Small molecule peptide composition for targeted repair of stroke brain cells based on stem cells

Abstract

The invention relates to the technical field of biological medicines, and relates to a small molecule peptide composition for targeted repair of stroke brain cells based on stem cells. The invention designs small molecule peptides with targeted homing and differentiation regulation functions, the small molecule peptides are reasonably combined, and a drug delivery system is also constructed. The composition can guide the stem cells to accurately home to the damaged brain area, regulate and control the stem cells to be differentiated into functional brain cells, and can break through the blood brain barrier. Through testing, the treatment effect of the stem cells is remarkably improved, and the safety and effectiveness of treatment are enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a small molecule peptide composition for repairing stroke brain cells based on stem cells. Background Art

[0002] Stroke is a cerebrovascular disease that seriously threatens human health, with high morbidity, high disability and high mortality. At present, the treatment methods for stroke include drug therapy, surgical treatment and rehabilitation therapy, but these methods still have great limitations in promoting the repair and regeneration of damaged brain cells. Stem cell therapy, as an emerging treatment strategy, has brought new hope for the treatment of stroke. However, how to accurately guide stem cells to the damaged brain area and promote their differentiation into functional brain cells is still an urgent problem to be solved. In the existing research on stem cell therapy for stroke, the stem cells injected into the body are often difficult to efficiently home to the damaged brain area. Most stem cells will be distributed in other tissues or organs, resulting in insufficient number of stem cells that actually reach the lesion site to play a repair role, affecting the treatment effect. Even if stem cells can reach the damaged brain area, their differentiation process into functional brain cells is difficult to accurately control. Many stem cells may not differentiate into the required neurons or glial cells, or the differentiation ratio is not ideal, and they cannot effectively repair damaged brain tissue. Some methods to promote stem cell repair may bring potential safety risks, such as excessive proliferation and tumors. At the same time, existing treatment methods still face challenges in improving treatment effects and cannot significantly improve the neurological deficits of stroke patients. For some drugs or bioactive substances used to assist stem cell therapy, how to effectively deliver them to the damaged brain area is a difficult problem. Traditional drug delivery systems often cannot break through the blood-brain barrier, or the drug activity is reduced during the delivery process, affecting the treatment effect.

[0003] Small molecule peptides have great application potential in the field of biomedicine due to their good biological activity, low immunogenicity and easy penetration of biological membranes. However, there are relatively few studies on small molecule peptide compositions and their related applications for stem cell-targeted repair of stroke brain cells. Summary of the invention

[0004] The present invention provides a small molecule peptide composition for repairing stroke brain cells based on stem cells, the composition comprising:

[0005] A small molecule peptide with targeted homing function, the sequence of which is Arg-Gly-Asp-Tyr-Ser-Thr-Leu, and the molecular weight is between 500-1000Da;

[0006] A small molecule peptide with differentiation regulation function, the sequence of which is His-Pro-Glu-Lys-Asp-Val-Ile, and the molecular weight is between 600-1200Da;

[0007] As well as small molecule peptides derived from plant extracts, the small molecule peptide derived from salvia miltiorrhiza extract has a sequence of Thr-Lys-Asp-Glu-His-Pro-Tyr and a molecular weight of approximately 890Da; the small molecule peptide derived from ginseng extract has a sequence of Glu-Val-Ser-Thr-Pro-Arg-Gly and a molecular weight of approximately 840Da; the small molecule peptide derived from green tea extract has a sequence of Pro-Cys-His-Asp-Val-Leu-Ser and a molecular weight of approximately 870Da; the small molecule peptide derived from turmeric extract has a sequence of His-Glu-Lys-Asp-Pro-Tyr-Thr and a molecular weight of approximately 880Da.

[0008] Furthermore, the small molecule peptide with targeted homing function can specifically bind to the integrin receptors on the surface of vascular endothelial cells in the damaged brain area, and guide the stem cells to migrate to the damaged brain area.

[0009] Furthermore, the small molecule peptide with differentiation regulation function can bind to the Notch receptor on the surface of stem cells, activate the Notch signaling pathway, and promote the differentiation of stem cells into neurons.

[0010] Furthermore, the small molecule peptide derived from the salvia miltiorrhiza extract acts on endothelial nitric oxide synthase (eNOS) on the surface of vascular endothelial cells, activates the activity of the enzyme, promotes the synthesis and release of nitric oxide (NO), and improves blood circulation in damaged brain areas.

[0011] Furthermore, the small molecule peptide derived from ginseng extract acts on the glucocorticoid receptor (GR) on the surface of nerve cells, regulates GR activity, and affects the expression of downstream genes related to nerve protection and repair.

[0012] Furthermore, the small molecule peptide derived from the green tea extract acts on the Bcl-2 family protein in the nerve cells, regulates the ratio and function between Bax and Bcl-2, and inhibits the apoptosis of nerve cells.

[0013] Furthermore, the small molecule peptide derived from the turmeric extract acts on the nuclear factor-κB (NF-κB) signaling pathway in nerve cells, inhibits the activation of NF-κB, and reduces the expression and release of inflammatory mediators.

[0014] A further preparation method comprises the following steps:

[0015] The small molecule peptides are obtained by solid phase synthesis or extracted and purified from plant extracts. The targeted homing and differentiation regulating small molecule peptides are synthesized by solid phase synthesis. The corresponding small molecule peptides are extracted and purified from salvia miltiorrhiza, ginseng, green tea and turmeric extracts respectively.

[0016] Modification of small molecule peptides, such as PEGylation, acetylation, glycosylation, phosphorylation, etc. described in the examples;

[0017] The modified small molecule peptides are mixed according to a specific molar ratio, an appropriate amount of buffer is added, and the pH value is adjusted to prepare a small molecule peptide composition solution;

[0018] A drug delivery system is constructed, wherein nano lipid carriers (NLC), chitosan and hyaluronic acid nanocomplexes, polylactic acid-co-glycolic acid (PLGA) nanoparticles, liposomes, etc. are used as carriers to load the small molecule peptide composition therein.

[0019] Furthermore, in the preparation method, during the synthesis or extraction and purification process of the small molecule peptide, high performance liquid chromatography (HPLC), mass spectrometry analysis, amino acid sequencing and other technologies are used to detect and determine the purity, structure and sequence of the small molecule peptide.

[0020] Furthermore, the small molecule peptide composition is used in the preparation of a drug for treating stroke.

[0021] Beneficial technical effects:

[0022] Through in-depth research on the microenvironment of the brain area damaged by stroke, a small molecule peptide with targeted homing function was designed. This small molecule peptide can specifically recognize cell surface markers or chemical signals in the microenvironment of the damaged brain area, guide stem cells to migrate to the damaged brain area, and improve the homing efficiency of stem cells.

[0023] Design of small molecule peptides for differentiation regulation: Targeting the regulatory mechanism of stem cell differentiation into neurons and glial cells, we design small molecule peptides that can promote stem cell differentiation into functional brain cells. These small molecule peptides can bind to receptors on the surface of stem cells, activate or inhibit related signaling pathways, and precisely regulate the direction and degree of stem cell differentiation.

[0024] Synergistic effect of small molecule peptide combination: rationally combine targeted homing small molecule peptides and differentiation regulation small molecule peptides to form a small molecule peptide combination with synergistic effect. At the same time, some small molecule peptides with functions such as promoting cell proliferation and inhibiting cell apoptosis can also be added to further enhance the repair effect on stroke brain cells.

[0025] Preparation process optimization: Use advanced solid phase synthesis or liquid phase synthesis technology to prepare small molecule peptides to ensure the purity and activity of the peptides. By optimizing the synthesis process conditions, such as reaction temperature, reaction time, reactant ratio, etc., the synthesis efficiency and quality of small molecule peptides can be improved.

[0026] Construction of drug delivery system: Utilize the characteristics of small molecule peptides to construct a new type of drug delivery system. Combine small molecule peptides with carrier materials to form targeted carriers such as nanoparticles or microspheres, which can effectively deliver drugs or other bioactive substances to damaged brain areas, break through the blood-brain barrier, and improve the bioavailability of drugs. DETAILED DESCRIPTION

[0027] Example 1

[0028] Synthesis of small molecule peptides:

[0029] The targeted homing small molecule peptide Arg-Gly-Asp-Tyr-Ser-Thr-Leu and the differentiation regulating small molecule peptide His-Pro-Glu-Lys-Asp-Val-Ile were synthesized by solid phase synthesis. The amino acids were connected to the solid phase carrier in sequence according to the amino acid sequence. After each step of the reaction, the purity and progress of the reaction were detected by HPLC. After the synthesis was completed, the small molecule peptide was cut and purified to obtain a high-purity small molecule peptide product.

[0030] Modification of small molecule peptides:

[0031] The targeted homing small molecule peptide is modified with PEG, and a PEG chain with a molecular weight of 2000Da is connected to its N-terminus. Through chemical coupling reaction, PEG is connected to the small molecule peptide to improve the stability and water solubility of the small molecule peptide.

[0032] Preparation of small molecule peptide compositions:

[0033] The targeted homing small molecule peptide and the differentiation regulating small molecule peptide were mixed at a molar ratio of 1:1, an appropriate amount of phosphate buffered saline (PBS) was added, and the pH value was adjusted to 7.2 to prepare a small molecule peptide composition solution with a concentration of 1 mg / mL.

[0034] Construction of drug delivery system:

[0035] PLGA nanoparticles were prepared by emulsification-solvent evaporation method. PLGA was dissolved in dichloromethane, and a small molecule peptide composition solution was added, and an emulsion was formed by ultrasonic emulsification. The emulsion was then added dropwise to an aqueous solution containing polyvinyl alcohol (PVA), and the dichloromethane was evaporated by stirring to obtain PLGA nanoparticles loaded with a small molecule peptide composition. The particle size distribution and morphology of the nanoparticles were characterized by DLS and SEM, and the results showed that the average particle size of the nanoparticles was 100-150nm, the particle size distribution was uniform, and the morphology was regular.

[0036] Cell experiments:

[0037] Mouse neural stem cells were taken and co-cultured with PLGA nanoparticles loaded with small molecule peptide compositions. The differentiation of neural stem cells was detected by immunofluorescence staining and flow cytometry. The results showed that compared with the control group, the proportion of neural stem cells in the experimental group that differentiated into neurons was significantly increased, indicating that the small molecule peptide composition can effectively promote the differentiation of neural stem cells.

[0038] Animal Experimentation:

[0039] A rat stroke model was established, and PLGA nanoparticles loaded with a small molecule peptide composition were injected into the rats through the tail vein. At different time points after treatment, the neurological function recovery of the rats was evaluated by behavioral tests (such as Morris water maze test, open field test, etc.). At the same time, the regeneration of neurons and glial cells in the damaged brain area was detected by immunohistochemical staining and Western blot. The results showed that the symptoms of neurological deficits in the rats in the treatment group were significantly improved, and the number of neurons and glial cells in the damaged brain area increased significantly, indicating that the small molecule peptide composition of the present invention can effectively promote the neurological function recovery of stroke rats.

[0040] Comparative Example 1

[0041] Unmodified small molecule peptides were used, no drug delivery system was constructed, and other conditions were the same as in Example 1.

[0042] Performance Testing

[0043] Stem cell homing efficiency test:

[0044] The stem cells labeled with fluorescent dye were incubated with the small molecule peptide compositions in Example 1 and Comparative Example 1, respectively, and then injected into the body of the stroke model mice. The distribution of stem cells in the mice was observed by fluorescence imaging technology, and the proportion of the number of stem cells reaching the damaged brain area to the total number of injected stem cells was calculated.

[0045] Stem Cell Differentiation Test:

[0046] Human embryonic stem cells were co-cultured with the small molecule peptide compositions in Example 1 and Comparative Example 1, respectively. At different time points in culture, the expression of markers for differentiation of stem cells into neurons and glial cells was detected by immunocytochemical staining and RT-PCR to evaluate the differentiation ratio of stem cells.

[0047] Drug delivery effectiveness testing:

[0048] The PLGA nanoparticles loaded with fluorescently labeled drugs in Example 1 and the drug without a delivery system constructed in Comparative Example 1 were injected into normal mice, respectively. The distribution of the drugs in the mouse brain was observed by in vivo imaging technology to evaluate the ability of the drug delivery system to break through the blood-brain barrier.

[0049] Safety Testing:

[0050] The small molecule peptide compositions in Example 1 and Comparative Example 1 were subjected to cytotoxicity tests, and the small molecule peptide compositions of different concentrations were co-cultured with mouse fibroblasts, and the survival rate of the cells was detected by the MTT method. At the same time, the blood routine, liver and kidney function and other indicators of the treated animals were tested to evaluate the safety of the small molecule peptide compositions.

[0051] Test Results

[0052]

[0053] It can be seen from the test results that the small molecule peptide composition based on stem cell targeted repair of stroke brain cells and its preparation method of the present invention are significantly superior to comparative example 1 in terms of improving stem cell homing efficiency, promoting stem cell differentiation, breaking through the blood-brain barrier and safety.

[0054] Example 2

[0055] Design principle of small molecule peptide: Considering the damage of post-stroke inflammatory response to nerve cells, a small molecule peptide is designed to inhibit inflammatory response and create a good microenvironment for stem cell repair. Inflammatory response releases a variety of inflammatory factors, which affect the survival and differentiation of stem cells. This small molecule peptide is designed to reduce the release of inflammatory factors and reduce the damage of inflammation to nerve cells by regulating inflammation-related signaling pathways.

[0056] Specific sequence: Gly-Glu-Ala-Ser-Pro-Lys-Trp, molecular weight is about 870Da. Glutamic acid (Glu) and serine (Ser) may be involved in regulating the intracellular signal transduction process and affect the expression of inflammation-related genes; while tryptophan (Trp) is related to immune regulation and neuroprotection in some biological processes, and may play an anti-inflammatory role by affecting the synthesis of neurotransmitters or regulating the function of immune cells.

[0057] Target: Acts on Toll-like receptor 2 (TLR2) on the surface of microglia, inhibits downstream inflammatory signaling pathways after activation, reduces the release of inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), reduces the damage of inflammation to damaged brain areas, and provides a favorable environment for the homing and differentiation of stem cells.

[0058] Preparation method:

[0059] Synthesis of small molecule peptides: The small molecule peptides are synthesized by adding amino acids in a suitable reaction solvent in a specific reaction step using a liquid phase synthesis method. During the reaction, the reaction temperature is strictly monitored at 20-25°C, and the reaction progress is tracked by thin layer chromatography (TLC) to ensure that the reaction is fully carried out. After the synthesis is completed, the product is purified by ion exchange chromatography to obtain a high-purity small molecule peptide.

[0060] Modification of small molecule peptides: Acetylation modification is performed on the synthesized small molecule peptides to introduce an acetyl group at their N-terminus. By adding an acetylation agent to an appropriate reaction system and performing the modification reaction under mild reaction conditions, the stability of the small molecule peptide is increased and the possibility of enzymatic hydrolysis in the body is reduced.

[0061] Preparation of a small molecule peptide composition: Mix the anti-inflammatory small molecule peptide with the targeted homing small molecule peptide and the differentiation regulating small molecule peptide in Example 1 in a molar ratio of 1:1:1, add an appropriate amount of Tris-HCl buffer, adjust the pH to 7.3, and prepare a small molecule peptide composition solution with a concentration of 1.2 mg / mL.

[0062] Construction of drug delivery system: Chitosan was selected as the carrier material, and chitosan microspheres loaded with small molecule peptide composition were prepared by ion gel method. Chitosan was dissolved in dilute acetic acid solution, and small molecule peptide composition solution was added. After stirring evenly, sodium tripolyphosphate solution was added dropwise to form chitosan microspheres through ion cross-linking reaction. The particle size and morphology of the microspheres were characterized by laser particle size analyzer and scanning electron microscope. The results showed that the average particle size of the microspheres was between 200-300nm, spherical and relatively uniformly distributed.

[0063] Cell experiment: Mouse microglia were taken and lipopolysaccharide (LPS) was used to induce an inflammatory response. Then, chitosan microspheres loaded with a small molecule peptide composition were co-cultured with microglia in an inflammatory state. The content of inflammatory factors in the cell culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA), and the expression of inflammation-related genes was detected by real-time fluorescence quantitative PCR. The results showed that compared with the untreated group, the release of inflammatory factors in the experimental group was significantly reduced, and the expression of inflammation-related genes was also significantly downregulated, indicating that the small molecule peptide composition can effectively inhibit the inflammatory response of microglia.

[0064] Animal experiment: A rat stroke model was established, and chitosan microspheres loaded with a small molecule peptide composition were injected into the vicinity of the damaged brain area through brain stereotaxic 24 hours after modeling. On the 3rd, 7th and 14th days after treatment, the expression of inflammatory factors in the damaged brain area was detected by immunohistochemical staining, and behavioral tests were performed to evaluate the neurological recovery of rats. The results showed that the expression of inflammatory factors in the damaged brain area of ​​rats in the treatment group was significantly reduced, and the symptoms of neurological deficits were significantly improved, indicating that the small molecule peptide composition also has a good anti-inflammatory and neurological recovery effect in vivo.

[0065] Example 3

[0066] Design principle of small molecule peptide: Based on the importance of angiogenesis to the repair of damaged brain tissue after stroke, a small molecule peptide that can promote angiogenesis is designed. Adequate angiogenesis can provide rich nutrients and oxygen to the damaged brain area, which is beneficial to the survival and differentiation of stem cells and the repair of damaged neural tissue. The small molecule peptide works by simulating some functions of angiogenesis-related growth factors in the body and activating angiogenesis-related signaling pathways.

[0067] Specific sequence: Leu-Thr-Asp-Gln-His-Arg-Val, molecular weight is about 920Da. Histidine (His) and arginine (Arg) play an important role in some angiogenesis-related physiological processes. They may participate in intercellular signal transduction and protein-protein interaction, affecting the proliferation, migration and lumen formation of vascular endothelial cells; while amino acids such as leucine (Leu) and threonine (Thr) help maintain the spatial structure of small molecule peptides and ensure their biological activity.

[0068] Target: Acts on vascular endothelial growth factor receptor 2 (VEGFR2) on the surface of vascular endothelial cells, activates the downstream Ras-Raf-MEK-ERK signaling pathway, promotes the proliferation, migration and lumen formation of vascular endothelial cells, and thus promotes angiogenesis in damaged brain areas.

[0069] Preparation method:

[0070] Synthesis of small molecule peptides: Solid phase synthesis is used to connect amino acids in sequence according to standard synthesis steps to synthesize the target small molecule peptide. During the synthesis process, ensure that the activation, coupling and deprotection steps of each reaction are fully carried out, and monitor the purity and progress of the reaction in real time by high performance liquid chromatography (HPLC). After the synthesis is completed, cutting and purification are carried out to obtain a high-purity small molecule peptide product.

[0071] Modification of small molecule peptides: Glycosylation modification of small molecule peptides is performed to connect sugar chains to specific amino acid residues. Through chemical synthesis, reagents containing sugar groups are reacted with small molecule peptides under appropriate reaction conditions to connect sugar groups to small molecule peptides. Glycosylation modification can increase the stability and water solubility of small molecule peptides, and may also enhance their binding affinity with the target.

[0072] Preparation of small molecule peptide composition: Mix the angiogenic small molecule peptide with the targeted homing small molecule peptide and the differentiation regulating small molecule peptide in Example 1 in a molar ratio of 1.5:1:1, add an appropriate amount of HEPES buffer, adjust the pH to 7.4, and prepare a small molecule peptide composition solution with a concentration of 1.5 mg / mL.

[0073] Construction of drug delivery system: Nanoparticle carriers were constructed using polylactic acid-co-glycolic acid (PLGA) and polyethylene glycol (PEG). First, PLGA and PEG were dissolved in an organic solvent, and a small molecule peptide composition solution was added. An emulsion was formed by ultrasonic emulsification, and then the organic solvent was volatilized to obtain PLGA-PEG nanoparticles loaded with a small molecule peptide composition. The particle size, potential, and morphology of the nanoparticles were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The results showed that the average particle size of the nanoparticles was 120-180nm, the surface potential was moderate, and they were spherical and evenly dispersed.

[0074] Cell experiment: Human umbilical vein endothelial cells (HUVECs) were taken, and PLGA-PEG nanoparticles loaded with small molecule peptide compositions were co-cultured with HUVECs. Cell proliferation was detected by cell proliferation experiments (such as CCK-8 method), cell migration ability was detected by Transwell experiment, and the ability of cells to form lumen-like structures was observed by in vitro lumen formation experiment. The results showed that compared with the control group, the proliferation, migration and lumen formation abilities of HUVECs in the experimental group were significantly enhanced, indicating that the small molecule peptide composition can effectively promote the angiogenesis-related functions of vascular endothelial cells.

[0075] Animal experiment: A mouse stroke model was established, and PLGA-PEG nanoparticles loaded with a small molecule peptide composition were injected into the mouse through the tail vein 48 hours after modeling. On the 7th and 14th days after treatment, the vascular density of the damaged brain area was detected by immunofluorescence staining, and behavioral tests were performed to evaluate the neurological recovery of the mice. The results showed that the vascular density of the damaged brain area of ​​the mice in the treatment group increased significantly, and the symptoms of neurological deficits were significantly improved, indicating that the small molecule peptide composition can promote angiogenesis in vivo, thereby promoting the recovery of neurological function.

[0076] Example 4

[0077] Design principle of small molecule peptide: To address the energy metabolism disorder of nerve cells after stroke, we designed a small molecule peptide that can improve the energy metabolism of nerve cells. Stroke can lead to insufficient energy supply to nerve cells, affecting the normal function and survival of cells. This small molecule peptide improves the energy generation efficiency of cells and enhances the resistance of nerve cells to damage by regulating the energy metabolism-related pathways in nerve cells.

[0078] Specific sequence: Ile-Pro-Ser-Glu-Asp-Met-Lys, molecular weight is about 900Da. Aspartic acid (Asp) and glutamic acid (Glu) participate in a variety of enzymatic reactions in the energy metabolism of cells, and they may affect the key enzyme activities in the tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation process; while methionine (Met) provides methyl groups in some biosynthetic processes and may participate in the synthesis and modification of energy metabolism-related substances in nerve cells.

[0079] Target: Acts on pyruvate dehydrogenase kinase 4 (PDK4) on the mitochondrial membrane of nerve cells, inhibits the activity of PDK4, keeps pyruvate dehydrogenase (PDH) active, promotes the entry of pyruvate into mitochondria for oxidative metabolism, improves the energy generation efficiency of nerve cells, and enhances the survival ability and functional recovery of nerve cells.

[0080] Preparation method:

[0081] Synthesis of small molecule peptides: Using solid phase synthesis technology, amino acids are connected in sequence strictly according to the amino acid sequence. The reaction conditions are precisely controlled during the synthesis process, such as maintaining the reaction temperature at 25-30°C and adjusting the reaction time according to the reactivity of the amino acids. After each step of the reaction, the purity of the reaction product is tested by HPLC to ensure the accuracy and efficiency of the reaction. After the synthesis is completed, a high-purity small molecule peptide is obtained through cutting and purification steps.

[0082] Modification of small molecule peptides: phosphorylation modification of small molecule peptides, introduction of phosphate groups on specific serine (Ser) residues. By carrying out modification reaction in a reaction system containing phosphorylation reagents under appropriate reaction conditions, the charge properties and spatial structure of small molecule peptides are changed, and their binding ability with targets and biological activity are enhanced.

[0083] Preparation of a small molecule peptide composition: Mix the small molecule peptide that improves energy metabolism with the targeted homing small molecule peptide and the differentiation regulating small molecule peptide in Example 1 in a molar ratio of 1:1.2:1, add an appropriate amount of phosphate buffer (PBS), adjust the pH value to 7.2, and prepare a small molecule peptide composition solution with a concentration of 1.3 mg / mL.

[0084] Construction of drug delivery system: Liposomes are used as carriers to encapsulate the small molecule peptide composition inside the liposomes. Liposomes are prepared by thin film dispersion method, where lipid materials such as phospholipids are dissolved in organic solvents, the organic solvents are removed by rotary evaporation to form a lipid film, and then the small molecule peptide composition solution is added for hydration. After ultrasonic treatment, liposomes loaded with the small molecule peptide composition are obtained. The particle size, morphology and membrane structure of the liposomes were characterized by dynamic light scattering and cryo-etching electron microscopy. The results showed that the average particle size of the liposomes was between 150-200nm, spherical and with a complete membrane structure.

[0085] Cell experiment: Rat primary neurons were taken, and the liposomes loaded with the small molecule peptide composition were co-cultured with the neurons. The energy metabolism and cell damage of the neurons were evaluated by detecting indicators such as the intracellular ATP content, lactate dehydrogenase (LDH) release, and mitochondrial membrane potential. At the same time, the morphology and distribution of mitochondria in the cells were observed using a fluorescence microscope. The results showed that compared with the control group, the ATP content in the neurons in the experimental group increased significantly, the LDH release decreased significantly, the mitochondrial membrane potential remained stable, and the mitochondrial morphology and distribution were more normal, indicating that the small molecule peptide composition can effectively improve the energy metabolism of neurons and reduce cell damage.

[0086] Animal experiment: A rat stroke model was established, and 36 hours after modeling, the liposomes loaded with the small molecule peptide composition were injected into the rats through the cerebroventricular cavity. On the 3rd, 7th and 14th days after treatment, the energy metabolism-related indicators of the nerve cells in the damaged brain area (such as ATP content, glucose uptake, etc.) were detected, and behavioral tests were performed to evaluate the recovery of the rats' neurological function. The results showed that the energy metabolism of the nerve cells in the damaged brain area of ​​the rats in the treatment group was significantly improved, and the symptoms of neurological deficits were significantly alleviated, indicating that the small molecule peptide composition can effectively improve the energy metabolism of nerve cells in vivo and promote the recovery of neurological function.

[0087] Example 5

[0088] Design principle of small molecule peptide: Considering the oxidative stress damage of nerve cells after stroke, a small molecule peptide with antioxidant function is designed. Oxidative stress will produce a large amount of reactive oxygen species (ROS), leading to lipid peroxidation, protein and DNA damage of nerve cells, affecting the survival and function of nerve cells. This small molecule peptide can protect nerve cells from damage by removing ROS and inhibiting the oxidative stress response.

[0089] Specific sequence: Pro-Gly-Cys-His-Thr-Asp-Val, molecular weight is about 880Da. Cysteine ​​(Cys) contains sulfhydryl (-SH) and has strong reducing property. It can directly react with ROS and reduce it to harmless substances, thus playing an antioxidant role; histidine (His) plays an important role in some antioxidant enzymes and may participate in the active center structure of antioxidant enzymes, or indirectly affect the antioxidant process by regulating the pH value in cells.

[0090] Target: Acts on the antioxidant enzyme system in nerve cells, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), etc., by interacting with these antioxidant enzymes, enhancing their activity, improving the antioxidant capacity of nerve cells, reducing the accumulation of ROS, and protecting nerve cells from oxidative stress damage.

[0091] Preparation method:

[0092] Synthesis of small molecule peptides: Solid phase synthesis is used to synthesize small molecule peptides step by step according to the amino acid sequence. During the synthesis process, attention should be paid to protecting the thiol group of cysteine ​​to prevent it from being oxidized during the reaction. By optimizing the reaction conditions, such as selecting appropriate protecting groups and reaction solvents, the smooth progress of the synthesis reaction is ensured. After the synthesis is completed, the purity and structure of the small molecule peptide are identified by high performance liquid chromatography and mass spectrometry to obtain a high-purity small molecule peptide product.

[0093] Modification of small molecule peptides: Methylation modification of small molecule peptides, introducing methyl groups on certain amino acid residues. By adding methylating agents to a specific reaction system and carrying out modification reactions under mild reaction conditions, the spatial structure and physicochemical properties of small molecule peptides are changed, and their stability and antioxidant activity are improved.

[0094] Preparation of small molecule peptide composition: Mix the antioxidant small molecule peptide with the targeted homing small molecule peptide and the differentiation regulating small molecule peptide in Example 1 in a molar ratio of 1:1:1.2, add an appropriate amount of MES buffer, adjust the pH to 6.8, and prepare a small molecule peptide composition solution with a concentration of 1.1 mg / mL.

[0095] Construction of drug delivery system: mesoporous silica nanoparticles (MSNs) modified with polydopamine (PDA) were used as carriers. First, MSNs were prepared, and then dopamine was self-polymerized and deposited on the surface of MSNs under alkaline conditions to form PDA-modified MSNs. The small molecule peptide composition was loaded onto PDA-MSNs, and the small molecule peptide was fixed by physical adsorption and electrostatic action. The morphology, pore size and specific surface area of ​​PDA-MSNs were characterized by transmission electron microscopy, nitrogen adsorption-desorption isotherm and other techniques. The results showed that PDA-MSNs had a regular spherical structure, an average particle size between 100-150nm, a uniform pore size distribution, and a large specific surface area, which was conducive to the loading and release of small molecule peptides.

[0096] Cell experiment: Mouse neuroblastoma cells (Neuro-2a) were taken, oxidative stress damage was induced with hydrogen peroxide (H2O2), and then PDA-MSNs loaded with small molecule peptide composition were co-cultured with cells with oxidative stress damage. The oxidative stress damage and antioxidant capacity changes of cells were evaluated by detecting indicators such as intracellular ROS level, malondialdehyde (MDA) content and antioxidant enzyme activity. At the same time, the apoptosis rate of cells was detected by flow cytometry. The results showed that compared with the untreated group, the intracellular ROS level and MDA content in the experimental group were significantly reduced, the antioxidant enzyme activity was significantly enhanced, and the cell apoptosis rate was significantly decreased, indicating that the small molecule peptide composition can effectively reduce the oxidative stress damage of nerve cells and protect cells from apoptosis.

[0097] Animal experiment: A mouse stroke model was established, and PDA-MSNs loaded with a small molecule peptide composition were injected into the mouse through the tail vein 24 hours after modeling. On the 3rd, 7th and 14th days after treatment, the oxidative stress-related indicators (such as ROS level, MDA content, antioxidant enzyme activity, etc.) in the damaged brain area were detected, and behavioral tests were performed to evaluate the neurological recovery of the mice. The results showed that the oxidative stress level in the damaged brain area of ​​the mice in the treatment group was significantly reduced, and the symptoms of neurological deficits were significantly improved, indicating that the small molecule peptide composition can effectively reduce oxidative stress damage in vivo and promote the recovery of neurological function.

[0098] Example 6

[0099] Small molecule peptide design principles:

[0100] After a stroke, the synaptic connections between nerve cells are damaged, resulting in obstruction of nerve signal transmission, which seriously affects the recovery of nerve function. The design of this small molecule peptide is based on the goal of promoting the formation and functional recovery of nerve cell synapses. Through in-depth research on the molecular mechanisms of nerve cell synaptic plasticity, it was found that some signaling pathways and proteins play a key role in the formation, development and functional maintenance of synapses. For example, neuregulin 1 (NRG1) and its receptor ErbB2-ErbB4 signaling pathway play an important regulatory role in the axon growth, dendritic branching, and synapse formation and function of nerve cells. This small molecule peptide is designed to mimic some of the functions of NRG1, and by activating this signaling pathway, it promotes the repair and reconstruction of nerve cell synapses, thereby improving nerve function.

[0101] Specific sequence:

[0102] After a lot of experimental research and screening, the sequence of the small molecule peptide was determined to be Ser-Asp-Glu-Lys-Pro-Ile-Leu, with a molecular weight of about 860Da. Serine (Ser) and aspartic acid (Asp) often serve as phosphorylation sites in the process of intracellular signal transduction, and participate in the regulation of various signal pathways. They may play a role in activating downstream signals after the small molecule peptide binds to the cell surface receptor; Glutamate (Glu) is an important component of neurotransmitters and is crucial for neural signal transmission. It may participate in regulating the function of synapses; Lysine (Lys) plays an important role in protein-protein interactions and may help the small molecule peptide bind to receptors or other related proteins; Proline (Pro) and isoleucine (Ile) help maintain the specific spatial structure of the small molecule peptide, ensuring its effective binding to the target and biological activity; Leucine (Leu) also plays an important role in the structural domains of some proteins, which may affect the overall stability and function of the small molecule peptide.

[0103] Target:

[0104] This small molecule peptide acts on the neuregulin 1 (NRG1) receptor on the surface of nerve cells. When the small molecule peptide binds to the NRG1 receptor, it can activate the downstream ErbB2-ErbB4 signaling pathway. Specifically, after the receptor is activated, it will trigger a series of phosphorylation cascade reactions, activating a variety of signaling molecules in the cell, such as phosphatidylinositol-3 kinase (PI3K), mitogen-activated protein kinase (MAPK), etc. These signaling molecules further regulate the expression of related genes, promote the axon growth, dendritic branching and synthesis and expression of synaptic proteins of nerve cells, thereby increasing the number and function of synapses, promoting signal transmission between nerve cells, and helping to restore damaged nerve function.

[0105] Preparation method:

[0106] Synthesis of small molecule peptides: prepared by solid phase synthesis. First, the first amino acid is covalently linked to a solid phase carrier (such as polystyrene resin), and then other amino acids are added in sequence according to the designed sequence. In each step of amino acid addition, the reaction conditions need to be strictly controlled. For example, the reaction temperature is maintained at 25-30°C to ensure the activity of the amino acid and the stability of the reaction; the reaction time is adjusted according to the reactivity of different amino acids, generally ranging from 1 to 3 hours, to ensure that each step of the reaction is fully carried out. After each amino acid is added, the purity and progress of the reaction are detected by high performance liquid chromatography (HPLC) to ensure the accuracy of the reaction. After the synthesis is completed, the small molecule peptide is separated from the solid phase carrier by a cleavage reaction, and then purified by reverse phase high performance liquid chromatography (RP-HPLC) to remove impurities and unreacted amino acids to obtain a high-purity small molecule peptide product.

[0107] Modification of small molecule peptides: Acetylation modification is performed on the synthesized small molecule peptides. An acetyl group is introduced at the N-terminus of the small molecule peptide to increase its stability and reduce the possibility of enzymatic hydrolysis in the body. The small molecule peptide is dissolved in a suitable organic solvent (such as dimethyl sulfoxide, DMSO), and an appropriate amount of acetylation reagent (such as acetic anhydride) and catalyst (such as pyridine) are added to react at room temperature for 1-2 hours. After the reaction is completed, the unreacted reagents and by-products are removed by dialysis or column chromatography to obtain an acetylated small molecule peptide.

[0108] Preparation of small molecule peptide composition: The modified small molecule peptide is mixed with the small molecule peptide with targeted homing function (such as Arg-Gly-Asp-Tyr-Ser-Thr-Leu) and the small molecule peptide with differentiation regulation function (such as His-Pro-Glu-Lys-Asp-Val-Ile) in Example 1 at a molar ratio of 1:1:1. Add an appropriate amount of phosphate buffer (PBS), adjust the pH value to 7.2, and prepare a small molecule peptide composition solution with a concentration of 1 mg / mL. PBS buffer has good buffering capacity, can maintain the stability of the small molecule peptide composition, and has low toxicity to cells and organisms.

[0109] Construction of drug delivery system: Polylactic acid-co-glycolic acid (PLGA) was selected as the carrier material, and PLGA nanoparticles loaded with small molecule peptide compositions were prepared by emulsification-solvent evaporation method. PLGA was dissolved in an organic solvent such as dichloromethane to form an organic phase; the small molecule peptide composition solution was mixed with an aqueous solution containing an emulsifier (such as polyvinyl alcohol, PVA) to form an aqueous phase. Under the action of ultrasound, the organic phase was slowly added dropwise to the aqueous phase to form a stable emulsion. The emulsion was then added dropwise to a large amount of aqueous solution containing PVA. Under stirring conditions, the organic solvent gradually evaporated, and PLGA gradually aggregated in the aqueous phase to form nanoparticles, encapsulating the small molecule peptide composition. The particle size distribution and morphology of the nanoparticles were characterized by dynamic light scattering (DLS) and scanning electron microscopy (SEM). DLS results showed that the average particle size of the nanoparticles was between 100-150nm, and the particle size distribution was uniform; SEM images showed that the nanoparticles were spherical and had a smooth surface.

[0110] Cell experiment: Rat primary nerve cells were cultured. PLGA nanoparticles loaded with small molecule peptide compositions were co-cultured with nerve cells, and a control group (only nerve cells were cultured without adding nanoparticles) and other control groups (such as only adding PLGA nanoparticles without small molecule peptide loading) were set up. On the 3rd, 5th and 7th days of culture, the axon growth length, the number of dendritic branches and the expression of synaptic proteins (such as synapsin) of nerve cells were detected by immunofluorescence staining. At the same time, the electrophysiological characteristics of nerve cells, such as the frequency and amplitude of action potentials, were detected by patch clamp technology to evaluate the functional state of nerve cells. The results showed that compared with the control group, the axon growth of nerve cells in the experimental group increased significantly, the number of dendritic branches increased significantly, the expression of synaptic proteins was significantly upregulated, and the frequency and amplitude of action potentials of nerve cells were also closer to normal levels, indicating that the small molecule peptide composition can effectively promote the formation of nerve cell synapses and functional recovery.

[0111] Animal experiment: A rat stroke model was established, and the ischemic stroke model was prepared by occluding the middle cerebral artery of the rat by suture embolism. 24 hours after modeling, PLGA nanoparticles loaded with small molecule peptide composition were injected into the rat through the tail vein. On the 3rd, 7th, 14th and 28th days after treatment, behavioral tests, such as modified neurological deficit score (mNSS) and Morris water maze test, were performed to evaluate the neurological recovery of the rats. At the same time, at different time points after treatment, the brain tissue of the rats was taken for immunohistochemical staining and Western blot detection to observe the axon growth, dendritic branching and synaptic protein expression changes of the nerve cells in the damaged brain area. The results showed that the mNSS score of the rats in the treatment group gradually decreased. In the Morris water maze test, the escape latency of the rats was significantly shortened, and the number of crossing the platform increased, indicating that the neurological function of the rats was significantly improved. The results of immunohistochemical staining and Western blot showed that the number of axons and dendrites of nerve cells in the damaged brain area increased, and the expression of synaptic proteins was significantly upregulated, further proving that the small molecule peptide composition can effectively promote the repair and reconstruction of nerve cell synapses in vivo and improve neural function.

[0112] Example 7

[0113] Design principle of small molecule peptide: Get inspiration from Danshen, a commonly used Chinese medicine for promoting blood circulation and removing blood stasis. The active ingredients in Danshen have multiple effects such as improving blood circulation and anti-oxidation. This small molecule peptide is designed to simulate the function of the active ingredients in Danshen, and promote the repair and regeneration of nerve cells by regulating the microenvironment of nerve cells. Considering the efficacy of Danshen in improving brain blood circulation, it is speculated that it may be achieved by affecting the function of vascular endothelial cells. Therefore, the designed small molecule peptide hopes to act on vascular endothelial cells, regulate related signal pathways, and then improve the blood supply of nerve cells and promote their repair.

[0114] Specific sequence: The small molecule peptide sequence obtained from the Danshen extract after separation, screening and identification is Thr-Lys-Asp-Glu-His-Pro-Tyr, with a molecular weight of about 890Da. Threonine (Thr) and lysine (Lys) play a role in some cell-to-cell interactions and signal transduction processes, and may help the binding of small molecule peptides to cell surface receptors; histidine (His) and tyrosine (Tyr) have potential roles in regulating the physiological functions and signal transduction of cells, and may be involved in signal pathways related to angiogenesis and neuroprotection.

[0115] Target: Endothelial nitric oxide synthase (eNOS) acting on the surface of vascular endothelial cells. By binding to eNOS, the activity of the enzyme is activated, promoting the synthesis and release of nitric oxide (NO). NO has the effects of vasodilation and inhibiting platelet aggregation, which can improve blood circulation in damaged brain areas, provide more oxygen and nutrients for nerve cells, and promote the repair and functional recovery of nerve cells.

[0116] Preparation method:

[0117] Extraction and purification of small molecule peptides: Select high-quality Danshen medicinal materials, and obtain Danshen crude extracts through conventional steps such as crushing and ethanol extraction. Then use chromatographic techniques such as silica gel column chromatography and high-performance liquid chromatography to separate and purify the crude extracts. Determine the sequence and structure of the target small molecule peptide through mass spectrometry analysis, amino acid sequencing, and other means to obtain high-purity small molecule peptides.

[0118] Modification of small molecule peptides: The extracted small molecule peptides are PEGylated, and a polyethylene glycol (PEG) chain with a suitable molecular weight (such as 5000Da) is connected to its N-terminus. The connection between PEG and small molecule peptides is achieved through chemical coupling reaction. PEGylation can increase the water solubility of small molecule peptides, prolong their circulation time in the body, and reduce immunogenicity.

[0119] Preparation of a small molecule peptide composition: Mix the small molecule peptide derived from Salvia miltiorrhiza extract with the targeted homing small molecule peptide and the differentiation regulating small molecule peptide in Example 1 in a molar ratio of 1:1.1:1, add an appropriate amount of citrate buffer, adjust the pH to 6.5, and prepare a small molecule peptide composition solution with a concentration of 1.2 mg / mL.

[0120] Construction of drug delivery system: Nano lipid carrier (NLC) was used as a carrier. Lipid materials such as phospholipids and fatty acids were mixed with a small molecule peptide composition solution, and NLC loaded with a small molecule peptide composition was prepared by high-pressure homogenization. The particle size and surface morphology of NLC were characterized by dynamic light scattering and atomic force microscopy. The results showed that the average particle size of NLC was between 100-150nm, spherical and smooth on the surface.

[0121] Cell experiment: Human umbilical vein endothelial cells (HUVECs) were taken, and the NLC loaded with the small molecule peptide composition was co-cultured with HUVECs. The effect of small molecule peptides on eNOS activity was evaluated by detecting the content of NO in the cell culture supernatant and using Western blot to detect the expression and phosphorylation level of eNOS protein. At the same time, the Transwell experiment was used to detect the migration ability of cells. The results showed that compared with the control group, the release of NO in the experimental group increased significantly, the phosphorylation level of eNOS protein increased, and the cell migration ability was enhanced, indicating that the small molecule peptide composition can effectively activate eNOS and promote the function of vascular endothelial cells.

[0122] Animal experiment: A rat stroke model was established, and NLC loaded with a small molecule peptide composition was injected into the rat through the tail vein 36 hours after modeling. On the 7th and 14th days after treatment, the blood perfusion of the damaged brain area was detected by laser Doppler blood flowmetry, and behavioral tests were performed to evaluate the neurological recovery of the rats. The results showed that the blood perfusion of the damaged brain area of ​​the rats in the treatment group was significantly improved, and the symptoms of neurological deficits were significantly alleviated, indicating that the small molecule peptide composition can effectively improve brain blood circulation in vivo and promote the recovery of neurological function.

[0123] Example 8

[0124] Design principle of small molecule peptide: Designed based on the neuroprotective effect of ginseng extract. Ginseng contains a variety of active ingredients, such as ginsenosides, which have antioxidant, anti-inflammatory, and promotion of nerve cell proliferation and differentiation functions. This small molecule peptide aims to screen out peptides with similar functions from ginseng extract, and enhance the damage resistance and repair ability of nerve cells by regulating the metabolism and signaling pathways of nerve cells. Considering the role of ginseng in regulating the neuroendocrine system and immune function, it is speculated that it may be achieved by affecting certain receptors on the surface of nerve cells. Therefore, the designed small molecule peptide hopes to act on specific receptors on the surface of nerve cells, regulate signal transduction within the cells, and promote the repair and regeneration of nerve cells.

[0125] Specific sequence: After in-depth research and screening of ginseng extracts, the small molecule peptide sequence obtained is Glu-Val-Ser-Thr-Pro-Arg-Gly, with a molecular weight of about 840Da. Glutamic acid (Glu) and serine (Ser) play an important role in cell metabolism and signal transduction, and may participate in a variety of biochemical reactions in nerve cells; arginine (Arg) and glycine (Gly) play a potential role in some neuroprotective and immunomodulatory processes, and may protect nerve cells by regulating the redox state of cells and immune response.

[0126] Target: Glucocorticoid receptor (GR) acting on the surface of nerve cells. When small molecule peptides bind to GR, they can regulate the activity of GR and affect the expression of a series of downstream genes related to nerve protection and repair. For example, it can upregulate the expression of antioxidant enzyme genes to enhance the antioxidant capacity of nerve cells; regulate the expression of inflammation-related genes to reduce the inflammatory response of nerve cells; promote the secretion of neurotrophic factors such as nerve growth factor, and promote the survival and regeneration of nerve cells.

[0127] Preparation method:

[0128] Extraction and purification of small molecule peptides: Select high-quality ginseng medicinal materials, and obtain ginseng extracts after preliminary treatments such as water extraction and alcohol precipitation. Then use ultrafiltration, gel filtration chromatography and other technologies to further separate and purify the extracts. The structure and sequence of the target small molecule peptides are determined by amino acid analysis, nuclear magnetic resonance and other means to obtain high-purity small molecule peptides.

[0129] Modification of small molecule peptides: Acetylation modification is performed on the extracted small molecule peptides to introduce an acetyl group at their N-terminus. In a suitable reaction system, an acetylation reagent is added and the modification reaction is performed under mild reaction conditions to increase the stability of the small molecule peptides and reduce their degradation in the body.

[0130] Preparation of a small molecule peptide composition: Mix the small molecule peptide derived from ginseng extract with the targeted homing small molecule peptide and the differentiation regulating small molecule peptide in Example 1 in a molar ratio of 1:1:1.3, add an appropriate amount of HEPES buffer, adjust the pH to 7.4, and prepare a small molecule peptide composition solution with a concentration of 1.3 mg / mL.

[0131] Construction of drug delivery system: Nanocomplexes were constructed using chitosan and hyaluronic acid as carriers. Chitosan was dissolved in a dilute acetic acid solution, and hyaluronic acid was dissolved in physiological saline. The two were then mixed and a small molecule peptide composition solution was added to form a nanocomplex through electrostatic interaction. The morphology and surface potential of the nanocomplex were characterized using a scanning electron microscope and a zeta potential analyzer. The results showed that the nanocomplex was spherical, with an average particle size between 150-200nm and a positive charge on the surface, which was conducive to its interaction with the cell surface.

[0132] Cell experiment: Mouse neural stem cells were taken, and the nanocomplex loaded with the small molecule peptide composition was co-cultured with the neural stem cells. The effect of small molecule peptides on neural stem cells was evaluated by detecting the activity of antioxidant enzymes, the expression of inflammatory factors, and the secretion of nerve growth factor in the cells. At the same time, immunofluorescence staining was used to detect the differentiation of neural stem cells. The results showed that compared with the control group, the activity of antioxidant enzymes in the experimental group was significantly enhanced, the expression of inflammatory factors was reduced, the secretion of nerve growth factor was increased, and the differentiation ratio of neural stem cells to neurons was increased, indicating that the small molecule peptide composition can effectively promote the proliferation, differentiation and neuroprotection of neural stem cells.

[0133] Animal experiment: A mouse stroke model was established, and the nanocomplex loaded with the small molecule peptide composition was injected into the damaged brain area through brain stereotaxic 24 hours after modeling. On the 3rd, 7th and 14th days after treatment, the expression of antioxidant enzymes, inflammatory factors and nerve growth factors in the damaged brain area was detected by immunohistochemical staining, and behavioral tests were performed to evaluate the neurological recovery of mice. The results showed that the expression of antioxidant enzymes in the damaged brain area of ​​the mice in the treatment group increased, the expression of inflammatory factors decreased, the expression of nerve growth factor was upregulated, and the symptoms of neurological deficits were significantly improved, indicating that the small molecule peptide composition can effectively play a role in neuroprotection and repair in vivo.

[0134] Example 9

[0135] Design principle of small molecule peptides: Look for small molecule peptides with neuroprotective and repairing effects from green tea extracts. Green tea is rich in tea polyphenols and other bioactive ingredients, which have antioxidant, anti-inflammatory, anti-apoptotic and other effects. The design idea of ​​this small molecule peptide is to simulate the mechanism of action of the active ingredients in green tea, and to reduce the damage to nerve cells and promote their repair by regulating the redox balance and signal pathways in nerve cells. Considering the role of green tea in inhibiting the apoptosis of nerve cells, it is speculated that it may be achieved by affecting the apoptosis-related signal pathways in the cells. Therefore, the designed small molecule peptide hopes to act on the apoptosis-related proteins in nerve cells, regulate the apoptosis process of cells, and promote the survival of nerve cells.

[0136] Specific sequence: After studying and screening green tea extracts, the determined small molecule peptide sequence is Pro-Cys-His-Asp-Val-Leu-Ser, with a molecular weight of about 870Da. Proline (Pro) and cysteine ​​(Cys) play an important role in maintaining the structure and function of proteins, and the sulfhydryl group of cysteine ​​also has antioxidant capacity; histidine (His) and aspartic acid (Asp) play a role in cell metabolism and signal transduction, and may be involved in regulating cell apoptosis-related signaling pathways; leucine (Leu) and serine (Ser) help maintain the spatial structure and biological activity of small molecule peptides.

[0137] Target: Acts on Bcl-2 family proteins in nerve cells, especially Bax and Bcl-2. By interacting with Bax and Bcl-2, it regulates the ratio and function between them. Specifically, it may inhibit the pro-apoptotic activity of Bax and promote the anti-apoptotic function of Bcl-2, thereby reducing the apoptosis of nerve cells and promoting the survival and repair of nerve cells.

[0138] Preparation method:

[0139] Extraction and purification of small molecule peptides: Select high-quality green tea leaves, and obtain green tea crude extracts through water extraction, filtration, concentration and other steps. Then use reversed-phase high-performance liquid chromatography, ion exchange chromatography and other technologies to separate and purify the crude extracts. The sequence and structure of the target small molecule peptides are determined by mass spectrometry analysis, amino acid sequencing and other means to obtain high-purity small molecule peptides.

[0140] Modification of small molecule peptides: Glycosylation modification is performed on the extracted small molecule peptides, and sugar chains are connected to specific amino acid residues. Through chemical synthesis, reagents containing sugar groups are reacted with small molecule peptides under appropriate reaction conditions to connect sugar groups to small molecule peptides. Glycosylation modification can increase the stability and water solubility of small molecule peptides, and may also enhance their binding affinity with the target.

[0141] Preparation of a small molecule peptide composition: The small molecule peptide derived from green tea extract was mixed with the targeted homing small molecule peptide and the differentiation regulating small molecule peptide in Example 1 at a molar ratio of 1.2:1:1, an appropriate amount of phosphate buffer (PBS) was added, the pH value was adjusted to 7.2, and a small molecule peptide composition solution with a concentration of 1.1 mg / mL was prepared.

[0142] Construction of drug delivery system: Polylactic acid-co-glycolic acid (PLGA) nanoparticles were used as carriers. PLGA was dissolved in an organic solvent, and a small molecule peptide composition solution was added. An emulsion was formed by ultrasonic emulsification, and then the organic solvent was volatilized to obtain PLGA nanoparticles loaded with the small molecule peptide composition. The particle size and morphology of PLGA nanoparticles were characterized by dynamic light scattering and transmission electron microscopy. The results showed that the average particle size of PLGA nanoparticles was between 120-180nm, spherical and uniformly distributed.

[0143] Cell experiment: Rat primary neurons were taken, and the oxygen-glucose deprivation (OGD) model was used to induce neuronal apoptosis. Then, the PLGA nanoparticles loaded with the small molecule peptide composition were co-cultured with the damaged neurons. The apoptosis rate of cells was detected by flow cytometry, and the expression levels of Bax and Bcl-2 proteins were detected by Western blot. The results showed that compared with the control group, the apoptosis rate of cells in the experimental group was significantly reduced, the expression of Bcl-2 protein was upregulated, and the expression of Bax protein was downregulated, indicating that the small molecule peptide composition can effectively inhibit the apoptosis of neurons.

[0144] Animal experiment: A rat stroke model was established, and PLGA nanoparticles loaded with a small molecule peptide composition were injected into the rats through the tail vein 48 hours after modeling. On the 7th and 14th days after treatment, TUNEL staining was used to detect the apoptosis of nerve cells in the damaged brain area, and behavioral tests were performed to evaluate the recovery of the rats' neurological function. The results showed that the apoptosis of nerve cells in the damaged brain area of ​​the rats in the treatment group was significantly reduced, and the symptoms of neurological deficits were significantly improved, indicating that the small molecule peptide composition can effectively inhibit the apoptosis of nerve cells in vivo and promote the recovery of neurological function.

[0145] Example 10

[0146] Design principle of small molecule peptides: Small molecule peptides are designed based on the biological activity of turmeric extracts. Curcumin, the main active ingredient in turmeric, has multiple effects such as anti-inflammatory, antioxidant, and neuroprotective. The design of this small molecule peptide aims to obtain peptides with similar functions from turmeric extracts, and promote the repair and regeneration of nerve cells by regulating the inflammatory response and oxidative stress state of nerve cells. Considering the role of turmeric in regulating immune and inflammation-related signaling pathways, it is speculated that it may be achieved by affecting certain inflammatory mediators and signaling molecules in cells. Therefore, the designed small molecule peptide hopes to act on the inflammation-related signaling pathways in nerve cells, inhibit the inflammatory response, and reduce the damage to nerve cells.

[0147] Specific sequence: The small molecule peptide sequence isolated and identified from turmeric extract is His-Glu-Lys-Asp-Pro-Tyr-Thr, with a molecular weight of about 880Da. Histidine (His) and glutamic acid (Glu) play an important role in cell metabolism and signal transduction, and may be involved in regulating the expression of inflammation-related genes; lysine (Lys) and aspartic acid (Asp) play a role in some protein-protein interactions and signal transduction processes, and may affect the release of inflammatory mediators and the activation of inflammatory signaling pathways; proline (Pro), tyrosine (Tyr) and threonine (Thr) help maintain the spatial structure and biological activity of small molecule peptides.

[0148] Target: Acts on the nuclear factor-κB (NF-κB) signaling pathway in nerve cells. By interacting with key proteins in the NF-κB signaling pathway, it inhibits the activation of NF-κB and reduces the expression and release of inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), thereby alleviating the inflammatory response of nerve cells and protecting nerve cells from inflammatory damage.

[0149] Preparation method:

[0150] Extraction and purification of small molecule peptides: Select high-quality turmeric medicinal materials, and obtain turmeric crude extracts through steps such as crushing and ethanol extraction. Then use silica gel column chromatography, preparative high-performance liquid chromatography and other technologies to separate and purify the crude extracts. The sequence and structure of the target small molecule peptide are determined by mass spectrometry analysis, amino acid sequencing and other means to obtain high-purity small molecule peptides.

[0151] Modification of small molecule peptides: Phosphorylation modification is performed on the extracted small molecule peptides to introduce phosphate groups on specific serine (Ser) or threonine (Thr) residues. In a reaction system containing a phosphorylation reagent, the modification reaction is carried out under appropriate reaction conditions to change the charge properties and spatial structure of the small molecule peptides, thereby enhancing their binding ability to the target and their biological activity.

[0152] Preparation of a small molecule peptide composition: The small molecule peptide derived from turmeric extract was mixed with the targeted homing small molecule peptide and the differentiation regulating small molecule peptide in Example 1 in a molar ratio of 1:1.1:1, an appropriate amount of Tris-HCl buffer was added, and the pH value was adjusted to 7.3 to prepare a small molecule peptide composition solution with a concentration of 1.2 mg / mL.

[0153] Construction of drug delivery system: Liposomes are used as carriers to encapsulate the small molecule peptide composition inside the liposomes. Liposomes are prepared by thin film dispersion method, where lipid materials such as phospholipids are dissolved in organic solvents, the organic solvents are removed by rotary evaporation to form a lipid film, and then the small molecule peptide composition solution is added for hydration. After ultrasonic treatment, liposomes loaded with the small molecule peptide composition are obtained. The particle size, morphology and membrane structure of the liposomes were characterized by dynamic light scattering and cryo-etching electron microscopy. The results showed that the average particle size of the liposomes was between 150-200nm, spherical and with a complete membrane structure.

[0154] Cell experiment: Mouse glial cells were taken, lipopolysaccharide (LPS) was used to induce an inflammatory response, and then the liposomes loaded with the small molecule peptide composition were co-cultured with glial cells in an inflammatory state. The content of inflammatory mediators in the cell culture supernatant was detected by ELISA, and the expression of inflammation-related genes was detected by real-time fluorescence quantitative PCR. The results showed that compared with the control group, the release of inflammatory mediators in the experimental group was significantly reduced, and the expression of inflammation-related genes was significantly downregulated, indicating that the small molecule peptide composition can effectively inhibit the inflammatory response of glial cells.

[0155] Animal experiment: A mouse stroke model was established, and liposomes loaded with small molecule peptide compositions were injected into the mice through the cerebroventricular cavity 36 hours after modeling. On the 3rd, 7th and 14th days after treatment, the expression of inflammatory mediators in the damaged brain area was detected by immunohistochemical staining, and behavioral tests were performed to evaluate the neurological recovery of mice. The results showed that the expression of inflammatory mediators in the damaged brain area of ​​the mice in the treatment group was significantly reduced, and the symptoms of neurological deficits were significantly improved, indicating that the small molecule peptide composition can effectively inhibit the inflammatory response in vivo and promote the recovery of neurological function.

Claims

1. A small molecule peptide composition based on stem cell targeted repair of stroke brain cells, characterized in that: The composition comprises at least one of the following: A small molecule peptide with targeted homing function, the sequence of which is Arg-Gly-Asp-Tyr-Ser-Thr-Leu, and the molecular weight is between 500-1000Da; A small molecule peptide with differentiation regulation function, whose sequence is His-Pro-Glu-Lys-Asp-Val-Ile and molecular weight is between 600-1200Da; As well as small molecule peptides derived from salvia miltiorrhiza extract, whose sequence is Thr-Lys-Asp-Glu-His-Pro-Tyr and whose molecular weight is about 890Da; small molecule peptides derived from ginseng extract, whose sequence is Glu-Val-Ser-Thr-Pro-Arg-Gly and whose molecular weight is about 840Da; small molecule peptides derived from green tea extract, whose sequence is Pro-Cys-His-Asp-Val-Leu-Ser and whose molecular weight is about 870Da; small molecule peptides derived from turmeric extract, whose sequence is His-Glu-Lys-Asp-Pro-Tyr-Thr and whose molecular weight is about 880Da.

2. The small molecule peptide composition according to claim 1, characterized in that: The small molecule peptide with targeted homing function can specifically bind to the integrin receptors on the surface of vascular endothelial cells in the damaged brain area, and guide the stem cells to migrate to the damaged brain area.

3. The small molecule peptide composition according to claim 1, characterized in that: The small molecule peptide with differentiation regulation function can bind to the Notch receptor on the surface of stem cells, activate the Notch signaling pathway, and promote the differentiation of stem cells into neurons.

4. The small molecule peptide composition according to claim 1, characterized in that: The small molecule peptide derived from the salvia miltiorrhiza extract acts on endothelial nitric oxide synthase on the surface of vascular endothelial cells, activates the activity of the enzyme, promotes the synthesis and release of nitric oxide (NO), and improves blood circulation in damaged brain areas.

5. The small molecule peptide composition according to claim 1, characterized in that: The small molecule peptide derived from ginseng extract acts on the glucocorticoid receptor on the surface of nerve cells, regulates GR activity, and affects the expression of downstream genes related to nerve protection and repair.

6. The small molecule peptide composition according to claim 1, characterized in that: The small molecule peptide derived from green tea extract acts on Bcl-2 family proteins in nerve cells, regulates the ratio and function between Bax and Bcl-2, and inhibits nerve cell apoptosis.

7. The small molecule peptide composition according to claim 1, characterized in that: The small molecule peptide derived from the turmeric extract acts on the nuclear factor-κB (NF-κB) signaling pathway in nerve cells, inhibits the activation of NF-κB, and reduces the expression and release of inflammatory mediators.

8. A method for preparing a small molecule peptide composition based on stem cell targeted repair of stroke brain cells, characterized in that: The following steps are involved: The small molecule peptides are obtained by solid phase synthesis or extracted and purified from plant extracts. The targeted homing and differentiation regulating small molecule peptides are synthesized by solid phase synthesis. The corresponding small molecule peptides are extracted and purified from salvia miltiorrhiza, ginseng, green tea and turmeric extracts respectively. Modifying small molecule peptides, including PEGylation, acetylation, glycosylation, and phosphorylation; The modified small molecule peptides are mixed in a ratio of 1:1, an appropriate amount of buffer is added, and the pH value is adjusted to prepare a small molecule peptide composition solution; A drug delivery system is constructed, wherein nano lipid carriers, chitosan and hyaluronic acid nanocomplexes, polylactic acid-glycolic acid copolymer nanoparticles, and liposomes are used as carriers to load the small molecule peptide composition therein.

9. The preparation method according to claim 8, characterized in that: During the synthesis or extraction and purification of small molecule peptides, high performance liquid chromatography, mass spectrometry analysis and amino acid sequencing are used to detect and determine the purity, structure and sequence of small molecule peptides.

10. Use of the small molecule peptide composition according to any one of claims 1 to 7 in the preparation of a drug for treating cerebral stroke.

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