Application of platelet factor 4 in preparation of medicine for treating acute ischemic stroke

Abstract

The invention relates to the technical field of biomedicine, in particular to application of a platelet factor 4 in preparation of a medicine for treating acute ischemic stroke. The platelet factor 4 can be used for remarkably relieving neurological impairment in the acute stage of ischemic stroke and reducing the infarct volume. For the early intervention of the acute ischemic stroke, a good treatment potential is shown, and a new drug target and a treatment strategy are provided for the treatment of the acute ischemic stroke.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedical technologies, and in particular, to the use of platelet factor 4 in the preparation of a medicament for treating acute ischemic stroke. Background Art

[0002] Ischemic stroke (IS) is caused by the sudden interruption of cerebral blood flow due to cerebral thrombosis or embolism, which in turn leads to irreversible neuronal necrosis, focal neurological deficits, and functional impairments. Ischemic stroke accounts for approximately 71% of all stroke types, is the second leading cause of death globally, and is also the main cause of long-term disability. Currently, the treatment of ischemic stroke mainly relies on achieving early reperfusion through tissue plasminogen activator (tPA)-mediated intravenous thrombolysis (IVT) and endovascular thrombectomy (EVT). Although IVT is widely used in IS patients, its treatment window is limited to 4.5 - 6 hours, and it has poor efficacy for large artery occlusion, achieving recanalization in less than 20% of cases. In contrast, EVT can achieve a recanalization rate of up to 80% in patients with large vessel occlusion, but less than 50% of patients can regain functional independence, and more than 15% of patients ultimately die. Therefore, it is urgent to explore new treatment approaches to extend the treatment window and improve the long-term recovery of IS patients, especially by means of biomolecules or proteins to exert neuroprotective effects, which has become a research hotspot. Summary of the Invention

[0003] To solve the above technical problems, the present disclosure provides the use of platelet factor 4 in the preparation of a medicament for treating acute ischemic stroke.

[0004] In a first aspect, the present disclosure provides the use of platelet factor 4 in the preparation of a medicament for treating acute ischemic stroke.

[0005] Platelet factor 4 (PF4) is a secreted protein mainly released from activated platelet α-granules.

[0006] Ischemic stroke can be divided into three stages: acute stage, subacute stage, and chronic stage. The acute stage refers to the earliest stage after the onset of stroke symptoms in patients, usually lasting for several hours to several days (commonly defined as within 7 days). In this stage, the brain tissue suffers ischemic damage due to the obstruction of blood circulation, resulting in limited oxygen and energy supply to the brain tissue, and further leading to ischemic and hypoxic necrosis of neurons, disruption of the blood-brain barrier, and inflammatory cascade reaction. The subacute stage is between the acute stage and the chronic stage, usually lasting for several days to several weeks. In this stage, the cerebrovascular system and nervous system of the patient begin to show a certain degree of repair process, but the inflammatory reaction persists, and it is accompanied by the initial initiation of brain tissue repair and neural plasticity. The chronic stage refers to the stage several weeks to several months after the occurrence of stroke. In this stage, neural plasticity and functional recovery of the patient gradually become dominant, and rehabilitation treatment is crucial. Different degrees of sequelae may be left, such as motor, sensory, language, cognitive impairment, etc.

[0007] The following are the preferred technical solutions of the present disclosure, but not the limitations of the technical solutions provided by the present disclosure. Through the following technical solutions, the technical objectives and beneficial effects of the present disclosure can be better achieved.

[0008] In a second aspect, the present disclosure provides a drug for treating acute ischemic stroke, including platelet factor 4.

[0009] As a preferred technical solution of the present disclosure, the dosage form of the drug is freeze-dried powder or injection.

[0010] As a preferred technical solution of the present disclosure, the drug further includes one or more pharmaceutically acceptable carriers or excipients.

[0011] As a preferred technical solution of the present disclosure, the pharmaceutically acceptable excipients include one or more of solubilizers, cosolvents, emulsifiers, flavoring agents, odor masking agents, coloring agents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, diluents, glidants, surfactants, or preservatives.

[0012] The technical solutions provided by the embodiments of the present disclosure have the following advantages compared with the prior art:

[0013] (1) The platelet factor 4 described in the present disclosure can significantly relieve the neurological deficit in the acute stage of ischemic stroke and reduce the infarct volume. For the early intervention of acute ischemic stroke, it shows good therapeutic potential, providing a new drug target and treatment strategy for the treatment of the acute stage of ischemic stroke; in addition, it also provides a research direction for the treatment of the chronic stage of ischemic stroke and can provide more treatment windows for clinical application;

[0014] (2) The platelet factor 4 described in the present disclosure can also be combined with other known stroke treatment drugs or therapies to form a combination treatment plan, improving the treatment effect;

[0015] (3) Moreover, based on the present disclosure, it helps to expand the application of platelet factor 4 in the preparation of drugs for treating different types of ischemic brain injuries (such as cerebral thrombosis, aneurysm rupture, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings herein are incorporated into the specification and form a part of the specification, showing embodiments consistent with the present disclosure, and are used together with the specification to explain the principles of the present disclosure.

[0017] To more clearly illustrate the technical solutions in the embodiments of the present disclosure or the prior art, the following will briefly introduce the accompanying drawings required for use in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 It is a graph showing the results of the modified Garcia neurological function scores of mice in the sham operation solvent administration control group, the model solvent administration control group, and the model PF4 administration group in the embodiments of the present disclosure;

[0019] Figure 2 It is a comparison graph of the contact time of mice in the sham operation solvent administration control group, the model solvent administration control group, and the model PF4 administration group in the sticky paper removal experiment in the embodiments of the present disclosure;

[0020] Figure 3 It is a comparison graph of the removal time of mice in the sham operation solvent administration control group, the model solvent administration control group, and the model PF4 administration group in the sticky paper removal experiment in the embodiments of the present disclosure;

[0021] Figure 4 It is a comparison graph of the misstep rate of mice in the sham operation solvent administration control group, the model solvent administration control group, and the model PF4 administration group in the grid walking experiment in the embodiments of the present disclosure;

[0022] Figure 5 It is an image of the movement trajectory of mice in the sham operation solvent administration control group, the model solvent administration control group, and the model PF4 administration group in the open field experiment in the embodiments of the present disclosure;

[0023] Figure 6 It is a comparison graph of the total movement distance of mice in the sham operation solvent administration control group, the model solvent administration control group, and the model PF4 administration group in the open field experiment in the embodiments of the present disclosure;

[0024] Figure 7The contrast graph of the average movement speed of the sham operation solvent administration control group mice, the model solvent administration control group mice, and the model PF4 administration group mice in the open field experiment in the embodiments of the present disclosure;

[0025] Figure 8 The contrast graph of the proportion of stationary movement time of the sham operation solvent administration control group mice, the model solvent administration control group mice, and the model PF4 administration group mice in the open field experiment in the embodiments of the present disclosure;

[0026] Figure 9 The contrast graph of the proportion of fast movement time of the sham operation solvent administration control group mice, the model solvent administration control group mice, and the model PF4 administration group mice in the open field experiment in the embodiments of the present disclosure;

[0027] Figure 10 The contrast graph of the number of inter-region transitions of the sham operation solvent administration control group mice, the model solvent administration control group mice, and the model PF4 administration group mice in the open field experiment in the embodiments of the present disclosure;

[0028] Figure 11 The representative MAP2 immunofluorescence staining graph of the cerebral infarction volume of the sham operation solvent administration control group mice, the model solvent administration control group mice, and the model PF4 administration group mice in the embodiments of the present disclosure;

[0029] Figure 12 The cerebral infarction volume result graph of the sham operation solvent administration control group mice, the model solvent administration control group mice, and the model PF4 administration group mice in the embodiments of the present disclosure.

[0030] It should be noted that in the figure, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. Detailed implementation manners

[0031] In order to be able to more clearly understand the above objects, features, and advantages of the present disclosure, the solutions of the present disclosure will be further described below. It should be noted that, without conflict, the embodiments of the present disclosure and the features in the embodiments can be combined with each other.

[0032] Many specific details are set forth in the following description in order to fully understand the present disclosure, but the present disclosure can also be implemented in other ways different from those described herein; obviously, the embodiments in the specification are only a part of the embodiments of the present disclosure, rather than all of the embodiments.

[0033] In a first aspect, an embodiment of the present disclosure provides an application of platelet factor 4 in the preparation of a drug for treating acute ischemic stroke.

[0034] Furthermore, in the future, it is necessary to evaluate the potential side effects of platelet factor 4 (such as blood coagulation, liver and kidney functions). If there are some stability or side effect problems with platelet factor 4 itself, PF4 can be improved through genetic engineering techniques or chemical modifications to enhance its efficacy or reduce adverse reactions.

[0035] In a second aspect, an embodiment of the present disclosure also provides a drug for treating acute ischemic stroke, which includes platelet factor 4.

[0036] As a preferred embodiment of the embodiment of the present disclosure, the dosage form of the drug is freeze-dried powder or injection.

[0037] As a preferred embodiment of the embodiment of the present disclosure, the drug further includes one or more pharmaceutically acceptable carriers or excipients.

[0038] As a preferred embodiment of the embodiment of the present disclosure, the pharmaceutically acceptable excipients include one or more of solubilizers, cosolvents, emulsifiers, flavoring agents, odor-correcting agents, coloring agents, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, diluents, glidants, surfactants or preservatives.

[0039] Next, the implementation schemes of the present disclosure will be described in detail in conjunction with embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present disclosure and should not be construed as limiting the scope of the present disclosure. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0040] Examples:

[0041] Animal experiments:

[0042] 1. Experimental animals:

[0043] Male C57BL / 6 mice, 2 - 3 months old, weighing 21 - 25 g, were randomly divided into 3 groups with the same number in each group, namely:

[0044] Sham operation solvent administration control group (Sham + PBS), model solvent administration control group (tMCAO + PBS), model PF4 administration group (tMCAO + PF4).

[0045] 2. Animal experiment method:

[0046] Establishment of an ischemic stroke mouse model with transient middle cerebral artery occlusion (tMCAO) reperfusion:

[0047] The mice in the model solvent administration control group (tMCAO+PBS) and the model PF4 administration group (tMCAO+PF4) were anesthetized with isoflurane through a face mask (5% isoflurane for induction of anesthesia and 1.5% isoflurane for maintenance of anesthesia). After successful anesthesia, the neck vessels were exposed. Under microscopic assistance, the ipsilateral common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated. Then, a 6-0 suture was inserted from the external carotid artery (ECA) into the internal carotid artery (ICA) until it reached the origin of the middle cerebral artery (MCA). The middle cerebral artery was occluded. After 1 hour of occlusion, the suture was slowly withdrawn to restore cerebral blood flow, i.e., reperfusion was achieved. The body temperature was maintained at 36.5-37.5 °C during the operation. After completing the surgical procedure, all the isolated vessels and tissues were repositioned, and the skin incision was carefully sutured with sterile sutures to ensure no residual bleeding. Then, the mice were placed in a cage under an infrared heating lamp until they recovered from anesthesia, and sufficient food and water were provided for them.

[0048] The sham operation solvent administration control group (Sham+PBS) received a sham operation. The specific operation was the same as that of the model group, but the operation only involved vessel isolation and no suture insertion.

[0049] The sham operation solvent administration control group (Sham+PBS): After receiving the sham operation, 100 μl of PBS solution (1×) was injected through the tail vein at 15 minutes, 24 hours, and 48 hours after the operation, respectively;

[0050] The model solvent administration control group (tMCAO+PBS): After successful modeling, 100 μl of PBS solution (1×) was injected through the tail vein at 15 minutes, 24 hours, and 48 hours after the operation, respectively;

[0051] The model PF4 administration group (tMCAO+PF4): After successful modeling, 100 μl of PF4 recombinant protein with a concentration of 2 μg / ml (product number: ab316401, purchased from Abcam) was injected through the tail vein at 15 minutes, 24 hours, and 48 hours after the operation, respectively.

[0052] 3. Detection methods:

[0053] 3.1 On the third day after successful modeling (i.e., after ischemic stroke / reperfusion), during which normal administration was carried out, the neurological function was evaluated in terms of sensory function and motor function, including the following methods:

[0054] (1) Modified Garcia neurological function score: Judgments were made from five manifestations: the lateral turning response of the animal, postural symmetry, forelimb extension movement, the ability to grasp and climb the iron cage, and the tactile reflex response on both sides of the body. The score for each manifestation was 0 - 3, and the higher the score, the less severe the neurological dysfunction. A total score of 15 indicated no neurological deficit.

[0055] (2) Sticker removal experiment: The sticker removal test is a classic method for measuring sensorimotor deficits. The mice were placed in a clean and transparent cage for 1 minute to adapt to the environment. Then, a sticky sticker (4 mm × 4 mm) was attached to the palmar side of the infarcted contralateral forepaw of the mice with the same force. Subsequently, the mice were gently placed back into the cage, and the time taken for the mice to touch and remove the sticker was recorded, with a maximum recording time of 120 seconds.

[0056] (3) Grid walking experiment: The grid walking experiment was used to evaluate motor coordination ability. The mice were placed on a grid (total size: 40 cm × 40 cm, small grid size: 2 cm × 2 cm, 50 cm above the ground). The mice were allowed to walk freely on the grid, and when the infarcted contralateral forelimb missed a step, it was defined as a misstep. The misstep rate was measured by the percentage of the number of missteps of the infarcted contralateral forelimb in the total number of steps.

[0057] (4) Open field experiment: The open field experiment was used to evaluate spontaneous motor ability. The mice were placed in an empty cube box (40 cm × 40 cm × 40 cm) located in a quiet and dark room, ensuring that the starting position was the same for each trial. A video monitoring device was used to record the movement for 5 minutes. The SMART V3.0 small animal behavior recording and analysis system was used to record the movement trajectory, total movement distance, average movement speed, percentage of stationary time, percentage of fast movement time, and number of movements, reflecting the spontaneous activity level of the mice.

[0058] 3.2 On the third day after successful modeling (while normal administration of drugs was carried out during this period), the cerebral infarction volume was evaluated, and the specific operations were as follows:

[0059] Animal perfusion and brain tissue fixation: The animals were perfused with 20 ml of normal saline at room temperature and 20 ml of 4% paraformaldehyde pre-cooled to 4°C. The mouse brain tissue was completely removed, dehydrated, and stored in a 4°C refrigerator for later use;

[0060] Freezing section of brain tissue: After embedding the mouse brain tissue, it was sectioned with a freezing microtome with a thickness of 60 μm. The obtained brain slices were immersed in a well plate containing 0.1 M PBS solution, and every 6th slice was placed in the same well. For example, the 1st, 7th, and 13th brain slices were placed in the same well, and the 2nd, 8th, and 14th brain slices were placed in the same well. The brain slices were stored in a 4°C refrigerator for later use;

[0061] Immunofluorescence staining: The antibody microtubule-associated protein 2 (MAP2) was used at a ratio of 1:500 to label neurons; brain slices from each animal in one well of the well plate were taken for staining;

[0062] Microscope photography: The sections were photographed with a fluorescence confocal microscope. The microscope parameters were adjusted to a magnification of 5× and a resolution of 512×512 pixels; after photographing, the images were exported and saved;

[0063] Calculation of cerebral infarction volume: The images taken by the microscope were imported into Image J software, the infarction area of each brain slice was circled, and the infarction area of a single brain slice was calculated through the software calculation module. Infarction volume = total infarction area × 60 μm × 6 (wells).

[0064] 4. Experimental results

[0065] 4.1 Platelet factor 4 can improve sensorimotor dysfunction in the acute phase of ischemic stroke in mice

[0066] To explore the effect of platelet factor 4 on neurological function in the acute phase of ischemic stroke, the present disclosure evaluated sensory function and motor function on the 3rd day after successful mouse modeling (while administering drugs normally), including the modified Garcia neurological score, sticky paper removal experiment, and grid walking experiment, where:

[0067] Figure 1 This is the graph of the modified Garcia neurological score results for the sham operation solvent-administered control group mice, model solvent-administered control group mice, and model PF4-administered group mice in the embodiments of the present disclosure;

[0068] Figure 2 This is the comparison graph of the contact time of the sham operation solvent-administered control group mice, model solvent-administered control group mice, and model PF4-administered group mice in the sticky paper removal experiment in the embodiments of the present disclosure;

[0069] Figure 3 This is the comparison graph of the removal time of the sham operation solvent-administered control group mice, model solvent-administered control group mice, and model PF4-administered group mice in the sticky paper removal experiment in the embodiments of the present disclosure;

[0070] Figure 4 This is the comparison graph of the misstep rate of the sham operation solvent-administered control group mice, model solvent-administered control group mice, and model PF4-administered group mice in the grid walking experiment in the embodiments of the present disclosure.

[0071] From Figure 1 It can be seen that compared with the model solvent-administered control group mice, the modified Garcia neurological score of the model PF4-administered group mice is significantly higher; fromFigure 2 and Figure 3 It can be seen that compared with the mice in the control group administered with the model solvent, the mice in the model PF4-administered group had significantly shorter contact time and removal time of the sticky paper; from Figure 4 It can be seen that compared with the mice in the control group administered with the model solvent, the mice in the model PF4-administered group had significantly lower misstep rate in the grid walking test. The results showed that PF4 could improve the neurological dysfunction in the acute stage of ischemic stroke in mice.

[0072] 4.2 Platelet factor 4 can improve the spontaneous motor ability of mice in the acute stage of ischemic stroke

[0073] In order to explore the effect of platelet factor 4 on the spontaneous motor ability in the acute stage of ischemic stroke, the open field test of mice was carried out on the 3rd day after successful establishment of the mouse model (administered with normal drugs during the period), where:

[0074] Figure 5 are the movement trajectory images of the mice in the sham operation solvent-administered control group, the model solvent-administered control group, and the model PF4-administered group of the present disclosure in the open field test;

[0075] Figure 6 is the comparison chart of the total movement distance of the mice in the sham operation solvent-administered control group, the model solvent-administered control group, and the model PF4-administered group of the present disclosure in the open field test;

[0076] Figure 7 is the comparison chart of the average movement speed of the mice in the sham operation solvent-administered control group, the model solvent-administered control group, and the model PF4-administered group of the present disclosure in the open field test;

[0077] Figure 8 is the comparison chart of the proportion of static movement time of the mice in the sham operation solvent-administered control group, the model solvent-administered control group, and the model PF4-administered group of the present disclosure in the open field test;

[0078] Figure 9 is the comparison chart of the proportion of fast movement time of the mice in the sham operation solvent-administered control group, the model solvent-administered control group, and the model PF4-administered group of the present disclosure in the open field test;

[0079] Figure 10 is the comparison chart of the number of transitions between regions of the mice in the sham operation solvent-administered control group, the model solvent-administered control group, and the model PF4-administered group of the present disclosure in the open field test.

[0080] From Figures 6 - 10It can be seen that, compared with the mice in the control group administered with the model solvent, the total movement distance of the mice in the model PF4-administered group increased, the average movement speed increased, the proportion of stationary time decreased, the proportion of fast movement time increased, and the number of transitions between regions increased. The results indicate that PF4 can improve the autonomous movement ability of mice in the acute stage of ischemic stroke.

[0081] 4.3 Platelet factor 4 can reduce the cerebral infarction volume in the acute stage of ischemic stroke in mice

[0082] Figure 11 This is a representative MAP2 immunofluorescence staining map of the cerebral infarction volume of the sham operation solvent-administered control group mice, the model solvent-administered control group mice, and the model PF4-administered group mice in the embodiments of the present disclosure;

[0083] Figure 12 This is a graph showing the results of the cerebral infarction volume of the sham operation solvent-administered control group mice, the model solvent-administered control group mice, and the model PF4-administered group mice in the embodiments of the present disclosure.

[0084] Through Figure 11 and Figure 12 it can be known that, compared with the mice in the model solvent-administered control group, the cerebral infarction volume of the mice in the model PF4-administered group was significantly reduced. The results indicate that PF4 can reduce the cerebral infarction volume in the acute stage of ischemic stroke in mice.

[0085] The above are only specific embodiments of the present disclosure, enabling those skilled in the art to understand or implement the present disclosure. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to these embodiments described herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A use of platelet factor 4 in the preparation of a drug for treating acute ischemic stroke.

2. A drug for treating acute ischemic stroke, characterized in that: Including platelet factor 4.

3. The drug according to claim 2, characterized in that The dosage form of the drug is lyophilized powder or injection.

4. The drug according to claim 2 or 3, characterized in that The medicine also includes one or more pharmaceutically acceptable carriers or excipients.

5. The drug according to any one of claims 2 to 4, characterized in that The pharmaceutically acceptable excipients include one or more of solubilizers, cosolvents, emulsifiers, flavoring agents, olfactory agents, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, pH regulators, stabilizers, diluents, glidants, surfactants or preservatives.

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