Use of pdgfb in the preparation of a medicament for treating cerebral hemorrhage and its secondary brain injury

Abstract

The application discloses application of PDGFB in preparation of a medicine for treating cerebral hemorrhage and secondary brain injury thereof. PDGFB promotes migration of oligodendrocyte precursor cells to the perihematoma after ICH, promotes myelin repair, thereby maintaining white matter integrity and improving recovery of nerve function, belongs to a new use of PDGFB in white matter repair after cerebral hemorrhage, provides a brand-new drug target for clinical treatment of secondary white matter injury after cerebral hemorrhage, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of PDGFB in the preparation of drugs for treating cerebral hemorrhage and its secondary brain injury. Background Technology

[0002] Intracerebral hemorrhage (ICH) is a significant type of hemorrhagic stroke, accounting for approximately 10%-15% of all stroke events. This disease is caused by the rupture of a blood vessel in the brain, allowing blood to enter the brain parenchyma and form a hematoma. Although its incidence is lower than that of ischemic stroke, statistics show that ICH accounts for a high proportion of stroke-related deaths and is often accompanied by severe neurological deficits, placing significant pressure on patients' quality of life, family care, and social medical resources.

[0003] Following a brain hemorrhage, the local hematoma directly compresses surrounding brain tissue, triggering a series of complex pathological changes, including changes in hematoma volume, release of blood components, and various secondary injury responses. These pathological processes interact, further exacerbating brain tissue damage. Currently, clinical treatment primarily includes symptomatic supportive medication and surgical removal of the hematoma, which can reduce the risk of death in the acute phase to some extent. However, even after hematoma removal, secondary brain injury may continue to progress, becoming a significant factor affecting the patient's long-term neurological recovery.

[0004] Following involuntary hemorrhage (ICH), white matter damage typically leads to myelin sheath destruction, affecting nerve impulse conduction velocity and consequently neurological function. Loss of myelin sheath and insufficient myelin formation are significant causes of neurological dysfunction. Therefore, promoting myelin formation is considered a potentially effective therapeutic strategy and a key process in white matter repair. Myelin sheaths are multilayered membrane structures formed by the repeated wrapping of axons by the membranous processes of oligodendrocytes. Myelin regeneration is initiated by the recognition of demyelinating lesions by oligodendrocytes (OPCs), which have the ability to differentiate into mature oligodendrocytes, thereby forming myelin sheaths and encapsulating axons, fundamental to maintaining white matter integrity.

[0005] In the central nervous system, the proliferation, migration, and differentiation of OPCs are regulated by other glial cells. As "observers" of the central nervous system, microglia are crucial for maintaining myelin regeneration and white matter health. Clinical studies have reported that patients with colony-stimulating factor 1 receptor mutations exhibit dysplasia of brain white matter when microglia are absent. Professor Veronique E. Miron's research group at the University of Edinburgh discovered that microglia in adult mice play a key role in homeostasis, demyelination, and myelin regeneration. Microglia regulate the proliferation, differentiation, and migration of OPCs through polarization and the secretion of factors such as osteopontin and HMGB1.

[0006] In ICH conditions, our previous studies found that the quantity and intensity of cell communication between microglia and OPCs were significantly downregulated during the acute phase of ICH. Specifically, the weakened interaction between platelet-derived growth factor B (PDGFB) and platelet-derived growth factor receptor α (PDGFRα) was particularly significant. However, to date, there are no studies on the migration relationship between PDGFB and OPCs in the context of intracerebral hemorrhage. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide the application of PDGFB in the preparation of drugs for treating cerebral hemorrhage and its secondary brain injury. Through experiments, it is further demonstrated that PDGFB promotes myelin repair by facilitating OPC migration, thereby alleviating white matter damage after ICH.

[0008] To address the problems in the existing technology, the technical solution adopted by this invention is as follows: Application of PDGFB in the preparation of drugs for the treatment of cerebral hemorrhage and its secondary brain injury.

[0009] Preferably, the PDGFB treats secondary brain injury by promoting the migration of oligodendrocyte precursor cells to the peri-hematoma area.

[0010] Preferably, the PDGFB promotes myelin repair after cerebral hemorrhage by enhancing the migration ability of oligodendrocyte precursor cells.

[0011] Preferably, the dosage of PDGFB is 1.6 μg / kg.

[0012] A pharmaceutical composition for treating cerebral hemorrhage and its secondary brain injury, comprising a therapeutically effective amount of PDGFB and a pharmaceutically acceptable carrier.

[0013] Preferably, the dosage form of the pharmaceutical composition is an injection. Beneficial effects

[0014] Compared with existing technologies, this invention relates to the application of PDGFB in the preparation of drugs for treating intracerebral hemorrhage and its secondary brain injury. It reveals for the first time a unique microenvironmental communication impairment mechanism in the acute phase of adult intracerebral hemorrhage (ICH) and achieves precise targeted rescue. Specifically, single-cell transcriptome sequencing and Western blotting experiments confirmed that after ICH, the quantity and intensity of cell communication between microglia and oligodendrocyte precursor cells (OPCs) around the hematoma specifically decline, with the most significant disruption being the PDGFB-PDGFRα signaling axis, and the expression of endogenous PDGFB in the local microenvironment showing time-dependent depletion. Based on this newly discovered pathophysiological feature, this invention targets and supplements exogenous PDGFB, precisely compensating for the specific factor deficiency in the local microenvironment after ICH. Unlike conventional methods that blindly screen for growth factors, this invention successfully restarts the microglia-OPC signaling interaction network, driving the chemotactic migration of OPCs to the hematoma periphery from the source, providing a highly specific and irreplaceable new therapeutic target for secondary white matter injury in adult intracerebral hemorrhage. The specific manifestations are as follows: (1) Combined with DMBP staining and electron microscopy G-ratio objectively confirmed that the PDGFB of the present invention can effectively reduce myelin damage around hematoma and comprehensively and significantly improve the neurobehavioral functions of mice such as motor coordination, sensory balance and spatial learning and memory.

[0015] (2) The present invention can exert significant neuroprotective and repair effects with only 1.6 μg / kg of local administration. It can exert significant neuroprotective and repair effects with extremely low dosage, effectively avoiding the systemic toxic side effects that may be caused by conventional high-dose administration, and has extremely high clinical safety.

[0016] (3) The PDGFB of the present invention is an endogenous growth factor with low immunogenicity and good biocompatibility. When it is combined with a pharmaceutically acceptable carrier to form a composition, its physicochemical properties are stable, and its advantages as a drug and its prospects for clinical translation are very clear. Attached Figure Description

[0017] Figure 1 (A) Single-cell sequencing analysis of changes in the quantity and intensity of microglia-OPC cell communication after ICH; Figure 1 (B) Cell chat analysis shows the changes in the interaction strength of each ligand pair between microglia and OPC; Figure 1 (C) shows the specific expression levels of PDGFB and PDGFRα in each cell population as mapped by single-cell sequencing; Figure 1 (D) shows the dynamic changes in PDGFB protein expression in the brain tissue surrounding hematoma in mice at different time points after ICH, as detected by Western blot. Figure 2 (A) is the procedure for a behavioral experiment; Figure 2 (B) After supplementing with PDGFB, the rotarod test was used to evaluate the motor coordination of the four groups of mice; Figure 2 (C) The Garcia score was used to evaluate the motor, sensory, and balance abilities of the four groups of mice. Figure 2 (DE) represents the open field test used to evaluate the autonomous exploratory behavior and stress levels of the four groups of mice. Figure 2 (FG) represents the Y-maze experiment comparing the spatial learning and memory abilities of four groups of mice; Figure 3 (A) Brain tissue sections of mice and control mice 12 hours after PDGFB orthotopic injection and DMBP staining to detect myelin damage around the hematoma; Figure 3 (BC) shows the electron microscopy and ultrastructural G-ratio of cerebral hemorrhage in mice and control mice 12 hours after PDGFB orthotopic injection; Figure 4 (A) Brain tissue sections of mice and control mice 12 hours after PDGFB orthotopic injection and NG2 staining to detect the distribution and quantity of OPC around the hematoma; Figure 4 (BC) shows the migration of primary OPC in the PDGFB intervention group and the control group and provides statistical results. Detailed Implementation

[0018] 1 Method 1.1 Laboratory Animals This animal experiment was authorized by the Ethics Committee of the First Affiliated Hospital of Soochow University and complied with ARRIVE guidelines. Adult male C57BL6 / J mice, weighing 20-25g, were purchased from the Chinese Academy of Sciences (Shanghai, China). Animals were housed under standard animal conditions: temperature 25±1℃, humidity 50-60%, 12-hour light / dark cycle, and free access to food and water. All animals were randomly assigned to different groups, and data were evaluated under blinded conditions.

[0019] 1.2 Drug Management For the treatment of cerebral hemorrhage, animals were randomly divided into four groups: (i) vehicle sham control, (ii) vehicle ICH control, (iii) PDGFB sham control, and (iv) PDGFB ICH treatment. Mice in groups (ii) and (iv) were stereotactically injected with recombinant PDGFB protein (1.6 μg / kg). The PDGFB used in the mice was purchased from MCE (HY-P73352, USA). On day 0, mice in groups (ii) and (iv) were stereotactically injected with autologous blood (30 μl) at the following locations: anteroposterior +0.1 mm, mid-lateral +2 mm, and dorsal-ventral -3.5 mm from the bregma. Groups (i) and (iii) underwent sham surgery without blood transfusion.

[0020] 1.3 Establishment of a mouse ICH model Adult male C57BL6 / J mice were anesthetized with isoflurane. Non-anticoagulated whole blood was collected from the medial canthal vein using a Hamilton 50 μl microsyringe without the addition of any anticoagulant. The head was fixed within a stereotactic frame, and the mice wore masks and were continuously anesthetized throughout the procedure. After a midline incision and skull exposure, 30 μl of autologous blood was injected into the striatum using precise stereotactic coordinates (2.0 mm left, 0.5 mm anterior, 3.5 mm relative to the bregma depth). The injection rate was 2 μl / min. After injection, the needle was left in place for 15 minutes to prevent backflow, then carefully removed at 1 mm / min, and the scalp was sutured. Postoperatively, the mice were placed in a warm chamber for 15 minutes to maintain body temperature and promote gradual recovery.

[0021] PDGFB was administered 60 minutes later. The total volume of a single intracerebral injection of PDGFB was strictly controlled at 2 μl (i.e., the working solution concentration was 20 ng / μl). Following the standard physicochemical properties of recombinant proteins and the supplier's (MCE) recommendations, the PDGFB lyophilized powder was first dissolved in sterile PBS (phosphate-buffered saline) containing 0.1% BSA (bovine serum albumin) to prepare a stock solution before use. Immediately before use, it was further diluted with sterile PBS to the required working concentration. During the procedure, a Hamilton microsyringe was used, and the PDGFB was injected slowly and uniformly at a rate of 0.2 μl / min. After injection, the microsyringe needle was left in place for 10 minutes to allow the drug to fully diffuse within the brain parenchyma before slowly withdrawing the needle to completely avoid reflux and ensure complete drug colonization in the target area surrounding the hematoma.

[0022] 1.4 Immunofluorescence analysis After perfusing mouse brains with 4% paraformaldehyde, the specimens were fixed and sectioned into 10 μm thick frozen sections. Antigen retrieval was performed using frozen section antigen retrieval buffer for 8 min. Then, a blocking step was performed at 37°C for 1 hour using QuickBlock™ blocking buffer for Immunol stain (Beyotime Biotechnology, China). Subsequently, primary antibodies were applied and incubated overnight at 4°C. The primary antibodies used were: Myelin Basic Protein antibody (1:300, #83683, CST, USA), DMBP antibody (1:300, 4229845, abcam, USA), PDGFB (1:200, NBP1-58279, novus, USA), PDGFRa (1:200, YA3824, MCE, USA), and Goat Iba1 (1:200, ab289874, abcam, USA). After washing three times with phosphate-buffered saline containing Tween 20 (PBST), the cells were incubated with secondary antibody at room temperature for 1 hour. After another three PBST washes, the nuclei were reverse stained with DAPI (Invitrogen, USA) to highlight the nuclei. Finally, the cells were observed under a fluorescence microscope. The secondary antibodies used were: Alexa Fluor™ 488 (1:300, A21202, Thermo Fisher), Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555 (1:300, AA31572, Thermo Fisher), and Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (1:300, A11055, Thermo Fisher).

[0023] 1.5 Behavioral Experiments Behavioral experiments included three behavioral assessment methods: rotata, Garcia score, open field, and Y-maze. A quiet laboratory environment was maintained to encourage spontaneous activity in the mice.

[0024] Rotary bar test: Mice were tested on an accelerated rotating rod, with the speed gradually increased from 5 rpm to 40 rpm over 300 seconds. Each mouse underwent three trials, and the average value was taken.

[0025] Modified Garcia Score: This invention uses the Modified Garcia Score to assess neurological deficits in mice. This scoring system is a modification of the standard established by Garcia et al. (see Stroke. 1995;26(4):627-634). Six parameters were assessed in mice before and 3, 7, and 14 days after brain hemorrhage surgery: spontaneous activity, proprioception, tactile response, limb symmetry, forelimb extension, and climbing. All tests were performed blinded, with scores given by researchers unaware of the group assignments, ranging from 0 to 3. The specific six assessment indicators and scoring details are as follows: A. Spontaneous activity: Observe the animal's free movement and exploration within the test cage for 5 minutes.

[0026] B. Symmetry of limb movement: Lift the animal by its tail to suspend it in the air and observe the extension and movement of its limbs.

[0027] To observe the animal's forelimb extension and walking, lift its tail so that its hind limbs are suspended in the air, leaving only its forelimbs touching the table. Observe its forelimb support and walking ability.

[0028] Climbing ability: Place the animal on an inclined or vertical grid / wire mesh with gripping points and observe its climbing ability and gripping strength of both forelimbs.

[0029] Proprioception: Use a blunt-tipped probe or cotton swab to touch the sides of the animal's trunk or lower abdomen.

[0030] Whisker tactile response: Gently touch the whiskers on both sides of the animal with a cotton swab or a fine needle and observe the reaction of the head and body.

[0031] Open field: Used to assess exploratory behavior and neuropsychiatric status in mice. The experimental setup measures 40×50×40 cm. 3 The plastic testing box was divided into a 4x4 grid at the bottom, with the four middle squares forming the central area. Mice were placed in the center, and spontaneous movement was recorded for 10 minutes. Average speed, total distance traveled, time spent in the central area, and number of entries were measured. Adaptation training was conducted for five consecutive days. Y-maze test: The maze consists of three identical arms in a Y-shape, each arm approximately 30cm long, 10cm wide, and 15cm high, with an angle of 120° between the arms. Each group included a control group and an experimental group, with 10 mice in each group. Each mouse was placed at the end of one arm and allowed to explore freely for 5 minutes. The order in which the mice entered each arm was recorded. The spontaneous alternation rate (the percentage of consecutive entries into all three arms out of the total possible entries) reflects spatial memory and spontaneous exploration ability; the frequency of entering new arms reflects the level of spatial memory.

[0032] 1.6 Primary cell culture: P0 newborn mice were sterilized and quickly cervically decapitated. The brains were then washed with pre-cooled HBSS, and the meninges and skull were removed. The brain hemispheres were chopped and placed in pre-warmed trypsin containing 0.25% EDTA at 37°C for 13 minutes, gently mixing every 4 minutes. Complete culture medium (DMEM / F) was then added. 12 Digestion was terminated with 10% FBS and 1% antibiotics. The cells were pipetted 30 times to ensure homogeneity, filtered to remove undigested tissue fragments, and centrifuged at 300×g for 5 minutes. Cells were resuspended in DMEM medium and cultured in Poly-D-lysine-coated flasks with OPCs medium (containing PDGF-AA and FGF2). On days 8-10, cells were shaken at 180 rpm and 37°C for 2 hours. The supernatant was removed, and the medium was replaced with fresh medium. The cells were then agitated at 250 rpm for 18 hours. The suspended OPCs were collected and transferred to uncoated culture dishes for 1 hour (to remove residual astrocytes / microglia). Cells were then cultured in medium containing 1% N2 and 1% B... 27 10 ng / mL PDGF-AA / FGF2 in OPCs medium (DMEM / F 12 +1% N2, 1% B 27 Resuspension inoculation with 10 ng / mL PDGF-AA / FGF2 + 1% double antibody.

[0033] 1.7 OPC Migration Experiment a. Compartment Coating: OPCs are highly adherent cells and have specific requirements for the substrate. The upper filter membrane of the Transwell compartment (usually with a pore size of 8 µm to allow cells to pass through) needs to be pre-coated with Poly-D-Lysine on the back side (i.e., the side facing out) or both sides, and then incubated overnight in an incubator before being washed with PBS for use.

[0034] b. Cell starvation treatment: 12 hours before the experiment, OPCs were replaced with serum-free or low-serum (and without high concentrations of chemokines) medium to eliminate signaling pathway activation under basal conditions, synchronize cells and increase their sensitivity to lower chamber chemokine signals.

[0035] c. Cell resuspension and counting: Digest the OPCs using a gentle digestion solution. OPCs are very fragile and must be handled with extreme care. After centrifugation, resuspend the cells in serum-free medium and adjust the cell density.

[0036] e. Plating and adding the drug to the lower chamber: Add culture medium containing Pdgfb (usually 500-600 µL) to the lower chamber.

[0037] Upper chamber: Gently place into the 24-well plate, avoiding the formation of air bubbles. Add 100-200 µL of the prepared OPC cell suspension to the upper chamber.

[0038] f. Incubation: Place the culture plate in a 37°C, 5% CO2 incubator for incubation.

[0039] g. Fixation and Staining: Remove the chamber and aspirate the liquid from the upper chamber. Gently wipe away any unmigrated cells from the inner side of the upper chamber's bottom membrane using a cotton swab. Fix the chamber in ice-cold methanol for 15-20 minutes. Stain with 0.1% crystal violet.

[0040] 1.8 Single-cell transcriptome sequencing and cell communication analysis Brain tissue surrounding hematoma and tissue from the sham-operated group during the acute phase of ICH in mice were extracted, and single-cell suspensions were prepared for 10xGenomics single-cell transcriptome sequencing. Cell clustering and dimensionality reduction analysis was performed using Seurat software to specifically extract microglia and oligodendrocyte precursor (OPC) subsets. The ligand-receptor interaction network, communication quantity, and strength between these two cell types were quantitatively analyzed using the CellChat software package.

[0041] 1.9 Western blot analysis Total protein was extracted from the brain tissue surrounding hematoma in mice at different time points after ICH (e.g., 1 day, 3 days, and 7 days post-surgery). After separation by SDS-PAGE gel electrophoresis, the protein was transferred to a PVDF membrane. The membrane was incubated with a PDGFB-specific primary antibody (Mouse anti-PDGF-B monoclonal antibody, Clone F-3, catalog number sc-365805, 1:300, Santa Cruz Biotechnology, USA). Dynamic changes in PDGFB protein expression were detected by ECL luminescence assay, with Tublin as an internal control.

[0042] 1.10 Statistical Analysis Data collection and analysis were performed blinded. Results are expressed as mean ± SD and statistical evaluation was performed using GraphPad Prism Software (version 9.4.1). One-way and two-way ANOVA were used for analysis of continuous variables among multiple groups. The Kolmogorov-Smirnov test was used to test the normality of all datasets for each group. p < 0.05 was considered significant.

[0043] 2 Results To investigate the specific microenvironmental molecular mechanisms of secondary white matter injury following ICH, we performed single-cell transcriptome sequencing on the hematoma tissues of ICH mice and control groups. Figure 1 As shown in Figure A, cell communication in the microenvironment undergoes significant changes after ICH, with both the number of interactions and interaction strength between microglia and OPCs showing a significant downward trend, suggesting that the signaling network between them collapses after cerebral hemorrhage. Further analysis of receptor-ligand pairs was conducted using CellChat. Figure 1 (Middle B), the results showed that among the many blocked communication pathways, the weakening of the PDGFB-PDGFRα signaling axis interaction was particularly significant. Combined with sequencing expression mapping ( Figure 1 As shown in Figure C, PDGFB is mainly secreted by specific cells in the microenvironment (such as microglia / macrophages), while its receptor PDGFRα is highly specifically expressed on the surface of OPCs. To verify this transcriptome-level finding, Western blotting was performed on perihemorrhagic brain tissue from mice at different time points after ICH. Figure 1 (D). The results confirmed that, with the progression of ICH, the expression level of endogenous PDGFB protein in the target area decreased significantly in a time-dependent manner.

[0044] Figure 2 As shown, (A) the behavioral experimental procedure. A mouse ICH model was established, and sham-operated group, ICH group, PDGFB solvent control group, and PDGFB group were set up for behavioral experiments, including rotarod, open field, Garcia score, and Y maze; (B) the rotarod test evaluated the motor coordination of the four groups of mice; (C) the Garcia score evaluated the motor, sensory, and balance of the four groups of mice; (DE) the open field test evaluated the autonomous exploratory behavior and tension of the four groups of mice; (FG) the Y maze test compared the spatial learning and memory abilities of the four groups of mice. After PDGFB supplementation, it was found that the neurobehavioral recovery in the acute phase of ICH in mice was improved, and PDGFB improved the neurological functional damage after ICH.

[0045] Figure 3(A) Brain tissue sections of mice and control mice 12 hours after PDGFB orthotopic injection and DMBP staining to detect myelin damage around the hematoma; Figure 3 (BC) shows the electron microscopy and ultrastructural G-ratio of cerebral hemorrhage 12 hours after orthotopic injection of PDGFB in mice and control mice. As can be seen from the figure, after administration of recombinant protein PDGFB, the DMBP staining signal around the hematoma in the acute phase of ICH in mice was significantly reduced, suggesting that PDGFB can alleviate myelin destruction.

[0046] Figure 4 (A) Brain tissue sections of mice and control mice 12 hours after PDGFB orthotopic injection and NG2 staining to detect the distribution and quantity of OPC around the hematoma; Figure 4 (BC) shows the migration and statistical analysis of primary OPC in the PDGFB intervention group and the control group. Figure 4 It can be seen that after administration of recombinant protein PDGFB, the number of OPCs around the hematoma in the acute phase of ICH in mice increased relatively; the migration ability of primary OPC cells was significantly enhanced.

[0047] In summary, this invention, through single-cell transcriptome sequencing and Western blotting experiments, confirms that after ICH (intracerebral hemorrhage), the quantity and intensity of cell communication between microglia and oligodendrocyte precursor cells (OPCs) around the hematoma specifically decline, with the most significant disruption being the PGFFB-PDGFRα signaling axis, and endogenous PGFFB expression in the local microenvironment showing time-dependent depletion. Based on this novel pathophysiological finding, this invention targets and supplements exogenous PGFFB, precisely compensating for the specific factor deficiency in the local microenvironment after ICH. Unlike conventional methods that blindly screen for growth factors, this invention successfully restarts the microglia-OPC signaling interaction network, driving the chemotactic migration of OPCs to the hematoma periphery from the source, providing a highly specific and irreplaceable new therapeutic target for secondary white matter injury in adult intracerebral hemorrhage.

Claims

1. Application of PDGFB in the preparation of drugs for the treatment of cerebral hemorrhage and its secondary brain injury.

2. The application according to claim 1, characterized in that, The PDGFB treats secondary brain injury by promoting the migration of oligodendrocyte precursor cells to the periphery of the hematoma.

3. The application according to claim 2, characterized in that, The PDGFB promotes myelin repair after cerebral hemorrhage by enhancing the migration ability of oligodendrocyte precursor cells.

4. The application according to claim 1, characterized in that, The dosage of PDGFB is 1.6 μg / kg.

5. A pharmaceutical composition for treating cerebral hemorrhage and its secondary brain injury, characterized in that, It comprises a therapeutically effective amount of PDGFB as described in claim 1 and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that, The dosage form of the pharmaceutical composition is an injection.

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