Dl-3-n-Butylphthalide attenuates early brain injury and delayed neurological dysfunction by regulating NLRP3 inflammasome after subarachnoid hemorrhage

doi:10.21203/rs.3.rs-3605148/v1

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Research Article

Dl-3-n-Butylphthalide attenuates early brain injury and delayed neurological dysfunction by regulating NLRP3 inflammasome after subarachnoid hemorrhage

https://doi.org/10.21203/rs.3.rs-3605148/v1

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Background

Subarachnoid hemorrhage (SAH) is a severe neurological event lacking of effective therapy. Early brain injury (EBI) and delayed neurological dysfunction are important cause in the poor prognosis of patients with SAH. NLRP3 inflammasome activation has been implicated in many inflammatory lesion pathogenesis including SAH. Dl-3-n-butylphthalide (NBP) has been reported to possess substantial anti-inflammatory properties, which is beneficial for various neurodegenerative diseases. However, the effect and molecular mechanisms of NBP on SAH have not been clearly identified. We designed this study to investigate the effect of NBP against EBI and delayed neurological dysfunction after SAH and to reveal the possible underlying mechanism.

Methods

The adult mice were subjected to endovascular perforation SAH model or sham operation. Mice were randomized to sham group, SAH group, or SAH + NBP group. The EBI (short-term study) was studied at 48 h post-SAH and delayed neurological dysfunction (long-term study) at 21 days post-SAH

Results

The results suggested that NBP evidently alleviated the EBI in mice at 48 h post-SAH, as shown by elevating neurological score, reducing brain edema, blood–brain barrier disruption, neuronal loss, and astrocyte aggregation, as well as ameliorating cerebral vasospasm. Moreover, NBP was able to improve long-term neurobehavioral functions and decrease neuronal apoptosis at 21 days after SAH. Significantly, NBP treatment also inhibited the expressions of NLRP3, caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in both EBI and delayed neurological dysfunction indued by SAH

Conclusions

Our findings suggested that NBP treatment exerts a profound neuroprotective effect against early brain injury and delayed neurological dysfunction induced by SAH, at least partially through regulating NLRP3 inflammasome signaling pathway.

Subarachnoid hemorrhage

NLRP3 inflammasome

neuroinflammation

early brain injury

Dl-3-n-Butylphthalide

Subarachnoid hemorrhage (SAH) is a devastating condition and accounts for about 10% of all human hemorrhagic stroke[1]. Early brain injury (EBI), which occurs within 3 days following SAH is the primary cause of the delayed neurological dysfunctions and poor outcomes for the high mortality in patients who suffer from SAH[1, 2]. In the past few decades, researchers have revealed many pathological mechanisms leading to EBI. The rupture of intracranial aneurysms initially contributes to emergency illness such as reduced cerebral blood flow, increased intracranial pressure, and brain edema[2, 3]. These events consecutively initiate various cascades of pathogenesis processes such as inflammation, apoptosis, oxidative stress, and blood–brain barrier disruption (BBB)[2, 4]. Among all these processes, overstimulation of inflammatory responses plays pivotal roles in the pathological progress of SAH-induced EBI, which may further exacerbate brain damage and lead to permanent neurological and cognitive impairment[5, 6]. Given the extensive injury induced by neuroinflammation, inhibition of inflammatory responses could be a promising strategy to reduce early brain injury and improve long-term neurological following SAH[7].

Nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome, as the most extensively characteristic inflammasome, is the best studied one among all kinds of inflammasomes and widely participate in the host defense response[8, 9]. NLRP3 inflammasome is considered a complex of cytoplasmic proteins containing NLRP3, caspase-1 and ASC[10]. The activation of NLRP3 inflammasome typically requires two conditions that its transcription surpasses a certain threshold and recruitments of caspase-1 and ASC. NLRP3 inflammasome activation can facilitate the maturation and secretion of interleukin 1β (IL-1β) and 18 (IL-18)[11]. Recently, mounting evidence has demonstrated that NLRP3 inflammasome is associated with onset and progression of many inflammatory diseases, including auto-immune and auto-inflammatory diseases, multiple sclerosis, metabolic disorders, as well as other brain injury disease[10, 12]. In SAH, the activation of NLRP3 inflammasome is essential for the modulation of pro-inflammatory cytokines, which has been implicated in neuron apoptosis, cerebral vasospasm and delayed neurological dysfunction[13]. Notably, interference with NLRP3 inflammasome activation by pharmacological treatment could attenuate brain injury and neurological deficits following SAH[14, 15]. Hence, inhibition NLRP3 inflammasome may be offer novel therapeutic avenues for SAH.

Dl-3-n-butylphthalide (NBP) is a favorable safe drug approved by the National Medical Products Administration to treat acute ischemic stroke since 2002 in China. It was observed that NBP could reduce infarct volume, BBB leakage, and neuronal cell death, and improve neurological function in stroke patients. Recently, many studies have indicated that NBP reduced the inflammatory response by inhibiting NLRP3 inflammasome activation in various neurodegenerative diseases, including ischemic stroke[16], Alzheimer’s disease[17], and Parkinson’s disease[18]. Unfortunately, there is no available data on the usage of NBP in preclinical and clinical SAH studies. The effect of NBP on the NLRP3 inflammasome during and after SAH remains unknown. In this study, we first explored the effect of NBP on NLRP3 inflammasome in early brain injury after SAH. Next, we further evaluated the role of NBP on NLRP3 inflammasome in long-term neurologic deficits following SAH.

Animals

A total of 88 healthy adult male C57BL/6J mice were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China), which were maintained on a 12-h light/dark cycle with temperature- and humidity-controlled in in specific pathogen free room. The mice were housed in animal care facilities and had free access to standard food and water.

Endovascular perforation SAH mice model

The endovascular perforation method was performed as previously reported[19]. Mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (30 mg/kg). Rectal temperature was kept at 37 ± 0.5°C using a heating pad during operation. A midline incision was made in the neck to expose the left common carotid artery bifurcation. The left external carotid artery was ligated and cut, leaving a tiny stump. Through the stump, a 4 − 0 monofilament nylon suture, was inserted into the internal carotid artery to a point at the bifurcation of the anterior and middle cerebral artery in the circle of Willis, where a resistance was encountered. Ultimately, the suture was advanced 2 mm further to perforated the right anterior cerebral artery and remained in place approximately 15 s before withdrawn, causing subarachnoid bleeding. Sham-operated mice underwent the same procedures but without perforation.

Study design and drug administration

Experiment 1 short-term study. We first evaluated the neuroprotective effect of NBP on early brain injury in the acute phase (48 h post-SAH) after SAH. Sixty-six male mice were randomly divided into three groups: sham groups (n = 18); SAH groups (n = 24); SAH + NBP groups (n = 24). NBP at a dosage of 10 mg/kg (dissolved in olive oil) were given by gastric gavage at 3 h and 6 h after SAH. On the 2nd day after operation, Mice were continuously gavaged with NBP twice a day.

Experiment 2 long-term study. We next examined the effects of NBP on long-term neurobehavioral functions in delayed phases (21 d post-SAH) following SAH. Twenty-two male mice were completely divided into three groups at random as followed: sham groups (n = 6); SAH groups (n = 8); SAH + NBP groups (n = 8). Mice were administered NBP (10 mg/kg, b.i.d.) by gastric gavage from day 0 to day 21 after SAH. Dosage and dosing strategy of NBP were chosen based on previous studies[17, 20].

SAH grade assay

The SAH grade was blindly evaluated according to a previously described grading scale [21]. The basal cistern was consisting of six segments, and each segment was graded from 0 to 3 depending on the amount of subarachnoid blood clot: grade 0, no subarachnoid blood; grade 1, minimal subarachnoid blood; grade 2, moderate blood clot with appreciable arteries; and grade 3, blood clot obliterating all arteries within the segment. The grade ranges from 0 to 18 after adding the scores from all six segments. Mice with SAH grading score ≤ 7, which had no prominent brain injury, were excluded from the study.

Neurological scores assay

The neurological scores were determined blindly at 3, 24 and 48 h after SAH by an independent investigator, using the modified Garcia test[21]. The evaluation consisted of six tests that can be scored from 0 to 3 or 1 to 3 as followed: spontaneous activity (0–3), symmetry of limbs (0–3), forelimb extension (0–3), climbing (1–3), body proprioception (1–3), and reaction to vibrissae (1–3). The scale of evaluation ranges from 3 to 18.

Brain water content assay

The brain water content was assessed through wet-dry method. Mice brain was quickly removed after being sacrificed and separated into 4 parts, including left hemisphere, right hemisphere, cerebellum and brain stem, and each part was immediately weighted (wet weight). Then, brain specimens were dehydrated in 100°C for 72 h to evaporate the brain water completely and get the dry weight. The percentage of water content was calculated as (wet weight-dry weight)/wet weight.

Blood-brain barrier disruption assay

At 48 h post-SAH, mice were injected intraperitoneally with Evan’s blue dye (4%, 10 µl/g) allowed to circulate for 3 h. The anesthetized mice were subjected to intracardiac perfusion with phosphate-buffered saline (PBS, 0.1 M) for cleaning intravascular Evan’s blue dye. Brain samples were harvested, weighted, and homogenized in PBS. Subsequently, brain homogenate was centrifuged at 15000 g for 30 min to obtain 0.5 ml of the resultant supernatant, and then incubated with same volume of 50% trichloroacetic acid. Mixtures were centrifuged and Evan’s blue dye in supernatant was measured using a spectrofluorophotometer at 610 nm.

Nissl staining and HE staining

At 48 h after SAH, mice were deeply anesthetized and transcardially perfused with PBS. The brain and brainstems were immediately harvested and fixed in 4% paraformaldehyde solution for 24 h. After dehydrating, the brain and brainstems were embedded in paraffin and cut into serial coronal sections (5 µm). Brain slices were stained with Nissl staining solution to assess neuronal loss. Brainstem sections was performed to HE staining for evaluating the degree of basilar artery vasospasm. The cerebral vasospasm was blindly assessed by using measurements of the wall thickness and luminal inner perimeter in basilar artery.

Immunohistochemistry and immunofluorescence staining

Brain coronal sections were deparaffined and rehydrated, and washed with PBS (0.01 M, pH 7.4). The sections were treated with 10 mmol/L sodium citrate-hydrochloric acid buffer and heated for 15 min, and then incubated with 3% hydrogen peroxide (H₂O₂) for 10 min at room temperature. The slices were blocked with 10% horse serum at room temperature for 1 h. The sections was then incubated with anti-GFAP (1:500, Abcam, Cambridge,UK) overnight at 4°C followed by incubating biotin-conjugated universal secondary antibody (Gene Tech, Shanghai, China) for 1 h at room temperature. Brain cell nucleus was counterstained with hematoxylin. For NeuN and TUNEL co-staining, the sections were subjected to NeuN antibody (1:1000, Millipore, MA, USA) at 4°C overnight and were then incubated with TUNEL staining with a kit (Beyotime Biotechnology, Shanghai, China). Image J software was used to analyze positive cells from six slices at similar coronal positions in mice by a viewer blinded to the experimental group.

Grip strength test

The mice were gently placed on the center of the grip plate, prompting the mice to grasp the grip plate, and then lightly pulled by the tail. Mice were detected three times and the average of the three results was used as the evaluation value. After the start of the formal experiment, mice were tested once a week[22].

Open-field (OF) test

The open field test is commonly performed to assess the spontaneous activities, emotional characteristics, exploring behaviors, or other behavioral in an open environment. After 21 days of surgery, mice were gently placed in the center of the box (50 × 50 × 50 cm). The behavior and movement trajectory of mice were recorded for 5 min by a video camera positioned above the box. The box was thoroughly cleaned by 75% alcohol solution after each mouse.

Novel object recognition (NOR) test

The novel object recognition test was used to measure the non-spatial working memory according to previously published protocols[23]. Firstly, mice were gently placed into an apparatus (50 × 50 × 50 cm) without any objects for 10 min per day for 2 continuous days to habituate to the environment. Then, two identical objects were placed parallel near one wall of the square box. Animals were placed singly in the apparatus and allowed to explore the objects for 10 min. After a one-hour interval, one of the old objects was replaced by a novel object and animals were allowed to explore for 5 min. The time each animal took to examine the familiar object and novel object was recorded by digital camera. The discrimination index was calculated according to the following expression: (novel object (s) - familiar object (s))/ (novel object (s) + familiar object (s)). The objects and the box were cleaned with ethanol (50%) after each individual trial to eliminate olfactory cues.

Western blot analysis

Brain tissue was homogenized with lysis buffer containing 1% PMSF and incubated for 30 min at 4°C. Mixture was centrifuged for 30 min at 15000 g, and the supernatant was collected and boiled. Protein concentrations was assessed with bicinchoninic acid (BCA) kit. Equal amounts of protein from different samples were loaded and separated on SDS-PAGE gel followed by transferred on methanol PVDF membranes. The membranes were blocked by 5% skim milk in Tris-buffered saline with Tween-20 (TBST) for 2 h at room temperature, and then incubated with the corresponding primary antibodies against NLRP3 (#ab263899, 1:1000, Abcam), ASC (#67824, 1:1000, Cell Signaling Technology), caspase-1 (#ab207802, 1:1000, Abcam), or GAPDH (#ab8245, 1:5000, Abcam) at 4°C overnight. After washing three times for 10 min in TBST, membranes were incubated with anti-rabbit IgG antibody and anti-mouse IgG antibody separately at room temperature for 1 h. Protein bands were visualized and quantified using the Carestream system (Carestream Health, Inc, USA).

Statistical analysis

All data were expressed as mean ± SEM and were analyzed using GraphPad Prism 8 software (GraphPad Software Inc.) and SPSS 13.0 statistic software (SPSS Inc., Chicago, IL, USA). Statistical analyses were conducted by one-way or two-way analysis of variance (ANOVA) followed by Tukey's test. P < 0.05 were considered statistically significant.

Effect of NBP on SAH grades and short-term neurological score at 48 h post-SAH

We first evaluated the neuroprotective effect of NBP on early brain injury (EBI) in the acute phase after SAH. Mice were administered NBP (10 mg/kg, b.i.d.) by gastric gavage from day 0 to day 2 after SAH. Neurological damage parameters including SAH grades, neurological score, brain edema, and blood brain barrier disruption were tested at 48 h post-SAH. As shown in Fig. 2A, the differences between SAH and SAH + NBP groups were of no statistical significance (P > 0.05), suggest that NBP treatment did not alter SAH grades. The neurological scores in each group were measured 3, 24 and 48 h after SAH. Endovascular perforation surgery leaded to a significant decrease in neurological score in mice, indicating neurological deficits. However, NBP treatment can obviously ameliorate neurological dysfunction compared with SAH group at 48 h post-SAH (P < 0.05, Fig. 2B).

NBP attenuates brain edema and blood brain barrier disruption at 48 h post-SAH

Water content assay and BBB disruption assay were used to assess the influence of NBP treatment on brain damage. Left hemisphere, right hemisphere, cerebellum, and brain stem were detected separately at same time point. Excluding brain stem, the brain water content was dramatically increased 48 h after SAH, while NBP administration significantly decreased the elevation of brain water content in both left hemisphere and right hemisphere (P < 0.05, Fig. 2C). The similar result can be found in BBB disruption assay. Notably, enhanced Evan’s blue extravasations in hemispheres and cerebellum were observed in SAH groups, which exposed the BBB injury. Nevertheless, NBP treatment markedly improved Evan’s blue extravasation as compared with SAH groups (P < 0.05, Fig. 2D).

NBP reduces neuronal loss and astrocyte aggregation at 48 h post-SAH

Nissl staining and immunohistochemical staining were performed to determine whether NBP could attenuate neuronal defect. The cortex and hippocampus of mice in SAH groups demonstrated obviously lower number of surviving neurons compare with sham groups. However, this reduction was significantly reversed by NBP treatment as compared to SAH groups (P < 0.05, Fig. 3A-D). Astrocytes assume a reactive phenotype in acute traumatic brain injury, which coincides with an up-regulation of the glial fibrillary acid protein (GFAP, also known as the scaffold of astrocytes). As shown in Fig. 3E-H, there were a substantial quantity increase of GFAP-positive cells in SAH groups compared with sham groups in cortex and hippocampus. By contrast, SAH + NBP groups have fewer GFAP-positive cells compared to SAH groups (P < 0.05).

NBP ameliorates cerebral vasospasm at 48 h post-SAH

The development of cerebral vasospasm is a leading contributor to unfavorable outcomes after SAH. The histological images of HE staining for the mice’ basilar arteries were applied to morphological change assessment of cerebral vasospasm, which is featured with a corrugated internal elastic lamina and a thickened vessel wall. In this study, there was a dramatic cerebral vasospasm 48 h after SAH as compared to sham groups (Fig. 4A), and the perimeter and the thickness of basilar arteries shown a significant statistic difference between sham groups and SAH groups by semi-quantitative analysis (P < 0.05, Fig. 4B and C). After NBP treatment, remarkable increased perimeter and decrease thickness of the arteries were detected (P < 0.05).

NBP inhibits NLRP3 inflammasome activation at 48 h post-SAH

The activation of NLRP3 inflammasome has been proven to plays a pivotal role in the progression of early brain injury (EBI) following SAH. To clarify the relationship between NLRP3 inflammasome and NBP treatment in SAH-induced EBI, we measured and analyzed the protein expression of NLRP3, ACS, and caspase-1 in the brain tissue of mice at 48 h post-SAH. Western blotting results showed that protein level of NLRP3 were remarkably up-regulated after SAH induction as compared to sham operation (P < 0.05, Fig. 5A-D), which was greatly relieved by NBP administration (P < 0.05). Similarly, prominently increased trends of ASC and caspase-1 were found in SAH groups, which were evidently reversed by NBP treatment (P < 0.05).

NBP improves long-term neurobehavioral functions 21 d after SAH

Based on the above results, we next evaluated the efficacy of NBP on long-term neurobehavioral functions 21 d after SAH. Grip strength test, open-field test, and novel object recognition test were performed to assess the delayed neurobehavioral dysfunction in 21 d after SAH. As shown in Fig. 6A, a significant reduction in the grip strength was observed in SAH groups in comparison to sham groups, while the NBP-treated animals exhibited an obviously increase in the grip strength compared to those of SAH group (P < 0.05). Next, in the open field test, the total moving distance was evaluated as main indicator. The result proved that total moving distance in SAH group was dramatically reduced compared to sham groups in 21 d post-SAH, but treatment with NBP significantly promoted the locomotor activity (P < 0.05, Fig. 6B). Furthermore, mice induced by SAH had lower discrimination index in comparison to sham mice. In contrast, discrimination index in NBP + SAH groups was memorably enhanced compared with the SAH group mice (P < 0.05), indicated that learning and cognitive ability of SAH mice were significantly improved by a treatment of NBP. Taken together, these finding suggested that NBP was able to improve delayed neurobehavioral dysfunction in SAH mice.

NBP suppresses NLRP3 inflammasome activation at 21 d after SAH

Based on the above findings that the treatment of NBP can inhibit NLRP3 inflammasome activation in the acute phase (48 h post-SAH) after SAH, we further examined the effects of NBP on NLRP3 inflammasome in delayed phases (21 d post-SAH) following SAH. Results in Fig. 6D-G shown that NLRP3, as well as ASC and caspase-1 expression levels were markedly elevated in the SAH group at 21 d following SAH relative to sham mice. However, the administration of NBP contributed to a dramatically reduction in NLRP3, ASC and caspase-1 compared with SAH groups (P < 0.05 or P < 0.01), which suggested the inhibition effort of NBP in NLRP3 inflammasome activation 21 d after SAH.

NBP decreases neuronal apoptosis 21 d after SAH

Neuronal cell apoptosis in the early and delayed phases following SAH is a hazardous factor for functional neurological deficits. Viable neurons and TUNEL-positive neurons were measured by using TUNEL-NeuN co-staining 21 d after SAH induction. As shown in Fig. 7, SAH induction signally decreased NeuN-positive cells and increased TUNEL-positive cells compared with sham groups, which were statistically verified by apoptosis index. After NBP treatment, NeuN-positive cells were significantly increased, in the meanwhile, NeuN -TUNEL co-staining positive cells were decreased in SAH + NBP groups as compared with SAH groups (P < 0.05), demonstrated that NBP treatment remarkably improved the viable neurons and reduced neuronal apoptosis 21 d after SAH.

Subarachnoid hemorrhage (SAH) is a life-threatening disease with poor outcomes [24]. Despite recent progress in endovascular surgery and microsurgical techniques, about 35% of SAH patients will die within 48 h of the onset event, and approximately one-third of survivors will experience long-term cognitive and neurological deficits[25]. Innovative therapeutic drugs with more advantages in terms of safety, efficacy, lower expense, and no ethical issues are urgently needed. Dl-3-n-butylphthalide (NBP), a compound extracted from celery seed, is favorable safe in clinical use for anti-cerebral ischemia[26]. The effect and mechanism of NBP on SAH are still unclear. In this study, we found that NBP treatment could attenuate early brain injury after SAH, which is characterized by improved short-term neurological deficits, brain edema and blood brain barrier disruption. In addition, we also observed that NBP significantly alleviated neuronal cell apoptosis, prevented neurodegeneration, and improved long-term neurobehavioral functions 21 days after SAH.

The brain damage caused by SAH occurs through a biphasic time process, characterized by distinctive pathological mechanisms in two phases. The first early phase, early brain injury (BEI), is believed to an acute brain injury within 3 days after aneurysm bleeding on the surface or bottom of the brain. Pathophysiologically, EBI has been implicated in blood-brain barrier (BBB) disorder, elevated intracranial pressure, reduced cerebral blood flow, neuroinflammation, and brain edema. These complex cellular cascades and brain vascular dysfunction drive neuron death and subsequent long-term neurological deficits. The delayed phase is characterized by delayed cerebral ischemia and neurological impairment, which often occur several weeks after SAH[27]. Despite the progress made in endovascular treatment and early diagnostics, approximately one-third of patients with SAH have different degrees of permanent neurological sequelae, including physical deficits and cognitive impairment[27, 28]. Therefore, improving early brain injury and long-term neurological dysfunction is crucial for the recovery of SAH patients. Hence, we first evaluated the effect of NBP on early brain injury in mice 48 h after SAH. Our results have shown that NBP was able to improve short-term neurological deficits, attenuate brain edema and blood brain barrier disruption, and ameliorate cerebral vasospasm. Moreover, NBP was found to reduce neuronal loss and astrocyte aggregation after SAH. Then, a long-term study was performed to investigate the efficacy of NBP on permanent neurological sequelae in mice 21 days after SAH. Interestingly, our data indicated that NBP significantly reduced neuronal cell apoptosis, prevented neurodegeneration, and improved long-term neurobehavioral functions 21 days after SAH. More importantly, NBP can not only alleviate early brain injury, but also improve long-term neurological dysfunction after SAH, which play a crucial role in overcoming poor outcome in SAH patients.

Inflammation responses were evidenced to play critical roles in the pathophysiology of SAH-induced early brain injury and the delayed brain deterioration. At the onset of SAH, blood is diffused at the site of bleeding and red blood cells breakdown and degrade over time. Degraded products, including hemoglobin, oxyhemoglobin heme, methemoglobin, and hemin, then deposit into subarachnoid space, which contribute to the release of pro-inflammatory cytokines and further trigger the inflammation cascade pathways[29]. Neuroinflammation evolves over time and is involve in the delayed brain injury after SAH[30]. Currently, there is a wealth of studies have indicated that NLRP3 inflammasome is predominantly released by microglia and is supposed to be the principal contributor of neuroinflammation[31]. NLRP3 inflammasome is a multiple protein complex that contains NLR protein cryopyrin, the adapter ASC and pro-caspase-1[32]. The activation of NLRP3 inflammasome has been proven to play an extremely important role in the progression of neurodegenerative diseases including Alzheimer's disease[33–35], Parkinson's disease[36], and cerebral ischemia [37, 38]. In addition, blood component derived from ruptured aneurysms, such as hemoglobin and heme, is believed to be a NLRP3 activator[39]. Recent studies have observed the activation of NLRP3 inflammasome after SAH, and inhibiting NLRP3 inflammasome activation might be a useful therapeutic intervention following SAH[14, 40]. Notably, previous studies reported that NBP could regulate NLRP3 inflammasome activation in Alzheimer's disease and Parkinson's disease[17, 18]. Thus, we speculated that NBP could alleviate early brain injury and long-term neurological dysfunction after SAH through the suppression of NLRP3 inflammasome activation. In fact, our findings indicated that NBP treatment markedly inhibited the NLRP3 inflammasome activation in both 48 h and 21 d post-SAH. The results confirm our hypothesis, that is, the mechanism of NBP against SAH insults is to inhibit NLRP3 inflammasome activation.

Our study demonstrates that NBP treatment exerts a profound neuroprotective effect against early brain injury and delayed neurological dysfunction induced by SAH, and this protection appears to be largely due to the inhibition of NLRP3 inflammasome.

SAH: Subarachnoid hemorrhage; NBP: Dl-3-n-butylphthalide; EBI: Early brain injury; NLRP3: Nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3; ASC: apoptosis-associated speck-like protein containing a caspase recruitment domain; GFAP: glial fibrillary acidic protein; IL-1β: Interleukin-1β; IL-18: Interleukin-18; BBB: Blood brain barrier; PBS: Phosphate-buffered saline; TBST: Tris-buffered saline with Tween-20

Ethics approval and consent to participate

All animal experimental protocols were performed in accordance with the National Institutes of Health guidelines (NIH, USA), and approved by the Ethics Committee of the Institute of Laboratory Animal Science at Jinan University.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request

Competing interests

The authors declare that they have no competing interests.

Funding

This work was partially supported by grants from Natural National Science Foundation of China (NSFC 82104135), China postdoctoral science foundation (2020M683207), Guangdong Research and Development project (2020A1515111207).

Authors' contributions

L.M.W. designed and supervised the project. F.F.G., S.J.Z. and D.C.C performed experiments, collected, and analyzed the data. S.J.Z. and D.C.C performed statistical analysis and prepared figure. L.M.W. and F.F.G. wrote the manuscript. All authors have read and approved the final manuscript.

Acknowledgements

Not applicable.

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No competing interests reported.

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Dl-3-n-Butylphthalide attenuates early brain injury and delayed neurological dysfunction by regulating NLRP3 inflammasome after subarachnoid hemorrhage

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Abstract

Background

Methods

Results

Conclusions

Figures

Introduction

Materials and methods

Animals

Endovascular perforation SAH mice model

Study design and drug administration

SAH grade assay

Neurological scores assay

Brain water content assay

Blood-brain barrier disruption assay

Nissl staining and HE staining

Immunohistochemistry and immunofluorescence staining

Grip strength test

Open-field (OF) test

Novel object recognition (NOR) test

Western blot analysis

Statistical analysis

Results

Effect of NBP on SAH grades and short-term neurological score at 48 h post-SAH

NBP attenuates brain edema and blood brain barrier disruption at 48 h post-SAH

NBP reduces neuronal loss and astrocyte aggregation at 48 h post-SAH

NBP ameliorates cerebral vasospasm at 48 h post-SAH

NBP inhibits NLRP3 inflammasome activation at 48 h post-SAH

NBP improves long-term neurobehavioral functions 21 d after SAH

NBP suppresses NLRP3 inflammasome activation at 21 d after SAH

NBP decreases neuronal apoptosis 21 d after SAH

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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