Use of prolyl 3-hydroxylase 2 in the preparation of a medicament for treating ischemic stroke
By using prolyl 3-hydroxylase 2 to prepare drugs, the activity and migration of cerebral microvascular endothelial cells are promoted, which solves the side effect problem of existing drugs for the treatment of ischemic stroke and achieves effective treatment of ischemic stroke.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HUAZHONG AGRI UNIV
- Filing Date
- 2025-08-15
- Publication Date
- 2026-05-29
Smart Images
Figure CN121059776B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically to the application of prolyl 3-hydroxylase 2 in the preparation of drugs for treating ischemic stroke. Background Technology
[0002] Stroke, also known as apoplexy or cerebrovascular accident, is an acute cerebrovascular disease with a rising incidence rate worldwide. According to the latest reports, more than 13 million new stroke cases occur annually, affecting one in four adults over the age of 25. Stroke is a broad term encompassing various central nervous system disorders caused by vascular factors, further subdivided into ischemic and hemorrhagic strokes. Ischemic stroke, involving infarction of the central nervous system leading to the death of brain, spinal cord, or retinal cells, accounts for 71% of all strokes worldwide.
[0003] Recent research on neurovascular networks has revealed the importance of close communication between neurons and blood vessels for brain function. Angiogenesis, the formation of new blood vessels, is a key protective mechanism for promoting nerve regeneration and functional recovery after stroke. Studies have shown that cerebral ischemia can induce transient angiogenesis, which is crucial for angiogenesis in the peri-infarct area, effectively reducing infarct volume, promoting neuronal survival, and restoring neurovascular network function. Increasing evidence suggests that endothelial cell angiogenesis plays a vital role in the repair of brain microvascular endothelial cells (BMECs) in ischemic stroke. Specifically, after stroke, endothelial cells can promote neurogenesis and improve neurological function by releasing microvesicles containing neural transcription factors, which can transform astrocytes into neural progenitor cells.
[0004] Vascular endothelial growth factor-A (VEGF-A), a core regulator of angiogenesis, plays a crucial role in the treatment of ischemic stroke (IS). However, clinical studies have shown that direct intervention with VEGF-A can easily induce side effects such as vascular structural abnormalities and blood-brain barrier leakage, limiting its therapeutic application. To date, the functional mechanisms and translational value of P3H2 in the treatment of ischemic stroke have not been explored. Summary of the Invention
[0005] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide an application of prolyl 3-hydroxylase 2 in the preparation of drugs for treating ischemic stroke, thereby solving the technical problem of side effects existing in current drugs for treating ischemic stroke.
[0006] To achieve the above-mentioned technical objectives, the present invention provides the application of prolyl 3-hydroxylase 2 in the preparation of drugs for treating ischemic stroke.
[0007] Furthermore, this invention also proposes the application of prolyl 3-hydroxylase 2 in the preparation of drugs that inhibit oxygen-glucose deprivation / reoxygenation-induced damage to brain microvascular endothelial cells.
[0008] In any embodiment, the drug alleviates the effects of oxygen-glucose deprivation / reoxygenation on the vitality of brain microvascular endothelial cells and the degree of oxidative damage by prolyl 3-hydroxylase 2.
[0009] In any embodiment, the drug promotes the migration, lumen formation and / or maintenance of the integrity of brain microvascular endothelial cells via prolyl 3-hydroxylase 2.
[0010] Furthermore, this invention also proposes the application of prolyl 3-hydroxylase 2 in the preparation of drugs for alleviating brain injury, reducing blood-brain barrier damage, relieving cerebral infarction, or relieving brain inflammation.
[0011] In any embodiment, the drug relieves brain injury caused by MCAO / R, reduces blood-brain barrier damage caused by MCAO / R, relieves cerebral infarction caused by MCAO / R, or relieves brain inflammation caused by MCAO / R by prolyl 3-hydroxylase 2.
[0012] In addition, the present invention also proposes a drug for treating ischemic stroke, comprising an effective ingredient that promotes the expression of prolyl 3-hydroxylase 2.
[0013] In any embodiment, the active ingredient is delivered via a carrier.
[0014] In any embodiment, the vector is a plasmid vector or a viral vector.
[0015] Compared with the prior art, the beneficial effects of the present invention include: the P3H2 gene proposed in the present invention can enhance the activity, migration ability, lumen formation ability and maintenance of integrity of brain microvascular endothelial cells, and at the same time has the effect of alleviating oxidative damage, and can treat ischemic stroke, thereby avoiding the side effects of existing ischemic stroke treatment drugs.
[0016] The P3H2 gene proposed in this invention can alleviate brain injury in MCAO / R mice and reduce blood-brain barrier damage. This invention expands the pharmaceutical application of the P3H2 gene. The P3H2 gene provided by this invention serves as a therapeutic target and method for ischemic stroke. Attached Figure Description
[0017] Figure 1 This is a graph showing the effect of OGD / R on P3H2 expression in HBMEC according to Example 1 of the present invention.
[0018] Figure 2 This is a graph showing the effect of P3H2 overexpression on HBMEC activity after OGD / R treatment in Example 2 of this invention.
[0019] Figure 3 This is a graph showing the effect of P3H2 overexpression on oxidative damage of HBMEC treated with OGD / R in Example 3 of the present invention (scale bar: 10 μm).
[0020] Figure 4 This is a graph showing the effect of P3H2 overexpression on the migration ability of HBMECs treated with OGD / R in Example 4 of this invention (scale bar: A-100μm; B-40μm).
[0021] Figure 5 This is a graph showing the effect of P3H2 overexpression on maintaining the integrity of HBMEC treated with OGD / R in Example 5 of the present invention.
[0022] Figure 6 This is a graph showing the effect of P3H2 overexpression on the lumen formation ability of OGD / R treated HBMECs in Example 6 of the present invention (scale bar: 100 μm).
[0023] Figure 7 This is a graph showing the effect of P3h2 overexpression on cerebral infarction in MCAO / R mice in Example 7 of this invention.
[0024] Figure 8 This is a graph showing the effect of P3h2 overexpression on alleviating brain inflammation in MCAO / R mice in Example 8 of this invention.
[0025] Figure 9 This is a diagram showing the results of P3h2 overexpression promoting the repair of the blood-brain barrier in MCAO / R mice in Example 9 of this invention. Detailed Implementation
[0026] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60–120 and 80–110 are listed for a specific parameter, it is understood that ranges of 60–110 and 80–120 are also expected. Furthermore, if minimum range values of 1 and 2 are listed, and if maximum range values of 3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.
[0027] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.
[0028] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).
[0029] This specific embodiment provides the application of prolyl 3-hydroxylase 2 (i.e., P3H2) in the preparation of drugs for treating ischemic stroke.
[0030] This specific embodiment also provides the application of prolyl 3-hydroxylase 2 in the preparation of a drug that inhibits oxygen-glucose deprivation / reoxygenation-induced damage to brain microvascular endothelial cells.
[0031] In some embodiments, the drug alleviates the effects of oxygen-glucose deprivation / reoxygenation on the vitality of brain microvascular endothelial cells and the degree of oxidative damage through prolyl 3-hydroxylase 2.
[0032] In some embodiments, the drug promotes the migration, lumen formation, and / or maintenance of the integrity of brain microvascular endothelial cells via prolyl 3-hydroxylase 2.
[0033] Furthermore, this specific embodiment proposes the application of prolyl 3-hydroxylase 2 in the preparation of drugs to alleviate brain injury, reduce blood-brain barrier damage, alleviate cerebral infarction, or alleviate brain inflammation.
[0034] In some embodiments, the drug relieves brain damage caused by MCAO / R, reduces blood-brain barrier damage caused by MCAO / R, relieves cerebral infarction caused by MCAO / R, or relieves brain inflammation caused by MCAO / R by prolyl 3-hydroxylase 2.
[0035] Furthermore, this specific embodiment also proposes a drug for treating ischemic stroke, including an effective ingredient that promotes the expression of prolyl 3-hydroxylase 2.
[0036] In some embodiments, the active ingredient is delivered via a vector, which is a plasmid vector or a viral vector.
[0037] This invention reveals a novel pharmaceutical application of the P3H2 gene in the treatment of ischemic stroke. It exerts its therapeutic effects through multiple mechanisms, including enhancing the activity of cerebral microvascular endothelial cells, promoting cell migration and lumen formation, maintaining vascular integrity, and alleviating oxidative damage. Based on these functional characteristics, the P3H2 gene can serve as a potential target for ischemic stroke treatment, providing a theoretical basis and technical pathway for developing novel treatment strategies.
[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0039] In this invention, the terms "some embodiments," "this embodiment," and examples are used to describe a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.
[0040] If the application documents contain similar descriptions such as "first / second", the following explanation shall be added: In the following description, the terms "first / second / third" are used only to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first / second / third" may be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.
[0041] In this embodiment, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, object A and / or object B can represent three situations: object A exists alone, object A and object B exist simultaneously, and object B exists alone.
[0042] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.
[0043] In the following embodiments, the nucleotide sequence of the P3H2 mRNA is as follows:
[0044]
[0045] Example 1: Effect of OGD / R (oxygen-glucose deprivation / reoxygenation) on P3H2 expression in HBMEC
[0046] This embodiment relates to the application of P3H2 in the preparation of drugs for treating ischemic stroke, specifically involving the detection of the effect of OGD / R on P3H2 expression in HBMEC using quantitative real-time PCR (qRT-PCR) and Western blotting. The experimental methods are as follows:
[0047] The following method was used to establish an in vitro cell model of oxygen-glucose deprivation / reoxygenation (OGD / R): HBMEC cells were seeded into culture plates and cultured in RPMI-1640 complete medium containing serum and penicillin-streptomycin at 37°C and 5% CO2. When the cells reached 70-80% confluence, the medium was replaced with glucose-free medium, and the cells were cultured in a hypoxic chamber at 37°C, 5% CO2, and 95% N2 for 4 h. After the treatment, the cells were replaced with complete medium for reoxygenation for 24 h. Subsequently, cells were collected for qRT-PCR and Western blotting to detect P3H2 expression levels.
[0048] like Figure 1 As shown, OGD / R treatment inhibited the expression level of P3H2 in HBMEC cells.
[0049] Example 2: Effect of P3H2 overexpression on the activity of OGD / R-treated HBMECs (brain microvascular endothelial cells)
[0050] This embodiment involves the CCK-8 assay to detect the effect of P3H2 overexpression on the activity of HBMEC treated with OGD / R. The experimental method is as follows:
[0051] An OGD / R in vitro cell model was constructed, and cells were transfected with the P3H2 overexpression plasmid. Transfection efficiency was detected by qRT-PCR and Western blotting. Cell viability was measured using a CCK-8 assay kit. The OD value at 450 nm for each well was recorded using a microplate reader.
[0052] This study established four cell models: the OE-NC group (untreated normal cells transfected with a negative control plasmid), the OE-P3H2 group (untreated cells transfected with a P3H2 overexpression plasmid), the OGD / R+OE-NC group (OGD / R model cells transfected with a negative control plasmid), and the OGD / R+OE-P3H2 group (OGD / R model cells transfected with a P3H2 overexpression plasmid). This grouping design included controls for gene expression regulation under basal and pathological conditions.
[0053] like Figure 2 As shown, P3H2 overexpression alleviated the downregulation of HBMEC activity caused by OGD / R.
[0054] Example 3: Effect of P3H2 overexpression on oxidative damage of OGD / R treated HBMECs
[0055] This embodiment involves the detection of the effects of malondialdehyde (MDA) and reactive oxygen species (ROS) on the oxidative damage of P3H2 overexpression on OGD / R-treated HBMECs. The experimental methods are as follows:
[0056] An OGD / R in vitro cell model was constructed and transfected with a P3H2 overexpression plasmid. The MDA content and ROS levels in the cells were detected using an MDA content assay kit and a ROS probe, respectively. MDA detection involved recording the optical density (OD) values at 532 nm and 600 nm using a microplate reader, while ROS detection involved recording the fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm using a microplate reader.
[0057] like Figure 3 As shown, P3H2 overexpression alleviated the oxidative damage to HBMEC caused by OGD / R.
[0058] Example 4: Effect of P3H2 overexpression on the migration ability of OGD / R-treated HBMECs
[0059] This embodiment involves the scratch assay and Transwell assay to detect the effect of P3H2 overexpression on the migration ability of OGD / R-treated HBMECs. The experimental methods are as follows:
[0060] An OGD / R in vitro cell model was constructed and transfected with a P3H2 overexpression plasmid.
[0061] The scratch method involved streaking the surface with a pipette tip after OGD / R treatment, observing and photographing the result under a microscope, and recording this as 0h. After culturing in serum-free medium for 24h, the surface was observed and photographed under a microscope, recording this as 24h. Measurement software was used to calculate the recovery of the scratches.
[0062] The Transwell assay involves seeding cells into Transwell chambers (8 μm pore size) after OGD / R treatment. The upper chamber contains serum-free culture medium, while the lower chamber contains complete culture medium. After 24 hours of normal culture, cells are stained with 0.1% crystal violet, observed and photographed under a microscope, and the number of migrated cells is calculated using metrology software.
[0063] like Figure 4 As shown, P3H2 overexpression alleviated the decrease in HBMEC migration ability caused by OGD / R.
[0064] Example 5: Effect of P3H2 overexpression on the maintenance of integrity of OGD / R-treated HBMECs
[0065] This embodiment relates to the effect of P3H2 overexpression on the maintenance of integrity of OGD / R-treated HBMECs by permeability detection. The experimental method is as follows:
[0066] An OGD / R in vitro cell model was constructed. Cells were seeded into Transwell chambers (0.4 μm pore size) and transfected with the P3H2 overexpression plasmid. After OGD / R treatment, the upper layer of the chamber was replaced with medium containing 20 μg / mL FITC-Dextran, and the lower chamber medium was replaced with 650 μL of sterile phosphate-buffered saline (PBS). After incubation at 37°C and 5% CO2 for 1 h, the PBS in the lower chamber was collected, and the fluorescence intensity in the PBS was measured using a microplate reader at an excitation wavelength of 492 nm and an emission wavelength of 518 nm. Simultaneously, Western blotting was performed on cells to detect the expression levels of tight junction proteins (ZO-1, Claudin-5, and Occludin).
[0067] like Figure 5 As shown, P3H2 overexpression alleviates the damage to HBMEC integrity caused by OGD / R.
[0068] Example 6: Effect of P3H2 overexpression on lumen formation ability of OGD / R treated HBMECs
[0069] This embodiment involves a lumen formation assay to detect the effect of P3H2 overexpression on the lumen formation ability of OGD / R-treated HBMECs. The experimental method is as follows:
[0070] An OGD / R in vitro cell model was constructed, and cells were transfected with a P3H2 overexpression plasmid. After treatment, HBMEC cells were cultured in serum-free medium and the cell density was adjusted to 2 × 10⁶ cells / year. 4One specimen per well was inoculated into a 96-well plate coated with substrate gel and incubated at 37°C in a 5% CO2 incubator for 6 hours. After incubation, the specimens were observed and photographed under a microscope, and the number of fulcrums in the lumen was measured using metrology software, followed by statistical analysis.
[0071] like Figure 6 As shown, P3H2 overexpression alleviated the effect of OGD / R on the lumen formation ability of HBMEC.
[0072] Example 7: Effects of P3h2 overexpression on cerebral infarction in MCAO / R mice
[0073] This embodiment involves the effect of TTC detection on cerebral infarction in MCAO / R mice. The experimental method is as follows:
[0074] Healthy 5-week-old male C57BL / 6 mice were selected and injected via tail vein with (5E+13 / mouse) AAV-P3h2 to induce P3h2 overexpression in brain vessels. The control group was injected with AAV-Vehicle empty vector virus. Four weeks after injection, mice were anesthetized with 2% isoflurane, followed by maintenance anesthesia with 1.3% isoflurane, and the mice were fixed in a supine position. After hair removal and disinfection of the neck, the skin was incised in the middle of the left neck, and the muscles and glands were separated to expose the common carotid artery. The vagus nerve was carefully separated and the artery was clamped. The external carotid artery (ECA) and internal carotid artery (ICA) were separated along the common carotid artery toward the head. The distal end of the ECA was ligated, and a small incision was made below the ligation point. A suture was inserted to the clamping point, turned and advanced to the ICA, continuing to penetrate about 8-9 mm until the middle cerebral artery encountered slight resistance and stopped. The wound was sutured and the mice were placed in a 28°C incubator. After 1 hour of ischemia, the thrombus was removed and reperfused. The ECA opening was ligated and the wound was sutured. After the mouse regained consciousness, it was returned to the rearing cage, and the MCAO / R model was completed.
[0075] Twenty-four hours after reperfusion, mice were anesthetized with 1% sodium pentobarbital (50 mg / kg), decapitated, and their brains were placed on ice and cut into four 2 mm thick coronal sections using a brain mold. The sections were stained with 1% TTC solution (covered with coverslips) and incubated at 37°C in the dark for 20 min. After staining, the sections were fixed overnight with 4% paraformaldehyde. Images were taken the next day for infarction assessment.
[0076] This study established three animal models: the Sham group (sham operation group, without induced cerebral ischemia, injected with an empty AAV vector as a control); the MCAO / R+AAV-Vehicle group (MCAO / R model induced, injected with an empty AAV vector); and the MCAO / R+AAV-P3h2 group (MCAO / R model induced, injected with an AAV overexpression vector carrying the P3h2 gene). This grouping design included controls for gene expression regulation under basal and pathological conditions.
[0077] like Figure 7As shown, P3h2 overexpression alleviated the effects of cerebral infarction in MCAO / R mice.
[0078] Example 8: Effect of P3h2 overexpression on alleviating brain inflammation in MCAO / R mice
[0079] This embodiment involves a qRT-PCR experiment to detect the effect of P3h2 overexpression on brain inflammation in MCAO / R mice. The experimental method is as follows:
[0080] The P3h2 gene was overexpressed in the brains of MCAO / R mice by tail vein injection of AAV virus. Four weeks after AAV virus injection, a mouse MCAO / R model was constructed. After reperfusion for 24 hours, RNA was extracted from mouse brain tissue and reverse transcribed into cDNA. Changes in inflammatory factors in the mouse brain were detected by qRT-PCR.
[0081] like Figure 8 As shown, P3h2 overexpression alleviated the effects of brain inflammation in MCAO / R mice.
[0082] Example 9: Effects of P3h2 overexpression on the blood-brain barrier in MCAO / R mice
[0083] This embodiment involves Evans blue and Western blotting experiments to detect the effect of P3h2 overexpression on the blood-brain barrier in MCAO / R mice. The degree of blood-brain barrier damage was assessed using the Evans blue extravasation method. The experimental methods are as follows:
[0084] First, mice were injected with 4 mL / kg of 2% Evans blue solution (dissolved in PBS) via the tail vein and allowed to circulate in vivo for 2 hours. Subsequently, the mice were perfused with PBS to remove intravascular dye, and intact brain tissue was harvested for photographing to qualitatively observe Evans blue extravasation. Next, the ischemic side of the brain tissue was isolated and weighed, and a suitable amount of PBS was added for mechanical homogenization to prepare a homogenate. The homogenate was centrifuged at 4°C and 13,000 rpm for 30 minutes, and the supernatant was collected. An equal volume of trichloroacetic acid was added to the supernatant, and the reaction was carried out overnight (18-24 hours) at 4°C. After the reaction, the mixture was centrifuged again at 4°C and 13,000 rpm for 30 minutes, and the final supernatant was collected. The absorbance (OD value) of the supernatant was measured at 620 nm using a microplate reader to quantitatively reflect the amount of Evans blue leakage caused by blood-brain barrier damage.
[0085] like Figure 9 As shown, P3h2 overexpression promotes the repair of the blood-brain barrier in MCAO / R mice.
[0086] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. Application of the active ingredient overexpressing prolyl 3-hydroxylase 2 in the preparation of drugs for treating ischemic stroke.
2. The application according to claim 1, characterized in that, The active ingredient is a plasmid vector or a viral vector.