A weight loss pharmaceutical composition containing mesenchymal stem cells and a preparation method and application thereof

Abstract

The application discloses a polypeptide composition for treating obesity and related metabolic diseases and application thereof. The polypeptide composition comprises polypeptide 1 and polypeptide 2, wherein the amino acid sequence of the polypeptide 1 is shown as SEQ ID NO. 1, can be specifically combined with the CD105 receptor on the surface of MSCs and the AdipoR1 receptor of adipocytes; the amino acid sequence of the polypeptide 2 is shown as SEQ ID NO. 2, can target adipose tissue and regulate glycolipid metabolism. The polypeptide composition is combined with fat-derived mesenchymal stem cells (MSCs) and applied to a high-fat-diet obese mouse model, and the results show that the composition has a significant synergistic effect in reducing body fat, improving the morphology of adipose tissue, regulating serum metabolic factors and inflammatory factors, and has no obvious organ toxicity. The application provides a new effective strategy and a safe and reliable pharmaceutical composition for treating obesity and related metabolic diseases.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a weight-loss drug composition containing mesenchymal stem cells, its preparation method, and its application. Background Technology

[0002] Obesity, a metabolic disease with a rising global incidence, has become a core risk factor for major chronic diseases such as diabetes, cardiovascular disease, and fatty liver. Current mainstream weight-loss drugs work by suppressing appetite, reducing fat absorption, or increasing energy expenditure, but they generally suffer from insufficient targeting, significant side effects (such as gastrointestinal reactions and cardiovascular toxicity), and high weight rebound rates after discontinuation. For example, long-term use of orlistat can lead to malabsorption of fat-soluble vitamins, and sibutramine has been banned in many countries due to increased cardiovascular risk. While novel GLP-1 receptor agonists are highly effective, they have limitations in injection convenience and gastrointestinal side effects, hindering the clinical application of traditional weight-loss strategies.

[0003] Mesenchymal stem cells (MSCs) exhibit unique advantages in obesity treatment due to their multi-lineage differentiation potential, immunomodulatory functions, and paracrine effects. Studies have shown that MSCs can improve the obesity-related inflammatory microenvironment by inhibiting adipocyte differentiation, promoting the browning of white adipose tissue, and regulating macrophage polarization. However, existing MSC-based weight loss strategies have two major drawbacks: first, MSCs have low homing efficiency in vivo and tend to remain in organs such as the lungs and liver after intravenous injection, making it difficult to accumulate in adipose tissue and exert a sustained effect; second, single MSC therapy has limited regulatory effects on lipid metabolism and cannot effectively block key pathogenic pathways in the obesity process (such as resistin-mediated insulin resistance). Although existing technologies attempt to improve the efficacy of MSCs through targeted delivery systems (such as liposomes and nanoparticles) or gene functionalization (such as overexpression of leptin), the former suffers from poor biocompatibility and complex preparation processes, while the latter faces safety risks and ethical controversies related to gene manipulation. In addition, although monoclonal antibodies against resistin can improve insulin sensitivity, their overall efficacy is limited because they do not bind to the homing properties of MSCs and therefore do not promote the catabolism of adipocytes.

[0004] To address the aforementioned technical bottlenecks, this invention innovatively designs a composite pharmaceutical composition containing MSCs. This composition achieves technological innovation at both the cellular localization and molecular signal regulation levels, significantly enhancing weight loss efficacy and reducing side effects. Summary of the Invention

[0005] The present invention aims to provide a polypeptide composition for the treatment of obesity and related metabolic diseases and its application. By rationally designing the polypeptide structure and combining it with MSCs, effective treatment of obesity and related metabolic diseases can be achieved.

[0006] (I) Composition of the polypeptide composition

[0007] The polypeptide composition of this invention comprises polypeptide 1 and polypeptide 2. The amino acid sequence of polypeptide 1 is shown in SEQ ID NO. 1, and it achieves specific binding to the CD105 receptor on the surface of MSCs and the AdipoR1 receptor on adipocytes through a unique structural design. Specifically, the primary structure design of polypeptide 1 includes: inserting the CD105 binding core sequence Pro-Arg-Pro (PRP) at amino acid positions 3-5 at the N-terminus as the CD105 targeting region; using the Trp-Tyr (WY) hydrophobic pair at amino acid positions 11-12 at the C-terminus as the AdipoR1 targeting region; and connecting the dual targeting motifs through a flexible amino acid (Glu-Asp-Val-Leu-Ile), while retaining the terminal Cys to form a disulfide bond. This structure enables peptide 1 to exhibit highly specific binding ability to CD105 and AdipoR1 in molecular docking. The cyclization structure significantly enhances its spatial conformation matching, and the binding energy is increased by more than 40% compared with the linear conformation. Moreover, the KD values ​​for both CD105 and AdipoR1 are in the nanomolar range (CD105: 2.3 nM, AdipoR1: 1.8 nM).

[0008] The amino acid sequence of peptide 2 is shown in SEQ ID NO.2. Its design aims to achieve a dual function of "targeting adipose tissue and regulating glucose and lipid metabolism." The N-terminal targeting region, based on structure-activity relationship analysis of the FABP4 inhibitor BMS309403, was optimized to obtain the CRGLYRKG sequence, enhancing its hydrophobic binding to FABP4. A flexible (GGGS) 3-linker was used in the linker region to ensure that the two functional domains function independently. Molecular simulations confirmed that the two functional domains of peptide 2 can form stable bindings with FABP4 and GLP-1R, respectively, with the linker region maintaining high flexibility and no spatial conflict. SPR experiments showed that peptide 2 has a KD of 78 nM for FABP4; cAMP experiments showed that its EC50 reaches 6.7 nM, exhibiting receptor agonist activity comparable to natural GLP-1, and effectively regulating glucose and lipid metabolism.

[0009] (II) Combined application of peptide compositions with MSCs

[0010] The above-mentioned polypeptide composition was combined with mouse adipose-derived mesenchymal stem cells (MSCs) for the treatment of obesity and related metabolic diseases. In animal experiments, a high-fat diet obesity model was established using 6-week-old SPF-grade C57BL / 6J male mice. The obese mice were randomly divided into groups and treated differently. The experimental group was injected with a combination of MSCs, polypeptide 1, and polypeptide 2 (CMP1 group). Results showed that the CMP1 group exhibited significant advantages in reducing body fat, improving adipose tissue morphology, and regulating serum metabolic and inflammatory factors. Compared with the control group, the CMP1 group showed a 36.3% reduction in body fat percentage and a significant decrease in the average diameter of adipocytes; serum TG, TC, and LDL-C levels were significantly reduced, while HDL-C levels were significantly increased; resistin and leptin levels were decreased, while adiponectin levels were increased, comprehensively improving insulin sensitivity and reducing the body's inflammatory response. Simultaneously, all liver and kidney function indicators in the CMP1 group were within the normal range, and HE pathological sections showed no abnormalities such as necrosis, fatty degeneration, or edema in liver, kidney, and cardiac cells, indicating that this combination therapy has good safety.

[0011] The polypeptide composition described in this invention and its combined application with mouse adipose-derived mesenchymal stem cells (MSCs) exhibit significant targeting, synergistic effects, safety, and broad application prospects. Polypeptide 1 and polypeptide 2 can specifically bind to relevant receptors or targets, enabling the composition to precisely target the desired effect. The combined application of polypeptides 1 and 2 with MSCs shows significant synergistic effects in reducing body fat, improving adipose tissue morphology, and regulating blood lipids and inflammatory metabolic factors. Animal experiments have confirmed that this combination therapy has no significant organ toxicity, with normal liver and kidney function indicators and no abnormal histopathological findings. This invention provides a novel and effective strategy and drug composition for obesity, type 2 diabetes, and other obesity-related metabolic diseases, with great potential for clinical translation. Detailed Implementation

[0012] 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. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0013] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0014] Example 1: Design and Study of Peptides

[0015] Experiment 1: Design, Synthesis and Functional Verification of Peptide 1

[0016] I. Experimental Objective

[0017] The specific binding ability of peptide 1 (specific amino acid sequence as shown in SEQ ID NO.1) to CD105 receptor on the surface of MSCs and AdipoR1 receptor on adipocytes through cyclization conformation was verified.

[0018] II. Structural Design and Conformation Optimization Experiment

[0019] 1. Primary Structural Design Steps

[0020] (1) Target motif screening, as detailed below:

[0021] CD105 targeting region: CD105 binds to the core sequence Pro-Arg-Pro (PRP) and inserts into the 3rd-5th amino acids at the N-terminus of the peptide.

[0022] AdipoR1 targeting region: Based on the structure of the AdipoR1 ligand binding domain, the Trp-Tyr(WY) hydrophobic pair is selected as the core, located at amino acid positions 11-12 at the C-terminus.

[0023] Linkage region design: A flexible amino acid (Glu-Asp-Val-Leu-Ile) is used to link the dual targeting motifs, while retaining the terminal Cys to form a disulfide bond.

[0024] (2) Sequence synthesis strategy: The Fmoc solid-phase synthesis method is used, and amino acids are sequentially linked from the N end to the C end. Cys is protected by the Trt group, and finally disulfide bonds are formed through oxidative cyclization.

[0025] 2. Three-dimensional conformational modeling and molecular docking

[0026] (1) Homologous modeling (Modeller 10.3):

[0027] CD105 template: Select PDB 5YWO (resolution) The missing regions of human CD105 extracellular segment were filled using Loop Modeling.

[0028] AdipoR1 template: Select PDB 3WXV (resolution) A model of the extracellular binding domain was constructed using the transmembrane region of mouse AdipoR1.

[0029] Initial peptide structure: Based on the disulfide bond precyclization conformation, a β-turn-linked bicyclic structure was generated using PyMOL.

[0030] (2) Molecular docking (AutoDockVina):

[0031] Receptor pretreatment: removal of ligands and water molecules, and addition of Kollman charge.

[0032] Polypeptide conformation: It can form two conformations: linear and cyclized (the cyclized conformation fixes the Cys1-Cys16 disulfide bonds).

[0033] Docking parameters: The search space was set as the CD105 active pocket (residues 300-500) and the AdipoR1 transmembrane hydrophobic pocket (residues 200-350), with a grid resolution of [missing information].

[0034] 3. The specific results are shown in Table 1. The cyclized polypeptide (i.e. polypeptide 1) specifically binds to CD105 and AdipoR1 through the Pro-Arg-Pro and Trp-Tyr motifs, respectively. The binding energy is increased by more than 40% compared with the linear conformation, which proves that disulfide bond cyclization significantly enhances the spatial conformation matching degree.

[0035] Table 1 Molecular docking data

[0036]

[0037] III. Experiments on the Synthesis and Purification of Polypeptide 1

[0038] 1. Solid-phase synthesis: The designed polypeptide 1 was sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis, and the purity (HPLC purity) was 98.7%.

[0039] 2. Structural verification data

[0040] MALDI-TOF-MS: Measured molecular weight 1789.1 Da (theoretical value 1789.3 Da), error <0.1%.

[0041] Disulfide bond verification: Ellman's reagent method showed that the free thiol group was <5%, proving that the cyclization was complete.

[0042] IV. Affinity Measurement Experiment

[0043] 1. Experimental Procedure

[0044] Chip fabrication: CM5 chip amino-coupled CD105 extracellular segment (1000RU) and AdipoR1 extracellular segment (800RU), EDC / NHS activated surface.

[0045] Sample analysis: Peptide 1 was diluted in a 1.56-100 nM gradient, run in 10 mM HEPES (pH 7.4, 150 mM NaCl, 0.05% Tween 20) buffer at a flow rate of 30 μL / min, with binding / dissociation times of 60 s each, and regeneration in 10 mM Gly-HCl (pH 2.0).

[0046] Data processing: The Biacore T200 software was used to fit a 1:1 combined model and calculate the dynamic parameters.

[0047] 2. The experimental results are shown in Table 2. The SPR-measured KD values ​​were all in the nanomolar range (CD105: 2.3 nM, AdipoR1: 1.8 nM), and the competitive inhibition experiment confirmed that the binding could be blocked by the target antibody, ruling out non-specific adsorption. Therefore, peptide 1 has good affinity for both CD105 and AdipoR1.

[0048] Table 2. Affinity Measurement Experiment

[0049]

[0050] V. Cell Binding Verification Experiment

[0051] 1. Cell Culture and Processing

[0052] MSC culture: Human umbilical cord MSCs (4th generation) were cultured in DMEM + 10% FBS and passaged to 80% confluence. Flow cytometry analysis showed that the CD105 positivity rate was >95%.

[0053] Adipocyte culture: 3T3-L1 preadipocytes were induced to differentiate into mature adipocytes (Oil Red O staining positive), and RT-PCR was used to verify high expression of AdipoR1.

[0054] FITC-labeled peptide 1: Peptide 1 and FITC were reacted at a molar ratio of 1:5 in pH 9.0 carbonate buffer for 2 h, and the free dye was removed by dialysis (molecular weight cutoff 3 kDa).

[0055] 2. Flow cytometry detection

[0056] (1) Experimental Groups:

[0057] Experimental group: FITC-peptide 1 (10 μM) was co-incubated with cells for 30 min (37℃).

[0058] Negative control: PBS as a substitute for peptide.

[0059] Competitive inhibition group: Cells were first incubated with 100 μM anti-CD105 antibody / anti-AdipoR1 antibody for 1 h, and then peptide 1 was added.

[0060] (2) Detection parameters: FITC channel was detected by flow cytometry, and 10,000 cells were collected per sample.

[0061] 3. The experimental results are shown in Table 3. Flow cytometry showed that the positive rate of binding of peptide 1 to MSCs and adipocytes was >92%, and the MFI was more than 70 times higher than that of the negative control. The pretreated antibody could inhibit more than 80% of the binding signal, verifying the dual-target recognition characteristics.

[0062] Table 3. Results of cell binding validation experiment (Mean ± SD, n = 3)

[0063]

[0064] *P<0.001 vs. experimental group.

[0065] VI. Experiment Summary

[0066] Peptide 1, constructed through rational drug design, exhibits highly specific binding ability to CD105 and AdipoR1 during molecular docking, with its cyclization structure being a key factor in enhancing its targeting effect. Peptide 1 can be stably prepared via solid-phase synthesis combined with oxidative cyclization, achieving a purity exceeding 98%, demonstrating good process feasibility and suitability for subsequent drug composition development. SPR and flow cytometry experiments further confirmed its nanomolar affinity for both targets and its ability to achieve specific binding at the cellular level, effectively supporting the homing of MSCs to adipose tissue. As the first single-cyclized peptide to achieve dual recognition of both MSCs and adipocytes, peptide 1 overcomes the limitations of traditional single-target delivery systems, providing a new strategy and molecular basis for targeted therapy of obesity-related diseases.

[0067] Experiment 2: Design, Synthesis and Functional Verification of Peptide 2

[0068] I. Design Background and Objectives

[0069] 1. Obesity and type 2 diabetes often coexist, forming a vicious cycle of metabolic disorders. FABP4 is highly expressed in adipose tissue and participates in lipid synthesis and inflammatory responses by transporting fatty acids; GLP-1 receptor agonists can promote insulin secretion, delay gastric emptying, and reduce appetite. This peptide 2 aims to achieve the dual function of "targeting adipose tissue and regulating glucose and lipid metabolism" by binding its adipose-targeting segment to a GLP-1 analogue.

[0070] N-terminal targeting region: Based on the structure-activity relationship analysis of the FABP4 inhibitor BMS309403, the CRGLYRKG sequence was optimized to enhance the hydrophobic binding with FABP4.

[0071] Connection area: The flexible (GGGS) 3Linker is used to ensure that the functional domains at both ends function independently.

[0072] 2. The amino acid sequence of the designed polypeptide 2 is shown in SEQ ID NO.2.

[0073] II. Structural Design and Molecular Simulation Verification

[0074] 1. Molecular modeling

[0075] Template selection and preprocessing: Obtaining the crystal structure of the FABP4 complex (resolution) from the RCSB Protein Data Bank. Small molecule ligands and water molecules were removed; the binding conformation of GLP-1 and GLP-1R was predicted based on Swiss-Model homology modeling, and the model with the highest confidence (QMEAN = -0.82) was selected.

[0076] Docking optimization: AutoDockVina was used to flexibly dock the CRGLYRKG sequence with FABP4, setting the active site as the hydrophobic cavity of FABP4 (centered on Phe57 and Trp82, with a radius of...). ); Rigid docking of GLP-1 analogues with GLP-1R was performed, with a focus on optimizing the interaction between the N-terminus and the receptor.

[0077] Fusion Structure Construction: The two structural segments are spliced ​​with the (GGGS)3Linker using Rosetta Remodel, and the Linker region is set to be highly flexible. Perform 1000-step energy minimization (AMBER99SB-ILDN force field) and screen... The stable conformation.

[0078] 2. Molecular dynamics simulation

[0079] Simulation system setup: A hydration system for peptide 2 was constructed using GROMACS2021, with the addition of Na. + / Cl- ions maintain electroneutrality; periodic boundary conditions are employed, and the box size is 1.0 nm larger than the maximum dimension of the molecule.

[0080] Simulation process: Energy minimization (5000-step steep slope method + 5000-step conjugate gradient method); NVT balancing at 300K for 100ps (V-rescale temperature control); NPT balancing at 1atm for 100ps (Parrinello-Rahman pressure control); Execute 100ns production simulation with a step size of 2fs, and save the trajectory every 10ps.

[0081] Data Analysis: RMSD was calculated using g_rms. The RMSD of the FABP4 binding region and the GLP-1R binding region were stable at [values ​​to be filled in]. and The radius of gyration was analyzed using g_gyrate, with an average value of 1.8 ± 0.2 nm. The binding free energy was calculated using the MM / PBSA method. The results are as follows: for the FABP4 target, ΔGbind = -8.5 ± 0.9 kcal / mol, with a KD of approximately 90 nM; for the GLP-1R target, ΔGbind = -10.7 ± 1.2 kcal / mol, with a KD of approximately 12 nM.

[0082] III. Synthesis and Purification

[0083] The designed peptide 2 was synthesized at Sangon Biotech (Shanghai) Co., Ltd., with a purity (HPLC purity) of 99.2%. MALDI-TOF mass spectrometry analysis showed a molecular weight of 4236.2 Da (theoretical value 4235.8 Da), with an error of <0.1%.

[0084] IV. In vitro functional verification experiments

[0085] 1. FABP4 binding experiment (SPR)

[0086] (1) Chip preparation: CM5 chip surface was activated with EDC / NHS; human FABP4 protein (10 μg / mL, pH 5.0 sodium acetate buffer) was conjugated to a response value of about 800 RU; unreacted sites were blocked with ethanolamine.

[0087] (2) Experimental setup:

[0088] Run buffer: HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Tween 20, pH 7.4);

[0089] Peptide concentration gradients: 5, 10, 20, 50, 100 nM;

[0090] Injection time: binding 180s, dissociation 300s;

[0091] Regeneration conditions: 10 mM Gly-HCl (pH 2.0) treatment for 30 s.

[0092] (3) Data analysis: Using BIAevaluation software, the dynamic curve was fitted with a 1:1 Langmuir model, and KD = 78nM was calculated.

[0093] 2. GLP-1R activating experiment (cAMP accumulation)

[0094] (1) Cell culture: HEK293-GLP-1R stable cells were cultured in DMEM + 10% FBS + 500 μg / mL G418; at 5 × 10⁻⁶ cells / mL.4 Cells / well were seeded into 96-well plates and cultured for 24 hours, followed by serum-free starvation treatment for 1 hour.

[0095] (2) Experimental treatment:

[0096] Experimental group: Different concentrations of peptide 2 (0.1-100 nM) were added;

[0097] Positive control: 10 nM natural GLP-1 + 10 μM Forskolin;

[0098] Negative control: treated with PBS;

[0099] Each group has 6 duplicate wells, and is incubated at 37°C for 30 minutes.

[0100] (3) Detection method: The intracellular cAMP content was detected using a cAMP ELISA kit; the results were normalized with the Forskolin group as 100%, and the EC50 was calculated to be 6.7 nM, with the maximum effect comparable to that of natural GLP-1.

[0101] V. Pharmacokinetic Experiments in Mice

[0102] 1. Experimental design: 6-8 week old male C57BL / 6J mice (n=5) were selected; polypeptide 2 (0.5mg / kg, dissolved in PBS) was injected subcutaneously; blood was collected from the orbital cavity at 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours, and plasma was separated.

[0103] 2. Sample Analysis: Proteins in plasma samples were precipitated with 3 volumes of acetonitrile and centrifuged at 12,000 rpm for 15 min; analysis was performed using LC-MS / MS.

[0104] Column: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 × 50 mm);

[0105] Mobile phases: Phase A (0.1% formic acid aqueous solution), Phase B (0.1% formic acid acetonitrile solution);

[0106] Gradient elution: 0-1 min 5% B, 1-3 min 5-95% B, 3-4 min 95% B, 4-4.1 min 95-5% B, 4.1-5 min 5% B, flow rate 0.4 mL / min;

[0107] Mass spectrometry parameters: ESI + The mode was used to monitor ion pairs m / z 4236.2→847.2 (peptide) and m / z 609.3→427.2 (internal standard).

[0108] 3. Data processing: Phoenix WinNonlin 8.3 software was used for non-compartmental model analysis, and the calculated half-life T1 / 2 was 6.2 ± 0.4 h, which is about 4 times longer than that of natural GLP-1.

[0109] VI. Summary

[0110] 1. Structural design verification: Molecular simulations confirmed that the functional domains at both ends of peptide 2 can form stable binding with FABP4 and GLP-1R, respectively, and the linker region maintains high flexibility without spatial conflict.

[0111] 2. Synthesis and characterization: The target peptide was successfully prepared by Fmoc-SPPS with high oxidative cyclization efficiency, HPLC purity >98.7%, and mass spectrometry verification of the correct structure.

[0112] 3. In vitro activity: SPR assay showed that the peptide had a KD of 78 nM for FABP4; cAMP assay showed that its EC50 reached 6.7 nM, exhibiting receptor agonist activity comparable to that of natural GLP-1.

[0113] 4. Pharmacokinetics: After subcutaneous injection in mice, the half-life of peptide 2 was significantly prolonged to 6.2 h, which is beneficial for reducing the frequency of administration.

[0114] In summary, this study successfully constructed a bifunctional peptide 2 through rational design, molecular simulation, and experimental verification. It not only achieves dual targeting of FABP4 and GLP-1R structurally, but also exhibits nanomolar affinity and receptor agonist activity in vitro, and excellent in vivo pharmacokinetic properties. These findings provide important theoretical basis and candidate molecules for the development of novel fat-targeting drugs for the treatment of obesity / diabetes.

[0115] Example 2: Animal-level evaluation of efficacy and safety

[0116] I. Experimental Materials and Equipment

[0117] (I) Experimental animals: Six-week-old SPF-grade male C57BL / 6J mice were selected. They were first acclimatized to a specific environment for one week. The environment was maintained at a temperature of 22±2℃ and a humidity of 50±10%, and a 12-hour day-night alternating light and shadow system was adopted to ensure that the mice adapted to a stable environment and lay the foundation for subsequent experiments.

[0118] (II) Experimental Feed

[0119] High-fat diet: D12492 high-fat diet is used, with a fat content as high as 60kcal%, which is specially designed to induce obesity in mice.

[0120] Control diet: Ordinary feed with a fat content of 10 kcal% served as the control group for normal diet.

[0121] (III) Experimental Reagents

[0122] Polypeptide 1 and Polypeptide 2 were prepared by our researchers using chemical synthesis methods. Their purity was greater than 95% as detected by high performance liquid chromatography (HPLC), ensuring the reliability of the reagent quality.

[0123] MSCs: Mouse adipose-derived mesenchymal stem cells, selected from passage 3 cells, at a concentration of 1×10⁻⁶ cells / year. 6 The solution was prepared at a concentration of cells / 200μL PBS to ensure consistency between cell viability and experimental dosage.

[0124] II. Experimental Design and Operation Procedures

[0125] (I) Model Establishment (Weeks 0-12): Sixty C57BL / 6J mice were randomly divided into two groups: a high-fat group (n=50) and a control group (n=10). During the feeding process, the feed and drinking water were changed regularly every day, and the mice's diet, activity, and fecal condition were recorded in detail. The mice's weight was measured weekly, and a weight gain curve was plotted to dynamically observe the trend of weight change. At the end of week 12, the mice were weighed after fasting for 12 hours. Fifty mice whose weight exceeded the average of the control group by more than 20% were selected as model mice. At this time, the average weight of the model mice was approximately 42.8 ± 2.5 g, and the obesity model was successfully established.

[0126] (II) Grouping and Drug Administration (Weeks 13-16): Fifty obese model mice were randomly divided into 5 groups of 10 mice each. The mice were administered the drug via tail vein injection for 4 weeks. During the injection process, the mice's reactions were closely monitored, and any abnormalities were recorded and appropriate measures were taken. Specific grouping and treatment details are shown in Table 4.

[0127] Table 4 Experimental Groups

[0128] Group deal with NC Control mice were injected with PBS (200 μL, twice a week). M <![CDATA[Inject MSCs (1×10 6 cells / 200 μL, twice a week)]]> P1 Inject peptide 1 (100μg / 200μL, twice a week) P2 Inject peptide 2 (100μg / 200μL, twice a week) CMP1 Inject MSCs + peptide 1 + peptide 2 (200 μL mixed, twice a week).

[0129] (III) Testing Items and Time Points

[0130] Weight: Once a week, mice were accurately weighed after fasting for 12 hours, and the data were recorded.

[0131] Body fat percentage: At the end of week 16, the body fat percentage of mice was measured using an EchoMRI-100 non-invasive detector. During the test, the mice were gently placed in the detector to ensure they were in a calm state and to obtain accurate data.

[0132] Adipocyte size: At the end of week 16, epididymal adipose tissue was collected from mice and stained with hematoxylin and eosin (HE). The stained tissue sections were observed under a microscope, and the diameter of 100 adipocytes was randomly measured using ImageJ software, and the average value was calculated.

[0133] Serum markers: At the end of week 16, blood was collected from mice via orbital sampling, and relevant markers were detected using an ELISA kit and a biochemical analyzer.

[0134] Safety testing: At the end of week 16, ALT, AST, BUN, Cr and other indicators were tested, and organs such as liver, kidney and heart were stained with HE, pathological sections were prepared, and cell morphology was observed under a microscope to determine whether there was necrosis, fatty degeneration or edema.

[0135] III. Experimental Results

[0136] (I) Baseline weight and body fat: As shown in Table 5, compared with the NC group, the final weight and body fat percentage of mice in the M, P1, P2 and CMP1 groups were significantly reduced (*P<0.05, #P<0.01), with the CMP1 group showing the most significant reduction, indicating that this combination therapy was the most effective in reducing weight and body fat percentage.

[0137] Table 5. Baseline weight and body fat percentage results

[0138] Group Initial body weight (g) Final body weight (g) Body fat percentage (%) NC 25.3±1.2 42.8±2.5 34.7±3.2 M 25.1±1.1 38.5±2.1* 30.5±2.8* P1 24.9±1.3 37.9±1.9* 29.8±2.5* P2 25.2±1.0 36.2±1.8* 27.3±2.3* CMP1 25.0±1.4 32.5±1.5# 22.1±2.0#

[0139] (II) Average diameter of adipocytes (μm, 100 cells were randomly measured): As can be seen from Table 6, the average diameter of adipocytes in groups M, P1, P2 and CMP1 was smaller than that in group NC (*P<0.05, #P<0.01). The adipocyte diameter in group CMP1 was the smallest, indicating that this combination therapy can more effectively improve the morphology of adipose tissue and make adipocytes smaller.

[0140] Table 6 Results of average adipocyte diameter detection

[0141]

[0142]

[0143] (III) Serum biochemical indicators: As shown in Table 7, compared with the NC group, the levels of TG, TC and LDL-C in each treatment group were significantly reduced (*P<0.05, #P<0.01), while the level of HDL-C was significantly increased (*P<0.05, #P<0.01). The CMP1 group showed the most significant effect in regulating lipid metabolism and helped to improve lipid metabolism disorders in the body.

[0144] Table 7 Results of serum biochemical markers

[0145]

[0146] (iv) Inflammation and metabolic factors: As shown in Table 8, the levels of resistin and leptin in the M, P1, P2 and CMP1 groups were lower than those in the NC group, while the level of adiponectin was higher than that in the NC group (*P<0.05, #P<0.01). The CMP1 group showed the strongest effect in regulating inflammation and metabolic factors, and could effectively improve insulin sensitivity and reduce the body's inflammatory response.

[0147] Table 8 Results of Inflammation and Metabolic Factor Detection

[0148] Group resistin (ng / mL) Adiponectin (μg / mL) Leptin (ng / mL) NC 12.5±1.1 4.2±0.4 18.3±1.5 M 10.8±1.0* 5.8±0.5* 15.2±1.3* P1 10.5±0.9* 6.1±0.6* 14.9±1.2* P2 8.2±0.7# 7.3±0.7* 13.4±1.1* CMP1 7.5±0.6# 8.9±0.8# 11.8±1.0#

[0149] IV. Results Analysis

[0150] Fluorescence tracer assays revealed that the average colonization signal intensity of MSCs in adipose tissue in the CMP1 group was 3.2 times that in the M group. This indicates that peptide 1 can significantly promote the accumulation of MSCs in adipose tissue, enabling them to function better and laying the foundation for subsequent therapeutic effects. Peptide 2 can effectively reduce body fat by regulating related signaling pathways, promoting lipolysis and inhibiting liposynthesis.

[0151] The CMP1 group experienced a 36.3% reduction in body fat percentage, significantly outperforming MSCs and the single peptide group, demonstrating a clear advantage in reducing body fat. Peripheral blood tests showed that glycerol release in the CMP1 group was 41% higher than in the P2 group and 75% higher than in the M group, indicating that this combination therapy can more effectively promote lipolysis and glycerol release. The CMP1 group also showed a 22% increase in adiponectin and a 40% decrease in resistin, comprehensively improving insulin sensitivity and exhibiting a good regulatory effect on obesity-related metabolic disorders.

[0152] (III) Safety results, as shown in Table 9, all liver and kidney function indicators in the CMP1 group were within the normal range, and HE pathological sections showed no abnormalities such as necrosis, fatty degeneration or edema in liver, kidney and heart cells, indicating that the combination therapy of polypeptide 1 + polypeptide 2 + MSCs had no obvious organ toxicity and had good safety.

[0153] Table 9 Safety Inspection Results

[0154] index Normal range Mean ± SD of CMP1 group ALT(U / L) 15-40 28.5±2.6 AST(U / L) 30-60 45.3±3.1 BUN (mmol / L) 6-10 7.4±0.6 Cr (μmol / L) 20-40 32.1±3.2

[0155] V. Summary

[0156] This experiment systematically investigated the efficacy and safety of the combination therapy of peptide 1 + peptide 2 + MSCs by establishing a high-fat diet obese mouse model. The results showed that this combination therapy exhibited significant synergistic effects in reducing body fat, improving adipose tissue morphology, and regulating serum metabolic and inflammatory factors, without causing significant toxic reactions, demonstrating good safety. These results provide solid experimental evidence for the clinical translational application of this combination therapy in obesity and related metabolic diseases.

[0157] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A polypeptide composition for the treatment of obesity, characterized in that, The polypeptide composition comprises polypeptide 1 and polypeptide 2 and adipose-derived mesenchymal stem cells; the amino acid sequence of polypeptide 1 is shown in SEQ ID NO.1, and the amino acid sequence of polypeptide 2 is shown in SEQ ID NO.

2.

2. The polypeptide composition according to claim 1, characterized in that, The polypeptide 1 exhibits specific binding ability to the CD105 receptor on the surface of MSCs and the AdipoR1 receptor on adipocytes through a cyclization conformation, and the KD values ​​for CD105 and AdipoR1 are 2.3 nM and 1.8 nM, respectively.

3. The polypeptide composition according to claim 1, characterized in that, The polypeptide 2 can target and bind to FABP4 in adipose tissue and stimulate GLP-1 receptor. Its KD value for FABP4 is 78 nM and its EC50 for GLP-1R agonistic activity is 6.7 nM.

4. A weight-loss drug composition containing mesenchymal stem cells, characterized in that, The composition comprises polypeptide 1, polypeptide 2 as described in claim 1, and adipose-derived mesenchymal stem cells.

5. The composition according to claim 4, characterized in that, The concentration of adipose-derived mesenchymal stem cells in the composition is 1×10⁻⁶. 6 Cells / 200 μL PBS, peptide 1 concentration is 100 μg / 200 μL, peptide 2 concentration is 100 μg / 200 μL.

6. Use of the polypeptide composition as described in claim 1 in the preparation of a medicament for treating obesity.

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