Application of heat shock protein in preparation of medicine for treating subarachnoid hemorrhage

Abstract

The invention relates to the technical field of bioengineering, and discloses an application of a heat shock protein in preparation of a medicine for treating subarachnoid hemorrhage, and the heat shock protein is HSP60. Wherein the expression of HSP60 is up-regulated after subarachnoid hemorrhage of a mouse, and NLRP3 inflammasome assembly is promoted by activating a TLR4 / MyD88 / NF-kappa B signal channel in cells, so that pyroptosis of microglial cells is induced, and early brain injury is aggravated; the invention further provides a product for treating / inhibiting subarachnoid hemorrhage. The invention discloses a key effect of HSP60 on induction of pyroptosis of microglial cells through a TLR4 / MyD88 / NF-kappa B-NLRP3 signal axis in SAH-induced early brain injury for the first time, and proves that mizoribine can effectively relieve neuroinflammation and brain injury by inhibiting HSP60.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to the application of a heat shock protein in the preparation of drugs for treating subarachnoid hemorrhage. Background Technology

[0002] Subarachnoid hemorrhage (SAH) is an acute cerebrovascular disease with extremely high mortality and disability rates. Among the many factors influencing the prognosis of SAH patients, early brain injury (EBI) is particularly crucial. EBI is generally defined as acute global brain injury occurring within 72 hours of SAH onset, and its severity is closely related to patient mortality, neurological deficits, and the occurrence of subsequent complications. The pathological mechanisms of EBI are extremely complex, involving multiple processes such as cerebral ischemia, inflammation, oxidative stress, and apoptosis. These processes work together to ultimately lead to neuronal death and brain tissue damage. The established core pathophysiological responses include ischemic injury and energy metabolism disorders, blood-brain barrier disruption, neuroinflammation, oxidative stress and ferroptosis, and pyroptosis.

[0003] Existing technology: Studies on the neuroprotective effects of SAHA on SAH models by regulating the HDAC1 / HSP70 / TDP-43 axis have disclosed the neuroprotective effects and related mechanisms of SAHA in SAH models, and whether HSP70 can inhibit TDP-43 cytoplasmic accumulation and reduce neuronal axonal damage in SAH models. However, HSP70 inhibitors can develop resistance after long-term use. How to further develop therapeutic targets for subarachnoid hemorrhage is a problem that needs to be addressed in this type of targeted therapy.

[0004] Therefore, a thorough understanding of the molecular network mechanisms of EBI is crucial for developing effective strategies for early intervention in SAH. Simultaneously, how to connect basic research findings with clinical applications through translational research to overcome current treatment bottlenecks is also a pressing issue that needs to be addressed with existing technologies. Summary of the Invention

[0005] The purpose of this invention is to provide the application of heat shock protein in the preparation of drugs for treating subarachnoid hemorrhage, so as to solve the technical problem of further providing therapeutic targets for subarachnoid hemorrhage in the prior art.

[0006] To solve the above-mentioned technical problems, the present invention specifically provides the following technical solution: This invention provides the application of a heat shock protein in the preparation of a drug for treating subarachnoid hemorrhage, wherein the heat shock protein is HSP60; In particular, HSP60 expression was upregulated after subarachnoid hemorrhage in mice, which promoted the assembly of NLRP3 inflammasomes by activating the TLR4 / MyD88 / NF-κB signaling pathway in cells, thereby inducing microglia pyroptosis and aggravating early brain injury.

[0007] As a preferred embodiment of the present invention, the product includes a product for treating heat shock proteins, which reduces neurological deficits, cerebral edema, blood-brain barrier disruption, and neuronal damage in mice following subarachnoid hemorrhage by inhibiting the expression of HSP60.

[0008] As a preferred embodiment of the present invention, the product contains at least imidazoribine.

[0009] This invention provides a product for treating / inhibiting subarachnoid hemorrhage, the product being a mixed solution composed of imidazolidin and a solvent.

[0010] In a preferred embodiment of the present invention, the solvent is physiological saline.

[0011] As a preferred embodiment of the present invention, the concentration of imidazoribine in the mixed solution is 20-100 mg / kg.

[0012] In a preferred embodiment of the present invention, the concentration of imidazoribine in the mixture is 100 mg / kg.

[0013] As a preferred embodiment of the present invention, the mixed solution is administered via intracranial injection.

[0014] This invention provides an application of the product in alleviating neurological deficits, cerebral edema, blood-brain barrier disruption, and / or neuronal damage following subarachnoid hemorrhage in mice.

[0015] As a preferred embodiment of the present invention, the following steps are included: The mixed solution was administered via lateral ventricle injection six hours after subarachnoid hemorrhage in mice; The injection rate was 0.5 μL / min.

[0016] Compared with the prior art, the present invention has the following advantages: This invention reveals for the first time the key role of HSP60 in inducing microglial pyroptosis through the TLR4 / MyD88 / NF-κB–NLRP3 signaling axis in SAH-induced early brain injury, and confirms that imidazolidin can effectively reduce neuroinflammation and brain injury by inhibiting HSP60. This invention further provides the application of imidazolidin in the preparation of drugs for treating subarachnoid hemorrhage. When an imidazolidin solution of about 100 mg / kg is applied to diseased mice, it can inhibit the extracellular expression of HSP60, effectively block its cascade reaction, significantly reduce microglial pyroptosis, and alleviate neuroinflammatory-related pathological damage. Attached Figure Description

[0017] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0018] Figure 1 This invention provides a flowchart for establishing in vivo and in vitro subarachnoid hemorrhage models and exploring the functional role of HSP60; Figure 2 (AC) provides a statistical graph of temporal expression changes of HSP60 in the early stages of brain injury detected by Western blotting. Figure 2 (DF) provides a statistical graph of temporal expression changes of NLRP3 in the early brain injury stage detected by Western blotting in this invention; Figure 3 (A, C) are statistical graphs provided by this invention for detecting the pyroptosis-related protein pro-Caspase-1 and its cleaved active form Cleaved Caspase-1 in in vitro and in vivo SAH models using Western blotting. Figure 3 (B, D) are dynamic statistical graphs of the full length of GSDMD (GSDMD-F) and its segmentation form GSDMD-N; Figure 4 A provides a statistical graph of the results of evaluating the inhibitory effect of different concentrations of imidazolidin on HSP60 expression by Western blotting. Figure 4 B provides a statistical graph of the CCK-8 assay showing that imidazolidin can restore the viability of microglia stimulated by OxyHB in a concentration-dependent manner. Figure 4 C. This invention provides a statistical chart of optimal concentration under in vitro conditions confirmed by Western blotting. Figure 5 Figures A, E, and I provide statistical graphs for detecting the expression of key proteins (NLRP3, pro-Caspase-1 / Cleaved Caspase-1, and ASC) in the NLRP3 inflammasome pathway using Western blotting, as described in this invention. Figure 5Figures B, F, and J represent statistical graphs of protein expression changes in microglia and related target proteins in in vitro and in vivo models, obtained by immunofluorescence double staining, and the results of Western blotting. Figure 6 C, G, and K are statistical graphs of cellular experimental results for detecting key proteins (NLRP3, pro-Caspase-1 / Cleaved Caspase-1, ASC) of the NLRP3 inflammasome pathway using Western blotting, as provided in this invention. Figure 6 The D, H, and L values ​​represent the changes in protein expression of microglia and related target proteins in in vivo and in vitro models, as well as the statistical results of Western blot analysis, obtained by immunofluorescence double staining. Figure 7 Figures A and C provide a statistical graph for detecting the expression of GSDMD-N, the active form of GSDMD, a key molecule mediating cell membrane pore formation, according to the present invention. Figure 7 Figures B and D are statistical graphs of positive signals of imidazoribine group target proteins in microglia analyzed by immunofluorescence co-localization. Figure 7 E is a transmission electron microscope image of the ultrastructure of pyroptosis-related structures observed after treatment with imidazolidin (blue arrows for cell membrane pore formation; red arrows for mitochondrial swelling). Figure 7 F represents a staining pattern used to assess the membrane integrity of primary microglia through immunofluorescence staining. Figure 8 A and B are statistical graphs showing the results of detecting the expression of inflammatory cytokines after microglia pyroptosis using Western blotting, provided by this invention. Figure 8 The CF graph represents the statistical results of IL-18 and IL-1β concentrations detected by ELISA. Figure 8 G and H are statistical graphs showing the number of M1-positive microglia and M2-positive microglia detected by immunofluorescence. Figure 9 Figures A and B are statistical graphs showing the results of detecting the expression of blood-brain barrier tight junction proteins ZO-1 and Occludin by Western blotting in this invention. Figure 9 C represents the results of brain water content measurement; Figure 9 The DF is a statistical graph of neurological function assessments (modified Garcia score, balance beam walking test, water maze test); Figure 9 G and H are statistical graphs of neuronal damage assessment results using TUNEL and Nissl staining. Figure 10 A, E, and I are statistical graphs showing the results of detecting the expression of key pathway proteins (TLR4, MyD88, p-NF-κB) in SAH mice by Western blotting in this invention. Figure 10B, F, and J are statistical graphs showing the results of immunofluorescence co-localization analysis of the activation of TLR4, MyD88, and p-NF-κB in microglia and the protein results of each pathway. Figure 11 The C, G, and K values ​​provide a statistical chart of experimental results for OxyHB stimulation of microglia in this invention. Figure 11 The D, H, and L values ​​are used to present a statistical chart of the in vitro experimental results of the effect of imidazolidin on microglia provided by this invention. Figure 12 This invention provides a schematic diagram of the mechanism by which HSP60 mediates microglia pyroptosis after SAH. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Within minutes of subarachnoid hemorrhage (SAH), hemoglobin degradation products (especially heme) in the subarachnoid blood can activate microglia via pattern recognition receptors (such as Toll-like receptor 4, TLR4), polarizing them into a pro-inflammatory phenotype (M1 type). Activated M1 microglia release large amounts of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-18 (IL-18), triggering a severe inflammatory cascade. This excessive inflammatory response not only directly damages neurons but also disrupts the integrity of the blood-brain barrier, promoting vasogenic cerebral edema and thus exacerbating end-stage renal disease (EBI).

[0021] Pyroptosis is a novel programmed pro-inflammatory cell death mechanism discovered in recent years, mediated by inflammasome activation and Gasdermin family proteins (such as GSDMD). Unlike apoptosis, pyroptosis is characterized by the formation and rupture of pores in the cell membrane, leading to the massive release of inflammatory contents and triggering a strong local and systemic immune response. In pathological conditions of the central nervous system, activated microglia are the primary cell type most susceptible to pyroptosis.

[0022] Specifically, after Toll-like receptors (such as TLR4) on the surface of microglia recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), the myeloid differentiation factor 88 (MyD88)-dependent pathway activates nuclear factor κB (NF-κB) signaling, initiating the transcription of pro-inflammatory genes (including NLRP3, pro-IL-1β, and pro-IL-18). Subsequently, NOD-like receptor heat protein domain-associated protein 3 (NLRP3), apoptosis-associated speckle-like protein (ASC), and pro-caspase-1 assemble to form the NLRP3 inflammasome complex. This complex catalyzes the autocleavage of pro-caspase-1, generating enzymatically active caspase-1. Activated caspase-1 plays two key roles: first, it cleaves and activates precursor cytokines, converting pro-IL-1β and pro-IL-18 into mature, biologically active forms, promoting their massive secretion; second, it specifically cleaves GSDMD, generating its N-terminal domain (GSDMD-N). GSDMD-N translocates to the cell membrane and oligomerizes to form transmembrane pores, leading to increased cell membrane permeability. This triggers ion influx (such as Na⁺, Cl⁻, and H₂O), disrupts osmotic balance, and ultimately causes cell swelling and lytic necrosis. This process is accompanied by the massive release of intracellular inflammatory cytokines (such as IL-1β and IL-18), further amplifying local and systemic inflammatory responses.

[0023] In the pathological cascade of subacute acute hemorrhage (SAH), microglial phenotypic transformation is a core driver of neuroinflammation. Mechanistic studies have shown that Toll-like receptor 4 (TLR4) on the surface of microglia plays a crucial regulatory role in the initiation and maintenance of neuroinflammation by activating nuclear factor κB (NF-κB) signaling. The endogenous danger signal (DAMP) released from damaged tissue—heat shock protein 60 (HSP60)—specifically binds to TLR4 receptors on microglia, thereby triggering an NF-κB-dependent inflammatory cascade. HSP60 is a highly conserved molecular chaperone that not only plays a fundamental role in maintaining proper protein folding but also exerts pleiotropic effects in stress responses, inflammation activation, cell death regulation, and disease progression. Under physiological conditions, HSP60 is constitutively expressed, while its expression is significantly upregulated in pathological microenvironments (such as precancerous lesions and neurodegenerative diseases).

[0024] However, the specific mechanism by which HSP60 mediates microglial activation in SAH remains unclear and requires further investigation.

[0025] This invention provides the application of a heat shock protein in the preparation of a drug for treating subarachnoid hemorrhage, wherein the heat shock protein is HSP60.

[0026] This invention proposes that HSP60 expression is upregulated after subarachnoid hemorrhage in mice, which promotes NLRP3 inflammasome assembly by activating the TLR4 / MyD88 / NF-κB signaling pathway, thereby inducing microglia pyroptosis and aggravating early brain injury.

[0027] Specifically, after subarachnoid hemorrhage, the expression of heat shock protein 60 (HSP60) increases, which upregulates the expression of NLRP3 inflammasome by activating the TLR4 / MyD88 / NF-κB signaling axis and catalyzes the cleavage of pro-caspase-1 into active caspase-1.

[0028] Activated caspase-1 promotes the release of mature IL-1β and IL-18 by cleaving pro-IL-1β and pro-IL-1β, while simultaneously inducing oligomerization of the N-terminal domain (GSDMD-N) of GasderminD (GSDMD), ultimately driving microglia pyroptosis. This process significantly exacerbates the neuroinflammatory response following subarachnoid hemorrhage (SAH) and worsens brain damage.

[0029] The proposed HSP60-TLR4 / MyD88 / NF-κB could provide a new molecular target for neuroprotective strategies against subarachnoid hemorrhage.

[0030] Targeted inhibition of HSP60 reduced pyroptosis-related cell membrane pores, significantly reduced microglial pyroptosis, significantly alleviated the degradation of tight junction proteins (ZO-1, occludin), improved blood-brain barrier integrity, reduced cerebral edema, decreased neuronal apoptosis, and ultimately improved neurological function.

[0031] Crucially, this inhibitor is mizoribine, which can effectively inhibit HSP60, thereby blocking the cascade reaction of the TLR4 / MyD88 / NF-κB signaling pathway, significantly reducing neurological deficits, cerebral edema, blood-brain barrier disruption, and neuronal damage after subarachnoid hemorrhage.

[0032] The administration methods of mizoribine include internal injection. Specifically, mizoribine is prepared into a drug solution with physiological saline and injected directly into the skull of mice to achieve rapid drug delivery.

[0033] The concentration of imidazoribine in the drug solution can be selected from several concentrations, with a preferred concentration of 100 mg / kg. In in vitro studies, the concentration of imidazoribine can be selected from 20 μmol / L, 40 μmol / L, and 80 μmol / L.

[0034] The following examples further verify the pathogenesis of HSP60 in subarachnoid hemorrhage and its specific applications.

[0035] 1. Preparation of experimental animals and SAH model Male C57BL / 6J mice (weighing 25±2g, aged 6-7 weeks) were provided by the Laboratory Animal Center of Yijishan Hospital, Wannan Medical College. All experimental procedures were approved by the Animal Ethics Committee of Yijishan Hospital, Wannan Medical College (Approval No.: WNMC-AWE-2024224) and strictly followed the "Guidelines for the Householding and Use of Laboratory Animals" issued by the National Institutes of Health (NIH). Mice were housed in a barrier environment with a temperature controlled at 25±1℃, a relative humidity of 55±5%, and a light / dark cycle of 12 hours of light / 12 hours of darkness, with free access to food and water.

[0036] 2. Method for establishing the SAH model - Prechiasmatic cistern injection method Mice were anesthetized by intraperitoneal injection of pentobarbital (40 mg / kg), and the depth of anesthesia was confirmed by the loss of corneal reflex. Mice were fixed prone on a stereotaxic apparatus, and a 1 cm incision was made along the sagittal line of the scalp. The subcutaneous fascia was bluntly dissected. A 1 mm diameter cranial foramen was drilled at the midline, 4.5 mm posterior to the anterior fontanelle, to expose the dura mater. 50 μL of arterial blood was collected from the anesthetized donor mouse via cardiac puncture. Using a micro-injection needle (Hamilton, #81000, USA), the needle was inserted vertically through the cranial foramen into the prechiasmatic cistern to a depth of 3.5 mm, and 50 μL of arterial blood was slowly injected at a rate of 0.5 μL / min. After injection, the needle was retained for 2 minutes before being slowly withdrawn to prevent blood reflux. The cranial foramen was sealed with bone wax, the scalp was sutured in layers, and disinfected with iodine. Mice in the sham-operated group underwent all the same procedures except for the lack of blood injection. Postoperatively, mice were placed on a 37°C heated blanket for 45 minutes for recovery before being returned to standard cages. Neurological deficits were assessed using a modified Garcia score, and mice with a score <6 were excluded.

[0037] 3. Cell culture and in vitro model construction methods Primary microglia were isolated from the brain tissue of newborn mice within 24 hours of birth: the meninges were carefully removed under a microscope, preserving the cortical tissue; the brain tissue was minced and placed in an ice-cold culture dish, further minced, and transferred to trypsin digestion solution (Solarbio, #T1300, China), and digested in a 37°C incubator for 5 minutes. Digestion was terminated by adding high-glucose DMEM medium (Gibco, #6125123, USA) containing 10% fetal bovine serum (Opcel, #BS-1102, China); the digested tissue was transferred to a new culture dish and thoroughly ground, filtered through a 40 μm cell sieve, centrifuged at 1000×g for 5 minutes, resuspended, and seeded into culture flasks for primary culture. Cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (Solarbio, #P1400, China), with a full medium change on day 3 and a half medium change on day 7. After 10 days of adherent culture, suspended microglia were collected using a constant-temperature shaker (150 rpm, 37°C, 1 hour), centrifuged, and then seeded into experimental culture plates. In this study, primary microglia were stimulated with 25 μmol / L oxyhemoglobin (OxyHb; Solarbio, #H8020, China) to construct an in vitro SAH model.

[0038] 4. Drug treatment methods Because HSP60 plays an important physiological role in maintaining cellular homeostasis, complete gene knockout may lead to severe developmental defects. Therefore, this study used the small molecule immunomodulator imidazoribine to selectively inhibit HSP60 activity.

[0039] Mizoribine (MCE, #HY-17470, China) was prepared into different concentration gradients with physiological saline. Mice were anesthetized by intraperitoneal injection of pentobarbital (40 mg / kg) and fixed on a stereotaxic apparatus. A hole was drilled in the skull (coordinates: 1.5 mm lateral and 1.0 mm posterior to the anterior fontanelle, depth 3.5 mm), and a 10 μL Hamilton syringe (Hamilton, #80900, USA) was inserted into the lateral ventricle. The drug was administered at a constant rate of 0.5 μL / min using an infusion pump. After injection, the needle was retained for 10 minutes to prevent backflow of the drug, and then slowly withdrawn over 5 minutes. The skull hole was then completely sealed with bone wax.

[0040] Validate the design: All animals were randomly assigned to four independent experimental protocols, with randomization performed upon inclusion in each experiment. In immunofluorescence staining and imaging experiments, the selection of slides for microscopic evaluation was kept blinded.

[0041] 1. Temporal expression analysis of HSP60 and pyroptosis-related proteins To assess the temporal changes in the expression of HSP60, GSDMD / GSDMD-N, NLRP3, and pro-Caspase-1 / Caspase-1 after subarachnoid hemorrhage (SAH), animals were randomly divided into six groups (sham-operated group, 6-hour post-SAH group, 12-hour post-SAH group, 24-hour post-SAH group, 48-hour post-SAH group, and 72-hour post-SAH group), with 10 mice in each group. After establishing the SAH model, mice were sacrificed at specified time points (6, 12, 24, 48, and 72 hours post-SAH); six mice from each group were selected for Western blot analysis, and the remaining mice were used for immunofluorescence staining. In in vitro experiments, microglia were stimulated with OxyHb, and the grouping strategy was consistent with that in animal experiments.

[0042] 2. Screening for optimal dose of mizoribine and assessment of neurological function To determine the optimal dose of imidazoribine, mice were randomly divided into 7 groups (sham-operated group, SAH group, SAH + solvent group, SAH + imidazoribine 50 mg / kg group, SAH + imidazoribine 100 mg / kg group, SAH + imidazoribine 150 mg / kg group, and SAH + imidazoribine 200 mg / kg group), with 10 mice in each group. Six hours after SAH, imidazoribine was administered via intraventricular injection, and mice were sacrificed at predetermined time points. Six mice from each group were selected for Western blotting and immunohistochemical analysis to evaluate the HSP60 inhibitory effect and determine the optimal dose.

[0043] In in vitro experiments, the CCK-8 assay (cell counting kit-8) was first used to determine the concentration range of imidazolidin that could improve cell viability. Then, three concentrations of 20 μmol / L, 40 μmol / L, and 80 μmol / L were used to screen the optimal dose for inhibiting HSP60 release from microglia.

[0044] To assess the effects of HSP60 on neurological function in SAH mice, animals were randomly divided into four groups (sham-operated group, SAH group, SAH + solvent group, and SAH + imidazoribine group), with 50 mice in each group. Imidazolidin was administered via intraventricular injection 6 hours after SAH. Neurological function scores, balance beam walking test, Morris water maze test, and brain water content were measured 24 hours after SAH. Six mice from each group were randomly selected for Western blot analysis of tight junction proteins (ZO-1, Occludin).

[0045] 3. The role of HSP60 in microglial pyroptosis and neuronal injury To investigate the crucial role of HSP60 in microglial pyroptosis after subarachnoid hemorrhage (SAH) and the impact of pyroptosis on neurons, we detected related biomarkers such as ASC, NLRP3, GSDMD / GSDMD-N, pro-Caspase-1 / Caspase-1, pro-IL-1β / IL-1β, and CD86 / CD206. Mice were randomly divided into four groups (sham-operated group, SAH group, SAH + solvent group, and SAH + imidazoribine group), with 50 mice in each group. Western blotting, immunofluorescence, ELISA, and transmission electron microscopy were used to detect related biomarkers. TUNEL staining and Nissl staining were used to assess the correlation between microglial pyroptosis and neuronal damage. For each detection method, six mice were randomly selected from each group. In in vitro experiments, microglia were divided into three groups (control group, OxyHb group, and OxyHb + imidazoribine group), and pyroptosis-related biomarkers were also assessed.

[0046] 4. HSP60-mediated signaling pathway mechanism of microglia pyroptosis To investigate the potential mechanism by which HSP60 mediates microglia pyroptosis after subarachnoid hemorrhage (SAH), mice were randomly divided into four groups (sham-operated group, SAH group, SAH + solvent group, and SAH + imidazoribine group), with 20 mice in each group. Western blotting and immunofluorescence were used to detect related signaling markers such as TLR4, MyD88, P65, and p-P65. For each detection method, 6 mice were randomly selected from each group. The in vitro experimental grouping was consistent with Experimental Design 3, and Western blotting and immunofluorescence were also used to detect molecular signaling markers.

[0047] 5. Brain water content measurement The degree of cerebral edema was assessed using the wet-dry weight method: Mice in each group were sacrificed immediately after SAH, and the whole brain was quickly harvested and weighed immediately (wet weight); the brain tissue was placed in a 60℃ constant temperature oven and baked for 24 hours until completely dry, then cooled to room temperature and weighed (dry weight). The formula for calculating brain water content is: [(wet weight - dry weight) / wet weight] × 100%.

[0048] Behavioral assessment 1. Modified Garcia Neurological Function Score Short-term neurological deficits were assessed using a modified Garcia scoring system, which covers six behavioral dimensions: voluntary activity, axial sensation, forelimb symmetry and extension, climbing ability, trunk tactile response, and whisker-evoked turning response. Each dimension was scored from 0 to 3 (3: normal function; 0: severe deficit), with a total score ranging from 3 to 18. Lower scores indicate more severe neurological impairment.

[0049] 2. Balance beam walking test Motor coordination and balance were assessed using a standardized balance beam apparatus (10 mm in diameter and 1 m in length). Each mouse underwent three independent tests, and the average score was recorded. The scoring criteria were strictly defined as follows: -0 points: Unable to hold onto the balance beam or falls immediately; -1 point: Able to grab the balance beam but not move (staying for ≥60 seconds); -2 points: Movement distance <10cm, falling within 60 seconds; -3 points: Able to pass the balance beam, but slipped or fell during the process; -4 points: Successfully passed the balance beam 31 without slipping or falling.

[0050] 3. Morris water maze experiment The Morris water maze was used to assess spatial learning and memory functions. One week after SAH surgery, mice were tested for five consecutive days: For the first four days, mice were placed in water from different starting points and asked to find a platform hidden underwater, with each test lasting a maximum of 60 seconds; if a mouse did not find the platform within 60 seconds, it was gently guided to the platform and allowed to stay there for 5 seconds. On the fifth day, an exploration test was conducted: the platform was removed, and the mice were allowed to explore freely in the target quadrant. The mice's movement trajectories were recorded using a video tracking system (EthoVisionXT, Noldus, Netherlands), and the escape latency and swimming path were quantitatively analyzed.

[0051] Other methods: 1. Immunofluorescence staining (IF) After establishing the model, mice were injected with ice-bathed PBS buffer (KGL, #KGL2206-500, China) via cardiac perfusion, followed by injection of 4% paraformaldehyde fixative (Biomiky, #MK014A, China). The whole brain was isolated and fixed in 4% paraformaldehyde at 4°C for 24 hours. The brain tissue was then dehydrated in 15% and 30% sucrose solutions until it settled. After removing surface moisture, 12 μm thick coronal frozen sections were prepared using a cryostat. The sections were air-dried at 37°C for 30 minutes, and the tissue areas were circled with an anti-circling pen. The sections were washed three times with PBS (5 minutes each time), permeated with 0.3% Triton X-100 (Beyotime, #P0096, China) at room temperature for 30 minutes, and then blocked with immunofluorescence blocking solution (Beyotime, #P0260, China) for 1 hour. Add primary antibody and incubate overnight at 4°C in a humidified chamber; wash 3 times with PBS (5 minutes each time), add species-matched fluorescent secondary antibody at room temperature and incubate for 2 hours, wash again with PBS, and mount with anti-fluorescence quenching mounting medium containing DAPI (Beyotime, #P0131, China).

[0052] In in vitro experiments, microglia grown on polylysine-coated coverslips or glass-bottomed culture dishes were fixed with 4% paraformaldehyde, permeabilized, blocked, and incubated with primary antibody overnight at 4°C, followed by incubation with fluorescent secondary antibody at room temperature for 2 hours. After washing with PBS, the slides were mounted with DAPI and fluorescent imaging was performed using a Pannoramic MIDI fluorescence microscope.

[0053] 2. Nissl staining method The same sectioning procedure as immunofluorescence staining was used, and Nissl staining was performed strictly according to the instructions of the toluidine blue staining kit (Solarbio, #G1430, China): the sections were immersed in toluidine blue solution for 15 minutes, rinsed with distilled water to remove excess dye; differentiated in Nissl differentiation solution for 2 minutes, rapidly dehydrated with anhydrous ethanol, cleared with xylene, and mounted with neutral resin; and imaged using a PannoracicMIDI slide scanner.

[0054] 3. Immunohistochemistry (IHC) Tissue samples were fixed with 4% paraformaldehyde and endogenous peroxidase activity was blocked with 3% hydrogen peroxide. The samples were then blocked with 5% bovine serum albumin (BSA) for 30 minutes at room temperature to block non-specific binding sites. Specific primary antibodies were incubated overnight at 4°C in a humidified chamber. The samples were washed three times with PBS (5 minutes each time) and incubated with biotin-labeled secondary antibody for 30 minutes at room temperature. After washing again with PBS, the samples were incubated with streptavidin-horseradish peroxidase complex for 20 minutes at room temperature. DAB chromogenic reagent was used for staining (1-3 minutes), and the staining process was observed and controlled under a microscope. Cell nuclei were counterstained with hematoxylin for 1 minute. The sections were dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. The stained sections were scanned using a PannoramicMIDI slide scanner.

[0055] 4. Cell viability analysis The dose-dependent effect of imidazolidin on microglia viability was quantitatively assessed using the CCK-8 assay (NCMBiotech, #C6005, China), and its optimal effective concentration was determined. Cells treated with different concentrations of imidazolidin were seeded into 96-well plates and cultured for 24 hours. CCK-8 solution was then added, and the cells were incubated for another 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader, and the percentage of cell viability was calculated using the untreated control group as a reference.

[0056] 5. TUNEL assay After processing the sections according to the immunofluorescence staining procedure (including fixation, permeabilization, blocking, and primary antibody incubation), neuronal apoptosis was detected using a one-step TUNEL apoptosis detection kit (Beyotime, #C1089, China) according to the instructions: NeuN immunolabeled frozen sections were incubated at room temperature in the dark with TUNEL reaction solution containing fluorescein-dUTP for 60 minutes; after thorough washing with PBS, the sections were mounted with antifluorescence quenching containing DAPI; and imaging was performed using a Pannoramic MIDI fluorescence microscope with excitation / emission wavelengths of approximately 550 / 570 nm (detecting TUNEL signals). Apoptotic events in NeuN-positive neurons were assessed through the DAPI channel.

[0057] 6. Western blotting Brain tissue and cell samples were lysed on ice for 30 minutes using RIPA lysis buffer (Beyotime, #P0013B, China) containing a mixture of protease and phosphatase inhibitors (Beyotime, #P1045, China); centrifuged at 14,000 rpm for 15 minutes, and the supernatant was collected as the total protein extract. Protein concentration was determined using a BCA protein quantification kit (Beyotime, #P0010, China), and samples were standardized to the same concentration. 20-50 μg of protein sample was mixed with loading buffer and boiled at 100°C for 10 minutes to denature the protein; proteins were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to a PVDF membrane (Merck Millipore, #IPVH00010, USA) using a wet transfer system under constant current. After transfer, the membrane was blocked for 2 hours at room temperature with TBST containing 5% skim milk (Tris buffered saline solution containing 0.1% Tween-20). The membrane was then washed three times with TBST (10 minutes each time) and incubated overnight at 4°C with an appropriately diluted primary antibody. The next day, the membrane was washed three times with TBST (10 minutes each time) and incubated for 2 hours at room temperature with a species-specific horseradish peroxidase (HRP)-labeled secondary antibody. Finally, the membrane was washed three times with TBST (10 minutes each time), and then incubated with an enhanced chemiluminescence (ECL) substrate (Merck Millipore, #WKLS0500, USA). The chemiluminescence signal was captured and analyzed using a Western blot imaging system. Detailed information on the primary and secondary antibodies used is shown in Table 1.

[0058] 7. Enzyme-linked immunosorbent assay (ELISA) Commercial mouse IL-1β ELISA kits (Yuanju Biotechnology, #YJ301814, China) and IL-18 ELISA kits (Ruier Gene, #RGM180-2, China) were used to detect the levels of IL-1β and IL-18 in mouse brain homogenate and microglial cell suspension after SAH. All experimental steps were strictly followed according to the kit instructions.

[0059] 8. Transmission electron microscopy technique The ultrastructural features of pyroptosis include cell swelling, chromatin margination and condensation, cell membrane bubbling, cytoplasmic vacuolation, and endoplasmic reticulum expansion, ultimately leading to cell membrane perforation and rupture. 32 To assess microglia pyroptosis, the aforementioned morphological changes were observed using transmission electron microscopy: Brain tissue samples were immediately immersed in 2.5% glutaraldehyde solution (Biosharp, #BL911A, China) for initial fixation (room temperature, 2-4 hours); washed three times with PBS (10 minutes each time), fixed with 1% osmium tetroxide (OsO4) solution at room temperature for 2 hours, and washed three more times with PBS (10 minutes each time); the tissue blocks were dehydrated by gradient ethanol (50%, 70%, 80%, 90%, 95%, 100%) and acetone, then infiltrated in a mixture of acetone and epoxy resin embedding agent (SPI, #90529-77-4, USA) for about 12 hours, followed by embedding and polymerization at 60°C for 48 hours; ultrathin sections of 70 nm thickness were prepared using an ultramicrotome, double-stained with uranium acetate and lead citrate, and the characteristic ultrastructure of microglia pyroptosis was observed under a transmission electron microscope (FEITecnaiG2, USA).

[0060] 9. Statistical Analysis Methods Data analysis and visualization were performed using GraphPad Prism 9 software (GraphPad Software, USA). All quantitative data are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and Tukey's post-hoc test was used to analyze differences between groups. A p-value < 0.05 was considered statistically significant.

[0061] The antibodies provided by this invention are shown in Table 1: Table 1 Antibody name Source company Dilution ratio Application method Product number HSP60 CST (USA) 1:1000 WB 12165 TLR4 abmat (China) 1:1000 WB P7215 MyD88 CST (USA) 1:1000 WB 4283T NF-κBP65 abmat (China) 1:1000 WB T50345 p-NF-κBP65 Boosen (China) 1:1000 WB / P 8235-RR NLRP3 abmat (China) 1:1000 WB P002R3 Caspase-1 abmat (China) 1:1000 WB / IF P784R2 ASC CST (USA) 1:1000 WB 6724 GSDMD abmat (China) 1:2000 WB / IF PU24937 IL-1β abmat (China) 1:1000 WB / IF P052R1 NeuN CST (USA) 1:200 IF 9403 ZO-1 Boosen (China) 1:1000 WB / IHC 2173AP Occludin abcam (USA) 1:1000 WB ab28984 CD86 abmat (China) 1:200 IF TD325 CD206 Boosen (China) 1:200 IF 815-R β-actin Boosen (China) 1:1000 WB 8115-R GAPDH Boosen (China) 1:1000 WB 816405R Fluorescent secondary antibody Invitrogen (USA) 1:400 IF A221207 / A22147 / A32766 Results Explanation 1. HSP60 expression is upregulated in ipsilateral cortex and OxyHB-stimulated microglia after SAH, and its expression dynamics are correlated with microglia pyroptosis. To investigate the pathological role of HSP60 in early brain injury (EBI) after SAH, the expression of HSP60 in ipsilateral cortical brain tissue of mice in each group was first detected.

[0062] The temporal expression results of HSP60 and NLRP3 in in vivo and in vitro SAH models are shown in [reference needed]. Figure 2 As shown, Western blot analysis revealed that HSP60 protein levels began to rise 6 hours after SAH, reached a significant peak at 24 hours, and then gradually decreased. Figure 2A). Immunofluorescence co-localization analysis showed that, compared with the sham-operated group, HSP60 expression in microglia of SAH mice was significantly increased ( Figure 2 B).

[0063] In in vitro experiments, primary microglia were stimulated with OxyHB to simulate SAH conditions. Western blot results showed that HSP60 expression was significantly upregulated 24 hours after OxyHB stimulation. Figure 2 C), an increase in HSP60 fluorescence intensity was also observed in immunofluorescence assay (C). Figure 2 E).

[0064] To elucidate the role of microglial pyroptosis in the epidermal endothelial brain (EBI) after subarachnoid hemorrhage (SAH), the expression of pyroptosis-related markers (NLRP3, Caspase-1, and GSDMD) in the ipsilateral cortex was detected. Western blot analysis showed that the expression of these proteins was significantly increased 24 hours after SAH. Figure 2 D、 Figure 3 A, Figure 3 B); In in vitro experiments, OxyHB stimulation for 24 hours also significantly increased the protein levels of NLRP3, Caspase-1, and GSDMD in primary microglia (B). Figure 2 F, Figure 3 C Figure 3 D).

[0065] The above results indicate that there is a clear temporal correlation between HSP60 upregulation after SAH and microglia pyroptosis, suggesting that HSP60 regulates the microglia pyroptosis process.

[0066] 2. Imidazolidin can effectively inhibit the upregulation of HSP60 in SAH-induced early brain injury. Imidazolidin is an immunosuppressant that potently inhibits HSP60 expression. To determine the optimal dose for HSP60 inhibition in a SAH-induced EBI model, concentration gradients were established in vivo for evaluation. Western blot results showed that 100 mg / kg imidazolidin had the most significant inhibitory effect on HSP60 expression. Figure 4 A).

[0067] In in vitro experiments, the concentration range of imidazolidin was first determined using the CCK-8 assay. The results showed that 20 μmol / L, 40 μmol / L, and 80 μmol / L imidazolidin could significantly alleviate the OxyHB-induced decrease in microglial cell viability. Figure 4 B); Subsequent Western blot analysis showed that 80 μmol / L imidazoribine had the best inhibitory effect on HSP60 expression (B). Figure 4 C).

[0068] 3. Inhibition of HSP60 can alleviate NLRP3 inflammasome-induced microglial pyroptosis after SAH. The inflammasome complex composed of NLRP3, ASC, and pro-caspase-1 is a key regulator of pyroptosis. Western blot analysis showed that, compared with the SAH+saline group, imidazolidin inhibition of HSP60 significantly reduced the protein expression of NLRP3, Caspase-1, and ASC in mice in the SAH+imidazolidin group. Figure 5 A, 5E, 5I); co-staining of microglia with these target proteins using immunofluorescence also confirmed that the number of positive cells was significantly reduced after treatment with imidazolidin. Figure 5 B, 5F, 5J).

[0069] In vitro experiments yielded consistent results: Western blotting showed that imidazolidin treatment effectively inhibited OxyHB-induced upregulation of NLRP3, Caspase-1, and ASC. Figure 6 C Figure 6 G, Figure 6 K), immunofluorescence assays of primary microglia also support this conclusion. Figure 6 D、 Figure 6 H, Figure 6 L).

[0070] To further investigate the relationship between HSP60 and microglia pyroptosis, the expression of GSDMD, a key molecule mediating cell membrane pore formation, and its active form GSDMD-N was detected. Western blotting showed that, compared with the SAH+ saline group, imidazolidin significantly reduced the expression of GSDMD and GSDMD-N. Figure 7 A); Immunofluorescence also showed a decrease in GSDMD positive signal in microglia in the imidazolidin group ( Figure 7 B).

[0071] In in vitro experiments, imidazolidin treatment also reduced the expression of GSDMD and GSDMD-N in the OxyHB group. Figure 7 C), the immunofluorescence results were consistent with those ( Figure 7 D). Transmission electron microscopy revealed that the brain tissue of mice in the SAH group and the SAH+saline group showed signs of cell membrane integrity disruption and mitochondrial swelling, while treatment with imidazolidin could improve these changes. Figure 7 E); In primary microglia, imidazolidin also reduced OxyHB-induced cell membrane rupture ( Figure 7 F).

[0072] The above results indicate that imidazolidin can inhibit HSP60 and effectively reduce microglia pyroptosis.

[0073] 4. Inhibition of HSP60 can reduce neuroinflammation after SAH and decrease the activation of pro-inflammatory microglia. Pyroptosis leads to the release of inflammatory factors. Western blot analysis showed that IL-1β levels were significantly increased after SAH, while treatment with imidazolidin reduced its expression. Figure 8 A); In in vitro experiments, imidazolidin also inhibited OxyHB-induced upregulation of IL-1β ( Figure 8 B). ELISA testing confirms that, regardless of whether it is in vivo ( Figure 8 C, 7D) or in vitro ( Figure 8 E, 7F), and imidazoribine can reduce the levels of IL-1β and IL-18.

[0074] Microglia can polarize into pro-inflammatory M1 type and anti-inflammatory M2 type. 33 Immunofluorescence co-staining showed an increase in the number of CD86-positive (M1 type) microglia after SAH, and treatment with imidazolidin could reverse this trend. Figure 8 G); Conversely, treatment with imidazolidin increased the number of CD206-positive (M2 type) microglia (G). Figure 8 H).

[0075] The above results indicate that inhibiting HSP60 can reduce neuroinflammation, decrease M1 microglia activation, and promote M2 polarization.

[0076] 5. Inhibition of HSP60 can improve neuronal damage and neurobehavioral prognosis after SAH, and reduce blood-brain barrier disruption. Western blot analysis showed that the expression of tight junction proteins (occludin, ZO-1) decreased after SAH, and treatment with imidazolidin could reverse this downregulation trend. Figure 9 A, 8B). Brain water content measurements showed that brain water content was significantly increased in the SAH group, and treatment with imidazolidin reduced cerebral edema ( ). Figure 9 C).

[0077] Neurobehavioral assessments (modified Garcia score, balance beam walking test, Morris water maze) showed that mice in the SAH + saline group had significant functional deficits compared to the sham-operated group; while imidazoribine treatment improved the Garcia score ( Figure 9 D) Improve balance beam walking performance ( Figure 9 E), and improves spatial learning and memory abilities in water maze tests by shortening the escape latency and swimming path length. Figure 9 F).

[0078] TUNEL and Nissl staining showed that SAH+saline mice had widespread neuronal apoptosis and morphological damage, while treatment with imidazolidin could alleviate these damages. Figure 9 G).

[0079] The above results indicate that HSP60 is involved in the neuronal damage process after SAH, and that imidazoribine exerts its neuroprotective effect by inhibiting HSP60.

[0080] 6. HSP60 mediates microglia pyroptosis via the TLR4 / MyD88 / NF-κB signaling axis. Western blot analysis showed that the expression of TLR4, MyD88, and phosphorylated NF-κB (p-NF-κB) increased after SAH, while the expression of these proteins decreased significantly after imidazolidin inhibition of HSP60. Figure 10 A, Figure 10 E, Figure 10 I); Immunofluorescence co-localization confirmed that the expression of these signaling molecules was enhanced in microglia after SAH, and that imidazolidin treatment could attenuate their expression ( Figure 10 B. Figure 10 F, Figure 10 J).

[0081] In in vitro experiments, OxyHB stimulation also upregulated the expression of TLR4, MyD88, and p-NF-κB, and imidazolidin could inhibit this effect. Figure 11 C Figure 11 G, Figure 11 K), and immunofluorescence results also support this conclusion. Figure 11 D、 Figure 11 H, Figure 11 L).

[0082] 7. Mechanism of HSP60-mediated microglia pyroptosis A schematic diagram illustrating the mechanism of HSP60-mediated microglia pyroptosis after SAH is shown below. Figure 12 As shown, HSP60 expression was significantly upregulated in the early brain injury stage after subarachnoid hemorrhage (SAH). HSP60 activated the TLR4 / MyD88 / NF-κB signaling pathway in microglia, inducing upregulation of NLRP3 expression and accumulation of inflammatory cytokine precursors such as pro-IL-1β and pro-IL-18. NLRP3 further promoted the assembly of NLRP3 inflammasomes, generating enzymatically active CleavedCaspase-1 by cleaving pro-Caspase-1. Activated Caspase-1 cleaved cytokine precursors into mature active forms (IL-1β and IL-18), and simultaneously cleaved Gasdermin D (GSDMD) to generate an N-terminal fragment (GSDMD-N). GSDMD-N formed pores on the cell membrane, mediating the massive release of mature inflammatory cytokines, ultimately triggering and exacerbating microglial pyroptosis, significantly aggravating early brain injury after SAH.

[0083] In summary, these results indicate that HSP60 mediates microglial pyroptosis after SAH by activating the TLR4 / MyD88 / NF-κB pathway. In vivo administration of imidazolidin significantly alleviated the downregulation of blood-brain barrier tight junction proteins ZO-1 and Occludin, effectively reducing cerebral edema, improving neurological deficits, decreasing neuronal apoptosis, and improving Nissl body integrity in mice after SAH, thus exhibiting neuroprotective effects.

[0084] Results analysis: 1. Heat shock protein 60 (HSP60) is a key molecular chaperone encoded by the nuclear gene HSPD1 on chromosome 2. It is mainly located in the mitochondrial matrix and works synergistically with the co-chaperone protein HSP10 to promote the correct folding of nascent proteins, repair misfolded proteins, and maintain mitochondrial protein homeostasis. HSP60 expression can be significantly upregulated under chemical, biological, or environmental stress conditions.

[0085] This invention elucidates the role of HSP60 in early brain injury (EBI) following subarachnoid hemorrhage (SAH). Results showed that HSP60 expression peaked 24 hours after SAH. Following SAH, HSP60 binds to Toll-like receptor 4 (TLR4) on the surface of microglia, activating the downstream NF-κB signaling pathway, significantly upregulating NLRP3 inflammasome expression, and subsequently inducing caspase-1-dependent pyroptosis. This process promotes the massive release of pro-inflammatory cytokines such as IL-1β and IL-18, ultimately exacerbating EBI.

[0086] Pyroptosis is a programmed cell death process mediated by Gasdermin proteins, characterized by cell membrane perforation and the release of inflammatory factors. The NLRP3 inflammasome, as a key molecular platform, activates caspase-1, which in turn cleaves GasderminD (GSDMD) to generate a pore-forming domain (GSDMD-N) and promotes the maturation of IL-1β and IL-18. HSP60 upregulates pro-IL-1β and NLRP3 expression through the TLR4 / MyD88 / NF-κB signaling pathway, promoting NLRP3 inflammasome assembly, activating caspase-1, and inducing microglia pyroptosis.

[0087] The proposed HSP60-TLR4 / MyD88 / NF-κB could provide a new molecular target for neuroprotective strategies against subarachnoid hemorrhage.

[0088] 2. After HSP60 inhibition, immunofluorescence co-labeling showed a decrease in the number of NLRP3, caspase-1, GSDMD, and IL-1β-positive microglia. Transmission electron microscopy ultrastructural analysis further confirmed the reduction of pyroptosis-related cell membrane pores, significantly reduced the degradation of tight junction proteins (ZO-1, occludin), improved blood-brain barrier integrity, reduced cerebral edema, decreased neuronal apoptosis, and ultimately improved neurological function. This provides a theoretical basis for a therapeutic strategy targeting the HSP60 / TLR4 / NLRP3 signaling axis.

[0089] 3. In vitro and in vivo experiments showed that imidazolidin can directly target HSP60 and inhibit its expression, and indirectly inhibit the activation of NLRP3 inflammasome, the NF-κB signaling pathway and the expression of downstream pro-inflammatory cytokines (IL-18, IL-1β). The expression of TLR4 and its downstream signaling molecules (MyD88, phosphorylated p65, NLRP3, pro-IL-1β) decreased in a dose-dependent manner, effectively alleviating microglia pyroptosis.

[0090] 4. This invention uses imidazoribine to specifically inhibit HSP60, which can significantly reduce neurological deficits, cerebral edema, blood-brain barrier disruption and neuronal damage after SAH in mice. The optimal dose of imidazoribine in in vivo experiments was determined to be 100 mg / kg and 80 μmol / L in in vitro experiments through concentration gradient experiments.

[0091] This study reveals for the first time the key role of HSP60 in inducing microglial pyroptosis through the TLR4 / MyD88 / NF-κB–NLRP3 signaling axis in SAH-induced early brain injury, and confirms that imidazoribine can effectively reduce neuroinflammation and brain injury by inhibiting HSP60.

[0092] The above embodiments are merely exemplary embodiments of this application and are not intended to limit this application. The scope of protection of this application is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to this application within its substance and scope of protection, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of this application.

Claims

1. The application of a heat shock protein in the preparation of drugs for treating subarachnoid hemorrhage, characterized in that, The heat shock protein is HSP60; In particular, HSP60 expression was upregulated after subarachnoid hemorrhage in mice, which promoted the assembly of NLRP3 inflammasomes by activating the TLR4 / MyD88 / NF-κB signaling pathway in cells, thereby inducing microglia pyroptosis and aggravating early brain injury.

2. The application of the heat shock protein according to claim 1 in the preparation of a drug for treating subarachnoid hemorrhage, characterized in that, This includes products for treating heat shock proteins, which reduce neurological deficits, cerebral edema, blood-brain barrier disruption, and neuronal damage in mice following subarachnoid hemorrhage by inhibiting HSP60 expression.

3. The application of the heat shock protein according to claim 1 in the preparation of a drug for treating subarachnoid hemorrhage, characterized in that, The product contains at least imidazoribine.

4. A product for treating / inhibiting subarachnoid hemorrhage as described in any one of claims 1-3, characterized in that, The product is a mixed solution composed of imidazoribine and a solvent.

5. The product according to claim 4, characterized in that, The solvent is physiological saline.

6. The product according to claim 4, characterized in that, The concentration of imidazoribine in the mixed solution is 20-100 mg / kg.

7. The product according to claim 6, characterized in that, The concentration of imidazoribine in the mixture is 100 mg / kg.

8. The product according to claim 4, characterized in that, The mixed solution is administered via intracranial injection.

9. The use of the product as described in any one of claims 4-8 in alleviating neurological deficits, cerebral edema, blood-brain barrier disruption, and / or neuronal damage following subarachnoid hemorrhage in mice.

10. The application according to claim 9, characterized in that, Includes the following steps: The mixed solution was administered via lateral ventricle injection six hours after subarachnoid hemorrhage in mice; The injection rate was 0.5 μL / min.

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