NLRP3 Inammasome Activation Attenuates Neuronal Apoptosis by Upregulating Autophagy through AMPK/Beclin-1 Pathway after Intracerebral Hemorrhage with Ventricular Extension in Rats

In brain, NLRP3 inammasome, mainly derived from macrophage/microglia, is involved in proinammatory and neurodecits after hemorrhage, and autophagy is vital for neuronal homeostasis and functions. Accumulating evidence suggested that NLRP3 inammasome and autophagy played an important role in intracerebral hemorrhage (ICH). Thus, this study was designed to further explore the pathogenesis of neurodecits after in posthemorrhagic hydrocephalus. Methods Autologous blood injection model was induced to mimic ICH with ventricular extension (ICH-IVH) in Sprague-Dawley rats. To elucidate the underlying mechanism, the NLRP3 inammasome inhibitor MCC950 was administered abdominally at 1 h after ICH-IVH. Magnetic resonance imaging, neurobehavioral tests, immunouorescence, western blotting, Fluoro-Jade C- staining, Tunel staining, and Quantitative RNA Sequencing were performed.


Abstract
Background In brain, NLRP3 in ammasome, mainly derived from macrophage/microglia, is involved in proin ammatory and neurode cits after hemorrhage, and autophagy is vital for neuronal homeostasis and functions. Accumulating evidence suggested that NLRP3 in ammasome and autophagy played an important role in intracerebral hemorrhage (ICH). Thus, this study was designed to further explore the pathogenesis of neurode cits after in posthemorrhagic hydrocephalus.

Autologous blood injection model was induced to mimic ICH with ventricular extension (ICH-IVH) in
Sprague-Dawley rats. To elucidate the underlying mechanism, the NLRP3 in ammasome inhibitor MCC950 was administered abdominally at 1 h after ICH-IVH. Magnetic resonance imaging, neurobehavioral tests, immuno uorescence, western blotting, Fluoro-Jade C-staining, Tunel staining, and Quantitative RNA Sequencing were performed.

Results
In the acute phase of ICH-IVH, both the expression of NLRP3 in ammasome and the autophagy of neurons were upregulated. The activated NLRP3 in macrophage/microglia promoted the release of IL-1β to extracellular, which contributed to excessive autophagy, leading to neurons apoptosis both in vivo and in vitro. AMPK/Beclin-1 pathway played an important role in NLRP3-related neurons autophagy. Using MCC950(NLRP3 in ammasome speci c inhibitor) treatment after ICH-IVH signi cantly reduced ventricles dilation, improved neurofunction, down-regulated the release of IL-1β, and alleviated neuroin ammation and excessive autophagy.

Conclusions
Our nding demonstrated that NLRP3 in ammasome activated in microglia/macrophage aggravated neurological outcomes and neuronal apoptosis by upregulating autophagy after ICH-IVH, which was partly mediated by the AMPK/Beclin-1 pathway. Therefore, inhibiting the activation of NLRP3 may be a potential therapeutic strategy for the neurode cits of ICH-IVH patients.

Background
Intracerebral hemorrhage (ICH) has a high morbidity and mortality and is associated with severe longterm disability [1,2], however, there is still no effective treatment so far [3]. About 40% of ICH break into the ventricle (ICH-IVH), and more than half of ICH-IVH patients will develop into varying degrees of hydrocephalus, which make the unfavorable outcome even worse [2,4,5]. Although there are many hypotheses about the pathogenesis of hydrocephalus after ICH-IVH, such as blood-clot blockage, barrier impairment, in ammation and blood components [6], due to the rare experimental veri cation, the exact pathogenesis remains unclear.
Once ICH-IVH occurred, the resident microglia/macrophage and recruited peripheral leukocytes were soon activated to release proin ammation cytokines, causing neuroin ammation and brain injury at acute phase [7][8][9]. The NLRP3 in ammasome, highly expressed in microglia/macrophage, has been proved to participate in a variety of pathological process such as psychiatric and neurodegenerative disorders [10,11]. By activation of NLRP3 in ammasome can induce cytokines, such as IL-1β, which was a new cardiovascular risk biomarker [12]. Accumulating evidences that NLRP3 in ammasome could relieve neuroin ammation and reduce cell death in early brain injury [13,14] make the NLRP3 in ammasomeinduced anti-in ammatory treatment be a potential strategy.
Autophagy is an evolutionarily conserved intracellular process to maintain cellular homeostasis by the phagosome and lysosomal pathways [15]. Abnormal autophagy has been found to play an important role in the pathogenic process of a variety of neurodegenerative diseases [16][17][18] Herein, we hypothesized that autophagy might have a role in ICH-IVH. Since in ammation and autophagy were closely related, we further explored the relationship between NLRP3 in ammasome (an important molecular regulator in in ammation) activation in microglia/macrophage and autophagy in neurons after ICH-IVH. To address this aim, a rat model of ICH with ventricular extension and PC12 cells were used.

Animals
All experimental procedures were approved by the institutional animal care and use committee of the third military university and were performed according to the guide for the care and use of laboratory animals of the national institutes of health and reported in compliance with the ARRIVE (animal research: reporting of in vivo experiments) guidelines. A total of 261 male rats (weight 220-250g) were housed in a temperature and humidity controlled room under a standard 12-h light/dark cycle for a minimum of 3 days before ICH-IVH induction and were provided free access to food and water.

ICH-IVH Model and Groups
ICH-IVH was induced by autologous arterial blood injection into the right peri-lateral ventricles as previously described [23]. Brie y, rats were anesthetized with pentobarbital (40 mg/kg, IP). The right femoral artery was catheterized as a source of blood sample. A cranial burr hole (1mm) was drilled after rats were positioned in a stereotaxic frame. Aliquots of 200μl nonheparinized arterial blood was infused into the right caudate nucleus (coordinates: 0.2mm posterior, 2.2mm lateral, and 5.0 mm ventral to the bregma) at a rate of 14μl/min using a microinfusion pump. The burr hole was sealed with bone wax and skin incision was closed with sutures after the needle was removed.
Rats were randomly divided into the following 3 group: sham, ICH-IVH, and MCC950. The sham group had only a needle insertion. MCC950 group received MCC950(10mg/kg, IP; MCE, USA) at 1 hour after ICH-IVH model. ICH-IVH group was given an equal volume of phosphate-buffered saline (PBS) at the same time.
Cell Culture and Treatment PC12 neurons cells of rats (ScienCell, USA) were used for in vitro study. PC12 cells were cultured in Dulbecco's Modi ed Eagle Medium (DMEM) supplemented with 10%(v/v) fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37℃ in a humidi ed atmosphere of 5% CO 2 . After the completion of cell processing, IL-1β (Novoprotein, China) was added into DMEM and diluted to different concentrations (0μg/L, 10μg/L, 20μg/L). PC12 cells were then treated with different dosages of L-1β for 24h.

Apoptosis Assay
Apoptosis was detected using ANNEXIN V-FITC/PI cell apoptosis detection kit (CST, USA). PC12 cells from different groups were digested with trypsin but without EDTA, resuspended in the blinding buffer, and stained with Annexin V-FITC for 15 min and PI for 5 min. The results were analyzed by ow cytometry (Canto2, BD, USA).

Immuno uorescence Staining
Under deep anesthesia, rats were sacri ced by transcardial perfusion with 100ml normal saline followed by 50ml 4% neutral buffered Paraformaldehyde. Brains were xed in 4% neutral buffered paraformaldehyde for 24h at 4℃ followed by 25% and 30% sucrose solution until brains were dehydrated fully. Then brains were cut into 10μm thick coronal sections using a cryostat (LM3050S, Leica, Germany) after being frozen at -80℃. Slides were washed with 0.01M of PBS 3 times for 10 min and then incubated in 0.3% Triton X-100 for 30min at room temperature. After being blocked with 5% BSA for 1h at room temperature, the sections were incubated with primary antibody at 4℃ overnight as follows

Cell Counting
Cell counting was performed on brain coronal sections. Three high-power images (×40 magni cation) were taken in SVZ using a digital camera. Interested positive cells were counted from 4 areas in each brain section by two researchers in a blinded manner.

Neurobehavior Assessment
On day 3, 7, and 14 after ICH-IVH, the modi ed Neurological Severity Score (mNSS) and corner turn were used to evaluate the motor, sensory, and balance functions of animals as previously described [24]. The mNSS was divided into 3 levels based on the range of scores: 1 to 6 indicated mild injury, 7 to12 indicated moderate injury, and 13 to18 indicated severe injury. In the corner turn test, each rat was allowed to proceed into a corner (the angle of 30°) for 10 times with at least 30s intervals between every trial. The rats need turn to right or left, and the percentage of right turns was calculated to assess the neurofunction.
Magnetic Resonance Imaging and Volume Measurement Rats were anesthetized with 2% iso urane/air mixture throughout MRI examination. The MRI scans were performed in a 7.0-T Varian MR scanner (Bruker, USA) with a T2 fast spin-echo sequence using a view eld of 35mm×35mm and 17 coronal slices (1.0mm thickness). Volumes were calculated as previously described [25]. Bilateral ventricles were outlined, and the areas were measured using Image J (NIH, USA). The volumes were assessed by calculating the areas of all slices and multiplying by the section thickness. All image analyses were performed by two observers in blinded manner.

Quantitative RNA Sequencing
Rats were euthanized on day 3 after ICH-IVH. RNA-Seq experiments were performed according to manufacturer's protocol, and data were analyzed by LC Biotech. Brie y, total RNA was extracted from the SVZ tissue using TRIzol reagent, and the quanti ed and puri ed total RNA were used to reversetranscribed to generate cDNAs, which were used to synthesize U-labeled second-stranded DNAs. The ligated products were ampli ed with PCR, and the average inset size for the nal cDNA library was 300 bp (50 bp). The expression levels of all transcripts were evaluated by calculating the fragments per kilobase per million reads. The threshold of signi cantly differential expression was set to p<0.05 and |log 2 (fold change) |≥1. The Gene Orthology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEEG) database were used to explore the biological pathways.

Statistical Analysis
All data were presented as mean ± SD. Data were analyzed by investigators blinded to experimental treatments. All analyses were performed using GraphPad Prism 8 (GraphPad software). We determined each sample size by power analysis using a signi cance level of α=0.05 with 80% power to detect statistical differences. Statistical evaluation of the data was performed by analysis of variance (ANOVA), followed by Tukey multiple-comparison post hoc analysis. Statistical signi cance was de ned as p<0.05.

Ventricular Dilation and Neurode cits after ICH-IVH
Compared with sham group, ICH-IVH led to obvious ventricles extension by time prolonged (sham vs ICH-IVH, p<0.01, Fig.1A). By calculating the ventricles volumes, we found that MCC950(NLRP3 in ammasome speci c inhibitor) treatment signi cantly reduced ventricles dilation at 3, 7, and 14 days after ICH-IVH (ICH-IVH vs MCC950, p<0.05, Fig.1B). In addition, ICH-IVH group started to occur obvious hydrocephalus at 3 days after ICH-IVH. The mNSS and corner test were used to assess neurofunction after ICH-IVH. Rats had severe neurode cits after ICH-IVH and MCC950 treatment improved neurofunction at 3 and 7 days ( Fig.1C and Fig.1D). As time goes on, inhibiting NLRP3 in ammasome gradually loss its function to improve neurode cits according to mNSS and corner test results at 14 days after ICH-IVH ( Fig.1C and Fig.1D). Since the ventricle extension and neurode cits occurred obviously on day 3 after ICH-IVH, we chose this time point (day 3 after ICH-IVH) to do further research.
NLRP3 In ammasome was Activated after ICH-IVH in SVZ The expression of NLRP3 and NLRP3 in ammasome-related cytokines caspase-1 and IL-1β in SVZ were signi cantly increased on day 3 after ICH-IVH (sham vs ICH-IVH, p<0.01, Fig.2A-D), all of which were signi cantly decreased by MCC950 treatment (ICH-IVH vsMCC950, p<0.01, Fig.2A-D). Immuno uorescence staining was performed to assess NLRP3 in ammasome related cytokines caspase-1 and IL-1β. What's more, both caspase-1 and IL-1β positive cells in SVZ were obviously increased after ICH-IVH, and MCC950 treatment decreased this up-regulation ( Fig.2E-G). Combined with neurobehavioral tests result, we demonstrated that NLRP3 in ammasome depended in ammation contributed to neurode cits after hemorrhage.

MCC950 Inhibited Microglia/macrophage Activation after ICH-IVH
On day 3 after ICH-IVH, the immuno uorescence staining showed that NLRP3 was colocalized with microglia (Iba1) and macrophage (CD68) in SVZ ( Fig.3A and Fig.3D). After ICH-IVH, microglia and macrophage were obviously activated, which was re ected by the increase of cells number (Fig.3B and Fig.3E). MCC950 treatment signi cantly inhibited the activation of microglia and macrophage ( Fig.3B and Fig.3E). In addition, NLRP3 positive microglia/macrophage increased after ICH-IVH, and using inhibitor MCC950 decreased NLRP3 activation in microglia and macrophage ( Fig.3C and Fig.3F). These results showed that NLRP3 activated in microglia/macrophage after ICH-IVH in SVZ, and NLRP3 related in ammation how to act to neurons and in uence neurons function need to explore.
Neurons Autophagy were Upregulated after ICH-IVH in SVZ According to previous suppose, we assessed autophagy in neurons. Autophagy was highly activated in neurons after ICH-IVH, the autophagy process speci c markers LC3B and p62 were used to evaluated autophagy levels. Firstly, autophagy process was proved located in neurons after ICH-IVH. the autophagy process marker LC3B positive neurons increased after ICH-IVH, and MCC950 treatment decreased LC3B positive neurons in SVZ ( Fig.4A and Fig.4C). The autophagosome degradation stage marker p62 also was observed in neurons which showed that p62 positive neurons decreased after ICH-IVH and MCC950 treatment inhibited this process ( Fig.4B and Fig.4D). Then, the LC3B expression level increased and p62 expression level decreased in SVZ after ICH-IVH, using MCC950 to inhibit NLRP3 could reverse both LC3B and p62 expression levels ( Fig.4E-H). These results indicated that neurons occurred severe autophagy after ICH-IVH, and inhibiting NLRP3 in ammasome reduced neurons autophagy.

Excessive autophagy mediated neurons apoptosis after ICH-IVH in SVZ
Excessive autophagy was observed in neurons after ICH-IVH. TUNEL staining was used to assess cells apoptosis. Compared with sham group, ICH-IVH group had more cells apoptosis in SVZ. MCC950 treatment decreased cells apoptosis (sham vs ICH-IVH, p<0.01; ICH-IVH vs MCC950, p<0.05, Fig.5A and Fig.5B). FJC staining was used to evaluate neuron condition. There were more damaged neurons (FJC positive cells) after ICH-IVH in SVZ, and MCC950 treatment reversed this trend (sham vs ICH-IVH, p<0.01; ICH-IVH vs MCC950, p<0.01, Fig.5C and Fig.5D). lavish autophagy mediated neuron apoptosis after ICH-IVH, and NLRP3 in ammasome contributed to this process. Using MCC950 to inhibit NLRP3 activation could prevent neurons apoptosis, relieve the ventricular dilatation, and improve neruode cits after ICH-IVH.

Effect of MCC950 on Transcriptional Phenotype after ICH-IVH in SVZ
To explore the relationship between NLRP3 in ammasome and neuron autophagy, transcriptome sequencing was used. Compared with sham group, 565 genes expression changed (518 genes upregulated, 47 genes downregulated) (Fig.6A and Fig.6B). Heat-map of different expression genes after ICH-IVH contained NLRP3, autophagy, and apoptosis related genes (Fig.6C). According to GO enrichment, we found in ammatory response and innate immune response had obvious change (Fig.S1A). Focus on KEEG pathway enrichment, phagosome pathway changed drastically (36 genes expression changed in this pathway) (Fig.S1B). Compared with ICH-IVH group, MCC950 group had 167 genes expression changed ( Fig.6D and Fig.6E), and heat-map of different expression genes after MCC950 treatment was used to select obviously related genes, which contained possible pathways that NLRP3 in ammasome mediated neuron autophagy through (Fig.6F). Combined with GO enrichment pathway and KEEG enrichment pathway, AMPK/Beclin-1 pathway was selected to further explore ( Fig.S1C and Fig.S1D). The transcriptome sequencing results also supported our previous conclusion.
IL-1β accelerated to excessive autophagy in PC12 cells IL-1β which could be secreted to extracellular from microglia/macrophage was the major cytokines after NLRP3 activated. Three dosages of IL-1β (0μg/L; 10μg/L; 20μg/L) was used to intervene PC12 cells which is a kind of neurons. Compared with vehicle group, there are more LC3B-and p62-positive neurons after IL-1β treated ( Fig.8A and Fig.8B). the expression of LC3B and p62 indicated the autophagy level upregulated after IL-1β intervened and reached to peak at a dose of 10μg/L (Fig.8C-E). However, IL-1β (20μg/L) intervened PC12 cells, the autophagy related proteins had lower expression level than IL-1β(10μg/L) group (Fig.8C-E). These results demonstrated IL-1β could upregulate autophagy in neurons as an important cytokine released after NLRP3 activated.

IL-1β mediated neurons apoptosis in PC12 cells
To explore whether IL-1β treatment in uenced neurons function, we evaluated the apoptosis of neurons. Compared with vehicle group, the apoptosis related protein cleaved-caspase3 had higher expression level, and increased along with the dose of IL-1β( Fig.9A and Fig.9D). The results of TUNEL staining and apoptosis assay showed the same conclusion (Fig.9B-C and Fig.9E). The results indicated that IL-1β leaded to neurons apoptosis by upregulating the autophagy level.

Discussion
In this study, we found that activation of NLRP3 in microglia/macrophage after ICH-IVH aggravated neurons apoptosis by upregulating autophagy through AMPK/Beclin-1 pathway in SVZ. Besides, IL-1β whose secretion was mainly promoted by NLRP3 activation mediated autophagy-induced PC12 cells apoptosis (Fig. 10). This study provide compelling evidence that NLRP3-mediated neurons apoptosis played an important role in the pathogenesis of ICH-IVH.
After ICH, resident glial cells were activated and circulating immune cells were recruited to participate in the occurrence and development of neuroin ammation[26-28]. Microglia was the resident macrophage distributed in brain that could be rapidly activated to mediate neuroin ammation in response to pathological conditions, including hypoxia, infection, and brain tissue injury [29]. Besides directly attack to neurons, the polarized microglia could also damage the neurons indirectly by changing the microenvironment via releasing neurotoxicity factors such as IL-1β or recruiting other neurotoxicity cells such as macrophages to amplify in ammatory response [30]. In ammation cytokines(IL-1β, IL-6, IL-27, and TNF-α) mainly released from microglia/macrophage and neutrophils during neuroin ammation aggravated secondary injury to neurons [14,31,32]. Thus, inhibiting neuroin ammation might play a protective role in ICH.

Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3(NLRP3)
in ammasome, the apoptosis-associated speck-like protein, has been proposed as a crucial mediator in innate immunity [33]. The activated NLRP3 could cleave pro-IL-1β and pro-IL-18 into their mature and functional form, resulting in the activation of subsequent in ammatory response [33,34]. Recent studies indicated NLRP3 in ammasome could also polarize microglia and exacerbate ischemia/hemorrhageinduced brain injury [35,36]. NLRP3 activation aggravate neuronal cell death and behavioral de cits, Knockdown or downregulating NLRP3 could improve neurological functions of ICH animals [37]. In this study, we found that NLRP3 was widely activated in microglia/macrophage on day 3 after ICH-IVH, while the neuroin ammation was supposed to reach to the peak at the same time. In addition, we found that using NLRP3 inhibitor MCC950 could improve cognitive and motor function of ICH rats. Individual neuronal cells and their synapses constituted neuronal circuits control all kinds of neurofunction.
Furthermore, to explore how the NLRP3 activation in microglia/macrophage in uences cognitive and motor function after hemorrhage, we aim to neurons.
As the terminally differentiated cells, neurons did not divide and replicate themselves, which was the main reason why a severe injury in brain was often di cult to recover [38]. Autophagy was a conserved intracellular process to degrade dysfunctional organelles and protein aggregates, and played an important role in maintaining neuronal homeostasis [38]. Much of the evidence to support this derives from studies adjusting autophagy in neurons and observing neurodegeneration, especially in AD and PD [39][40][41]. Normal autophagy was considered as a protective factor against neurodegeneration, infection, and brain injury disease [42], however, autophagy disfunction was associated with increased susceptibility of neurons to ischemic injury. It was reported that the activation of autophagy was coincide with axonal swelling of PC12 cells when nerve growth factor was deprived or cells were in excitotoxicity, suggesting a close relationship between autophagic process and neurite degeneration [43]. In addition, neurotoxin exposure induced apoptosis with a concomitant increase of autophagy ux in primary cortical neurons [44]. All of these evidences indicated that autophagy ux participated in neuronal injury in many kinds of CNS diseases. However, the role of autophagy after ICH still remained controversy. Studies found that autophagy-related disorders promoted the occurrence of stroke in some cases [45], and autophagy exacerbated brain injury after ICH. Autophagic cell death of neurons after ICH was con rmed by using conditional knockout Atg7 mice [46]. Although autophagy was involved in promoting brain injury during the acute phase of ICH, studies showed that autophagy had a neuroprotective function via clearing up the accumulation of cell rubbish [47], and the anti-neuronal apoptosis effects was related to the enhancement of autophagy [48]. Several studies showed that ICH induced autophagy of immune cells, especially microglia/macrophage, contributing to improvement of outcomes by regulating in ammation [49][50][51]. Hence, we aimed to explore the relationship between neuronal apoptosis and autophagy in acute phase of ICH, and to prove that excessive neuronal autophagy aggravated neuronal apoptosis, which contributed to neurode cits after ICH-IVH.
NLRP3 activation in microglia/macrophage was the key process to adjust neuronal autophagy, and according to the RNA sequencing results, we found that NLRP3 mediated neuronal autophagy through AMPK/Beclin-1 pathway. Since NLRP3 activation could promote the release of cytokines, among which IL-1β played the most important role among these factors [52,53], we further explored the role of IL-1β, and found that IL-1β was the key factor mediating excessive autophagy and neuronal apoptosis after NLRP3 activation by using PC12 cells in vitro.
Several limitations need to be mentioned in this study. First, NLRP3 plays multifunctional roles in adaptive immunity, and NLRP3 in ammasome is activated in many kinds of neural cells. Further research is needed to investigate the other mechanisms underlying the neuroprotective effects of NLRP3 in secondary brain injury after ICH-IVH. Second, since NLRP3 is not only expressed on microglia/macrophage as our immuno uorescence staining showed, further study about the roles of NLRP3 on other CNS cells, such as astrocytes, is necessary. Third, how the neuronal autophagy affects neuronal function after hemorrhage and the related mechanism associated with NLRP3 should be more deeply evaluated. Recent studies and our present study have demonstrated the effectiveness of inhibiting NLRP3 expression by using MCC950 [54,55]. However, the time window and stability of MCC950 are very limited. This study only focused on the early pathophysiological changes (3 days) in SVZ after ICH-IVH, the longer time research is needed in the further study. At the same time, the effects of NLRP3 on speci c cell types require more precise gene-editing techniques, such as the use of cre/loxp rat to edit NLRP3 genes in speci c cells.
In sum, we found that NLRP3 was activated to promote the release of IL-1β to extracellular from microglia/macrophage after ICH-IVH, which mediated neuronal apoptosis by upregulating autophagy through AMPK/Beclin-1 pathway. These results provide a potential therapeutic target to ICH-IVH patients in acute stage.

Conclusions
In conclusion, we demonstrated that NLRP3 in ammasome activation in microglia/macrophage attenuated neuroin ammation and neuronal apoptosis after ICH-IVH, which was mediated by upregulating autophagy through AMPK/Beclin-1 signaling pathway in neurons. At least in part, NLRP3 related extracellular cytokine IL-1β contributed to this process. Therefore, NLRP3 activation may be a potential therapeutic strategy in the management of ICH-IVH patients.         (n=6/group). Data were represented as mean ± SD, **P < 0.01 and *P < 0.05 versus Sham group, ##P < 0.01 10μg/L group versus 20μg/L group.

Supplementary Files
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