DDX3X aggravates neuronal pyroptosis and inhibits autophagy via NLRP3 inflammasome after experimental subarachnoid hemorrhage

doi:10.21203/rs.3.rs-4877582/v1

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DDX3X aggravates neuronal pyroptosis and inhibits autophagy via NLRP3 inflammasome after experimental subarachnoid hemorrhage

https://doi.org/10.21203/rs.3.rs-4877582/v1

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Neuronal cell death was considered as the symbol of early brain injury after subarachnoid hemorrhage (SAH), but the mechanism was still unclear. DDX3X, a member of DEAD-box helicase family, has a major effect on many biological processes, such as immune inflammation, growth and development, by mediating RNA metabolism. Recent study reported that DDX3X regulated the balance of NLRP3 inflammasome and stress granules in stress condition, but the role of DDX3X in SAH was still unknown. Therefore, we conduced the in-vivo and in-vitro experiments for the role and mechanism of DDX3X in SAH. Western blot and immunofluorescence showed that SAH promoted DDX3X expression, especially in the cortical neurons. After up-regulation of DDX3X, NLRP3 inflammasome was activated and autophagy was inhibited, resulting in the neurological dysfunction and damage of blood brain barrier. Further rescue experiments confirmed that DDX3X aggravated neuronal pyroptosis and inhibited autophagy via NLRP3 inflammasome after SAH. Hence, DDX3X might be a potential therapeutic target for early brain injury of SAH.

DDX3X

pyroptosis

NLRP3

neuron

subarachnoid hemorrhage

Subarachnoid hemorrhage (SAH) is a common cerebrovascular disease, accounting for approximately 5% of all strokes[1]. SAH places a serious burden on the national economy, due to the significant patient disability and mortality[1–3]. In the process of SAH, especially early brain injury after SAH, various pathophysiological events, including oxidative stress, neuroinflammation, blood-brain barrier (BBB) dysfunction, and neuronal cell death, were considered as the main mechanisms of cerebral injury[3, 4].

The DEAD (Asp-Glu-Ala-Asp)-box helicase family is the largest helicase family in eukaryotes, in which DDX3X is one of the members[5, 6]. As a ubiquitously expressed ATP-dependent RNA helicase, DDX3X has a major effect on many biological processes, such as g transcription, nuclear export, stress granule dynamics, and mRNA translation, based on its function in RNA metabolism[5, 6]. In central nervous system diseases, DDX3X was also reported associated with neuronal outcome of spinal cord injury and neurogenesis during fetal cortical development[7, 8]. But the role of DDX3X in hemorrhagic stroke was still unclear. Hence, we investigated the expression pattern and the effect on neurological functions of DDX3X. Recent research revealed that DDX3X drove the process of NLRP3 inflammasome and stress granule assembly, which was a rheostat-like mechanistic paradigm for regulating live-or-die cell-fate decisions under stress conditions[9]. Therefore, we suggested a hypothesis that DDX3X mediated neuronal cell fate via the NLRP3 inflammasome-related pathway in SAH.

In this study, in-vivo and in-vitro SAH models were established to explore the role of DDX3X. Firstly, the temporal expression pattern of DDX3X were investigated. And then, we conducted the up and down regulation of DDX3X to explore whether DDX3X had an effect on SAH. Finally, the molecular mechanisms, especially the NLRP3 inflammasome-related pathways, such as pyroptosis and NLRP3 inflammasome, were further investigated.

2.1 Experimental animals

All of the animal experiments were performed in accordance with the guidelines of the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85 − 23, revised 1996) and approved by the ethics committee of Shenyang General Hospital. In vivo experiments were carried out using 8-week-old, male C57BL6 mice (weight, 19.0–22.0 g). Mice were bred and kept under standard specific pathogen free conditions with food and water ad libitum.

2.2 SAH model

The SAH endovascular perforation model was created as previously reported[10]. In brief, the mice were anesthetized using pentobarbital sodium at a dose of 40 mg/kg intraperitoneal. After exposure of the left common carotid artery(CCA), external carotid artery (ECA) and internal carotid artery(ICA), the ECA was ligated and transected with a 2-mm stump. Next, we carefully inserted a 5 − 0 sharpened monofilament nylon wire from ECA into the left ICA and advanced approximately 3 mm until resistance was felt. At this point, the sutures of the distal portion was removed from the ligation point while ensuring patency of ICA. The same surgical procedure was carried out on sham-operted animals except that the suture was not advanced into the internal carotid artery bifurcation.

2.3 Intracerebroventricular injection

For operation of intracerebroventricular injection, a guide cannula was implanted into the lateral ventricle as described by Li et al[10]. In brief, a bone havest drill was used to make a hole with a diameter of 2.0 mm in the skull. Then, a micro syringe (Microliter 701, Hamilton Company, USA) was stereotactically inserted into the left lateral ventricle, located about 3.0 mm below the horizontal plane of the bregma. 1 h after induction of SAH, a total 2 µL volume of DDX3X recombinant protein solution (TP526484, OriGene, China), or glyburide solution (HY-15206, MCE, USA) was infused at a rate of 0.2 µL/min, whereas 500 pmol/2 µL of DDX3X shRNA or scrambled siRNA was injected at the same rate, 48 h before the induction of SAH.

2.4 Neurological outcome assessment

Modified Garcia’s method was performed 24 h and 72 h after SAH induction to evaluate the neurological deficits in each group[11]. Briefly, the modified Garcia scale is an 18-point scoring system that consists of 6 tests covering spontaneous activity (0–3), symmetry in the movement of four limbs (0–3), forepaw outstretching (0–3), climbing (1–3), body proprioception (1–3), and response to vibrissae touch (1–3), in which a higher Garcia score indicates better neurological function.

2.5 Brain water content

The brain samples of rats were immediately collected, weighed and recorded as brain wet weight. Following, the samples were subjected to desiccation at 55°C for 48 h in an oven and weighted to determine dry weight. The percentage of water content was calculated according to the formula: ([wet weight - dry weight]/wet weight) × 100%.

2.6 Cell culture and treatment

HT-22 cells (Institute of Biochemistry and Cell Biology, SIBS, CAS, China) were maintained in DMEM (11965092, Gibco, USA) supplemented with 10% fetal bovine serum (10091, Gibco, USA) and 1% penicillin-streptomycin (15140122, Sigma-Aldrich, USA) at 37°C in a humidified incubator containing 5% CO₂. To establish in vitro neuronal SAH model, the cells were exposed to oxyhemoglobin (OxyHb, 25µM) for 24 h for subsequent experiments.

2.7 Lentivirus construction

The DDX3X short hairpin RNA lentivirus (ShDDX3X), and empty vector (EV) were constructed by Hanbio Techonology Company, Shanghai, China.

2.8 Western blotting

Total proteins of cells and tissues were extracted by RIPA lysis buffer (R0278, Sigma-Aldrich, USA). Total protein lysate (30 µg protein) was separated by using 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. The blotted membranes were subsequently blocked with 10% serum in 10 mM PBS (pH 7.4) at 37 ℃ for 1 h, and then incubated overnight at 4 ℃ with primary antibodies. After being washed thrice with TBST, the HRP-labeled secondary antibodies (diluted 1:3000 in TBST) were incubated for 2 h at room temperature. Finally, the images were captured by using a Molecular Imager System, and the protein bands were quantified with ImageJ software. The information of antibody application in western blotting was shown in the supplemental table 1.

2.9 Statistical analysis

Data are shown as the means ± the standard error of the mean (SEM). Comparisons between the different groups were performed using Chi-square tests and one-way ANOVA, followed by Tukey's multiple comparisons test. GraphPad Prism 8.0 software (GraphPad Software, Inc.) was used to analyze the data, and differences at p < 0.05 were considered statistically significant.

3.1 DDX3X is activated after SAH

Firstly, we established the SAH model in mice and collected the brain samples at different time point after SAH. The in-vivo and in-vitro experimental designs were shown in the supplemental Fig. 1. The expression of DDX3X increased 6h after SAH and reached its peak at 24h (Fig. 1A). From 24h to 72h after SAH, DDX3X expression gradually decreased (Fig. 1A). Following, we conducted the SAH model in HT-22 neuronal cell line to further explore the expression of DDX3X at different time point after OxyHb treatment. Similarly, the expression of DDX3X raised 6h after SAH in HT-22 cells (Fig. 1B). The cellular experiments confirmed that DDX3X reached the expression peak 24h after SAH (Fig. 1B). The in-vivo DDX3X/NeuN staining showed that the DDX3X protein was mainly expressed in neurons, and SAH promoted the neuronal DDX3X expression (Fig. 1C). In short, the in-vivo and in-vitro results demonstrated that DDX3X was activated after SAH and increased to the top after 24h.

3.2 DDX3X promotes pyroptosis and inhibits autophagy in vivo after SAH

For further exploring the role of DDX3X in SAH, we added DDX3X recombinant protein in mice via intracerebroventricular injection. The group treated with DDX3X recombinant protein manifested as higher DDX3X expression (Supplemental Fig. 2A-B). The in-vivo results identified that 2µg/kg was the proper concentration of DDX3X recombinant protein (Supplemental Fig. 2A-B). Equally, the in-vitro experiments confirmed that 200ng/mL DDX3X recombinant protein should be uses in the following experiments (Supplemental Fig. 2C-D).

It was reported that DDX3X was tightly associated with the establishment of NLRP3 inflammasome[9]. Hence, we detected the expressions of NLRP3 inflammasome-related proteins (NLRP3, ASC, Caspase1 and IL1), and found that inhibiting DDX3X by ShDDX3X reduced the expression of all NLRP3, ASC, Caspase1 and IL1 (Fig. 2A), which indicated that the expression changes of NLRP3 inflammasome were related to DDX3X after SAH. Conversely, overexpression of DDX3X by recombinant protein significantly promoted the expressions of NLRP3 inflammasome- related proteins (Fig. 2A). It was also found that mice in the SAH group got less modified Garcia score, comparing with that in the sham group (Fig. 2B). And mice treated with ShDDX3X showed better neurological outcome, while mice treated with DDX3X recombinant protein showed worse neurological outcome after SAH (Fig. 2B). In addition, the results of brain water content corresponded to similar regulation (Fig. 2C), demonstrating DDX3X aggravated the BBB disruption after SAH. Generally, the above results confirmed that DDX3X participated in the neuronal damage after SAH, but the mechanism was unclear. Thus, we conducted immunofluorescence co-staining of NeuN (the marker of neurons) and PI (the marker of pyroptosis). It was found that SAH promoted the number of pyroptosis in neurons (Fig. 2D-E). And ShDDX3X decreased the rate of pyroptosis neurons, while DDX3X recombinant protein increased that.

As a normal physiological mechanism, autophagy transports cytoplasmic contents to lysosomes for degradation, and promotes cell survival under stress[12], which was reported tightly associated with SAH[13]. Therefore, we detected the expression of autophagy-related proteins, including Beclin1 and LC3B. The results showed that SAH promoted the process of autophagy, which was reversed by DDX3X overexpression (Fig. 3A). And inhibiting DDX3X by ShDDX3X also increased the expression of Beclin1 and LC3B-Ⅱ (Fig. 3A). For further exploring the autophagy in neurons, we conducted the NeuN and LC3B (the marker of autophagy) co-staining. The results of immunofluorescence were consistent with those of western blot (Fig. 3B-C).

3.3 DDX3X promotes neuronal pyroptosis and inhibits autophagy in vitro after OxyHb

Moreover, the role of DDX3X in SAH cellular model was also established, and NLRP3 inflammasome-related proteins, including NLRP3, ASC, Caspase1 and IL1, were detected. Results showed that ShDDX3X reduced the expression of NLRP3 inflammasome- related proteins, while DDX3X raised that (Fig. 4A). Meanwhile, the number of PI positive neurons in OxyHb + ShDDX3X group was less than that in OxyHb group (Fig. 4B-C). Results of CCK8 assay demonstrated that the cellular survival rate of OxyHb + ShDDX3X group was also higher than that of OxyHb group (Fig. 4D). And the neurons in OxyHb + DDX3X group showed less cellular survival rate and more PI positive neurons than that in OxyHb group (Fig. 4B-D). In short, the in-vivo and in-vitro results demonstrated that DDX3X promotes pyroptosis after SAH.

The protein expressions of Beclin1 and LC3B showed that the autophagy was activated by ShDDX3X, while DDX3X reversed the increasing expression of Beclin1 and LC3B-Ⅱ after SAH (Fig. 5A). Meanwhile, treatment of OxyHb in HT-22 cells increased the relative number of LC3B + cells (Fig. 5B-C). And more LC3B + neurons were observed in OxyHb + ShDDX3X group, while less LC3B + neurons in OxyHb + DDX3X group, compared with OxyHb group (Fig. 5B-C). Generally, the in-vivo and in-vitro results demonstrated that DDX3X inhibits autophagy after SAH.

3.4 DDX3X aggravates neuronal pyroptosis and inhibits autophagy via NLRP3 inflammasome after SAH

It was reported that DDX3X played a significant role in driving NLRP3 inflammasome[9]. Therefore, DDX3X recombinant protein and glyburide, NLRP3 inhibitor, was applied to investigate the relation between DDX3X and NLRP3. The in-vivo results revealed that the expression of ASC, Caspase1 and IL1 in SAH + DDX3X + glyburide group was less than that in SAH + DDX3X group (Fig. 6A), indicating that DDX3X promoted NLRP3 inflammasome after SAH. And the mice treated with DDX3X recombinant protein and glyburide showed higher modified Garcia score and less brain water content than SAH + DDX3X group (Fig. 6B-C). Besides, the addition of glyburide partly reversed the increase of PI positive cells after only DDX3X treatment (Fig. 6D-E). Moreover, the addition of glyburide increased the protein expression of Beclin1 and LC3B-Ⅱ, compared with SAH + DDX3X group (Fig. 7A). And the results of NeuN and LC3B co-staining also confirmed the results of western blot (Fig. 7B-C).

The in-vitro results also revealed that the addition of glyburide had no influence on the expression of DDX3X, and reversed the increasing of NLRP3 expression by DDX3X recombinant protein (Fig. 8A). And the NLRP3 inflammasome-related expression of OxyHb + DDX3X + glyburide group was less than that of OxyHb + DDX3X group (Fig. 8A). CCK8 assay showed that glyburide also increased the cellular survival rate and decreased the number of PI positive neurons (Fig. 8B-D). And the addition of glyburide partly reversed the expression decreasing of Beclin1 and LC3B-Ⅱ (Fig. 9A). And the results of NeuN and LC3B co-staining also confirmed the results of western blot (Fig. 9B-C). In summary, the results demonstrated that DDX3X aggravates neuronal pyroptosis and inhibits autophagy via NLRP3 inflammasome after SAH.

In this study, we performed the in-vivo and in-vitro experiments to verify the role of DDX3X in SAH. Firstly, we found that SAH promoted the increasing expression of DDX3X, which reached the expressing peak 24h after SAH. By up and down regulation, it was revealed that DDX3X aggravated the BBB disruption and neurological functional damage after SAH. In this process, pyroptosis and NLRP3 inflammasome were activated, while autophagy was inhibited. The rescue experiment confirmed that DDX3X aggravates neuronal pyroptosis and inhibits autophagy via NLRP3 inflammasome, which was the reason of neuronal damage after SAH.

As an ATP-dependent RNA helicase, the role of DDX3X was mainly explored in cancer and neurodevelopment[6, 7, 14]. Besides, DDX3X could act as a modifier of RAN translation to participate in neurodegeneration diseases[15]. Actually, DDX3X both played an important role in neuron and glia. In the HT-22 cells treated with ketamine, DDX3X was activated by lncRNA TUG1 and promoted BAG5 expression to exacerbate KET-induced neurotoxicity[16]. Moreover, DDX3X mediated the activation of the NLRP3 inflammasome, including caspase1 and henceforward generation of interleukin-1β and of other proinflammatory cytokines, finally resulting in the astrogliosis[17]. And it was reported that aluminum caused microglial activation and neuroinflammation via DDX3X-NLRP3 pathway[18]. Owing to the relationship with NLRP3, DDX3X took effect on the activation of glia and inflammation. The role of DDX3X in NLRP3 inflammasome were always reported in immunocytes, but the relationship between DDX3X and NLRP3 in neurons was still unclear. Therefore, we conducted this study and found that DDX3X mediated pyroptosis via NLRP3 inflammasome in neurons after SAH.

As a part of the innate immune system, inflammasomes involves multimeric protein complexes sensing various environmental and cellular stress signals[4, 19]. The oligomerization of NLRP3 inflammasome triggered the helical fibrillar assembly of ASC, resulting in caspase1 activation and release of the mature cytokine IL1[4, 19–21]. The inflammatory responses involving microglial/macrophage activation and neutrophil infiltration had been considered as the main cause of neurological dysfunctions of early brain injury after SAH[22]. Recent research revealed that the activation of NLRP3 inflammasome in microglia promoted brain edema and neuroinflammation, leading to short term neurobehavioral dysfunctions after SAH[4]. But the mechanism of NLRP3 inflammasome activation was still unclear. The damage of biologic redox equilibrium, especially reactive oxygen species, was found to activate NLRP3 inflammasome and promote release of inflammatory cytokines, resulting in the M1 polarization of microglia/macrophage[23]. The phosphorylation of NF-κB p65 promoted its nuclear translocation and activated NLRP3 inflammasome[2]. Besides, triggering receptor expressed on myeloid cells 1 (TREM1), a glycoprotein of the immunoglobulin superfamily, was another molecular mechanism of NLRP3 inflammasome activation[22]. In this study, we found DDX3X, an ATP-dependent RNA helicase, might be another molecular mechanism of NLRP3 inflammasome activation and pyroptosis. But the mode of action, such as direct or indirect interact, and transcriptional regulation, needed further investigation in the following researches. The potential therapeutic effect of NLRP3 in both the acute and delayed phases following SAH was explored in a profound study[21]. In the acute phase (24h after SAH), NLRP3 inhibition attenuated cerebral edema, tight junction disruption, microthrombosis, and microglial reactive morphology shift[21]. In the delayed phase (5d after SAH), NLRP3 inhibition ameliorated middle cerebral artery vasospasm and sensorimotor deficits[21], which demonstrated the association between NLRP3-mediated neuroinflammation and cerebrovascular dysfunction in both the early and delayed phases after SAH, and put forward a novel potential therapeutic target.

As a new cell death mechanism, pyroptosis has been found and widely explored in programmed neuronal cell death. Pyroptosis was widespread in diseases of the central nervous system, which was involved in the repair, aging, tumor, cerebral hemorrhage, and ischemia[13, 24, 25]. It was reported that the process of inflammasome activation and subsequent pyroptosis are initiated by the endogenous damage-associated molecular pattern (DAMP) stimulations after SAH[25, 26]. The activation of caspase1 was the symbol of the maturation of inflammasome, which triggered the maturation and release of interleukin, as well as the cleavage of Gasdermin D (GSDMD)[2, 22]. The activation of GSDMD promoted pore formation in the plasma membrane and further facilitated interleukin release and osmotic cell lysis, namely, pyroptosis[13, 25, 26]. In the area of SAH, pyroptosis as a novel type of programmed neuronal cell death, recently got multiple attention. It was revealed that neuronal cell death and neuroinflammation were the main cause of early brain injury after SAH, which was mediated by pyroptosis[24]. Neuronal pyroptosis was associated with neurological deficits, which represented decrease of modified Garcia score and behavior disorder in beam balance, rotarod and Morris water maze test[2]. Generally, pyroptosis had been considered as one of the significant types of neuronal programmed cell death. In this study, we found that SAH promoted neurological deficits and BBB disruption via neuronal pyroptosis, which was consistent with previous researches. Besides, pyroptosis of glia also participated in SAH and aggravated neuronal damage via promoting neuroinflammation. Fang et al reported that pyroptosis- related proteins were activated in human CSF after SAH[25]. And the activation of pyroptosis in astrocytes after SAH promoted the production/release of tissue factors and neuroinflammation, finally resulting in neurological deficits[25]. In microglia, GSDMD- dependent pyroptosis was activated after SAH, and aggravated the neuronal injury and brain edema[26]. And inhibiting GSDMD or IL1 in microglia could improve neurological deficits, mitigate brain water content, and preserve brain-blood barrier integrity 24 h after SAH[22], which revealed that pyroptosis of microglia might be another therapeutic target of SAH. Actually, the activation of pyroptosis and NLRP3 inflammasome also reported associated with microglia polarization[23]. The roles of pyroptosis and NLRP3 inflammasome in nerve cells were still unclear, needed further investigations.

Autophagy is an intracellular process mediating delivery of cytosolic constituents to maintain the cellular homeostasis[27]. And autophagy was also recognized as the cellular protective mechanism[27], therefore, stress condition, such as hypoxia, ischemia, and mechanical damage, would activate autophagy. The relationship between NLRP3 and autophagy were explored to clarify the regulatory relationships of inflammation and autophagy. On the one hand, it was revealed that autophagy inhibited NLRP3 inflammasome and inflammation by reducing mitochondrial ROS releasing[27–29]. On the other hand, overexpression of core molecules of NLRP3 inflammasome elevated autophagy and influenced the amount of the LC3-II protein[27], which was consistent with our finding. Meanwhile, the regulation of NLRP3 and autophagy was also clarified in cerebral diseases. It was demonstrated that impairing microglial autophagy aggravates pro-inflammatory responses to LPS and exacerbates MPTP-induced neurodegeneration by modulating NLRP3 inflammasome responses[30, 31]. In this study, we found that DDX3X was the activation of NLRP3-dependent pyroptosis and the inhibition of autophagy. Further, the autophagy inhibition of DDX3X was found associated with NLRP3, which demonstrated that promoting autophagy might be a potential therapeutic strategy for SAH. The therapeutic effects of other drugs such as fluoxetine and Mer tyrosine kinase were reported dependent on increasing autophagy and used in SAH treatment[32, 33].

Actually, there were some limitations in this study. The interaction form between DDX3X and NLRP3 inflammasome was not further explored in this study. In the following research, whether DDX3X directly promoted NLRP3 inflammasome by interaction would be investigated. And the concrete binding sites and action modes between them needed to be clear. If they were indirectly interacted, the molecular mechanism should be verified in the following studies.

In conclusion, our data provided new evidence that pyroptosis and autophagy of neurons are the crucial players in the pathophysiology of SAH. And DDX3X aggravates neuronal pyroptosis and inhibits autophagy via NLRP3 inflammasome, which was the cause of neurological deficits and BBB disruption after SAH. The finding provided the theoretical basis that neuronal pyroptosis and autophagy participated in SAH and might be the potential therapeutic target of SAH. And this study firstly clarified the role of DDX3X in subarachnoid hemorrhage, providing a new perspective of molecular mechanism in SAH.

Acknowledgements We are very grateful to other laboratory staff for their support and cooperation in our experiments, and to the director of the laboratory for his encouragement and guidance to our work.

Author Contribution G.Z. H, Y.W. W and Y. H designed the study and drafted the manuscript for content; G.Z. H, Y. H, Y.W. H and Z.L. S had done experiments and obtained data; X.Y. Y, P.Y. P, Y.S. D, and G.B. L analyzed the data. All authors reviewed the manuscript.

Funding This project was sponsored by National Natural Science Foundation of China (82071481). Sponsor names: Yushu Dong.

Data Availability The authors confirm that the findings of this study are supported by the data. The data are available from the corresponding author.

Ethics Approval and Consent to Participate This study was conducted in accordance with the ARRIVE guidelines and approved by the Animal and Ethics Committee of the General Hospital of the PLA Northern Theater Command of China. All experimental procedures were conducted according to the ARRIVE guidelines. The protocols for the animal experiments were performed according to the Animal and Ethics Review Committee of our institution.

Consent for Publication Not applicable.

Conflict of Interest The authors declare no conflicts of interest.

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No competing interests reported.

Supplementaltable1.docx
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Graphical abstract After SAH, DDX3X was activated in both in-vivo and in-vitro neurons. DDX3X aggravated neuronal pyroptosis and inhibited autophagy via NLRP3 inflammasome.
OriginalImagesforBlots.pdf
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Supplemental Fig. 1 Experimental design.
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Supplemental Fig. 2 DDX3X recombinant protein promoted DDX3X expression in vivo and in vitro. (A) Western blot detection of DDX3X in mice brain samples. (B) The result of western blot was recorded. (C) Western blot detection of neuronal DDX3X. (B) The result of western blot was recorded. Data are shown as the means ± SEM, n = 3 per group. *p < 0.05 vs. Sham or Ctrl. # p < 0.05 vs. SAH or OxyHb.

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DDX3X aggravates neuronal pyroptosis and inhibits autophagy via NLRP3 inflammasome after experimental subarachnoid hemorrhage

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Experimental animals

2.2 SAH model

2.3 Intracerebroventricular injection

2.4 Neurological outcome assessment

2.5 Brain water content

2.6 Cell culture and treatment

2.7 Lentivirus construction

2.8 Western blotting

2.9 Statistical analysis

3. Results

3.1 DDX3X is activated after SAH

3.2 DDX3X promotes pyroptosis and inhibits autophagy in vivo after SAH

3.3 DDX3X promotes neuronal pyroptosis and inhibits autophagy in vitro after OxyHb

3.4 DDX3X aggravates neuronal pyroptosis and inhibits autophagy via NLRP3 inflammasome after SAH

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

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