Atractylodes lancea exosome loaded with atractylenolide II, extraction method of atractylodes lancea exosome and application of atractylodes lancea exosome in preparation of medicine for preventing and treating myocardial injury

Abstract

The invention discloses rhizoma atractylodis exosomes loaded with atractylenolide II, an extraction method of the rhizoma atractylodis exosomes and application of the rhizoma atractylodis exosomes in preparation of medicines for preventing and treating myocardial injury, and belongs to the technical field of biology. The extraction method of the rhizoma atractylodis exosome comprises the following steps: mixing rhizoma atractylodis and a PBS (Phosphate Buffer Solution), squeezing to obtain rhizoma atractylodis juice, filtering, centrifuging to remove precipitate and reserve supernatant, centrifuging to discard the supernatant and reserve precipitate at the bottom of a tube, blowing and beating the precipitate in a centrifugal tube by using the PBS, resuspending, filtering the suspension, and drying to obtain the rhizoma atractylodis exosome. And then concentrating to obtain the atractylodes lancea exosome. The invention also provides the atractylodes lancea exosome loaded with atractylenolide II, or application of the atractylodes lancea exosome in preparation of medicines for preventing and treating myocardial injury. The atractylodes lancea exosome loaded with atractylenolide II or the atractylodes lancea exosome provided by the invention can remarkably recover mitochondrial energy metabolism so as to protect myocardial cells, and a brand new application prospect is provided for treatment of myocardial diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an Atractylodes lancea exosome loaded with atractylodes lactone II, its extraction method, and its application in the preparation of drugs for preventing and treating myocardial injury. Background Technology

[0002] Cardiovascular disease (CVD) is one of the leading causes of death worldwide, with its incidence and mortality rates remaining high due to population aging. Statistics show that CVD causes approximately 17.9 million deaths annually, accounting for 32% of global deaths, while acute cardiac events such as septic cardiomyopathy and ischemia-reperfusion injury account for over 40% of deaths among critically ill patients. The pathogenesis of CVD is extremely complex, involving multiple factors, including genetic factors, lifestyle (such as smoking, lack of exercise, and unhealthy diet), metabolic disorders (such as diabetes and high cholesterol), and environmental factors. Mitochondrial dysfunction is considered one of the key pathological bases of CVD. Clinical and animal studies have consistently confirmed that the "energy-inflammation double whammy" caused by infection or ischemia is the core link in myocardial damage: on the one hand, circulating endotoxins (LPS) surge; on the other hand, local glucose / fatty acid supply is interrupted, leading to carbon starvation (CS). These two factors synergistically simulate the real-world scenario of "infection-ischemia," rapidly inducing mitochondrial dysfunction in cardiomyocytes. However, the specific molecular mechanisms have not yet been systematically elucidated, and interventions targeting this pathological axis are lacking. Therefore, developing a drug delivery system that combines "energy metabolism reprogramming and mitochondrial quality control" using a cardiomyocyte model of mitochondrial synergistic damage by LPS and carbon starvation is a technical bottleneck that urgently needs to be overcome in the field of acute cardiac injury.

[0003] Traditional Chinese medicine (TCM) possesses unique advantages in the prevention and treatment of cardiovascular diseases. Its multi-component, multi-target, and holistic regulatory approach can fundamentally regulate the body's internal environment, particularly exerting its unique therapeutic effects by modulating mitochondrial function. In recent years, exosomes, as important mediators of intercellular communication, have received widespread attention due to their potential applications in disease treatment. Exosomes are nanoscale vesicles secreted by cells, with diameters between 30-150 nm, capable of carrying bioactive molecules such as proteins, nucleic acids (e.g., mRNA, miRNA), and lipids, playing a crucial role in intercellular communication and disease regulation. Exosomes can regulate mitochondrial function by delivering mitochondrial-related molecules (e.g., mtDNA, miRNA). Plant-derived exosome-like nanovesicles (PELNs) have become a research hotspot in the modernization of TCM delivery platforms due to their triple advantages of "natural targeting, high biocompatibility, and engineerability." Atractylodes lancea (Thunb.) DC. is a perennial herb of the Asteraceae family. It has a pungent and bitter taste, is warm in nature, and enters the spleen, stomach, and liver meridians. The *Compendium of Materia Medica* records that it "treats wind-cold-dampness arthralgia, dead muscle, spasms, jaundice, stops sweating, clears heat, and aids digestion." Modern phytochemical studies have shown that Atractylodes lancea is rich in sesquiterpene lactones (atractylodes lactones I / II / III), alkenes, polysaccharides, and volatile oils, exhibiting significant antioxidant, anti-inflammatory, immunomodulatory, and metabolic regulatory activities. However, the oral bioavailability of Atractylodes lancea's active ingredients is low (<5%) and its water solubility is poor, meaning its therapeutic potential in acute and critical conditions such as myocardial ischemia and septic cardiomyopathy is far from being realized. Atractylodes lancea-derivedexosomes (AL-Exo) have a diameter of 50-150 nm, and their membrane surface is enriched with Atractylodes lancea-specific glycoproteins and phosphatidic acids. The active ingredients of Atractylodes lancea can be delivered through its exosomes, which is expected to become a new and efficient delivery system for active ingredients in traditional Chinese medicine, providing new ideas and methods for the modernization of traditional Chinese medicine.

[0004] Atractylenolide II (AT-II) is a diterpene lactone active ingredient with the molecular formula C2. 15 H 20O2 has extremely low toxicity. Its traditional efficacy focuses on "strengthening the spleen and replenishing qi, drying dampness and promoting diuresis," while modern pharmacological studies have revealed that it also possesses multi-target activities such as antioxidant, anti-apoptotic, calcium homeostasis regulation, and metabolic regulation, and has been used in models for neuroprotection, fatty liver, and atherosclerosis. However, systematic research on AT-II in mitochondrial dysfunction-induced cardiomyopathy remains lacking. AT-II has poor water solubility (logP ≈ 3.8), and traditional tablets or injections have insufficient cardiac targeting, making it difficult to achieve effective concentrations in the mitochondria of the lesion. Therefore, clarifying the role of AT-II in cardiomyocyte mitochondrial dysfunction and constructing a platform for efficient delivery of this small molecule are crucial steps in filling the current technological gap and are also key prerequisites for developing drugs for myocardial injury. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide an Atractylodes lancea exosome loaded with atractylodes lactone II, its extraction method, and its application in the preparation of drugs for preventing and treating myocardial injury, thereby solving the technical problem of how to effectively prevent and treat myocardial injury in the prior art.

[0006] To achieve the above-mentioned technical objectives, the present invention provides a method for extracting Atractylodes lancea exosomes, comprising the following steps:

[0007] Atractylodes lancea juice was obtained by mixing Atractylodes lancea and PBS buffer, then filtered, and then centrifuged multiple times at different speeds from low to high to remove the precipitate and retain the supernatant. Then, the supernatant was discarded by ultracentrifugation, and the precipitate at the bottom of the tube was retained. The precipitate in the centrifuge tube was then blown with PBS buffer and resuspended, then the suspension was filtered, and then concentrated to obtain the Atractylodes lancea exosomes.

[0008] In any embodiment, the multiple differential centrifugations include: centrifuging at 1000-1500 x g for 8-10 min, discarding the precipitate and retaining the supernatant; centrifuging at 3000-3500 x g for 25-30 min, discarding the precipitate and retaining the supernatant; and centrifuging at 10,000-10500 x g for 55-60 min, discarding the precipitate and collecting the supernatant.

[0009] In addition, the present invention also proposes an Atractylodes lancea exosome, which is prepared by the above extraction method.

[0010] Furthermore, the present invention also proposes the application of the above-mentioned Atractylodes exosomes in the preparation of Atractylodes exosomes loaded with Atractylodes lactone II.

[0011] In any embodiment, the above application includes the following steps: mixing atractylodes lactone II and atractylodes exosomes at a mass ratio of (2-2.5):1 in PBS buffer, followed by sonication for 1-1.5 min to obtain a mixture; incubating the mixture at 4-5°C for 20-24 h, centrifuging and concentrating to collect atractylodes exosomes loaded with atractylodes lactone II.

[0012] The present invention also proposes the use of the above-mentioned Atractylodes exosomes loaded with Atractylodes lactone II, or the use of Atractylodes exosomes in the preparation of drugs for the prevention and treatment of myocardial injury.

[0013] In any embodiment, the myocardial injury is LPS-carbon starvation synergistic myocardial injury.

[0014] In any embodiment, the drug prevents and treats myocardial injury by restoring mitochondrial energy metabolism in cardiomyocytes, clearing mitochondrial ROS, and / or stabilizing mitochondrial membrane potential.

[0015] Compared with the prior art, the beneficial effects of the present invention include: the Atractylodes exosomes loaded with Atractylodes lactone II proposed in the present invention can significantly restore mitochondrial energy metabolism and thus protect cardiomyocytes, providing a new application prospect for the treatment of myocardial diseases. Attached Figure Description

[0016] Figure 1 This is a flowchart of the extraction of Atractylodes lancea exosomes in Embodiment 1 of the present invention.

[0017] Figure 2 This is an electron microscope image of the exosomes of Atractylodes lancea in Example 2 of the present invention.

[0018] Figure 3 This is the particle size distribution of Atractylodes lancea exosomes in Example 2 of the present invention.

[0019] Figure 4 This is a graph showing the detection results of the effects of atractylodes lactone II, atractylodes exosomes, and atractylodes exosomes loaded with atractylodes lactone II on the viability of H9c2 cells in Example 4 of this invention.

[0020] Figure 5 This is a graph showing the detection results of Atractylodes lactone II, Atractylodes exosomes, and Atractylodes exosomes loaded with Atractylodes lactone II on the mitochondrial ROS clearance capacity of H9c2 cells in the LPS+cs model in Example 5 of the present invention.

[0021] Figure 6 This is a graph showing the detection results of Atractylodes lactone II, Atractylodes exosomes, and Atractylodes exosomes loaded with Atractylodes lactone II on the ability of H9c2 cells in the LPS+cs model to restore mitochondrial membrane potential in Example 6 of the present invention.

[0022] Figure 7 This is a graph showing the detection results of Atractylodes macrocephala lactone II, Atractylodes lancea exosomes, and Atractylodes lancea exosomes loaded with Atractylodes macrocephala lactone II on the ATP synthesis recovery ability of H9c2 cells in the LPS+cs model in Example 7 of the present invention. Detailed Implementation

[0023] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60~120 and 80~110 are listed for a specific parameter, it is also expected that ranges of 60~110 and 80~120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1~3, 1~4, 1~5, 2~3, 2~4, and 2~5. In this application, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0024] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0025] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0026] This specific embodiment provides a method for extracting Atractylodes lancea exosomes, including the following steps:

[0027] Atractylodes lancea juice was obtained by mixing Atractylodes lancea and PBS buffer, then filtered, and then centrifuged multiple times at different speeds from low to high to remove the precipitate and retain the supernatant. Then, the supernatant was discarded by ultracentrifugation, and the precipitate at the bottom of the tube was retained. The precipitate in the centrifuge tube was then blown with PBS buffer and resuspended, then the suspension was filtered, and then concentrated to obtain the Atractylodes lancea exosomes.

[0028] In some embodiments, the multiple differential centrifugations include: centrifuging at 1000-1500 x g for 8-10 min, discarding the precipitate and retaining the supernatant; centrifuging at 3000-3500 x g for 25-30 min, discarding the precipitate and retaining the supernatant; and centrifuging at 10,000-10500 x g for 55-60 min, discarding the precipitate and collecting the supernatant.

[0029] Furthermore, this specific embodiment also proposes an Atractylodes lancea exosome, which is prepared by the above extraction method.

[0030] This specific embodiment also proposes the application of Atractylodes lancea exosomes in the preparation of Atractylodes lancea exosomes loaded with atractylodes lactone II, including the following steps: Atractylodes lactone II and Atractylodes lancea exosomes are mixed in PBS buffer at a mass ratio of (2-2.5):1, and then sonicated for 1-1.5 min to obtain a mixture; the mixture is incubated at 4-5℃ for 20-24 h, centrifuged and concentrated to obtain Atractylodes lancea exosomes loaded with atractylodes lactone II.

[0031] Furthermore, this specific embodiment also proposes the application of the above-mentioned Atractylodes exosomes loaded with atractylodes lactone II in the preparation of drugs for preventing and treating myocardial injury.

[0032] In some embodiments, the myocardial injury is LPS-carbon starvation synergistic myocardial injury.

[0033] In some embodiments, the drug prevents and treats myocardial injury by restoring mitochondrial energy metabolism in cardiomyocytes, clearing mitochondrial ROS, and / or stabilizing mitochondrial membrane potential.

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] In this invention, the terms "some embodiments," "this embodiment," and examples are used to describe a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.

[0036] If the application documents contain similar descriptions such as "first / second", the following explanation shall be added: In the following description, the terms "first / second / third" are used only to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first / second / third" may be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0037] In this embodiment, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, object A and / or object B can represent three situations: object A exists alone, object A and object B exist simultaneously, and object B exists alone.

[0038] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0039] Example 1: Extraction of Atractylodes lancea exosomes

[0040] This embodiment proposes a method for extracting exosomes from Atractylodes lancea, including the following steps:

[0041] 1. Pretreatment and impurity removal: Thoroughly wash and dry the surface of the Atractylodes lancea sample, removing any dirt and epidermis. Place the sample in a fume hood to air dry. Extract the juice using a juicer by mixing 1xPBS (pH 7.4) at a volume ratio of 1.5:1 (mL / g) of Atractylodes lancea. Avoid prolonged operation to prevent temperature rise. Perform preliminary filtration with gauze to remove residue. Collect the filtrate and aliquot it into 50mL centrifuge tubes.

[0042] 2. Differential centrifugation: At 4°C, perform the following centrifugation steps in sequence to remove cell debris and impurities: 1000xg, 10 min, discard the precipitate and retain the supernatant; 3000xg, 30 min, discard the precipitate and retain the supernatant; 10,000xg, 60 min, discard the precipitate and collect the supernatant.

[0043] 3. Ultracentrifugation: After differential centrifugation, the supernatant is subjected to ultracentrifugation (error ≤ 0.001g). The conditions are set as follows: 4℃, 150,000xg, 180min. The supernatant is discarded, and the precipitate at the bottom of the tube is retained.

[0044] 4. Collect exosomes: After ultracentrifugation, discard the supernatant, and resuspend the precipitate in the centrifuge tube by blowing it with PBS buffer.

[0045] 5. Filtration and concentration: The suspension is filtered through a 0.22μm filter membrane in a clean bench to obtain the sterilized Atractylodes lancea exosome suspension. After ultrafiltration and concentration with a 100kDa ultrafiltration tube, it can be used for characterization and in vivo and in vitro experiments.

[0046] Example 2: Characterization of Atractylodes lancea exosomes

[0047] 1. Determination of total protein concentration in Atractylodes lancea exosomes using the BCA method

[0048] 1.1 The total protein concentration of the Atractylodes lancea exosomes prepared in Example 1 was determined using the BCA Protein Assay Kit (Enhanced Version), purchased from BioSharp. Experimental Principle: Under alkaline conditions, the peptide bonds in protein molecules can react with Cu... 2+ The reaction occurs to form a complex, and Cu 2+ Reduced to Cu + BCA reagent can specifically react with Cu. + These components combine to form a stable, colored complex with maximum light absorption at a wavelength of 562 nm. The intensity of the complex's color is directly proportional to the protein concentration; therefore, the protein content can be determined by measuring its absorbance.

[0049] 1.2 Preparation of BCA working solution: Mix reagent A and reagent B at a volume ratio of 50:1 to prepare BCA working solution. A precipitate will form when the two are mixed; the precipitate will disappear after thorough mixing.

[0050] 1.3 Microplate assay procedure: Add an appropriate volume of the sample to be tested (4 μl) to the microplate, and bring the volume to 20 µl with PBS. Add 200 µl of BCA working solution to the microplate, mix well, and incubate at 37°C for 30 min.

[0051] 1.4 Measure the absorbance at 562 nm and record the readings. Use the absorbance of a sample without BSA as a blank control.

[0052] 1.5 The protein concentration in the sample was calculated using a standard curve plotted with A562 as the ordinate and BSA content as the abscissa, based on the determination of protein standards.

[0053] 2. Observation of Atractylodes exosome morphology using transmission electron microscopy

[0054] 2.1 Ensure the copper mesh surface is clean. Take 20 μl of Atractylodes lancea exosome suspension and evenly drop it onto the carbon membrane copper mesh, ensuring the sample is evenly covered on the carbon membrane. Let it stand for 3 minutes to allow the exosomes to adsorb onto the carbon membrane. Use filter paper to absorb excess liquid.

[0055] 2.2 Immerse the copper mesh in a 1% phosphotungstic acid solution for 1-2 minutes, dry at room temperature, and observe under a transmission electron microscope. Images are then acquired and analyzed. Results are as follows: Figure 1 As shown, the exosomes extracted by this invention exhibit a clear cup-shaped membrane vesicle structure in the electron microscope field of view.

[0056] 3. Measurement of Atractylodes lancea exosome particle size using dynamic laser scattering instrument

[0057] 3.1 Rinse the dynamic light scattering cuvette 2-3 times with Atractylodes lancea exosome suspension.

[0058] 3.2 Carefully add 10 μl of Atractylodes lancea exosome suspension into a dynamic light scattering cuvette, avoiding the generation of air bubbles.

[0059] 3.3 Place the dynamic light scattering cuvette in the dynamic laser scattering instrument (DLS), adjust the parameters, and set the default room temperature to 25℃ and the measurement time to 1 min.

[0060] 3.4 Start the measurement program. The instrument will automatically perform particle size distribution measurement, and the results will be as follows: Figure 1 As shown, the particle size distribution of the Atractylodes lancea exosomes extracted in this invention is 110-190 nm.

[0061] Example 3: Atractylodes lactone II loaded into Atractylodes lancea exosomes

[0062] Atractylodes lactone II and Atractylodes exosomes were mixed in PBS at a mass ratio of 2:1. The mixture was sonicated at 20% amplitude for 1 min (2 s on, 2 s off). The mixture was incubated at 4°C for 24 h, and excess free atractylodes lactone II was removed by centrifugation. The mixture was then concentrated by ultrafiltration using a 100 kDa ultrafiltration tube to collect Atractylodes exosomes loaded with atractylodes lactone II (AT-EXO). The loading process was confirmed by high-performance liquid chromatography.

[0063] Example 4: Detection of the effects of atractylodes lactone II, atractylodes exosomes, and atractylodes exosomes loaded with atractylodes lactone II on H9c2 cell viability.

[0064] 1. Experimental materials and reagents:

[0065] 1.1 Experimental reagents: DMEM high glucose medium, fetal bovine serum, 0.25% trypsin, PBS phosphate buffer, CCK-8 kit (Biosharp, BS350B).

[0066] 1.2 Experimental instruments and materials: CO2 incubator, clean bench, culture dishes, centrifuge tubes, cell counting chamber, pipette, disposable pipette tips, 96-well plate, rat cardiomyocytes H9c2 (laboratory-preserved cells).

[0067] 2. Experimental steps:

[0068] 2.1 Trypsin digestion of cultured H9c2 cells, cell count.

[0069] 2.2 H9C2 cells were passaged and seeded into 96-well plates at a seeding density of 8 × 10⁶ cells / well. 3 One cell adheres to the wall overnight.

[0070] 2.3 The mixture was treated with 20 μM of atractylodes lactone II (AT-II), 25 mg / mL of atractylodes exosomes (AL-EXO), and 25 mg / mL of atractylodes exosomes loaded with atractylodes lactone II (AT-EXO) for 24 h.

[0071] 2.4 Subsequently, the cells were co-incubated with CCK-8 solution (Biosharp, BS350B). 10 μL of CCK-8 solution and 100 μL of fresh culture medium were added to each well. The control group and reaction time were set according to the manufacturer's instructions. After color development, the absorbance was measured at 450 nm.

[0072] Example 5: Detection of the mitochondrial ROS scavenging capacity of H9c2 cells in the LPS+cs model by atractylodes lactone II, atractylodes exosomes, and atractylodes exosomes loaded with atractylodes lactone II.

[0073] 1. Experimental materials and reagents:

[0074] 1.1 Experimental reagents: DMEM high glucose medium, sugar-free, sodium glutamate-free, and pyruvamide-free DMEM medium, fetal bovine serum, 0.25% trypsin, PBS phosphate buffer, LPS (lipopolysaccharide).

[0075] 1.2 Experimental instruments and materials: CO2 incubator, clean bench, culture dishes, centrifuge tubes, cell counting chamber, cell slides, pipettes, disposable pipette tips, 12-well plates, rat cardiomyocytes H9c2 (laboratory-preserved cells).

[0076] 2. Experimental steps:

[0077] 2.1 Trypsin digestion of cultured H9c2 cells, cell count.

[0078] 2.2 Passage H9C2 cells into 12-well plates. A cell crawling slide should be placed at the bottom of each well beforehand. The seeding density should be 3 × 10⁶ cells / well. 5 One cell adheres to the wall overnight.

[0079] 2.3 The mixture was treated with 20 μM of atractylodes lactone II (AT-II), 25 mg / mL of atractylodes exosomes (AL-EXO), and 25 mg / mL of atractylodes exosomes loaded with atractylodes lactone II (AT-EXO) for 24 h.

[0080] 2.4 Replace the medium with sugar-free, sodium glutamate-free, and acetone-free DMEM medium supplemented with 10% fetal bovine serum, and add 100 ng / mL LPS to induce LPS+cs modeling for 3 h.

[0081] 2.5 MitoSOX (purchased from MCE) mitochondrial ROS probe was diluted 1:1000 with serum-free DMEM basal medium and incubated with cell smears at 37°C in the dark for 30 min.

[0082] 2.6 Wash twice with PBS phosphate buffer, fix cells with 4% paraformaldehyde at room temperature for 30 min.

[0083] 2.7 After fixation, the cell slides were washed twice with PBS phosphate-buffered saline (PBS) to prepare an adhesion slide. 8 μL of DAPI-containing anti-fluorescence quencher was added. The fixed cell slides were then removed, ensuring the cell-containing side was in contact with the DAPI. The slides were fixed and mounted with mounting medium, stained at room temperature in the dark for 10-15 minutes, and then photographed using a confocal microscope. Mitochondrial ROS is a red fluorescence channel, with an excitation wavelength of 510 nm and an emission wavelength of 580 nm.

[0084] Example 6: Detection of the ability of atractylodes lactone II, atractylodes exosomes, and atractylodes exosomes loaded with atractylodes lactone II to restore mitochondrial membrane potential in H9c2 cells in the LPS+cs model.

[0085] 1. Experimental materials and reagents:

[0086] 1.1 Experimental reagents: DMEM high glucose medium, sugar-free, sodium glutamate-free, and acetoamide-free DMEM medium, fetal bovine serum, 0.25% trypsin, PBS phosphate buffer, LPS (lipopolysaccharide), enhanced mitochondrial membrane potential detection kit (JC-1) (Beyotime C2003S).

[0087] 1.2 Experimental instruments and materials: CO2 incubator, clean bench, culture dishes, centrifuge tubes, cell counting chamber, pipette, disposable pipette tips, copolymerization dish, rat cardiomyocytes H9c2 (laboratory-preserved cells).

[0088] 2. Experimental steps:

[0089] 2.1 Trypsin digestion of cultured H9c2 cells, cell count.

[0090] 2.2 Passage H9C2 cells to copolymer dishes, seeding at a density of 3 × 10⁶ cells / mL. 5 One cell adheres to the wall overnight.

[0091] 2.3 The mixture was treated with 20 μM of atractylodes lactone II (AT-II), 25 mg / mL of atractylodes exosomes (AL-EXO), and 25 mg / mL of atractylodes exosomes loaded with atractylodes lactone II (AT-EXO) for 24 h.

[0092] 2.4 Replace the medium with sugar-free, sodium glutamate-free, and acetoamide-free DMEM medium supplemented with 10% fetal bovine serum, and add 100 ng / mL LPS to induce LPS+cs modeling for 3 h.

[0093] 2.5 Wash twice with PBS phosphate buffer, add 2.5 μM JC-1 to the cell culture medium, and incubate at 37°C for 20 minutes.

[0094] 2.6 After incubation at 37℃, remove the supernatant and wash twice with JC-1 staining buffer.

[0095] 2.7 Add cell culture medium and take confocal images. When detecting JC-1 monomers, the excitation wavelength was set to 490 nm and the emission wavelength to 530 nm; when detecting JC-1 polymers, the excitation wavelength was set to 525 nm and the emission wavelength to 590 nm. The appearance of green fluorescence indicates a decreased mitochondrial membrane potential, and the cell is likely in the early stages of apoptosis. The appearance of red fluorescence indicates a relatively normal mitochondrial membrane potential and a relatively normal cell condition.

[0096] Example 7: Detection of the ability of atractylodes lactone II, atractylodes exosomes, and atractylodes exosomes loaded with atractylodes lactone II to restore ATP synthesis in H9c2 cells in the LPS+cs model.

[0097] 1. Experimental materials and reagents:

[0098] 1.1 Experimental reagents: DMEM high glucose medium, sugar-free, sodium glutamate-free, and pyruvamide-free DMEM medium, fetal bovine serum, 0.25% trypsin, PBS phosphate buffer, LPS (lipopolysaccharide), and enhanced ATP assay kit (Beyotime S0027).

[0099] 1.2 Experimental instruments and materials: CO2 incubator, clean bench, culture dishes, centrifuge tubes, cell counting chamber, pipette, disposable pipette tips, 12-well plate, rat cardiomyocytes H9c2 (laboratory-preserved cells).

[0100] 2. Experimental steps:

[0101] 2.1 Trypsin digestion of cultured H9c2 cells, cell count.

[0102] 2.2 Passage H9C2 cells into 12-well plates, seeding at a density of 3 × 10⁶ cells / well. 5 One cell adheres to the wall overnight.

[0103] 2.3 The mixture was treated with 20 μM of atractylodes lactone II (AT-II), 25 mg / mL of atractylodes exosomes (AL-EXO), and 25 mg / mL of atractylodes exosomes loaded with atractylodes lactone II (AT-EXO) for 24 h.

[0104] 2.4 Replace the medium with sugar-free, sodium glutamate-free, and acetone-free DMEM medium supplemented with 10% fetal bovine serum, and add 100 ng / mL LPS to induce LPS+cs modeling for 3 h.

[0105] 2.5 Wash once with PBS phosphate buffer, digest with trypsin and collect cells by centrifugation.

[0106] 2.6 Add 100 μL of lysis buffer, sonicate to lyse the cells, and centrifuge to collect the supernatant.

[0107] 2.7 Prepare the reaction mixture according to the manufacturer's instructions. Add 120 μL of the reaction mixture to each well of a black-backed 96-well plate and record the luminescent RLU using a Biotek Cytation 5 microplate reader. ATP concentration was calculated using a standard curve (0–10 μM), and the ATP level was normalized to total protein content (nmol / mg protein), which was determined using a BCA assay kit (Biosharp, BL1054S).

[0108] Experimental Results and Conclusions

[0109] Based on the above experimental objectives and procedures, the experimental data, conclusions, and other information obtained should match the initial experimental objectives and demonstrate that the specific experimental steps achieved the intended effect.

[0110] 1. Extraction and characterization of exosomes from Atractylodes lancea

[0111] Combination Figure 1 , Figure 2 and Figure 3 The exosomes extracted by this invention have complete morphological structure and particle size consistent with the size of exosomes.

[0112] 2. Atractylodes lancea exosomes loaded with atractylodes lactone II have a more significant promoting effect on the viability of H9c2 cells.

[0113] Combination Figure 4 Cell viability assays revealed that treatment of H9c2 cells with AT-II, AL-EXO, and AT-EXO for 24 hours all promoted cell growth. Specifically, AT-EXO showed a 40% higher promotion of cell growth viability compared to AT-II and a 20% higher promotion compared to AL-EXO. These results indicate that, compared to either agent acting alone, Atractylodes lancea exosomes loaded with atractylodes lactone II had a more significant promoting effect on H9c2 cell viability.

[0114] 3. Atractylodes lancea exosomes loaded with atractylodes lactone II showed a more significant restorative effect on mitochondrial dysfunction in H9c2 under LPS+cs conditions.

[0115] Cardiomyocyte survival is highly dependent on the functional integrity of mitochondria. Mitochondrial dysfunction is a core mechanism of cardiomyocyte injury, specifically manifested as: mitochondrial membrane potential collapse (decreased ΔΨm); excessive production of reactive oxygen species (ROS), leading to oxidative damage; mitochondrial dynamics imbalance (abnormal fusion / division); and dysregulation of mitophagy. This study investigated whether LPS+cs treatment of H9c2 cells, using changes in intracellular mitochondrial ROS and mitochondrial membrane potential, significantly protected H9c2 cells from mitochondrial damage under LPS+cs (i.e., LPS-carbon starvation) stress by loading atractylodes lactone II onto Atractylodes lancea exosomes.

[0116] Figure 5 The results showed that LPS+cs significantly activated the production of mitochondrial ROS in H9c2 cells, AT-II reduced mitochondrial ROS accumulation by about 30%, AL-EXO reduced mitochondrial ROS accumulation by about 50%, and AT-EXO significantly reduced mitochondrial ROS accumulation by about 80% under LPS+cs conditions.

[0117] Figure 6 The results showed that LPS+cs significantly reduced the mitochondrial membrane potential of H9c2 cells, AT-II restored about 20% of the mitochondrial membrane potential, AL-EXO restored about 27% of the mitochondrial membrane potential, and AT-EXO significantly restored about 81% of the mitochondrial membrane potential under LPS+cs conditions.

[0118] The results above indicate that, compared to the effects of either ingredient alone, the atractylodes lactone II loaded on Atractylodes lancea exosomes has a more significant repair effect on mitochondrial damage in H9c2 cells under LPS+cs stress.

[0119] 4. Atractylodes lancea lactone II loaded on Atractylodes lancea exosomes has a more significant restorative effect on the energy metabolism of H9c2 under LPS+cs conditions.

[0120] Mitochondrial function is closely related to cellular ATP metabolism. Mitochondrial dysfunction leads to a decrease in the activity of some ATPases, reducing ATP production and causing an energy crisis. Figure 7 Cellular ATP concentration measurements revealed that LPS+cs significantly reduced ATP concentration in H9c2 cells, indicating insufficient ATP synthesis and an imbalance in energy metabolism. AT-II restored approximately 40% of the ATP concentration, and AL-EXO restored approximately 80% of the mitochondrial membrane potential, while AT-EXO significantly restored approximately 160% of the mitochondrial membrane potential under LPS+cs conditions. These results indicate that, compared to the individual effects of either agent, Atractylodes lancea exosomes loaded with Atractylodes lactone II have a more significant restorative effect on energy metabolism in H9c2 cells under LPS+cs stress.

[0121] In summary, atractylodes lactone II can be loaded into Atractylodes lancea exosomes, and the two work synergistically to repair mitochondrial dysfunction and energy metabolism imbalance in cardiomyocytes across species, providing a new approach and method for the development and delivery of drugs for myocardial diseases.

[0122] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for extracting exosomes from Atractylodes lancea, characterized in that, Includes the following steps: Atractylodes lancea juice was obtained by mixing and pressing with PBS buffer, then filtered, and then centrifuged multiple times at different speeds from low to high to remove the precipitate and retain the supernatant. Then, the supernatant was discarded by ultracentrifugation, and the precipitate at the bottom of the tube was retained. The precipitate in the centrifuge tube was then blown with PBS buffer and resuspended, then the suspension was filtered, and then concentrated to obtain the Atractylodes lancea exosomes.

2. The method for extracting Atractylodes lancea exosomes according to claim 1, characterized in that, The multiple differential centrifugations include: centrifuging at 1000-1500 xg for 8-10 min, discarding the precipitate and retaining the supernatant; centrifuging at 3000-3500 xg for 25-30 min, discarding the precipitate and retaining the supernatant; and centrifuging at 10,000-10500 xg for 55-60 min, discarding the precipitate and collecting the supernatant.

3. An Atractylodes lancea exosome, characterized in that, It is prepared by the extraction method according to any one of claims 1-2.

4. The use of the Atractylodes exosomes according to claim 3 in the preparation of Atractylodes exosomes loaded with atractylodes lactone II.

5. The application according to claim 4, characterized in that, Includes the following steps: Atractylodes lactone II and Atractylodes exosomes were mixed in PBS buffer at a mass ratio of (2-2.5):1, and then sonicated for 1-1.5 min to obtain a mixture. The mixture was then incubated at 4-5℃ for 20-24 h, centrifuged and concentrated to obtain Atractylodes exosomes loaded with atractylodes lactone II.

6. The use of Atractylodes lancea exosomes loaded with Atractylodes lactone II, or the use of Atractylodes lancea exosomes in the preparation of drugs for the prevention and treatment of myocardial injury.

7. The application according to claim 6, characterized in that, The myocardial injury described was caused by LPS-carbon starvation synergistic myocardial injury.

8. The application according to claim 6, characterized in that, The drug prevents and treats myocardial damage by restoring mitochondrial energy metabolism in cardiomyocytes, clearing mitochondrial ROS, and / or stabilizing mitochondrial membrane potential.

Images