Application of camel milk exosome in preparation of medicine for treating heart diseases

Abstract

The invention relates to an application of camel milk exosome in preparation of a medicine for treating heart diseases. According to the invention, the camel milk exosome is used for preparing the medicine for treating heart diseases, especially for relieving chronic heart failure, and a brand-new, safe and effective treatment scheme is provided for protecting the heart.

Description

Technical Field

[0001] This invention belongs to the field of cardiac disease treatment technology, and specifically relates to the application of camel milk exosomes in the preparation of drugs for treating cardiac diseases. Background Technology

[0002] Chronic heart failure (CHF) refers to a persistent state of heart failure that can be stable, worsening, or decompensated. It manifests as decreased myocardial contractility, increased cardiac load, leading to an acute drop in cardiac output, increased pulmonary circulation pressure, and increased peripheral circulatory resistance. This results in pulmonary congestion and pulmonary edema, which may be accompanied by tissue and organ insufficiency and cardiogenic shock, with left ventricular failure being the most common manifestation. In recent years, exosomes have become a prominent emerging research hotspot in the biomedical field. Exosomes are nanoscale vesicles secreted by cells, possessing natural drug-carrying properties, low immunogenicity, and targeted delivery capabilities. They can carry various bioactive molecules such as proteins and nucleic acids, precisely regulating the function of receptor cells.

[0003] CN116459317B discloses the application of Gastrodia elata exosomes in the preparation of drugs for protecting against myocardial injury caused by doxorubicin. The extracted Gastrodia elata exosomes significantly inhibit apoptosis of H9c2 cardiomyocytes after doxorubicin treatment, thereby reducing cardiac damage caused by doxorubicin. CN117482203B discloses the use of ginger exosomes in the preparation of drugs for treating ischemic heart disease. The prepared ginger exosomes, while removing the pungent and irritating substances from ginger, possess the same pharmacological effects as ginger, exhibiting anti-inflammatory and reactive oxygen species reduction functions. They significantly improve the cell viability of H9c2 cardiomyocytes after hypoxia treatment and inhibit H9c2 cardiomyocyte death, ROS production, and LDH release after hypoxia treatment, reducing the expression of inflammatory factors in cardiomyocytes caused by hypoxia treatment, thereby reducing cardiomyocyte damage caused by hypoxia treatment.

[0004] However, all of the aforementioned exosomes are plant-derived. In contrast, animal-derived exosomes, such as cow's milk and camel's milk exosomes, are more promising research directions due to their wide availability and stable composition. Currently, research on camel milk exosomes in cardiac diseases and their mechanisms of action remains very limited. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide an application of camel milk exosomes in the preparation of drugs for treating heart diseases. It is the first time that camel milk exosomes have been proposed to be used in the preparation of drugs for treating heart diseases, especially for relieving chronic heart failure, providing a new, safe and effective treatment option for protecting the heart.

[0006] This invention provides the application of camel milk exosomes in the preparation of drugs for treating heart disease.

[0007] Preferably, the camel milk exosomes are derived from camel milk whey.

[0008] Preferably, the camel milk exosomes significantly reduce cardiac tissue cell apoptosis, alleviate cardiac tissue fibrosis, reduce cardiac tissue necrosis, and reduce acute cardiac injury.

[0009] Preferably, the method for preparing the camel milk exosomes includes the following steps:

[0010] S1. Centrifuge fresh camel milk and extract the middle layer liquid after centrifugation as whey;

[0011] S2. Filter the whey obtained in S1, centrifuge the filtrate again, collect the gel-like precipitate at the bottom of the tube and break it up.

[0012] S3. Resuspend the precipitate after S2 in buffer solution, centrifuge the resuspended solution again, collect the gel-like precipitate at the bottom of the tube and break it up.

[0013] S4. Resuspend the precipitate after S3 in buffer solution. Filter the resulting resuspended solution through a coarse filter membrane and a sterile filter membrane. Collect the filtrate to obtain camel milk exosomes.

[0014] Furthermore, the centrifugation in step S1 specifically involves centrifuging at 10,000g to 15,000g for 20 to 40 minutes.

[0015] Furthermore, the filtration in step S2 specifically involves using a filter membrane with a pore size of 70–100 μm.

[0016] Furthermore, the centrifugation in steps S2 and S3 specifically involves centrifuging at 100,000–120,000 g for 1–2 hours.

[0017] Preferably, the particle size range of the camel milk exosomes is 50-200 nm.

[0018] Preferably, the dosage of the camel milk exosomes is (1-10)×10⁻⁶. 12 particles / kg.

[0019] Preferably, the heart disease includes coronary heart disease, cardiomyopathy, arrhythmia, or heart failure.

[0020] Preferably, the heart failure includes chronic heart failure.

[0021] The present invention also provides a medicament for treating heart disease, the medicament comprising camel milk exosomes and a pharmaceutically acceptable carrier.

[0022] Preferably, the dosage form of the drug includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powders for injection, and injections.

[0023] Beneficial effects

[0024] This invention, through experiments, has shown that administration via camel milk exosomes can significantly reduce cardiac tissue cell apoptosis, alleviate cardiac tissue fibrosis, decrease cardiac tissue necrosis, and reduce acute cardiac injury, demonstrating excellent overall performance and achieving a cardioprotective therapeutic effect. Specifically, it works through the following mechanisms:

[0025] 1. Regulation of Apoptosis: Camel milk exosomes can precisely regulate intracellular apoptosis-related signaling pathways, significantly reducing the apoptosis rate of tissue cells. By inhibiting the expression of pro-apoptotic proteins and enhancing the activity of anti-apoptotic proteins, they maintain the stability of the intracellular environment, enabling cardiomyocytes to maintain normal physiological function and survival.

[0026] 2. Inhibition of Fibrosis Progression: Camel milk exosomes effectively reduce the degree of fibrosis in cardiac tissue and decrease excessive deposition of extracellular matrix, thereby blocking the progression of cardiac fibrosis. By regulating the cytokine network, they promote the degradation and remodeling of fibrous tissue, gradually restoring the cardiac tissue structure to normal.

[0027] 3. Reduced Necrosis: Camel milk exosomes significantly reduced the necrosis rate of cardiac tissue. This is achieved by improving blood circulation in the heart, increasing oxygen and nutrient supply to cardiomyocytes, and mitigating the damage to cardiomyocytes caused by ischemia and hypoxia. Simultaneously, it enhances the antioxidant capacity of cardiomyocytes, scavenges free radicals, and reduces oxidative stress damage to cells, thereby effectively protecting cardiac tissue from further destruction.

[0028] In summary, the therapeutic effects of camel milk exosomes are not only reflected in the improvement of pathological indicators, but more importantly, in the protection and repair of overall cardiac function. Through the synergistic effect of the above-mentioned multiple mechanisms, the heart can maintain normal function in chronic heart failure, promote the regeneration and repair of cardiomyocytes, and ultimately achieve the therapeutic effect of protecting the heart, providing a safe and effective new strategy for the treatment of chronic heart failure. Attached Figure Description

[0029] Figure 1 The image shows the particle size distribution of camel milk exosomes prepared in Example 1.

[0030] Figure 2 The image shows a transmission electron microscope (TEM) image of the camel milk exosomes prepared in Example 1.

[0031] Figure 3 H&E staining images of the hearts of mice in the control group, model group, and exosome treatment group.

[0032] Figure 4 MASSON staining images of the hearts of mice in the control group, model group, and exosome treatment group.

[0033] Figure 5 MASSON staining analysis of the hearts of mice in the control group, model group, and exosome treatment group.

[0034] Figure 6 TUNEL staining images of the hearts of mice in the control group, model group, and exosome treatment group.

[0035] Figure 7 TUNEL staining analysis of the hearts of mice in the control group, model group, and exosome treatment group.

[0036] Figure 8 CD31 staining images of the hearts of mice in the control group, model group, and exosome treatment group.

[0037] Figure 9 CD31 staining analysis of the hearts of mice in the control group, model group, and exosome treatment group.

[0038] Figure 10 The images show CD206 / 86 staining of the hearts of mice in the control group, model group, and exosome treatment group.

[0039] Figure 11 CD206 / 86 staining analysis of the hearts of mice in the control group, model group, and exosome treatment group.

[0040] Figure 12 Principal component analysis diagrams of proteomics in mice from the control group, model group, and exosome treatment group.

[0041] Figure 13 The top ten proteins in the proteomics of mice in the control group, model group, and exosome treatment group, along with their correlation heatmaps.

[0042] Figure 14 This is a statistical chart showing the percentage of differentially expressed proteins in the proteomics of mice in the control group, model group, and exosome treatment group.

[0043] Figure 15 Volcano plot of differentially expressed proteins in the proteomics of mice in the control group, model group, and exosome treatment group.

[0044] Figure 16 Bar chart showing GO analysis of proteomics in mice of the control group, model group, and exosome treatment group.

[0045] Figure 17 Bubble graphs showing GO analysis of proteomics in mice from the control group, model group, and exosome treatment group. Detailed Implementation

[0046] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0047] Example 1

[0048] Preparation and Detection of Camel Milk Exosomes

[0049] 1.1 Preparation of camel milk exosomes

[0050] Step 1: Separate the whey. Divide 500mL of fresh camel milk into high-speed centrifuge tubes, centrifuge at 13000g for 30 minutes, and take the middle layer liquid to obtain whey.

[0051] Step 2: Filtration to obtain exosomes. After filtering the whey through a 70μm filter membrane, the filtrate was dispensed into ultracentrifuge tubes, centrifuged at 100,000g for 1.5h, the supernatant was discarded, and the gel-like precipitate at the bottom of the tube was collected and crushed into pieces.

[0052] Step 3: Remove impurities. Transfer the precipitate from the previous step to a new centrifuge tube, add PBS buffer to 50 mL, and then centrifuge at 100,000 g for 1.5 h. Discard the supernatant, collect the gel-like precipitate at the bottom of the tube, and break it into pieces.

[0053] Step 4: Dissolution and sterilization. Resuspend the clump of precipitate in 40 mL of PBS buffer and dissolve it thoroughly into a homogeneous liquid by pipetting or homogenizing. Then, filter it through a 70 μm filter to remove insoluble impurities and a 0.22 μm filter membrane for sterilization. Collect the filtrate to obtain purified camel milk exosomes.

[0054] 1.2 NTA test results

[0055] NTA (Nano-Tracking Analysis) technology for exosomes uses laser scattering microscopy to record the Brownian motion trajectory of nanoparticles in solution and calculates particle size and concentration using the Stokes-Einstein equation.

[0056] The prepared exosomes were diluted with PBS at a volume ratio of 1:1000. Using a 1ml syringe, 1ml of the exosome dilution solution was slowly injected into the NTA device tubing. The detection parameters were set, and the detection was started. The results are as follows: Figure 1 As shown, the average particle size is 166.6 nm, and the concentration is 1.33 × 10⁻⁶. 12 Particles / mL.

[0057] 1.3 Electron Microscopy Observation

[0058] Take 20 μl of the exosome suspension prepared in step 1.1 and pipette it onto a copper grid. Allow it to absorb naturally for 5-10 minutes. Then, use filter paper to remove excess droplets and let it air dry slightly. Next, pipette 20 μl of a 2% phosphotungstic acid solution and pipette it onto the copper grid. Let it stand for 3-5 minutes for staining. Remove excess droplets with filter paper and let it air dry under an incandescent lamp. Finally, observe and photograph the sample under a transmission electron microscope. The resulting transmission electron microscope micrographs are shown below. Figure 2 As shown, this result is consistent with the NTA detection result.

[0059] Example 2

[0060] The therapeutic effects of camel milk exosomes on chronic heart failure

[0061] 1. Experimental animals: 8-week-old male mice of the C57 strain.

[0062] 2. Experimental grouping and dosing regimen

[0063] Experimental Groups: Mice were randomly divided into three groups: control group, model group, and exosome treatment group. The model group and exosome treatment group were used to establish an animal model of ischemic acute myocardial infarction. The specific modeling method was as follows: Myocardial infarction was induced by permanent ligation of the left anterior descending coronary artery (LAD) (isoflurane anesthesia, open-chest exposure of the heart, LAD ligation with 6-0 silk suture). Postoperative observation lasted one month. The marker of heart failure was an ejection fraction below 50% after 28 days, indicating successful modeling. The control group did not have coronary artery ligation (n=10); the model group received saline via gavage after coronary artery ligation (n=10); and the exosome treatment group received exosomes via gavage after coronary artery ligation (n=10).

[0064] Administration regimen: Starting one month post-surgery, once the ejection fraction is confirmed to be less than 50%, administer via gavage daily for 8 weeks. Dosage: 2 × 10⁻⁶ 12 particles / kg.

[0065] 3. Experimental Results

[0066] 3.1 H&E staining

[0067] Hematoxylin and eosin (H&E) staining was performed on samples from the apex of the heart from each experimental group in step 2 (the infarcted area was taken from the apex). The results are as follows. Figure 3As shown, in the control group, the endocardium, myocardium, and epicardium of the heart tissue were clearly defined, the myocardial fibers (orange arrows) were uniformly stained, the cell boundaries were clear, the course was consistent, and no obvious abnormalities were observed in the interstitium; no obvious inflammatory cell infiltration was observed. The black boxes indicate magnified areas with no obvious lesions; in the model group, a large area of ​​myocardial cell necrosis and disappearance was observed in the myocardial layer, replaced by proliferating connective tissue; in the exosome treatment group, the endocardial structure of the heart tissue was clear, the myocardial layer occasionally showed focal disappearance of myocardial cells, replaced by proliferating connective tissue; occasional lymphocyte and granulocyte infiltration was observed.

[0068] 3.2 MASSON staining

[0069] Step 2: Heart tissue samples from each experimental group were collected and subjected to MASSON staining. MASSON staining, a classic connective tissue staining method, provides a direct and effective means of detecting fibrosis. In fibrotic tissue, MASSON staining will show distinct red or purple areas. These areas represent the proliferation and deposition of collagen fibers, a typical manifestation of fibrotic lesions. By observing the distribution and extent of these areas, the degree and extent of fibrosis can be preliminarily determined. The results of MASSON staining of heart tissue samples are as follows: Figure 4 The analysis results are shown below. Figure 5 The control group showed no obvious lesions; the model group had severe cardiac basement membrane fibrosis and increased collagen fibers; the exosome treatment group showed reduced cardiac basement membrane fibrosis and basically normal collagen fibers.

[0070] 3.3 TUNEL staining

[0071] In step 2, heart tissue samples from each experimental group were collected and subjected to TUNEL staining. TUNEL staining is a commonly used method for detecting DNA breaks in apoptosis. TUNEL-positive cells indicate the presence of DNA breaks, a key characteristic of apoptosis. By counting the number of positive cells, the proportion of apoptotic cells can be assessed. The TUNEL staining results are shown below. Figure 6 The study observed the number of apoptotic cells by taking samples from the LAD ligated infarct area, specifically the anterior wall of the heart, near the apex, and from the region near the edge of the myocardial infarction, focusing on areas where apoptosis was the primary feature. Figure 7 As shown, the control group had no obvious apoptotic positive cells, while the model group had a large number of positive cells and a high apoptosis rate; the exosome treatment group had fewer apoptotic positive cells.

[0072] 3.4 CD31 staining of blood vessels (to observe angiogenesis)

[0073] CD31 staining of blood vessels is a classic and commonly used immunohistochemical method in pathology and histology for observing and quantifying vascular endothelial cells, thereby assessing vascular density and angiogenesis. CD31, also known as platelet endothelial cell adhesion molecule-1, is a transmembrane glycoprotein primarily expressed on the surface of vascular endothelial cells. It participates in endothelial cell adhesion, leukocyte migration, and angiogenesis signal transduction. Due to its specific high expression on endothelial cells, it has become one of the most commonly used and reliable markers for identifying vascular endothelium. Figure 8 As shown, in the exosome-treated group of mice, collateral circulation formed after myocardial ischemia, and increased neovascularization was detected, which was close to that in the control group. Figure 9 ).

[0074] 3.5 Staining with CD86 & CD206 markers, macrophage polar molecules

[0075] Macrophage polarization is typically identified using specific molecular markers. Molecular markers for different polarization phenotypes (such as M1, M2, and their subtypes) can be detected using techniques such as immunofluorescence staining, flow cytometry, and immunohistochemistry. M1 macrophages (classical activation): usually induced by IFN-γ and LPS, exhibiting pro-inflammatory, anti-tumor, and antimicrobial activity; CD86 is one of their important surface markers. M2 macrophages (alternative activation): usually induced by IL-4, IL-13, and IL-10, exhibiting anti-inflammatory, tissue repair, angiogenesis, and tumor growth-promoting activity; CD206 is one of their most characteristic surface markers. Results are as follows... Figure 10 and Figure 11 As shown, CD206 is a red fluorescent label and CD86 is a green fluorescent label. The number of CD206 / CD86 positive cells in mice treated with exosomes increased significantly, indicating the start of the repair phase and promoting angiogenesis and collagen deposition.

[0076] In conclusion, compared with the model group, the exosome treatment group showed a significant decrease in tissue necrosis rate. Figure 3 ), degree of tissue fibrosis ( Figure 5 ) and the degree of tissue cell apoptosis ( Figure 7 ) significantly decreased, and neovascularization increased ( Figure 9 This indicates that camel milk exosomes have a cardioprotective effect.

[0077] Example 3

[0078] Proteomic results of camel milk exosome therapy for chronic heart failure

[0079] 1. Experimental animals: 8-week-old male mice of the C57 strain.

[0080] 2. Sample collection method: Two mice were collected from each of the control group (N), model group (M), and exosome treatment group (L). The infarcted area at the apex of the heart was taken, and the heart was circumferentially dissected (two hearts were pooled) for testing.

[0081] 3. Technical Introduction: Proteomics studies proteins as research objects, investigating the composition and changes of proteins in cells, tissues, body fluids, or organisms. Essentially, it studies protein characteristics at a large scale, including protein expression levels, post-translational modifications, and protein-protein interactions, thereby gaining a holistic and comprehensive understanding of disease development, cellular metabolism, and other processes at the protein level. Label-free proteomics quantitative technology is a novel protein quantification technique that does not rely on isotope labels. This technique analyzes peptides formed by trypsin digestion of proteins using liquid chromatography-mass spectrometry (LC-MS). It does not require expensive stable isotope labels as internal standards; it only needs to analyze the mass spectrometry data generated during large-scale protein identification, comparing the signal intensity of corresponding peptides in different samples to perform relative quantification of the corresponding proteins.

[0082] 4. Analysis Results

[0083] 4.1 Principal Component Analysis (PCA) is also commonly used to assess between-group differences and within-group sample replication. PCA uses linear algebra calculations to perform dimensionality reduction and principal component extraction on the samples, using protein quantification results as variables, to examine between-group differences. Figure 12 ).

[0084] 4.2 Sample Correlation Analysis

[0085] Biological replication demonstrates that the biological experimental procedures involved are not accidental but reproducible. This also ensures more reliable results for subsequent differential protein analyses. The correlation of gene expression levels between samples is an important indicator of experimental reliability and the appropriateness of sample selection. The closer the correlation coefficient is to 1, the higher the similarity of expression patterns between samples. Figure 13 ).

[0086] 4.3 Results of Differentially Expressed Proteins Between Groups

[0087] 4.3.1 Results of Differentially Expressed Protein Analysis

[0088] To determine the significance of differential protein expression, if each sample group has at least three biological or technical replicates, the relative protein quantification values ​​are log2 transformed and the pval is calculated using the limmaR package. If each sample group has fewer than three biological or technical replicates, the significance A method is used to calculate the pval. The percentage of differentially expressed proteins is also calculated. Figure 14 ).

[0089] 4.3.2 Volcano plot of differentially expressed proteins

[0090] Protein differential expression analysis, based on the required sample pairs for comparison, uses the ratio of each protein's expression to the mean of all biological replicate quantitative values ​​within the compared sample pairs as the fold change (FC). When FC ≥ 1.5 / 2.0 and pval ≤ 0.05, the protein is upregulated; when FC ≤ 0.67 / 0.5 and pval ≤ 0.05, the protein is downregulated. Figure 15 ).

[0091] 4.4 GO enrichment analysis

[0092] The principle of GO enrichment analysis is to perform GO term functional annotation on differentially expressed protein-coding genes, calculate the number of differentially expressed protein-coding genes in each GO term, and use a hypergeometric test to compare the annotation results with the genomic background to screen for differentially expressed protein-coding genes that significantly enrich GO terms. The calculation formula is as follows:

[0093] P=1-∑i=0m-1(Mi)(N-Mn-i)(Nn)P=1-∑i=0m-1(Mi)(N-Mn-i)(Nn);

[0094] Where N is the number of genes with GO functional annotations;

[0095] n is the number of differentially expressed protein-coding genes in N;

[0096] M represents the number of genes annotated with a specific GO term;

[0097] m represents the number of differentially expressed protein-coding genes for a specific GO term.

[0098] Multiple hypothesis testing was performed on the calculated p-values, and GO terms with a q-value ≤ 0.05 were defined as significantly enriched GO terms for differentially expressed protein-coding genes. (Bar chart) Figure 16 This shows the distribution of GO functions related to differentially expressed protein-coding genes at the GO Level 2 functional level. (Bubble chart) Figure 17 The results are displayed as the top 30 ranked by enrichment level (x-axis: Rich Factor). The y-axis represents the GO term name, the dot color indicates the GO enrichment significance (q-value), the dot shape indicates which of the three major categories of the GO database the corresponding GO entry belongs to, and the dot size represents the number of differentially expressed protein-coding genes mapped to this GO term.

[0099] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. The application of a camel milk exosome in the preparation of a drug for treating heart disease.

2. The application according to claim 1, characterized in that, The camel milk exosomes are derived from camel milk whey.

3. The application according to claim 1, characterized in that, The camel milk exosomes significantly reduced cardiac tissue cell apoptosis, alleviated cardiac tissue fibrosis, reduced cardiac tissue necrosis, and reduced acute cardiac injury.

4. The application according to claim 1, characterized in that, The method for preparing camel milk exosomes includes the following steps: S1. Centrifuge fresh camel milk and extract the middle layer liquid after centrifugation as whey; S2. Filter the whey obtained in S1, centrifuge the filtrate again, collect the gel-like precipitate at the bottom of the tube and break it up. S3. Resuspend the precipitate after S2 in buffer solution, centrifuge the resuspended solution again, collect the gel-like precipitate at the bottom of the tube and break it up. S4. Resuspend the precipitate after S3 in buffer solution. Filter the resulting resuspended solution through a coarse filter membrane and a sterile filter membrane. Collect the filtrate to obtain camel milk exosomes.

5. The application according to claim 1, characterized in that, The particle size range of the camel milk exosomes is 50-200 nm.

6. The application according to claim 1, characterized in that, The dosage of the camel milk exosomes used is (1-10)×10 12 particles / kg.

7. The application according to claim 1, characterized in that, The heart disease mentioned includes coronary heart disease, cardiomyopathy, arrhythmia, or heart failure.

8. The application according to claim 7, characterized in that, The heart failure mentioned includes chronic heart failure.

9. A drug for treating heart disease, characterized in that, The drug comprises camel milk exosomes as described in any one of claims 1-8 and pharmaceutically acceptable carriers.

10. The medicament according to claim 9, characterized in that, The dosage form of the drug includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powders for injection, and injections.

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