Human mesenchymal stem cell-derived mitochondrial active peptide library as well as construction method and application thereof

Abstract

The invention relates to a human mesenchymal stem cell-derived mitochondrial active peptide library as well as a construction method and application thereof. Specifically, the invention provides a mitochondrial active peptide library, and active peptides can be utilized in vivo through gastrointestinal digestion and absorption, liver metabolism and blood circulation, have obvious curative effects on cartilage regeneration and the like, and provide candidate drugs for regenerative medicine.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and more specifically, to a mitochondrial bioactive peptide library derived from human mesenchymal stem cells, its construction method, and its application. Background Technology

[0002] Human mesenchymal stem cells (MSCs) possess strong self-renewal capacity, multi-lineage differentiation potential, and paracrine function, making them an important biological resource in the field of regenerative medicine. Mitochondrial proteins play a crucial regulatory role in the application of MSCs to tissue regeneration. Meanwhile, bioactive peptides, due to their broad indications, high safety profile, and significant therapeutic effects, have become a popular target for drug development. Numerous studies have shown that mitochondrial bioactive peptides have significant effects in anti-aging and anti-inflammatory aspects, but their poor adherence is due to their inability to be taken orally.

[0003] Therefore, there is an urgent need in this field to develop mitochondrial bioactive peptides suitable for oral administration, or bioactive peptides that can be digested and absorbed in the gastrointestinal tract, metabolized in the liver, and utilized in the bloodstream, and have significant therapeutic effects on cartilage regeneration, thus providing candidate drugs for regenerative medicine. Summary of the Invention

[0004] The purpose of this invention is to provide an active peptide that can be digested and absorbed by the gastrointestinal tract, metabolized by the liver, and utilized in the bloodstream. It has significant therapeutic effects on cartilage regeneration and other aspects, and provides a candidate drug for regenerative medicine.

[0005] In a first aspect of the invention, there is provided the use of a pharmaceutical composition in the preparation of a fusion protein, a targeted drug, or a targeted delivery system that targets and expresses cGAS protein, said pharmaceutical composition comprising mitochondrial active peptides selected from the group consisting of:

[0006] (1) Mitochondrial polypeptide 1, the amino acid sequence of which is shown in SEQ ID NO.1: TEIIILATR;

[0007] (2) Mitochondrial polypeptide 2, the amino acid sequence of which is shown in SEQ ID NO.2: LIFAGK;

[0008] (3) Mitochondrial polypeptide 3, the amino acid sequence of which is shown in SEQ ID NO.3: LHLVLR;

[0009] (4) Mitochondrial polypeptide 4, the amino acid sequence of which is shown in SEQ ID NO.4: STLHLVLR;

[0010] (5) Mitochondrial polypeptide 5, the amino acid sequence of which is shown in SEQ ID NO.5: TLHLVLR;

[0011] (6) Mitochondrial polypeptide 6, the amino acid sequence of which is shown in SEQ ID NO.6: TLHLVLRLRGGAKKRKKKS;

[0012] (7) Mitochondrial polypeptide 7, the amino acid sequence of which is shown in SEQ ID NO.7: VAAMLLLLSA; or

[0013] (8) An amino acid sequence in which one or more amino acids are deleted, added and / or substituted in any of (1)-(7) but its mitochondrial active peptide function remains unchanged.

[0014] In another preferred embodiment, the mitochondrial active peptide is a mitochondrial active peptide derived from human mesenchymal stem cells.

[0015] In another preferred embodiment, the mitochondrial active peptide is a cGAS-targeting peptide.

[0016] In another preferred embodiment, the interaction force score between the mitochondrial active peptide and the cGAS protein molecule is less than -7, more preferably less than -9, and even more preferably less than -11.

[0017] In another preferred embodiment, the pharmaceutical composition is administered orally.

[0018] In another preferred embodiment, the fusion protein, targeted drug, or targeted delivery system targets cells expressing mitochondrial proteins.

[0019] In another preferred embodiment, the cells expressing mitochondrial proteins include tumor cells and immune cells.

[0020] In another preferred embodiment, the fusion protein, targeted drug, or targeted delivery system targets cells expressing the cGAS protein.

[0021] In another preferred embodiment, the cells expressing cGAS protein include inflammatory cells, chondrocytes, tumor cells, and immune cells.

[0022] In another preferred embodiment, the targeted drug is a drug that inhibits cGAS expression and / or a drug for treating osteoarthritis.

[0023] In a second aspect of the present invention, a method for preparing mitochondrial bioactive peptides is provided, the method comprising the following steps:

[0024] (i) Extracting mitochondrial proteins from human mesenchymal stem cells,

[0025] (ii) The mitochondrial proteins are digested in vivo and in vitro via the gastrointestinal tract to obtain mitochondrial protein digests;

[0026] (iii) By simulating small intestinal absorption and liver metabolism in vitro and in vivo, mitochondrial bioactive peptides selected from the following groups were obtained:

[0027] (1) Mitochondrial polypeptide 1, the amino acid sequence of which is shown in SEQ ID NO.1: TEIIILATR;

[0028] (2) Mitochondrial polypeptide 2, the amino acid sequence of which is shown in SEQ ID NO.2: LIFAGK;

[0029] (3) Mitochondrial polypeptide 3, the amino acid sequence of which is shown in SEQ ID NO.3: LHLVLR;

[0030] (4) Mitochondrial polypeptide 4, the amino acid sequence of which is shown in SEQ ID NO.4: STLHLVLR;

[0031] (5) Mitochondrial polypeptide 5, the amino acid sequence of which is shown in SEQ ID NO.5: TLHLVLR;

[0032] (6) Mitochondrial polypeptide 6, the amino acid sequence of which is shown in SEQ ID NO.6: TLHLVLRLRGGAKKRKKKS;

[0033] (7) Mitochondrial polypeptide 7, the amino acid sequence of which is shown in SEQ ID NO.7: VAAMLLLLSA; or

[0034] (8) An amino acid sequence in which one or more amino acids are deleted, added and / or substituted in any of (1)-(7) but its mitochondrial active peptide function remains unchanged.

[0035] In another preferred embodiment, the screening includes the following steps:

[0036] Peptide sequences coexisting with components that are easily absorbed by small intestinal epithelial cells and metabolized by liver cells and components that enter the bloodstream were screened. cGAS was used as the target for screening, and then mitochondrial active peptides targeting cGAS were synthesized by solid-phase synthesis technology.

[0037] In a third aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0038] (a) The first active ingredient, which includes active peptides selected from the group consisting of:

[0039] (1) Mitochondrial polypeptide 1, the amino acid sequence of which is shown in SEQ ID NO.1: TEIIILATR;

[0040] (2) Mitochondrial polypeptide 2, the amino acid sequence of which is shown in SEQ ID NO.2: LIFAGK;

[0041] (3) Mitochondrial polypeptide 3, the amino acid sequence of which is shown in SEQ ID NO.3: LHLVLR;

[0042] (4) Mitochondrial polypeptide 4, the amino acid sequence of which is shown in SEQ ID NO.4: STLHLVLR;

[0043] (5) Mitochondrial polypeptide 5, the amino acid sequence of which is shown in SEQ ID NO.5: TLHLVLR;

[0044] (6) Mitochondrial polypeptide 6, the amino acid sequence of which is shown in SEQ ID NO.6: TLHLVLRLRGGAKKRKKKS;

[0045] (7) Mitochondrial polypeptide 7, the amino acid sequence of which is shown in SEQ ID NO.7: VAAMLLLLSA; or

[0046] (8) An amino acid sequence in which one or more amino acids are deleted, added and / or substituted in any of (1)-(7) but its mitochondrial active peptide function remains unchanged.

[0047] (b) The second active ingredient contains other drugs that inhibit cGAS expression.

[0048] In another preferred embodiment, the pharmaceutical composition is used to treat inflammation of chondrocytes or tissues.

[0049] In another preferred embodiment, the pharmaceutical composition is used to treat osteoarthritis.

[0050] In another preferred embodiment, the pharmaceutical composition is administered to animals selected from the group consisting of rodents or primates.

[0051] In another preferred embodiment, the subject is a patient with chondrocyte or tissue inflammation or osteoarthritis.

[0052] In another preferred embodiment, the first active ingredient accounts for 1-99 wt% of the total weight of the pharmaceutical composition; more preferably, 10-90 wt%; and even more preferably, 30-70 wt%.

[0053] In another preferred embodiment, the pharmaceutical composition may be a single compound or a mixture of multiple compounds.

[0054] In another preferred embodiment, the pharmaceutical composition is in the form of an oral or non-oral dosage form.

[0055] In another preferred embodiment, the oral dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.

[0056] In another preferred embodiment, the non-oral dosage form is an injection or syringe.

[0057] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injections, inhalers, tinctures, powders, granules, capsules, oral liquids, tablets, pills, suspensions, emulsions, lozenges, or drops.

[0058] In a fourth aspect of the invention, a medicine box is provided, the medicine box comprising:

[0059] (I) A first formulation located within a first container, the first formulation comprising (a) a mitochondrial active peptide or a pharmaceutically acceptable salt thereof as a first active ingredient, and (b) a pharmaceutically acceptable carrier; wherein the mitochondrial active peptide is selected from the group consisting of:

[0060] (1) Mitochondrial polypeptide 1, the amino acid sequence of which is shown in SEQ ID NO.1: TEIIILATR;

[0061] (2) Mitochondrial polypeptide 2, the amino acid sequence of which is shown in SEQ ID NO.2: LIFAGK;

[0062] (3) Mitochondrial polypeptide 3, the amino acid sequence of which is shown in SEQ ID NO.3: LHLVLR;

[0063] (4) Mitochondrial polypeptide 4, the amino acid sequence of which is shown in SEQ ID NO.4: STLHLVLR;

[0064] (5) Mitochondrial polypeptide 5, the amino acid sequence of which is shown in SEQ ID NO.5: TLHLVLR;

[0065] (6) Mitochondrial polypeptide 6, the amino acid sequence of which is shown in SEQ ID NO.6: TLHLVLRLRGGAKKRKKKS;

[0066] (7) Mitochondrial polypeptide 7, the amino acid sequence of which is shown in SEQ ID NO.7: VAAMLLLLSA; or

[0067] (8) An amino acid sequence in which one or more amino acids are deleted, added and / or substituted in any of (1)-(7), but whose mitochondrial active peptide function remains unchanged.

[0068] (II) An optional second formulation located within a second container, the second formulation comprising (c) a cGAS inhibitor as a second active ingredient; and

[0069] (III) Instructions for use, which state that the medicine box is used for the treatment of cartilage damage or osteoarthritis-related diseases.

[0070] In another preferred embodiment, the cGAS inhibitor comprises: a small molecule compound or a combination thereof for inhibiting Stat3 expression.

[0071] In another preferred embodiment, the first and second formulations are independent of each other.

[0072] In another preferred embodiment, the first formulation is a lyophilized formulation or a liquid formulation.

[0073] In another preferred embodiment, the kit further includes (III) an optional third preparation located in a third container, the third preparation containing other treatments for cartilage damage or osteoarthritis.

[0074] In a fifth aspect of the invention, the use of the pharmaceutical composition according to the third aspect of the invention in the preparation of a targeted medicament for treating tumors, including solid tumors and hematologic malignancies, is provided.

[0075] In a sixth aspect of the invention, the use of the pharmaceutical composition according to the third aspect of the invention in the preparation of a medicament for inhibiting cGAS expression is provided.

[0076] In a seventh aspect of the invention, the use of the pharmaceutical composition according to a third aspect of the invention in the preparation of a medicament selected from the group consisting of:

[0077] (1) Drugs used to treat inflammation of chondrocytes or tissues.

[0078] (2) Drugs used to treat osteoarthritis.

[0079] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0080] Compared with the prior art, the present invention has the following technical effects:

[0081] (1) This invention extracts mitochondrial proteins from human mesenchymal stem cells to obtain seven bioactive peptides that can be utilized in vivo through gastrointestinal digestion and absorption, liver metabolism, and blood circulation, based on an in vivo and in vitro digestion-absorption-metabolism model.

[0082] (2) In this invention, the cyclized GMP-AMP synthase cGAS is used as the target protein, and the active peptide TLHLVLRLRGGAKKRKKKS (Thr-Leu-His-Leu-Val-Leu-Arg-Leu-Arg-Gly-Gly-Ala-Lys-Lys-Arg-Lys-Lys-Ser) with the best interaction force is screened by molecular docking.

[0083] (3) This invention provides a mitochondrial active peptide that has significant therapeutic effects on cartilage regeneration and other aspects, providing a candidate drug for regenerative medicine. Attached Figure Description

[0084] Figure 1 The figures shown represent the characterization results of mitochondrial proteins derived from human mesenchymal stem cells in embodiments of the present invention. Specifically, 1 part represents the stock solution of mitochondrial proteins derived from human mesenchymal stem cells in 12 15 cm culture dishes with a concentration of 80-90%; 1 / 2 part represents half the concentration of the stock solution of mitochondrial proteins derived from human mesenchymal stem cells in 12 15 cm culture dishes; and 1 / 4 part represents one-quarter the concentration of the stock solution of mitochondrial proteins derived from human mesenchymal stem cells in 12 15 cm culture dishes.

[0085] Figure 2 The figure shown represents the characterization results of mitochondrial proteins and their in vitro and in vivo digestive metabolites derived from human mesenchymal stem cells in this embodiment of the invention. MP represents mitochondrial proteins; AP represents poorly absorbed components; BL represents easily absorbed but poorly metabolized components; blood, small intestine, and large intestine of 10 adult C57BL / 6j mice were collected 4 hours after gavage administration of mitochondrial proteins; BX represents components entering the bloodstream; DX represents large intestine metabolic components; and XX represents small intestine metabolic components.

[0086] Figure 3 The display shows the total ion flow of mitochondrial proteins derived from human mesenchymal stem cells.

[0087] Figure 4 The display shows the total ion flux of mitochondrial protein digestion metabolites derived from human mesenchymal stem cells. In the cell co-culture model of Caco-2 / HepG2 small intestinal absorption and liver metabolism based on Transwell plates, ym-ap represents poorly absorbed components (red), and ym-bl represents components that are easily absorbed but poorly metabolized (green). Four hours after mitochondrial protein was administered to 10 adult C57BL / 6j mice via gavage, blood, small intestine, and large intestine were collected. ym-bx represents components entering the bloodstream (blue), ym-dx represents components remaining in the large intestine (yellow-green), and ym-xx represents components remaining in the small intestine (black).

[0088] Figure 5 The diagram shown is a schematic representation of the structure of 1-TEIIILATR (Thr-Glu-Ile-Ile-Ile-Leu-Ala-Thr-Arg), a mitochondrial polypeptide derived from human mesenchymal stem cells that can be utilized in vivo according to an embodiment of the present invention.

[0089] Figure 6 The diagram shown is a schematic representation of the structure of 2-LIFAGK (Leu-Ile-Phe-Ala-Gly-Lys), a mitochondrial polypeptide derived from human mesenchymal stem cells that can be utilized in vivo according to an embodiment of the present invention.

[0090] Figure 7 The diagram shown is a schematic representation of the structure of the human mesenchymal stem cell-derived mitochondrial polypeptide 3-LHLVLR (Leu-His-Leu-Val-Leu-Arg), which can be utilized in vivo in an embodiment of the present invention.

[0091] Figure 8 The diagram shown is a schematic representation of the structure of the human mesenchymal stem cell-derived mitochondrial polypeptide 4-STLHLVLR (Ser-Thr-Leu-His-Leu-Val-Leu-Arg), which can be utilized in vivo in an embodiment of the present invention.

[0092] Figure 9 The diagram shown is a schematic representation of the structure of 5-TLHLVLR (Thr-Leu-His-Leu-Val-Leu-Arg), a mitochondrial polypeptide derived from human mesenchymal stem cells that can be utilized in vivo in this embodiment of the invention.

[0093] Figure 10 The diagram shows the structure of the human mesenchymal stem cell-derived mitochondrial polypeptide 6-TLHLVLRLRGGAKKRKKKS (Thr-Leu-His-Leu-Val-Leu-Arg-Leu-Arg-Gly-Gly-Ala-Lys-Lys-Arg-Lys-Lys-Ser), which can be utilized in vivo in this embodiment of the invention.

[0094] Figure 11 The diagram shown is a schematic representation of the structure of 7-VAAMLLLLSA (Val-Ala-Ala-Met-Leu-Leu-Leu-Leu-Ser-Ala), a mitochondrial polypeptide derived from human mesenchymal stem cells that can be utilized in vivo in this embodiment of the invention.

[0095] Figure 12 The figure shown is a diagram of the molecular docking results between the mitochondrial polypeptide 6-TLHLVLRLRGGAKKRKKKS (abbreviated as MP) and the cGAS protein in an embodiment of the present invention.

[0096] Figure 13 The figure shown is a graph illustrating the protein inhibition effect of mitochondrial polypeptide 6-TLHLVLRLRGGAKKRKKKS (MP) on cGAS in an embodiment of the present invention.

[0097] Figure 14The image shown is an illustration of the results of mitochondrial polypeptide 6-TLHLVLRLRGGAKKRKKKS (MP) staining of chondrocytes with Alixin Blue in an embodiment of the present invention.

[0098] Figure 15 The figure shown is a graph illustrating the efficacy evaluation results of mitochondrial polypeptide 6-TLHLVLRLRGGAKKRKKKS (MP) on cartilage function in the embodiments of the present invention. Detailed Implementation

[0099] To make the objectives, technical solutions, beneficial effects, and significant advancements of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, all the described embodiments are only some embodiments of the present invention, and not all embodiments; based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0100] the term

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0102] As used herein, “including” or “containing” includes “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0103] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents involved in this invention are commercially available.

[0104] Example 1: Screening and Identification of Bioactive Peptides

[0105] This embodiment involves the screening and identification of bioactive peptides. The specific experimental procedure is as follows:

[0106] 1. Human mesenchymal stem cell culture:

[0107] Human mesenchymal stem cells were cultured in 15 cm culture dishes using serum-free mesenchymal stem cell culture medium (Yokang, catalog number: NC0103+NC0103.S) until the culture dish reached 80%–90% (1 x 10⁻⁶) of the dish surface area.7 ~2x10 7 (12 15cm culture dishes were cultured).

[0108] 2. Mitochondrial isolation:

[0109] Mitochondria were extracted from 12 human mesenchymal stem cells in culture dishes using the Thermo Scientific Mitochondria Isolation Kit (Thermo Scientific, catalog number: 89874).

[0110] 3. Mitochondrial protein extraction:

[0111] Mitochondria from human mesenchymal stem cells were diluted with ultrapure water at 4°C (mitochondrial volume: ultrapure water = 1:9). The mitochondria were then sonicated using an ultrasonic cell disruptor (40 kHz, 10 minutes, 5 seconds on / 5 seconds off). The mixture was then stirred at 4°C with a magnetic stirrer for 1 hour. After centrifugation at 14000×g for 25 minutes, the supernatant was collected to obtain the mitochondrial protein solution. The solution was then freeze-dried at -80°C for 1 hour or -20°C overnight for 3 days to obtain mitochondrial protein powder.

[0112] Take an appropriate amount of mitochondrial protein powder, dilute it with ultrapure water to 4 mg / mL, add loading buffer, perform Coomassie brilliant blue staining after electrophoresis, and characterize the mitochondrial proteins.

[0113] Coomassie Brilliant Blue electrophoresis results showed that mitochondrial proteins in human mesenchymal stem cells were enriched and widely distributed. Figure 1 ).

[0114] 4. In vitro screening of bioactive peptides: After 2 hours of in vitro gastrointestinal digestion (using an experimental automated gastrointestinal digestion device, patent number: ZL 2020 2 2054404.2), enzyme inactivation was performed, followed by freeze-drying to obtain mitochondrial protein digest. Then, in vitro simulation of small intestinal absorption and liver metabolism was conducted (using a Caco-2 / HepG2 cell co-culture model based on Transwell plates; Caco-2 cells simulated human small intestinal epithelial cells, cultured for 21 days in the apical side (AP) of the Transwell plate; HepG2 cells simulated human liver cells, cultured for 2 days in the basolateral (BL) of the Transwell plate). The AP and BL sides were freeze-dried separately; the AP side contained mitochondrial protein digest, and the BL side contained metabolites of transported and absorbed mitochondrial proteins. In vivo screening of bioactive peptides: Ten 4-6 month old C57BL / 6J mice were administered mitochondrial protein at a concentration of 1 mg / mL via gavage (0.5 mL per mouse). Four hours later, blood, small intestine, and large intestine samples were collected, specifically the components entering the bloodstream (BX), large intestine metabolic components (DX), and small intestine metabolic components (XX), which were then freeze-dried. Appropriate amounts of each component were diluted with ultrapure water to 4 mg / mL, loading buffer was added, and electrophoresis was performed followed by Coomassie brilliant blue staining for characterization of each component. Figure 2 ).

[0115] 5. Identification of mitochondrial proteins and their bioactive peptides: Appropriate amounts of each component from steps 3 and 4 above were subjected to liquid chromatography-mass spectrometry (LC-MS / MS, (1) Q Exactive mass spectrometer (Thermo Scientific); (2) Dionex Ultimate 3000 RSLCnano liquid chromatography (Thermo Scientific)). The results are shown below:

[0116] Figure 3 Total ion flux of mitochondrial proteins derived from human mesenchymal stem cells.

[0117] Figure 4 The total ion flux of mitochondrial protein digestion metabolites derived from human mesenchymal stem cells (based on a Transwell plate-based small intestine absorption-liver metabolism cell co-culture model of Caco-2 / HepG2, ym-ap represents components that are not easily absorbed (red), ym-bl represents components that are easily absorbed but not easily metabolized (green); blood, small intestine and large intestine of 10 adult C57BL / 6j mice were collected 4 hours after mitochondrial protein was administered by gavage, ym-bx represents components that entered the blood circulation (blue), ym-dx represents components remaining in the large intestine (yellow-green), ym-xx represents components remaining in the small intestine (black)).

[0118] The raw mass spectrometry file was processed and converted using MM File Conversion software to obtain an MGF format file. Then, the human mesenchymal stem cell database in uniprot was searched using MASCOT (http: / / www.matrixscience.com / ), and peptide sequences coexisting with both the component easily absorbed by small intestinal epithelial cells and metabolized by liver cells (BL) and the component entering the bloodstream (BX) were identified from the search results. These peptide sequences are as follows:

[0119] 1. Mitochondrial polypeptide 1-TEIIILATR (Thr-Glu-Ile-Ile-Ile-Leu-Ala-Thr-Arg, SEQ ID NO: 1) Figure 5 ), molecular weight 1029.24 Da;

[0120] 2. Mitochondrial peptide 2-LIFAGK (Leu-Ile-Phe-Ala-Gly-Lys, SEQ ID NO: 2) Figure 6 ), molecular weight 647.82 Da;

[0121] 3. Mitochondrial polypeptide 3-LHLVLR (Leu-His-Leu-Val-Leu-Arg, SEQ ID NO: 3) Figure 7 ), 749.95 Da;

[0122] 4. Mitochondrial polypeptide 4-STLHLVLR (Ser-Thr-Leu-His-Leu-Val-Leu-Arg, SEQ ID NO: 4) Figure 8 ), molecular weight 938.14 Da;

[0123] 5. Mitochondrial polypeptide 5-TLHLVLR (Thr-Leu-His-Leu-Val-Leu-Arg, SEQ ID NO: 5) Figure 9 ), molecular weight 851.06 Da;

[0124] 6. Mitochondrial polypeptide 6-TLHLVLRLRGGAKKRKKKS (Thr-Leu-His-Leu-Val-Leu-Arg-Leu-Arg-Gly-Gly-Ala-Lys-Lys-Arg-Lys-Lys-Lys-Ser, SEQ ID NO: 6) Figure 10 ), molecular weight 2189.73 Da;

[0125] 7. Mitochondrial polypeptide 7-VAAMLLLLSA (Val-Ala-Ala-Met-Leu-Leu-Leu-Leu-Ser-Ala, SEQ ID NO: 7) Figure 11 ), molecular weight 1001.29 Da.

[0126] This invention can effectively screen and identify all bioactive peptides from mitochondrial proteins derived from human mesenchymal stem cells that can be utilized in vivo, thereby constructing a peptide library. Then, based on target proteins of related diseases, molecular docking is performed to screen for peptides with optimal interactions from the peptide library, which are then synthesized using solid-phase synthesis technology for functional verification.

[0127] Example 2: Study on the inhibitory effect of mitochondrial active peptides on cGAS

[0128] Cyclic GMP-AMP synthase cGAS plays a key role in the pathogenesis of osteoarthritis by participating in chondrocyte or tissue inflammatory responses and degradation. If peptide sequences that inhibit cGAS can be screened from the mitochondrial bioactive peptide library, it will provide a new potential strategy for the treatment of osteoarthritis.

[0129] 1. The three-dimensional structures of bioactive peptides were plotted using Molecular Docking (Discovery Studio was used to create the 3D structures). Then, the Schrödinger LigPrep module was used to generate single, low-energy, chiral 3D structures, simultaneously producing molecules with various ionization states, topological isomers, stereochemical configurations, and cyclic conformations. The cGAS protein structure was downloaded from the uniprot database (PDBID: 6lrk). It was then visualized using PyMOL, followed by dehydration, hydrogenation, charge calculation, and nonpolar hydrogen synthesis using Mgtools 1.5.6. The ligand and receptor were then saved as PDBQT files. Autodock Vina 1.1.2 was used to dock mitochondrial bioactive peptides derived from human mesenchymal stem cells with the cGAS protein. The intermolecular interaction forces were simulated using computer simulations; a value below -7 indicated a strong interaction, with lower values ​​indicating stronger interactions. The binding energy scores of each bioactive peptide with cGAS are shown in Table 1 below.

[0130] Table 1

[0131]

[0132] The results showed that mitochondrial active peptides 1-7, as determined by binding energy scores, all exhibited strong interactions with cGAS (scores below -7); among them, active peptide 6-TLHLVLRLRGGAKKRKKKS showed the strongest interaction with cGAS (score -11.980).

[0133] 2. 100 mg of mitochondrial active peptide 6-TLHLVLRLRGGAKKRKKKS was synthesized using solid-phase synthesis technology.

[0134] 3. Incubate mouse primary chondrocytes to 80%–90% of the area of ​​a 6-well cell culture plate (approximately 1 x 10⁻⁶ cells per well). 6 ~2x10 6 After [number] cells were collected, they were divided into 5 groups: the Ctrl group (healthy primary chondrocytes); the IL-1β group (incubated with 10 ng / mL IL-1β for 2 hours to construct a primary chondrocyte inflammatory injury model); the 0.1 mM MP group (incubated with 10 ng / mL IL-1β and 0.1 mM active peptide 6-TLHLVLRLRGGAKKRKKKS for 2 hours); the 0.5 mM MP group (incubated with 10 ng / mL IL-1β and 0.5 mM active peptide 6-TLHLVLRLRGGAKKRKKKS for 2 hours); and the 1 mM MP group (incubated with 10 ng / mL IL-1β and 1 mM active peptide 6-TLHLVLRLRGGAKKRKKKS for 2 hours). Western blotting of cGAS protein was performed on the proteins from each group.

[0135] The results showed that the mitochondrial bioactive peptide 6-TLHLVLRLRGGAKKRKKKS significantly downregulated cGAS protein levels in a concentration-dependent manner. Figure 13 ).

[0136] Similarly, mitochondrial active peptides 1-5 and mitochondrial active peptide 7 can also significantly downregulate cGAS protein levels in a concentration-dependent manner.

[0137] 4. Primary mouse chondrocytes were seeded into 96-well cell culture plates, 1 x 10⁶ cells per well. 4 Cells were divided into 6 groups: Ctrl group (healthy primary chondrocytes); IL-1β group (10 ng / mL IL-1β); 0.1 mM MP group (10 ng / mL IL-1β and 0.1 mM mitochondrial active peptide TLHLVLRLRGGAKKRKKKS); 0.2 mM MP group (10 ng / mL IL-1β and 0.2 mM active peptide TLHLVLRLRGGAKKRKKKS); 0.5 mM MP group (10 ng / mL IL-1β and 0.5 mM mitochondrial active peptide TLHLVLRLRGGAKKRKKKS); and 1 mM MP group (10 ng / mL IL-1β and 1 mM mitochondrial active peptide TLHLVLRLRGGAKKRKKKS). After incubation for 3 days, the cells were fixed with 4% paraformaldehyde and stained with alexandrite blue.

[0138] The results showed that as the concentration of mitochondrial active peptides increased, the expression level of glycosaminoglycans in chondrocytes significantly increased, suggesting that mitochondrial active peptides promote cartilage formation. Figure 14 ).

[0139] 5. Incubate mouse primary chondrocytes to 80%–90% of the area of ​​a 6-well cell culture plate (approximately 1 x 10⁻⁶ cells per well). 6 ~2x10 6 After [number] cells were collected, they were divided into 6 groups: Ctrl group (healthy primary chondrocytes); IL-1β group (incubated with 10 ng / mL IL-1β for 2 hours to construct a primary chondrocyte inflammatory injury model); 0.1 mM MP group (incubated with 10 ng / mL IL-1β and 0.1 mM active peptide 6-TLHLVLRLRGGAKKRKKKS for 2 hours); 0.2 mM MP group (incubated with 10 ng / mL IL-1β and 0.2 mM active peptide 6-TLHLVLRLRGGAKKRKKKS for 2 hours); 0.5 mM MP group (incubated with 10 ng / mL IL-1β and 0.5 mM active peptide 6-TLHLVLRLRGGAKKRKKKS for 2 hours); and 1 mM MP group (incubated with 10 ng / mL IL-1β and 1 mM active peptide 6-TLHLVLRLRGGAKKRKKKS for 2 hours). RNA was extracted from each component in a 6-well plate and reverse transcribed, then real-time quantitative PCR was performed to measure key indicators of cartilage function (ACAN, COL2A1, MMP13, and MMP3).

[0140] The results showed that the mitochondrial bioactive peptide 6-TLHLVLRLRGGAKKRKKKS upregulated the transcriptional levels of ACAN and COL2A1 and decreased the expression levels of MMP13 and MMP3. This suggests that the mitochondrial bioactive peptide 6-TLHLVLRLRGGAKKRKKKS significantly improved cartilage function in a concentration-dependent manner. Figure 15 This allows for the treatment of osteoarthritis.

[0141] Similarly, mitochondrial active peptides 1-5 and mitochondrial active peptide 7 can also upregulate the transcriptional levels of ACAN and COL2A1, reduce the expression levels of MMP13 and MMP3, improve cartilage function, and have a concentration-dependent effect, thereby treating osteoarthritis.

[0142] The preferred embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and the devices and structures not described in detail should be understood as being implemented in a conventional manner in the art. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the scope of the present invention. This does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the present invention's technical solutions still fall within the protection scope of the present invention.

Claims

1. The use of a pharmaceutical composition in the preparation of a fusion protein, a targeted drug, or a targeted delivery system for cells that target the expression of cGAS protein, characterized in that, The pharmaceutical composition comprises mitochondrial active peptides selected from the group consisting of: (1) Mitochondrial polypeptide 1, the amino acid sequence of which is shown in SEQ ID NO.1: TEIIILATR; (2) Mitochondrial polypeptide 2, the amino acid sequence of which is shown in SEQ ID NO.2: LIFAGK; (3) Mitochondrial polypeptide 3, the amino acid sequence of which is shown in SEQ ID NO.3: LHLVLR; (4) Mitochondrial polypeptide 4, the amino acid sequence of which is shown in SEQ ID NO.4: STLHLVLR; (5) Mitochondrial polypeptide 5, the amino acid sequence of which is shown in SEQ ID NO.5: TLHLVLR; (6) Mitochondrial polypeptide 6, the amino acid sequence of which is shown in SEQ ID NO.6: TLHLVLRLRGGAKKRKKKS; (7) Mitochondrial polypeptide 7, the amino acid sequence of which is shown in SEQ ID NO.7: VAAMLLLLSA; or (8) An amino acid sequence in which one or more amino acids are deleted, added and / or substituted in any of (1)-(7) but its mitochondrial active peptide function remains unchanged.

2. The application according to claim 1, characterized in that, The pharmaceutical composition is administered orally.

3. The application according to claim 1, characterized in that, The cells expressing cGAS protein include inflammatory cells, chondrocytes, tumor cells, and immune cells.

4. The application according to claim 1, characterized in that, The targeted drug is a drug that inhibits cGAS expression and / or a drug for treating osteoarthritis.

5. A method for preparing mitochondrial bioactive peptides, characterized in that, The method includes the following steps: (i) Extracting mitochondrial proteins from human mesenchymal stem cells, (ii) The mitochondrial proteins are digested in vitro via the gastrointestinal tract to obtain mitochondrial protein digests; (iii) Mitochondrial bioactive peptides selected from the following groups were obtained by simulating small intestinal absorption and liver metabolism in vitro: (1) Mitochondrial polypeptide 1, the amino acid sequence of which is shown in SEQ ID NO.1: TEIIILATR; (2) Mitochondrial polypeptide 2, the amino acid sequence of which is shown in SEQ ID NO.2: LIFAGK; (3) Mitochondrial polypeptide 3, the amino acid sequence of which is shown in SEQ ID NO.3: LHLVLR; (4) Mitochondrial polypeptide 4, the amino acid sequence of which is shown in SEQ ID NO.4: STLHLVLR; (5) Mitochondrial polypeptide 5, the amino acid sequence of which is shown in SEQ ID NO.5: TLHLVLR; (6) Mitochondrial polypeptide 6, the amino acid sequence of which is shown in SEQ ID NO.6: TLHLVLRLRGGAKKRKKKS; (7) Mitochondrial polypeptide 7, the amino acid sequence of which is shown in SEQ ID NO.7: VAAMLLLLSA; or (8) An amino acid sequence in which one or more amino acids are deleted, added and / or substituted in any of (1)-(7) but its mitochondrial active peptide function remains unchanged.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a) The first active ingredient, which includes active peptides selected from the group consisting of: (1) Mitochondrial polypeptide 1, the amino acid sequence of which is shown in SEQ ID NO.1: TEIIILATR; (2) Mitochondrial polypeptide 2, the amino acid sequence of which is shown in SEQ ID NO.2: LIFAGK; (3) Mitochondrial polypeptide 3, the amino acid sequence of which is shown in SEQ ID NO.3: LHLVLR; (4) Mitochondrial polypeptide 4, the amino acid sequence of which is shown in SEQ ID NO.4: STLHLVLR; (5) Mitochondrial polypeptide 5, the amino acid sequence of which is shown in SEQ ID NO.5: TLHLVLR; (6) Mitochondrial polypeptide 6, the amino acid sequence of which is shown in SEQ ID NO.6: TLHLVLRLRGGAKKRKKKS; (7) Mitochondrial polypeptide 7, the amino acid sequence of which is shown in SEQ ID NO.7: VAAMLLLLSA; or (8) An amino acid sequence in which one or more amino acids are deleted, added and / or substituted in any of (1)-(7) but its mitochondrial active peptide function remains unchanged. (b) The second active ingredient contains other drugs that inhibit cGAS expression.

7. A medicine box, characterized in that, The medicine box contains: (I) A first formulation located within a first container, the first formulation comprising (a) a mitochondrial active peptide or a pharmaceutically acceptable salt thereof as a first active ingredient, and (b) a pharmaceutically acceptable carrier; wherein the mitochondrial active peptide is selected from the group consisting of: (1) Mitochondrial polypeptide 1, the amino acid sequence of which is shown in SEQ ID NO.1: TEIIILATR; (2) Mitochondrial polypeptide 2, the amino acid sequence of which is shown in SEQ ID NO.2: LIFAGK; (3) Mitochondrial polypeptide 3, the amino acid sequence of which is shown in SEQ ID NO.3: LHLVLR; (4) Mitochondrial polypeptide 4, the amino acid sequence of which is shown in SEQ ID NO.4: STLHLVLR; (5) Mitochondrial polypeptide 5, the amino acid sequence of which is shown in SEQ ID NO.5: TLHLVLR; (6) Mitochondrial polypeptide 6, the amino acid sequence of which is shown in SEQ ID NO.6: TLHLVLRLRGGAKKRKKKS; (7) Mitochondrial polypeptide 7, the amino acid sequence of which is shown in SEQ ID NO.7: VAAMLLLLSA; or (8) An amino acid sequence in which one or more amino acids are deleted, added and / or substituted in any of (1)-(7), but whose mitochondrial active peptide function remains unchanged. (II) An optional second formulation located within a second container, the second formulation comprising (c) a cGAS inhibitor as a second active ingredient; and (III) Instructions for use, which state that the medicine box is used for the treatment of cartilage damage or osteoarthritis-related diseases.

8. The use of the pharmaceutical composition according to claim 6 in the preparation of a targeted drug for treating tumors, characterized in that, The tumors include solid tumors and hematologic tumors.

9. The use of the pharmaceutical composition according to claim 6 in the preparation of a medicament for inhibiting cGAS expression.

10. The use of the pharmaceutical composition according to claim 6 in the preparation of a medicament selected from the group consisting of: (1) Drugs used to treat inflammation of chondrocytes or tissues; (2) Drugs used to treat osteoarthritis.

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