Application of natural protein molecule in preparation of medicine for resisting Parkinson's syndrome

Abstract

The invention belongs to the technical field of biological medicine, discloses application of a natural protein molecule in preparation of a medicine for resisting Parkinson's syndrome, and particularly discloses a protein molecule which comprises A-B, A comprises A-chain polypeptide or functional variants of the A-chain polypeptide, and B comprises A-chain polypeptide or functional variants of the A-chain polypeptide. B comprises a B-chain polypeptide or a functional variant thereof; the A is in disulfide bridging with the B; the amino acid sequence of the A-chain polypeptide is as shown in SEQ ID NO: 1; the amino acid sequence of the B-chain polypeptide is as shown in SEQ ID NO: 2. The invention discloses a natural protein molecule and application thereof in treatment and / or prevention of Parkinson's syndrome for the first time, the protein molecule can show antioxidant capacity by activating GSH in cells and inhibiting ROS formation, and meanwhile, the protein molecule can also show antioxidant capacity by activating mitochondrial activity and anti-GFAP activity in an MPP-induced brain injury environment, so that the protein molecule can be used for treating and / or preventing Parkinson's syndrome. The natural protein molecule has the medical effect of relieving and treating Parkinson's syndrome.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of a natural protein molecule in the preparation of an anti-Parkinson's disease drug. Background Art

[0002] Parkinson's disease is a chronic, progressive neurodegenerative disorder with an age-related onset. Globally, Parkinson's disease affects approximately 6.2 million people annually and causes approximately 117,000 deaths. Clinical manifestations of Parkinson's disease primarily include: 1. symptoms caused by progressive dopaminergic system dysfunction; 2. symptoms caused by dysfunction of non-dopaminergic systems; and 3. symptoms caused by therapeutic medications. These clinical manifestations are major factors affecting patients' quality of life.

[0003] In recent years, numerous clinical reports have confirmed the close relationship between oxidative stress and the development and progression of Parkinson's disease. Oxidative stress is caused by an imbalance between oxidative and antioxidant functions in the body. Under the influence of oxidative stress, the body produces a large number of reactive oxygen species (ROS), triggering cellular damage. The generation of ROS leads to dopaminergic neuronal loss in the brains of Parkinson's patients. Decreased dopamine metabolism and glutathione levels can both lead to the production of large amounts of ROS. Therefore, as oxidative stress increases, the degree of neuronal damage caused by substantia nigra dopamine increases significantly. Notably, extensive evidence indicates that glutathione exists in two forms, reduced (GSH) and oxidized (GSSG), with reduced glutathione predominating under physiological conditions. Reduced glutathione is a crucial antioxidant in the brain, effectively scavenging free radicals and protecting dopamine neurons, thereby controlling the progression of Parkinson's disease. In addition, a number of clinical studies have confirmed that glial fibrillary acidic protein (GFAP) is considered to be a marker of astrocyte activation. When the central nervous system is damaged, astrocytes release GFAP into the peripheral blood. Studies have also shown that the plasma GFAP level in patients with Parkinson's disease is higher than that in the healthy control group, and is associated with impaired cognitive function in patients with Parkinson's disease. At the same time, during autopsies of Parkinson's patients, it was found that microglia in the substantia nigra were activated, and microglia in other brain regions (hippocampus and cortex) also increased significantly, confirming that pro-inflammatory cytokines increased in the substantia nigra system of Parkinson's patients, thereby damaging the blood-brain barrier. At the same time, after testing the mitochondria in the prefrontal lobe of Parkinson's patients, it was found that the level of carbonyl proteins in the mitochondrial components increased significantly, confirming that the mitochondrial components can cause oxidative damage to the body.

[0004] While there's currently no cure for Parkinson's disease, several medications, surgeries, and integrated multidisciplinary therapies can alleviate symptoms. Medications for motor symptoms include L-dopa, dopamine receptor agonists, and monoamine oxidase inhibitors. The choice of medication depends on the severity of the disease. L-dopa is a commonly used anti-Parkinson's disease medication, but it only temporarily relieves symptoms and can cause side effects that can impact patients' quality of life. Summary of the Invention

[0005] Based on the above technical problems, the present invention aims to provide a new application of natural protein, mainly for preparing drugs or excipients for treating Parkinson's syndrome.

[0006] The first aspect of the present invention aims to provide a protein molecule.

[0007] The second aspect of the present invention aims to provide a biological material related to the protein molecule of the first aspect of the present invention.

[0008] The third aspect of the present invention is to provide a pharmaceutical composition.

[0009] The fourth aspect of the present invention aims to provide applications of the protein molecule of the first aspect of the present invention, the biomaterial of the second aspect of the present invention, or the pharmaceutical composition of the third aspect of the present invention.

[0010] The fifth aspect of the present invention aims to provide a product.

[0011] The sixth aspect of the present invention aims to provide a method.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] The first aspect of the present invention provides a protein molecule comprising AB, wherein:

[0014] A includes an A chain polypeptide or a functional variant thereof;

[0015] B includes a B chain polypeptide or a functional variant thereof;

[0016] A and B are disulfide bridged;

[0017] The amino acid sequence of the A chain polypeptide is the polypeptide shown in SEQ ID NO: 1.

[0018] The amino acid sequence of the B chain polypeptide is shown in SEQ ID NO: 2.

[0019] In some embodiments of the present invention, the cysteine ​​residue at position 9 in the A chain polypeptide is covalently bound to the cysteine ​​residue at position 10 in the B chain polypeptide via a disulfide bond, and the cysteine ​​residue at position 22 in the A chain polypeptide is covalently bound to the cysteine ​​residue at position 22 in the B chain polypeptide via a disulfide bond.

[0020] In some embodiments of the present invention, the protein molecule is derived from regenerative tissues such as testicles and ovaries of animals such as pigs, whales, and tunicates; it can also be artificially synthesized, for example, by chemical synthesis or biosynthesis.

[0021] The second aspect of the present invention provides a biomaterial related to the protein molecule of the first aspect of the present invention, wherein the biomaterial is any one of a1) to a12):

[0022] a1) a nucleic acid molecule encoding the protein molecule of the first aspect of the present invention;

[0023] a2) an expression cassette containing the nucleic acid molecule described in a1);

[0024] a3) a recombinant vector containing the nucleic acid molecule described in a1);

[0025] a4) a recombinant vector containing the expression cassette described in a2);

[0026] a5) a recombinant microorganism containing the nucleic acid molecule described in a1);

[0027] a6) a recombinant microorganism containing the expression cassette described in a2);

[0028] a7) a recombinant microorganism containing the recombinant vector described in a3);

[0029] a8) a recombinant microorganism containing the recombinant vector described in a4);

[0030] a9) a transgenic animal cell line containing the nucleic acid molecule described in a1);

[0031] a10) a transgenic animal cell line containing the expression cassette described in a2);

[0032] a11) a transgenic animal cell line containing the recombinant vector described in a3);

[0033] a12) a transgenic animal cell line containing the recombinant vector described in a4);

[0034] The transgenic animal cell line does not contain reproductive material.

[0035] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0036] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a non-viral vector.

[0037] In some embodiments of the present invention, the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector.

[0038] In some embodiments of the present invention, the non-viral vector comprises a plasmid vector.

[0039] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a phage, a cosmid, an F cosmid, or an artificial chromosome.

[0040] In some embodiments of the present invention, the plasmid vector may be any plasmid, and the viral vector may be any virus.

[0041] The third aspect of the present invention provides a pharmaceutical composition comprising the protein molecule of the first aspect of the present invention or the biomaterial of the second aspect of the present invention.

[0042] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0043] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a color and flavor regulator, a solvent, a solubilizer, a cosolvent, an emulsifier, an absorption promoter, a surfactant, an antioxidant, an excipient, a stabilizer, a flavoring agent, a sustained-release agent, a metal complexing agent, an inert gas, a preservative, a filler, a local analgesic, a pH regulator, and an isotonic or isotonic regulator.

[0044] In some embodiments of the present invention, the diluent includes, but is not limited to, at least one of starches, sugars, celluloses, and inorganic salts. The wetting agent includes, but is not limited to, at least one of water, ethanol, and propanol. The binder includes, but is not limited to, at least one of starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, and polyethylene glycol. The disintegrant includes, but is not limited to, at least one of microcrystalline cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, cross-linked polyvinyl pyrrolidone, surfactants (such as sodium dodecylbenzene sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan or polysorbate, etc.), and effervescent disintegrants. The lubricant includes, but is not limited to, at least one of talc, calcium stearate, magnesium stearate, magnesium lauryl sulfate, micropowdered silica gel, calcium carbonate, sodium bicarbonate, and polyethylene glycol. The color and flavor modifier includes, but is not limited to, at least one of pigments, flavorings, sweeteners, mucilages, and flavor correctors. The solvent includes, but is not limited to, at least one of water, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil, ethyl acetate, and vegetable oil (such as soybean oil, castor oil, peanut oil, blended oil, etc.). The solubilizing agent includes, but is not limited to, at least one of Tweens, glycerols, polyoxyethylene fatty alcohol ethers, soaps, sulfates, and sulfonates. The cosolvent includes, but is not limited to, at least one of organic acids and their salts, amide and amine compounds, inorganic salts, polyethylene glycol, povidone, and glycerol. The emulsifier includes, but is not limited to, at least one of Spans, Tweens, glycerols, benzyls, glycerol fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, tragacanth, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxide, silicon dioxide, and bentonite. The suspending agent includes, but is not limited to, at least one of glycerin, syrup, gum arabic, gum tragacanth, agar, sodium alginate, cellulose derivatives, povidone, carbopol, polyvinyl alcohol, and thixotropic gel. The antioxidant includes, but is not limited to, at least one of sulfites, pyrosulfites, bisulfites, ascorbic acid, gallic acid, and their esters. The metal chelating agent includes, but is not limited to, at least one of disodium ethylenediaminetetraacetic acid and polycarboxylic acid compounds; the inert gas includes, but is not limited to, at least one of nitrogen and carbon dioxide. The preservative includes, but is not limited to, at least one of parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, and volatile oils. The filler includes, but is not limited to, at least one of starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, or glucose. The flavoring agent includes, but is not limited to, at least one of aspartame, sucralose, flavoring, stevia, acesulfame potassium, citric acid, or sodium saccharin. The local analgesic includes but is not limited to at least one of benzyl alcohol, chlorobutanol, lidocaine, and procaine.The pH adjuster includes, but is not limited to, at least one of hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate, and citrate. The isotonic or isotonic adjuster includes, but is not limited to, at least one of glucose, sodium chloride, sodium citrate, sorbitol, and xylitol.

[0045] In some embodiments of the present invention, the pharmaceutical composition may further comprise a combination drug, including a drug for treating or preventing Parkinson's disease-related diseases.

[0046] In this case, the at least two active ingredients may be administered together or separately and as part of the same pharmaceutical preparation or as different preparations.Thus, the composition used in the context of the present invention may be used in combination with a drug for preventing Parkinson's disease-related diseases. The combination drugs include tolcapone, fluvoxamine, rubidium chloride, cycloastragenol, plasmin, baclofen, cinacalcet, mexiletine, levodopa, talipexol, piribedil, rotigotine, bromocriptine, pergolide, cabergoline, lisuride, pramipexole, ropinirole, apomorphine, selegiline, rasagiline, ganglioside GM1, citicoline, droxidopa mazatrol, promethazine, quetiapine, procyclidine, o-phenhydramine, domperidone, benztropine, trihexyphenidyl, biperiden, clozapine, desipramine, citalopram, nortriptyline, paroxetine, atomoxetine, venlafaxine, amantadine, donepezil, rivastigmine and megin.

[0047] In some embodiments of the present invention, the parkinsonism comprises at least one of Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, corticobasal degeneration, diffuse Lewy body disease, parkinsonism-dementia, X-linked dystonia-parkinsonism, and secondary parkinsonism.

[0048] The fourth aspect of the present invention provides the use of the protein molecule of the first aspect of the present invention, the biomaterial of the second aspect of the present invention, or the pharmaceutical composition of the third aspect of the present invention in any one of b1) to b4):

[0049] b1) Preparation of medicaments for the treatment and / or prevention of Parkinson's disease;

[0050] b2) Antioxidant;

[0051] b3) preparing antioxidant products;

[0052] b4) Preparation of products for the treatment and / or prevention of inflammation.

[0053] In some embodiments of the present invention, the parkinsonism comprises at least one of Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, corticobasal degeneration, diffuse Lewy body disease, parkinsonism-dementia, X-linked dystonia-parkinsonism, and secondary parkinsonism.

[0054] In some embodiments of the present invention, the anti-oxidation includes inhibiting the formation of reactive oxygen species and / or enhancing the relative activity of cellular mitochondria.

[0055] In some embodiments of the present invention, the anti-inflammatory effect includes inhibiting the expression of glial fibrillary acidic protein and / or inhibiting the expression of tyrosine hydroxylase.

[0056] In some embodiments of the present invention, the product includes a reagent, a medicine, a food or a health food.

[0057] In some embodiments of the present invention, the drug can be administered orally, parenterally (e.g., intravenously, intramuscularly, intraperitoneally), intracranially, intrathecally, intraarterially (e.g., via the carotid artery), rectally, cutaneously, nasally, vaginally, by inhalation, through the skin (patch), or ophthalmically, and the effective dose administered is a pharmaceutically acceptable dose.

[0058] In some embodiments of the present invention, the dosage form of the drug is selected from tablets (such as ordinary tablets, double-layer tablets, multi-layer tablets, sustained-release tablets, single-chamber controlled-release tablets, double-chamber controlled-release tablets, microporous controlled-release tablets, sublingual tablets, orally rapidly disintegrating tablets, dispersible tablets, enteric-coated tablets), pills, patches, powders, suspensions, gels, emulsions, creams, granules, solutions, nanoparticles, drops, capsules (such as ordinary capsules, sustained-release capsules, controlled-release capsules, capsules containing micropellets or small tablets, pH-dependent capsules containing micropellets or small tablets, gastrointestinal composite capsules), enemas, suppositories, granules, injections, sprays and injections.

[0059] In some embodiments of the present invention, the cells include but are not limited to human neuroblastoma cells and cerebral cortical astrocytes.

[0060] The fifth aspect of the present invention provides a product comprising the protein molecule of the first aspect of the present invention, the biomaterial of the second aspect of the present invention, or the pharmaceutical composition of the third aspect of the present invention.

[0061] In some embodiments of the present invention, the product has at least one of the functions c1) to c5):

[0062] c1) Antioxidant;

[0063] c2) inhibiting the formation of reactive oxygen species;

[0064] c3) Enhance the relative activity of cell mitochondria;

[0065] c4) inhibiting the activity of glial fibrillary acidic protein;

[0066] c5) enhance the activity of reduced GSH;

[0067] In some embodiments of the present invention, the cells include but are not human neuroblastoma cells or cerebral cortical astrocytes.

[0068] In some embodiments of the present invention, the product includes a reagent, a medicine, a food or a health food.

[0069] The sixth aspect of the present invention provides a method comprising the step of treating with the protein molecule of the first aspect of the present invention, the biomaterial of the second aspect of the present invention, or the pharmaceutical composition of the third aspect of the present invention;

[0070] The method is at least one of d1) to d3):

[0071] d1) A method for enhancing cellular antioxidant activity:

[0072] d2) a method for inhibiting cellular ROS formation;

[0073] d3) a method for increasing the relative activity of mitochondria;

[0074] The method is not intended for disease diagnosis or treatment.

[0075] In some embodiments of the present invention, the cells include but are not limited to human neuroblastoma cells and cerebral cortical astrocytes.

[0076] In some embodiments of the present invention, the treated reaction system contains 10 to 1000 ng / mL of protein molecules.

[0077] The beneficial effects of the present invention are:

[0078] The present invention discloses a natural protein molecule derived from regenerative tissues such as the testicles and ovaries of animals such as pigs, whales, and tunicates. This natural protein molecule exhibits antioxidant capacity by activating GSH in cells and inhibiting ROS formation. It can also activate mitochondrial activity and inhibit GFAP activity in the setting of MPP-induced brain damage, indicating that this natural protein molecule has the medical effect of alleviating and treating Parkinson's disease. Furthermore, the preparation method of this natural protein molecule is simple and efficient, and its preparation into an anti-Parkinson's disease drug will provide an important drug option for the clinical treatment of the disease.

[0079] Specifically, the present invention establishes a model for detecting anti-Parkinson's syndrome activity, including an SH-SY5Y cell model to detect the production of GSH antioxidants, the formation of reactive oxygen free radicals (ROS), and the efficacy of inducing mitochondrial activity and inhibiting GFAP activity in the MPP environment of mouse cortical astrocytes. After a large amount of screening, it was found that a natural protein molecule from regenerative tissues such as testicles and ovaries of animals such as pigs, whales, and tunicates can induce the production of GSH in SH-SY5Y cell populations, and GSH plays an important antioxidant role in cells. At the same time, the inventors also found that this natural protein molecule can reduce the formation of ROS and enhance mitochondrial activity; and in a mouse model, the inventors have experimentally demonstrated that this natural protein molecule can relieve and treat Parkinson's syndrome in the mouse brain by inducing reduced GSH activity, inhibiting oxidation, inhibiting GFAP activity, and enhancing mitochondrial activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 Figure 2 is the growth of GSH induced by natural protein in SH-SY5Y cells; A is the cytotoxicity experimental result of natural protein on SH-SY5Y cells, and B is the activity of natural protein in inducing GSH in SH-SY5Y cells. In the figure, * represents P < 0.05.

[0081] Figure 2 The natural protein demonstrated the ability to inhibit ROS formation and enhance mitochondrial activity in a mouse model under the environment of MPP-induced brain cell damage; among them, A shows the cytotoxicity experimental results of the natural protein on mouse cortical astrocytes, B shows the effect of the natural protein on ROS formation, and C shows the effect of the natural protein on mitochondrial activity induced by cortical astrocytes. In the figure, * represents P < 0.05, and ** represents P < 0.01.

[0082] Figure 3 This is a natural protein that inhibits glial fibrillary acidic protein (GFAP) in mouse cortical astrocytes. Scale bars in panel A are 500 μm, and in panel B are 100 μm. DETAILED DESCRIPTION

[0083] The present invention is further described in detail below through specific examples.

[0084] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0085] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0086] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0087] Unless otherwise specified, the percentage contents mentioned in the present invention refer to mass percentage for solid-liquid mixture and solid-solid mixture, and refer to volume percentage for liquid-liquid mixture.

[0088] The percentage concentrations mentioned in the present invention, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0089] The temperature parameters in the present invention, unless otherwise specified, allow for either constant temperature treatment or treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range controlled by the instrument.

[0090] The term "amino acid residue" is intended to indicate the amino acid residues comprised in the group consisting of alanine (Ala or A), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) and tyrosine (Tyr or Y) residues.

[0091] "Polypeptide," "peptide," and "protein" are used interchangeably herein and include a molecular chain of two or more amino acids linked by peptide bonds. The terms do not refer to the specific length of the product. The terms include post-translational modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like. In addition, protein fragments, analogs, mutant or variant proteins, fusion proteins, and the like are included within the meaning of polypeptide. The terms also include molecules comprising one or more amino acid analogs or non-standard or unnatural amino acids that can be synthesized or recombinantly expressed using known protein engineering techniques. In addition, the fusion proteins of the present invention can be derivatized as described herein using well-known organic chemistry techniques.

[0092] "Functional variant" refers to a variant of a polypeptide or protein that has substantial or significant sequence identity with a polypeptide or protein and retains at least one biological activity of the polypeptide or protein. Functional variants of a polypeptide or protein can be prepared based on the present disclosure by means known in the art. Functional variants may include one or more modifications to the amino acid sequence of a polypeptide or protein. In certain embodiments, the modifications change one or more physicochemical properties of the polypeptide or protein, for example, by improving the thermal stability of the polypeptide or protein, changing substrate specificity, changing the optimal pH, and reducing immunogenicity, etc. In certain embodiments, the modifications change one or more biological properties of the polypeptide or protein, provided that they do not destroy or impair all biological properties of the polypeptide or protein. In the case of polypeptide variants of the present invention, a functional variant is a variant that displays at least some of its natural activity.

[0093] According to some embodiments of the present invention, the functional variant of a polypeptide or protein comprises one or more amino acid substitutions to a polypeptide or protein, and the substitution does not significantly affect the biological activity of the polypeptide or protein, preferably a conservative amino acid substitution. Conservative amino acid substitutions include but are not limited to: amino acid substitutions in the basic amino acid group (arginine, lysine and histidine), amino acid substitutions in the polar amino acid group (glutamic acid and aspartic acid), amino acid substitutions in the hydrophobic amino acid (leucine, isoleucine and valine) group, amino acid substitutions in the aromatic amino acid (phenylalanine, tryptophan and tyrosine) group, and amino acid substitutions in the small amino acid (glycine, alanine, serine, threonine and methionine) group. Non-standard or non-natural amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid and α-methylserine) can also or as an alternative to replace the standard amino acid residues in a polypeptide or protein. For example, proline can be substituted by 4-hydroxyproline, lysine can be substituted by 5-hydroxylysine; histidine can be substituted by 3-methylhistidine, serine can be substituted by homoserine, and lysine can be substituted by ornithine.

[0094] When referring to the polypeptides of the present invention, the terms "fragment", "variant", "derivative" and "analog" include any polypeptide that retains at least some of the receptor binding properties of the corresponding wild-type polypeptide. Fragments of the polypeptides of the present invention include proteolytic fragments and deletion fragments, and also include polypeptides with altered amino acid sequences due to amino acid substitutions, deletions or insertions. Variants can be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be produced using mutagenesis techniques known in the art. Variant polypeptides can contain conservative or non-conservative amino acid substitutions, deletions or additions. Variant polypeptides may also be referred to herein as "polypeptide analogs". As used herein, a "derivative" of a polypeptide refers to a target polypeptide having one or more residues chemically derivatized by reaction of functional side groups.

[0095] The term "parkinsonism" defines a class of progressive neurodegenerative disorders characterized by resting tremor and / or bradykinesia associated with rigidity, postural instability, loss of postural reflexes, flexed posture, and / or freezing (when the feet briefly "stick" to the ground). Examples of parkinsonian disorders include Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, cortical-basal ganglionic degeneration, diffuse Lewy body disease, Parkinson-dementia, X-linked dystonia-parkinsonism, and secondary parkinsonism (due to environmental causes, such as toxins, drugs, encephalitis, brain tumors, head trauma, normal pressure hydrocephalus).

[0096] Parkinson's disease is the most common form of Parkinson's syndrome. Parkinson's disease (PD) is such a neurodegenerative disease, which causes motor and non-motor manifestations and is characterized by extensive degeneration of dopaminergic neurons in the substantia nigra system. The motor manifestations of PD are attributable to the degeneration of dopaminergic neurons in the substantia nigra. They include tremor, hypokinesia (e.g., bradykinesia, akinesia, rigidity) postural instability, gait abnormalities and dysphagia. Non-motor symptoms include autonomic and neuropsychiatric disorders such as loss of smell, or sleep abnormalities. In the context of the present invention, the term PD includes any of the above-mentioned disease manifestations.

[0097] As used herein, the term "treat" refers to obtaining a desired pharmacological and / or physiological effect. The effect may be complete or partial prevention of the occurrence or onset of a disease or its symptoms, partial or complete alleviation or relief of a disease and / or its symptoms, and / or partial or complete cure of a disease and / or its symptoms, including: (a) preventing the occurrence or onset of a disease in a subject, who may have the factors of the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., blocking its formation; and (c) alleviating the disease and / or its symptoms, i.e., causing the disease and / or its symptoms to subside or disappear.

[0098] "Effective dose" or "effective amount" refers to the amount of a component or its related compound that is sufficient to achieve the prevention and / or treatment of a disease when administered to a mammal or other subject (companion animal, e.g., dog, cat, etc.; farm animal, e.g., cattle, sheep, pig, horse, etc.; experimental animal, e.g., rat, mouse, guinea pig, etc.) for the treatment of a disease. The "therapeutically effective dose" will vary depending on the component of the protein molecule pathway or its related compound used, the severity of the disease and / or its symptoms of the subject to be treated, as well as the age, weight, etc.

[0099] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0100] Example 1 Extraction and preparation of a natural animal protein

[0101] Regenerative tissues, such as testicles and ovaries, from animals such as pigs, whales, and tunicates are ground, pulverized, and then gelatinized for use as initial samples. The samples are then added to 70% acetone, reacted at 4°C for 5 hours, and centrifuged (3000 rpm) for 15 minutes to remove solid impurities. The supernatant is then reacted at 4°C for 60 hours, after which acetone is added to the supernatant to a concentration of 90%, allowing solid protein to precipitate to the bottom. After removing the solution, including the acetone, the protein product is extracted from the sample using a 25 mM CH₃COONH₄ solution in deionized water and dialysis to remove macromolecular compounds. Macromolecular impurities are then removed using column chromatography (using 0.2 M saline as the mobile phase in carboxymethyl cellulose (CM-cellulose) at pH 5.5 and pH 7.8, respectively, to remove larger molecular impurities). The sample is then collected and further lyophilized and purified to obtain native protein with a purity of 90% or higher for use as a test sample.

[0102] After determination, the natural animal protein is a double-chain structure, and the specific amino acid sequence is:

[0103] A chain: RMTLSEKCCQVGCIRKDIARLC (SEQ ID NO: 1);

[0104] Chain B: ZRTNDLIKACGRELVRLWVEICGSVRWGQSAL (SEQ ID NO: 2).

[0105] The natural protein structure is shown below:

[0106]

[0107] Subsequently, a model for detecting anti-Parkinson's syndrome activity will be further established to deeply test the activity of this naturally extracted protein.

[0108] Example 2 Natural protein induces the production of reduced GSH

[0109] Experimental Principle

[0110] SH-SY5Y cells are commonly used as an in vitro model for neuronal function and cell differentiation. They have been used to study various areas of neuroscience, including Parkinson's disease and Alzheimer's disease. This study used this cell population to investigate the anti-Parkinson's disease activity of natural proteins by measuring their antioxidant activity.

[0111] Experimental procedures

[0112] Cell culture:

[0113] SH-SY5Y cells (CRL-2266, ATCC) were cultured in DMEM (Thermo Scientific, Waltham, MA) supplemented with 10% (vol / vol) fetal bovine serum (FBS, Thermo Scientific, Waltham, MA) and 1% (vol / vol) penicillin / streptomycin (P / S; Thermo Scientific, Waltham, MA) at 37°C in a 5% CO2 atmosphere.

[0114] Mouse primary cortical astrocytes were prepared from mice at postnatal day 0 (P0). The main steps were as follows: P0 mice were decapitated, the cortex was removed and digested with TrypLE express (Thermo Scientific, Waltham, MA) at 37°C for 15 minutes with agitation. 0.1 mg / mL DNase I, 2.5 mM MgCl2 and 0.5 mM CaCl2 were then added and incubated for 5 minutes. The digested cortex was precipitated and washed twice with DMEM, and then the cells were dispersed by stimulation with a flamed fine glass Pasteur pipette. The dispersed cells were then centrifuged at 300 x g for 7 minutes and then washed twice with DMEM + 10% (vol / vol) FBS + 1% (vol / vol) P / S. The cells were suspended in DMEM + 10% (vol / vol) FBS + 1% (vol / vol) P / S and the cells were plated at a concentration of 1×10 6 The cells were cultured in a 10 cm dish. The culture medium was changed on the 7th day (DIV) of in vitro culture and daily starting from the 8th day (DIV).

[0115] Cytotoxicity assay:

[0116] Ten thousand SH-SY5Y cells were dispersed in a 96-well tissue culture plate, with 10,000 cells per well. Before treating the cells, the cells were pretreated with different concentrations of native protein samples (final concentrations of 0 μM, 10 μM, 50 μM, 100 μM, 1000 μM) for 24 hours, and then treated with 5 μM rotenone for 24 hours. In order to evaluate the viability of the treated cells, 10 μL of 5 mg / mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added to the cells and incubated in an incubator. After incubation for 2 hours, 110 μL of 10% sodium dodecyl sulfate (SDS) in 0.01N hydrochloric acid was added to the wells and incubated overnight to dissolve the formed formazan. crystals, and then measure the absorbance at 570 nm using a microplate reader.

[0117] Sample activity detection:

[0118] 200,000 SH-SY5Y cells were dispersed in 12-well tissue culture plates. Before treating the cells, the cells were pretreated with different concentrations of native protein samples (final concentrations of 100 μM and 1000 μM) for 24 hours, and then treated with 1.25, 2.5 or 5 μM rotenone for 24 hours. The enzyme cycling method described by Rahman et al. (https: / / pubmed.ncbi.nlm.nih.gov / 17406579 / ) was used to measure the intracellular reduced / oxidized glutathione (GSH) concentration. Briefly, the cells were washed with ice-cold PBS and incubated with 100 μL of 3% sulfosalicyclic acid at 4°C for 10 minutes. 30 μL of cell lysate (SC-3812, Santa Cruz Biotechnology) was reacted with 180 μL of enzyme reaction mixture. The enzyme reaction mixture contained 0.630 mM 5,5'-dinitrobenzoic acid (DTNB), 530 μM β-nicotinamide adenine dinucleotide phosphate (β-NADPH), and 525 mU / mL glutathione reductase (GR) in phosphate buffer (100 mM Na₂HPO₄ / NaH₂PO₄, 50 mM Na₂EDTA) preheated to 37°C. The reaction was immediately monitored by absorbance at 415 nm, recorded every minute for 10 times. To measure oxidized glutathione concentration, 50 μL of cell lysate was mixed with 5 μL of 20% 2-vinylpyridine and 60% triethanolamine and allowed to react at room temperature for 10 minutes. The reaction was then carried out as described above. A standard curve of reduced and oxidized glutathione was prepared using a serial dilution of reduced glutathione and oxidized glutathione. Protein concentration of the cell lysate was measured using a Bradford Protein Assay Kit (23200, Thermo Fisher Scientific) according to the manufacturer's instructions.

[0119] Experimental results

[0120] The results are as follows Figure 1 As shown in Figure 2, compared with the positive control (Rotenone), the effects of various concentrations of the native protein on the survival rate in SH-SY5Y cells were not significant, indicating that the native protein at a concentration of 0 to 1000 μM did not have significant toxicity in the cells ( Figure 1 Compared with the blank control (PBS), the addition of a certain concentration (100-1000 ng / mL) of natural protein samples can induce GSH activity in cells ( Figure 1 Middle B) shows that the natural protein sample can be expressed in cells to inhibit Parkinson's disease activity by inducing GSH intermediates.

[0121] Example 3 Natural protein samples inhibit ROS formation and enhance mitochondrial relative activity in mouse cerebral cortical astrocytes

[0122] Experimental Principle

[0123] ROS production and the relative activity of mitochondria are also closely related to Parkinson's disease. Therefore, detecting the relative activity of the two in natural protein samples can further determine the biological activity of the sample in inhibiting Parkinson's disease.

[0124] Experimental operation

[0125] Mouse primary cortical astrocytes (prepared as in Example 2) were dispersed in 96-well plates at a density of 10,000 cells per well at DIV14. The cells were pretreated with native protein samples or PBS blank controls for 24 hours before treatment, and then treated with 500 or 1000 μM 1-methyl-4-phenylpyridinium (MPP+) iodide for 24 hours. After treatment, cell viability was measured using PrestoBlue cell viability reagent (A13261, Thermo Fisher Scientific) according to the manufacturer's instructions. The cells were then washed with Hank's balanced salt solution (HBSS) preheated to 37°C and then incubated at 37°C for 30 minutes in HBSS containing 0.5 μg / mL Hoechst 33342, 25 μM H2DCF-DA, 250 nM TMRE, and 0.1 μM MitoTracker Deep Red FM. After washing twice, live images were captured using a Zeiss Celldiscoverer 7, and fluorescence readings were measured using a fluorescence microplate reader.

[0126] Experimental results

[0127] The results are as follows Figure 2 As shown, the natural protein sample can inhibit ROS formation and enhance the relative activity of mitochondria in mouse cortical astrocytes. In the positive control group (PBS), different concentrations (10-1000ng / mL) of protein samples had no effect on cell viability, indicating that the protein sample had no obvious toxicity in this cell population; MPP can affect oxidative phosphorylation in mitochondria, causing ATP depletion and cell death, and is therefore toxic ( Figure 2 In the MPP environment with different concentrations, the natural protein sample can inhibit the formation of ROS at a concentration of 10-1000 ng / mL ( Figure 2 In the presence of MPP, different concentrations of natural protein samples (10-1000 ng / mL) can induce mitochondrial activity in rat cortical astrocytes ( Figure 2Middle C).

[0128] Example 4: Natural protein samples inhibit tyrosine hydroxylase activity in mouse cerebral cortical astrocytes

[0129] Experimental Principle

[0130] Research has found that glial fibrillary acidic protein (GFAP), a marker of astrocyte activation, is associated with impaired cognitive function in Parkinson's disease patients. This study aims to investigate the effect of natural protein samples from the brains of mice on alleviating cognitive impairment in Parkinson's disease patients.

[0131] Experimental operation

[0132] All animal experiments and breeding procedures were conducted in accordance with animal protocols approved by the Animal Ethics Committee of the Hong Kong University of Science and Technology (HKUST). Animals were housed in the HKUST laboratory animal facility under a 12-h light / 12-h dark cycle, maintained at room temperature between 20 and 24°C, and with a relative humidity of 40% to 70%. Two to five animals were housed per cage and had free access to food. Natural protein was added to drinking water bags, and mice were given free access to food. Mice were orally administered with 25 μg / kg body weight of natural protein sample or PBS as a blank control daily. The amount of natural protein sample added to the drinking water bag was calculated based on the mice's daily water intake. After 8 days of protein sample treatment, mice were sacrificed, and brains were dissected and fixed overnight at 4°C in 4% paraformaldehyde solution, followed by treatment with 30% sucrose for 2 days. Brains were then embedded in OCT solution, and 25 μm coronal sections were cut. Brain sections were stained with antibodies against tyrosine hydroxylase or glial fibrillary acidic protein, followed by staining with fluorescently labeled secondary antibodies and DAPI. The stained brain sections were then observed using a Zeiss Axio Scan.Z1 microscope.

[0133] Experimental results

[0134] The results are as follows Figure 3 As shown, the marker glial fibrillary acidic protein (GFAP) is stained red. Compared to the blank control group, GFAP levels were significantly decreased in the groups treated with native protein. This demonstrates that the native protein sample (25 μg / kg) used in this experiment can effectively inhibit GFAP activity in the mouse brain, thereby demonstrating clinical activity in alleviating Parkinson's disease.

[0135] Example 5

[0136] A drug for treating Parkinson's syndrome comprises the natural protein prepared in Example 1, and the drug is in the form of an granule.

[0137] Example 6

[0138] A drug for treating Parkinson's syndrome comprises the natural protein prepared in Example 1, and the drug is in the form of granules.

[0139] Example 7

[0140] A drug for treating Parkinson's syndrome comprises the natural protein prepared in Example 1, and the drug is in the form of a capsule.

[0141] Example 8

[0142] A drug for treating Parkinson's syndrome, comprising the natural protein prepared in Example 1, wherein the drug is in the form of a tablet.

[0143] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0144] The above-described embodiments merely illustrate several implementations of the present invention, facilitating a specific and detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the scope of the present invention, all of which fall within the scope of protection of the present invention.

[0145] It should be understood that any technical solution obtained by a person skilled in the art through logical analysis, reasoning, or limited experimentation based on the technical solution provided by the present invention is within the scope of protection of the appended claims. Therefore, the scope of protection of the patent for this invention shall be based on the content of the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. A protein molecule comprising AB, wherein: A includes A chain polypeptide or a functional variant thereof; B includes a B chain polypeptide or a functional variant thereof; A and B are disulfide bridged; The amino acid sequence of the A chain polypeptide is the polypeptide shown in SEQ ID NO: 1; The amino acid sequence of the B chain polypeptide is shown in SEQ ID NO:

2.

2. The protein molecule according to claim 1, characterized in that The ninth cysteine ​​residue in the A chain polypeptide is covalently bonded to the tenth cysteine ​​residue in the B chain polypeptide via a disulfide bond, and the twenty-second cysteine ​​residue in the A chain polypeptide is covalently bonded to the twenty-second cysteine ​​residue in the B chain polypeptide via a disulfide bond.

3. A biomaterial related to the protein molecule according to claim 1 or 2, wherein the biomaterial is any one of a1) to a12): a1) a nucleic acid molecule encoding the protein molecule according to claim 1 or 2; a2) an expression cassette containing the nucleic acid molecule described in a1); a3) a recombinant vector containing the nucleic acid molecule described in a1); a4) a recombinant vector containing the expression cassette described in a2); a5) a recombinant microorganism containing the nucleic acid molecule described in a1); a6) a recombinant microorganism containing the expression cassette described in a2); a7) a recombinant microorganism containing the recombinant vector described in a3); a8) a recombinant microorganism containing the recombinant vector described in a4); a9) a transgenic animal cell line containing the nucleic acid molecule described in a1); a10) a transgenic animal cell line containing the expression cassette described in a2); a11) a transgenic animal cell line containing the recombinant vector described in a3); a12) a transgenic animal cell line containing the recombinant vector described in a4); The transgenic animal cell line contains no reproductive material.

4. A pharmaceutical composition comprising the protein molecule according to claim 1 or 2 or the biomaterial according to claim 3.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable excipient; Preferably, the pharmaceutically acceptable excipients include at least one of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a color and flavor regulator, a solvent, a solubilizer, a cosolvent, an emulsifier, an absorption promoter, a surfactant, an antioxidant, an excipient, a stabilizer, a flavoring agent, a sustained-release agent, a metal complexing agent, an inert gas, a preservative, a filler, a local analgesic, a pH regulator, and an isotonic or isotonic regulator.

6. Use of the protein molecule according to any one of claims 1 or 2, the biomaterial according to claim 3, or the pharmaceutical composition according to claim 4 or 5 in any one of b1) to b4): b1) Preparation of drugs for the treatment and / or prevention of Parkinson's disease; b2) Antioxidant; b3) preparing antioxidant products; b4) Preparation of products for the treatment and / or prevention of inflammation.

7. The use according to claim 6, characterized in that: The parkinsonism syndrome includes at least one of Parkinson's disease, progressive supranuclear palsy, multiple system atrophy, corticobasal degeneration, diffuse Lewy body disease, parkinsonism-dementia, X-linked dystonia-parkinsonism and secondary parkinsonism syndrome.

8. The use according to claim 6, characterized in that: The anti-oxidation includes inhibiting the formation of reactive oxygen species and / or enhancing the relative activity of cell mitochondria; and / or, the anti-inflammatory includes inhibiting the expression of glial fibrillary acid protein and / or inhibiting the expression of tyrosine hydroxylase.

9. A product comprising the protein molecule of claim 1 or 2, the biomaterial of claim 3 or the pharmaceutical composition of claim 4 or 5; Preferably, the product has at least one of the functions c1) to c5): c1) Antioxidant; c2) inhibiting the formation of reactive oxygen species; c3) Enhance the relative activity of cell mitochondria; c4) inhibiting the expression of glial fibrillary acid protein; c5) Enhance the activity of reduced GSH.

10. A method comprising the step of treating with the protein molecule of claim 1 or 2, the biomaterial of claim 3, the pharmaceutical composition of claim 4 or 5, or the product of claim 9; The method is at least one of d1) to d3): d1) A method for improving the antioxidant effect of cells: d2) a method for inhibiting cellular ROS formation; d3) A method for enhancing the relative activity of mitochondria.

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