Use of OGR1 as a target in the preparation of drugs for treating cerebral ischemia

Abstract

This invention relates to the field of biomedical technology, specifically to the application of OGR1 as a target in the preparation of drugs for treating cerebral ischemia. This invention utilizes a viral vector to overexpress OGR1 in neurons surrounding infarcts. Ogr1 Treatment with OGR1 agonists can significantly promote the recovery of motor function and nerve repair after cerebral ischemia, and the treatment time window is at least 3-7 days after cerebral ischemia, which significantly enhances the treatment effect of cerebral ischemia.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of OGR1 as a target in the preparation of drugs for treating cerebral ischemia. Background Technology

[0002] Ischemic stroke, also known as cerebral ischemia, is an acute cerebrovascular disease caused by cerebral artery occlusion, leading to cerebral ischemia and hypoxia, and consequently, neurological deficits. It is characterized by five major features: high incidence, high disability rate, high mortality rate, high recurrence rate, and high economic burden. Cerebral ischemia can be divided into three phases based on its course: acute (0-3 days), subacute (3 days to several weeks), and chronic (several weeks to several months). In the acute phase, rapidly restoring blood flow perfusion (such as intravenous thrombolysis and mechanical thrombectomy) is the core strategy for reducing neurological damage; however, its clinical application is limited by key challenges such as a narrow treatment time window (only a few hours), the risk of hemorrhage transformation, and ineffective recanalization. Entering the subacute phase, the endogenous neural repair process begins—enhancing neural plasticity and promoting neural network reconstruction through synapsis and axonal regeneration, becoming a critical rehabilitation window for promoting neurological function recovery. Although different stages of cerebral ischemia present unique pathological mechanisms and treatment challenges, and the potential for endogenous neural repair in the subacute phase offers important directions for expanding the treatment time window and developing novel intervention strategies, current interventions from the subacute to the chronic phase primarily rely on physical rehabilitation training, which has limitations such as a long recovery period and limited functional improvement. Currently, there are no effective therapeutic drugs to promote neural repair at this stage. Ovarian cancer G protein-coupled receptor 1 (OGR1, also known as GPR68) is a proton-sensitive G protein-coupled receptor expressed in various tissues and cell types. Activated OGR1 is widely involved in biological processes such as neuroinflammation, cellular immunity, and apoptosis. In recent years, OGR1 has been shown to be mostly expressed in neurons in the brain. Recent studies have indicated that OGR1 plays a neuroprotective role in the acute phase of cerebral ischemia, but whether this target is a therapeutic target for neural repair in the subacute to chronic phases after cerebral ischemia remains unreported.

[0003] Tirazepam is a selective OGR1 agonist and currently has no clinical indication for the treatment of cerebral ischemia. Summary of the Invention

[0004] To address the shortcomings of the existing technologies, this invention provides an application of OGR1 as a target in the preparation of drugs for treating cerebral ischemia. This invention utilizes a viral vector to overexpress OGR1 in neurons in the peri-infarct region. Ogr1 Treatment with OGR1 agonists can significantly promote the recovery of motor function from the subacute to the chronic phase of cerebral ischemia, and significantly increase the density of immature dendritic spines, a neurorepair marker in the peri-infarct area, with a treatment time window of at least 3-7 days after cerebral ischemia.

[0005] To achieve the above objectives, in a first aspect, the present invention provides the application of OGR1 as a target in the preparation of drugs for treating cerebral ischemia, wherein the drug application stage is from the subacute to the chronic phase of cerebral ischemia.

[0006] Preferably, the drug regulates OGR1 overexpression.

[0007] Preferably, the drug is an OGR1 agonist.

[0008] Preferably, the OGR1 agonist is thiamethoxam.

[0009] Secondly, the present invention provides a recombinant virus, which can overexpress... Ogr1 .

[0010] Preferably, the recombinant virus is selected from one of recombinant adeno-associated virus, recombinant lentivirus, and recombinant adenovirus.

[0011] Preferably, the recombinant virus is AAV-hSyn- Ogr1 Virus.

[0012] Thirdly, the present invention provides the use of the recombinant virus as described in the second aspect in the preparation of a drug for treating cerebral ischemia, wherein the drug is used in the subacute to chronic phase of cerebral ischemia.

[0013] Fourthly, the present invention provides a pharmaceutical preparation for treating cerebral ischemia, the pharmaceutical preparation containing the recombinant virus and / or OGR1 agonist described in the second aspect.

[0014] Preferably, the OGR1 agonist is tirazepam.

[0015] Preferably, the dosage forms of the pharmaceutical preparation include tablets, pills, capsules, powders, drops, lyophilized preparations, granules, suspensions, syrups, decoctions, injections, and oral solutions.

[0016] In the above technical solution, this invention utilizes wild-type C57 mice to demonstrate for the first time, in a photochemically induced cerebral ischemia (PTI) model, that OGR1 is an important target for promoting the recovery of neurological function after cerebral ischemia. This is achieved by injecting mice containing... Ogr1 Adeno-associated virus vector AAV-hSyn- Ogr1 (HBAAV2 / 9-Syn-m-Gpr68-3xflag-ZsGreen, designed and manufactured by Shanghai Hanheng Biotechnology Co., Ltd., China), overexpressed in neurons in the peri-infarct region. Ogr1It significantly promoted the recovery of motor function in patients with cerebral ischemia and significantly increased the density of immature dendritic spines, a neural repair marker in the peri-infarct area, and remained effective even 7 days after ischemia. Extending the treatment window for cerebral ischemia to 7 days significantly enhanced the treatment effect in these patients. Simultaneously, it was also confirmed that OGR1-activated tiazem effectively promoted the recovery of motor function in the subacute phase of cerebral ischemia, significantly increased the density of immature dendritic spines, a neural repair marker in the peri-infarct area, and remained effective even 3 days after ischemia.

[0017] This invention proposes a novel method to promote the recovery of neurological function after cerebral ischemia by targeting neurons. Furthermore, this method of overexpressing the therapeutic target holds promise for providing new treatment options for the rehabilitation of patients in the subacute to chronic phases of cerebral ischemia.

[0018] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0019] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0020] Figure 1 Western blot image of AAV virus expression verification in an experiment exploring the beneficial effects of OGR1 overexpression in neurons.

[0021] Figure 2 Statistical graph of OGR1 protein expression level verified by AAV virus expression in an exploratory experiment to explore the beneficial role of OGR1 overexpression in neurons, *P<0.05.

[0022] Figure 3 AAV virus localization map (scale bar: 50 μm) used to verify AAV virus expression in an exploratory experiment to explore the beneficial effects of OGR1 overexpression in neurons.

[0023] Figure 4 Line graphs showing the sliding rate of mice in each group during an exploratory experiment to explore the beneficial effects of OGR1 overexpression in neurons. ***P<0.001 vs PTI group, ###P<0.001 vs negative virus PTI group.

[0024] Figure 5 Asymmetric index line graphs of mice in each group in an exploratory experiment to explore the beneficial effects of OGR1 overexpression in neurons. *P<0.05, **P<0.01, ***P<0.001 vs PTI group, #P<0.05, ###P<0.001 vs negative virus PTI group.

[0025] Figure 6 Immunofluorescence typical images of the number of microglia (Iba-1) in the cerebral cortex of mice in each group during an exploratory experiment to explore the beneficial effects of overexpressing neuronal OGR1 (scale bar: 50 μm).

[0026] Figure 7 Statistical graph of the number of microglia (Iba-1) in the cerebral cortex of mice in each group during the exploratory experiment to explore the beneficial effects of overexpressing neuronal OGR1. *P<0.05, **P<0.01, ***P<0.001.

[0027] Figure 8 Typical diagrams of neuronal tree mutations (Golgi staining) in each group of mice during an exploratory experiment to explore the beneficial effects of overexpressing OGR1 in neurons (scale bar: 100 μm).

[0028] Figure 9 A schematic diagram showing the changes in neuronal dendritic complexity in different groups of mice during an exploratory experiment to explore the beneficial effects of overexpressing OGR1 in neurons.

[0029] Figure 10 Statistical graph of total dendritic length of mice in each group during the exploratory experiment to explore the beneficial effects of OGR1 overexpression in neurons. *P<0.05, **P<0.01, ***P<0.001.

[0030] Figure 11 Statistical plot of dendritic complexity of mice in each group during the exploratory experiment to explore the beneficial effects of OGR1 overexpression in neurons. **P<0.01, ***P<0.001.

[0031] Figure 12 Statistical plot of total dendritic spine density of mice in each group during the exploratory experiment to explore the beneficial effects of overexpressing OGR1 in neurons. **P<0.01,***P<0.001.

[0032] Figure 13 Statistical graph of mature dendritic spine density in mice from different groups during an exploratory experiment to explore the beneficial effects of OGR1 overexpression in neurons. **P<0.01.

[0033] Figure 14 Statistical graph of immature dendritic spine density in mice from different groups during an exploratory experiment to explore the beneficial effects of overexpressing OGR1 in neurons. **P<0.01,***P<0.001.

[0034] Figure 15 Statistical graph of infarct volume in mice from different groups during an experiment exploring the beneficial effects of overexpressing OGR1 in neurons.

[0035] Figure 16 OGR1 agonist LeazepamLine graphs of the sliding rate of mice in each group during the exploratory experiment to explore the beneficial effects, ***P<0.001 vs PTI group, ###P<0.001 vs inhibitor group.

[0036] Figure 17 OGR1 agonist Leazepam Asymmetry index line graphs of mice in each group during the exploratory experiment to explore the beneficial effects, ***P<0.001 vs PTI group, ###P<0.001 vs inhibitor group.

[0037] Figure 18 OGR1 agonist Leazepam Typical images of neuronal tree mutations (Golgi staining) in each group of mice during the exploratory experiment that demonstrated the beneficial effects (scale bar: 50 μm).

[0038] Figure 19 OGR1 agonist Leazepam A schematic diagram showing the changes in the dendritic complexity of neurons in different groups of mice during an exploratory experiment to explore their beneficial effects.

[0039] Figure 20 OGR1 agonist Leazepam Statistical diagram of total dendritic length of mice neurons in each group during the exploratory experiment to explore its beneficial effects, ***P<0.001.

[0040] Figure 21 OGR1 agonist Leazepam Statistical graph of dendritic complexity of mice neurons in each group during the exploratory experiment to explore the beneficial effects, ***P<0.001.

[0041] Figure 22 OGR1 agonist Leazepam Statistical graph of total dendritic spine density of mice in each group during the exploratory experiment to explore its beneficial effects. ***P<0.001.

[0042] Figure 23 OGR1 agonist Leazepam Statistical graph of mature dendritic spine density of mice in each group during the exploratory experiment to explore its beneficial effects. ***P<0.001.

[0043] Figure 24 OGR1 agonist Leazepam Statistical graph of the density of immature dendritic spines in the neurons of mice in each group during the exploratory experiment to explore the beneficial effects, ***P<0.001.

[0044] Figure 25 OGR1 agonist Leazepam Statistical graph of neuronal infarct volume in each group of mice during the exploratory experiment to explore its beneficial effects. Detailed Implementation

[0045] The present invention will be described in detail below through examples. It should be understood that the embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0046] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0047] In the following examples, the medicines and pharmaceuticals are all commercially available products.

[0048] Example 1

[0049] I. Experimental Methods

[0050] 1. Establishment of a photochemically induced cerebral ischemia model (PTI model)

[0051] This invention uses C57 / BL6 wild-type (WT) mice (purchased from the Experimental Animal Center of Shanxi Medical University) and bred there. Male WT mice aged 7-8 weeks were used in a PTI model, simulating mouse brain injury using a cold light source. Mice were generally anesthetized by intraperitoneal injection of 4% sodium pentobarbital (45 mg / kg). A circular area with a diameter of 3 mm was marked with the Bregma (anterior fontanelle) as the origin, centered 1.5 mm to the right and 0 mm forward. A light source with a diameter of 2 mm and an intensity of 26,000 Lux was placed at the center of the circle. Freshly prepared rose red (100 mg / kg) was injected intraperitoneally. After 5 minutes, the light source was lowered by 0.6 mm and irradiated continuously for 20 minutes to establish the PTI model. The sham-operated group received the same surgical procedure but received an equal volume of saline intraperitoneally and did not receive rose red. The wound was sutured. A heating pad was used to maintain the mice's body temperature at 36-37°C until they fully recovered.

[0052] 2. Treatment with the OGR1 agonist tiazem and the OGR1 inhibitor OGM

[0053] The treatment methods using the OGR1 agonist tiazem and the OGR1 inhibitor OGM (Ogremorphin (OGM) is an OGR1 inhibitor with anti-inflammatory and anti-tumor activities. Ogremorphin can inhibit the migration of human melanoma cells and induce ferroptosis in glioblastoma cells; it was purchased from MCE) in this invention are entirely consistent. Both tiazem and OGM were administered intranasally starting on day 3 after PTI, every other day, 10 μL each time, continuing until day 14. sulfur Xipan The dosage was 32 μg / animal, and the OGM concentration was 2 μmol / L. In this invention, the OGR1 inhibitor (OGM) was administered within... Leazepam Perform the administration half an hour beforehand to ensure the correct sequence of drug action.

[0054] 3. Grid Experiment

[0055] Three days prior to PTI modeling, mice underwent acclimatization training. They were placed on a grid platform measuring 35 cm × 35 cm with a grid spacing of 2 cm × 2 cm and a height of 50 cm for 20 minutes, twice daily. A soft pad was placed under the grid platform to prevent injury from slipping. Motor function was evaluated by performing a grid inspection test on the mice 1 day before PTI and at 3, 7, 14, 21, and 28 days after PTI. The number of times the left forelimb slipped within 50 steps on the grid was recorded, and the slip rate was calculated as (slipped steps / total steps) × 100%. After each mouse's data collection, the grid platform was wiped with 75% alcohol. Data collection was performed at the same time each time.

[0056] 4. Cylinder Experiment

[0057] On day 1 before PTI and on days 3, 7, 14, 21, and 28 after PTI, mice were placed inside a transparent acrylic cylinder with a diameter of 13 cm and a height of 16 cm. The number of times each mouse touched the wall with its left forelimb, right forelimb, or both forelimbs during 20 standing-up maneuvers was recorded. The asymmetry index was calculated by analyzing the number of wall touches to assess the symmetry of forelimb use: Asymmetry Index = (Number of right forelimb wall touches - Number of left forelimb wall touches) / (Number of left forelimb wall touches + Number of right forelimb wall touches + Number of both forelimb wall touches) × 100%. After each mouse's collection, the cylinder was wiped with pure water. All collections were performed at the same time interval.

[0058] 5.AAV-hSyn- Ogr1 Intracerebral injection

[0059] The AAV virus injected in this invention includes: AAV-GFAP-CON negative virus (HBAAV2 / 9-Syn-ZsGreen) and AAV-hSyn- Ogr1 Positive virus (HBAAV2 / 9-Syn-m-Gpr68-3xflag-ZsGreen). Wild-type C57BL / 6 male mice were generally anesthetized after anesthesia with sodium pentobarbital (45 mg / kg, ip). The mice were fixed on a stereotaxic instrument, and the scalp was cut approximately 2 cm along the midline of the brain to fully expose the skull. The skull surface was wiped dry with sterile cotton swabs. Referring to the mouse brain stereotaxic atlas, the Bregma point was used as the origin, and three injection sites for the AAV vector in the right brain parenchyma were precisely marked: (right anterior fontanelle 2.5 mm, anterior 0 mm, depth 1 mm), (right anterior fontanelle 1.5 mm, anterior 0.33 mm, depth 0.88 mm), and (right anterior fontanelle 1.5 mm, posterior 0.66 mm, depth 0.88 mm). A cranial drill was then used to drill holes at these sites. The AAV carrier was injected into the brain parenchyma using a microinfusion pump at a rate of 0.1 μl / min and an injection volume of 1 μl / site. After 10 minutes of injection at each site, the needle was stopped for 10 minutes before being slowly removed. Finally, the scalp was sutured and disinfected. The mouse was removed from the adapter, and its body temperature was maintained at 36-37°C using a heating pad until it was fully awake.

[0060] 6. Immunofluorescence staining

[0061] One week after establishing the PTI model in mice, AAV viral vector was injected. Four weeks after PTI, brain tissue was perfused and collected. The tissue was then fixed in 4% paraformaldehyde and dehydrated in a gradient of 15% and 30% sucrose solutions until it settled completely. The brain tissue was cut into a series of 20 μm thick coronal sections using a cryostat. The brain sections were repaired with sodium citrate buffer (0.01 M, pH 6.0) at 80 °C for 30 min, permeated with 10% Triton X-100 for 30 min, blocked with 10% donkey serum at room temperature for 2 h, and incubated overnight at 4 °C with primary antibodies rabbit anti-NeuN (1:200, Sigma, Abn78), rabbit anti-GFAP (1:2000, Abcam, Ab278054), and goat anti-Iba-1 (1:400, Novus Biologicals, NB100-1028). Subsequently, the slides were incubated with the corresponding secondary antibodies, donkey anti-rabbit IgG Alexa Fluor 594 (1:400, Yeasen, 34212ES60) and donkey anti-sheep IgG Alexa Fluor 594 (1:400, Yeasen, 34312ES60), at room temperature for 2 hours. Finally, they were stained with 4',6-diamino-2-phenylindole (DAPI) solution and incubated at room temperature for 10 minutes, then mounted with anti-fluorescence attenuation mounting medium. Fluorescence signals in the peri-infarct region were captured using a Leica fluorescence microscope to observe the co-localization of AAV virus ZsGreen green fluorescence signals with NeuN, GFAP, and Iba-1 red fluorescence signals, as well as changes in the number of microglia in the peri-infarct region.

[0062] 7. Western blot

[0063] Three weeks after AAV vector injection in mice, tissue samples were collected. Mice were euthanized by cervical dislocation, and the brains were quickly decapitated. Cortical tissue from the AAV-injected region was separated. The tissue was lysed in RIPA lysis buffer containing protease and phosphatase inhibitors. Equal volumes of proteins were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated with 5% skim milk at room temperature for 2 hours, then placed on primary antibodies (mouse anti-flag, 1:1000, Hanheng Biotechnology, HB-ADF-0100 / 020) and rabbit anti-GAPDH, 1:6000, Bioworld, Ap0063, and incubated overnight at 4°C. Subsequently, it was incubated with goat anti-mouse (1:5000, Boster, Ba1050) and goat anti-rabbit (1:5000, Boster, Ba1054) horseradish peroxidase (HRP) secondary antibodies at room temperature for 2 hours. The OGR1-flag or GAPDH immunoblot images were visualized using the ECL hypersensitive chemiluminescence kit on the Bio-Rad imaging system.

[0064] 8. Golgi staining

[0065] Golgi staining was performed using the FD Rapid Golgi Astain Kit (PK401). Mice euthanized 28 days after PTI by cervical dislocation, and brain tissue was rinsed and fixed in a solution of equal proportions of Solution A and Solution B at room temperature in the dark for 2 weeks. The tissue was then transferred to Solution C and fixed at room temperature in the dark for 3 days. After rapid freezing with dry ice and isopentane, 80 μm thick sections were cut and fixed at room temperature in the dark for 3 days. Sections were rinsed with Milli-Q water and placed in a mixture of Solution D, Solution E, and Milli-Q (mixed in a 1:1:2 ratio). Sections were rinsed again with Milli-Q water and dehydrated using graded ethanol solutions (50%-100%). The sections were cleared with xylene and mounted with neutral resin. Sections were photographed using a Leica fluorescence microscope, and ImageJ was used to calculate the total dendritic length, branching complexity, and dendritic spine density of neurons.

[0066] II. Experimental Design

[0067] 1. Exploratory experiments on the beneficial effects of overexpressing OGR1 in neurons

[0068] Wild-type mice were injected with no virus, negative virus (AAV-hSyn-CON), or positive virus (AAV-hSyn-CON). Ogr1 Brain tissue was harvested 3 weeks after injection, and Western blot analysis was used to verify the results. Ogr1Whether it was successfully overexpressed in the cerebral cortex tissue of wild-type mice. Meanwhile, to verify... Ogr1 To determine whether the virus is specifically overexpressed in neurons, AAV viral vector was injected one week after PTI, and the brain was harvested 28 days after injury. Frozen sections were then subjected to immunofluorescence staining to observe the colocalization of cells successfully expressing AAV viral vector (i.e., ZsGreen positive cells) with neuronal marker NeuN, microglia marker Iba-1, and astrocyte marker GFAP.

[0069] To observe the use of positive virus (AAV-hSyn- Ogr1 Enhance neurons in the peri-infarct area of ​​mice. Ogr1 To investigate whether the expression of [a specific substance] can promote functional rehabilitation in mice after cerebral ischemia, we randomly divided mice into four groups: a negative virus control group (Sham+AAV-hSyn-CON), a negative virus PTI group (PTI+AAV-hSyn-CON), and a positive virus control group (Sham+AAV-hSyn-CON). Ogr1 ), positive virus PTI group (PTI+ AAV-hSyn- Ogr1 The AAV viral vector was injected one week after PTI. Grid and cylinder tests were performed on day 1 before PTI and on days 3, 7, 14, 21, and 28 after PTI to assess the recovery of motor function in each group of mice. Twenty-eight days after PTI, tissue samples were collected from each group of animals, and Iba-1 immunofluorescence staining was used to observe and statistically analyze changes in the number of microglia in the peri-infarct area and assess overexpressing neurons. Ogr1 The effect on microglial cell activation was investigated. Golgi staining was also used to observe the total dendritic length, branching complexity, and dendritic spine density of neurons in the peri-infarct region.

[0070] 2. OGR1 agonist Leazepam Exploratory experiments that play a beneficial role

[0071] In order to explore Leazepam To investigate whether mice can promote functional recovery after cerebral ischemia, and whether this promoting effect is mediated through the OGR1 target, we randomly divided mice into 5 groups: control group (Sham group), PTI group, agonist group (PTI+). Leazepam The group consisting of three groups: a PTI+OGM group and an agonist-inhibitor combination group. Leazepam +OGM group). Leazepam Both OGM and OGR1 inhibitors are administered intranasally every other day, starting 3 days after PTI. However, OGM requires administration only after… LeazepamThe procedure was performed half an hour before administration to ensure the order of drug action. Grid and cylinder tests were conducted one day before PTI and on days 3, 7, and 14 after PTI to assess the recovery of motor function in each group of mice. Fourteen days after PTI, tissue samples were collected from each group of animals, and Golgi staining was used to observe the total length of dendrites, branching complexity, and dendritic spine density in the peri-infarct area.

[0072] III. Experimental Results

[0073] 1. An exploratory experiment on the beneficial effects of overexpressing OGR1 in neurons; results are shown in […]. Figure 1-15 .

[0074] pass Figure 1 , Figure 2 We discovered AAV-hSyn- Ogr1 OGR1-flag overexpression was successful in this group. Figure 3 It is evident that the green fluorescent protein ZsGreen expressed by the AAV virus completely co-localizes with the neuronal marker NeuN, but does not overlap with the microglial marker Iba-1 or the neuronal marker NeuN. Therefore, this AAV viral vector can be selectively expressed on neurons. Ogr1.

[0075] pass Figure 4 and Figure 5 Behavioral assessment revealed that the positive viral PTI group (PTI+ AAV-hSyn- Ogr1 In mice, neurons overexpress... Ogr1 Afterward, it significantly reduced the slippage rate and asymmetry index at 14, 21, and 28 days post-PTI. However, it did not significantly improve motor function in mice treated with the virus-negative virus.

[0076] Furthermore, we examined the number of microglia in the peri-infarct area. For example... Figure 6 and Figure 7 As shown, the number of microglia increased significantly after PTI compared to the control group, and neurons overexpressed [the virus]. Ogr1 It significantly reduced the number of microglia in mice 28 days after PTI. These results further indicate that neuronal overexpression Ogr1 It can inhibit excessive activation of microglia, reduce microglia-mediated neuroinflammation, and thus promote the recovery of neurological function after PTI in mice.

[0077] After Golgi staining, we found that in the negative PTI group (PTI+ AAV-hSyn-CON) and the positive PTI group (PTI+ AAV-hSyn-CON)... Ogr1 In the positive PTI group, the overall morphological recovery of neurons was better. Figure 8-10 ), with higher complexity ( Figure 11-13 Immature dendritic spines have a higher density. Figure 14 This indicates that OGR1 overexpression significantly enhances the neural repair capacity of the peri-infarct area. Furthermore, OGR1 overexpression had no significant effect on infarct volume. Figure 15 This indicates that the recovery of neurological function in OGR1-overexpressing mice depends on OGR1-mediated neurorepair mechanisms, rather than neuroprotective mechanisms. Therefore, it is reasonable to conclude that using AAV virus to overexpress neuronal... Ogr1 It can promote the recovery of nerve function and nerve repair in the subacute and chronic phases after PTI.

[0078] 2. An exploratory experiment on the beneficial effects of the OGR1 agonist tiazem was conducted, and the results are shown in [the table below]. Figure 16-25 .

[0079] pass Figure 16 and Figure 17 Behavioral assessments revealed that mice in the agonist group (PTI + tiazem) showed a significant reduction in gait slip rate and asymmetry index at 7 and 14 days post-PTI administration of the OGR1 agonist tiazem. However, mice in the inhibitor group (PTI + OGM) and the combination of agonist and inhibitor group (PTI + tiazem + OGM) showed no significant improvement in motor function. This indicates that tiazem promotes the recovery of motor function after PTI in mice, and that this recovery effect is mediated through the OGR1 target.

[0080] Following Golgi staining, we found that the overall neuronal morphology recovered better 14 days after PTI in the agonist group (PTI + tiazem). Figure 18-20 ), with higher complexity ( Figure 21-22 Immature dendritic spines have a higher density. Figure 24 This effect can be reversed by the OGR1 inhibitor OGM. Figure 24 This indicates that tiazem significantly enhances the neural repair capacity of the peri-infarct area by activating OGR1. Furthermore, tiazem had no significant effect on the volume of the cerebral infarction. Figure 25 Furthermore, the density of mature dendritic thorns did not change significantly. Figure 23 This indicates that the effect of tiazem activating OGR1 in promoting neurological function recovery in mice depends on an OGR1-mediated neurorepair mechanism, rather than a neuroprotective mechanism. Therefore, tiazem can promote neurological function recovery and repair from the subacute to chronic phase after PTI by activating OGR1. In summary, this invention is the first to clearly demonstrate that overexpression of OGR1 on neurons can promote neurological function recovery and repair. Ogr1 The OGR1 agonist tiazem can promote the recovery of motor function after PTI, promote nerve repair after cerebral ischemia, and improve neurological dysfunction. Its therapeutic window can be extended to 3-7 days after PTI. Therefore, this invention effectively overcomes the various shortcomings of the prior art and has high industrial application value.

[0081] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0082] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0083] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. The application of an OGR1 agonist, tiazem, in the preparation of drugs for treating cerebral ischemia, characterized in that, The drug is applied in the subacute to chronic phases of a photochemically induced cerebral ischemia model. 2.AAV-hSyn- Ogr1 The application of viruses in the preparation of drugs for treating cerebral ischemia is characterized by, The drug is applied during the subacute to chronic phase of a photochemically induced cerebral ischemia model; the AAV-hSyn- Ogr1 The virus can overexpress OGR1.

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