Use of DDAH1 in preparation of drugs for treating ischemic stroke

Abstract

The application discloses application of dimethylarginine dimethylaminohydrolase 1 (DDAH1) in a drug for treating and preventing ischemic stroke.

Description

Technical Field

[0001] This invention relates to the application of dimethylarginine dimethylaminohydrolase 1 (DDAH1) in the treatment and prevention of ischemic stroke. Background Technology

[0002] Acute ischemic stroke is characterized by high recurrence, high mortality, and high disability rates. With advancements in science and medicine, significant progress has been made in stroke prevention and early intervention, leading to a decrease in mortality and a stable incidence rate. Recent epidemiological statistics suggest that stroke has become one of the leading causes of public health threats. However, current clinical treatment strategies are limited to timely restoration of blood flow and reperfusion in the early stages of injury to reduce the size of the infarct. However, recent studies have found that after acute ischemic brain injury, the proliferation, differentiation, migration, and maturation of endogenous new neurons in specific brain regions, namely the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ), are enhanced. Therefore, activated endogenous neuroregeneration may represent a new approach and method for the intervention and treatment of acute stroke.

[0003] DDAH1 is widely distributed in various tissues, and current research focuses on its regulatory role in cardiovascular diseases, diabetes, and other conditions. A clinical study on DDAH1 in stroke showed that promoter polymorphism is associated with the proportion of stroke and coronary heart disease patients. This promoter polymorphism leads to loss of function at both the mRNA and protein levels of DDAH1 and is positively correlated with the incidence of thrombotic stroke and coronary heart disease. However, the therapeutic effect of DDAH1 on ischemic stroke, especially its protective effect on cognitive function, has not been reported. Summary of the Invention

[0004] This invention provides the application of DDAH1 in the treatment and prevention of ischemic stroke, specifically involving the protective effect of DDAH1 on ischemic stroke and its protective effect on secondary nerve damage by promoting nerve regeneration.

[0005] Technical solution

[0006] This invention utilizes Cre / LoxP technology to construct DDAH1 knockout (KO) mice, DDAH1 overexpression (TG) mice, and wild-type C57BL / 6J (WT) control mice to establish a middle cerebral artery embolism model. The protective effect of DDAH1 against acute ischemic brain injury was analyzed through neurological scoring and infarct volume measurement. Furthermore, the protective effect of DDAH1 against secondary neurological damage following acute stroke and its regulatory role in neurogenesis were analyzed through water maze tests and immunofluorescence staining of newly formed neurons.

[0007] DDAH1 knockout exacerbated neurological functional impairment following cerebral ischemia-reperfusion injury in mice. Correspondingly, 2,3,5-triphenyltetrazolium chloride (TTC) staining showed that DDAH1 knockout increased infarct volume after cerebral ischemia-reperfusion injury in mice. The water maze test showed that DDAH1 knockout also exacerbated learning and memory impairment after cerebral ischemia-reperfusion injury in mice. DDAH1 overexpressing mice showed the opposite trend to DDAH1 knockout mice in terms of neurological function, infarct volume, and the water maze test. Immunofluorescence experiments showed that DDAH1 promoted neuronal regeneration after cerebral ischemia-reperfusion injury in mice.

[0008] This invention discloses the protective effects of DDAH1 on neurological function, infarct volume, and learning and memory function in acute cerebral ischemia-reperfusion injury. It was found that DDAH1 can not only protect against acute ischemic stroke injury but also repair stroke-related damage by promoting neuronal regeneration after ischemic injury. Therefore, DDAH1 can serve as a target and protein-based drug for the treatment of ischemic stroke. Attached Figure Description

[0009] Figure 1 Neurological scores and infarct volume changes after cerebral ischemia-reperfusion injury in mice of different genotypes. In the figures, a represents the changes in neurological scores after cerebral ischemia-reperfusion injury in mice of different genotypes; b is a representative plot of TTC staining results after injury; and c is a statistical plot of infarct volume stained with TTC. (WT group, n=6; KO and TG groups, n=7; mean ± standard error; one-way ANOVA) * (P < 0.05)

[0010] Figure 2 Results of the water maze test after cerebral ischemia-reperfusion injury in mice of different genotypes. Where 'a' represents the time required for mice to find the platform in the first 4 days (Sham group, WT group, KO group, and TG group, n=8, mean ± standard error, two-way ANOVA). * P < 0.05 **P < 0.01 relative to the Sham group, # P < 0.05 (relative to the WT group); b represents the number of times the mouse passed the plateau on day 5. (Sham group, WT group, KO and TG groups, n = 8, mean ± standard error, one-way ANOVA) ** P < 0.01 relative to the Sham group, # P < 0.05 relative to the WT group.

[0011] Figure 3 Immunofluorescence staining results after cerebral ischemia-reperfusion injury in mice of different genotypes. Figure a shows representative images of newly generated neurons in the SVZ, striatum, and dentate gyrus (DG) regions of the hippocampus; figure b shows the number of cells co-stained with BrdU and NeuN in the striatum; and figure c shows the number of cells co-stained with 5-bromodeoxyuridine (BrdU) and neuron-specific nucleoprotein (NeuN) in the dentate gyrus. (Sham group, WT group, KO group, and TG group, n=4, mean ± standard error, one-way ANOVA) * P < 0.05, relative to the Sham group, # P < 0.05 (relative to the WT group). Detailed Implementation

[0012] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0013] Example 1: Protective effect of DDAH1 against ischemic stroke and cerebral infarction

[0014] I. Experimental Methods

[0015] 1. Mouse source. Wild-type mice were C57BL / 6J mice, obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd., and used as control groups for DDAH1 KO and TG mice. DDAH1 KO mice were obtained from Jackson Laboratory in the United States, and DDAH1 TG mice were obtained from Nanjing Saiye Biotechnology Co., Ltd.

[0016] 2. Acute transient middle cerebral artery occlusion (MCAO) model in mice. Mice were placed in a supine position on a heated pad to maintain a constant rectal temperature (37±0.5℃). They were anesthetized with 3% isoflurane for induction and 1.5% for maintenance anesthesia, respectively. Hair was shaved from the front of the neck, and a longitudinal incision of approximately 1.5 cm was made in the skin at the front of the neck. The right common carotid artery, external carotid artery, and internal carotid artery were bluntly dissected and ligated. A suture was inserted into the common carotid artery at the opening of the external carotid artery, slightly retracted, and rotated approximately 180°. The suture was then inserted into the internal carotid artery approximately 0.9-1 cm. The procedure was timed for 1 hour, after which the suture was removed, and the free end of the external carotid artery was electrocoagulated to restore blood flow. The control group (Sham) mice underwent the same procedures as the experimental group, except that no suture was inserted. During ischemia, local blood flow was monitored using laser speckle Doppler flow imaging. A decrease in local blood flow to 20% of the baseline was considered a successful ischemia marker. Throughout the entire procedure, the room temperature was maintained at 24-25℃.

[0017] 3. Neurological scoring. The Longa score was used to assess the neurological deficits in mice following ischemia-reperfusion injury. A score of 0 indicated no neurological damage, 1 indicated left forelimb flexion, 2 indicated body rotation to the left, 3 indicated body tilting to the left, and 4 indicated inability to move spontaneously and decreased level of consciousness.

[0018] 4. TTC staining. The injured mouse brain was removed and placed in ice-cold PBS at -20°C for 10 minutes. It was then placed in a mold for sectioning. The brain sections were incubated in 1% TTC solution at 37°C for 10 minutes, followed by incubation on the reverse side for 5-7 minutes. They were then placed in 4% paraformaldehyde solution for 2-3 hours. The brain sections were scanned, and the infarct volume of each section was calculated. The corrected infarct volume for each brain section was calculated as: Corrected infarct volume = Healthy brain volume - Non-infarcted volume on the operated side. The percentage of total infarct volume was calculated as: ∑ Corrected infarct volume / ∑ Healthy brain volume x 100%.

[0019] II. Experimental Results

[0020] 1. DDAH1 alleviates neurological function impairment after MCAO ( Figure 1 a) Compared to WT mice, DDAH1 knockout significantly improved neurological scores, while DDAH1 overexpression mice showed the opposite trend, indicating that DDAH1 has a protective effect on neurological function after MCAO.

[0021] 2. DDAH1 reduces infarct volume after MCAO ( Figure 1 (b, c) Compared to WT mice, KO mice showed an increase in infarct volume, while TG mice showed the opposite trend, indicating that DDAH1 has a certain promoting effect on the reduction of infarct volume after MCAO.

[0022] Example 2: The promoting effect of DDAH1 on neurogenesis and cognitive function recovery after ischemic stroke

[0023] I. Experimental Methods

[0024] 1. Mouse source. As described in Example 1, wild-type mice were C57BL / 6J mice, obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd., and used as control groups for DDAH1 KO and TG mice. DDAH1 KO mice were obtained from Jackson Laboratory, USA, and DDAH1 TG mice were obtained from Nanjing Saiye Biotechnology Co., Ltd.

[0025] 2. Mouse MCAO model. The model was established as described in Example 1.

[0026] 3. Water Maze Test. The water maze test was conducted daily at 9:00 AM from day 24 to 28 after MCAO. The first four days were for hidden platform training (the platform was placed approximately 1-2 cm below the water surface). The last day was a probe trial, where the platform was removed. During the experiment, the water temperature was heated and maintained at 19-20°C. Mice entered the water facing the maze wall, and a 90-second timer was started. If the mouse did not find the platform within the time limit, it was guided to swim to the platform and stay there for 30 seconds. If it found the platform during this time, it also stayed there for 30 seconds. Two different entry points were tested each day, and the time required to find the platform and the movement trajectory were recorded. On the last day, the platform was removed, and the mice again entered the water from both entry points and swam for 90 seconds, recording the time the mouse spent in the quadrant where the platform was located.

[0027] 4. Immunofluorescence experiment. Mice were anesthetized by intraperitoneal injection of 5% chloral hydrate and fixed on a mouse board, ventral side up. The abdominal and thoracic cavities of the mice were opened with scissors to expose the abdominal organs and heart. The perfusion pump was turned on, and a needle was inserted into the left ventricle through the apex of the mouse heart to remove the right atrial appendage. The perfusion pump flow rate was 5 ml / min, and phosphate-buffered saline (PBS) was used for perfusion for 13 minutes, followed by 4% paraformaldehyde perfusion for 20 minutes. After perfusion, the brain was removed. The removed mouse brain was placed in 4% paraformaldehyde and fixed at 4°C in the dark for 24 hours. The solution was replaced with 20% sucrose solution and dehydrated until the mouse brain settled to the bottom of the bottle. The dehydrated mouse brain was placed on a mouse brain mold, and the cerebellum was removed. The removed mouse brain was placed in embedding medium and frozen in dimethyl-butane pre-cooled with liquid nitrogen. The specimen was cut into 20 μm sections using a cryostat. After incubating the slides at room temperature for 15 minutes, rinse the specimen three times with PBS for 2 minutes each time. Antigen retrieval: Place in a citrate buffer box and heat in a microwave for 6 minutes. After heating, allow to return to room temperature to expose the antigen sites. DNA denaturation: Wash with PBS for 2 minutes, then with 2M hydrochloric acid at 37°C for 30 minutes, followed by 0.1M boric acid solution. Rinse the specimen three times with PBS for 2 minutes each time. Block with blocking solution for 30 minutes. Incubate with primary antibody at 4°C for 72 hours. On the fourth day, incubate at room temperature, rinse three times with 1×PBS for 10 minutes each time. Incubate with secondary antibody for 2 hours, then store in a humidified chamber at room temperature, protected from light. Stain with 4'6-diamidion-2-phenylindole (DAPI) for 10 minutes. Rinse three times with PBS for 10 minutes each time. Gently dry the area around the specimen, add one drop of mounting solution, and cover with a coverslip. Observe and photograph the desired areas using a laser confocal microscope. The primary and secondary antibodies required for the experiment are listed in Table 1.

[0028] Table 1: Primary and Secondary Antibodies Required for Immunofluorescence Staining

[0029]

[0030] II. Experimental Results

[0031] 1. DDAH1 promotes the recovery of learning and memory function after MCAO ( Figure 2 Compared to the sham-operated group, the MCAO group showed significant impairment in learning and memory function in the water maze test. DDAH1 knockout exacerbated this phenomenon, while DDAH1 overexpression could reverse the functional decline caused by MCAO.

[0032] 2. DDAH1 promotes the regeneration of mature neurons in the striatum and dentate gyrus of the hippocampus on the affected side after MCAO. Figure 3 Compared to the Sham group, the MCAO group showed ischemic striatum on the affected side. Figure 3 b) and hippocampal dentate gyrus ( Figure 3c) The number of newly formed mature neurons increased, while the number of newly formed neurons in the dentate gyrus of the hippocampus on the affected side of the KO group mice was significantly lower than that in the WT group mice. In contrast, the TG group mice had more newly formed mature neurons in the dentate gyrus of the hippocampus on the affected side.

Claims

1. Application of dimethylarginine dimethylamine hydrolase 1 in the preparation of drugs for treating learning and memory impairment after ischemic stroke.

2. The application according to claim 1, characterized in that, The ischemic stroke mentioned above is caused by local cerebral ischemia.

3. The application of the pharmaceutical composition in the preparation of a drug for treating learning and memory impairment after ischemic stroke, wherein, The pharmaceutical composition is prepared with dimethylarginine dimethylamine hydrolase 1 and one or more pharmaceutically acceptable carriers.

4. The application as described in claim 3, characterized in that, The composition is formulated into any clinically acceptable dosage form.

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