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

Abstract

The invention relates to an application of camel milk exosome in preparation of a medicine for treating kidney diseases. According to the invention, the camel milk exosome is used for kidney diseases, especially for relieving acute renal failure, and a brand-new and safe treatment scheme is provided for kidney protection; the camel milk exosome is simple in preparation process, good in stability, high in safety and good in application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of the treatment of kidney diseases, and particularly relates to the application of camel milk exosomes in the preparation of drugs for treating kidney diseases. Background Art

[0002] In recent years, exosomes have become a hot topic in the emerging research field of biomedicine. Exosomes are nanoscale vesicles secreted by cells, with natural drug-loading properties, low immunogenicity, and targeted delivery capabilities. They can carry various bioactive molecules such as proteins and nucleic acids, and precisely regulate the functions of recipient cells. In the research on the treatment of kidney diseases, a research team from the School of Medicine of Jiangsu University and the Jiangsu Key Laboratory of Laboratory Medicine affiliated to it published in the journal "Am J Transl Res" that exosomes derived from human umbilical cord mesenchymal stem cells can transfer 14-3-3ζ by adjusting the ATG16L protein, thereby significantly enhancing autophagy and effectively preventing cisplatin-induced acute kidney injury. CN114728024B discloses exosomes isolated from precursor cells of induced pluripotent stem cell-derived mesenchymal stem cells pretreated or not pretreated with a pretreatment substance as an active ingredient, and the exosomes show further improved preventive or therapeutic effects on kidney diseases. However, due to the extraction and purification process of exosomes being affected by various factors such as sample source, treatment method, and experimental conditions, there may be significant differences between different batches of exosomes. Such differences may affect the quality and function of exosomes, and thus affect their consistency and reliability in experimental and clinical applications.

[0003] In contrast, animal-derived exosomes, such as bovine milk and camel milk exosomes, have become a more promising research direction due to their wide sources and stable components. Among them, camel milk exosomes have attracted the attention of many researchers due to their unique biocompatibility and high safety. Existing research has shown that camel milk exosomes have cytotoxic effects on various cancer cell lines, while having relatively low toxicity to normal cells. Although camel milk exosomes have shown certain advantages in related research, the current research on kidney diseases and their mechanisms of action is still very scarce. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide the application of camel milk exosomes in the preparation of drugs for treating kidney diseases, and for the first time, propose to use camel milk exosomes for kidney diseases, especially to relieve acute renal failure, providing a new and safe treatment plan for protecting the kidneys.

[0005] On the one hand, the present invention provides the application of camel milk exosomes in the preparation of drugs for treating kidney diseases.

[0006] Preferably, the camel milk exosomes are derived from camel milk whey.

[0007] Preferably, the camel milk exosomes significantly reduce apoptosis of kidney tissue cells, alleviate the degree of kidney tissue fibrosis, reduce the necrosis rate of kidney tissue, and alleviate acute kidney injury.

[0008] Preferably, the method for preparing the camel milk exosomes comprises the following steps:

[0009] S1. Centrifuge fresh camel milk, and extract the middle layer liquid after centrifugation as whey;

[0010] S2. Filter the whey obtained in S1, centrifuge the obtained filtrate again, collect the gel-like precipitate at the bottom of the tube and break it;

[0011] S3. Resuspend the precipitate after breaking in S2 with a buffer solution (such as PBS buffer solution), centrifuge the obtained resuspension again, collect the gel-like precipitate at the bottom of the tube and break it;

[0012] S4. Resuspend the precipitate after breaking in S3 with a buffer solution (such as PBS buffer solution), filter the obtained resuspension through a coarse filter membrane (70 μm) and a sterile filter membrane (0.22 μm), and collect the filtrate to obtain camel milk exosomes.

[0013] Further, the centrifugation in the step S1 is specifically: centrifuging at 10,000 g to 15,000 g for 20 to 40 minutes.

[0014] Further, the filtration in the step S2 is specifically: using a filter membrane with a pore size of 70 to 100 μm.

[0015] Further, the centrifugation in the steps S2 and S3 is specifically: centrifuging at 100,000 to 120,000 g for 1 to 2 hours.

[0016] Preferably, the particle size range of the camel milk exosomes is 50 - 200 nm.

[0017] Preferably, the dosage of the camel milk exosomes is (1 - 10) * 10 12 particles / kg.

[0018] Preferably, the kidney diseases include nephritis, renal fibrosis or renal failure.

[0019] Preferably, the renal failure is acute renal failure, including acute renal failure caused by ischemia.

[0020] On the other hand, the present invention provides a drug for treating kidney diseases, and the drug comprises the camel milk exosomes as described above and a pharmaceutically acceptable carrier.

[0021] Preferably, the dosage form of the drug includes at least one of tablets, capsules, pills, powders, granules, suspensions, oral solutions, powder injections and injections.

[0022] The present invention proposes to apply camel milk exosomes to the treatment of kidney diseases, especially acute renal failure. By optimizing the exosome purification process, highly pure camel milk exosomes can be obtained quickly and efficiently. With the help of an animal model of ischemic acute renal failure, its effects in reducing the total amount of urinary creatinine and urea, inhibiting renal cell apoptosis and fibrosis are verified, opening up a new way for the treatment of acute renal failure.

[0023] Beneficial effects

[0024] The present invention proposes to prepare and administer drugs for kidney diseases with camel milk exosomes, especially for acute renal failure, which can very effectively relieve acute kidney injury, including significantly reducing renal tissue cell apoptosis, reducing the degree of renal tissue fibrosis, and significantly reducing the necrosis rate of renal tissue. The comprehensive performance is excellent, achieving the curative effect of protecting the kidneys. It acts through the following mechanisms:

[0025] 1. Regulation of apoptosis: Camel milk exosomes can precisely regulate the apoptosis-related signaling pathways in cells and significantly reduce the apoptosis rate of renal tissue cells. By inhibiting the expression of pro-apoptotic proteins and enhancing the activity of anti-apoptotic proteins at the same time, the stability of the intracellular environment is maintained, enabling renal cells to maintain normal physiological functions and survival states.

[0026] 2. Inhibition of fibrosis process: Camel milk exosomes effectively reduce the degree of renal tissue fibrosis, can inhibit the activation and proliferation of renal stellate cells, and reduce the excessive deposition of extracellular matrix, thereby blocking the progression of renal fibrosis. By regulating the cytokine network, it promotes the degradation and remodeling of fibrous tissues, gradually restoring the normal structure of the renal tissue.

[0027] 3. Relief of necrosis degree: Camel milk exosomes significantly reduce the necrosis rate of renal tissue. By improving the blood circulation of the kidneys, increasing the oxygen supply and nutrient supply to renal cells, and reducing the damage of ischemia and hypoxia to renal cells. At the same time, it enhances the antioxidant capacity of renal cells, scavenges free radicals, and reduces the damage of oxidative stress to cells, thereby effectively protecting renal tissue from further damage.

[0028] Generally speaking, the therapeutic effect of camel milk exosomes is not only reflected in the improvement of pathological indicators, but more importantly, in the protection and repair of the overall renal function. Through the synergistic action of the above-mentioned multiple mechanisms, the kidneys can maintain normal metabolic, detoxification and synthetic functions in the state of acute kidney injury, promote the regeneration and repair of renal cells, and finally achieve the curative effect of protecting the kidneys, providing a safe and effective new strategy for the treatment of acute renal failure. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Particle size distribution results of camel milk exosomes prepared in Example 1

[0030] Figure 2 Transmission electron microscopy test image of camel milk exosomes prepared in Example 1

[0031] Figure 3 A-D: H&E staining images of the kidneys of rats in the camel milk exosome treatment group and the control group; the scale bar in the figure is 100 microns

[0032] Figure 4 Analysis of the effect of camel milk exosomes on the change of H&E staining in rat kidneys

[0033] Figure 5 A-D: TUNEL staining images of the kidneys of rats in the camel milk exosome treatment group and the control group; the scale bar in the figure is 100 microns

[0034] Figure 6 Analysis of TUNEL staining of camel milk exosomes in rat kidneys

[0035] Figure 7 A-D: MASSON staining images of the kidneys of rats in the camel milk exosome treatment group and the control group; the scale bar in the figure is 100 microns

[0036] Figure 8 Analysis of MASSON staining of camel milk exosomes in rat kidneys

[0037] Figure 9 A-D: PAS staining images of the kidneys of rats in the camel milk exosome treatment group and the control group; the scale bar in the figure is 100 microns

[0038] Figure 10 Analysis of PAS staining of camel milk exosomes in rat kidneys

[0039] Figure 11 A-D: Effect of camel milk exosomes on the total amount of urinary urea (BUN) in animals with acute renal failure model of ischemia-reperfusion Detailed implementation mode

[0040] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application

[0041] Example 1

[0042] Preparation and physical sign detection of camel milk exosomes

[0043] 1.1 Preparation of Camel Milk Exosomes

[0044] Step 1: Separation of Whey: Pour 500 mL of fresh camel milk into a high-speed centrifuge tube, centrifuge at 13,000 g for 30 minutes, take the middle layer liquid to obtain camel milk whey.

[0045] Step 2: Filtration to Obtain Exosomes: After filtering the whey through a 70 μm filter membrane, divide the filtrate into ultracentrifuge tubes, centrifuge at 100,000 g for 1.5 h, discard the supernatant, take the gel-like precipitate at the bottom of the tube, and crush it into pieces.

[0046] Step 3: Removal of Impurities: Transfer the precipitated pieces from the previous step to a new ultracentrifuge tube, make up the PBS buffer to 50 mL, then centrifuge at a high speed of 100,000 g for 1.5 h, discard the supernatant, take the gel-like precipitate at the bottom of the tube, and crush it into pieces.

[0047] Step 4: Dissolution and Sterilization: Resuspend the precipitated pieces with 40 mL of PBS buffer, fully dissolve them into a uniform liquid by pipetting or homogenizing, and then sequentially filter through a 70 μm coarse filter membrane to remove insoluble impurities and a 0.22 μm sterile filter membrane for sterilization, and collect the filtrate to obtain purified camel milk exosomes.

[0048] 1.2 NTA Detection Results

[0049] For the NTA detection of exosomes, the nanoparticle tracking analysis (NTA) technology uses laser scattering microscopy imaging technology to record the Brownian motion trajectories of nanoparticles in solution, and calculates the particle size and particle concentration through the Stokes-Einstein equation. This technology can measure particle sizes in the range of 30 - 1000 nm, so it can provide more accurate particle size data and is currently the most mainstream method for measuring exosome size.

[0050] Dilute the prepared camel milk exosomes with PBS at a volume ratio of 1:1000. Use a 1 mL syringe to draw 1 mL of the exosome dilution, slowly inject it into the NTA device pipeline, set the detection parameters, start the detection, and the results are as Figure 1 shown. The average particle size is 166.6 nm, and the concentration is 1.33×10 12 Particles / mL.

[0051] 1.3 Electron Microscopy Observation

[0052] Take 20 μl of the prepared camel milk exosome suspension sample in step 1.1, pipette it onto a copper mesh and let it adsorb naturally for 5 - 10 minutes. Then use a filter paper strip to remove the excess liquid droplets and let it air-dry slightly. Pipette 20 μl of 2% phosphotungstic acid solution onto the copper mesh and let it stand for 3 - 5 minutes for staining. Use a filter paper strip to remove the excess liquid droplets and dry it under an incandescent lamp. Finally, observe and take pictures under a transmission electron microscope. The obtained transmission electron microscope micrograph is as Figure 2 shown, and this result is consistent with the NTA detection result.

[0053] Example 2

[0054] Effect of Camel Milk Exosomes on Carbon Tetrachloride-Induced Acute Renal Failure

[0055] Ischemic Acute Kidney Injury (IAKI) is a common type of acute kidney injury (AKI) clinically, mainly caused by insufficient renal blood perfusion, commonly seen in shock, major surgeries (such as cardiac or large vascular surgeries), severe dehydration, sepsis, etc. Its core pathophysiological mechanism is renal ischemia-reperfusion injury (IRI), involving tubular epithelial cell necrosis, inflammatory response, oxidative stress, and microcirculation disorders. The ischemic acute renal failure model is a commonly used experimental method for studying the mechanism of renal ischemia-reperfusion injury and is widely used in drug screening, pathological mechanism exploration, and treatment strategy evaluation.

[0056] 1. Experimental animals and reagents: 24 male SD rats, 7 - 8 weeks old, SPF grade. The experimental animals were purchased from Vital River Laboratories and raised in Hanshu Biomedical Co., Ltd. The rats were housed in an IVC system in a barrier facility.

[0057] 2. Experimental grouping and dosing regimen

[0058] Randomly divide the 24 mice into 4 groups: A - control group, B - acute renal failure model group, C - low-dose exosome treatment group, D - high-dose exosome treatment group, with 6 mice in each group.

[0059] Construction of an animal model of ischemic acute renal failure: On the 3rd day of drug administration, after the drug was administered in the morning, the rats were completely anesthetized and fixed in the supine position on the operating table. After depilating the abdomen, it was disinfected with iodophor and deiodized with 75% alcohol. A longitudinal incision was made along the midline of the abdomen to open the abdominal cavity. First, the right kidney was exposed, and the right renal pedicle and right ureter were separated. The entire renal pedicle and right ureter were ligated with 4-0 silk thread and the right kidney was removed; the left kidney was exposed and the left renal pedicle was separated. After the renal pedicle was clamped with a non-traumatic hemostatic clip for 60 minutes, the clip was loosened to restore renal perfusion. At this time, the color of the kidney first changed from bright red to pale or dark red, and then turned bright red again, indicating successful reperfusion. After observing that there was no bleeding or oozing in the surgical field, it was sutured layer by layer and the abdominal cavity was closed. The anesthesia, opening of the abdominal cavity, etc. in the sham group were the same as above. After exposing the left and right kidneys, the renal capsule was bluntly dissected. After the operation, 0.1 ml of gentamicin and meloxicam were given subcutaneously to the animals for analgesia and prevention of infection. The animals were placed on a warming blanket in the lateral position and returned to the breeding cage after being fully awake. Postoperative care was provided for 3 days.

[0060] Specific grouping and treatment methods: Groups A and B were gavaged with an equal volume of PBS buffer, 5 ml / kg, twice a day; Group C was intravenously injected with low-dose camel milk exosomes (1*10 11 particles / kg), twice a day; Group D was intravenously injected with high-dose camel milk exosomes (1*10 12 particles / kg), twice a day. On the 3rd day of drug administration, an acute renal failure model was established. Among them, Group A was gavaged with an equal volume of normal saline once, 2.5 ml / kg; the other three groups were gavaged with 50% carbon tetrachloride (volume ratio of carbon tetrachloride to olive oil 1:1) once, 2.5 ml / kg. Endpoint sample collection: Samples of the heart, kidney, lung, kidney, and whole brain were taken. Half of each tissue sample was snap-frozen in liquid nitrogen and stored at -80°C, and the other half was fixed with 4% PFA.

[0061] 3. Experimental results

[0062] 3.1 H&E staining

[0063] The renal cell samples of each experimental group in Step 2 were collected for H&E staining. The results were as Figure 3 shown. A was the control group with no obvious lesions; B was the model group with vacuolar lesions in the renal tubules and a large number of inflammatory cell infiltrations in the renal cortex; C was the low-dose treatment group with a small amount of inflammatory cell infiltrations in the renal cortex and a reduction in vacuolar lesions in the renal tubules; D was the high-dose treatment group with fewer inflammatory cells in the renal cortex and rare vacuolar lesions in the renal tubules.

[0064] 3.2 TUNEL staining

[0065] The renal cell samples of each experimental group in Step 2 of collection were subjected to TUNEL staining. The TUNEL staining technique is a commonly used method for detecting DNA fragmentation during apoptosis. Cells with positive TUNEL staining indicate DNA fragmentation, which is a key feature of apoptosis. By counting the number of positive cells, the proportion of apoptotic cells can be evaluated. The results of TUNEL analysis are as Figure 5 shown. Group A is the control group, with no obvious apoptotic positive cells; Group B is the model group, where positive cells appear and are numerous, and the apoptotic ratio is high; Group C is the low-dose treatment group, with a decrease in apoptotic positive cells and a decrease in vacuolar lesions in the renal tubules; Group D is the high-dose treatment group, and the positive cells also decrease significantly.

[0066] 3.3 Masson staining

[0067] The renal tissue samples of each experimental group in Step 2 of collection were subjected to Masson staining. As a classic connective tissue staining method, Masson staining provides an intuitive and effective means for detecting fibrosis. In fibrotic tissues, Masson staining will show obvious red or purple areas. These areas represent the proliferation and deposition of collagen fibers, which are typical manifestations of fibrotic lesions. By observing the distribution and scope of these areas, the degree and scope of fibrosis can be preliminarily judged. The results of Masson analysis of the renal tissue samples are as Figure 7 shown: Group A is the control group, with no obvious lesions; Group B is the model group, with severe fibrosis of the glomerular basement membrane and an increase in collagen fibers in the renal tubules; Group C is the low-dose treatment group, with a reduction in fibrosis of the glomerular basement membrane and the collagen fibers in the renal tubules being basically normal; Group D is the high-dose treatment group, with a reduction in fibrosis of the glomerular basement membrane and the collagen fibers in the renal tubules being normal. The fibrosis ratio of the rat kidneys in the exosome treatment group is lower than that in the acute renal failure model group. This result indicates that camel milk exosomes can alleviate the degree of renal fibrosis.

[0068] 3.4 PAS staining

[0069] PAS staining is a commonly used histochemical staining method, especially suitable for renal pathological examination, because it can clearly show polysaccharide-rich structures such as the basement membrane, mesangial matrix, glycogen, and mucinous substances, which helps to evaluate the lesions of the glomeruli, renal tubules, and interstitium. The results are as Figure 9 shown. Group A is the control group, with no obvious lesions; Group B is the model group, with multiple red-stained mucins seen in the renal tubular epithelial cells; Group C is the low-dose treatment group, with a reduction in red-stained mucins in the renal tubular epithelial cells; Group D is the high-dose treatment group, with a reduction in red-stained mucins in the renal tubular epithelial cells.

[0070] 3.5 Biochemical indicators:

[0071] After surgical ischemia modeling, urine samples were collected at 24 h, 48 h, 72 h, and 120 h to detect the levels of uric acid, urea, and creatinine, and urinary microalbumin was detected by biochemical analysis.

[0072] Terminal sample collection: Take samples of heart, liver, lung, kidney, and whole brain. Half of each tissue sample is snap-frozen in liquid nitrogen and stored at -80°C, and the other half is fixed with 4% PFA.

[0073] Test results: As Figure 11 shown in A - D, at the 72h and 120h time points after single kidney ischemia-reperfusion, the total 24-hour urinary creatinine of the animals in the model group increased significantly, while the total urinary creatinine was significantly reduced in the camel milk exosome administration group.

[0074] In summary, compared with the positive renal failure group in group B, in groups C and D of the camel milk exosome administration group, the degree of apoptosis of renal tissue cells ( Figure 6 ) and the degree of renal tissue fibrosis were significantly reduced ( Figure 8 ), the necrosis rate of renal tissue was significantly decreased ( Figure 4 ), and urinary creatinine was significantly reduced.

[0075] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various deformations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. Use of camel milk exosomes in the preparation of drugs for treating kidney diseases.

2. The application according to claim 1, wherein 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 reduce apoptosis of kidney tissue cells, alleviate the degree of fibrosis of kidney tissue, reduce the necrosis rate of kidney tissue, and alleviate acute kidney injury.

4. The application according to claim 1, wherein The preparation method of the camel milk exosomes comprises 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 obtained filtrate again, collect the gel-like precipitate at the bottom of the tube and break it; S3. Resuspend the precipitate broken in S2 with a buffer solution, centrifuge the obtained resuspended solution again, collect the gel-like precipitate at the bottom of the tube and break it; S4. Resuspend the precipitate broken in S3 with a buffer solution, filter the obtained resuspended solution through a coarse filter membrane and a sterile filter membrane, and 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 is (1 - 10) * 10 12 particles / kg.

7. The application according to claim 1, characterized in that The kidney diseases include nephritis, renal fibrosis or renal failure.

8. The application according to claim 7, characterized in that, The renal failure is acute renal failure.

9. A drug for treating kidney diseases, characterized in that, The drug comprises the camel milk exosomes as described in any one of claims 1-8 and a pharmaceutically acceptable carrier.

10. The drug 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, powder injections and injections.

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