Application of LIAS gene in preparation of subarachnoid hemorrhage diagnosis and treatment product

Abstract

The embodiment of the invention discloses application of an LIAS gene in preparation of a product for diagnosing and treating subarachnoid hemorrhage. The copper death related gene after subarachnoid hemorrhage is screened out through information analysis, the copper death related protein LIAS in cerebrospinal fluid of a subarachnoid hemorrhage patient is detected, and it is determined that the copper death related protein LIAS is related to the disease severity of the patient; an SAH mouse model is constructed, the effects of promoting cell copper death and enhancing oxidative stress of LIAS in SAH are researched, and the process can be inhibited by applying RVG / RBCEVs to load siRNA (RVG / RBCEVs / siRNA) of LIAS. The effectiveness of RVG / RBCEVs / siRNA on SAH post-brain injury control is good for promoting neurological function recovery after brain injury and improving SAH prognosis.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biotechnology, and specifically to an application of a LIAS gene in preparing a subarachnoid hemorrhage diagnosis and treatment product. Background Art

[0002] Subarachnoid hemorrhage (SAH) is a very harmful hemorrhagic cerebrovascular disease, mainly caused by rupture of intracranial aneurysms. It has a high mortality rate among various cerebrovascular diseases, accounting for about 5% of all stroke cases and ranking second among diseases that cause death in stroke patients. About 30% of survivors will be left with permanent disabilities, which seriously affects their quality of life. From the perspective of pathological mechanism, brain damage after SAH mainly includes early brain injury (EBI) and cerebral vasospasm, among which EBI is the key pathogenic factor, involving multiple physiological dysfunctions such as inflammatory damage, increased brain edema, and neuronal apoptosis.

[0003] At the same time, the role of copper in neurological diseases has attracted much attention. A large number of studies have shown that copper deficiency is directly related to a variety of neuronal diseases such as stroke, Alzheimer's disease, Menkes disease, and traumatic brain injury. The brain is rich in copper because of its extremely high metabolic and signaling needs, and copper has multiple functions in the central nervous system (CNS). However, copper overload can trigger excessive production of reactive oxygen species (ROS), causing oxidative damage, weakening the brain's ability to scavenge hydroxyl free radicals, and leading to copper death mediated by mitochondrial ROS.

[0004] Although copper is closely associated with a variety of neurological diseases, there is still a lack of research on its correlation with SAH, and there are no relevant reports. Therefore, finding markers related to SAH treatment is of great significance for a deeper understanding of the pathogenesis of SAH and the development of effective treatments. Summary of the invention

[0005] To this end, an embodiment of the present invention provides an application of a LIAS gene in preparing a product for diagnosing and treating subarachnoid hemorrhage.

[0006] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] According to a first aspect of an embodiment of the present invention, the present invention provides use of the LIAS gene in preparing a subarachnoid hemorrhage diagnosis product.

[0008] LIAS is a key protein in the lipoic acid (LA) biosynthesis pathway that coordinates two [4Fe-4S] iron-sulfur proteins, which is essential for its activity. [Fe-S] clusters are a ubiquitous group of coenzymes found in proteins in most mammalian organelles such as mitochondria, cytoplasm, and nucleus. Their functions are diverse, ranging from electron transfer to sulfur donors or gene expression regulation. [Fe-S] clusters are composed only of iron and inorganic sulfur and have different nuclei, mainly including [2Fe-2S] and [4Fe-4S] clusters. [Fe-S] clusters are required for enzymes to remain active, such as LIAS. LIAS, like mitochondrial aconitase, carries a [4Fe-4S] cluster and participates in the tricarboxylic acid cycle.

[0009] Furthermore, the diagnostic product includes a reagent for detecting the expression level of the LIAS gene.

[0010] According to a second aspect of the embodiments of the present invention, the present invention provides use of the LIAS gene in preparing a product for treating subarachnoid hemorrhage.

[0011] Furthermore, the therapeutic goal is achieved by inhibiting neuronal copper death associated with subarachnoid hemorrhage and reducing oxidative stress.

[0012] According to a third aspect of the embodiments of the present invention, the present invention provides a drug for treating subarachnoid hemorrhage, wherein the drug comprises a LIAS gene expression inhibitor.

[0013] Furthermore, the LIAS gene expression inhibitor is siRNA, and the siRNA sequence is:

[0014] Sense strand: 5′-CCGGAUAUUUGGAAGAUAUTT-3′;

[0015] Antisense strand: 5′-AUAUCUUCCAAAUAUCCGGTT-3′.

[0016] Subarachnoid hemorrhage (SAH) is an important cause of death and disability in early brain injury (EBI), which is characterized by neuronal death. In this study, first, through bioinformatics analysis, it was found that three CRGs were expressed at elevated levels in SAH mice compared with the Sham group. Then, the feasibility of measuring the differential expression levels of the three CRGs in the SAH mouse model and measuring the LIAS protein level in the cerebrospinal fluid (CSF) of SAH patients was determined. Immunofluorescence staining revealed that LIAS protein was localized in the neurons of SAH mice. Then, a brain-targeted siRNA delivery system RVG-RBCEVs was developed, which successfully delivered therapeutic nucleic acids (LIAS siRNA) to neurons. Finally, the study found that after SAH occurred, LIAS siRNA achieved the therapeutic purpose by inhibiting neuronal copper death and attenuating oxidative stress.

[0017] The embodiments of the present invention have the following advantages:

[0018] The present invention screens out copper death-related genes after subarachnoid hemorrhage through bioinformatics analysis, detects copper death-related protein LIAS in the cerebrospinal fluid of patients with subarachnoid hemorrhage, and determines that it is related to the severity of the patient's condition; constructs a SAH mouse model, studies the role of LIAS in promoting cell copper death and enhancing oxidative stress in SAH, and uses RVG / RBCEVs to load LIAS siRNA (RVG / RBCEVs / siRNA) to inhibit this process. RVG / RBCEVs / siRNA is effective in controlling brain damage after SAH, promoting neurological function recovery after brain damage, and improving SAH prognosis. The present invention provides a theoretical basis and experimental basis for determining new targets for the clinical treatment of SAH, and provides new ideas and methods for effective intervention of brain damage after SAH. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0020] Figure 1Identification of CRGs in SAH. A. Heat map showing copper death-related genes between SAH and control tissues in mice. B. Volcano plot showing copper death-related genes in vivo, where the x-axis represents log2 (difference fold) (logFC) and the y-axis represents -log10 (corrected p value). C. Relative concentration of copper in the brain of SAH model and control group. D. Expression of LIAS, PLAT and LIPT2 mRNA in the brain of SAH model and control group (n=10). E, F. Western blot detection and quantitative analysis of LIAS protein levels in the brain of SAH model and control group. All quantitative data are expressed as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

[0021] Figure 2 It was shown that LIAS is mainly expressed in neurons and can be used as a clinical biomarker for SAH. A. Relative concentration of copper in cerebrospinal fluid (CSF) of SAH patients. B. LIAS level in CSF of SAH patients (ELISA test results). This study included 46 patients, and CSF was obtained from all patients on the third day of onset. C. Correlation analysis between LIAS level and disease severity (WFNS score) in SAH patients. D. Immunofluorescence staining of brain tissue of SAH mice showed that LIAS was mainly localized in neurons. Scale bar, 100 μm. All quantitative data are expressed as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

[0022] Figure 3 Figure 2. Modification and characterization of RBCEVs. A. Schematic diagram of coupling RVG and Cy5.5 fluorescent groups to the RBCEVs membrane by a two-step reaction. B. Transmission electron microscopy images of unmodified RBCEVs and RVG-RBCEVs. The scale bar is 100 nm. C. Western blot analysis results of CD63, TSG101, Alix and HBA relative to GAPDH in red blood cells and RBCEVs. D. Particle size distribution of unmodified RBCEVs and RVG-RBCEVs measured based on nanoparticle tracking analysis (NTA). E. Fluorescence images of unmodified RBCEVs and RVG-RBCEVs. Green represents DIO-stained lipid membrane and red represents Cy5.5. The scale bar is 5 mm.

[0023] Figure 4Showing the characteristics of RVG-RBCEVs / siRNA and its ability to target SAH lesion areas. A. Schematic diagram of electroporation to load siRNA onto vesicles. B. TEM of unmodified RBCEVs / siRNA and RVG-RBCEVs / siRNA. Scale bar is 100 nm. C. Particle size distribution of unmodified RBCEvs / siRNA and RVG-RBCEvs / siRNA based on NTA measurement. D. Relative concentration of siRNA in the electroporation supernatant.

[0024] Figure 5 RVG-RBCEVs / siRNA showed that RVG-RBCEVs / siRNA reduced the expression of LIAS, a gene related to copper death in vivo. A. Changes in LIAS mRNA expression in each group (n=6). B. LIAS protein expression level and quantitative analysis in each group. C. Relative concentration of copper levels in brain tissue of SAH mice after RVG-RBCEVs / siRNA treatment. D. Western blotting verification results of lipidated proteins Lip-DLAT and Lip-DLST levels. The internal reference was the expression of β-Actin, GAPDH or β-tubulin, and the data were expressed as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

[0025] Figure 6 RVG-RBCEVs / siRNA regulates mitochondrial function in SAH mice. A, B. GSH and MDA content assays were used to determine the mitochondrial oxidative stress levels in brain tissue of SAH mice in each group. C. Mitochondrial respiratory control rate was detected. D. Mitochondrial membrane potential was measured using a JC-1 probe. Scale bar: 100 μm. E. TEM was used to observe the occurrence of copper death in neurons (scale bar: 1 μm, black arrows indicate normal mitochondria, and red arrows indicate abnormal mitochondria). All quantitative data are expressed as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

[0026] Figure 7Figure 3 Effects of RVG-RBCEVs / siRNA treatment on neuroprotection, brain edema, neurodegeneration, and blood-brain barrier disruption in mice after SAH. A. The modified Garcia score was used to evaluate the sensory and motor functions of each group (n=18). B. The chimney behavior test was used to evaluate the motor function of each group of mice (n=18). C. The beam balance test was used to evaluate the motor integration and coordination ability of each group of mice (n=18). D. Changes in brain water content in each group (n=6). E. Quantitative analysis of Evanblue dye extravasation (n=6). F, G. FJC staining and quantitative analysis of the number of cortical FJC-positive cells. Scale bar, 50 μm. All quantitative data are expressed as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001).

[0027] Figure 8 Schematic diagram showing the effect of RVG-RBCEVs / siRNA on alleviating copper death in SAH mice. DETAILED DESCRIPTION

[0028] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Patient source and animal model construction

[0030] Patients: In this study, 46 patients with aSAH and World Federation of Neurosurgeons (WFNS) score <Ⅴ underwent lumbar puncture, lumbar external drainage, or external ventricular drainage in the Department of Neurosurgery, the First Affiliated Hospital of Wannan Medical College from October 2023 to May 2024, and cerebrospinal fluid (CSF) samples were collected. Inclusion criteria were consecutive adult patients with aSAH and WFNS grade <Ⅴ within 24 hours after admission. Exclusion criteria were as follows: non-aneurysmal SAH, end-stage renal or liver disease, systemic or CSF infectious diseases, and loss of consciousness on admission. Patients with inguinal hernia who underwent spinal anesthesia before surgery served as the control group (n=10). Immediately after collection, CSF biospecimens were centrifuged at 500 rpm and 4°C for 10 minutes. The CSF supernatant was transferred into EP tubes. Subsequently, CSF biospecimens were immediately frozen and stored at -80°C for analysis. The initial clinical status was obtained using the WFNS grade. The study was conducted in strict accordance with the Declaration of Helsinki. This study (2023IRB115) was approved by the institutional ethics committee, and participants were informed to provide consent or valid proxy before the study.

[0031] Animal model: All mice (male, C57BL / 6J, 8-10 weeks, 21-26 g) were purchased from CollectivePharmachem Biotechnology Co., Ltd. All mouse experiments were performed in accordance with the guidelines of the National Institutes of Health and approved by the Laboratory Animal Welfare and Ethics Committee of the First Affiliated Hospital of Wannan Medical College (approval number: LLSC-2023-112). The mice were anesthetized with 2% isoflurane, and a sharp nylon monofilament (6-0) was gently inserted from the external carotid artery to the end of the internal carotid artery with a slight breakthrough feeling, and then the nylon monofilament was removed to obtain the SAH model. The sham group also underwent the same surgical procedure without puncturing the artery. After the SAH mice were killed at the designated time points, the brain tissue around the temporal base hemorrhage was sampled and analyzed.

[0032] Detection method:

[0033] RNA isolation and qRT-PCR: Total RNA was extracted from RBCEVs or brain tissues according to the RNeasy universal kit (Qiagen, Germany). The mRNA level was analyzed using the FastKing One-Step RT-qPCR Kit (SYBR Green, Tiangen Biotech, China). Total RNA was reverse transcribed into cDNA using the miRcute Plus miRNA First-Strand cDNA Kit (Tiangen Biotech, China). Subsequently, qRT-PCR was performed using the miRcute Plus miRNAqPCR Kit (SYBR Green; Tiangen Biotech, China) to analyze the level of siRNA. All PCR reactions were set up in triplicate, and the mRNA level was normalized using the expression level of β-actin as a reference. The primer sequences are shown in Table 1. The PCR reaction results of mRNA expression were calculated using the 2-ΔΔCt method.

[0034] Table 1

[0035]

[0036] Western blotting: Red blood cells or brain tissues were lysed with RIPA lysis buffer containing protease inhibitors, and then the protein supernatant was extracted by centrifugation. Subsequently, the protein sample concentration was determined using a BCA protein concentration assay kit (Beyotime, Shanghai, China). The protein supernatant was mixed with 5x Protein Loading Buffer and denatured at 95°C for 10 min. Two-color pre-stained protein markers (Epizyme Biotech, Shanghai, China) and protein samples were electrophoretically separated on 10% SDS-PAGE (Epizyme Biotech, Shanghai, China). After the electrophoresis, the electrophoresis was transferred to a PVDF membrane (Millipore, USA). The PVDF membrane was blocked in 5% skim milk at room temperature for 2 h, and the blocked PVDF membrane was incubated with specific primary antibodies at 4°C overnight. The primary antibodies included: rabbit anti-LIAS (1:1000, ab246917, Abcam, USA), rabbit anti-DLAT (1:1000, HA721267, HUABIO, China), rabbit anti-DLST (1:10000, ab177934, Abcam, USA), rabbit anti-β-tubulin (1:10000, ET1602-4, Huaan Biotechnology, USA, China), rabbit anti-LipoicAcid (1:1000, ab1604-4, Abcam, USA). cam, USA), rabbit anti-TSG101 (1:50000, ab133586, Abcam, USA), rabbit anti-CD63 (1:50000, ab134045, Abcam, USA), rabbit anti-ALIX (1:1000, ab275377, Abcam, USA), rabbit anti-HBA (1:1000, ab92492, Abcam, USA), rabbit anti-GAPDH (1:10000, ET1601-4, HUABIO, China). The PVDF membrane was washed three times with TBST and then incubated with enzyme-labeled anti-rabbit secondary antibody (1:50000, HA1001 HUABIO, China) at room temperature for 1 hour. Finally, the bands were visualized using an ultrasensitive ECL chemiluminescence kit (Beyotime, Shanghai) and quantified using Image J software (USANIH).

[0037] Enzyme-linked immunosorbent assay (ELISA): The concentration of LIAS in CSF was quantitatively detected using a specific ELISA kit (E16215h, ELAab, China). The final concentration of LIAS was calculated using the OD value.

[0038] Copper level detection: The copper concentration in CSF and brain tissue homogenate was quantified using a copper colorimetric assay kit (E-BC-K300-M, Elabscience Biotechnology, China). The operation was as follows: 15 μL of CSF sample or 100 mg / ml brain homogenate supernatant was added to 230 μL of colorant working solution, the mixture was incubated at 37°C for 5 min, and the absorbance (580 nm) was measured using a microplate reader (MD, Shanghai).

[0039] Immunofluorescence staining: Paraffin sections of mouse brain tissue were dewaxed, rehydrated, and then antigen retrieval was performed in modified sodium citrate antigen retrieval solution (Beyotime, Shanghai, China). Subsequently, the sections were incubated with hydrogen peroxide for 15 minutes at room temperature to block endogenous peroxidase activity, then incubated with Triton X-100 for 15 minutes to permeabilize the membrane, and then blocked with 5% BSA for 1 hour. Next, the sections were incubated with primary antibodies at 4°C overnight. The primary antibodies were rabbit anti-LIAS (1:1000, ab246917, Abcam, USA), goat anti-IBA-1 (1:1000, ab289874, Abcam, USA), mouse anti-NEUN (1:50, HA601111, HUABIO, China), and mouse anti-GFAP (1:50, EM140707, HUABIO, China). Afterwards, the slides were rinsed 3 times with PBS and then incubated with fluorescently conjugated secondary antibodies for 1 hour at room temperature. Secondary antibodies are: anti-goat iFluor TM 488 (1:1000, HA1131, HUABIO, China), anti-mouse iFluor TM 488 (1:5000, HA1125, HUABIO, China) and anti-rabbit iFluor TM 594 (1:5000, HA1122, HUABIO, China). Finally, anti-fluorescence quenching blocking solution containing DAPI (Beyotime, Shanghai, China) was added to block the slides. An independent observer obtained images under a fluorescence microscope (AXIO observer 3, Zeiss, Germany).

[0040] Glutathione (GSH) and malondialdehyde (MDA) content determination: GSH and MDA content were quantified using GSH assay kit (S0053, Beyotime, Shanghai, China) and lipid peroxidation MDA assay kit (S0131S, Beyotime, Shanghai, China). Brain tissue was mechanically homogenized at 200 mg / ml in 0.9% saline and centrifuged at 12,000 rpm for 15 min at 4°C. The resulting supernatant was mixed with GSH working solution at room temperature, and the absorbance (412 nm) was measured after 5 min. 0.2 mL of thiobarbituric acid reagent was added to 0.1 mL of supernatant. The mixture was reacted at 100°C for 15 min, cooled to room temperature in a water bath, and centrifuged at 1000 g for 10 min. Subsequently, 0.2 mL of supernatant was added to a 96-well plate, and the absorbance was measured at 532 nm using an ELISA analyzer.

[0041] Mitochondrial isolation and respiratory control ratio (RCR) determination: Mitochondria were extracted by tissue mitochondrial isolation kit (C3606, Tianyu, Shanghai, China) according to the manufacturer's instructions. Mitochondrial respiratory function was measured using an Oxygraph-2K respirometer (Oroboros, Innsbruck, Austria). State 3 and state 4 respiration were established, and RCR (ratio of state 3 to state 4) was calculated, which reflects the ability of mitochondrial oxidative phosphorylation.

[0042] Mitochondrial membrane potential measurement: Mitochondrial membrane potential was measured using the JC-1 detection kit (C2003S, Tianyu, Shanghai, China) according to the manufacturer's instructions. Frozen fresh tissue sections were incubated with JC-1 staining solution at 37°C in the dark for 20 min, then washed twice with staining buffer, and images were taken under a fluorescence microscope (Olympus, Tokyo, Japan).

[0043] Brain edema assessment: Brain edema was assessed by measuring brain water content (wet weight / dry weight). At 24 h after SAH, mice were anesthetized and the brain tissue was completely removed. The wet weight (WW) of the brain sample was first weighed, and then the brain sample was dried at 100°C for 72 h to obtain the dry weight (DW). The brain water content was calculated using the formula: (WW-DW) / WW×100%.

[0044] Blood-brain barrier damage detection: 24 hours after SAH, mice were injected with Evan blue dye (5 ml / kg, Sigma-Aldrich). One hour later, mice were euthanized, and brain samples were collected and homogenized in 50% trichloroacetic acid. Evan blue dye in the supernatant was measured at 620 nm using a fluorescence spectrophotometer.

[0045] Fluoro-Jade C staining: FJC staining (AG325, Millipore, Germany) was used to identify neurodegeneration and evaluate damaged neurons as previously described. The frozen sections were reheated with double distilled water, dried at room temperature, and incubated with 0.0001% FJC solution for 30 min in the dark. The slides were dried at 60 ° C for 5 minutes, cleared in xylene for 5 minutes, and then sealed with neutral glue. Neuronal degeneration was imaged by fluorescence microscopy.

[0046] Neurological function score: In this experiment, the modified Garcia score (mGS) was used by an independent observer to detect the degree of neurological damage in mice. The modified Garcia score includes spontaneous activity (0-3 points), symmetry of limb movement (0-3 points), forepaw extension (0-3 points), climbing (1-3 points), body proprioception after trunk contact (1-3 points) and response to whisker stimulation (1-3 points). The modified Garcia score uses a scale of 3-18 points, and higher scores indicate better neurological function.

[0047] Chimney Test (CT): The chimney test is used to evaluate the motor ability of mice. A 45 cm long and 53 mm diameter transparent plastic tube is placed horizontally on a table and the mouse is placed at the mouth of the tube. When the mouse reaches the end of the tube, the tube is placed vertically with the mouse's head facing downward. The time required for the mouse to reach the 20 cm mark on the tube is recorded. If the mouse cannot complete the task within 1 minute, it is judged that there is a problem with its motor ability.

[0048] Balance beam test (BBT): The score range of the balance beam test is 0-5 points: no movement within 40 seconds and falling from the balance beam (0 points); no movement within 40 seconds but not falling from the balance beam (1 point); moving no more than half the length of the balance beam within 40 seconds (2 points); moving more than half the length of the balance beam within 40 seconds (3 points); reaching any platform at both ends of the balance beam within 40 seconds (4 points); reaching any platform at both ends of the balance beam within 25 seconds (5 points). The higher the score, the better the mouse's motor integration and coordination ability.

[0049] Statistical analysis: All statistical analyses were performed using Prism 6.0 (GraphPad Software, USA) and MedCalc version 19.0.4 (Broekstraat 529030, Mariakerke, Belgium). All data are presented as mean ± SD. Before analysis, each data set was tested for distribution normality using the Kolmogorov-Smirnov test. The differences between the two groups were evaluated using the Mann-Whitney U and / or Student's t test. Multiple comparisons of data from more than two groups were performed using the Kruskal-Wallis test or one-way analysis of variance. The correlation between LIAS levels and WFNS scores was evaluated using the Spearman correlation coefficient analysis. P < 0.05 was considered statistically significant.

[0050] Example 1

[0051] (1) Data collection and identification of copper death-related genes (CRGs)

[0052] The transcriptome data of SAH in GSE79416 were retrieved from GEO (https: / / www.ncbi.nlm.nih.gov / geo / ) to determine the gene expression data. This study identified 36 copper death-related genes (CRGs) ( Figure 1 A).

[0053] To further evaluate the differences in CRGs between groups, we used the ConsensusCluster-Plus1.60.0 package in R software (version 4.0.3) for global statistical analysis and screened for differentially analyzed genes (adjusted p value < 0.001). The results showed that 14 genes were upregulated, indicating that copper metabolism may be dysregulated in SAH ( Figure 1 B). Further evaluation of the differences revealed that |log2(fold-change)|>0.5 for LIAS, PLAT, and LIPT2 genes, P<0.001.

[0054] To verify the results of mRNA expression profiling, the mRNA expression of 20 brain samples was analyzed, including SAH mouse samples (n = 10) and sham-operated (Sham) mouse samples (n = 10). qRT-PCR results confirmed that in the mouse SAH model, the expression of three mRNAs, LIAS, PLAT, and LIPT2, was statistically significant compared with the Sham group ( Figure 1 C). In addition, the study found that the mRNA levels of LIAS, PLAT, and LIPT2 peaked at 12-24 hours after SAH, while the copper level peaked at 24 hours after SAH compared with the Sham group ( Figure 1 D). In addition, the expression of Lias protein was detected at different time points after SAH, and the results showed that the level of Lias showed a gradual increase ( Figure 1 E, F).

[0055] (2) Relationship between LIAS and severity of SAH

[0056] The WFNS score was used to assess the severity of EBI after SAH. The baseline characteristics of the patients are summarized in Table 2. After SAH, the levels of LIAS and copper in CSF of SAH patients were significantly increased within 3 days compared with the control group ( Figure 2 A, B). To further study the relationship between LIAS and WFNS score levels, the results showed that in the CSF of SAH patients, the LIAS level was positively correlated with the severity of SAH ( Figure 2 C). Immunofluorescence staining was used to study the localization of Lias protein in the brain tissue of SAH mice, and it was found that Lias protein was localized in neurons in the basal temporal lobe, but not in microglia and astrocytes ( Figure 2 D).

[0057] Table 2

[0058]

[0059] Example 2

[0060] The brain-targeting vector rabies virus glycoprotein-modified erythrocyte extracellular vesicles (RVG / RBCEVs) were loaded with siRNA of LIAS.

[0061] (1) Preparation of RVG-RBCEVs

[0062] The preparation of engineered vesicles RVG-RBCEVs is divided into two parts: Figure 3 A). First, water-soluble DBCO-sulfo-NHS forms a covalent bond with RBCEV (red blood cell-derived extracellular vesicle) surface proteins or phosphatidylethanolamine amino groups. Next, DBCO-coupled RBCEV and RVG9-azide peptide form a stable triazole bond through copper-free click chemistry to produce RVG-RBCEVs. At the same time, Cy5.5 azide is also coupled to the DBCO group to achieve tracking of RBCEV.

[0063] Transmission electron microscopy (TEM) was used to observe the morphology of unmodified RBCEVs and RVG-RBCEVs. Figure 3B). Next, nanoparticle tracking analysis (NTA) was used to evaluate the size distribution of unmodified RBCEVs and RVG-RBCEVs. The results showed that the average diameter of unmodified RBCEVs was around 86 nm, while the average diameter of RVG-RBCEVs increased to 134 nm ( Figure 3 C). Western blot analysis showed that CD63, TSG101, ALIX and the red blood cell marker protein hemoglobin A (HBA) were expressed in purified RBCEVs ( Figure 3 D). To verify the successful synthesis of RVG-RBCEVs, fluorescence microscopy showed that DIO-labeled RBCEVs were green and cy5.5 coupled to DBCO-RBCEVs were red. The overlap of the two colors confirmed that RVG was successfully coupled to the RBCEV surface ( Figure 3 E).

[0064] (2) Synthesis of siRNA

[0065] Based on the LIAS gene, siRNA that inhibits the expression of the LIAS gene was screened out, and its sequence is as follows:

[0066] Sense strand: 5'-CCGGAUAUUUGGAAGAUAUTT-3' (SEQ ID No. 9);

[0067] Antisense strand: 5'-AUAUCUUCCAAAUAUCCGGTT-3' (SEQ ID No. 10).

[0068] Negative control siRNA was also set up and synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0069] (3) Preparation of RVG-RBCEVs / siRNA

[0070] RVG-RBCEVs with a total protein concentration of 20 μg (using BCAAssay kit, Shanghai Biyuntian, China) were mixed with 20 μl Lias siRNA or negative control siRNA in 180 μl nucleofection buffer (Cell Line nucleoector kit V, Amaxa) and transferred to the nucleoector TM Electroporation was performed at 350 V and 150 μF in a IIs / 2b apparatus. To remove unbound siRNA, RVG-RBCEVs were washed by sequential ultracentrifugation in PBS (4°C).

[0071] (4) Characterization of RVG-RBCEVs / siRNA

[0072] Lias siRNA was loaded into unmodified RBCEVs and RVG-RBCEVs ( Figure 4 A). The morphology and size distribution of unmodified RBCEVs / siRNA and RVG-RBCEVs / siRNA were observed by TEM and NTA ( Figure 4 B, C). To evaluate the effect of siRNA encapsulation in RVG-RBCEVs, the RNA concentration in the waste solution after electroporation (Lias siRNA or NC control concentration) was detected using a NanoDrop ND-2000 spectrophotometer. Compared with the total siRNA solution, the concentration of Lias siRNA or NC control in the supernatant without siRNA was significantly reduced ( Figure 4 D).

[0073] Example 3

[0074] RVG-RBCEVs / siRNA can reduce the expression of LIAS, a key factor related to copper growth in vivo

[0075] RVG-RBCEVs / siRNA (abbreviated as R-EVs / siR) or RVG-RBCEVs / siRNANC (abbreviated as R-EVs / NC) were injected 2 h after SAH, and then the mice were killed and brain tissues were harvested 24 h after SAH. We measured the expression level of LIAS in the brain of mice after SAH. After SAH, the levels of LIAS protein and mRNA increased, and after RVG-RBCEVs / siRNA treatment, the levels of LIAS protein and mRNA decreased ( Figure 5 A, B). However, after RVG-RBCEVs / siRNA treatment, copper levels were significantly decreased ( Figure 5 C). Lip-DLAT and Lip-DLST were both elevated in SAH brain tissue, and RVG-RBCEVs / siRNA injection resulted in a more significant decrease ( Figure 5 D). The above results indicate that RVG-RBCEVs / siRNA treatment can inhibit the expression and activity of LIAS and reduce the protein levels of Lip-DLAT and Lip-DLST.

[0076] Example 4

[0077] RVG-RBCEVs / siRNA regulates mitochondrial function in SAH mice

[0078] GSH is a factor that is crucial in the regulation of mitochondrial reactive oxygen species (ROS) and plays a role in protecting neurons in the copper death pathway, but its expression is low in SAH. Figure 6As shown in A ("—" represents the SAH group, the same below), SAH caused changes in the expression of GSH in the basal surface of the temporal lobe of SAH mice. In contrast, RVG-RBCEVs / siRNA had the most significant effect on increasing the GSH content in the basal surface of the inferior temporal lobe of SAH mice, while the administration of RVG-RBCEVs (abbreviated as R-EVs) and RVG-RBCEVs / NC did not cause significant differences in GSH. In addition, malondialdehyde (MDA) was detected in the lesions after SAH, which reflects the degree of lipid peroxidation, which is the main feature of abnormal copper metabolism. The MDA level in the basal surface of the temporal lobe of SAH mice was significantly upregulated, while RVG-RBCEVs / siRNA significantly downregulated the MDA content in the basal surface of the temporal lobe of SAH mice ( Figure 6 B). The respiratory control ratio (RCR) value is an effective method to evaluate the integrity of mitochondrial respiratory structure and function. Figure 6 As shown in C, RCR decreased after SAH, and RCR increased significantly after RVG-RBCEVs / siRNA treatment. The above indicated that mitochondrial ATP synthesis and respiratory disorders were inhibited after SAH, and RVG-RBCEVs / siRNA treatment could alleviate mitochondrial dysfunction. JC-1 staining was used to detect mitochondrial membrane potential, which is a landmark event in the early stage of cell death. After SAH, J aggregates decreased and monomers increased. RVG-RBCEVs / siRNA treatment attenuated the membrane potential collapse induced by SAH ( Figure 6 D). To understand the effect of RVG-RBCEVs / siRNA treatment, transmission electron microscopy was used to observe the mitochondrial size and number of mitochondrial ridges after SAH. The number of mitochondrial ridges in the SAH group was reduced compared with the Sham group. However, under RVG-RBCEVs / siRNA treatment, the number of mitochondrial ridges increased significantly ( Figure 6 E).

[0079] Example 5

[0080] Effects of RVG-RBCEVs / siRNA treatment on neuroprotection, brain edema, neurodegeneration and BBB disruption in mice after SAH

[0081] To investigate the protective effect of RVG-RBCEVs / siRNA treatment on SAH mice, we tested the neurological and behavioral scores, neurodegeneration, BBB disruption, and brain water content in SAH mice. Figure 7 As shown in A, the neurological score of the SAH group was lower than that of the Sham group. After RVG-RBCEVs / siRNA treatment, the neurological score of the mice recovered. In CT, the motor ability of SAH mice was severely impaired compared with the Sham group, while the performance of SAH mice was significantly improved after RVG-RBCEVs / siRNA treatment ( Figure 7B). BBT evaluated the motor integration and coordination of mice and found that RVG-RBCEVs / siRNA had a stronger protective effect than RVG-RBCEVs and RVG-RBCEVs / NC ( Figure 7 C). Similarly, the degree of brain edema in the RVG-RBCEVs / siRNA-treated group was significantly reduced compared with the RVG-RBCEVs and RVG-RBCEVs / NC groups ( Figure 7 D). The level of Evans blue extravasation was significantly increased in SAH mice compared with the Sham group. In contrast, RVG-RBCEVs / siRNA treatment attenuated Evans blue extravasation compared with the RVG-RBCEVs and RVG-RBCEVs / NC groups ( Figure 7 E). In addition, neurodegeneration was evaluated by FJC staining, and the results showed that neurodegeneration in SAH mice, RVG-RBCEVs, and RVG-RBCEVs / NC groups was higher than that in the Sham group, while RVG-RBCEVs / siRNA significantly reduced neurodegeneration in mice after SAH ( Figure 7 F, G).

[0082] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. Application of LIAS gene in the preparation of diagnostic products for subarachnoid hemorrhage.

2. The use according to claim 1, characterized in that: The diagnostic product includes a reagent for detecting the expression level of the LIAS gene.

3. Application of LIAS gene in the preparation of products for the treatment of subarachnoid hemorrhage.

4. The use according to claim 3, characterized in that: The therapeutic goal is achieved by inhibiting neuronal copper death associated with subarachnoid hemorrhage and reducing oxidative stress.

5. A drug for treating subarachnoid hemorrhage, characterized in that: The drugs include LIAS gene expression inhibitors.

6. The drug for treating subarachnoid hemorrhage according to claim 5, characterized in that: The LIAS gene expression inhibitor is siRNA, and the siRNA sequence is: Sense strand: 5′-CCGGAUAUUUGGAAGAUAUTT-3′; Antisense strand: 5′-AUAUCUUCCAAAUAUCCGGTT-3′.

Images