Application of miRNA in preparation of medicine for preventing and treating Parkinson's disease

Abstract

The invention discloses an application of miRNA (micro Ribonucleic Acid) in preparation of a medicine for preventing and treating Parkinson's disease, and particularly provides an application of miRNA, which is miR-363-3p, miR-146b-3p, miR-24-2-5p, miR-26a-5p and miR-22-3p and plays a role in neuroprotection in SKP-SC-EVs (SKP-SC-EVs). Wherein the miR-146b-3p has the best effect of improving the activity of damaged nerve cells, and can recover TCA cycle key enzyme down-regulation caused by neurotoxic substances, and inhibition of the miR-146b-3p can lead to cell activity weakening with increased SKP-SC-EVs. The invention provides miRNA for protecting dopaminergic neurons by adjusting mitochondrial functions and energy metabolism, can be applied to preparation of drugs for preventing and treating Parkinson's disease, and is beneficial to prevention and treatment of Parkinson's disease and other neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to the application of miRNA in the preparation of drugs, and particularly to the application of miRNA in the preparation of drugs for preventing and treating Parkinson's disease, belonging to the field of biomedical technology. Background Art

[0002] Parkinson's disease (PD) is a common neurodegenerative disease with more than 10 million patients globally, and it is expected to exceed 17 million by 2040. The clinical manifestations of PD include motor symptoms (such as bradykinesia, rigidity, and resting tremor) and non-motor symptoms (such as olfactory dysfunction, depression, and sleep disorders). Its pathological features are mainly the progressive loss of dopaminergic neurons in the substantia nigra of the midbrain and the formation of Lewy bodies formed by the aggregation of α-synuclein. The pathogenesis of PD is complex, involving multiple factors such as mitochondrial dysfunction, neuroinflammation, and oxidative stress, among which mitochondrial dysfunction plays a central role in the pathology of PD. Currently, levodopa is still the main treatment drug for PD, and other drugs such as dopamine receptor agonists and MAO-B inhibitors are also commonly used to relieve symptoms. However, although the existing treatment methods can relieve some symptoms and delay the progression of the disease, no significant breakthrough has been achieved in protecting dopaminergic neurons.

[0003] Extracellular vesicles (EVs), as a double-membrane nanostructure secreted by cells, have the ability to cross the blood-brain barrier and show great potential in the treatment of neurodegenerative diseases. Studies have shown that EVs from multiple sources have neuroprotective effects in PD models. Skin-derived precursor cells (SKPs), due to their self-renewal and multi-directional differentiation potential, have become an ideal cell source in regenerative medicine. The inventors' previous research confirmed (Patent Invention Application Publication No. CN 115554316A) that SKPs can differentiate into Schwann cells (SCs), and the EVs secreted by them (SKP-SC-EVs) show good effects in promoting peripheral nerve regeneration. In addition, the conditioned medium of SKP-SCs can reduce the aggregation of α-synuclein by activating the PI3K / AKT / mTOR pathway and inhibiting autophagy, suggesting its potential value in the treatment of neurodegenerative diseases.

[0004] Currently, it is not clear which substances play a neuroprotective role in SKP-SC-EVs, which limits the prevention and treatment of PD and other neurodegenerative diseases. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide the application of miRNA in the preparation of drugs for preventing and treating Parkinson's disease.

[0006] Technical solution: The present invention provides the use of miRNA in the preparation of a drug for preventing and treating Parkinson's disease, and the miRNA is miR-363-3p, miR-146b-3p, miR-24-2-5p, miR-26a-5p or miR-22-3p.

[0007] In order to explore the specific substances that play a neuroprotective role in SKP-SC-EVs, the inventors analyzed the levels of microRNA (miRNA, miR) in SKP-SC-EVs and studied each of the top ten miRNAs with the highest content one by one. After treating cells with synthetic miRNA mimics, the ten miRNAs were overexpressed respectively to explore their therapeutic effects on SH-SY5Y cells damaged by the neurotoxin MPP + By comparing the cell viability of each group measured by CCK-8, it was found that miR-363-3p, miR-146b-3p, miR-24-2-5p, miR-26a-5p and miR-22-3p could improve the viability of SH-SY5Y cells damaged by MPP +

[0008] Preferably, the miRNA is derived from the vesicles secreted by Schwann cells induced from skin precursor cells (SKP-SC-EVs).

[0009] Preferably, the role of the miRNA in the drug for treating Parkinson's disease is to protect nerves.

[0010] Preferably, the nerve is a dopaminergic neuron.

[0011] Preferably, the way to protect nerves is to improve mitochondrial function, inhibit neuronal apoptosis or reduce dopaminergic neuron loss.

[0012] Preferably, the mechanism of improving mitochondrial function is to up-regulate the key enzymes of the TCA cycle, promote the synthesis of NADH, restore the ATP level and reduce ROS accumulation.

[0013] Preferably, the key enzymes of the TCA cycle are OGDH, MDH, IDH.

[0014] NADH is one of the important metabolites in the tricarboxylic acid cycle, and α-ketoglutarate dehydrogenase (OGDH), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) are the three key enzymes that generate NADH in the tricarboxylic acid cycle.

[0015] Preferably, the mechanism of inhibiting neuronal apoptosis is to regulate the expression of mitochondrial transcription factor TFAM and maintain the integrity of mitochondrial DNA.​

[0016] Preferably, the drug comprises miR-146b-3p and an adjuvant.

[0017] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0018] 1. The present invention provides miRNAs that play a neuroprotective role in SKP-SC-EVs, and it is found that miR-363-3p, miR-146b-3p, miR-24-2-5p, miR-26a-5p, and miR-22-3p can improve the viability of damaged nerve cells.

[0019] 2. miR-146b-3p has the best effect in improving the viability of damaged nerve cells; inhibiting miR-146b-3p will weaken the increased cell viability of SKP-SC-EVs; miR-146b-3p can also restore the downregulation of key enzymes in the TCA cycle caused by neurotoxic substances.

[0020] 3. The present invention provides miRNAs that protect dopaminergic neurons by regulating mitochondrial function and energy metabolism, which can be applied to the preparation of drugs for preventing and treating Parkinson's disease, and is beneficial to the prevention and treatment of Parkinson's disease and other neurodegenerative diseases. Description of the Drawings

[0021] Figure 1 Characteristics of SKP-SC-EVs isolated in Example 1;

[0022] Figure 2 For SKP-SC-EVs to be able to protect MPP + -damaged SH-SY5Y cells;

[0023] Figure 3 For SKP-SC-EVs to be able to restore the mitochondrial function of MPP + -damaged SH-SY5Y cells;

[0024] Figure 4 For SKP-SC-EVs to be able to regulate the expression of TFAM and the generation of reactive oxygen species in MPP + -damaged SH-SY5Y cells;

[0025] Figure 5 For SKP-SC-EVs to be able to relieve the motor dysfunction and olfactory defects of Parkinson's disease mice induced by MPTP;

[0026] Figure 6 For SKP-SC-EVs to be able to prevent mitochondrial dysfunction in the midbrain of Parkinson's disease mice induced by MPTP;

[0027] Figure 7 To analyze the midbrain metabolomics of mice with MPTP-induced Parkinson's disease affected by SKP-SC-EVs;

[0028] Figure 8 To demonstrate that miR-146b-3p enriched in SKP-SC-EVs has a protective effect on MPP+-damaged SH-SY5Y cells. Detailed implementation

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0030] Example 1: Isolation and characterization of SKP-SC-EVs

[0031] SKP-SCs cells (constructed and preserved in our laboratory) were cultured in DMEM / F12 (Gibco, USA) medium containing 10% FBS (Gibco, USA) and maintained in a humidified incubator at 37°C with 95% air and 5% carbon dioxide. The supernatant of cryopreserved SKP-SCs cells was thawed in water at 37°C. SKP-SC-EVs were isolated and extracted using the exoEasy Maxi Kit (QIAGEN, GER).

[0032] SKP-SC-EVs were diluted at a ratio of 1:1000 with 0.01M PBS (CORNING, USA), and the vesicle size and concentration were measured using NTA ( Figure 1 A-B). Electron microscopy showed that the morphology of SKP-SC-EVs presented a cup-shaped, and the morphological characteristics were consistent with the basic morphological characteristics of EVs ( Figure 1 C). Western Blot was used to detect the expression of vesicle marker proteins such as Calnexin, HSP70, CD9, CD81, and TSG101 in SKP-SC-EVs ( Figure 1 D).

[0033] Example 2: In vitro neuroprotective effect of SKP-SC-EVs

[0034] SH-SY5Y cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM medium and divided into a control group, an MPP+-damaged group, and an MPP++SKP-SC-EVs treatment group.

[0035] Establishment of an MPP+-induced SH-SY5Y cell PD model: One day in advance, seed 2×104 SH-SY5Y cells per well in a 96-well plate. On the second day, after the cells adhere to the wall, discard the supernatant. Add different concentrations of MPP+ to each well in different experimental groups. The final concentration gradients are set to 5 mM, 10 mM, 20 mM, and 40 mM, and the final volume per well is 100 μl. After culturing in a 37°C carbon dioxide incubator for 24 h, discard the supernatant and add freshly prepared CCK-8 (Vazyme, A311-01 / 02) reaction solution (90 μl DMEM + 10 μl CCK-8 corresponding to each well). After incubating at 37°C for about 30 min, measure OD450 with an enzyme-labeled instrument. Select the MPP+ concentration (20 mM) when the cell viability is 50% as the final concentration for subsequent cell model construction ( Figure 2 A).

[0036] Effect of SKP-SC-EVs pretreatment on cell viability and apoptosis: Seed SH-SY5Y cells at 2×104 cells per well in a 96-well plate. After the cells adhere to the wall, pretreat the cells with SKP-SC-EVs at concentrations of 106, 107, 108, and 109 Particles / mL for 24 h, and then damage the cells with 20 mM MPP+ for 24 h. Detect cell viability by CCK8, and it is found that the cell viability damaged by MPP+ can be restored after pretreatment with SKP-SC-EVs at a concentration of 1×109 Particles / mL ( Figure 2 B-C). Therefore, in subsequent experiments, we selected SKP-SC-EVs at a concentration of 1×109 Particles / mL to pretreat SH-SY5Y cells. Detect cell apoptosis by AnnexinV-FITC / PI staining method, and the results show that the apoptosis rate of the SKP-SC-EVs pretreatment group is significantly lower than that of the MPP+ treatment group ( Figure 2 D-E).

[0037] Detection of mitochondrial function and ATP content: Pretreat SH-SY5Y cells with 109 Particles / mL SKP-SC-EVs for 24 hours, and then treat them with 20 mM MPP+. Observe the mitochondrial morphology by transmission electron microscopy (TEM). The results show that compared with the MPP+ treatment group, the mitochondrial morphology in the SKP-SC-EVs pretreatment group is significantly improved ( Figure 3 A), and the number of mitochondria is significantly increased ( Figure 3B). Add lysis buffer to the mitochondrial precipitate and resuspend the mitochondria corresponding to each dish with 200 μl. Pipette to mix well and centrifuge at 4°C for 5 min at 12,000 g. Dilute the ATP standard solution to 0, 0.01, 0.03, 0.1, 0.3 μM with the ATP detection lysis solution to prepare for standard curve establishment. Dilute the ATP detection reagent with the ATP detection reagent diluent in a 1:9 ratio to prepare the ATP detection working solution. In a transparent 96-well plate, first add 100 μL of the ATP detection working solution to each well and let it stand at room temperature for 5 min, with a 1-well interval between each sample-adding well. Add 20 μL of the supernatant and standard product of each group to the 96-well plate respectively, mix well appropriately, and detect the chemiluminescence intensity of each well with a microplate reader. Calculate the ATP concentration in the mitochondria of each group according to the established standard curve. Compared with the MPP+-treated group, the ATP level in the SKP-SC-EVs pretreatment group was significantly increased ( Figure 3 C). SKP-SC-EVs pretreatment significantly decreased the expression of the mitochondrial function-related protein TFAM in cells ( Figure 4 A-C), and decreased the generation of the oxidative stress product ROS ( Figure 4 D-E).

[0038] Example 3: In vivo neuroprotective effect of SKP-SC-EVs

[0039] Randomly divide C57BL / 6 mice (purchased from the Experimental Animal Center of Nantong University) into 4 groups: Control group, Control+EVs group, MPTP+Buffer group, and MPTP+EVs group. There are 28 mice in each group, and they are housed in the animal house for 7 d to get familiar with the environment. The MPTP+Buffer group and the MPTP+EVs group are intraperitoneally injected with 20 mg / kg of MPTP according to body weight, and the Control group and the Control+EVs group are intraperitoneally injected with normal saline, and the administration is continuous for 7 d. Behavioral training starts on the 4th day of administration, including rotarod, hanging, and olfactory behavioral tests.

[0040] Rotarod experiment: Continuously train the mice for 3 d in advance. Place the mice on the rotarod, set the initial speed at 4 rpm, the maximum speed at 40 rpm, and reach the maximum speed in 5 min, and record the time when the mice fall. Record 5 times for each mouse.

[0041] Hanging experiment: Continuously train the mice for 3 d in advance. Fix a clothes hanger 30 cm above the ground. The mice grasp the bottom of the clothes hanger with their front paws, and record the state of the mice grasping the clothes hanger within 1 min. Getting all four paws on the clothes hanger scores 3 points, getting three paws on scores 2 points, getting two paws on scores 1 point, and not getting on scores 0 point. Record 5 times for each mouse.

[0042] Olfactory experiment: Mice were allowed to get familiar with the smell of cheese sticks 3 days in advance. Mice were fasted 1 day before the olfactory experiment started. In a new cage box of 50 cm × 30 cm, 3 cm of new bedding was laid, the cheese sticks were hidden at the bottom of the bedding, the mice were placed in the center of the cage box, and the time for the mice to find the cheese sticks was recorded, with each mouse recorded 5 times.

[0043] After the behavioral tests were completed, the Control+EVs group and the MPTP+EVs group were given nasal instillation of SKP-SC-EVs once every 3 days for 4 times. SKP-SC-EVs were diluted with Buffer XE, and the dosage of vesicles for each mouse was 1×109 Particles. Dilute SKP-SC-EVs with Buffer XE, hold the mouse with its head down and nose up, use a pipette to suck a small amount of SKP-SC-EVs, and drop it at the mouse's nostril. After observing that the droplet was inhaled by the mouse, repeat the operation until the dosage was completed. After every 2 instillations of SKP-SC-EVs, a behavioral test was conducted ( Figure 5 A).

[0044] The results showed that nasal administration of SKP-SC-EVs could reduce the decrease in the number of TH-positive neurons in the midbrain of model mice ( Figure 5 B-E), increase the time the PD model mice stayed on the rotarod and the hanging score, and reduce the time for the PD model mice to find food ( Figure 5 F-H), indicating that SKP-SC-EVs could not only enhance the motor function of PD mice but also promote the recovery of non-motor functions, suggesting that SKP-SC-EVs could improve the behavioral disorders of PD model mice. The electron microscopy results showed that SKP-SC-EVs could improve the pathological changes of mitochondria in the midbrain of PD mice induced by MPTP and inhibit the decrease in the number of mitochondria ( Figure 6 A-B), increase the ATP content and the expression of TFAM ( Figure 6 C-F). Metabolomics analysis was used to detect the metabolites in the midbrain. The results showed that compared with the MPTP treatment group, there were significant changes in the metabolites in the midbrain of the SKP-SC-EVs treatment group, and 14 metabolites including reduced coenzyme I (NADH), gluconic acid, methyl deoxyadenylate, thyronamine, glucuronic acid, homoserine, isoleucine, galactitol, etc. were up-regulated ( Figure 7 A-E). NADH is one of the important metabolites in the tricarboxylic acid cycle, and α-ketoglutarate dehydrogenase (OGDH), malate dehydrogenase (MDH), and isocitrate dehydrogenase (IDH) are the three key enzymes that generate NADH in the tricarboxylic acid cycle. SKP-SC-Evs could significantly increase the expression of the above three key enzymes in both in vivo and in vitro models ( Figure 7 F-K).

[0045] Example 4: Functional verification of miRNA

[0046] Previously, the inventors' research found that SKP-SC-EVs contain 279 known miRNAs, and the top 10 miRNAs with the highest expression levels are miRNA-30a-5p, let-7i-5p, miRNA-21-5p, miRNA-363-3p, miRNA-27b-3p, miRNA-146b-3p, miRNA-30e-5p, miRNA-24-2-5p, miRNA-26a-5p, and miRNA-22-3p. These 10 miRNAs were sorted by abundance ( Figure 8 A), and their functions were verified. miRNA mimics and corresponding miRNA inhibitors were both synthesized by Ribobio (Table 1), and the control miR was the 22-nt nucleotide sequence micrON mimic NC#22 (miR1N0000001-1-5).

[0047] Table 1 miRNA sequence information

[0048]

[0049]

[0050] miRNA transfection: One day before miR treatment, seed 2×10⁴ SH-SY5Y cells per well (100 μl final volume) in a 96-well plate. After the cells adhered, discard the supernatant. Add miRNA transfection medium to each well, and the preparation method is as follows: According to the Lipofectamine RNAiMAX liposome transfection reagent instruction manual, 20 μl of Opti-MEM medium corresponds to each well, and 0.2 μl of RNAiMAX is used per well. First, gently mix 10 μl of Opti-MEM medium corresponding to each well with miRNA mimic or miRNA inhibitor (50 nM final concentration per well), and gently mix 10 μl of Opti-MEM medium corresponding to each well with RNAiMAX, and let it stand for 5 min. Add the RNAiMAX dilution to the miRNA dilution and gently mix, and let it stand at room temperature for 15 min. Add 79.55 μl of PS-free DMEM medium corresponding to each well to the mixture to prepare the transfection medium. Discard the medium in the 96-well plate on the day of adding miRNA. The control group and the MPP+ group add 100 μl of PS-free DMEM medium as a control. After culturing for 24 h, the control group adds the corresponding concentration of DMSO, and the other groups add 100 μl of 5 mM MPP+ solution for treatment. After 24 h, perform a cell viability test. The results show that these miRNAs have no significant effect on normally cultured SH-SY5Y cells ( Figure 8B); Pretreatment with miR-363-3p, miR-146b-3p, miR-24-2-5p, miR-26a-5p, and miR-22-3p can increase the viability of MPP+-damaged SH-SY5Y cells ( Figure 8 C).

[0051] Among the above miRNAs that can increase the viability of MPP+-damaged SH-SY5Y cells, cells treated with miR-146b-3p showed the highest cell viability. miR-146b-3p is a mature strand of microRNA-146b and belongs to the miR-146 family. Previous studies have shown that it plays an important role in various biological processes, especially in cancer, inflammation, and immune regulation. Further functional verification of miR-146b-3p was carried out.

[0052] An miR-146b-3p inhibitor was used to detect whether this miRNA has neuroprotective effects in a PD model. SH-SY5Y cells were seeded in 96-well plates at a density of 2×104 cells / well. After the cells adhered, they were pretreated with SKP-SC-EVs at a concentration of 109 Particles / mL. In the inhibitor group, an miR-146b-3p inhibitor at a final concentration of 50 nM was added and incubated for 24 h, and then the cells were damaged with 20 mM MPP+ for 24 h. The results of CCK8 detection of cell viability showed that the increased cell viability after SKP-SC-EVs pretreatment was attenuated by the miR-146b-3p inhibitor ( Figure 8 D). The results of fluorescence quantitative PCR showed (primer information is shown in Table 2) that miR-146b-3p could upregulate the decrease in OGDH and IDH caused by MPP+ damage ( Figure 8 E-G), indicating that miR-146b-3p may be an effective miRNA with potential neuroprotective effects in SKP-SC-EVs.

[0053] Table 2 Fluorescence quantitative PCR primer information

[0054]

[0055]

[0056] In the table, M: mouse; H: human.

Claims

1. Use of miRNA in the preparation of a medicament for preventing and treating Parkinson's disease, characterized in that, The miRNA is miR-363-3p, miR-146b-3p, miR-24-2-5p, miR-26a-5p or miR-22-3p.

2. The application according to claim 1, characterized in that The miRNA is derived from vesicles secreted by Schwann cells induced from skin precursor cells (SKP-SC-EVs).

3. The application according to claim 1 or claim 2, characterized in that, The role of the miRNA in the drug for treating Parkinson's disease is to protect nerves.

4. The application according to claim 3, wherein The nerves are dopaminergic neurons.

5. The application according to claim 3, characterized in that, The way to protect nerves is to improve mitochondrial function, inhibit neuronal apoptosis or reduce dopaminergic neuron loss.

6. The application according to claim 5, characterized in that, The mechanism of action for improving mitochondrial function is to upregulate the key enzymes of the TCA cycle, promote NADH synthesis, restore ATP levels and reduce ROS accumulation.

7. The application according to claim 6, characterized in that, The key enzymes of the TCA cycle are OGDH, MDH, IDH.

8. The application according to claim 5, characterized in that, The mechanism of action for inhibiting neuronal apoptosis is to regulate the expression of mitochondrial transcription factor TFAM and maintain the integrity of mitochondrial DNA.

9. The application according to claim 1, characterized in that, The drug includes miR-146b-3p and excipients.

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