (B-D) Quantification of protein levels of DRP1, NLRP3 and cleaved caspase-1. Mdivi-1 significantly reduced SAH-induced upregulation of DRP1, NLRP3 and cleaved caspase-1. E: Mdivi-1 treatment decreased the expression of ROS. (n = 6, **p b 0.01 vs. sham group; #p b 0.05, ##p b 0.01 vs. vehicle group). Bars represent mean ± SD.
another that involves the mitochondrial fission protein DRP1 (X. Wang et al. 2014). In our study, we found the upregulation of RIP1 and RIP3 protein together with the increasing of NLRP3 inflammasome protein expression at 24 h after SAH. Moreover the administration of Nec-1 reduced the protein expression of RIP1, RIP3 and NLRP3 inflammasome simultaneously, suggesting an inhibitory effect of Nec-1 on RIP1-RIP3 activation and subsequent inflammatory response following SAH. It has been proved that TNF stimulates RIP1 kinase activity to drive formation of the RIP1-RIP3 complex, which acts as a scaffold for RIP3 kinase activation. The presence of Nec-1 abolishes the formation of the RIP1– RIP3 complex and affects the activation of RIP3 (Vandenabeele et al., 2010). This may explain why RIP1 and RIP3 were simultaneously inhibited by Nec-1, as a specific inhibitor of RIP1. However, how the RIP1-RIP3 complex is activated after SAH induction needs to be further studied. We investigated the role of DRP1 and mitochondrial ROS to study the mechanism of RIP1-RIP3 induced NLRP3 inflammasome activation. Emerging evidence suggests that mitochondrial dysfunction and excessive production of ROS may be associated with the activation of NLRP3 inflammasome (Heid et al., 2013). The regulation of mitochondrial dynamics is controlled by specific proteins such as mitofusions (Mfn1 and Mfn2), optic atrophy-1(OPA1) for fusion and DRP1 for fission (Liesa et al., 2009; Purnell and Fox, 2013). After translocated into mitochondrial outer-membrane from cytoplasm, DRP1 initiates mitochondrial fission and leads to mitochondrial dysfunction (Chan, 2006). However, the role of DRP1 and mitochondrial fission in inflammasome
activation is complex. It was reported that the activation of AMP-activated protein kinase (AMPK) inhibited mitochondrial fission via DRP1 and thus blocked mitochondrial ROS-associated endoplasmic reticulum stress and the NLRP3 inflammasome activation (A. Li et al. 2016; Li et al. 2015b). In addition, DRP1-knockdown cells containing elongated mitochondria induced NLRP3 activation in response to classical ATP or nigericin stimulation (Park et al., 2015). Our results showed that DRP1 were up-regulated after SAH induction, and thereby caused mitochondria damage, the release of ROS and the activation of NLRP3 inflammasome. Mdivi-1 reduced the expression of DRP1, alleviated mitochondria damage and attenuated the expression of ROS and NLRP3 inflammasome. We demonstrated a role of DRP1 in the modulation of mitochondrial dysfunction, accumulation of ROS and the activation of NLRP3 inflammasome. DRP1 activation is regulated by serine phosphorylation of DRP1 and it is reported that mitochondrial fission is induced by phosphorylation of DRP1 at serine 616 residue, leading to mitochondrial dysfunction (Y. Li et al. 2016). DRP1 has also been proposed to be downstream of PGAM5, which recruits DRP1 and activates its GTPase by dephosphorylating Ser637 of DRP1 to promote mitochondrial fission and necrosis (Wang et al., 2012). In our study, we found the upregulation of pDRP1 together with the increasing level of RIP1, RIP3 at 24 h after SAH, and the expression of p-DRP1 could be inhibited by Nec-1, the inhibitor of RIP1. It suggested a role of RIP1-RIP3 complex in the activation of DRP1. Our results were consistent with the recent study that revealed a specific role for the RIP1-RIP3-DRP1 pathway in RNA virus–induced
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Fig. 6. Effects of Mdivi-1 treatment on SAH induced mitochondria damage observed by transmission electron microscope. (A) Normal mitochondria (white arrows) were found in sham group. (B) Severe mitochondria damage appeared in the SAH + vehicle group. Black arrows indicate mitochondria vacuolization and white arrows indicate autophagosomes with mitochondria. (C) In SAH + Mdivi-1 (3.6 mg/kg) group, as white arrows show, mitochondria gathered and exhibited vague cristae and dense membrane (Scale bar = 1 μm).
Fig. 7. Double immunofluorescence staining for RIP1/Iba-1, RIP3/Iba-1 and DRP1/ Iba-1 in the left basal cortex at 24 h after SAH. (A) Colocalization of RIP1 and Iba-1. (B) Colocalization of RIP3 and Iba-1. (C) Colocalization of DRP1 and Iba-1 (Scale bar = 50 μm, Scale bar = 5 μm for smaller figure).
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activation of the NLRP3 inflammasome (X. Wang et al. 2014). However, the relation between RIP1-RIP3 complex and DRP1 after SAH remained to be further investigated. Our study also found that RIP1, RIP3 and DRP1 were mainly expressed with the activation of microglia cells. This is in accordance with the previous reports that indicated the role of microglia cells in innate inflammatory response in CNS (Hanamsagar et al., 2011) and the studies suggesting that NLRP3 was mainly expressed in microglia cells (Li et al. 2015a; Ma et al., 2014). This further confirmed the role of RIP1-RIP3-DRP1 in the activation of NLRP3 inflammasome following SAH. In addition, brain edema after SAH reflects disruption of the blood brain barrier, which can be resulted from microglia activation and inflammatory reaction (Li et al. 2015a). Besides inhibition of inflammation, previous studies have demonstrated that Nec-1 and mdivi-1 can prevent the endothelial dysfunction (Strilic et al., 2016; Tanner et al., 2017) and protect BBB permeability (King et al., 2014; Wu et al., 2017). In the present study, we found that Nec-1 and Mdivi-1 could attenuate brain edema through inhibiting inflammation after SAH. There are still some limitations in this study. We evaluated the neuroprotective functions of Nec-1 and Mdivi-1 at 24 h after SAH induction, while the long-term outcomes have not been investigated and need further study. In addition, the relation between RIP1-RIP3 complex and DRP1 has not been completely elucidated and the interaction between inflammation and necroptosis after SAH also need further study. In conclusion, the present study demonstrates the RIP1-RIP3-DRP1 pathway in regulating the activation of NLRP3 inflammasome in EBI after SAH. Nec-1 and Mdivi-1 can decrease brain edema and improve neurological function by suppressing inflammation after SAH. It provides a potential for the treatment of SAH. Acknowledgements This work was supported by National Natural Science Foundation of China (81500992, 81371433), and Natural Science Foundation of Zhejiang Province (LQ16H090002) and Medical and health key project of Zhejiang Province (2016RCA015). References Abais, J.M., Xia, M., Zhang, Y., Boini, K.M., Li, P.L., 2015. Redox regulation of NLRP3 inflammasomes: ROS as trigger or effector? Antioxid. Redox Signal. 22, 1111–1129. Caner, B., Hou, J., Altay, O., Fujii, M., Zhang, J.H., 2012. Transition of research focus from vasospasm to early brain injury after subarachnoid hemorrhage. J. Neurochem. 123 (Suppl. 2), 12–21. Chan, D.C., 2006. Mitochondria: dynamic organelles in disease, aging, and development. Cell 125, 1241–1252. Chen, G., Shaw, M.H., Kim, Y.G., Nunez, G., 2009. NOD-like receptors: role in innate immunity and inflammatory disease. Annu. Rev. Pathol. 4, 365–398. Chen, S., Ma, Q., Krafft, P.R., Hu, Q., Rolland 2nd, W., Sherchan, P., Zhang, J., Tang, J., Zhang, J.H., 2013. P2X7R/cryopyrin inflammasome axis inhibition reduces neuroinflammation after SAH. Neurobiol. Dis. 58, 296–307. Cho, Y.S., Challa, S., Moquin, D., Genga, R., Ray, T.D., Guildford, M., Chan, F.K., 2009. Phosphorylation-driven assembly of the RIP1-RIP3 complex regulates programmed necrosis and virus-induced inflammation. Cell 137, 1112–1123. Dong, Y., Fan, C., Hu, W., Jiang, S., Ma, Z., Yan, X., Deng, C., Di, S., Xin, Z., Wu, G., Yang, Y., Reiter, R.J., Liang, G., 2016. Melatonin attenuated early brain injury induced by subarachnoid hemorrhage via regulating NLRP3 inflammasome and apoptosis signaling. J. Pineal Res. 60, 253–262. Etminan, N., 2015. Aneurysmal subarachnoid hemorrhage—status quo and perspective. Transl. Stroke Res. 6, 167–170. van Gijn, J., Kerr, R.S., Rinkel, G.J., 2007. Subarachnoid haemorrhage. Lancet 369, 306–318. Hanamsagar, R., Torres, V., Kielian, T., 2011. Inflammasome activation and IL-1beta/IL-18 processing are influenced by distinct pathways in microglia. J. Neurochem. 119, 736–748. Heid, M.E., Keyel, P.A., Kamga, C., Shiva, S., Watkins, S.C., Salter, R.D., 2013. Mitochondrial reactive oxygen species induces NLRP3-dependent lysosomal damage and inflammasome activation. J. Immunol. 191, 5230–5238. Jo, E.K., Kim, J.K., Shin, D.M., Sasakawa, C., 2016. Molecular mechanisms regulating NLRP3 inflammasome activation. Cell. Mol. Immunol. 13, 148–159. Kang, T.B., Yang, S.H., Toth, B., Kovalenko, A., Wallach, D., 2013. Caspase-8 blocks kinase RIPK3-mediated activation of the NLRP3 inflammasome. Immunity 38, 27–40. King, M.D., Whitaker-Lea, W.A., Campbell, J.M., Alleyne Jr., C.H., Dhandapani, K.M., 2014. Necrostatin-1 reduces neurovascular injury after intracerebral hemorrhage. Int J Cell Biol 2014, 495817.
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