Dendrobium exosome composition loaded with miR-146a and miR-124 and preparation method and application thereof
By using Dendrobium officinale exosome composition loaded with miR-146a and miR-124, the problems of blood-brain barrier penetration and stability were solved, achieving effective treatment of neuroinflammation and exhibiting significant anti-inflammatory and antioxidant functions.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGXI ZHENDING BIOMEDICAL RES CO LTD
- Filing Date
- 2025-10-09
- Publication Date
- 2026-05-12
AI Technical Summary
Existing delivery systems have difficulty penetrating the blood-brain barrier, and miR-146a and miR-124 are prone to degradation during delivery, posing challenges to the treatment of central nervous system inflammation.
Using Dendrobium officinale exosomes as carriers, miR-146a and miR-124 were loaded using electroporation technology to construct an EXE-miR-146a-124 composition. By utilizing the natural nanosize and antioxidant function of exosomes, delivery across the blood-brain barrier and enhanced stability were achieved.
It achieves significant anti-inflammatory and antioxidant effects, inhibits neuroinflammation through multiple pathways, and improves the stability and targeting of miR-146a and miR-124, making it suitable for the treatment of neuroinflammatory diseases of the central or peripheral nervous system.
Smart Images
Figure CN120960248B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, its preparation method, and its application. Background Technology
[0002] Central nervous system diseases (such as ischemic stroke) are often accompanied by chronic neuroinflammation, with the cytokine storm caused by excessive microglial activation being a core pathological mechanism. MicroRNAs (miRNAs), non-coding RNAs approximately 22 nt in length, achieve post-transcriptional gene silencing by targeting the 3'-UTR region of mRNA, and have become key regulators of the inflammatory network. miR-146a, as a key anti-inflammatory factor, can inhibit excessive microglial activation, thereby alleviating neuroinflammation. miR-124, primarily expressed in the nervous system, is a key negative regulator of central nervous system inflammation. It targets the transcription factor C / EBP-α, which is a key driver of microglial (macrophage) activation and pro-inflammatory cytokine expression. Decreased miR-124 expression leads to excessive microglial activation; therefore, restoring miR-124 levels is an important direction for the treatment of neuroinflammation. However, existing delivery systems face two major bottlenecks: ① Difficulty in penetrating the blood-brain barrier: synthetic vectors (such as liposomes) have difficulty effectively penetrating the blood-brain barrier (BBB); ② Insufficient stability and safety: viral vectors pose an immunogenicity risk, while free miRNAs are easily degraded by serum nucleases.
[0003] Plant exosomes, with their natural nanoscale size, low immunogenicity, high biocompatibility, and potential BBB penetration, are ideal CNS delivery vectors and have been explored for drug delivery. However, loading miR-146a and miR-124 still faces challenges, such as low loading efficiency and easy degradation of miRNAs during delivery. Therefore, there is an urgent need to develop an efficient and stable method for loading miR-146a and miR-124 onto plant exosomes to overcome the shortcomings of existing technologies.
[0004] Dendrobium officinale exosomes are naturally derived nanoparticles (approximately 30-150 nm in size) derived from plant cells. They possess excellent biocompatibility, low immunogenicity, and ease of large-scale production. They can effectively neutralize excess reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced intracellularly, reducing oxidative stress damage to lipids, proteins, and DNA, thereby systematically enhancing the cell's antioxidant defense system and offering potential for combating oxidative damage-related diseases. Based on this, this invention develops a Dendrobium officinale exosome composition simultaneously loaded with miR-146a and miR-124, which treats neuroinflammation through anti-inflammatory effects and the exosomes' own antioxidant functions. Summary of the Invention
[0005] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, its preparation method, and its application. Dendrobium officinale exosomes possess antioxidant functions, while miR-146a and miR-124 have anti-inflammatory functions and are easily degraded. Dendrobium officinale exosomes can provide a delivery carrier for miR-146a and miR-124, improving their stability and anti-inflammatory function, ultimately leading to the development of a drug for treating neuritis and related diseases.
[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, comprising Dendrobium officinale exosomes and anti-inflammatory factor miR-146a and nervous system regulatory factor miR-124.
[0007] The present invention also provides a method for preparing the Dendrobium officinale exosome composition loaded with miR-146a and miR-124, comprising the following steps:
[0008] S1. Extraction of exosomes from Dendrobium officinale
[0009] S101, Raw material pretreatment
[0010] Take fresh Dendrobium officinale stems, rinse them thoroughly with sterile PBS solution, remove the nodes and epidermis, and cut them into 0.5cm pieces. 3 Thin slices were crushed into powder and then soaked in water for 12-48 hours to obtain a Dendrobium officinale suspension.
[0011] S102, Preparation of Dendrobium officinale exosomes
[0012] The Dendrobium officinale suspension obtained from S101 was centrifuged stepwise, the supernatant was discarded, and the resulting precipitate was resuspended in PBS solution pre-cooled to 4°C and centrifuged to obtain Dendrobium officinale exosomes.
[0013] S2, Dendrobium officinale exosomes loaded with miR-146a and miR-124
[0014] The anti-inflammatory factor miR-146a and the nervous system regulator miR-124 were mixed to obtain a loading; then, Dendrobium officinale exosomes were mixed with the loading in a low-conductivity electroporation buffer to obtain a mixture; the mixture was then subjected to electroporation conversion to obtain a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, abbreviated as EXE-miR-146a-124.
[0015] Preferably, in S101, 2-year-old fresh Dendrobium officinale is selected.
[0016] Preferably, the stepwise centrifugation in S102 includes: primary centrifugation, 2000g, 20-30min, to remove cell debris; secondary centrifugation, 10000g, 30-40min, to obtain large vesicles; and tertiary ultracentrifugation, 4℃, 110000g, 50-90min.
[0017] Preferably, the centrifugation conditions after resuspending the PBS solution in S102 are: 110000g, 50-90min, for a total of 2-3 centrifugations.
[0018] Preferably, the nucleotide sequence of miR-146a in S2 is 5'-UGAGAACUGAAUUCCAUGGGUU-3', and the nucleotide sequence of miR-124 is 5'-UAAGGCACGCGGUGAAUGCCAA-3'.
[0019] Preferably, the low-conductivity electroporation buffer in S2 is siPORT™ siRNA Electroporation Buffer.
[0020] Preferably, the molar ratio of miR-146a and miR-124 in the loading in S2 is 1:(1-10).
[0021] The concentration of Dendrobium officinale exosomes in the mixture was 1×10⁻⁶. 7 ~1×10 8 cells / mL;
[0022] The concentration of the loaded material in the mixture is 50 nM.
[0023] Preferably, the parameters for electroporation conversion in S2 are: 0.1-1.5kV perforation pulse voltage, 100-400μF capacitance, 4°C operation throughout the electroporation process to reduce thermal effects, and 37°C repair for 30 minutes after the pulse to promote membrane pore closure.
[0024] The present invention also provides an application of the Dendrobium officinale exosome composition loaded with miR-146a and miR-124, wherein the Dendrobium officinale exosome composition loaded with miR-146a and miR-124 is used for the treatment of neuritis and related diseases.
[0025] This invention has significant technical advantages compared to existing technologies:
[0026] This invention provides a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, and its preparation method. For the first time, it combines naturally bioactive Dendrobium officinale exosomes with miR-146a and miR-124, which have synergistic anti-inflammatory effects, to construct the pharmaceutical composition EXE-miR-146a-124. This composition exhibits good anti-inflammatory and antioxidant functions, making it suitable for treating neuroinflammatory diseases of the central or peripheral nervous system. Compared with existing technologies, this invention not only provides a safe and efficient miRNA delivery system but also endows it with neuroprotective and anti-inflammatory effects, offering a novel, naturally derived intervention for the treatment of inflammatory diseases of the nervous system, with the following advantages:
[0027] (1) Significant anti-inflammatory effect: inhibits neuroinflammatory response through multiple pathways.
[0028] miR-146a reduces the expression of inflammatory factors (such as TNF-α, IL-6, and IL-1β) by inhibiting the TLR4 / NF-κB signaling pathway; miR-124 promotes the transformation of microglia / Mϕ into M2-type immunomodulatory cells, reducing neurotoxic inflammation; EXE-miR-146a-124 can significantly reduce inflammation levels and improve the tissue microenvironment in a neuritis model.
[0029] (2) It has strong antioxidant capacity and can alleviate oxidative stress damage.
[0030] Dendrobium officinale exosomes are rich in antioxidants such as polyphenols and polysaccharides, which can effectively reduce ROS, NO and MDA levels and protect neurons from oxidative stress damage; and miR-146a and miR-124 can indirectly regulate the expression of antioxidant enzymes such as SOD and CAT.
[0031] (3) Achieve cross-blood-brain barrier (BBB) delivery and target brain tissue.
[0032] Dendrobium officinale exosomes have small particle sizes (30-150nm) and can be delivered into the central nervous system through the nasal cavity or vein. Moreover, Dendrobium officinale exosomes are of natural origin and have an immune "camouflage" effect, which can increase BBB permeability.
[0033] (4) High stability and targeting
[0034] The synergistic effect of miR-146a and miR-124 resulted in a greater reduction in inflammatory factors and a more significant neuroprotective effect than when used alone. Compared with miR-146a or miR-124 alone, exosome encapsulation of miR-146a and miR-124 improved their stability and targeting, and extended the duration of therapeutic effect.
[0035] (5) High safety, no immunotoxic side effects
[0036] Exosomes extracted from Dendrobium officinale, a plant used for both medicinal and edible purposes, are natural, low in toxicity, and low in immunogenicity. They do not require the use of artificial synthetic materials (such as cationic polymers, viral vectors, PEG, etc.), and long-term use does not cause significant liver or kidney toxicity or immune reactions, making them suitable for the treatment of chronic neuroinflammatory diseases.
[0037] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description
[0038] Figure 1 This is the result of the total antioxidant capacity test of EXE-miR-146a-124 in Example 4 of the present invention;
[0039] Figure 2 This is the result of the expression level of the antioxidant enzyme SOD in the antioxidant function test of EXE-miR-146a-124 in Example 4 of the present invention;
[0040] Figure 3 This is the result of the expression level of the antioxidant substance MDA in the antioxidant function test of EXE-miR-146a-124 in Example 4 of the present invention;
[0041] Figure 4 This refers to the TNF-α level result in the anti-inflammatory function test of EXE-miR-146a-124 in Example 5 of the present invention;
[0042] Figure 5 This refers to the results of the anti-inflammatory function test of EXE-miR-146a-124 in Example 5 of the present invention, which showed the level of the pro-inflammatory cytokine IL-6.
[0043] Figure 6 This refers to the level of the pro-inflammatory cytokine IL-1β in the anti-inflammatory function test of EXE-miR-146a-124 in Example 5 of this invention;
[0044] Figure 7 This is the result of the CD206 mRNA expression level test of EXE-miR-146a-124 in Example 6 of the present invention;
[0045] Figure 8 It is the blood-brain barrier (BBB) permeability of EXE-miR-146a-124 in Embodiment 7 of the present invention. Detailed Implementation
[0046] The miR-146a and miR-124 used in this invention were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The nucleotide sequence of miR-146a is 5'-UGAGAACUGAAUUCCAUGGGUU-3', and the nucleotide sequence of miR-124 is 5'-UAAGGCACGCGGUGAAUGCCAA-3'.
[0047] The low-conductivity electroporation buffer used was siPORT™ siRNA Electroporation Buffer (Ambion / Thermo Fisher), supplied by Applied Biosystems (Ambion).
[0048] Example 1
[0049] This embodiment describes the preparation of a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, including the following steps:
[0050] S1. Extraction of exosomes from Dendrobium officinale
[0051] S101, Raw material pretreatment
[0052] Take the stems of fresh 2-year-old Dendrobium officinale, rinse three times with sterile PBS solution, remove nodes and epidermis, and cut into 0.5cm pieces. 3 Thin slices were pulverized into powder using a pulverizer and then soaked in water for 24 hours to obtain a Dendrobium officinale suspension.
[0053] S102, Preparation of Dendrobium officinale exosomes
[0054] The Dendrobium officinale suspension obtained from S101 was subjected to a series of centrifugations to remove impurities, specifically: primary centrifugation, 2000g, 20min, to remove cell debris; secondary centrifugation, 10000g, 30min, to obtain large vesicles; tertiary ultracentrifugation, 4℃, 110000g, 70min; then the supernatant was discarded, and the resulting precipitate was resuspended in PBS solution pre-cooled to 4℃ and centrifuged at 110000g for 70min, for a total of 2 centrifugations, to obtain Dendrobium officinale exosomes.
[0055] S2, Dendrobium officinale exosomes loaded with miR-146a and miR-124
[0056] The anti-inflammatory factor miR-146a and the nervous system regulatory factor miR-124 were mixed at a molar ratio of 1:1 to obtain the loading; then 1×10 7One Dendrobium officinale exosome and 200 ng of loading were mixed in 1 mL siPORT™ siRNA Electroporation Buffer to obtain a mixture; the mixture was then subjected to electroporation conversion with the following parameters: 1 kV poration pulse voltage, 300 μF capacitance, 4°C operation throughout the electroporation process to reduce thermal effects, and 37°C repair for 30 min after the pulse to close the membrane pores, resulting in a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, abbreviated as EXE-miR-146a-124.
[0057] Example 2
[0058] This embodiment describes the preparation of a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, including the following steps:
[0059] S1. Extraction of exosomes from Dendrobium officinale
[0060] S101, Raw material pretreatment
[0061] Take the stems of fresh 2-year-old Dendrobium officinale, rinse three times with sterile PBS solution, remove nodes and epidermis, and cut into 0.5cm pieces. 3 Thin slices were pulverized into powder using a pulverizer and then soaked in water for 12 hours to obtain a Dendrobium officinale suspension.
[0062] S102, Preparation of Dendrobium officinale exosomes
[0063] The Dendrobium officinale suspension obtained from S101 was subjected to a series of centrifugations to remove impurities, specifically: primary centrifugation, 2000g, 30min, to remove cell debris; secondary centrifugation, 10000g, 40min, to obtain large vesicles; tertiary ultracentrifugation, 4℃, 110000g, 90min; then the supernatant was discarded, and the resulting precipitate was resuspended in PBS solution pre-cooled to 4℃ and centrifuged at 110000g for 90min. This process was repeated three times to obtain Dendrobium officinale exosomes.
[0064] S2, Dendrobium officinale exosomes loaded with miR-146a and miR-124
[0065] The anti-inflammatory factor miR-146a and the nervous system regulatory factor miR-124 were mixed at a molar ratio of 1:5 to obtain the loading; then 5 × 10 7One Dendrobium officinale exosome and 200 ng of loading were mixed in 1 mL siPORT™ siRNA Electroporation Buffer to obtain a mixture; the mixture was then subjected to electroporation conversion with the following parameters: 1.5 kV poration pulse voltage, 100 μF capacitance, 4°C operation throughout the electroporation process to reduce thermal effects, and 37°C repair for 30 min after pulse to close the membrane pores, resulting in a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, abbreviated as EXE-miR-146a-124.
[0066] Example 3
[0067] This embodiment describes the preparation of a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, including the following steps:
[0068] S1. Extraction of exosomes from Dendrobium officinale
[0069] S101, Raw material pretreatment
[0070] Take the stems of fresh 2-year-old Dendrobium officinale, rinse three times with sterile PBS solution, remove nodes and epidermis, and cut into 0.5cm pieces. 3 Thin slices were pulverized into powder using a pulverizer and then soaked in water for 48 hours to obtain a Dendrobium officinale suspension.
[0071] S102, Preparation of Dendrobium officinale exosomes
[0072] The Dendrobium officinale suspension obtained from S101 was subjected to a series of centrifugations to remove impurities, specifically: primary centrifugation, 2000g, 20min, to remove cell debris; secondary centrifugation, 10000g, 30min, to obtain large vesicles; tertiary ultracentrifugation, 4℃, 110000g, 50min; then the supernatant was discarded, and the resulting precipitate was resuspended in PBS solution pre-cooled to 4℃ and centrifuged at 110000g for 50min, for a total of 2 centrifugations, to obtain Dendrobium officinale exosomes.
[0073] S2, Dendrobium officinale exosomes loaded with miR-146a and miR-124
[0074] The anti-inflammatory factor miR-146a and the nervous system regulatory factor miR-124 were mixed at a molar ratio of 1:10 to obtain the loading; then 1×10 8One Dendrobium officinale exosome and 200 ng of loading were mixed in 1 mL siPORT™ siRNA Electroporation Buffer to obtain a mixture; the mixture was then subjected to electroporation conversion with the following parameters: 0.5 kV pore pulse voltage, 400 μF capacitance, 4°C operation throughout the electroporation process to reduce thermal effects, and 37°C repair for 30 min after the pulse to close the membrane pores, resulting in a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, abbreviated as EXE-miR-146a-124.
[0075] Example 4
[0076] The antioxidant capacity of EXE-miR-146a-124 was determined by the ABTS rapid method.
[0077] The principle of the ABTS method for determining antioxidant capacity: ABTS is oxidized to green ABTS under the action of an oxidizing agent. ·+ ABTS in the presence of antioxidants ·+ The generation of ABTS will be suppressed, and ABTS will be measured at 414 nm or 734 nm. ·+ The total antioxidant capacity of a sample can be determined and calculated from its absorbance. Trolox is a vitamin E analog with similar antioxidant capacity to vitamin E and is used as a reference for the total antioxidant capacity of other antioxidants. For example, the total antioxidant capacity of Trolox is 1. At the same concentration, the antioxidant capacity of other substances is expressed as a multiple of their antioxidant capacity compared to Trolox.
[0078] Preparation of ABTS working solution: ABTS stands for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). After preparation, the ABTS working stock solution should be stored at room temperature in the dark for 12-16 hours before use and is stable for 2-3 days. Before use, dilute the ABTS working stock solution with PBS or 80% ethanol to prepare the ABTS working solution. The absorbance of the ABTS working solution should be subtracted from the corresponding PBS or 80% ethanol blank control. 734 The value is 0.7 ± 0.05, corresponding to A. 405 The concentration is around 1.4. When the sample to be tested is water-soluble, it is diluted with PBS, and the dilution factor of the ABTS working stock solution is approximately 30-50 times. When the sample to be tested is not water-soluble, it is diluted with 80% ethanol, and the dilution factor of the ABTS working stock solution is approximately 35-55 times.
[0079] The measurement steps are as follows:
[0080] (1) Preparation of the sample to be tested:
[0081] Prepare 10 μL of EXE-miR-146a-124 sample and set three sample concentrations: 1 × 10⁻⁶. 7 exosomes / mL, 5×10 7 exosomes / mL, 1×10 8 One exosome / mL. Preparation method is the same as in Example 1.
[0082] (2) Preparation for standard curve determination:
[0083] Dilute the standards with PBS to make 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5 mM solutions of 10 mM Trolox.
[0084] (3) Determination of antioxidant capacity:
[0085] a. Add 200 μL of ABTS working solution to each well of the 96-well plate.
[0086] b. Add 10 μL of PBS solution to the blank control well, add 10 μL of Trolox standard solution of different concentrations to the standard curve detection well, and add 10 μL of EXE-miR-146a-124 sample of different exosome concentrations to the sample detection well, and mix gently.
[0087] c. Measure A after incubating at room temperature for 2-6 minutes. 734 .
[0088] d. Calculate the antioxidant capacity of the sample based on the standard curve; if the absorbance measured by the sample is outside the range of the standard curve, the sample needs to be appropriately diluted or concentrated before measurement.
[0089] e. Methods of expressing antioxidant capacity:
[0090] When using Trolox as a standard for total antioxidant capacity testing, the antioxidant capacity of the sample is directly expressed by the molar concentration of Trolox.
[0091] The antioxidant function test results of EXE-miR-146a-124 are as follows: Figure 1 As shown, the results indicate that EXE-miR-146a-124 can enhance antioxidant capacity and alleviate oxidative stress damage.
[0092] Figure 2 This is the result of the expression level of the antioxidant enzyme SOD. Figure 3The results show the expression levels of the antioxidant MDA. This indicates that miR-146a and miR-124 can indirectly regulate the expression of antioxidant enzymes such as SOD and CAT; the exosomes of *Dendrobium officinale* are rich in antioxidants such as polyphenols and polysaccharides, effectively reducing ROS, NO, and MDA levels, and protecting neurons from oxidative stress damage.
[0093] Example 5
[0094] The anti-inflammatory function of EXE-miR-146a-124 was determined by enzyme-linked immunosorbent assay (ELISA), as follows:
[0095] (1) Establishment of a cell model of neuritis
[0096] Microglia were seeded into 96-well plates (approximately 1 × 10⁶ cells per well). 4 Cells / well), cultured at 37°C and 5% CO2 for 24 hours.
[0097] Inflammation induction: Replace with culture medium containing LPS and stimulate for 6 hours.
[0098] (2) Remove the LPS-containing culture medium and add a sample concentration of 1×10⁻⁶. 8 EXE-miR-146a-124, 1×10 8 EXE-miR-146a, 1×10 8 Fresh culture medium treated with EXE-miR-124 was cultured for another 24 hours.
[0099] 1×10 8 The preparation method of EXE-miR-146a is the same as in Example 3, except that miR-124 is not added; 1×10 8 The preparation method of EXE-miR-124 is the same as in Example 3, except that miR-146a is not added.
[0100] (3) Sample collection
[0101] Collect the cell supernatant into centrifuge tubes, centrifuge at 3000 rpm for 10 min at 4°C to remove cell debris, aliquot the supernatant, and store at -80°C (avoid repeated freeze-thaw cycles).
[0102] (4) The concentration of inflammatory factors was determined by ELISA.
[0103] Results of TNF-α level test for inflammatory factor as follows Figure 4 As shown, the IL-6 level test results are as follows: Figure 5 As shown, the IL-1β level test results are as follows: Figure 6As shown in the results, miR-146a significantly reduced inflammation levels and improved the tissue microenvironment in the neurological model by inhibiting the TLR4 / NF-κB signaling pathway and reducing the expression of inflammatory factors (such as TNF-α, IL-6, and IL-1β). The synergistic effect of EXE-miR-146a-124 was greater than that of single EXE-miR-146a or EXE-miR-124 treatment, resulting in a greater reduction in inflammatory factors and a more significant neuroprotective effect. Compared with single miRNAs, exosome-encapsulated miR-146a and miR-124 improved stability and targeting, and prolonged the duration of therapeutic effect. EXE-miR-146a-124 can inhibit neuroinflammatory responses through multiple pathways and has a significant anti-inflammatory effect.
[0104] Example 6
[0105] EXE-miR-146a-124 promotes the transformation from M0 microglia to M2 microglia. The experimental procedure is as follows:
[0106] 1. Material preparation
[0107] Microglia (BV2 microglia line), 1 mL contains 1×10 8 One EXE-146a-124 sample.
[0108] 2. Microglia treatment and polarization
[0109] Microglia (BV2) were seeded into 6-well plates to achieve 70-80% confluence.
[0110] Incubate with unstimulated complete culture medium for 24 hours to maintain the M0 resting state, then add 1 mL of a solution containing 1×10⁻⁶ cells / mL. 8 Incubate EXE-146a-124 cells for 24-48 hours and collect the cells.
[0111] 3. SM2 type polarization assessment
[0112] The qRT-PCR method for detecting CD206 mRNA expression levels is as follows:
[0113] (1) Extract total RNA from cells;
[0114] (2) Reverse transcription: 1 μg of total RNA was reverse transcribed into cDNA using the PrimeScript™ RT reagent Kit (Takara), strictly following the instructions.
[0115] (3) qPCR: The qPCR was performed on a real-time quantitative PCR instrument using the BeyoFast™ SYBR Green One-Step qRT-PCR Kit (Shanghai Beyotime Biotechnology Co., Ltd.). The primers are as follows:
[0116]
[0117] The reaction system consisted of: 10 μL qPCR Mix (2×), 1 μL primer F, 1 μL primer R, 1 μL cDNA template, and ddH2O to a final volume of 20 μL.
[0118] The reaction program was as follows: pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 10 seconds, annealing at 60°C for 20 seconds, 40 cycles; extension at 72°C for 30 seconds.
[0119] Figure 7 This is a graph showing the results of CD206 mRNA expression level detection. Elevated CD206 mRNA expression level indicates that miR-124 promotes the transformation of microglia / Mϕ into M2 type immunomodulatory cells and reduces neurotoxic inflammation.
[0120] Example 7
[0121] The study on EXE-miR-146a-124 crossing the blood-brain barrier was conducted as follows:
[0122] 1. Constructing an in vitro BBB model
[0123] hCMEC cells were seeded in the upper chamber of a Transwell (0.4 μm membrane), and endothelial culture medium was added to the lower chamber. After 5-7 days of culture, a dense monolayer was formed, and the transmembrane resistance was measured using a TEER meter (>150 Ω·cm). 2 (Qualified).
[0124] 2. Sample addition treatment
[0125] Add 1 mL of 1×10 to the upper chamber 8 EXE-miR-146a-124 exosomes labeled with DiI were incubated for 6 hours; control group: LNP-miR-146a-124.
[0126] 3. Sampling and Detection
[0127] Culture medium was collected from the lower chamber, and the fluorescence signal was detected using an ELISA reader.
[0128] Figure 8This is a graph showing the permeability of EXE-miR-146a-124 across the blood-brain barrier (BBB). Dendrobium officinale exosomes have small particle sizes (30-150 nm) and can be delivered into the central nervous system via the nasal cavity or vein. The EXE-miR-146a-124 of this invention can achieve cross-BBB delivery, targeting brain tissue. Furthermore, since Dendrobium officinale exosomes are of natural origin, they possess an immune "camouflage" effect, which can enhance BBB permeability.
[0129] In summary, this invention provides a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, and its preparation method. The Dendrobium officinale exosomes possess antioxidant functions, while miR-146a and miR-124 exhibit anti-inflammatory functions and are readily degradable. The Dendrobium officinale exosomes can provide a delivery carrier for miR-146a and miR-124, enhancing their stability and anti-inflammatory function, ultimately leading to the development of a drug for treating neuritis and related diseases. This invention not only provides a safe and efficient miRNA delivery system but also endows it with neuroprotective and anti-inflammatory effects, offering a novel, naturally derived intervention for the treatment of inflammatory diseases of the nervous system.
[0130] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.
Claims
1. A method for preparing a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, characterized in that, The Dendrobium officinale exosome composition loaded with miR-146a and miR-124 includes Dendrobium officinale exosomes, anti-inflammatory factor miR-146a, and nervous system regulator miR-124. Its preparation method includes the following steps: S1. Extraction of exosomes from Dendrobium officinale S101, Raw material pretreatment Take fresh Dendrobium officinale stems, rinse them thoroughly with sterile PBS solution, remove the nodes and epidermis, and cut them into 0.5cm pieces. 3 Thin slices were crushed into powder and then soaked in water for 12-48 hours to obtain a Dendrobium officinale suspension. S102, Preparation of Dendrobium officinale exosomes The Dendrobium officinale suspension obtained from S101 was centrifuged stepwise, the supernatant was discarded, and the resulting precipitate was resuspended in PBS solution pre-cooled to 4°C and centrifuged to obtain Dendrobium officinale exosomes. S2, Dendrobium officinale exosomes loaded with miR-146a and miR-124 Anti-inflammatory factor miR-146a and nervous system regulator miR-124 were mixed at a molar ratio of 1:(1-10) to obtain a loading; then, *Dendrobium officinale* exosomes were mixed with the loading in a low-conductivity electroporation buffer to obtain a mixture, wherein the concentration of *Dendrobium officinale* exosomes in the mixture was 1×10⁻⁶. 7 ~1×10 8 The concentration of the loaded material was 50 nM; the mixture was then subjected to electroporation conversion with the following parameters: poration pulse voltage 0.1-1.5kV, capacitance 100-400μF, electroporation operation at 4℃ throughout to reduce thermal effects, and repair at 37℃ for 30 min after pulse to promote membrane pore closure, resulting in a Dendrobium officinale exosome composition loaded with miR-146a and miR-124, abbreviated as EXE-miR-146a-124; The nucleotide sequence of miR-146a is 5'-UGAGAACUGAAUUCCAUGGGUU-3', and the nucleotide sequence of miR-124 is 5'-UAAGGCACGCGGUGAAUGCCAA-3'.
2. The method according to claim 1, characterized in that, S101 selects 2-year-old fresh Dendrobium officinale.
3. The method according to claim 1, characterized in that, The stepwise centrifugation described in S102 includes: primary centrifugation, 2000g, 20-30min, to remove cell debris; secondary centrifugation, 10000g, 30-40min, to obtain large vesicles; and tertiary ultracentrifugation, 4℃, 110000g, 50-90min.
4. The method according to claim 1, characterized in that, The centrifugation conditions after resuspending the PBS solution in S102 are: 110000g, 50-90min, for a total of 2-3 centrifugations.
5. The method according to claim 1, characterized in that, The low-conductivity electroporation buffer mentioned in S2 is siPORT™ siRNA Electroporation Buffer.
6. The use of the Dendrobium officinale exosome composition loaded with miR-146a and miR-124 as described in claim 1 in the preparation of a medicament for treating neuritis.