Use of roxburgh rose exosome in preparation of medicine for treating neurodegenerative diseases

Abstract

The application provides application of a roxburgh rose exosome in preparation of a medicine for treating a neurodegenerative disease, and belongs to the technical field of biological medicine. The roxburgh rose exosome presents an oval vesicle structure, the particle size is 100-200 nm, the ZETA potential is-38.27+ / -0.58 mV, can effectively inhibit the inflammatory reaction of microglia cells induced by LPS, and can remove excessive intracellular ROS, and has good anti-inflammatory and antioxidant effects in the treatment of neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of prickly pear exosomes in the preparation of drugs for treating neurodegenerative diseases. Background Technology

[0002] Neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, and Huntington's disease) are characterized by progressive damage and loss of neurons, primarily affecting the central nervous system and ultimately leading to cognitive and motor dysfunction. Their common pathological mechanism involves the synergistic effect of oxidative stress and chronic inflammation. Oxidative stress plays a key driving role in the disease process; the accumulation of excessive free radicals can induce lipid peroxidation, protein denaturation, and DNA damage, accelerating neuronal apoptosis and creating a vicious cycle by activating a neuroinflammatory cascade, thereby further promoting disease progression.

[0003] Exosomes are nanoscale vesicles rich in bioactive components such as proteins, nucleic acids, and lipids, capable of mediating intercellular communication. Due to their advantages such as natural origin, high biocompatibility, low immunogenicity, and strong ability to cross biological barriers, exosomes have become an important research vehicle for drug delivery in neurodegenerative diseases. Studies have shown that exosomes can load various therapeutic molecules and cross the blood-brain barrier to achieve targeted delivery to neurons or glial cells, thereby regulating cellular function and improving the pathological microenvironment, demonstrating promising therapeutic potential.

[0004] Rosa rugosa, a plant belonging to the genus Rosa of the Rosaceae family, is rich in vitamin C, vitamin E, and various flavonoids and polyphenols, exhibiting significant antioxidant and anti-inflammatory effects. It holds potential value in the prevention and treatment of neurodegenerative diseases. Rosa rugosa-derived exosomes not only inherit its natural active ingredients but also possess the highly efficient delivery characteristics of exosomes, allowing them to intervene in disease progression through the synergistic regulation of oxidative stress and inflammatory responses. Based on these unique advantages, Rosa rugosa exosomes hold promise as a potential candidate strategy for the prevention and treatment of neurodegenerative diseases, warranting further in-depth research and development. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the application of prickly pear exosomes in the preparation of drugs for treating neurodegenerative diseases.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This application provides an application of prickly pear exosomes, specifically in the preparation of drugs for treating neurodegenerative diseases.

[0007] The method for preparing prickly pear exosomes in this application includes: adding cleaned prickly pear fruit to PBS buffer at 4°C for juicing, and then centrifuging the filtered suspension three times, including ultracentrifugation and density gradient centrifugation, to obtain prickly pear exosomes.

[0008] The present invention has the following beneficial effects: (1) The prickly pear exosomes provided in this application can significantly inhibit LPS-induced microglial cell activation and inflammatory response, clear excess ROS in cells, thereby reducing oxidative stress damage and showing good anti-inflammatory and antioxidant effects in the prevention and treatment of neurodegenerative diseases.

[0009] (2) The prickly pear exosomes provided in this application have a typical elliptical vesicle structure with clear membrane boundaries, uniform overall morphology, good structural integrity, and conform to the classic morphological characteristics of exosomes.

[0010] (3) The prickly pear exosomes provided in this application have an average particle size of 150.1 nm, high particle concentration and good dispersion, and uniform particle size distribution, which conforms to the general characteristics of exosome nanoparticles and is conducive to their transport and penetration in the body.

[0011] (4) The ZETA potential of the prickly pear exosomes provided in this application is -38.27±0.58mV, which has good stability and helps to maintain its dispersibility and functional activity in the physiological environment. Attached Figure Description

[0012] Figure 1 This is a TEM (Transmission Electron Microscope) image of the prickly pear exosomes prepared in Example 1 of this application; Figure 2 This is a particle size distribution diagram of the prickly pear exosomes prepared in Example 1 of this application; Figure 3 This is a potential distribution diagram of prickly pear exosomes prepared in Example 1 of this application; Figure 4 The images show the effects of prickly pear exosomes on inflammatory factors in BV2 cells. In the images, A shows the effect of prickly pear exosomes on TNFα, B shows the effect of prickly pear exosomes on IL6, C shows the effect of prickly pear exosomes on CXCL10, and D shows the effect of prickly pear exosomes on CCL5. Figure 5 The graph shows the effect of prickly pear exosomes on ROS levels in BV2 cells; where Figure A represents the ROS level in the control group, Figure B represents the ROS level in the model group, and Figure C represents the ROS level in the prickly pear exosome group. Detailed Implementation

[0013] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0014] Example 1 This application provides a method for preparing prickly pear exosomes, the method comprising: Cleaned prickly pear fruits and 4°C pre-cooled PBS buffer (phosphate buffer saline) were added to a juicer and juiced 5 times with a 10-second stirring time followed by a 10-second interval. After filtration, a homogeneous suspension was obtained.

[0015] The suspension was centrifuged for 15 minutes at 2500g, the precipitate was removed, and the supernatant was collected. The supernatant was then centrifuged for 20 minutes at 6000g, the precipitate was removed, and the supernatant was collected again. The supernatant was then centrifuged for 30 minutes at 10000g, the precipitate was removed, and the supernatant was collected again. This supernatant was centrifuged for 70 minutes at 100000g, and the precipitate was resuspended in PBS buffer to form a resuspension. The resuspension was then centrifuged for 120 minutes at 100000g using sucrose solutions of concentrations of 15%, 30%, 45%, and 60%, respectively. The bands of precipitates with concentrations of 30-45% sucrose were collected; these bands were identified as prickly pear exosomes.

[0016] In this embodiment, after washing the prickly pear exosomes with PBS buffer, the pellet was centrifuged at 100,000 g for 60 min and the precipitate was collected. The precipitate was resuspended in sterile PBS buffer, filtered through a 0.22 μm filter membrane, and stored at -80°C.

[0017] Example 2 In this embodiment, transmission electron microscopy and a nanoparticle tracking analyzer were used to characterize the prickly pear exosomes extracted in Example 1, including morphology, particle size, and ZETA potential analysis. The results are shown in the appendix. Figure 1 -Appendix Figure 3 As shown.

[0018] From the appendix Figure 1 -Appendix Figure 3 As can be seen, the prickly pear exosomes extracted in Example 1 exhibit an elliptical vesicle structure with good morphological and structural integrity; the average particle size of the prickly pear exosomes is 150.1 nm, with uniform particle size distribution and high particle concentration, which is conducive to their transport and penetration in the organism; the zeta potential of the prickly pear exosomes is -38.27 ± 0.58 mV, which shows good stability.

[0019] Example 3 Example 3 of this application describes the anti-inflammatory and antioxidant effects of prickly pear exosomes extracted in Example 1 on neurodegenerative diseases. The specific details are as follows: 1. Anti-inflammatory test Overactivation of microglia is a key initiating factor in the inflammatory cascade response in neurodegenerative diseases. By examining the inhibitory effects of prickly pear exosomes on inflammatory factors such as TNFα, IL6, CXCL10, and CCL5 in BV2 microglia, the therapeutic efficacy of this treatment for neurodegenerative diseases can be evaluated.

[0020] BV2 cells were divided into three groups: a control group, a model group, and a prickly pear exosome group. The prickly pear exosome group was incubated with prickly pear exosomes (20 μM) for 1 h. The model group and the prickly pear exosome group were stimulated with LPS (1 μg / mL) for 3 h. The control group was incubated with the corresponding volume of culture medium. After incubation, the expression levels of inflammatory factors TNFα, IL6, CXCL10, and CCL5 in microglia of each group were measured by RT-qPCR. The results are shown in the attached figure. Figure 4 As shown.

[0021] From the appendix Figure 4 As can be seen, compared with the control group, the expression of inflammatory factors TNFα, IL6, CXCL10, and CCL5 was significantly increased in the model group, indicating that LPS stimulation of BV2 cells successfully induced inflammation. Compared with the model group, the expression of inflammatory factors TNFα, IL6, CXCL10, and CCL5 was significantly decreased in the prickly pear exosome group, indicating that prickly pear exosomes can downregulate the mRNA levels of TNFα, IL6, CXCL10, and CCL5, significantly inhibit the excessive activation of microglia, and thus block the neuroinflammatory cascade and exert a neuroprotective effect.

[0022] 2. Antioxidant detection Oxidative stress is a key pathological feature in the development of neurodegenerative diseases, especially in the degeneration of dopaminergic neurons. By examining the effect of prickly pear exosomes on LPS-induced intracellular ROS levels in BV2 microglia, its potential interventional role in neurodegenerative diseases can be effectively evaluated.

[0023] This application divides BV2 cells into a blank group, a model group, and a prickly pear exosome group. The prickly pear exosome group was pre-incubated with prickly pear exosomes (20 μM) for 1 h, followed by stimulation with LPS (1 μg / mL) for 3 h in both the model and prickly pear exosome groups. The blank group received an equal volume of culture medium as a control. After treatment, DCFH-DA fluorescent probe staining was used, and flow cytometry was used to quantitatively detect intracellular ROS levels in each group, yielding the results. Figure 5 .

[0024] From the appendix Figure 5As can be seen, compared with the control group, the ROS level in BV2 cells of the model group was significantly increased; after intervention with prickly pear exosomes, the ROS level in the prickly pear exosome group was significantly reduced. These results indicate that prickly pear exosomes can effectively remove excess ROS in cells, alleviate the damage of oxidative stress to mitochondrial function, and thus inhibit the neuroinflammatory cascade.

[0025] As can be seen from the above, the prickly pear exosomes provided in this application have a significant effect on inhibiting LPS-induced microglial inflammatory response and reducing oxidative stress, providing experimental evidence for their application in neurodegenerative diseases.

[0026] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. Application of prickly pear exosomes in the preparation of drugs for treating neurodegenerative diseases.

2. The application according to claim 1, characterized in that, The preparation method of the prickly pear exosomes includes: adding cleaned prickly pear fruit to 4℃ PBS buffer and juicing; filtering the suspension and then centrifuging it three times, including ultracentrifugation and density gradient centrifugation, to obtain prickly pear exosomes.

3. The application according to claim 2, characterized in that, The three centrifugations consist of: a first centrifugation at 2500g for 15 minutes, a second centrifugation at 6000g for 20 minutes, and a third centrifugation at 10000g for 30 minutes.

4. The application according to claim 2, characterized in that, The ultracentrifugation conditions were: centrifugal force of 100,000 g and centrifugation time of 70 min; after ultracentrifugation, the sample was resuspended in PBS buffer.

5. The application according to claim 2, characterized in that, The density gradient centrifugation includes: centrifuging with sucrose solutions of concentrations of 15%, 30%, 45%, and 60% at a centrifugal force of 100,000 g for 120 min; the band precipitate of the 30-45% sucrose layer is prickly pear exosomes.

6. The application according to claim 2, characterized in that, The prickly pear exosomes were washed sequentially with PBS buffer, centrifuged at 100,000g for 60 min, resuspended in sterile PBS buffer, filtered through a 0.22μm filter membrane, and then stored at -80℃.

7. The application according to claim 2, characterized in that, The particle size of the prickly pear exosomes is 100-200 nm, and the ZETA potential is -38.27±0.58 mV.

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