Application of active decapeptide in preparation of medicine for preventing or treating cerebral apoplexy

Abstract

The invention provides application of active decapeptide in preparation of a medicine for preventing or treating cerebral apoplexy, and belongs to the technical field of biological medicine, the active decapeptide with the amino acid sequence as shown in SEQ ID NO.1 is applied to prevention or treatment of cerebral apoplexy for the first time, the active decapeptide can effectively inhibit formation of cerebral thrombosis of zebra fish with ischemic cerebral apoplexy, and the active decapeptide can be used for preventing or treating cerebral apoplexy. The brain blood flow supply is recovered; meanwhile, the active decapeptide can significantly reduce the hemorrhagic area and hemorrhagic rate of the zebra fish brain with hemorrhagic stroke, repair brain vascular injury and inhibit the occurrence and development of hemorrhagic stroke. In addition, the active decapeptide has small toxic and side effects, and is of great significance for improving the clinical curative effect of cerebral apoplexy and reducing the medication risk when being used for research and development of novel drugs for resisting cerebral apoplexy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of an active decapeptide in preparing a drug for preventing or treating stroke. Background Art

[0002] Stroke is an acute cerebrovascular disease caused by blockage or sudden rupture of a cerebral blood vessel, resulting in ischemia and hypoxia of the brain tissue. Clinically, it manifests primarily as weakness on one side of the limbs, slurred speech or difficulty expressing oneself, facial numbness, and facial paralysis. Based on its pathological mechanism, stroke can be divided into two major categories: ischemic stroke (accounting for approximately 80%) and hemorrhagic stroke (accounting for approximately 20%). Currently, surgical or medical interventions are available for ischemic stroke. However, mechanical thrombectomy and other procedures have a narrow treatment window (typically 4.5–6 hours) and are costly, while also associated with risks such as vascular injury, bleeding, and re-thrombosis. Consequently, clinical treatment options include antiplatelet, anticoagulant, thrombolytic, and drugs that improve cerebral circulation and metabolism. However, these drugs are often associated with the risk of intracranial and gastrointestinal bleeding and have strict limitations in terms of treatment window and applicable population. For hemorrhagic stroke, surgical removal of the hematoma is the primary approach in clinical practice, but this can be detrimental and can lead to numerous postoperative complications. Commonly used medications, such as mannitol and furosemide, cannot directly remove the hematoma pressure and can cause side effects such as renal impairment, electrolyte imbalance, and blood volume fluctuations. Therefore, developing effective anti-stroke drugs with minimal side effects is crucial for improving clinical efficacy and reducing medication risks.

[0003] The zebrafish genome shares 87% homology with the human genome. Compared to mammalian models, zebrafish possess advantages such as rapid development, robust reproductive capacity, embryonic transparency, and ease of gene editing. The zebrafish coagulation system and neurovascular unit structure are highly similar to those of mammals, enabling accurate simulation of the pathological features of mammalian stroke. Furthermore, the transparency of zebrafish larvae allows for direct observation of cerebral thrombosis and hematoma formation following hemorrhage. Using transgenic fluorescently labeled zebrafish, changes in the brain's vascular network and neuronal damage during stroke can be directly observed. Therefore, the zebrafish model can be used to rapidly screen and discover anti-stroke active molecules for new drug development.

[0004] The invention patent, publication number "CN111423495A" (application number CN202010313278.X), discloses five Rhizoma Cirrhosae peptides that have the ability to protect against oxidative stress. It was found that all five peptides can independently scavenge ROS production, reduce macrophage aggregation in zebrafish, inhibit the production of angiotensin-converting enzyme in blood vessels, and inhibit the production of inflammatory cytokines, thereby repairing damage caused by oxidative stress. Currently, research on the activity of Rhizoma Cirrhosae-derived peptides focuses primarily on anti-inflammatory, antibacterial, anti-cancer, and auditory hair cell protection, but has not yet explored the field of cerebrovascular disease. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides an application of an active decapeptide in the preparation of a drug for preventing or treating stroke.

[0006] The technical solutions of the present invention are as follows: An active decapeptide is used in the preparation of a drug for preventing or treating stroke, wherein the amino acid sequence of the active decapeptide is shown in SEQ ID NO.1.

[0007] Preferably, the stroke is ischemic stroke disease and hemorrhagic stroke disease.

[0008] Preferably, the active decapeptide prevents or treats ischemic stroke by inhibiting cerebral thrombosis and restoring cerebral blood supply.

[0009] Preferably, the active decapeptide prevents or treats hemorrhagic stroke by inhibiting cerebral hemorrhage and repairing cerebral vascular damage.

[0010] An active decapeptide is used in the preparation of an anti-cerebral thrombosis drug, wherein the amino acid sequence of the active decapeptide is shown as SEQ ID NO.1.

[0011] An active decapeptide is used in the preparation of an anti-cerebral hemorrhage drug, wherein the amino acid sequence of the active decapeptide is shown as SEQ ID NO.1.

[0012] Beneficial effects This invention, for the first time, applies an active decapeptide with the amino acid sequence shown in SEQ ID NO.1 to the prevention or treatment of stroke. This active decapeptide can effectively inhibit the formation of thrombi in the brains of zebrafish with ischemic stroke and restore cerebral blood flow. Furthermore, this active decapeptide can significantly reduce the area and rate of hemorrhage in the brains of zebrafish with hemorrhagic stroke, repair brain vascular damage, and inhibit the occurrence and progression of hemorrhagic stroke. Furthermore, this active decapeptide has minimal toxic and side effects, and its use in the development of new anti-stroke drugs is of great significance for improving the clinical efficacy of stroke and reducing medication risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Results for zebrafish brain image collection; Figure 2 This is the statistical result of thrombus area in zebrafish brain; Figure 3 The statistical results of the average staining intensity of red blood cells in the zebrafish brain; Figure 4 This is a diagram of zebrafish brain blood flow dynamics; Figure 5 This is the statistical result of zebrafish brain blood flow velocity; Figure 6 Results for zebrafish brain image collection; Figure 7 This is the statistical result of zebrafish brain hemorrhage rate; Figure 8 This is the statistical result of the hemorrhage area in the zebrafish brain; Figure 9 Results of zebrafish brain vascular image acquisition. DETAILED DESCRIPTION

[0014] The following describes the process in conjunction with specific embodiments: Example 1: Anti-ischemic stroke effect of active decapeptide 1. Zebrafish Sample Pretreatment The zebrafish used in this patent were housed according to standardized procedures. Male and female individuals were maintained separately in a recirculating water system at a constant temperature of 28 ± 0.5°C, under a 14-hour light / 10-hour dark cycle, and fed brine shrimp and paramecium twice daily. Before the experiment began, mature, healthy zebrafish were placed in a spawning tank at a ratio of 2:2 female:male. The next day, before the lights were turned on, the baffles were removed, and ovulation was stimulated by light. Embryos were collected 2 hours later. After washing, the embryos were transferred to embryo culture medium (composition: 5 mmol / L NaCl, 0.17 mmol / L KCl, 0.33 mmol / L CaCl2, 0.33 mmol / L MgSO4) containing 0.5 mg / L methylene blue and cultured in a constant temperature, illuminated incubator at 28 ± 0.5°C. After 6 hpf (6 hours after fertilization), dead embryos were removed and phenylthiourea was added at a final concentration of 0.03 mg / mL to block melanin synthesis. The culture medium was replaced every 24 hours and dead individuals were removed to ensure a stable environment for embryonic development.

[0015] 2. Synthesis of active decapeptides The active decapeptide having the amino acid sequence shown in SEQ ID NO. 1 was synthesized by the Fmoc solid phase synthesis method described by Liu Zhennan et al. (Liu Zhennan, Huang Qiang. Fmoc solid phase synthesis method [J]. Journal of Guangxi University for Nationalities, 1999, 5 (2): 110-112).

[0016] 3. Experimental Methods Wild-type AB zebrafish were used as experimental animals. After decapping at 48 hpf, zebrafish were placed in 24-well plates and randomly divided into a blank control group, an ischemic stroke model group, an aspirin-positive control group, a low-concentration active decapeptide treatment group, a medium-concentration active decapeptide treatment group, and a high-concentration active decapeptide treatment group. Each group had three replicates, with 10 fish per replicate to ensure the reliability of the experimental data. The blank control group received zebrafish embryo culture medium, the ischemic stroke model group received ponatinib at a final concentration of 1.9 μmol / L, the aspirin-positive control group received ponatinib at a final concentration of 1.9 μmol / L and aspirin at a final concentration of 124.9 μmol / L, and the low-concentration, medium-concentration, and high-concentration active decapeptide treatment groups received ponatinib at a final concentration of 1.9 μmol / L and active decapeptide at a final concentration of 5 μmol / L, 10 μmol / L, or 20 μmol / L, respectively. The six groups of zebrafish were cultured in a constant-temperature incubator at 28 ± 0.5°C. After 24 h of culture, the culture medium was aspirated and the cells were stained with o-dianisidine for 10 min in the dark. The cells were then washed three times with dimethyl sulfoxide and fixed with 4% paraformaldehyde. Zebrafish brain images were collected under a Zeiss microscope. Image-Pro Plus software was used to calculate the thrombus area and the average red blood cell staining intensity in the zebrafish brain. Statistical analysis was performed using GraphPad Prism. The results are expressed as mean ± SEM. P < 0.05 indicated statistical significance.

[0017] 4. Experimental Results The experimental results are as follows Figures 1 to 3 As shown, aspirin represents the positive control group, 5 μmol / L, 10 μmol / L and 20 μmol / L represent the low concentration active decapeptide treatment group, the medium concentration active decapeptide treatment group and the high concentration active decapeptide treatment group, respectively. The zebrafish brain image acquisition results are as follows Figure 1 As shown, the cerebral thrombus is framed by a yellow dotted line, and the scale bar is 100 μm.

[0018] The statistical results of zebrafish brain thrombus area are as follows Figure 2 As shown, compared with the blank control group, #### P<0.0001; compared with the ischemic stroke model group, *** P<0.001, **** P<0.0001. Figure 2It was found that Ponatinib can induce an ischemic stroke model in zebrafish, which is manifested by a significant increase in the area of ​​cerebral thrombus; after treatment with active decapeptides at concentrations of 10 μmol / L and 20 μmol / L, the area of ​​cerebral thrombus in zebrafish was significantly reduced compared with the ischemic stroke model group, among which the inhibitory effect of 20 μmol / L active decapeptide on the area of ​​cerebral thrombus in zebrafish was better than that of the aspirin positive control group.

[0019] The statistical results of the average staining intensity of red blood cells in the zebrafish brain are as follows Figure 3 As shown, compared with the blank control group, #### P<0.0001; compared with the ischemic stroke model group, *** P<0.001, **** P<0.0001. Figure 3 It was found that Ponatinib can induce an ischemic stroke model in zebrafish, which is manifested by a significant increase in the average staining intensity of brain red blood cells; after treatment with active decapeptides at concentrations of 10 μmol / L and 20 μmol / L, the average staining intensity of zebrafish brain red blood cells was significantly reduced compared with the ischemic stroke model group.

[0020] The above results show that the active decapeptide can significantly reduce the thrombus area and the average staining intensity of red blood cells in the zebrafish brain, and has significant anti-ischemic stroke activity.

[0021] Example 2: Anti-ischemic stroke effect of active decapeptide The zebrafish sample pretreatment and active decapeptide synthesis methods were the same as in Example 1.

[0022] 1. Experimental Methods Wild-type AB zebrafish were used as experimental animals. 48 hpf zebrafish were grouped and treated the same way as in Example 1 until 72 hpf. Each group of zebrafish was then anesthetized by immersing them in a 0.3% tricaine solution for 40–90 seconds and then placed on a methylcellulose-coated glass slide in a natural prone position. Ten zebrafish were randomly selected from each group. A 10-second video of blood flow in the zebrafish brain vessels was recorded using the Zebralab blood flow system. Blood flow velocity in the video was analyzed using Zebra Blood software, and hemodynamic images were obtained. GraphPad Prism was used to analyze the blood flow velocity in the zebrafish brains of each group. Results are expressed as mean ± SEM, with P < 0.05 indicating statistical significance.

[0023] 2. Experimental Results The experimental results are as follows Figure 4 、 Figure 5As shown, aspirin represents the positive control group, 5 μmol / L, 10 μmol / L and 20 μmol / L represent the low concentration active decapeptide treatment group, the medium concentration active decapeptide treatment group and the high concentration active decapeptide treatment group, respectively. Zebrafish brain blood flow dynamics diagram Figure 4 As shown; the statistical results of zebrafish brain blood flow velocity are shown Figure 5 As shown, compared with the blank control group, #### P<0.0001; compared with the ischemic stroke model group, * P < 0.1, *** P<0.001, **** P<0.0001.

[0024] Depend on Figure 4~Figure 5 It was found that compared with the blank control group, the blood flow velocity in the zebrafish brain of the ischemic stroke model group was significantly slowed down or even stopped; compared with the ischemic stroke model group, the blood flow velocity in the zebrafish brain was significantly increased after treatment with the active decapeptide at concentrations of 10 μmol / L and 20 μmol / L.

[0025] The above results show that the active decapeptide can significantly improve the brain blood flow velocity of zebrafish with ischemic stroke and restore normal blood supply.

[0026] Example 3: Anti-hemorrhagic stroke effect of active decapeptide The zebrafish sample pretreatment and active decapeptide synthesis methods were the same as in Example 1.

[0027] 1. Experimental Methods Wild-type AB zebrafish were used as experimental animals. After decapping at 24 hpf, zebrafish were placed in 24-well plates and randomly divided into a blank control group, a hemorrhagic stroke model group, a valsartan positive control group, a low-concentration active decapeptide treatment group, a medium-concentration active decapeptide treatment group, and a high-concentration active decapeptide treatment group. Each group had three replicates, with 10 fish per replicate to ensure the reliability of the experimental data. The blank control group received zebrafish embryo culture medium, the hemorrhagic stroke model group received atorvastatin at a final concentration of 1.75 μmol / L, the valsartan positive control group received atorvastatin at a final concentration of 1.75 μmol / L and valsartan at a final concentration of 10 μmol / L, and the low-concentration, medium-concentration, and high-concentration active decapeptide treatment groups received atorvastatin at a final concentration of 1.75 μmol / L and active decapeptide at a final concentration of 5 μmol / L, 10 μmol / L, or 20 μmol / L, respectively. The six groups of zebrafish were cultured in a constant temperature incubator at 28 ± 0.5°C. After 12 h of culture, 10 zebrafish were randomly selected from each group. Zebrafish brain images were acquired using a Zeiss microscope. Image-Pro Plus software was used to measure the hemorrhage area in the zebrafish brain. Statistical analysis was performed using GraphPad Prism. The results are expressed as mean ± SEM. P < 0.05 indicated statistical significance.

[0028] 2. Experimental Results The experimental results are as follows Figures 6 to 8 As shown, valsartan represents the positive control group, 5 μmol / L, 10 μmol / L and 20 μmol / L represent the low concentration active decapeptide treatment group, the medium concentration active decapeptide treatment group and the high concentration active decapeptide treatment group, respectively. The zebrafish brain image acquisition results are as follows Figure 6 Scale bar is 200 μm.

[0029] The statistical results of zebrafish brain hemorrhage rate are as follows Figure 7 As shown, compared with the blank control group, #### P<0.0001; compared with the hemorrhagic stroke model group, **** P<0.0001. Figure 7 It was found that atorvastatin can induce a hemorrhagic stroke model in zebrafish, which is manifested by a significant increase in the brain hemorrhage rate of zebrafish; after treatment with active decapeptides at concentrations of 5 μmol / L, 10 μmol / L and 20 μmol / L, the brain hemorrhage rate of zebrafish was significantly reduced compared with the hemorrhagic stroke model group, among which the inhibitory effect of 20 μmol / L active decapeptide on the brain hemorrhage rate of zebrafish was better than that of the valsartan positive control group.

[0030] The statistical results of zebrafish brain hemorrhage area are as follows Figure 8 As shown, compared with the blank control group, #### P<0.0001; compared with the hemorrhagic stroke model group, **** P<0.0001. Figure 8 It was found that atorvastatin can induce a hemorrhagic stroke model in zebrafish, which is manifested by a significant increase in the hemorrhage area in the zebrafish brain; after treatment with active decapeptide at concentrations of 5 μmol / L, 10 μmol / L and 20 μmol / L, the hemorrhage area in the zebrafish brain was significantly reduced compared with the hemorrhagic stroke model group, among which after treatment with 20 μmol / L active decapeptide, the hemorrhage area in the zebrafish brain was close to 0.

[0031] The above results show that the active decapeptide can significantly reduce the hemorrhage rate and hemorrhage area in the zebrafish brain, and has significant anti-hemorrhagic stroke activity.

[0032] Example 4: Anti-hemorrhagic stroke effect of active decapeptide The zebrafish sample pretreatment and active decapeptide synthesis methods were the same as in Example 1.

[0033] 1. Experimental Methods choose Tg ( flk1:EGFP ) Zebrafish were used as experimental animals to test brain vascular integrity. 24 hpf zebrafish were grouped and treated the same way as in Example 3 until 48 hpf. Ten zebrafish were randomly selected from each group, and images of their brain vessels were captured using a Zeiss microscope to assess brain vascular loss.

[0034] 2. Experimental Results The experimental results are as follows Figure 9 As shown in the figure, valsartan represents the positive control group, 5 μmol / L, 10 μmol / L and 20 μmol / L represent the low concentration active decapeptide treatment group, the medium concentration active decapeptide treatment group and the high concentration active decapeptide treatment group, respectively. Figure 9 The red arrow indicates the part of the zebrafish brain where blood vessels are missing, and the yellow arrow indicates the part of the zebrafish brain where blood vessels are not missing; the scale bar is 200 μm. Figure 9 It was found that there were obvious defects in the brain blood vessels of zebrafish in the hemorrhagic stroke model group, but after treatment with 5 μmol / L, 10 μmol / L and 20 μmol / L of active decapeptide, the defects in the brain blood vessels of zebrafish were significantly restored.

[0035] The above results indicate that the active decapeptide can effectively repair brain vascular damage in zebrafish with hemorrhagic stroke and inhibit the occurrence and development of hemorrhagic stroke.

Claims

1. Use of an active decapeptide in the preparation of a drug for preventing or treating stroke, characterized in that: The amino acid sequence of the active decapeptide is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The cerebral stroke is ischemic cerebral stroke disease and hemorrhagic cerebral stroke disease.

3. The use according to claim 2, characterized in that The active decapeptide prevents or treats ischemic stroke by inhibiting cerebral thrombosis and restoring cerebral blood supply.

4. The use according to claim 2, characterized in that The active decapeptide prevents or treats hemorrhagic stroke by inhibiting cerebral hemorrhage and repairing cerebral vascular damage.

5. Use of an active decapeptide in the preparation of an anti-cerebral thrombotic drug, characterized in that: The amino acid sequence of the active decapeptide is shown in SEQ ID NO.

1.

6. Use of an active decapeptide in the preparation of an anti-cerebral hemorrhage drug, characterized in that: The amino acid sequence of the active decapeptide is shown in SEQ ID NO.1.

Images