Methods and agents for treating Huntington's disease

Abstract

Methods and medicaments for treating Huntington's disease are provided, which comprise administering to a subject a therapeutically effective amount of a component of the plasminogen activation pathway or a compound related thereto. Also provided are medicaments, pharmaceutical compositions, products, and kits for treating Huntington's disease, which include the component or compound.

Description

[Technical field]

[0001] The present invention relates to a treatment for Huntington's disease comprising administering to a subject an effective amount of a component of the plasminogen activation pathway or a related compound, such as plasminogen, to treat Huntington's disease. [Background technology]

[0002] Huntington's disease (HD), also known as Grand's chorea or Huntington's chorea, is an autosomal dominant neurodegenerative disease. Its causative gene is the IT-15 gene (also called the HTT gene) located on chromosome 4p16.3. This gene shows abnormal amplification of the cytosine-adenine-guanine (CAG) trinucleotide repeat sequence in Huntington's disease patients, and when its copy number exceeds 40, it has complete penetrance. Generally, the disease develops in middle age, and the main symptoms include progressive dyskinesia represented by chorea-like symptoms, cognitive decline, and psychiatric symptoms. Currently, second-generation antipsychotic drugs such as tetrabenazine and olanzapine are often used to suppress the symptoms of chorea, and antidepressants are often used to improve psychiatric symptoms such as depression. However, these drugs cannot slow the progression of the disease. Currently, research is being actively conducted to explore the cause of the disease by gene therapy, but there are many uncertainties in the method of gene therapy. Therefore, it has become a focus of research into this disease to find alternative ways to find more effective treatments. Summary of the Invention

[0003] Through research, the present invention has discovered that plasminogen can significantly improve Huntington's disease and its associated symptoms, for example, improve motor disorders, improve cognitive impairment, slow weight loss, protect undamaged neurons, promote the repair of damaged neurons, and / or improve neuropsychiatric symptoms in subjects with Huntington's disease, thereby providing an effective treatment for Huntington's disease.

[0004] Specifically, the present invention relates to the following:

[0005] In one aspect, the present application relates to a method of treating Huntington's disease comprising administering to a subject with Huntington's disease a therapeutically effective amount of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

[0006] In one aspect, the present application relates to the use of one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors in the preparation of a medicament for treating Huntington's disease.

[0007] In one aspect, the present application relates to an agent or pharmaceutical composition for treating Huntington's disease comprising one or more compounds selected from components of the plasminogen activation pathway, compounds that can activate plasminogen directly or indirectly by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activators, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of fibrinolysis inhibitors.

[0008] 2. The method, use, agent or pharmaceutical composition according to item 1, wherein the component of the plasminogen activation pathway is selected from plasminogen, recombinant human plasminogen, Lys-plasminogen, Glu-plasminogen, plasmin, plasminogen and plasmin variants and analogs containing one or more kringle domains and protease domains of plasminogen and plasmin, mini-plasminogen, mini-plasmin, micro-plasminogen, micro-plasmin, delta-plasminogen, delta-plasmin, plasminogen activators, tPA and uPA.

[0009] 3. The method, use, agent or pharmaceutical composition according to item 1, wherein the fibrinolysis inhibitor antagonist is an inhibitor of PAI-1, complement C1 inhibitor, alpha 2 antiplasmin or alpha 2 macroglobulin, such as an antibody.

[0010] 4. The method, use, medicament or pharmaceutical composition according to any one of items 1 to 3, wherein the compound has one or more effects selected from the following for a subject with Huntington's disease: improving or alleviating motor impairment, improving cognitive impairment, slowing weight loss, promoting repair of damaged neurons, improving neuropsychiatric symptoms, relieving anxiety, reducing GFAP expression in the hippocampus, reducing apoptosis in the hippocampus, promoting recovery of Nissl body numbers in cerebellar, hippocampal or striatal neurons, repairing damage to the hippocampus, striatum or olfactory tubercle, promoting BDNF expression in the hippocampus or striatum, and promoting myelination in the striatum.

[0011] 5. The method, use, medicament or pharmaceutical composition according to any one of items 1 to 4, wherein the compound is used in combination with one or more other medicaments or treatment methods.

[0012] Item 6. The method, use, medicament or pharmaceutical composition according to item 5, wherein the other medicament or treatment method is one or more selected from antidopaminergic agents, dopamine receptor inhibitors, antipsychotics (e.g., butyrylphenones and phenothiazines), gamma-aminobutyric acid transferase inhibitors, cell transplantation therapy, and gene therapy.

[0013] 7. The method, use, medicament, or pharmaceutical composition according to any one of items 1 to 6, wherein the compound is plasminogen.

[0014] 8. The method, use, agent, or pharmaceutical composition according to any one of items 1 to 7, wherein the plasminogen is human full-length plasminogen or a conservatively substituted variant thereof.

[0015] 9. The method, use, agent or pharmaceutical composition according to any one of items 1 to 7, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2 and still has plasminogen lysine binding activity or proteolytic activity.

[0016] 10. The method, use, agent or pharmaceutical composition according to any one of claims 1 to 7, wherein the plasminogen comprises a protein that consists of an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with sequence 14 and still has the proteolytic activity of plasminogen.

[0017] 11. The method, use, agent, or pharmaceutical composition according to any one of items 1 to 7, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, delta-plasminogen, or mutants thereof that retain the proteolytic activity of plasminogen.

[0018] 12. The method, use, agent, or pharmaceutical composition according to any one of claims 1 to 7, wherein the plasminogen comprises the amino acid sequence shown in sequence 2, 6, 8, 10 or 12, or comprises a conservatively substituted variant of the amino acid sequence shown in sequence 2, 6, 8, 10 or 12.

[0019] 13. The method, use, medicament, or pharmaceutical composition according to any one of items 1 to 12, wherein the compound is administered by nasal inhalation, aerosol inhalation, nasal drops, eye drops, ear drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g. via the carotid artery), and intramuscular administration.

[0020] In any one of the above embodiments of the present application, the plasminogen may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2, 6, 8, 10 or 12 and still have plasminogen activity, for example, lysine binding activity or proteolytic activity. In some embodiments, the plasminogen is a protein in which 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid is added, deleted, and / or substituted based on sequence 2, 6, 8, 10 or 12 and still have plasminogen activity, for example, lysine binding activity or proteolytic activity.

[0021] In some embodiments, the plasminogen is a protein that includes plasminogen fragments and still has plasminogen activity, such as lysine-binding activity or proteolytic activity. In some embodiments, the plasminogen is Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, δ-plasminogen, or a variant thereof that retains plasminogen activity, such as proteolytic activity. In the above embodiments, the plasminogen is natural or synthetic human plasminogen, or a variant or fragment thereof that still retains plasminogen activity, such as lysine-binding activity or proteolytic activity. In some embodiments, the plasminogen is an orthologue of human plasminogen from a primate or rodent, or a variant or fragment thereof that still retains plasminogen activity, such as lysine-binding activity or proteolytic activity. In some embodiments, the amino acid of the plasminogen is shown in sequence 2, 6, 8, 10, or 12. In some embodiments, the plasminogen is human natural plasminogen.

[0022] In some embodiments, the subject is a human. In some embodiments, the subject is deficient or lacking in plasminogen. In some embodiments, the deficient or lacking is congenital, inherited and / or localized.

[0023] In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier and plasminogen for use in the aforementioned methods. In some embodiments, the kit may be a prophylactic or therapeutic kit comprising (i) plasminogen for use in the aforementioned methods, and (ii) means for delivering the plasminogen to the subject. In some embodiments, the means is a syringe or a vial. In some embodiments, the kit further comprises a label or a protocol for instructing administration of the plasminogen to the subject to perform any of the aforementioned methods.

[0024] In some embodiments, the article of manufacture comprises a container containing a label and (i) plasminogen or a pharmaceutical composition containing plasminogen for use in the aforementioned methods, the label providing instructions for administering the plasminogen or composition to the subject to perform any of the aforementioned methods.

[0025] In some embodiments, the kit or article of manufacture further comprises one or more additional components or containers that contain other agents.

[0026] In some embodiments of the method, the plasminogen is administered systemically or locally, preferably by intravenous, intramuscular, or subcutaneous administration of plasminogen. In some embodiments of the method, the plasminogen is administered in combination with a suitable polypeptide carrier or stabilizer. In some embodiments of the method, the plasminogen is administered at a daily dose of 0.0001-2000 mg / kg, 0.001-800 mg / kg, 0.01-600 mg / kg, 0.1-400 mg / kg, 1-200 mg / kg, 1-100 mg / kg, 10-100 mg / kg (calculated per kilogram of body weight), or 0.0001-2000 mg / cm 2 , 0.001-800mg / cm 2 , 0.01-600mg / cm 2 , 0.1-400mg / cm 2 , 1-200mg / cm 2 , 1-100mg / cm 2 , 10-100mg / cm 2 (calculated per square centimeter of body surface area) and is preferably administered one or more times, preferably at least daily.

[0027] The present invention expressly covers all combinations of the technical features belonging to the embodiments of the present invention, and the technical configurations after the combinations are expressly disclosed in this application as if the above technical configurations were separately expressly disclosed. Moreover, the present invention also expressly covers the combinations between each embodiment and their elements, and the technical configurations after the combinations are expressly disclosed in this specification. [Brief description of the drawings]

[0028] [Figure 1]Figure 1 shows the statistical results of the total movement distance in the open field test of Huntington's disease model mice 7 days after plasminogen administration. As a result, the mice in the blank control group moved a certain distance during the experiment, the total movement distance of the mice in the solvent control group was significantly longer than that of the blank control group, and the total movement distance of the mice in the plasminogen administration group was significantly shorter than that of the solvent control group, and the difference was statistically significant (representing *P<0.05), and the total movement distance of the plasminogen administration group was close to that of the blank control group. This suggests that plasminogen can promote the recovery of spontaneous behavior in Huntington's disease model mice. [Diagram 2] FIG. 2 shows the statistical results of the resting time rate in the border zone of the open field test of Huntington's disease model mice 7 days after plasminogen administration. The results showed that the mice in the blank control group had a certain resting time rate in the border zone, the resting time rate in the border zone of the mice in the solvent control group was significantly smaller than that of the blank control group, and the resting time rate in the border zone of the mice in the plasminogen administration group was significantly larger than that of the solvent control group, and the statistical difference was highly significant (*** represents P<0.001), and the resting time rate in the border zone of the plasminogen administration group was close to the value of the blank control group. This suggests that plasminogen can reduce the anxiety behavior of Huntington's disease model mice. [Diagram 3] FIG. 3 shows the statistical results of the average moving speed in the central zone of the open field test of Huntington's disease model mice 7 days after plasminogen administration. The results showed that the mice in the blank control group had a constant moving speed in the central zone, the average moving speed in the central zone of the mice in the solvent control group was significantly smaller than that of the blank control group, and the average moving speed in the central zone of the mice in the plasminogen group was significantly larger than that of the solvent control group, and the statistical difference was significant (* represents P<0.05), and the moving speed in the central zone of the plasminogen administration group was close to that of the blank control group. This suggests that plasminogen can promote the recovery of spontaneous activity and avoidance behavior in Huntington's disease model mice and alleviate anxiety behavior. [Figure 4]FIG. 4 shows the statistical results of the average movement speed in the border zone of the open field test of Huntington's disease model mice 7 days after plasminogen administration. The results showed that the mice in the blank control group had a constant movement speed in the border zone, the average movement speed in the border zone of the mice in the solvent control group was significantly greater than that of the blank control group, and the average movement speed in the border zone of the mice in the plasminogen group was significantly smaller than that of the solvent control group, and the statistical difference was significant (* represents P<0.05), and the average movement speed in the border zone of the plasminogen administration group was close to that of the blank control group. This suggests that plasminogen can promote the recovery of spontaneous activity and avoidance behavior in Huntington's disease model mice and alleviate anxiety behavior. [Diagram 5] Figure 5A-C are diagrams of the movement trajectories in the open field test of Huntington's disease model mice 7 days after plasminogen administration. A is the blank control group, B is the solvent control group, and C is the plasminogen administration group. As a result, the mice in the blank control group had less movement in the central zone, more movement in the border zone, and a regular movement trajectory; compared with the blank control group, the movement in the central zone and the total movement distance of the mice in the solvent group were significantly increased, and the movement trajectory was disorderly and irregular; the movement in the central zone and the total movement distance of the mice in the plasminogen group were significantly less than those in the solvent group, and the movement trajectory was similar to that of the blank control group. This suggests that plasminogen can promote the recovery of spontaneous activity and avoidance behavior in Huntington's disease model mice. [Figure 6] Figure 6 shows the calculation results of the body weight ratio (percentage) (%) on day 7 and day 0 of Huntington's disease model mice 7 days after plasminogen administration. As a result, the body weight ratio of the blank control group was greater than 1, indicating a tendency for body weight to increase; the body weight ratio of the mice in the solvent control group was smaller than that of the blank control group; the body weight ratio of the mice in the plasminogen administration group was significantly higher than that of the solvent control group, and the statistical difference was close to significance (P = 0.07). This result suggests that plasminogen can promote weight gain in Huntington's disease model mice. [Figure 7]7A-D are diagrams showing the results of hippocampal GFAP immunohistochemistry of Huntington's disease model mice 7 days after plasminogen administration. A is the blank control group, B is the solvent control group, C is the plasminogen administration group, and D is the quantitative analysis result of the average optical density. The results showed that there was a certain amount of GFAP expression in the hippocampus of the mice in the blank control group, the expression of GFAP in the hippocampus of the mice in the solvent control group was significantly higher than that of the blank control group, and the statistical difference was highly significant (*** represents P<0.001), and the expression of GFAP in the hippocampus of the mice in the plasminogen administration group was significantly lower than that of the solvent control group, and the statistical difference was highly significant, and there was no statistical difference compared with the expression of GFAP in the hippocampus of the mice in the blank control group. This result suggests that plasminogen can reduce the expression of GFAP in the hippocampus of Huntington's disease model mice, inhibit the proliferation of astroglial cells, and promote the recovery of hippocampal damage. [Figure 8] Figure 8A-D shows the results of immunohistochemical staining of caspase-3 in the hippocampus of Huntington's disease model mice 7 days after plasminogen administration. A is the blank control group, B is the solvent control group, C is the plasminogen administration group, and D is the quantitative analysis result of the mean optical density. The results showed that the hippocampus of the blank control group expressed a certain amount of caspase-3 (marked with an arrow), the expression of caspase-3 in the hippocampus of the solvent control group was significantly increased, and the expression of caspase-3 in the hippocampus of the plasminogen administration group was significantly lower than that of the solvent control group, and the statistical difference was close to significance (P = 0.058). This result suggests that plasminogen can reduce the expression of caspase-3 and reduce the apoptosis of hippocampal cells in Huntington's disease model mice. [Figure 9]9A-D are diagrams showing the results of toluidine blue staining of the cerebellum of Huntington's disease model mice 7 days after plasminogen administration. A is the blank control group, B is the solvent control group, C is the plasminogen administration group, and D is the analysis result of the number of cerebellar Nissl bodies. As a result, a certain number of Nissl bodies were present in the cerebellar neurons of the blank control group (marked with an arrow), the number of Nissl bodies in the cerebellar neurons of the solvent control group was significantly higher than that of the blank control group, and the number of Nissl bodies in the cerebellar neurons of the mice in the plasminogen group was not significantly different from that of the blank control group, but was significantly lower than that of the solvent control group, and the difference was statistically significant (* represents P<0.05). This result suggests that plasminogen can promote the recovery of the number of Nissl bodies in the cerebellar neurons of Huntington's disease model mice. [Figure 10] Figure 10A-D shows the results of tar violet staining of the left hippocampus of Huntington's disease model mice 7 days after plasminogen administration. A is the blank control group, B is the solvent control group, C is the plasminogen administration group, and D is the analysis result of the number of hippocampal Nissl bodies. As a result, a certain amount of Nissl bodies was present in the left hippocampal neurons of the mice in the blank control group (marked with an arrow), the number of Nissl bodies in the left hippocampal neurons of the mice in the solvent group was significantly higher than that of the blank control group, and the number of Nissl bodies in the left hippocampal neurons of the mice in the plasminogen group was significantly lower than that of the solvent group, and the difference was statistically significant (* represents P<0.05). This result suggests that plasminogen can promote the recovery of the number of Nissl bodies in the left hippocampal neurons. [Figure 11]11A-D are diagrams showing the results of tar violet staining of the striatum of Huntington's disease model mice 7 days after plasminogen administration. A is the blank control group, B is the solvent control group, C is the plasminogen administration group, and D is the analysis result of the number of striatal Nissl bodies. As a result, a certain amount of Nissl bodies was present in the striatal neurons of the mice in the blank control group (marked with an arrow), the number of Nissl bodies in the striatal neurons of the mice in the solvent control group was significantly lower than that of the blank control group, and the number of Nissl bodies in the striatal neurons of the mice in the plasminogen group was significantly higher than that of the solvent control group, and the difference was statistically significant (* indicates P<0.05). This result indicates that plasminogen can promote the recovery of the number of striatal Nissl bodies. [Figure 12] Figure 12A-I shows the results of H&E staining of the hippocampus of Huntington's disease model mice 7 days after plasminogen administration. A, B, and C are blank control groups, D, E, and F are solvent control groups, and G, H, and I are plasminogen-administered groups. The results showed that the overall shape of the hippocampus of the mice in the blank control group was normal and the structure was intact; the CA1 region of the hippocampus of the mice in the solvent control group showed disordered arrangement of neurons, atrophy of nerve cells (marked with thin arrows), degeneration and necrosis of cells, and loosening and edema of the surrounding brain tissue (marked with triangles), and the nerve cell nuclei in multiple locations in the CA3 region were condensed and strongly stained (marked with thick arrows); in the plasminogen-administered group, the shape of each region of the hippocampus was restored to a certain extent, and some nerve cell nuclei in local areas of the CA3 and DG regions were condensed and strongly stained. This result suggests that plasminogen can repair hippocampal damage in Huntington's disease model mice. [Figure 13]Figure 13A-C shows the results of H&E staining of the striatum in Huntington's disease model mice 7 days after plasminogen administration. A is the blank control group, B is the solvent control group, and C is the plasminogen-administered group. As a result, the morphological structure of the striatum of the mice in the blank control group was normal, while the striatal neurons of the mice in the solvent group were degenerated, some cell nuclei were vacuolated (marked with arrows), and the arrangement of brain cells was disturbed; the mice in the plasminogen-administered group showed a significant improvement in the overall structure of the striatum, similar to the blank control group. This result suggests that plasminogen can repair striatal damage in Huntington's disease model mice. [Figure 14] 14A-C are diagrams showing the results of H&E staining of the olfactory tubercle in Huntington's disease model mice 7 days after plasminogen administration. A is the blank control group, B is the solvent control group, and C is the plasminogen-administered group. As a result, the olfactory tubercle of the brain of the mice in the blank control group had normal morphology and structure, the olfactory tubercle of the mice in the solvent group had severe glial cell infiltration (marked with arrows), and the olfactory tubercle glial cell infiltration of the mice in the plasminogen-administered group was significantly less than that of the solvent group. This result suggests that plasminogen can improve the brain olfactory tubercle damage in Huntington's disease model mice. [Figure 15] Figure 15A-D shows the results of hippocampal BDNF immunohistochemical staining of Huntington's disease model mice 7 days after plasminogen administration. A is the blank control group, B is the solvent control group, C is the plasminogen administration group, and D is the quantitative analysis result of the average optical density. The results showed that there was a certain amount of BDNF expression in the right hippocampus of the mice in the blank control group (marked with an arrow), the expression of BDNF in the right hippocampus of the mice in the solvent control group was slightly higher than that of the blank control group, and the expression of BDNF in the right hippocampus of the mice in the plasminogen group was significantly higher than that of the solvent control group, and the statistical difference was close to significance (P = 0.07). This result suggests that injury stimulates the expression of BDNF, and plasminogen can further promote the expression of BDNF, thereby promoting the recovery of hippocampal injury in Huntington's disease model mice. [Figure 16]Figure 16A-D shows the results of immunohistochemical staining of striatal BDNF in Huntington's disease model mice 7 days after plasminogen administration. A is the blank control group, B is the solvent control group, C is the plasminogen administration group, and D is the quantitative analysis result of the average optical density. The results showed that there was a certain amount of BDNF expression in the striatum of mice in the blank control group (marked with an arrow), the expression of BDNF in the striatum of mice in the solvent control group was slightly higher than that of the blank control group, and the expression of BDNF in the striatum of mice in the plasminogen administration group was significantly higher than that of the solvent control group, and the statistical difference was close to significance (P = 0.1). This result suggests that injury stimulates the expression of BDNF, and plasminogen can further promote the expression of BDNF, thereby promoting the recovery of striatal injury in Huntington's disease model mice. [Figure 17] Figure 17A-D shows the results of LFB staining in the striatum of Huntington's disease model mice 7 days after plasminogen administration. A is the blank control group, B is the solvent control group, C is the plasminogen administration group, and D is the quantitative analysis result of the average optical density. As a result, the striatal myelin structure of the mice in the blank control group was normal, the amount of striatal myelin protein of the mice in the solvent group was reduced and cavity degeneration was observed (marked with arrows), and the amount of striatal myelin protein of the mice in the plasminogen administration group was significantly higher than that of the solvent group, and the statistical difference was close to significance (P = 0.09). This result suggests that plasminogen can promote the formation of striatal myelin protein in Huntington's disease model mice. Detailed Description of the Invention

[0029] Huntington's disease (HD) is a monogenic autosomal dominant neurodegenerative disease with mHtt protein toxicity as the main pathogenic mechanism, and its main symptoms include progressive dyskinesia, cognitive decline, and psychiatric symptoms, which are typically manifested by chorea-like symptoms. When gene mutations cause an abnormal increase in CAG copy number, the amino-terminal (N-terminal) polyglutamine tract (polyQ) of the mutant Htt (mHtt) protein is extended, forming an abnormal conformation including a lamellar structure, which causes the mHtt protein to lose its normal function and acquire a toxic effect.

[0030] The "movement disorders" of Huntington's disease mainly include involuntary movement disorders (most commonly chorea) and voluntary movement disorders (including ataxia and slowness of movement). Progressive dyskinesia appears as sudden, rapid throbbing or spasms of the limbs, face, and trunk that are unknown and uncontrollable beforehand, and may also appear as uncontrollable slow movements. Examination reveals choreic involuntary movements and hypotonia. Choreic involuntary movements are the most prominent feature of the disease, most of which begin as brief, uncontrollable grimaces, nodding, and finger flexion and extension movements that resemble painless spasms but are slower and atypical. As the disease progresses, the involuntary movements become progressively worse, with typical eyebrow raising and head flexion, head turning when staring, the patient walks unsteadily, the gait becomes jumpy, the hand posture changes constantly, and the whole body moves in a dance-like manner. In the later stages of the disease, patients are unable to stand or walk due to involuntary movements of the whole body. As the disease progresses, the impairment of voluntary movements becomes increasingly evident, with clumsy, slow, and rigid movements, inability to maintain complex voluntary movements, dysphagia, hesitancy in speech, and dysarthria. Abnormal eye movements may also appear. In the advanced stages of the disease, a stupor with immobility of the limbs may occur.

[0031] Chorea dyskinesia is typical of adult-onset Huntington's disease. Juvenile patients with onset before age 20 years have immobile myotonia as the primary movement disorder, which may manifest as myotonia, myoclonus, and, in later stages, opistonia. Furthermore, unlike adults, approximately 50% of patients with juvenile Huntington's disease experience generalized epileptic seizures.

[0032] The "cognitive impairment" of Huntington's disease may appear many years before the motor symptoms and progress more slowly. Progressive dementia is another hallmark of Huntington's disease patients. Dementia may be characterized as subcortical in the early stages and as mixed cortical and subcortical dementia in the later stages.

[0033] Cognitive impairment may appear in the early stages of Huntington's disease. It begins with a decline in memory and arithmetic in daily life and at work, and patients have only mild impairment in recalling new information but significant deficits in recall. There are also changes in speech, such as decreased verbal fluency, mild difficulty in finding words, and dysarthria. Impaired oral fluency is one of the early cognitive impairments in Huntington's disease. In the middle and later stages of the disease, patients are unable to complete naming tests that require recall of rare words. Choreomorphic movement disorders often affect the tongue and lips, disrupting the rhythm and agility of pronunciation and interfering with the volume, rate, rhythm, and length of speech. Thus, articulation and rhythm disorders are prominent features of patients with this disease.

[0034] The "neuropsychiatric symptoms" of Huntington's disease can appear very early, with early symptoms such as depression, irritability and apathy, and more severe symptoms including delusions or schizophrenia-like symptoms.

[0035] The fibrinolytic system, also called the fibrinolytic system, is a system of a series of chemicals involved in the process of fibrinolysis (fibrinolysis), mainly including plasminogen (PLG), plasmin, plasminogen activators, and fibrinolysis inhibitors. Plasminogen activators include tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). t-PA is a serine protease and is synthesized by vascular endothelial cells. t-PA activates plasminogen, a process that mainly occurs in fibrin. Urokinase-type plasminogen activator (u-PA) is produced by renal tubular epithelial cells and vascular endothelial cells and can directly activate plasminogen without the need for fibrin as a cofactor. Plasminogen (PLG) is synthesized in the liver. When blood clots, PLG is adsorbed in large quantities to the fibrin net and activated to plasmin by the action of t-PA or u-PA to promote fibrinolysis. Plasminase (PL) is a serine protease that degrades fibrin and fibrinogen, hydrolyzes various coagulation factors V, VIII, X, VII, XI, II, etc., converts plasminogen to plasmin, hydrolyzes complement, etc. Fibrinolysis inhibitors include plasminogen activator inhibitor (PAI) and α2-antithyplasmin (α2-AP). There are two main forms of PAI, PAI-1 and PAI-2, which can specifically bind to t-PA in a 1:1 ratio and inactivate t-PA while activating PLG. α2-AP is synthesized in the liver and binds to PL in a 1:1 ratio to form a complex, thereby inhibiting PL activity. FXIII covalently binds α2-AP to fibrin, thereby weakening the susceptibility of fibrin to PL. Substances that inhibit the activity of the fibrinolysis system in vivo include PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin.

[0036] As used herein, the term "a component of the plasminogen activation pathway" refers to a 1. Plasminogen, Lys-plasminogen, Glu-plasminogen, micro-plasminogen, delta-plasminogen, their variants or analogs; 2. Plasmin and its variants or analogues; and 3. Plasminogen activators, including, for example, tPA and uPA, as well as tPA or uPA variants and analogs that contain one or more domains of tPA or uPA, such as one or more of the kringle domain and the proteolytic domain.

[0037] The above "variants" of plasminogen, plasmin, tPA and uPA include all naturally occurring genetic variants of human and other mammalian forms of these proteins, as well as proteins having, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1 amino acid added, deleted and / or substituted and still having plasminogen activity, plasmin activity, tPA or uPA activity. For example, a "variant" of plasminogen, plasmin, tPA or uPA includes, for example, mutants of these proteins resulting from substitution of 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 conservative amino acids.

[0038] The "plasminogen variant" of the present invention covers a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with sequence 2, 6, 8, 10 or 12 and still having plasminogen activity, e.g., lysine binding activity or proteolytic activity. For example, the "plasminogen variant" of the present invention can be a protein having 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 1 amino acid added, deleted and / or substituted based on sequence 2, 6, 8, 10 or 12 and still having plasminogen activity, e.g., lysine binding activity or proteolytic activity. In particular, the plasminogen variants of the present invention include all naturally occurring genetic variants of human and other mammalian forms of these proteins, as well as mutants of these proteins obtained by conservative substitution of, for example, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2, 1 amino acids.

[0039] The plasminogen of the present invention may be an orthologue of human plasminogen derived from a primate or rodent, or a variant which still retains plasminogen activity, e.g., lysine-binding activity or proteolytic activity, such as the plasminogen shown in sequences 2, 6, 8, 10 or 12, e.g., human native plasminogen shown in sequence 2.

[0040] The above-mentioned "analogs" of plasminogen, plasmin, tPA and uPA include compounds that provide substantially the same effect as plasminogen, plasmin, tPA or uPA, respectively.

[0041] The above "variants" and "analogs" of plasminogen, plasmin, tPA and uPA cover "variants" and "analogs" of plasminogen, plasmin, tPA and uPA that contain one or more domains (e.g., one or more kringle domains and a proteolytic domain). For example, "variants" and "analogs" of plasminogen cover plasminogen variants and analogs, such as mini-plasminogen, that contain one or more plasmin domains (e.g., one or more kringle domains and a proteolytic domain). "Variants" and "analogs" of plasmin cover plasmin "variants" and "analogs" such as mini-plasmin and delta-plasmin that contain one or more plasmin domains (e.g., one or more kringle domains and a proteolytic domain).

[0042] Whether the above-mentioned "variants" or "analogs" of plasminogen, plasmin, tPA or uPA have the activity of plasminogen, plasmin, tPA or uPA, respectively, or whether they exert substantially the same effect as plasminogen, plasmin, tPA or uPA, respectively, can be measured by the level of activated plasmin activity using methods known in the art, such as enzymography, ELISA (enzyme-linked immunosorbent assay) and FACS (fluorescence-activated cell sorting). For example, the measurement can be performed with reference to the methods described in the following documents:Ny,A.,Leonardsson,G.,Hagglund,A.C,Hagglof,P.,Ploplis,V.A.,Carmeliet,P. and Ny,T. (1999). Ovulation inplasminogen-deficient mice. Endocrinology 140,5030-5035;Silverstein RL, Leung LL, Harpel PC, Nachman RL (November 1984). “Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator”. J. Clin. Invest. 74 (5): 1625-33;Gravanis I, Tsirka SE (February 2008). “Tissue-type plasminogen activator as a therapeutic target in stroke”. Expert Opinion on Therapeutic Targets. 12 (2): 159-70;Geiger M, Huber K, Wojta J, Stingl L, Espana F, Griffin JH, Binder BR (Aug 1989). “Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo”. Blood. 74 (2): 722-8。

[0043] In some embodiments of the present invention, the "component of the plasminogen activation pathway" of the present invention is plasminogen. In some embodiments, the plasminogen is human full-length plasminogen or a conservative mutant thereof that retains its plasminogen activity (e.g., its lysine-binding activity or proteolytic activity). In some embodiments, the plasminogen is Glu-plasminogen, Lys-plasminogen, miniplasminogen, microplasminogen, δ-plasminogen or a variant thereof that retains their plasminogen activity (e.g., their lysine-binding activity or proteolytic activity). In some embodiments, the plasminogen is natural or synthetic human plasminogen or a conservative mutant or fragment thereof that still retains plasminogen activity (e.g., its lysine-binding activity or proteolytic activity). In some embodiments, the plasminogen is an orthologue of human plasminogen from a primate or rodent, or a conservative mutant or fragment thereof that still retains plasminogen activity. In some embodiments, the plasminogen comprises an amino acid sequence as shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen comprises a conservative substitution sequence of the amino acid sequence shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the amino acids of the plasminogen are shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is a conservative substitution variant of the plasminogen shown in sequence 2, 6, 8, 10 or 12. In some embodiments, the plasminogen is human native plasminogen or a conservative variant thereof. In some embodiments, the plasminogen is human native plasminogen shown in sequence 2 or a conservative variant thereof.

[0044] "A compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway" refers to any compound capable of directly activating plasminogen or indirectly activating plasminogen by activating an upstream component of the plasminogen activation pathway, including, for example, tPA, uPA, streptokinase, saruplase, alteplase, reteplase, tenecteplase, anistreplase, monteplase, lanoteplase, pamiteplase, and staphylokinase.

[0045] The "antagonist of fibrinolysis inhibitors" of the present invention is a compound that antagonizes, weakens, blocks, or prevents the action of fibrinolysis inhibitors, such as PAI-1, complement C1 inhibitor, α2-antiplasmin, and α2-macroglobulin. Said antagonists are antibodies to PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or for example antisense RNA or miniRNA that blocks or downregulates the expression of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or compounds that occupy the binding site of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin but do not have the function of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin, or compounds that block the binding and / or activity domains of PAI-1, complement C1 inhibitor, α2-antiplasmin or α2-macroglobulin.

[0046] Plasmin is an important component of the plasminogen activation system (PA system). It is a broad-spectrum protease that can hydrolyze several components of the extracellular matrix (ECM), including fibrin, gelatin, fibronectin, laminin, and proteoglycans. Plasmin can also activate some pro-matrix metalloproteinases (pro-MMPs) to become active matrix metalloproteinases (MMPs). Thus, plasmin is an important upstream regulator of extracellular proteolysis. Plasmin is formed by proteolysis of two physiological PAs: tissue-type plasminogen activator (tPA) or urokinase plasminogen activator (uPA). Plasminogen is present at relatively high relative levels in plasma and other body fluids, and traditionally, it has been believed that the regulation of the PA system is mainly achieved by the synthesis and activity levels of PAs. The synthesis of PA system components is tightly regulated by different factors, such as hormones, growth factors, and cytokines. In addition, there are specific physiological inhibitors of plasmin and PAs. The main inhibitor of plasmin is α2-antiplasmin. The activity of PAs is simultaneously inhibited by plasminogen activator inhibitor-1 (PAI-1) of uPA and tPA, and is regulated by plasminogen activator inhibitor-2 (PAI-2), which mainly inhibits uPA. Some cells have a uPA-specific cell surface receptor (uPAR) with direct hydrolytic activity.

[0047] Plasminogen is a single-chain glycoprotein consisting of 791 amino acids with a molecular weight of approximately 92 kDa. It is synthesized mainly in the liver and is present in large amounts in extracellular fluids. The plasminogen content in plasma is approximately 2 μM. Thus, plasminogen is a large potential source of proteolytic activity in tissues and body fluids. There are two molecular forms of plasminogen: glutamate-plasminogen (Glu-plasminogen) and lysine-plasminogen (Lys-plasminogen). The naturally secreted and undegraded form of plasminogen has one amino-terminal (N-terminal) glutamic acid and is therefore called glutamate-plasminogen. However, in the presence of plasmin, glutamate-plasminogen is hydrolyzed to lysine-plasminogen at Lys76-Lys77. Compared to glutamate-plasminogen, lysine-plasminogen has a higher affinity for fibrin and can be activated by PA at a higher rate. The Arg560-Val561 peptide bond of the two forms of plasminogen is cleaved by uPA or tPA, leading to the formation of the disulfide-linked double-chain protease plasmin. The amino-terminal part of plasminogen contains five homologous tricycles, the so-called kringles, and the carboxyl-terminal part contains the protease domain. Some kringles contain a lysine-binding site that mediates the specific interaction of plasminogen with fibrin and its inhibitor α2-AP. The most recently discovered plasminogen is a 38 kDa fragment, containing kringlel-4, which is a potent inhibitor of angiogenesis. This fragment, named angiostatin, is generated from several protease-hydrolyzed plasminogens.

[0048] The main substrate of plasmin is fibrin, and fibrin lysis is a key point to prevent the formation of pathological thrombi. Plasmin also has substrate specificity for several components of the ECM, including laminin, fibronectin, proteoglycan, and gelatin, indicating that plasmin plays an important role in ECM reconstruction. Indirectly, plasmin also degrades other components of the ECM by converting some protease precursors, including MMP-1, MMP-2, MMP-3, and MMP-9, into active proteases. Therefore, it may be proposed that plasmin is an important upstream regulator of extracellular protein hydrolysis. Plasmin also has the ability to activate some latent forms of growth factors. In vitro, plasmin can also hydrolyze components of the complement system to release chemotactic complement fragments.

[0049] "Plasmin" is a very important enzyme present in blood, which hydrolyzes fibrin clots into fibrin degradation products and D-dimers.

[0050] "Plasminogen" is the enzyme precursor form of plasmin, which, based on the sequence in Swiss prot, is calculated as the amino acid sequence of natural human plasminogen including the signal peptide (Sequence 4), consisting of 810 amino acids, has a molecular weight of about 90 kD, is synthesized mainly in the liver, and is a glycoprotein that can circulate in the blood, and the cDNA sequence encoding said amino acid sequence is as shown in Sequence 3. Full-sized plasminogen contains seven domains, namely a serine protease domain located at the C-terminus, a Pan Apple (PAp) domain located at the N-terminus, and five Kringle domains (Kringle 1-5). With reference to the sequence in swiss prot, the signal peptide comprises residues Met1-Gly19, PAp comprises residues Glu20-Val98, Kringle1 comprises residues Cys103-Cys181, Kringle2 comprises residues Glu184-Cys262, Kringle3 comprises residues Cys275-Cys352, Kringle4 comprises residues Cys377-Cys454, and Kringle5 comprises residues Cys481-Cys560. According to the NCBI data, the serine protease domain comprises residues Val581-Arg804.

[0051] Glu-plasminogen is a natural full-size human plasminogen, and consists of 791 amino acids (not including a signal peptide consisting of 19 amino acids). The cDNA sequence encoding this sequence is as shown in sequence 1, and its amino acid sequence is as shown in sequence 2. In vivo, Lys-plasminogen is formed by hydrolysis at the 76th-77th amino acid positions of Glu-plasminogen, and is, for example, as shown in sequence 6, and the cDNA sequence encoding this amino acid sequence is as shown in sequence 5. Delta-plasminogen (δ-plasminogen) is a fragment of full-size plasminogen lacking the Kringle2-Kringle5 structure, and contains only Kringle1 and a serine protease domain (also called the protease domain (PD)). There is a literature that reports the amino acid sequence of δ-plasminogen (sequence 8), and the cDNA sequence encoding this amino acid sequence is, for example, sequence 7. Mini-plasminogen consists of Kringle5 and serine protease domains, and the literature reports residues Val443-Asn791 (starting with the Glu residue of the Glu-plasminogen sequence without the signal peptide), and its amino acid sequence is as shown in sequence 10, and the cDNA sequence encoding this amino acid sequence is as shown in sequence 9. However, literature reports that microplasminogen only contains a serine protease domain, and its amino acid sequence is residues Ala543-Asn791 (the Glu residue of the Glu-plasminogen sequence without the signal peptide is the starting amino acid), and patent document CN102154253A discloses that it contains residues Lys531-Asn791 (the Glu residue of the Glu-plasminogen sequence without the signal peptide is the starting amino acid), and in this patent application, the sequence of microplasminogen can be referred to patent document CN102154253A, and its amino acid sequence is as shown in sequence 12, and the cDNA sequence encoding the amino acid sequence is as shown in sequence 11.

[0052] The structure of full-length plasminogen is also described in a paper by Aisina et al. (Aisina RB, Mukhametova L I. Structure and function of plasminogen / plasmin system[J]. Russian Journal of Bioorganic Chemistry, 2014, 40(6):590-605). According to the above mentioned article by Aisina et al., plasminogen contains Kringle 1, 2, 3, 4, 5 domains and a serine protease domain (also called protease domain (PD)), Kringles are responsible for binding plasminogen to low and high molecular weight ligands (i.e., lysine binding activity), which results in the conversion of plasminogen to a more open configuration and makes it more accessible to activation, and the protease domain (PD) is residues Val562-Asn791, and tPA and UPA specifically cleave the Arg561-Val562 activating bond of plasminogen, thereby allowing plasminogen to form plasmin. Thus, the protease domain (PD) is the region that confers the proteolytic activity of plasminogen.

[0053] In the present invention, "plasmin", "fibrin plasmin", and "fibrous protein plasmin" can be used interchangeably and have the same meaning. "Plasminogen", "fibrin plasminogen", and "fibrous protein plasminogen" can be used interchangeably and have the same meaning.

[0054] In this application, the term "insufficiency" of plasminogen refers to the content or activity of plasminogen in the subject's body being lower than that of a normal person and being sufficiently low to affect the normal physiological function of the subject. The term "deficiency" of plasminogen refers to the content or activity of plasminogen in the subject's body being significantly lower than that of a normal person, the activity or expression being extremely low, and the normal physiological function can only be maintained by external supply.

[0055] Those skilled in the art can understand as follows. All technical configurations of plasminogen in the present invention can be applied to plasmin, so the technical configurations described in the present invention cover plasminogen and plasmin. In the circulation process, plasminogen takes a closed inactive conformation, but when it binds to a thrombus or cell surface, it becomes active plasmin with an open conformation under the mediation of plasminogen activator (PA). The active plasmin further hydrolyzes fibrin clots into fibrin degradation products and D-dimers, thereby dissolving the thrombus. Among them, the PAp domain of plasminogen contains an important epitope that maintains plasminogen in an inactive closed conformation, but the KR domain can bind to lysine residues on receptors and substrates. Several types of enzymes as plasminogen activators have already been known, including tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), kallikrein, and coagulation factor XII (Hagemann factor), etc.

[0056] "Plasminogen active fragment" refers to an active fragment that binds to lysine in the target sequence of a substrate (lysine binding activity), or an active fragment that exerts a proteolytic function (proteolytic activity), or a fragment that has both proteolytic activity and lysine binding activity. The plasminogen technical configuration of the present invention encompasses technical configurations that replace plasminogen with a plasminogen active fragment. In some embodiments, the plasminogen active fragment of the present invention comprises the serine protease domain of plasminogen or consists of the serine protease domain of plasminogen. In some embodiments, the plasminogen active fragment of the present invention comprises sequence 14, or comprises an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity to sequence 14, or consists of an amino acid sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99% identity to sequence 14. In some embodiments, the plasminogen active fragment of the present invention comprises one or more domains selected from Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5, or conservative substitution variants thereof, or consists of one or more domains selected from Kringle 1, Kringle 2, Kringle 3, Kringle 4, and Kringle 5, or conservative substitution variants thereof. In some embodiments, the plasminogen of the present invention comprises a protein comprising an active fragment of plasminogen as described above.

[0057] At present, the methods for measuring plasminogen and its activity in blood include measuring the activity of tissue plasminogen activator (t-PAA), measuring the activity of plasma tissue plasminogen activator antigen (t-PAAg), measuring the activity of plasma tissue plasminogen (plgA), measuring the activity of plasma tissue plasminogen antigen (plgAg), measuring the activity of plasma tissue plasminogen activator inhibitor, measuring the inhibitor antigen of plasma tissue plasminogen activator, and measuring the activity of plasma plasmin-antiplasmin complex (PAP). The most common measuring method is the chromogenic substrate method: streptokinase (SK) and a luminescent substrate are added to the plasma of the subject, and the PLG in the plasma of the subject becomes PLM under the action of SK, and the latter acts on the luminescent substrate, which is then measured by a spectrophotometer, and the increase in absorbance is directly proportional to the activity of plasminogen. In addition, immunochemical methods, gel electrophoresis, immunoturbidimetry, radial immunodiffusion, etc. can also be used to measure plasminogen activity in blood.

[0058] "Ortholog" refers to a homologue between different species, including both protein and DNA homologues, also called a direct lineage gene. It specifically refers to a protein or gene that is evolved from the same ancestral gene between different species. The plasminogen of the present invention includes human native plasminogen, and further includes orthologs or orthologs of plasminogens with plasminogen activity derived from different species.

[0059] A "conservative substitution variant" is a variant in which a given amino acid residue is modified but does not change the overall conformation and function of a protein or enzyme, including but not limited to those that substitute an amino acid in the amino acid sequence of a parent protein with an amino acid of similar properties (e.g., acidic, alkaline, hydrophobic, etc.). Amino acids with similar properties are known. For example, arginine, histidine, and lysine are hydrophilic alkaline amino acids and can be substituted for each other. Similarly, isoleucine is a hydrophobic amino acid and can be substituted by leucine, methionine, or valine. Therefore, the similarity of two proteins or amino acid sequences with similar functions may vary. For example, they have a similarity (identity) of 70% to 99% based on the MEGALIGN algorithm. "Conservative substitution variants" further include polypeptides or enzymes with an amino acid identity of 60% or more based on the BLAST or FASTA algorithm, preferably 75% or more, most preferably 85% or more, and most preferably 90% or more, and have the same or essentially similar properties or functions compared to the natural or parent protein or enzyme.

[0060] "Isolated" plasminogen is plasminogen protein that has been separated and / or recovered from its natural environment. In some embodiments, the plasminogen is (1) purified to greater than 90%, greater than 95%, or greater than 98% purity (by weight), e.g., greater than 99% (by weight), as determined by the Lowry method; (2) purified to at least 15 residues of N-terminal or internal amino acid sequence as determined by a spinning cup sequencer; or (3) purified to homogeneity, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions with Coomassie brilliant blue or silver staining. Isolated plasminogen includes plasminogen produced from recombinant cells by bioengineering techniques and separated by at least one further purification step.

[0061] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, including genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having modified peptide backbones. The terms also include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions containing leader sequences of heterologous and homologous origin (with or without an N-terminal methionine residue); and the like.

[0062] The definition of "percentage (%) of amino acid sequence identity" of a reference peptide sequence is the percentage of amino acid residues in a candidate sequence that are the same as those in the reference polypeptide sequence, after introducing gaps as necessary to achieve the maximum percentage sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity can be achieved by several methods within the skill of the art, for example by publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm that achieves the maximum alignment requirements for the full size of the sequences to be compared. However, for the purposes of the present invention, the percentage of amino acid sequence identity is obtained by the sequence comparison computer software ALIGN-2.

[0063] When comparing amino acid sequences by using ALIGN-2, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (or alternatively, a given amino acid sequence A that has or contains a certain % amino acid sequence identity to, with, or for a given amino acid sequence B) is calculated as follows: Fraction X / Y×100

[0064] where X is the number of amino acid residues that the sequence alignment program ALIGN-2 evaluates as identical and matching in its alignment of A and B, and Y is the total number of amino acid residues in B. It should be understood as follows: if the length of amino acid sequence A and the length of amino acid sequence B are not equal, then the % amino acid sequence identity of A to B will be different from the % amino acid sequence identity of B to A. Unless otherwise stated, all % amino acid sequence identity values ​​used in this text are as described in the previous paragraph and are obtained by the ALIGN-2 computer program.

[0065] As used herein, the term "treatment" refers to obtaining a desired pharmacological and / or physiological effect. The effect may be completely or partially preventing the onset or development of a disease or its symptoms, or partially or completely reducing the disease and / or its symptoms, and / or partially or completely curing the disease and / or its symptoms. It also includes: (a) preventing a disease from occurring in a subject, where the subject has the causative agent for the disease but has not been diagnosed with the disease; (b) inhibiting the disease or inhibiting its formation; and (c) attenuating a disease and / or its symptoms, i.e., causing a decrease or disappearance of the disease and / or its symptoms.

[0066] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), and the like.

[0067] A "therapeutically effective amount" or "effective amount" is an amount of a component of the plasminogen activation pathway or its related compound (e.g., plasminogen) that is capable of achieving said prevention and / or treatment of a disease when administered to a mammal or other subject and used to treat the disease. A "therapeutically effective amount" will vary according to the component of the plasminogen activation pathway or its related compound (e.g., plasminogen) used, the severity of the disease and / or symptoms, and the age, weight, etc., of the subject to be treated.

[0068] Preparation of the Plasminogen of the Invention For therapeutic use, plasminogen may be isolated and purified from nature or may be synthesized by standard chemical peptide synthesis techniques. When polypeptides are synthesized by chemical methods, the synthesis may be performed in liquid phase or solid phase. Solid phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support and the remaining amino acids in the sequence are added in order, is suitable for the chemical synthesis of plasminogen. Various forms of SPPS, e.g., Fmoc and Boc, can be used to synthesize plasminogen. The techniques used for solid-phase synthesis are described in: Barany and Solid-Phase Peptide Synthesis; pages 3-284, The Peptides: Analysis, Synthesis, Biology. Volume 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6: 3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12: 723-8. Briefly, small insoluble porous beads are treated with functional units on which the peptide chains are built. After repeated coupling / deprotection cycles, the free N-terminal amine of the attached solid phase is coupled with a single N-protected amino acid unit, which is then deprotected to expose a new N-terminal amine for linking with another amino acid. The peptide remains immobilized on the solid phase and is then excised.

[0069] The plasminogen of the present invention is produced by standard recombinant methods. For example, a nucleic acid encoding plasminogen is inserted into an expression vector and operably linked to control sequences in the expression vector. Expression control sequences include, but are not limited to, a promoter (e.g., a naturally associated promoter or a heterologous promoter), a signal sequence, an enhancer element, and a transcription termination sequence. Control of expression can be a eukaryotic promoter system in a vector, which is transformed or transfected into a eukaryotic host cell (e.g., COS or CHO cells). Once the vector is introduced into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and for collection and purification of the plasminogen.

[0070] Suitable expression vectors usually replicate in the host cell either free or as an integrated part of the host cell chromosomal DNA. Usually, expression vectors contain a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) which is useful for detecting those cells which have been transformed in vitro with the desired DNA sequence.

[0071] Escherichia coli is an example of a prokaryotic host cell for cloning a polynucleotide encoding an antibody of interest. Other suitable microbial hosts include bacilli, such as Bacillus subtilis and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can be generated, which usually contain expression control sequences (e.g., origins of replication) compatible with the host cell. There are also many known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the β-lactamase promoter system, or the phage lambda-derived promoter system. Promoters generally control expression and may further contain sequences, such as ribosome binding site sequences, to initiate transcription and translation when controlling gene sequences, as needed.

[0072] Other microorganisms, such as yeast, can also be used for expression. Yeast (e.g., Saccharomyces (S. cerevisiae) and Pichia are examples of suitable yeast host cells, in which suitable carriers include expression control sequences (e.g., promoters), origins of replication, termination sequences, etc., as necessary. Exemplary promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes for maltose and galactose utilization.

[0073] In addition to microorganisms, mammalian cells (e.g., mammalian cells cultured in in vitro cell culture) can also be used to express and produce the anti-Tau antibodies of the present invention (e.g., polynucleotides encoding such anti-Tau antibodies of interest). See, e.g., Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for use in these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and required processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression control sequences are promoters derived from rabbit immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

[0074] Once synthesized (chemically or recombinantly), the plasminogen according to the invention can be purified by standard procedures in the art, such as ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid chromatography (HPLC), gel electrophoresis, etc. The plasminogen is essentially pure, e.g., at least about 80% to 85% pure, at least about 85%-90% pure, at least about 90%-95% pure, or 98%-99% pure or even more pure, e.g., free of contaminants, such as cellular debris, large molecules other than the desired product, etc.

[0075] Drug combinations Plasminogen of the desired purity is mixed with pharmaceutical carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980)) as required to form a lyophilized formulation or aqueous solution to obtain the therapeutic combination. Acceptable carriers, excipients, and stabilizers are non-toxic to subjects at the required dosages and concentrations, and further include buffers such as phosphates, citrates, and other organic acids. Antioxidants include ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethylenediamine chloride; benzalkonium chloride). chloride), benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parahydroxybenzoic acid esters, such as methyl or propyl parahydroxybenzoic acid ester; pyrocatechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol; low molecular weight polypeptides (those having less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, aspartic acid, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates include glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, fucose or sorbitol; salt forming counterions, such as sodium; metal complexes (such as zinc-protein complexes); and / or non-ionic surfactants, such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). A preferred lyophilized anti-VEGF antibody formulation is described in WO97 / 04801, incorporated herein by reference.

[0076] The combination preparation of the present invention may contain one or more active compounds as required for the specific condition requiring treatment, preferably with complementary activities and no side effects, such as antihypertensives, antiarrhythmics, and antidiabetics.

[0077] The plasminogen of the present invention can be encapsulated in microcapsules made, for example, by aggregation techniques or interfacial polymerization, can be incorporated, for example, into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or can be incorporated into hydroxymethylcellulose or gel microcapsules and poly(methyl methacrylate) microcapsules in coarse emulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0078] The plasminogen of the invention to be used for in vitro administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes, prior to or after lyophilization and recombination.

[0079] The plasminogen of the present invention can be prepared in sustained release formulations. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers having defined shapes and containing the glycoprotein, such as membranes or microcapsules. Examples of sustained release matrices include polyesters, aqueous gels (e.g., poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981); Langer, Chem. Tech., 12:98-105 (1982)) or poly(vinyl alcohol), polylactide (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid (Sidman, et al., Biopolymers 22:547 (1983)), non-degradable ethylene-vinyl acetate (Langer, et al., same source as above), or degradable lactic acid-hydroxyacetic acid copolymers, e.g., Lupron. Depot™ (injectable microspheres composed of lactic acid-hydroxyacetic acid copolymer and leuprolide acetate), and poly D-(-)-3-hydroxybutyrate. Polymers such as ethylene-ethyl acetate and lactic acid-hydroxyacetic acid can release molecules sustainedly for over 100 days, but some aqueous gels release proteins for a shorter period of time. Rational strategies can be designed to stabilize proteins depending on the mechanism involved. For example, if the aggregation mechanism is the exchange of sulfur disulfide bonds to form intermolecular SS bonds, stabilization can be achieved by modifying sulfhydryl residues, freeze-drying from acidic solutions, controlling humidity, using appropriate additives, and developing specific polymer matrix compositions.

[0080] Dosage and dosage Administration of the pharmaceutical compositions of the present invention can be achieved by different modes, such as nasal inhalation, aerosol inhalation, nasal or eye drops, intravenous, intraperitoneal, subcutaneous, intracranial, intrathecal, intraarterial (e.g., via the carotid artery), intramuscular, and intrarectal administration.

[0081] Preparations for use in parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolytic replenishers, and the like. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, inert gases, and the like.

[0082] A medical practitioner may determine a dosage regimen based on various clinical factors. For example, as is known in the medical arts, the dosage regimen for any given patient is determined by several factors, including the patient's body type, body surface area, age, the specific compound being administered, sex, frequency and route of administration, overall health, and other drugs being administered at the same time. The dosage range of the pharmaceutical composition containing plasminogen of the present invention may be about 0.0001-2000 mg / kg of subject body weight daily, or about 0.001-500 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 10 mg / kg, 50 mg / kg, etc.). For example, the dosage may be 1 mg / kg body weight or 50 mg / kg body weight or in the range of 1-50 mg / kg, or at least 1 mg / kg. Doses higher or lower than this exemplary range are also covered, particularly when the above factors are taken into account. Doses intermediate to the above range are also within the scope of the present invention. Subjects may receive such doses daily, every other day, weekly, or according to any schedule determined by empirical analysis. An exemplary dose schedule is 0.01-100 mg / kg for several consecutive days. During the administration of the drug of the present invention, it is necessary to evaluate the therapeutic effect and safety in real time.

[0083] Product or drug kit One embodiment of the present invention relates to an article of manufacture or drug kit containing the plasminogen or plasmin of the present invention that can be used to treat cardiovascular disease caused by diabetes and its associated disorders. The article of manufacture preferably includes a container, a label, or a protocol. Suitable containers are bottles, vials, syringes, etc. The containers can be made of various materials, such as glass or plastic. The container contains a composition, the composition is effective to treat the disease or condition of the present invention, and has a sterile inlet (e.g., the container is an intravenous infusion pack or vial and includes a stopper that can be pierced by a hypodermic needle). At least one activator in the composition is plasminogen / plasmin. A label on or attached to the container describes the composition as being used to treat cardiovascular disease caused by diabetes and its associated disorders according to the present invention. The article of manufacture further includes a second container containing a pharmaceutical buffer, such as phosphate buffered saline, Ringer's solution, and glucose solution. It can further include other materials required from a commercial and user perspective, i.e., other buffers, diluents, filters, needles, and syringes. The article of manufacture also includes a protocol with instructions for use, including, for example, instructing a user of the composition to administer the plasminogen composition and other medications associated with treating the disease to a patient. EXAMPLES

[0084] The human plasminogen used in the following examples was derived from the plasma of a human donor and has been described in the following publications: Kenneth C Robbins, Louis Summaria, David Elwyn et al. Further Studies on the Purification and Characterization of Human Plasminogen and Plasmin. Journal of Biological Chemistry, 1965, 240(1):541-550; Summaria L, Spitz F, Arzadon L et al. Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins. J Biol Chem. 1976 Jun 25; 251(12):3693-9; HAGAN JJ, ABLONDI FB, DE RENZO EC. Purification and biochemical properties of human plasminogen. J Biol Chem. 1960 The plasminogen was purified from human donor plasma using an optimized process based on the method described in Apr;235:1005-10, and the purity of the isolated plasminogen was over 98%. Example 1

[0085] Example 1 relates to the improvement of spontaneous activity and avoidance behavior in Huntington's disease model mice by plasminogen. We took 6-week-old C57 male mice, weighed all mice 3 days before modeling, performed a simple open field test, and observed them for 5 minutes to exclude spontaneous differences in random animals. Finally, we determined 23 experimental animals. According to the total distance traveled by mice in the open field test, the mice were randomly divided into two groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were administered 125 μL of PBS solution by intraperitoneal injection, and the mice in the model group were administered 3-nitropropionic acid (3-NP) solution at 50 mg / kg body weight by intraperitoneal injection, twice a day (at 12-hour intervals) for 5 consecutive days to construct the Huntington's model. [1] Preparation of 3-NP solution: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg / ml. The day after modeling was completed was designated as day 0 of administration, and all mice were weighed and subjected to an open field test. The mice in the model group were randomly divided into two groups, a solvent control group (9 mice) and a plasminogen administration group (8 mice), according to the total distance traveled in the open field. The mice in the solvent control group were administered with solvent (10 mM sodium citrate solution, pH 7.4) at 0.1 ml / mouse via tail vein injection, and the mice in the plasminogen administration group were administered with human plasminogen at 1 mg / 0.1 ml / mouse via tail vein injection for 7 consecutive days. The open field test was performed on the 7th day of administration. 3-NP is a naturally occurring mycotoxin that irreversibly inhibits mitochondrial succinate dehydrogenase, crossing the blood-brain barrier and causing neurotoxicity, leading to neuronal cell death and mimicking HD symptoms in humans. [1] . Open field experiment During the experiment, the mouse was placed in the center of the bottom of the open field (40 x 40 x 40 cm), and was observed for 5 minutes with simultaneous photography and timing. Each mouse underwent three experiments. The Smart System is a complete and easy-to-use video tracking system for evaluating the behavior of experimental animals. It can record movement trajectories, activities, specific behaviors (turning, stretching, eating, etc.) and events, and perform calculations of various analysis parameters. In this experiment, the Smart3.0 system was used to record and analyze the mouse's movements. Parameters included total movement distance, boundary zone rest time rate, average movement speed in the central zone, and average movement speed in the boundary zone. After each experiment, the box was wiped with 70% alcohol to prevent odor preference. [1] . The design principle of the open field experiment is based on mouse avoidance. This refers to the tendency of mice to "stick to the walls" because they are afraid of open, unknown, and potentially dangerous places. Avoidance was assessed by the activity of mice in the peripheral areas of the field (four corners and four sides). Judging from the activity time in the periphery, which reflects avoidance, a shorter time indicates that the mouse tends to be more "adventurous," while a significantly longer activity time in the central zone indicates a lower level of avoidance and anxiety (depression). Total movement distance The total movement distance refers to the length of the movement trajectory within a specified test time. As a result, the mice in the blank control group moved a certain distance during the experiment, the total movement distance of the mice in the solvent control group was significantly longer than that of the blank control group, and the total movement distance of the mice in the plasminogen administration group was significantly shorter than that of the solvent control group, and the difference was statistically significant (representing *P<0.05), and the total movement distance of the mice in the plasminogen administration group was close to that of the blank control group (Figure 1). This indicates that plasminogen can promote the behavioral recovery of spontaneous activity in Huntington's disease model mice. Boundary Zone Rest Time Rate The border zone refers to the periphery of the open field (four corners and four sides). The border zone resting time rate is the ratio of the resting time in the border zone to the total resting time (including the border zone resting time and the central zone resting time). As a result, the mice in the blank control group had a certain proportion of resting time in the border zone, the proportion of resting time in the border zone of the mice in the solvent control group was significantly smaller than that of the blank control group, and the proportion of resting time in the border zone of the mice in the plasminogen administration group was significantly larger than that of the solvent control group, and the statistical difference was highly significant (*** represents P<0.001), and the proportion of resting time in the border zone of the plasminogen administration group was close to the value of the blank control group (Figure 2). This indicates that plasminogen can alleviate the anxiety behavior of Huntington's disease model mice. Average movement speed in the central zone The central zone refers to the central 20x20cm area of ​​the open field. The average movement speed in the central zone refers to the ratio of the total movement distance in the central zone to the total stay time in the central zone (including movement time and rest time). The results showed that the mice in the blank control group had a constant movement speed in the central zone, the average movement speed in the central zone of the mice in the solvent control group was significantly smaller than that of the blank control group, and the average movement speed in the central zone of the mice in the plasminogen group was significantly greater than that of the solvent control group, the statistical difference was significant (* represents P<0.05), and the movement speed in the central zone of the plasminogen-administered group was close to that of the blank control group (Figure 3). This indicates that plasminogen can promote spontaneous activity and alleviate anxiety behavior in Huntington's disease model mice. Average movement speed of the boundary zone The average movement speed in the border zone refers to the ratio of the total movement distance in the border zone to the total time spent in the border zone. As a result, the mice in the blank control group had a constant movement speed in the border zone, the average movement speed in the border zone of the mice in the solvent control group was significantly greater than that of the blank control group, and the average movement speed in the border zone of the mice in the plasminogen group was significantly smaller than that of the solvent control group, the statistical difference was significant (* represents P<0.05), and the average movement speed in the border zone of the plasminogen administration group was close to that of the blank control group (Figure 4). This suggests that plasminogen can promote spontaneous activity and alleviate anxiety behavior in Huntington's disease model mice. movement trajectory The results showed that the mice in the blank control group (Figure 5A) had less movement in the central zone, more movement in the border zone, and a regular movement trajectory; compared with the blank control group, the movement in the central zone and total movement distance of the mice in the solvent group (Figure 5B) were significantly increased, and the movement trajectory was chaotic and irregular; the movement in the central zone and total movement distance of the mice in the plasminogen group (Figure 5C) were significantly less than those in the solvent group, and the movement trajectory was similar to that of the blank control group. This suggests that plasminogen can promote the recovery of spontaneous activity and avoidance behavior in Huntington's disease model mice. Example 2

[0086] Example 2 relates to the effect of plasminogen on slowing weight loss in Huntington's disease model mice. We took 6-week-old C57 male mice, weighed all mice 3 days before modeling, performed a simple open field test, and observed them for 5 minutes to exclude spontaneous differences in random animals. Finally, we determined 23 experimental animals. According to the total distance traveled by mice in the open field test, the mice were randomly divided into two groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were administered 125 μL of PBS solution by intraperitoneal injection, and the mice in the model group were administered 3-nitropropionic acid (3-NP) solution at 50 mg / kg body weight by intraperitoneal injection, twice a day (at 12-hour intervals) for 5 consecutive days to construct the Huntington's model. [1] Preparation of 3-NP solution: 3-NP powder (Sigma, Catalog No. N5636) was dissolved in PBS solution to a concentration of 10 mg / ml. The day after modeling was completed was designated as day 0 of administration, and all mice were weighed and subjected to an open field test. The mice in the model group were randomly divided into two groups, a solvent control group (9 mice) and a plasminogen administration group (8 mice), according to the total distance traveled in the open field. The mice in the solvent control group were administered with solvent (10 mM citric acid-sodium citrate solution, pH 7.4) at 0.1 ml / mouse via tail vein injection, and the mice in the plasminogen administration group were administered with human plasminogen at 1 mg / 0.1 ml / mouse via tail vein injection for 7 consecutive days. The mice were weighed on the 7th day of administration, and the weight ratio (%) between day 7 and day 0 was calculated. Weight loss is a common non-neurological phenotype of Huntington's disease that is usually progressive and can lead to malnutrition or cachexia. [5] . As a result, the body weight ratio of the blank control group was greater than 1, indicating a tendency for body weight to increase; the body weight ratio of the mice in the solvent control group was smaller than that of the blank control group; the body weight ratio (%) of the mice in the plasminogen administration group was significantly higher than that of the solvent control group, and the statistical difference was close to significance (P = 0.07) (Figure 6). This result suggests that plasminogen can promote weight gain in Huntington's disease model mice. Example 3

[0087] Example 3 relates to the effect of plasminogen on the reduction of GFAP expression in the hippocampus of Huntington's disease model mice. We took 6-week-old C57 male mice, weighed all mice 3 days before modeling, performed a simple open field test, and observed them for 5 minutes to exclude spontaneous differences in random animals. Finally, we determined 23 experimental animals. According to the total distance traveled by mice in the open field test, the mice were randomly divided into two groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were administered 125 μL of PBS solution by intraperitoneal injection, and the mice in the model group were administered 3-nitropropionic acid (3-NP) solution at 50 mg / kg body weight by intraperitoneal injection, twice a day (at 12-hour intervals) for 5 consecutive days to construct the Huntington's model. [1]Preparation of 3-NP solution: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg / ml. The day after modeling was completed was designated as day 0 of administration, and all mice were weighed and subjected to an open field test. The mice in the model group were randomly divided into two groups, a solvent control group (9 mice) and a plasminogen administration group (8 mice), according to the total distance traveled in the open field. The mice in the solvent control group were administered with solvent (10 mM citric acid-sodium citrate solution, pH 7.4) at 0.1 ml / mouse via tail vein injection, and the mice in the plasminogen administration group were administered with human plasminogen at 1 mg / 0.1 ml / mouse via tail vein injection for 7 consecutive days. On the 7th day of administration, the mice were sacrificed, and the hippocampi were collected and fixed in 10% neutral formaldehyde solution for 24 to 48 hours. Fixed hippocampal tissue was dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The thickness of the tissue section was 3 μm. The sections were deparaffinized, rehydrated, and washed once with water. The tissue was circled with a PAP marker, incubated with 3% hydrogen peroxide for 15 min, and washed twice with 0.01 M PBS, for 5 min each time. Blocking was performed with 5% normal goat serum (Vector Laboratories, Inc., USA) for 30 min. When the time was up, the goat serum was discarded, and rabbit anti-GFAP antibody (ab7260, Abcam) was dropped and incubated overnight at 4°C, washed twice with 0.01 M PBS, for 5 min each time. Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 h, washed twice with 0.01 M PBS, for 5 min each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. They were dehydrated in a graded alcohol stream, cleared with xylene, and embedded in neutral rubber. Sections were observed under an optical microscope at 400x magnification. Glial fibrillary acidic protein (GFAP) is a characteristic intermediate filament protein of astrocytes, which participates in the formation of the cytoskeleton and maintains its tensile strength. Astrocytes are one of the major glial cells in the central nervous system. Studies have reported that Huntington's disease is accompanied by astroglial hyperplasia and increased activity, manifested as upregulated GFAP expression and cell hypertrophy. [6-7] . The results showed that there was a certain amount of GFAP expression in the hippocampus of the mice in the blank control group (Figure 7A), the expression of GFAP in the hippocampus of the mice in the solvent control group (Figure 7B) was significantly higher than that of the blank control group, and the statistical difference was highly significant (*** represents P<0.001) (Figure 7D), and the expression of GFAP in the hippocampus of the mice in the plasminogen group (Figure 7C) was significantly lower than that of the solvent control group, and the statistical difference was highly significant, and there was no statistical difference compared with the expression of GFAP in the hippocampus of the mice in the blank control group. This result indicates that plasminogen can reduce the expression of GFAP in the hippocampus of Huntington's disease model mice, inhibit the proliferation of astroglial cells, and have a neuroprotective effect. Example 4

[0088] Example 4 relates to the effect of plasminogen on reducing apoptosis in hippocampal cells of a Huntington's disease model mouse. We took 6-week-old C57 male mice, weighed all mice 3 days before modeling, performed a simple open field test, and observed them for 5 minutes to exclude spontaneous differences in random animals. Finally, we determined 23 experimental animals. According to the total distance traveled by mice in the open field test, the mice were randomly divided into two groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were administered 125 μL of PBS solution by intraperitoneal injection, and the mice in the model group were administered 3-nitropropionic acid (3-NP) solution at 50 mg / kg body weight by intraperitoneal injection, twice a day (at 12-hour intervals) for 5 consecutive days to construct the Huntington's model. [1]Preparation of 3-NP solution: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg / ml. The day after modeling was completed was designated as day 0 of administration, and all mice were weighed and subjected to an open field test. The mice in the model group were randomly divided into two groups, a solvent control group (9 mice) and a plasminogen administration group (8 mice), according to the total distance traveled in the open field. The mice in the solvent control group were administered with solvent (10 mM citric acid-sodium citrate solution, pH 7.4) at 0.1 ml / mouse via tail vein injection, and the mice in the plasminogen administration group were administered with human plasminogen at 1 mg / 0.1 ml / mouse via tail vein injection for 7 consecutive days. On the 7th day of administration, the mice were sacrificed, and the hippocampi were collected and fixed in 10% neutral formaldehyde solution for 24 to 48 hours. Fixed hippocampal tissue was dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The thickness of the tissue section was 3 μm. The sections were deparaffinized, rehydrated, and washed once with water. The tissue was circled with a PAP marker, incubated with 3% hydrogen peroxide for 15 min, and washed twice with 0.01 M PBS, for 5 min each time. Blocking was performed with 5% normal goat serum (Vector Laboratories, Inc., USA) for 30 min. When the time was up, the goat serum was discarded, and rabbit anti-caspase-3 antibody (BA2142, Boster Biological Technology) was dropped and incubated overnight at 4°C, washed twice with 0.01 M PBS, for 5 min each time. Goat anti-rabbit IgG (HRP) antibody (Abcam) secondary antibody was incubated at room temperature for 1 h, washed twice with 0.01 M PBS, for 5 min each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. They were dehydrated in a graded alcohol stream, cleared with xylene, and embedded in neutral rubber. Sections were observed under an optical microscope at 400x magnification. The caspase family plays a crucial role in the process of mediating apoptosis, among which caspase-3 is a key executioner molecule that functions in many pathways of apoptosis signaling. In the 3-NP Huntington's model, neuronal apoptosis was increased and caspase-3 expression was upregulated. The results showed that the hippocampus of the blank control group (Figure 8A) expressed a certain amount of caspase-3 (marked by arrows), the expression of caspase-3 in the hippocampus of the solvent control group (Figure 8B) was significantly increased, and the expression of caspase-3 in the hippocampus of the plasminogen-administered group (Figure 8C) was significantly lower than that of the solvent control group, with the statistical difference approaching significance (P = 0.058) (Figure 8D). This result suggests that plasminogen can reduce the expression of caspase-3 and reduce the apoptosis of hippocampal cells in Huntington's disease model mice. Example 5

[0089] Example 5 relates to the fact that plasminogen promotes the recovery of the number of Nissl bodies in cerebellar neurons of Huntington's disease model mice. We took 6-week-old C57 male mice, weighed all mice 3 days before modeling, performed a simple open field test, and observed them for 5 minutes to exclude spontaneous differences in random animals. Finally, we determined 23 experimental animals. According to the total distance traveled by mice in the open field test, the mice were randomly divided into two groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were administered 125 μL of PBS solution by intraperitoneal injection, and the mice in the model group were administered 3-nitropropionic acid (3-NP) solution at 50 mg / kg body weight by intraperitoneal injection, twice a day (at 12-hour intervals) for 5 consecutive days to construct the Huntington's model. [1]Preparation of 3-NP solution: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg / ml. The day after modeling was completed was designated as day 0 of administration, and all mice were weighed and subjected to an open field test. The mice in the model group were randomly divided into two groups, a solvent control group (9 mice) and a plasminogen administration group (8 mice), according to the total distance traveled in the open field. The mice in the solvent control group were administered with solvent (10 mM citric acid-sodium citrate solution, pH 7.4) at 0.1 ml / mouse via tail vein injection, and the mice in the plasminogen administration group were administered with human plasminogen at 1 mg / 0.1 ml / mouse via tail vein injection for 7 consecutive days. On the 7th day of administration, the mice were sacrificed, and the cerebella were collected and fixed in 10% neutral formaldehyde solution for 24 to 48 hours. Fixed cerebellar tissues were dehydrated in an alcohol gradient, cleared in xylene, and embedded in paraffin. The tissue sections were 3 μm thick. The sections were deparaffinized, rehydrated, and stained with 0.5% toluidine blue stain. They were dehydrated in an alcohol gradient, cleared in xylene, and mounted in neutral rubber. They were observed under a light microscope and photographed. Nissl bodies are one of the characteristic structures of neurons, and are the synthesis sites of structural and functional proteins in neurons. Their number and distribution are closely related to the functional state of neurons. The main chemical components of Nissl bodies are ribonucleic acid and protein, which are called extranuclear chromatin and have affinity for basic dyes such as toluidine blue, thionine, and tar violet, among which toluidine blue staining is the most commonly used traditional method for displaying Nissl bodies. [8] When neurological disorders occur, the state of Nissl bodies may become abnormal. The results showed that there was a certain number of Nissl bodies in the cerebellar neurons of the blank control group (Figure 9A) (marked with arrows), the number of Nissl bodies in the cerebellar neurons of the solvent control group (Figure 9B) was significantly higher than that of the blank control group, and the number of Nissl bodies in the cerebellar neurons of the mice in the plasminogen group (Figure 9C) was not significantly different from that of the blank control group, but was significantly lower than that of the solvent control group, and the difference was statistically significant (* represents P<0.05) (Figure 9D). This result suggests that plasminogen can promote the recovery of the number of Nissl bodies in the cerebellar neurons of Huntington's disease model mice. Example 6

[0090] Example 6 relates to the promotion of recovery of the number of Nissl bodies in hippocampal neurons of Huntington's disease model mice. We took 6-week-old C57 male mice, weighed all mice 3 days before modeling, performed a simple open field test, and observed them for 5 minutes to exclude spontaneous differences in random animals. Finally, we determined 23 experimental animals. According to the total distance traveled by mice in the open field test, the mice were randomly divided into two groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were administered 125 μL of PBS solution by intraperitoneal injection, and the mice in the model group were administered 3-nitropropionic acid (3-NP) solution at 50 mg / kg body weight by intraperitoneal injection, twice a day (at 12-hour intervals) for 5 consecutive days to construct the Huntington's model. [1]Preparation of 3-NP solution: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg / ml. The day after modeling was completed was designated as day 0 of administration, and all mice were weighed and subjected to an open field test. The mice in the model group were randomly divided into two groups, a solvent control group (9 mice) and a plasminogen administration group (8 mice), according to the total distance traveled in the open field. The mice in the solvent control group were administered with solvent (10 mM citric acid-sodium citrate solution, pH 7.4) at 0.1 ml / mouse via tail vein injection, and the mice in the plasminogen administration group were administered with human plasminogen at 1 mg / 0.1 ml / mouse via tail vein injection for 7 consecutive days. On the 7th day of administration, the mice were sacrificed, and the hippocampi were collected and fixed in 10% neutral formaldehyde solution for 24 to 48 hours. Fixed hippocampal tissue was dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The tissue sections were 3 μm thick. The sections were deparaffinized, rehydrated, and stained with 0.4% tar violet staining solution (manufacturer: Sinopharm Chemical Reagent Co., Ltd., batch number: 20181019) (pH = 3). The sections were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in neutral rubber. The sections were observed under an optical microscope at 400x magnification and photographed. Tar violet staining uses tar violet as the core dye, which has a photosensitive effect and can clearly reveal changes in Nissl bodies. The results showed that there was a certain amount of Nissl bodies (marked by arrows) in the left hippocampal neurons of the mice in the blank control group (Figure 10A), the number of Nissl bodies in the left hippocampal neurons of the mice in the vehicle group (Figure 10B) was significantly higher than that of the blank control group, and the number of Nissl bodies in the left hippocampal neurons of the mice in the plasminogen group (Figure 10C) was significantly lower than that of the vehicle group, and the difference was statistically significant (* indicates P<0.05) (Figure 10D). This result indicates that plasminogen can promote the recovery of the number of Nissl bodies in hippocampal neurons. Example 7

[0091] Example 7 relates to the effect of plasminogen on the recovery of the number of striatal Nissl bodies in Huntington's disease model mice. We took 6-week-old C57 male mice, weighed all mice 3 days before modeling, performed a simple open field test, and observed them for 5 minutes to exclude spontaneous differences in random animals. Finally, we determined 23 experimental animals. According to the total distance traveled by mice in the open field test, the mice were randomly divided into two groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were administered 125 μL of PBS solution by intraperitoneal injection, and the mice in the model group were administered 3-nitropropionic acid (3-NP) solution at 50 mg / kg body weight by intraperitoneal injection, twice a day (at 12-hour intervals) for 5 consecutive days to construct the Huntington's model. [1] Preparation of 3-NP solution: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg / ml. The day after modeling was completed was designated as day 0 of administration, and all mice were weighed and subjected to an open field test. The mice in the model group were randomly divided into two groups, a solvent control group (9 mice) and a plasminogen administration group (8 mice), according to the total distance traveled in the open field. The mice in the solvent control group were administered with solvent (10 mM citric acid-sodium citrate solution, pH 7.4) at 0.1 ml / mouse via tail vein injection, and the mice in the plasminogen administration group were administered with human plasminogen at 1 mg / 0.1 ml / mouse via tail vein injection for 7 consecutive days. On the 7th day of administration, the mice were sacrificed, and the striatum was collected and fixed in 10% neutral formaldehyde solution for 24 to 48 hours. Fixed striatal tissues were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. Tissue sections were 3 μm thick and were deparaffinized, rehydrated, and stained with 0.4% tar violet stain (pH=3). They were dehydrated in an alcohol gradient, permeabilized with xylene, and mounted in neutral rubber. Sections were observed under a light microscope at 400x magnification and photographed. The results showed that there was a certain amount of Nissl bodies in the striatal neurons of the mice in the blank control group (Figure 11A) (marked with arrows), the number of Nissl bodies in the striatal neurons of the mice in the vehicle group (Figure 11B) was significantly lower than that of the blank control group, and the number of Nissl bodies in the striatal neurons of the mice in the plasminogen group (Figure 11C) was significantly higher than that of the vehicle group, and the difference was statistically significant (* indicates P<0.05) (Figure 11D). This result indicates that plasminogen can promote the recovery of the number of striatal Nissl bodies. Example 8

[0092] Example 8 relates to the effect of plasminogen on reducing hippocampal damage in Huntington's disease model mice. We took 6-week-old C57 male mice, weighed all mice 3 days before modeling, performed a simple open field test, and observed them for 5 minutes to exclude spontaneous differences in random animals. Finally, we determined 23 experimental animals. According to the total distance traveled by mice in the open field test, the mice were randomly divided into two groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were administered 125 μL of PBS solution by intraperitoneal injection, and the mice in the model group were administered 3-nitropropionic acid (3-NP) solution at 50 mg / kg body weight by intraperitoneal injection, twice a day (at 12-hour intervals) for 5 consecutive days to construct the Huntington's model. [1]Preparation of 3-NP solution: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg / ml. The day after modeling was completed was designated as day 0 of administration, and all mice were weighed and subjected to an open field test. The mice in the model group were randomly divided into two groups, a solvent control group (9 mice) and a plasminogen administration group (8 mice), according to the total distance traveled in the open field. The mice in the solvent control group were administered with solvent (10 mM citric acid-sodium citrate solution, pH 7.4) at 0.1 ml / mouse via tail vein injection, and the mice in the plasminogen administration group were administered with human plasminogen at 1 mg / 0.1 ml / mouse via tail vein injection for 7 consecutive days. On the 7th day of administration, the mice were sacrificed, and the hippocampi were collected and fixed in 10% neutral formaldehyde solution for 24 to 48 hours. The fixed hippocampal tissue was dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The sections were 4 μm thick, deparaffinized, rehydrated, stained with hematoxylin and eosin (HE staining), fractionated in 1% hydrochloric acid alcohol, turned blue with ammonia water, dehydrated in an alcohol gradient, and mounted. The sections were observed under an optical microscope at 200x magnification. The results showed that the overall shape of the hippocampus of the mice in the blank control group (Figure 12A-C) was normal and the structure was intact; the CA1 region of the hippocampus of the mice in the solvent control group (Figure 12D-F) showed disordered arrangement of neurons, neuronal atrophy (marked by thin arrows), cell degeneration and necrosis, and loosening and edema of the surrounding brain tissue (marked by triangles), and the neuronal nuclei in multiple locations in the CA3 region were condensed and strongly stained (marked by thick arrows); in the plasminogen-treated group (Figure 12G-I), the shape of each region of the hippocampus was restored to a certain extent, and some localized neuronal nuclei in the CA3 and DG regions were condensed and strongly stained. This result indicates that plasminogen can repair hippocampal damage in Huntington's disease model mice. Example 9

[0093] Example 9 relates to the effect of plasminogen on reducing striatal damage in Huntington's disease model mice. We took 6-week-old C57 male mice, weighed all mice 3 days before modeling, performed a simple open field test, and observed them for 5 minutes to exclude spontaneous differences in random animals. Finally, we determined 23 experimental animals. According to the total distance traveled by mice in the open field test, the mice were randomly divided into two groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were administered 125 μL of PBS solution by intraperitoneal injection, and the mice in the model group were administered 3-nitropropionic acid (3-NP) solution at 50 mg / kg body weight by intraperitoneal injection, twice a day (at 12-hour intervals) for 5 consecutive days to construct the Huntington's model. [1] Preparation of 3-NP solution: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg / ml. The day after modeling was completed was designated as day 0 of administration, and all mice were weighed and subjected to an open field test. The mice in the model group were randomly divided into two groups, a solvent control group (9 mice) and a plasminogen administration group (8 mice), according to the total distance traveled in the open field. The mice in the solvent control group were administered with solvent (10 mM citric acid-sodium citrate solution, pH 7.4) at 0.1 ml / mouse via tail vein injection, and the mice in the plasminogen administration group were administered with human plasminogen at 1 mg / 0.1 ml / mouse via tail vein injection for 7 consecutive days. On the 7th day of administration, the mice were sacrificed, and the striatum was collected and fixed in 10% neutral formaldehyde solution for 24 to 48 hours. The fixed striatal tissue was dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. Sections were 3 μm thick, deparaffinized, rehydrated, stained with hematoxylin and eosin (HE staining), fractionated in 1% hydrochloric acid alcohol, bluing with ammonia water, dehydrated in an alcohol gradient, and mounted. Sections were observed under a light microscope at 200x magnification. The results showed that the morphological structure of the striatum of the mice in the blank control group (Figure 13A) was normal, while the striatal neurons of the mice in the vehicle group (Figure 13B) were degenerated, some cell nuclei were vacuolated (marked with arrows), and the arrangement of brain cells was disorganized; the mice in the plasminogen-treated group (Figure 13C) showed a significant improvement in the overall structure of the striatum, similar to the blank control group. This result indicates that plasminogen can repair striatal damage in Huntington's disease model mice. Example 10

[0094] Example 10 relates to the improvement of olfactory tubercle damage in Huntington's disease model mice by plasminogen. We took 6-week-old C57 male mice, weighed all mice 3 days before modeling, performed a simple open field test, and observed them for 5 minutes to exclude spontaneous differences in random animals. Finally, we determined 23 experimental animals. According to the total distance traveled by mice in the open field test, the mice were randomly divided into two groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were administered 125 μL of PBS solution by intraperitoneal injection, and the mice in the model group were administered 3-nitropropionic acid (3-NP) solution at 50 mg / kg body weight by intraperitoneal injection, twice a day (at 12-hour intervals) for 5 consecutive days to construct the Huntington's model. [1]Preparation of 3-NP solution: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg / ml. The day after modeling was completed was designated as day 0 of administration, and all mice were weighed and subjected to an open field test. The mice in the model group were randomly divided into two groups, a solvent control group (9 mice) and a plasminogen administration group (8 mice), according to the total distance traveled in the open field. The mice in the solvent control group were administered with solvent (10 mM citric acid-sodium citrate solution, pH 7.4) at 0.1 ml / mouse via tail vein injection, and the mice in the plasminogen administration group were administered with human plasminogen at 1 mg / 0.1 ml / mouse via tail vein injection for 7 consecutive days. On the 7th day of administration, the mice were sacrificed, and brain tissue was collected and fixed in 10% neutral formaldehyde solution for 24 to 48 hours. The fixed brain tissue was dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The sections were 3 μm thick, deparaffinized, rehydrated, stained with hematoxylin and eosin (HE staining), fractionated in 1% hydrochloric acid alcohol, bluing with ammonia water, dehydrated in an alcohol gradient, and mounted. The sections were observed under a light microscope at 200x magnification. Study reports that mice with Huntington's disease have damaged olfactory nerves, and that Huntington's patients suffer from smell loss [9] . The results showed that the olfactory tubercle of the brain of the mice in the blank control group (Figure 14A) had normal morphology and structure, the olfactory tubercle of the mice in the vehicle group (Figure 14B) had severe glial cell infiltration (marked with arrows), and the olfactory tubercle glial cell infiltration of the mice in the plasminogen administration group (Figure 14C) was significantly less than that of the vehicle group. This result indicates that plasminogen can improve the brain olfactory tubercle damage of Huntington's disease model mice. Example 11

[0095] Example 11 relates to the promotion of BDNF expression in the hippocampus of Huntington's disease model mice by plasminogen. We took 6-week-old C57 male mice, weighed all mice 3 days before modeling, performed a simple open field test, and observed them for 5 minutes to exclude spontaneous differences in random animals. Finally, we determined 23 experimental animals. According to the total distance traveled by mice in the open field test, the mice were randomly divided into two groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were administered 125 μL of PBS solution by intraperitoneal injection, and the mice in the model group were administered 3-nitropropionic acid (3-NP) solution at 50 mg / kg body weight by intraperitoneal injection, twice a day (at 12-hour intervals) for 5 consecutive days to construct the Huntington's model. [1]Preparation of 3-NP solution: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg / ml. The day after modeling was completed was designated as day 0 of administration, and all mice were weighed and subjected to an open field test. The mice in the model group were randomly divided into two groups, a solvent control group (9 mice) and a plasminogen administration group (8 mice), according to the total distance traveled in the open field. The mice in the solvent control group were administered with solvent (10 mM citric acid-sodium citrate solution, pH 7.4) at 0.1 ml / mouse via tail vein injection, and the mice in the plasminogen administration group were administered with human plasminogen at 1 mg / 0.1 ml / mouse via tail vein injection for 7 consecutive days. On the 7th day of administration, the mice were sacrificed, and the hippocampi were collected and fixed in 10% neutral formaldehyde solution for 24 to 48 hours. Fixed hippocampal tissue was dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The thickness of the tissue section was 3 μm. The sections were deparaffinized, rehydrated, and washed once with water. The tissue was circled with a PAP marker, incubated with 3% hydrogen peroxide for 15 min, and washed twice with 0.01 M PBS, for 5 min each time. Blocking was performed with 5% normal goat serum (Vector Laboratories, Inc., USA) for 30 min. When the time was up, the goat serum was discarded, and rabbit anti-BNDF antibody (PB9075, Boster Biological Technology) was dropped and incubated overnight at 4°C, washed twice with 0.01 M PBS, for 5 min each time. Goat anti-rabbit IgG (HRP) antibody (Vector laboratories, MP-7451) secondary antibody was incubated at room temperature for 30 min, washed twice with 0.01 M PBS, for 5 min each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. They were dehydrated in a graded alcohol stream, cleared with xylene, and embedded in neutral rubber. Sections were observed under an optical microscope at 400x magnification. BDNF, or brain derived neurotrophic factor, is a protein synthesized in the brain, widely distributed in the central nervous system, and plays an important role in the survival, differentiation, growth, and development of neurons during the development of the central nervous system. BDNF can prevent neurons from being damaged and dying, improve the pathological condition of neurons, and promote biological effects such as the regeneration and differentiation of damaged neurons.

[10] . The results showed that there was a certain amount of BDNF expression in the right hippocampus of the mice in the blank control group (Figure 15A) (marked with an arrow), the expression of BDNF in the right hippocampus of the mice in the vehicle group (Figure 15B) was slightly higher than that of the blank control group, and the expression of BDNF in the right hippocampus of the mice in the plasminogen administration group (Figure 15C) was significantly higher than that of the vehicle control group, and the statistical difference was close to significance (P=0.07) (Figure 15D). This result suggests that injury stimulates the expression of BDNF, and plasminogen can further promote the expression of BDNF, thereby promoting the recovery of hippocampal injury in Huntington's disease model mice. Example 12

[0096] Example 12 relates to the promotion of BDNF expression in the striatum of Huntington's disease model mice by plasminogen. We took 6-week-old C57 male mice, weighed all mice 3 days before modeling, performed a simple open field test, and observed them for 5 minutes to exclude spontaneous differences in random animals. Finally, we determined 23 experimental animals. According to the total distance traveled by mice in the open field test, the mice were randomly divided into two groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were administered 125 μL of PBS solution by intraperitoneal injection, and the mice in the model group were administered 3-nitropropionic acid (3-NP) solution at 50 mg / kg body weight by intraperitoneal injection, twice a day (at 12-hour intervals) for 5 consecutive days to construct the Huntington's model. [1]Preparation of 3-NP solution: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg / ml. The day after modeling was completed was designated as day 0 of administration, and all mice were weighed and subjected to an open field test. The mice in the model group were randomly divided into two groups, a solvent control group (9 mice) and a plasminogen administration group (8 mice), according to the total distance traveled in the open field. The mice in the solvent control group were administered with solvent (10 mM citric acid-sodium citrate solution, pH 7.4) at 0.1 ml / mouse via tail vein injection, and the mice in the plasminogen administration group were administered with human plasminogen at 1 mg / 0.1 ml / mouse via tail vein injection for 7 consecutive days. On the 7th day of administration, the mice were sacrificed, and the striatum was collected and fixed in 10% neutral formaldehyde solution for 24 to 48 hours. Fixed striatal tissues were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The thickness of the tissue sections was 3 μm. The sections were deparaffinized, rehydrated, and washed once with water. The tissues were circled with a PAP marker, incubated with 3% hydrogen peroxide for 15 min, and washed twice with 0.01 M PBS, for 5 min each time. Blocking was performed with 5% normal goat serum (Vector Laboratories, Inc., USA) for 30 min. When the time was up, the goat serum was discarded, and rabbit anti-BNDF antibody (PB9075, Boster Biological Technology) was dropped and incubated overnight at 4°C, washed twice with 0.01 M PBS, for 5 min each time. Goat anti-rabbit IgG (HRP) antibody (Vector laboratories, MP-7451) secondary antibody was incubated at room temperature for 30 min, washed twice with 0.01 M PBS, for 5 min each time. The sections were stained with a DAB kit (Vector Laboratories, Inc., USA), washed three times with water, counterstained with hematoxylin for 30 seconds, and rinsed with running water for 5 minutes. They were dehydrated in a graded alcohol stream, cleared with xylene, and embedded in neutral rubber. Sections were observed under an optical microscope at 400x magnification. The results showed that there was a certain amount of BDNF expression in the striatum of mice in the blank control group (Figure 16A) (marked with an arrow), the expression of BDNF in the striatum of mice in the vehicle group (Figure 16B) was slightly higher than that of the blank control group, and the expression of BDNF in the striatum of mice in the plasminogen administration group (Figure 16C) was significantly higher than that of the vehicle group, and the statistical difference was close to significance (P=0.1) (Figure 16D). This result suggests that injury stimulates the expression of BDNF, and plasminogen can further promote the expression of BDNF, thereby promoting the recovery of striatal injury in Huntington's disease model mice. Example 13

[0097] Example 13 relates to the promotion of regeneration of striatal myelin in Huntington's disease model mice by plasminogen. We took 6-week-old C57 male mice, weighed all mice 3 days before modeling, performed a simple open field test, and observed them for 5 minutes to exclude spontaneous differences in random animals. Finally, we determined 23 experimental animals. According to the total distance traveled by mice in the open field test, the mice were randomly divided into two groups, 6 mice in the blank control group and 17 mice in the model group. The mice in the blank control group were administered 125 μL of PBS solution by intraperitoneal injection, and the mice in the model group were administered 3-nitropropionic acid (3-NP) solution at 50 mg / kg body weight by intraperitoneal injection, twice a day (at 12-hour intervals) for 5 consecutive days to construct the Huntington's model. [1]Preparation of 3-NP solution: 3-NP powder (sigma, catalog number N5636) was dissolved in PBS solution to a concentration of 10 mg / ml. The day after modeling was completed was designated as day 0 of administration, and all mice were weighed and subjected to an open field test. The mice in the model group were randomly divided into two groups, a solvent control group (9 mice) and a plasminogen administration group (8 mice), according to the total distance traveled in the open field. The mice in the solvent control group were administered with solvent (10 mM citric acid-sodium citrate solution, pH 7.4) at 0.1 ml / mouse via tail vein injection, and the mice in the plasminogen administration group were administered with human plasminogen at 1 mg / 0.1 ml / mouse via tail vein injection for 7 consecutive days. On the 7th day of administration, the mice were sacrificed, and the striatum was collected and fixed in 10% neutral formaldehyde solution for 24 to 48 hours. The fixed striatal tissue was dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in paraffin. The cross-sectional sections of spinal cord tissue were 4 μm thick. The sections were deparaffinized, rehydrated, and stained with LFB using myelin staining solution. The sections were dehydrated in an alcohol gradient, permeabilized with xylene, and embedded in neutral rubber. The sections were observed under an optical microscope and photographed. LFB (luxol fast blue) staining is the staining of myelin using fast blue staining, which is an effective method for studying the localization of the corticospinal tract, myelin lesions, and morphological observation of injury and regeneration repair. [11-12] . As a result, the striatal myelin structure of the mice in the blank control group (Figure 17) was normal, the amount of striatal myelin protein of the mice in the vehicle group (Figure 17B) was reduced and cavity degeneration was observed (marked with arrows), and the amount of striatal myelin protein of the mice in the plasminogen administration group (Figure 17C) was significantly higher than that of the vehicle group, with the statistical difference approaching significance (P = 0.09) (Figure 17D). This result suggests that plasminogen can promote the formation of striatal myelin protein in Huntington's disease model mice. References: [1]Wang L , Wang J , Yang L , et al. Effect of Praeruptorin C on 3-nitropropionic acid induced Huntington’s disease-like symptoms in mice[J]. Biomedicine & Pharmacotherapy, 2017, 86:81-87. [2]Marchei P , Diverio S , Falocci N , et al. Breed differences in behavioural development in kittens[J]. Physiology & Behavior, 2009, 96(4-5):522-531. [3]Jacob W, Yassouridis A, Marsicano G, et al. Endocannabinoids render exploratory behaviour largely independent of the test aversiveness: role of glutamatergic transmission.[J]. Genes Brain & Behavior, 2010, 8(7):685-698. [4]Walz K, Spencer C, Kaasik K, et al. Behavioral characterization of mouse models for Smith-Magenis syndrome and dup(17)(p11.2p11.2)[J]. Human Molecular Genetics, 2004, 13(4):367. [5]Aziz N A , Burg J M M V D , Landwehrmeyer G B , et al. Weight loss in Huntington disease increases with higher CAG repeat number[J]. Neurology, 2009, 73(7):1506. [6]Hol E M, Pekny M. Glial fibrillary acidic protein (GFAP) and the astrocyte intermediate filament system in diseases of the central nervous system.[J]. Current Opinion in Cell Biology, 2015, 32(1):121-130. [7]Vagner T, Dvorzhak A, Wojtowicz A M, et al. Systemic application of AAV vectors targeting GFAP-expressing astrocytes in Z-Q175-KI Huntington's disease mice.[J]. Molecular & Cellular Neuroscience, 2016, 77:76-86. [8]Xie J X, Feng Y, Yuan J M, et al. Positive effects of bFGF modified rat amniotic epithelial cells transplantation on transected rat optic nerve[J]. Plos One, 2014, 10(3):e0119119. [9]Kohl Z, Regensburger M, Aigner R, et al. Impaired adult olfactory bulb neurogenesis in the R6 / 2 mouse model of Huntington’s disease[J]. BMC Neuroscience,11,1(2010-09-13), 2010, 11(1):114.

[10] Hyman C, Hofer M, Barde Y A, et al. BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra[J]. Nature, 1991, 350(6315):230-232.

[11] Sun SW, Liang HF, Trinkaus K et al. Noninvasive detection of cuprizone induced axonal damage and demyelination in the mouse corpus callosum. Magn Reson Med. 2006 Feb;55(2):302-8.

[12] Vallieres N1, Berard JL, David S et al. Systemic injections of lipopolysaccharide accelerates myelin phagocytosis during Wallerian degeneration in the injured mouse spinal cord. Glia. 2006 Jan 1;53(1):103-13.

Claims

1. A pharmaceutical composition for treating Huntington's disease comprising plasminogen, the plasminogen having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:2 and still having the proteolytic activity of plasminogen.

2. 2. The pharmaceutical composition of claim 1, wherein the plasminogen has one or more effects on the subject with Huntington's disease selected from the following: improving or alleviating motor impairment, improving cognitive impairment, slowing weight loss, promoting repair of damaged neurons, improving neuropsychiatric symptoms, relieving anxiety, reducing hippocampal GFAP expression, reducing hippocampal apoptosis, promoting recovery of Nissl body numbers in cerebellar, hippocampal or striatal neurons, repairing damage to the hippocampus, striatum or olfactory tubercle, promoting BDNF expression in the hippocampus or striatum, and promoting remyelination in the striatum.

3. The pharmaceutical composition of claim 1 or 2, wherein the plasminogen is used in combination with one or more other drugs or therapeutic methods.

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