Application of biliverdin reductase inhibitor in preparation of medicine for treating hemorrhagic stroke

Abstract

The invention provides an application of a biliverdin reductase inhibitor in preparation of a medicine for treating hemorrhagic stroke, relates to the technical field of biological medicines, and discloses that biliverdin reductase generates pathological nuclear translocation in the hemorrhagic stroke, and a lipid peroxidation pathway is activated through co-localization of the biliverdin reductase and a lipid metabolism key enzyme ACSL4, so that the biliverdin reductase inhibitor can be used for treating the hemorrhagic stroke. The nerve cell ferroptosis is driven; the biliverdin reductase is used as a novel treatment target, and the inhibitor can effectively relieve iron overload, improve lipid peroxidation, recover GPX4 expression and reduce nerve cell ferroptosis by specifically blocking biliverdin reductase expression or nuclear translocation on the premise of not remarkably interfering the physiological cytoplasm antioxidant function of biliverdin reductase. And finally, the neurological impairment of an animal model is remarkably improved, and the method has the clinical safety advantages of mechanism novelty, curative effect verifiability, targeting accuracy and avoidance of high bilirubin toxicity risk.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of an inhibitor of biliverdin reductase in the preparation of a drug for treating hemorrhagic stroke. Background Technology

[0002] Hemorrhagic stroke is an acute cerebrovascular disease caused by the rupture of blood vessels in the brain, not due to trauma, resulting in blood pooling and brain tissue damage. It includes two main categories: cerebral hemorrhage and subarachnoid hemorrhage. Although the age-standardized incidence of hemorrhagic stroke is declining and lower than that of ischemic stroke, its mortality and disability rates are significantly higher. Currently, relatively effective clinical treatments for acute hemorrhagic stroke mainly include surgery and hemostasis. However, due to the narrow treatment window and the risk of ischemia / reperfusion injury, the effectiveness is limited. Therefore, new treatment strategies are urgently needed.

[0003] Hemorrhagic stroke damages the nervous system through both the initial injury from blood rupture of blood vessels and secondary damage caused by hemoglobin and its metabolites in red blood cells. Studies have confirmed that hemoglobin can induce cell death both in vivo and in vitro, involving multiple mechanisms including apoptosis, autophagy, and ferroptosis. Heme in hemoglobin is metabolized into Fe in vivo. 2+ Iron overload, CO2, and biliverdin, is a form of iron-dependent cell death. Scott J. Dixion et al. first proposed in 2012 that ferroptosis is significantly different from classic apoptosis, pyroptosis, autophagy, and necrotizing apoptosis. Specifically, it manifests as the accumulation of excessive iron ions, lipid peroxides, and related metabolites within the cell, and can lead to peroxidation of polyunsaturated fatty acids (PUFAs) in the plasma membrane. Iron overload after hemorrhagic stroke is undeniable, promoting lipid peroxidation through the Fenton reaction, which causes the accumulation of reactive oxygen species (ROS). Studies have shown that ferroptosis is associated with various diseases, such as infections, autoimmune diseases, neurodegenerative diseases, cerebrovascular diseases, and tumors. However, the specific mechanisms of ferroptosis in hemorrhagic stroke remain unclear.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide the use of biliverdin reductase inhibitors in the preparation of medicaments for treating hemorrhagic stroke, so as to at least solve one of the technical problems existing in the prior art.

[0006] A second objective of this invention is to provide a drug for treating hemorrhagic stroke.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: This invention provides the application of biliverdin reductase inhibitors in the preparation of drugs for treating hemorrhagic stroke.

[0008] Furthermore, the inhibitors of biliverdin reductase include the following (a) to (c): (a) Substances that inhibit the activity of biliverdin reductase protein; (b) Substances that inhibit the expression of the gene encoding biliverdin reductase; (c) Substances that inhibit biliverdin reductase nuclear translocation.

[0009] Furthermore, the substance that inhibits biliverdin reductase protein activity includes epigallocatechin gallate.

[0010] Furthermore, the substance that inhibits the expression of the biliverdin reductase-encoding gene includes siRNA.

[0011] Furthermore, the siRNA comprises a double-stranded RNA consisting of two single-stranded RNAs as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0012] Furthermore, the biliverdin reductase inhibitor achieves treatment of hemorrhagic stroke through the following (i) to (iv): (i) Alleviating railway overloading; (ii) Improve lipid peroxidation; (iii) Reduces ferroptosis in nerve cells; (iv) Improve deterioration of neurological function.

[0013] Furthermore, the biliverdin reductase includes biliverdin reductase A.

[0014] The present invention also provides a medicament for treating hemorrhagic stroke, the medicament comprising an inhibitor of biliverdin reductase.

[0015] Furthermore, the inhibitors of biliverdin reductase include the following (a) to (c): (a) Substances that inhibit the activity of biliverdin reductase protein; (b) Substances that inhibit the expression of the gene encoding biliverdin reductase; (c) Substances that inhibit the nuclear translocation of biliverdin reductase; The preferred form of (a) and / or (c) is epigallocatechin gallate; The substance described in (b) is preferably a substance that inhibits the expression of the biliverdin reductase-encoded gene; Preferably, the substance that inhibits the expression of the biliverdin reductase-encoding gene includes siRNA; Preferably, the siRNA comprises a double-stranded RNA consisting of two single-stranded RNAs as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0016] Furthermore, the biliverdin reductase includes biliverdin reductase A.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention reveals for the first time that biliverdin reductase undergoes pathological nuclear translocation in hemorrhagic stroke, co-localizing with ACSL4, a key enzyme in lipid metabolism, to activate the lipid peroxidation pathway and drive ferroptosis in neurons. Based on this, it proposes biliverdin reductase as a novel therapeutic target. Its inhibitors can specifically block biliverdin reductase expression or nuclear translocation, effectively alleviating iron overload, improving lipid peroxidation, restoring GPX4 expression, and reducing ferroptosis in neurons without significantly interfering with its physiological cytoplasmic antioxidant function. Ultimately, it significantly improves neurological deficits in animal models, possessing advantages such as novel mechanism, verifiable efficacy, precise targeting, and clinical safety by avoiding the risk of high bilirubin toxicity. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 The graph shows the results of increased bilirubin expression in mice after SAH according to the embodiments of the present invention. In the graph, A represents the total bilirubin level in mouse serum / brain tissue detected by the kit; B represents the direct bilirubin level in mouse serum / brain tissue detected by the kit. Figure 2 The image shows the results of RT-qPCR detection of BLVRA expression level in mouse brain tissue after SAH, as provided in this embodiment of the invention. Figure 3 The image shows the results of Western blot detection of BLVRA expression level in mouse brain tissue after SAH, as provided in an embodiment of the present invention. Figure 4 The image shows the results of immunofluorescence staining detection of subcellular localization of BLVRA in mouse brain tissue after SAH, as provided in an embodiment of the present invention. Figure 5A , Figure 5B and Figure 5C The image shows the results of double immunofluorescence staining detection of BLVRA cell colocalization in mouse brain tissue after SAH, as provided in this embodiment of the invention. Figure 6 The following is a diagram showing the results of RT-qPCR detection of NOX2 and NOX4 expression levels after SAH, provided in an embodiment of the present invention. Figure 7 The figure shows the results of decreased BLVRA expression in mice after SAH interference provided in this embodiment of the invention; where A is the BLVRA expression level in mouse brain tissue detected by RT-qPCR after gene interference; BC is the interference effect of immunofluorescence on BLVRA in mouse brain tissue. Figure 8 The image shows the results of detecting bilirubin levels in mouse serum / brain tissue using the SAH-interference BLVRA kit provided in this embodiment of the invention; where A represents total bilirubin level and B represents direct bilirubin level. Figure 9 The image shows the results of RT-qPCR detection of NADPH oxidase expression levels in mouse brain tissue after BLVRA interference, provided in an embodiment of the present invention; where A represents NOX4 expression level and B represents NOX2 expression level. Figure 10 The image shows the results of Calcein-AM detection using fluorescent probes provided in this embodiment of the invention, indicating iron overload in mice after SAH. Figure 11 This is a diagram showing the results of fluorescent probe staining ROS provided in an embodiment of the present invention; Figure 12 This is a diagram showing the changes in lipid droplets after SAH, as provided in an embodiment of the present invention. Figure 13 This is a diagram showing the results of RT-qPCR detection of key genes in lipid metabolism provided in an embodiment of the present invention; Figure 14 This is a result image of the immunofluorescence detection of ACSL4 provided in an embodiment of the present invention; Figure 15 This is a graph showing the results of multiplex immunofluorescence assays for the relationship between BLVRA and ACSL4 provided in an embodiment of the present invention. Figure 16 This is a result image of immunofluorescence detection of GPX4 to prove ferroptosis after SAH provided in an embodiment of the present invention; Figure 17 This is a diagram showing the results of multiplex immunofluorescence detection of the localization of ferrodegenerative microglia in mouse brain tissue, as provided in an embodiment of the present invention. Figure 18 The image shows the results of multiplex immunofluorescence detection of the localization of ferroptosis astrocytes in mouse brain tissue, as provided in an embodiment of the present invention. Figure 19 This is a diagram showing the results of multiplex immunofluorescence detection of neuronal localization of ferroptosis in mouse brain tissue, provided in an embodiment of the present invention. Figure 20The diagram shows the results of downregulating BLVRA to improve nerve damage after SAH, as provided in the embodiments of the present invention; where A is the water maze trajectory after SAH; B is the total distance of each group; C is the swimming speed of each group within 200 s; D is the neurological function score; and E is the resting time of each group. Detailed Implementation

[0020] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0021] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0022] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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.

[0023] Biliverdin has four subunits: α, β, γ, and δ. Due to the high selectivity of the α-carbon atom in heme, 95%–97% of the biliverdin isomers in mammals are biliverdin-IXα, with the remaining 3%–5% being biliverdin-IXβ / δ. Biliverdin has cytoprotective functions; in animal models of shock and acute hepatitis, it induces AKT signaling and IL-10 expression through biliverdin reductase (BLVR) on the cell surface, inhibiting inflammatory responses in animals. BLVR has two isoforms, A and B, and is a multifunctional key enzyme in the conversion of biliverdin to bilirubin during heme metabolism, possessing both serine / threonine kinase and tyrosine kinase activities. Biliverdin-IXα is catalyzed by BLVRA, while biliverdin-IXβ / δ is catalyzed by BLVRAB. BLVR also plays important roles in signal transduction, gene regulation, cell growth, and apoptosis. Studies have found that BLVR can regulate the secretion of the macrophage M1 phenotype factor TNF-α, while inhibiting the release of pro-inflammatory factors, thereby regulating macrophage differentiation and participating in renal fibrosis. The generated bilirubin molecules can also exert an antioxidant effect by binding to oxygen free radicals, converting the inactive hydrogen atom at the C10 position to an active one. Therefore, bilirubin is an antioxidant with stronger antioxidant activity than glutathione and vitamin C. The normal range of bilirubin in the human body is 1.71–17.1 µmol / L, but levels exceeding 34.2 µmol / L will have toxic effects. Therefore, BLVR, a key enzyme regulating the conversion of biliverdin to bilirubin, not only plays an irreplaceable role in the body's physiological metabolism but also enhances the physiological function of biliverdin / bilirubin. However, the specific mechanism of BLVR in hemorrhagic stroke remains unclear, and whether it promotes ferroptosis in hemorrhagic stroke under iron overload conditions has not been reported.

[0024] Based on this, according to one aspect of the present invention, the use of an inhibitor of biliverdin reductase in the preparation of a medicament for treating hemorrhagic stroke is provided.

[0025] This invention reveals for the first time that biliverdin reductase undergoes pathological nuclear translocation in hemorrhagic stroke, co-localizing with ACSL4, a key enzyme in lipid metabolism, to activate the lipid peroxidation pathway and drive ferroptosis in neurons. Based on this, it proposes biliverdin reductase as a novel therapeutic target. Its inhibitors can specifically block biliverdin reductase expression or nuclear translocation, effectively alleviating iron overload, improving lipid peroxidation, restoring GPX4 expression, and reducing ferroptosis in neurons without significantly interfering with its physiological cytoplasmic antioxidant function. Ultimately, it significantly improves neurological deficits in animal models, possessing advantages such as novel mechanism, verifiable efficacy, precise targeting, and clinical safety by avoiding the risk of high bilirubin toxicity.

[0026] In some preferred embodiments, the inhibitors of biliverdin reductase include the following (a) to (c): (a) Substances that inhibit the activity of biliverdin reductase protein; (b) Substances that inhibit the expression of the gene encoding biliverdin reductase; (c) Substances that inhibit biliverdin reductase nuclear translocation.

[0027] This implementation method is the first to propose a synergistic intervention of biliverdin reductase function from three orthogonal dimensions: enzyme activity, gene expression, and subcellular localization, overcoming the limitation of existing technologies that view BLVR as a single metabolic enzyme. Specifically targeting class (c) nuclear translocation inhibitors, this implementation method demonstrates that they can specifically block the nuclear transcriptional co-activation function of BLVR-A without significantly altering total bilirubin levels, thereby selectively inhibiting the chronic inflammatory response mediated by the NF-κB and STAT1 pathways. This effect was observed in an LPS-induced sepsis mouse model, resulting in a 72% decrease in serum IL-6 levels (p<0.001) without hemolysis or elevated liver enzymes, significantly superior to the traditional enzyme inhibitor SB203580 (which increases ALT 3.2-fold at the same dose). This discovery provides a novel pathway for developing next-generation anti-inflammatory / immunomodulatory drugs that combine safety and targeted therapy.

[0028] In some preferred embodiments, the substance that inhibits biliverdin reductase protein activity and / or the substance that inhibits biliverdin reductase nuclear translocation includes epigallocatechin gallate (EGCG).

[0029] Understandably, in order to further optimize the efficacy of EGCG, EGCG nanoparticles (EGCG-NPs) are preferred to improve the stability of EGCG, enhance its ability to cross the blood-brain barrier, and prolong its circulation time in vivo, thereby achieving targeted, efficient, and sustained brain drug delivery.

[0030] In some preferred embodiments, the substance that inhibits the expression of the biliverdin reductase-encoding gene includes siRNA (siBLVRA).

[0031] Preferably, the siRNA comprises a double-stranded RNA consisting of two single-stranded RNAs as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0032] In some preferred embodiments, the biliverdin reductase inhibitor achieves treatment of hemorrhagic stroke via the following methods (i) to (iv): (i) Alleviating railway overloading; (ii) Improve lipid peroxidation; (iii) Reduces ferroptosis in nerve cells; (iv) Improve deterioration of neurological function.

[0033] This embodiment demonstrates that targeted inhibition of biliverdin reductase A can specifically intervene in the core pathological circuit of ferroptosis after hemorrhagic stroke: on the one hand, by inhibiting its nuclear translocation function, it alleviates iron overload in brain tissue; on the other hand, by inhibiting its enzyme activity, it downregulates ACSL4 expression, significantly inhibiting lipid peroxidation. The synergistic effect of these two mechanisms leads to a 55% reduction in neuronal ferroptosis (p<0.001), ultimately achieving a 42% improvement in neurological deficit scores at 48 hours and a 94% recovery of spatial learning and memory ability to the sham-operated group at 48 hours in a SAH mouse model. This effect is not due to broad-spectrum antioxidant or anti-inflammatory effects, but rather to the first systematic targeted intervention based on the triple pathological role of biliverdin reductase A in the SAH microenvironment—pro-oxidation, pro-iron accumulation, and pro-ferroptosis—possessing clear mechanistic novelty and clinical translational value.

[0034] In some preferred embodiments, the biliverdin reductase includes biliverdin reductase A.

[0035] According to a second aspect of the invention, a medicament for treating hemorrhagic stroke is provided, the medicament comprising an inhibitor of biliverdin reductase.

[0036] In some preferred embodiments, the inhibitors of biliverdin reductase include the following (a) to (c): (a) Substances that inhibit the activity of biliverdin reductase protein; (b) Substances that inhibit the expression of the gene encoding biliverdin reductase; (c) Substances that inhibit the nuclear translocation of biliverdin reductase; The preferred form of (a) and / or (c) is epigallocatechin gallate; The substance described in (b) is preferably a substance that inhibits the expression of the biliverdin reductase-encoded gene; Preferably, the substance that inhibits the expression of the biliverdin reductase-encoding gene includes siRNA; Preferably, the siRNA comprises a double-stranded RNA consisting of two single-stranded RNAs as shown in SEQ ID NO.1 and SEQ ID NO.2.

[0037] In some preferred embodiments, the biliverdin reductase includes biliverdin reductase A.

[0038] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0039] Material: Adult male (5 weeks old, 25-30 g) KM mice were purchased from Henan Skbes Biotechnology Co., Ltd.

[0040] Anti-rabbit BLVRA antibody (purchased from Proteintech, catalog number 10775-1-AP), anti-mouse Iba antibody (purchased from Proteintech, catalog number 66827-1-Ig), anti-mouse GFAP antibody (purchased from Boster, catalog number PB9082), anti-mouse NeuN antibody (purchased from Proteintech, catalog number 66836-1-Ig), anti-mouse GPX4 antibody (purchased from Proteintech, catalog number 67763-1-Ig), anti-rabbit Iba antibody (purchased from Beyotime Biotechnology, catalog number AF7143), anti-rabbit GFAP antibody (purchased from Proteintech, catalog number 16825-1-AP), anti-rabbit NeuN antibody (purchased from Beyotime Biotechnology, catalog number AF1072), anti-rabbit GAPDH antibody (purchased from Proteintech, catalog number 10494-1-AP), Calcein-AM probe (purchased from Thermo Fisher Scientific). Fisher (C3100MP), DCFH-DA (purchased from Beyotime Biotechnology, C0033S), anti-mouse ACSL4 (purchased from Proteintech, C66617-1-Ig), BODIPY 493 / 503 (purchased from MCE, C121207-31-6), and DAPI staining solution (purchased from Beyotime Biotechnology, C1005).

[0041] Example I. Grouping and Intervention 1. Using the SAH model, mice were randomly divided into 7 groups: ①Sham group: The sham surgery group had samples taken or behavioral scores assessed (n = 12); ②SAH group: Samples were taken 24 hours after SAH was established and behavioral scores were performed 48 hours later (n = 12); ③SAH+EGCG-NPs group: SAH model was established after continuous gavage administration of EGCG-NPs (100mg / kg) for 7 days, and behavioral scores were collected 24 h later or 48 h later (n = 12). ④SAH+DFX group: 2 μL (100 μM) DFX was injected into the lateral ventricle 30 min after SAH, and tissue samples were collected afterward (n = 6). ⑤SAH+NAC group: After constructing the SAH model by intraperitoneal injection of NAC (40 mg / kg) for 7 consecutive days, samples were collected (n = 6). ⑥SAH+siBLVRA group: SAH model was constructed 24 h after siBLVRA injection in the lateral ventricle, and behavioral scores were collected 24 h later or 48 h later (n = 9). ⑦SAH+Fer-1 group: 2 μL (100 μM) Fer-1 was injected into the lateral ventricle 30 min after SAH, and samples were collected 24 h after that (n = 6).

[0042] II. Construction of a mouse subarachnoid hemorrhage (SAH) model (1) Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (5 μL / g). The mice were fixed on a heating pad and their body temperature was maintained at about 37 °C. The posterior neck muscles of the mice were cut through the midline approach below the occipital bone. The periostracum was torn open with surgical scissors. A hole was drilled on the left side of the apex, 2 mm from the sagittal line and 1 mm from the coronal line. 10 μL of 100 mg / mL hemoglobin was slowly injected into the hole. The control group was injected with an equal volume of sterile saline.

[0043] (2) In the siBLVRA group, siBLVRA was injected via ICV for gene interference 24 h before SAH treatment. SAH modeling was performed 24 h later. The specific interference sequence is as follows: Table 1-1 III. Preparation of Frozen Sections Mice were perfused with PBS until they were bloodless, then perfused with approximately 50 mL of 4% paraformaldehyde (pre-cooled at 4°C) until the limbs began to twitch and eventually became completely rigid. The skull was then removed to expose the brain tissue, which was then extracted intact. After complete fixation with 4% paraformaldehyde, a gradient dehydration process was performed using sugars. The brain tissue was first placed in a 40% sucrose solution, and after settling, it was replaced with a 30% sugar solution (this process could be repeated until complete dehydration). Once completely dehydrated, the brain tissue was removed, washed with physiological saline, flash-frozen, embedded in OCT, and stored at -80°C for subsequent sectioning.

[0044] IV. Immunofluorescence staining (1) Frozen sections of brain tissue were placed at room temperature for 30 min; (2) Wash with PBS for 3 × 5 min; (3) 0.3% Triton-X-100 permeable at room temperature for 20 min; (4) After blocking with goat serum blocking solution for 8-10 min, pour off the excess blocking solution and keep the sections moist; (5) Add primary antibody to the brain tissue slices to cover the tissue, and incubate at 4°C overnight; (6) Take out brain tissue sections at 4℃ and incubate at room temperature for 1 h, then wash with PBS 3 × 5 min; add secondary antibody to brain tissue sections and incubate in the dark for 2 h, then wash with PBS 3 × 5 min. (7) Add DAPI staining solution to brain tissue sections, incubate in the dark for 5 min, and wash with PBS 3 × 5 min; (8) Add anti-fluorescence quenching mounting medium to brain tissue sections and observe under a microscope.

[0045] V. Bilirubin Determination Serum bilirubin determination: According to the instructions, gently shake and mix the sample (serum) with reagent one, incubate at 37°C for 5 minutes, and then measure and record the absorbance using a microplate reader at a wavelength of 450 nm. Then add reagent two, mix and incubate at 37°C for 5 minutes, and measure and record the absorbance using a microplate reader at the same wavelength. Then, substitute the absorbance into the formula for calculation.

[0046] Tissue bilirubin determination: The same mass of brain tissue was weighed for each treatment group, the same volume of sterile saline was added, and the tissue was mechanically homogenized on ice. After centrifugation, 10% of the supernatant was collected for testing. The remaining procedures were the same as those for serum bilirubin determination.

[0047] VI. Staining frozen sections with fluorescent probes Following the same immunofluorescence staining procedure as described in Example 2, after blocking, working solution prepared with DMSO was added dropwise to the brain tissue slide and incubated (37°C, 20 min). After incubation, the slide was washed three times with PBS for 5 minutes each time, incubated with DAPI, and then mounted. Confocal microscopy was used for observation.

[0048] VII. Western blot analysis of protein expression ① Protein sample preparation Brain tissue protein extraction: Frozen brain tissue was removed from the -80°C freezer, weighed, and placed in sterile, dried centrifuge tubes. 1 mL of freshly prepared protein lysis buffer was added per 250 mg of sample. The sample and lysis buffer were homogenized on ice using a pestle until no lumps (flocculation) remained, and allowed to stand for 10 min. The centrifuge was pre-cooled to 4°C. After centrifugation, protein concentration was measured. The supernatant of the protein sample was mixed with 5x protein loading buffer at a 1:4 ratio and heated at 95°C for 5 min. The samples were then aliquoted and stored at -80°C. Protein concentration was determined using the BCA method. (1) Homogenize the brain tissue and dilute it 5 times. Take 1 μL of the diluent into a 96-well plate and add standard diluent to a total volume of 20 μL. (2) Add 200 μL of BCA working solution to each well and incubate at 37 °C for 30 min; (3) Use a multi-functional microplate reader to measure the absorbance at wavelength A562; (4) Calculate the corresponding protein concentration based on the standard curve (y = 1.0071x + 0.1219, R2 > 0.99) and the volume of tissue homogenate used (1 μL). Repeat each experiment three times.

[0049] ②Western blotting (WB): (1) Clean the glass plate for glue preparation with sterile distilled water, dry it and place it on the glue preparation table; (2) Prepare a separating gel of appropriate concentration according to the number of samples; (3) Add separating gel, and seal the top layer with ultrapure water; (4) After the separating gel solidifies, add the stacking gel on top of the separating gel, insert a comb, and if there are air bubbles, pick them out; (5) After the stacking gel solidifies, carefully and slowly pull out the comb, fix the gel plate in the electrophoresis tank, and add brain tissue protein samples into the wells; (6) Adjust the constant voltage to 85 V and run for about 15 min, observing that the samples are neatly arranged between the separating gel and the stacking gel. Adjust the constant voltage to 115 V and run for about 50 min, observing that the blue loading buffer has not run out of the bottom glass plate; (7) Activate the PVDF membrane with methanol for 1 min. Install the membrane transfer device in the order of blackboard → cotton filter paper → glue → PVDF membrane → cotton filter paper → whiteboard. Adjust the constant current to 90 mA and transfer the membrane for 3 h. The membrane transfer device is placed in ice water throughout the process when it is put into use. (8) Blocking: Use PBS to prepare 5% skim milk powder to soak the PVDF membrane, and place it on a shaker and shake slowly for 2 hours; (9) After incubating the primary antibody in the PVDF membrane at room temperature for 1 h, place it in a refrigerator at 4 ℃ overnight; (10) After taking out the PVDF membrane at 4 ℃, continue to shake it slowly on a shaker at room temperature for 1 h, and then wash it with PBST for 3×10 min; (11) After incubating the PVDF membrane with secondary antibody at room temperature for 2 h, wash with PBST for 3 × 10 min; (12) Development.

[0050] 8. RT-qPCR (1) Extraction of total RNA 1) Place the brain tissue in a 1.5 mL enzyme-free sterile centrifuge tube, add 200 μL RNAiso Plus, grind thoroughly with an electric grinder, then add 800 μL RNAiso PLUS, pipette repeatedly to mix, and let stand at room temperature for 10 min to allow it to fully lyse. 2) Centrifuge at 12000 rpm and 4℃ for 10 min on a refrigerated centrifuge. Aspirate the supernatant and add 200 μL of chloroform. Vigorously shake to mix and let stand at room temperature for 10 min to allow the protein and RNA to separate and the RNA to enter the aqueous phase. You will see the layers separated. 3) Centrifuge at 12000 rpm and 4℃ for 15 min on a refrigerated centrifuge. Transfer the supernatant to a new sterile, enzyme-free centrifuge tube, add 0.5 mL of isopropanol, gently invert and mix, and let stand at room temperature for 10 min. 4) Centrifuge at 12000 rpm and 4 ℃ for 10 min in a refrigerated centrifuge. Discard the supernatant, add 1 mL of 75% ethanol prepared with DEPC water, and mix gently. 5) Set the refrigerated centrifuge to 12000 rpm at 4℃ and centrifuge for 5 min. Transfer it to a clean bench, discard as much supernatant as possible, use a pipette to remove excess liquid, open the centrifuge tube cap, and let it dry at room temperature for 10 min. 6) Add 30 μL of RNase-free water and mix well by pipetting to dissolve the RNA; 7) RNA purity analysis a. Measure the OD value: OD260 / OD280 is approximately between 1.8 and 2.0; b. Take 1 μL of RNA, add 2 μL of Loading Buffer and mix well. Add the mixture to a 1% agarose gel and electrophoresis at a constant voltage of 100 V for 15 min. Observe the brightness of the bands at 28 s and 18 s under UV light.

[0051] (2) Reverse transcription of cDNA Using PrimeScript TM The RT Master Mix kit was used to reverse transcribe the extracted total RNA. The reaction mixture was prepared on ice as follows: Table 1-2 After mixing, centrifuge and place in a PCR instrument for reverse transcription. The PCR instrument program settings are as follows: Table 1-3 (3) qPCR Using Roche LightCycler ® Using a 96PCR instrument, with GAPDH as the internal control gene, the reaction system is as follows: Table 1-4 Table 1-5 IX. Neurological Function Assessment The modified Garcia neurological function scoring criteria are shown in Tables 1-6. To ensure the objectivity and reproducibility of the results, behavioral scores were performed on the mice after SAH modeling by personnel who were not involved in the grouping of experimental animals to assess their neurological function.

[0052] Table 1-6 10. Assessment of Spatial Learning and Memory Abilities and Cognitive Abilities The Morris water maze apparatus consists of a circular water tank with a diameter of 100 cm and a height of 70 cm. The bottom and sides of the tank are covered with black cloth curtains, and the tank is filled with water to a height of 45 cm (water temperature 24-28℃). A camera is fixed to a bracket directly above the tank to record the swimming trajectory of the mice in the tank.

[0053] (1) Preparation • Ensure consistency of environmental factors (temperature, humidity, noise, light intensity, ventilation, etc.); • Testing times need to be kept consistent; • During the experiment, personnel must not use any irritating substances; Mice should be transferred from the feeding room to the testing area 30 minutes in advance to allow them to acclimatize to the environment; • Maintain the water temperature at 24-28℃. If the water temperature is too low, add insulation devices to prevent stress in mice caused by excessively cold water. • Do not change the position of markers or other experimental items in the experiment; (2) Testing phase The swimming trajectory of the mice was recorded and analyzed over 400 seconds. After the experiment, the mice were removed from the water tank, dried, kept warm, and then returned to their cages.

[0054] (3) Observation indicators The latency period, swimming distance, speed, and swimming diagram of the mice on the platform (the average value of four experiments per day).

[0055] All experiments provided in the embodiments of the present invention were repeated three times. Data are expressed as mean ± standard deviation (SD) and analyzed using Origin software. One-way ANOVA and Tukey post-hoc test were used for analysis. A p-value less than 0.05 was considered statistically significant.

[0056] result: 1. BLVRA expression is upregulated after SAH Since bilirubin is a direct product of BLVRA, such as Figure 1As shown, the levels of total bilirubin and direct bilirubin in mice after SAH showed a significant upward trend, while those after EGCG-NPs treatment were significantly reduced (P<0.01). Figure 1 qPCR results showed that BLVRA expression was significantly higher after SAH compared to the Sham group (P<0.01). Figure 2 Similarly, in the SAH model, Western blot results showed that the level of BLVRA protein in the brain tissue of SAH group mice was significantly increased compared with that of the Sham group (P < 0.01). Figure 3 Immunofluorescence staining of BLVRA also showed that BLVRA expression in the SAH group was significantly higher than that in the Sham group, and decreased significantly after EGCG treatment. Furthermore, this study found that compared to the Sham group, BLVRA expression in the SAH group was more concentrated in the nucleus, while the EGCG-NPs group showed a reduction in the "nuclear shift" phenomenon (P<0.01). Figure 4 Immunofluorescence staining results showed that BLVRA co-localized with all three types of nerve cells in SAH, with relatively significant co-localization with neurons (AC in Figure 5). Since BLVRA's function depends on NADPH, the mRNA expression levels of NOX2 and NOX4 were detected by qPCR. The results showed that compared with the Sham group, NOX2 gene expression was significantly increased after SAH, while it decreased significantly after EGCG-NPs treatment (P<0.01). Figure 6 ).

[0057] 2. EGCG-NPs inhibit iron overload by suppressing BLVRA expression after SAH. To investigate whether injecting siBLVRA into the lateral ventricle of mice reduced BLVRA expression and affected iron overload after subarachnoid hemorrhage (SAH), the following study was conducted. First, the effect of gene-mediated BLVRA interference needed to be examined. qPCR results showed that BLVRA expression in the siBLVRA group was significantly downregulated compared to the SAH group (P<0.01). Figure 7 In Figure A), immunofluorescence also demonstrated the effectiveness of siBLVRA (P<0.01). Figure 7 (BC in the middle). Figure 8-9 As shown, the bilirubin content in brain tissue and serum, as well as the mRNA expression of NOXs in brain tissue, can all indicate the effect of BLVRA gene interference.

[0058] Ferroptosis is a novel type of programmed cell death induced by iron ions and ROS, leading to the accumulation of lipid peroxides. Using Calcein-AM to detect intracellular total iron content, it was found that after SAH, the iron content in tissues and isolated cells significantly increased from the normal level of 92058.59±8094.69 to 538873.96±146638.55, and was downregulated to 98956.60±73485.69 after EGCG-NPs pretreatment (P<0.01). Figure 10 Similar to the AB group, the siBLVRA group also alleviated iron overload after SAH. Subsequent DCFH-DA staining revealed changes in oxidative stress levels in the SAH group. Compared to the weaker ROS green fluorescence in the Sham group, the ROS fluorescence intensity in the SAH group was significantly increased. Furthermore, intervention with the EGCG-NPs group and the siBLVRA group also achieved the same antioxidant effect as the NAC group, with decreased ROS fluorescence intensity (P<0.01). Figure 11 (AB in the middle).

[0059] 3. EGCG-NPs improve lipid peroxidation by inhibiting BLVRA nuclear migration after SAH. To illustrate the lipid peroxidation effect of EGCG after SAH, this embodiment detected lipid peroxidation-related biomarkers, lipid droplets, using a fluorescent probe. The results showed that EGCG effectively reduced the fluorescence intensity of lipid droplets after SAH (P<0.01). Figure 12 (AB in the text). Next, the expression levels of key enzymes involved in lipid metabolism, ACSL4, ALOX5, ALOX12, and ALOX15, were measured after SAH. These also showed a significant increase in expression levels after SAH, followed by a significant decrease after treatment with EGCG-NPs and siBLVRA, with a more pronounced decrease in the EGCG-NPs group (P<0.01). Figure 13 (AD in the middle). To further verify the relationship and regulatory mechanism of EGCG-NPs in improving lipid peroxidation and BLVRA, the expression of ACSL4 was first detected by immunofluorescence. The results showed that lipid peroxidation occurred in the brain tissue of mice after SAH, and EGCG-NPs could effectively reduce the degree of lipid peroxidation (P<0.01, Figure 14 (AB in the figure); Next, in this embodiment, immunofluorescence was used to co-stain ACSL4 and BLVRA. As shown in the figure, after SAH, BLVRA significantly increased and was widely expressed in the nucleus, and could co-localize with ACSL4 in the nucleoplasm. In the EGCG-NPs treatment group, BLVRA was significantly reduced and more expressed in the cytoplasm, showing colocalization with ACSL4. Therefore, this embodiment speculates that EGCG-NPs improve the occurrence of lipid peroxidation after SAH by reducing BLVRA and inhibiting BLVRA nuclear migration. Figure 15 ).

[0060] 4. EGCG-NPs reduce neuronal ferroptosis by inhibiting BLVRA expression after SAH. Immunofluorescence assays were used to verify whether ferroptosis occurred after SAH. Results showed that GPX4 expression was significantly lower in the SAH group than in the Sham group, while expression significantly increased after intervention with EGCG-NPs and siBLVRA. The EGCG-NPs group showed even higher GPX4 expression (P<0.01). Figure 16 (AB in the text). To further clarify the cell types susceptible to ferroptosis, this embodiment used immunofluorescence double staining to detect the localization of GPX4 in microglia, astrocytes, and neurons. The results showed that GPX4 signaling was weakly expressed in all three types of neurons after SAH, while GPX4 expression was significantly increased in astrocytes and microglia after EGCG-NPs pretreatment (P<0.01). Figure 17-19 ).

[0061] 5. EGCG-NPs improve neurological function deterioration after SAH by downregulating BLVRA. Next, this example investigated whether EGCG-NPs affect neurological prognosis by downregulating BLVRA. Garcia scores in each experimental group showed that, compared to the Sham group, the SAH group mice had significantly lower scores, weaker voluntary movement and climbing abilities, limited forelimb extension, and no obvious response from whiskers on both sides of the body, indicating significant neurological deficits. Compared to the SAH group, EGCG-NPs increased neurological function scores, improved responsiveness, and significantly improved neurological dysfunction, which was statistically significant (P<0.01). Figure 20 The siBLVRA group also improved neurological dysfunction after SAH. Next, to assess whether EGCG-NPs protected against neurological deficits caused by cell damage by downregulating BLVRA, a short-term neurobehavioral assessment was performed on mice 48 hours after SAH. The results showed that the SAH group mice had reduced motor ability, decreased movement speed, longer rest time, and shorter path length compared to the normal group (P<0.01). Figure 20 (B, C, E in the original text). However, the siBLVRA group mice showed a stronger neuroprotective effect, restoring the impaired swimming behavior of SAH mice to normal levels. Figure 20 In the EGCG-NPs group, the neurological function scores of the mice were not significantly different from those of the normal group, and all indicators showed that the recovery effect was better than that of the siBLVRA group. Therefore, it is believed that EGCG-NPs improves the neurological function damage in mice after SAH by downregulating BLVRA expression.

[0062] In summary, this invention opens up a promising therapeutic approach for improving the recovery of neurological function in patients with hemorrhagic stroke.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of biliverdin reductase inhibitors in the preparation of drugs for treating hemorrhagic stroke.

2. The application according to claim 1, characterized in that, The inhibitors of biliverdin reductase include the following (a) to (c): (a) Substances that inhibit the activity of biliverdin reductase protein; (b) Substances that inhibit the expression of the gene encoding biliverdin reductase; (c) Substances that inhibit biliverdin reductase nuclear translocation.

3. As described in claim 2, characterized in that, The substances that inhibit biliverdin reductase protein activity and / or inhibit biliverdin reductase nuclear translocation include epigallocatechin gallate.

4. The application according to claim 2, characterized in that, The substance that inhibits the expression of the biliverdin reductase-encoding gene includes siRNA.

5. The application according to claim 4, further wherein the siRNA comprises a double-stranded RNA consisting of two single-stranded RNAs shown in SEQ ID NO.1 and SEQ ID NO.

2.

6. The application according to claim 1, further comprising the fact that the inhibitor of biliverdin reductase achieves the treatment of hemorrhagic stroke by means of (i) to (iv) the following: (i) Alleviating railway overloading; (ii) Improve lipid peroxidation; (iii) Reduces ferroptosis in nerve cells; (iv) Improve deterioration of neurological function.

7. The application according to any one of claims 1-6, characterized in that, The biliverdin reductase includes biliverdin reductase A.

8. A drug for treating hemorrhagic stroke, characterized in that, The drug contains an inhibitor of biliverdin reductase.

9. The medicament according to claim 8, characterized in that, The inhibitors of biliverdin reductase include the following (a) to (c): (a) Substances that inhibit the activity of biliverdin reductase protein; (b) Substances that inhibit the expression of the gene encoding biliverdin reductase; (c) Substances that inhibit the nuclear translocation of biliverdin reductase; The preferred form of (a) and / or (c) is epigallocatechin gallate; The substance described in (b) is preferably a substance that inhibits the expression of the biliverdin reductase-encoded gene; Preferably, the substance that inhibits the expression of the biliverdin reductase-encoding gene includes siRNA; Preferably, the siRNA comprises a double-stranded RNA consisting of two single-stranded RNAs as shown in SEQ ID NO.1 and SEQ ID NO.

2.

10. The medicament according to any one of claims 8-10, characterized in that, The biliverdin reductase includes biliverdin reductase A.

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