Application of idebenone in preparation of medicines for treating prion diseases

Abstract

The invention relates to the technical field of virology, in particular to application of idebenone in preparation of medicines for treating prion diseases. It is found that idebenone can effectively relieve oxidative damage and neuronal apoptosis caused by prion viruses, further plays a role in relieving and treating the prion viruses and has the potential of serving as a medicine for relieving or treating the prion viruses; a further mechanism research shows that idebenone relieves oxidative damage and neuronal apoptosis caused by prion viruses by activating SIRT3-SOD2. The new application of idebenone provided by the invention provides a new medicine and a new method for treating prion.

Description

Technical Field

[0001] The invention relates to the technical field of virology, and in particular to application of idebenone in preparing prion disease drugs. Background Art

[0002] Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a class of chronic, progressive and fatal infectious neurodegenerative diseases that can infect humans and a variety of animals and can be transmitted between species. The pathogen causing prion disease is the abnormal form of prion protein PrP associated with the disease. Sc , which is composed of the body's normal cellular prion protein PrP C The histopathological features of prion disease are widespread spongiform encephalopathy, neuronal loss, gliosis, and accumulation of abnormal prion proteins and amyloid plaques in the central nervous system.

[0003] PrP 106-126 It is a peptide homologous to residues 106-126 of the human PrP protein, and its sequence is based on the PrP amyloid protein from patients with Gerstmann-Sträussler-Scheinker syndrome. 106-126 The sequence is highly conserved in different species and is closely related to the initiation of PrP C Conformational changes that promote PrP C PrP Sc The most critical region of the transformation is related to PrP 106-126 PrP Sc Possessing similar structural characteristics and biological activities. 106-126 and PrP Sc Common structural features of PrP include: (1) β-pleated sheet structure, (2) formation of toxic soluble oligomers, (3) high fibril-forming tendency, and (4) resistance to proteinase K digestion. 106-126 PrP Sc Similar biological activities include: (1) neurotoxicity, (2) induction of apoptosis, (3) gliosis, (4) autophagy, (5) ion channel formation, (6) oxidative stress, etc. 106-126 Usually as PrP Sc Model peptides for constructing cell models of prion diseases.

[0004] Idebenone (IDE) was originally developed by Takeda Pharmaceuticals in Japan in the 1980s as a coenzyme Q10 analog, and was launched in 1986 for clinical use. It has achieved good results in the treatment of Friedreich's ataxia, Leyden's hereditary optic neuropathy, cerebrovascular disease and other diseases. Idebenone is similar in structure to coenzyme Q10, so it has the ability to promote aerobic respiration in mitochondria. Idebenone is reduced to idebenol in the cytoplasm, which can transfer electrons to the cellular respiratory chain, thereby enhancing the ability of mitochondria to produce ATP. Idebenone is also a strong antioxidant and is very effective in scavenging ROS. However, there are no reports of using idebenone to treat prion diseases. Summary of the invention

[0005] The invention provides application of idebenone in preparing prion disease medicine.

[0006] The present invention aims to develop an effective ingredient with the potential to prepare a drug for treating prion disease. During the research and development process, the present invention found that idebenone has an effective mitigating effect on neuronal mitochondrial morphological damage, dysfunction and neuronal apoptosis in prion disease and further clarified the mechanism of action of idebenone on prion disease. The results of the study showed that idebenone can rescue neuronal mitochondrial morphological damage, dysfunction and neuronal apoptosis in prion disease cell models by activating the SIRT3-SOD2-mediated mitochondrial antioxidant pathway. Idebenone can be used to prepare drugs for treating prion disease and alleviate neurological damage caused by prions.

[0007] It is known that idebenone can relieve cognitive dysfunction in patients with Alzheimer's disease, improve cardiac function in patients with Friedreich's ataxia, alleviate physical, mental, emotional and cognitive symptoms in patients with cerebrovascular disease, and relieve vision loss in Leber's hereditary optic neuropathy. Unlike the manifestations of the above diseases, the symptoms of prion disease are mainly neurological symptoms such as progressive ataxia, tremor, dementia and behavioral disorders. The course of the disease is slow but progressive, and all end in death. There is no evidence that idebenone has a therapeutic effect in prion diseases. Based on the known efficacy of idebenone, it is difficult to expect that idebenone can relieve prion diseases.

[0008] Specifically, the present invention provides the technical solution described below.

[0009] In a first aspect, the present invention provides use of idebenone in preparing a drug for treating prion disease.

[0010] In a second aspect, the present invention provides use of an idebenone derivative or a composition comprising idebenone in the preparation of a medicament for treating prion disease.

[0011] In the present invention, the active ingredient of the drug may be only idebenone, or may contain other substances that have the effect of treating prion disease. In addition to the active ingredient, the drug may also contain excipients permitted in the pharmaceutical field, including but not limited to fillers, adsorbents, binders, lubricants, dispersants, wetting agents, disintegrants, etc. The dosage form of the drug includes but is not limited to tablets, granules, etc.

[0012] In the present invention, the medicament comprises a therapeutically effective dose of idebenone.

[0013] In the present invention, the prion disease is manifested as neuronal apoptosis, neuronal mitochondrial morphological damage and / or neuronal mitochondrial dysfunction.

[0014] In the present invention, idebenone treats prion disease by alleviating neuronal apoptosis, neuronal mitochondrial morphological damage and / or neuronal mitochondrial dysfunction caused by prions.

[0015] In the present invention, idebenone derivatives are substances derived from idebenone and still retain the efficacy of idebenone. These substances can be obtained by chemically modifying idebenone.

[0016] In the present invention, the composition containing idebenone may be composed of idebenone and other substances having the effect of treating prion disease. Exemplarily, the other substances having the effect of treating prion disease may be quinacrine, chlorpromazine hydrochloride, diphenylamine-2-sulfonic acid, etc.

[0017] In a third aspect, the present invention provides idebenone, its derivatives or compositions containing idebenone for use in the preparation of a method for treating prion protein PrP Sc Application of drugs in treating diseases of the nervous system.

[0018] Preferably, the pathogenic prion protein PrP Sc PrP 106-126 .

[0019] Specifically, the polypeptide PrP 106-126 The amino acid sequence (SEQ ID NO.1) is: KTNMKHMAGAAAAGAVVGGLG.

[0020] Preferably, the nervous system disease is manifested as neuronal apoptosis, neuronal mitochondrial morphological damage and / or neuronal mitochondrial dysfunction.

[0021] In a fourth aspect, the present invention provides use of idebenone, its derivatives or a composition comprising idebenone in preventing or alleviating neuronal damage or apoptosis caused by prions.

[0022] In a fifth aspect, the present invention provides the use of SIRT3 and / or SOD2 as drug targets for prion diseases in screening drugs for treating prion diseases.

[0023] The present invention found that the expression of SIRT3 and SOD2 in prion disease will be significantly reduced, the SIRT3-SOD2 pathway will be damaged, and the activity of antioxidant enzymes represented by SOD2 will be reduced. Sirtuin 3 (SIRT3) is an important drug effect target of idebenone and is a NAD-dependent + Deacetylase can regulate the redox balance of neurons in neurodegenerative diseases by deacetylation of multiple substrates. The present invention found that in a cell model of prion disease, idebenone can alleviate neuronal mitochondrial morphological damage, dysfunction and neuronal apoptosis in prion disease, and its mechanism of action is to upregulate the SIRT3-SOD2 pathway.

[0024] Preferably, the drug comprises an expression promoter of SIRT3 and / or SOD2.

[0025] The above-mentioned expression promoter is a substance that can promote the expression of the protein, and these substances can be compounds, mixtures, proteins or nucleic acids, etc.

[0026] In the present invention, the subject of the drug is a mammal, preferably a human, sheep, cattle or mouse.

[0027] The beneficial effects of the present invention include at least: the present invention finds that idebenone can effectively alleviate the oxidative damage and neuronal apoptosis caused by prions, thereby exerting an alleviating and therapeutic effect on prion diseases, and has the potential to be used as a drug for alleviating or treating prion diseases; further mechanism studies show that idebenone alleviates the oxidative damage and neuronal apoptosis caused by prions by activating SIRT3-SOD2. The new use of idebenone provided by the present invention provides a new drug and method for the treatment of prions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1In the embodiments of the present invention, idebenone can inhibit the decrease of SIRT3 expression in the prion disease cell model; wherein, A. protein immunoblotting method is used to detect SIRT3 protein levels at different times of modeling; B. SIRT3 protein levels are quantitatively analyzed at different times of modeling; C. SIRT3 protein levels are detected by protein immunoblotting method; D. SIRT3 protein levels are quantitatively analyzed.

[0030] Figure 2 The oxidative damage of prion disease cell model alleviated by idebenone in the embodiment of the present invention; wherein, A. mitochondrial superoxide level; B. ratio of reduced glutathione to oxidized glutathione; C. content of malondialdehyde, a lipid peroxidation product; D. mitochondrial DNA copy number; E. SOD activity; F. CAT activity.

[0031] Figure 3 The present invention shows that idebenone alleviates the mitochondrial morphological damage in the prion disease cell model; wherein, A. laser confocal microscopy was used to observe the mitochondrial morphology of N2a cells and the mitochondrial length of N2a cells; B. transmission electron microscopy was used to observe the mitochondrial morphology of N2a cells.

[0032] Figure 4 The mitochondrial dysfunction of the prion disease cell model alleviated by idebenone in the embodiment of the present invention; wherein, A. mitochondrial ATP level of N2a cells; B. mitochondrial membrane potential level of N2a cells.

[0033] Figure 5 The figure shows that idebenone alleviates the increase of TUNEL-positive cells in the prion disease cell model in the embodiment of the present invention; wherein, A. TUNEL-positive cell statistics; B. TUNEL-stained N2a cells were observed under laser confocal microscopy.

[0034] Figure 6 The present invention shows that idebenone affects the expression of apoptosis-related proteins in prion disease. A. Western blotting was used to detect apoptosis effector proteins cleaved caspase-3 and cleaved caspase-9; B. quantification of cleaved caspase-3; C. quantification of cleaved caspase-9.

[0035] Figure 1-Figure 6 In the table, control or ctrl represents the negative control group, PrP 106-126 PrP 106-126 Peptide treatment group, PrP 106-126 +IDE or IDE+PrP 106-126 Idebenone + PrP 106-126Peptide group, IDE represents idebenone treatment, SIRT3 siRNA, siRNA-Sirt3 or siSirt3 represent SIRT3 siRNA transfection group, NC siRNA, siRNA NC or siNC represent scrambled siRNA transfection group, siRNA-Sirt3+PrP 106-126 or siSirt3+PrP 106-126 Represents SIRT3 siRNA transfection + PrP 106-126 Peptide group, siRNA-Sirt3+PrP 106-126 +IDE or siSirt3+PrP 106-126 +IDE represents SIRT3 siRNA transfection + idebenone + PrP 106-126 Peptide group. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Example

[0038] 1. Materials and Methods

[0039] 1.1 Cell culture

[0040] Mouse neuroblastoma (N2a) cells preserved in the National Animal Spongiform Encephalopathy Laboratory were cultured in DMEM (HUANKE, China) containing 10% fetal bovine serum (Gibco, New York, USA) and 1% penicillin / streptomycin at 37 °C in a humidified incubator with 5% CO2. The N2a cells were confirmed to be mycoplasma-free by polymerase chain reaction.

[0041] 1.2 PrP 106-126 Preparation of peptides and idebenone

[0042] Using PrP 106-126 N2a cells were treated with peptides to construct a prion disease cell model.

[0043] PrP 106-126The peptide (amino acid sequence: KTNMKHMAGAAAAGAVVGGLG, purity 99%) was synthesized by Zhongke Yaguang Biotechnology Co., Ltd. (Beijing, China), dissolved in PBS, and rotated at 4°C for 24 h. The peptide storage concentration was 1 mM, the final experimental concentration was 150 μM, and the action time on N2a cells was 24 h. In this way, a prion disease cell model was obtained.

[0044] Idebenone (Solarbio, Beijing, China) solid powder was dissolved in anhydrous ethanol to prepare a 10 mM idebenone stock solution, which was stored at -20°C with a working concentration of 1 μM. 106-126 Add 2 hours before processing.

[0045] 1.3 siRNA, plasmid preparation and cell transfection

[0046] SIRT3 small interfering RNA (siRNA; sense: 5′- CGGCUCUAUACACAGAACA -3′, SEQ ID NO.2) was synthesized by Hongxun Biotechnology Co., Ltd. (Suzhou, China). PcDNA3.1(+)-SIRT3 plasmid was synthesized by Hongxun Biotechnology Co., Ltd. (Suzhou, China). DsRed-Mito plasmid was from Clontech. Plasmids and siRNAs were transfected into N2a cells using Lipofetamine3000 reagent (Invitgen, USA) in Opti-MEM (Invitgen, USA) according to the instructions, and subsequent experimental operations were performed 48 h later.

[0047] 1.4 Protein level detection by Western blot analysis

[0048] RIPA cell lysis buffer (containing 1 mmol / L PMSF, Solebao, China) was added to the cells treated with the experiment, and the cells were lysed on ice for 15 min, then centrifuged at 12000 g at 4°C, the supernatant was taken, and an appropriate amount of protein loading buffer was added, and the cells were denatured at 100°C for 10 min. An appropriate amount of sample was loaded, separated by 10% or 12% SDS-PAGE electrophoresis, wet-transferred using a 0.20 μm PVDF membrane, blocked with 5% skim milk powder for 1 h, incubated with the corresponding primary antibody overnight in a 4°C refrigerator, incubated with the corresponding secondary antibody for 1 h the next day, exposed with ECL chemiluminescent developer and visualized with a chemiluminescent imaging system (Tanon Science And Technology), and finally the grayscale value of the bands was presented using Image J software for statistical analysis.

[0049] The primary antibodies and dilutions used for detection are as follows: cleaved caspase-3 (1:1000, #9664, Cell Signaling Technology), cleaved caspase-9 (1:1000, 10380-1-AP, Proteintech), BCL-2 (1:1000, 26593-1-AP , Proteintech), cytochrome C (1:1000, 10993-1-AP, Proteintech), bax (1:1000, 50599-2-Ig, Proteintech), VDAC (1:1000, #4661, Cell Signaling Technology), SIRT3 (1:1000, #5490, Cell Signaling Technology), SOD2 (1:2000, 66474-1-Ig, Proteintech), acetyl K68 SOD2 (1:1000, ab137037, Abcam), tubulin (1:5000, 10094-1-AP, Proteintech). The secondary antibodies and dilutions used for detection were as follows: horseradish peroxidase-labeled affinity-purified goat anti-rabbit antibody (H+L) (1:10000, Proteintech, SA00001-2) and horseradish peroxidase-labeled affinity-purified goat anti-mouse antibody (H+L) (1:10000, Proteintech, SA00001-1).

[0050] 1.5 Mitochondrial membrane potential detection

[0051] The mitochondrial membrane potential of cells was detected using the JC-1 mitochondrial membrane potential detection kit (Beyotime, C2006). After the experimental treatment, N2a cells were collected in a test tube, washed three times with PBS, and then incubated with JC-1 at 37°C for 30 minutes. After the cells were washed three times with the washing buffer in the kit, they were detected using FACS Calibur (BD Biosciences, SanJose, CA, USA).

[0052] 1.6 ATP detection

[0053] ATP levels were detected using an enhanced ATP assay kit (Beyotime, S0027), and chemiluminescence was measured using a Spark multifunctional microplate reader (TECAN). The values ​​measured in the experimental group were normalized to those in the control group.

[0054] 1.7 Immunofluorescence staining

[0055] N2a cells were cultured using 24-well cell slides and subjected to corresponding experimental treatments. Fluorescent images of mitochondria were collected using an A1 confocal microscope (Nikon, Tokyo), and mitochondrial length was measured using Image J.

[0056] 1.8 Reverse transcription-real-time quantitative polymerase chain reaction (RT-qPCR)

[0057] Total RNA was extracted from N2a cells using the EASYSpin Plus kit (AidLab) according to the instructions. Reverse transcription was performed using HiScrip®III All-in-One RT SuperMix Perfect for qPCR (Vazyme, R333) to obtain cDNA. Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Q712) was used for amplification using an ABI StepOnePlus real-time fluorescence quantitative polymerase chain reaction analyzer (American Biosystems). The primer sequences are listed in Table 1 and were synthesized by Qingke Biotechnology Co., Ltd. (Beijing). GAPDH was used as an internal reference gene, and 2 -ΔΔCt Methods Relative gene expression levels were calculated.

[0058] 1.9 Determination of mitochondrial DNA copy number

[0059] The genomic DNA was extracted using the EasyPure Genomic DNA Kit (Transgen, EE101), and the mtDNA and nuclear DNA (nDNA) were determined by RT-qPCR. The method is shown in 1.8 above, and the primer sequences are shown in Table 1. The mitochondrial DNA copy number was determined by the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA), using 2 −ΔΔCt Methods Calculation, the measured values ​​of the experimental group were normalized with those of the control group.

[0060] Table 1 Primer sequences

[0061] 1.10 Cell viability assay

[0062] Cell viability kit-8 (CCK-8 kit, Beyotime, C0038) was used to evaluate the activity of N2a cells after experimental treatment. The absorbance of each sample at 450 nm was measured using a microplate reader (Thermo Fisher, USA). The measured values ​​of the experimental group were normalized with those of the control group.

[0063] 1.11 One-step TUNEL apoptosis assay

[0064] According to the instructions, a one-step TUNEL apoptosis detection kit (Beyotime, No. C1086) was used to detect the level of cell apoptosis, and an A1 confocal microscope (Nikon, Tokyo) was used to observe positive cells and collect images, followed by counting and analysis.

[0065] 1.12 Lipid peroxidation MDA detection

[0066] According to the instructions, cells were lysed using the MDA detection kit (Beyotime, S0131S), and 100 µL of sample / standard was added with 200 µL of MDA detection working solution and gently mixed; the plate was placed in a 100°C metal bath for 15 minutes. 10 µL of sample / standard was added to a 96-well plate. 300 µL of G250 staining solution (detergent-compatible) was added to each well, and the A was measured using an ELISA reader. 595 , calculate the MDA concentration of the sample according to the standard curve.

[0067] 1.13 GSH / GSSG detection

[0068] According to the instructions, GSH / GSSG was detected using a glutathione detection kit (Beyotime, S0053). The cell sample was blown off and collected in a centrifuge tube, and a protein removal reagent M solution 3 times the volume of the cell pellet was added, vortexed to mix, and the sample was quickly frozen and thawed twice, centrifuged at 10000 g for 10 minutes at 4°C, and the supernatant was used for the determination of total glutathione. 20µL of diluted GSH removal auxiliary solution was added to each 100µL sample, and vortexed to mix; 4µL of GSH removal reagent working solution was added to each 100µL sample, and vortexed to mix. React at 25°C for 60 minutes. 10µL of the sample / standard to be tested and 150µL of total glutathione detection working solution were added to the 96-well plate in sequence, mixed and incubated at room temperature for 5 minutes, and then 50µL of 0.5mg / mL NADPH was added. After 25 minutes, the sample was detected using an A412 microplate reader, and the content of total glutathione or GSSG in the sample was obtained by comparing the standard curve.

[0069] 1.14 Mitochondrial superoxide MitoSOX detection

[0070] According to the instructions, mitochondrial superoxide red fluorescent probe (Thermo Fisher, M36005) was used to detect mitochondrial superoxide MitoSOX. The cell samples were blown off and collected in a 1.5 mL centrifuge tube. The cells were resuspended with 1 mL of 2.5 µM MitoSOX working solution and incubated at 37°C for 20 minutes. The samples were washed three times with PBS buffer, and the cells were filtered into a flow cytometer tube with a cell strainer and detected using FACS Calibur (BD Biosciences, San Jose, CA, USA).

[0071] 1.15 Data Statistical Analysis

[0072] All results were performed with at least three replicates, and the replicates in each figure are represented by separate data points. Data are presented as mean ± standard error. Parametric data were analyzed using one-way analysis of variance (ANOVA) and Tukey's post hoc test. Prism 9.0 software (GraphPad Software, La Jolla, CA, USA) was used for analysis. * represents P < 0.5, ** represents P < 0.01, and *** represents P < 0.001.

[0073] 2. Results

[0074] 2.1 Idebenone can inhibit the decrease of SIRT3 expression in prion disease cell model

[0075] SIRT3 is a deacetylase that regulates neuronal mitochondrial quality control, especially mitochondrial biogenesis, in neurodegenerative diseases. It is also an important target for idebenone to exert its neuroprotective effect in prion diseases.

[0076] The results showed that the expression of SIRT3 in prion disease was significantly decreased, and the addition of idebenone could restore the expression of SIRT3 to a certain extent ( Figure 1 ).

[0077] 2.2 Idebenone alleviates mitochondrial oxidative stress by activating the SIRT3-SOD2 pathway

[0078] Oxidative stress is an early event in prion disease, and controlling oxidative stress is of great significance for maintaining the health of neurons. This paper explores the regulatory effect of idebenone on mitochondrial oxidative stress in a SIRT3-dependent manner. The experiment set up seven groups, namely, the negative control group (N2a cells, not transfected, not using PrP 106-126 Peptide treatment), PrP 106-126 Peptide treatment group (addition of PrP 106-126 Peptide treatment of N2a cells, a prion disease cell model), idebenone + PrP 106-126Peptide group (addition of idebenone to the culture of prion disease cell model), SIRT3 siRNA transfection group (transfection of SIRT3 siRNA in N2a cells), SIRT3 siRNA transfection + PrP 106-126 Peptide group (transfection of SIRT3 siRNA in prion disease cell model), SIRT3 siRNA transfection + idebenone + PrP 106-126 Peptide group (SIRT3 siRNA was transfected in the prion disease cell model and treated with idebenone) and scrambled siRNA transfection group (scrambled siRNA and SIRT3 siRNA were synthesized under the same conditions, and their sequences were not complementary to the mRNA sequence of SIRT3, which was used to exclude the effects of adding siRNA itself on various biological indicators). The present invention detected a total of six indicators of cellular oxidative stress: mitochondrial superoxide level, lipid peroxidation level, reduced glutathione ratio, mitochondrial DNA copy number, superoxide dismutase (SOD) activity, and catalase (CAT) activity. The experimental results showed that in the prion disease cell model, all six indicators of oxidative stress were expressed, indicating that oxidative stress occurred in neurons in prion disease; and the addition of idebenone could reverse the oxidative damage of neuronal mitochondria in prion disease ( Figure 2 ).

[0079] In summary, in prion disease, idebenone can alleviate mitochondrial oxidative damage by activating the SIRT3-SOD2 pathway.

[0080] 2.3 Idebenone can alleviate neuronal mitochondrial morphological damage and dysfunction in prion disease

[0081] The mitochondrial morphology was observed by laser confocal microscopy and compared with the control group (untransfected, without PrP 106-126 Compared with the peptide treatment, the mitochondrial length of the prion disease cell model was significantly reduced; the addition of idebenone restored the mitochondrial length and morphology to a certain extent ( Figure 3 ).

[0082] Mitochondrial membrane potential (MMP) and ATP level are two basic markers of mitochondrial function. 106-126 The levels of mitochondrial membrane potential and ATP in the prion disease cell model were significantly reduced in N2a cells treated with idebenone. After specific transfection of SIRT3 siRNA, the effects of idebenone on mitochondrial morphological damage and mitochondrial function recovery were inhibited ( Figure 4 ).

[0083] In summary, idebenone can alleviate neuronal mitochondrial dysfunction in a prion disease cell model in a SIRT3-dependent manner.

[0084] 2.4 Idebenone can alleviate neuronal apoptosis in prion disease cell models

[0085] The protective effect of idebenone on neuronal apoptosis in prion disease was further explored. The present invention found that compared with the control group, PrP 106-126 Peptide treatment increased the proportion of TUNEL-positive cells, while the addition of idebenone reduced the proportion of TUNEL-positive cells ( Figure 5 In addition, protein immunoblotting was used to detect apoptosis-related proteins cleaved caspase-3, cleaved caspase-9, and caspase-9, and it was found that the addition of idebenone increased the levels of cleaved caspase-3 and cleaved caspase-9 and decreased caspase-9 in the prion disease cell model, all of which returned to levels similar to those of the control group ( Figure 6 ). This suggests that idebenone inhibits apoptosis in the prion disease cell model.

[0086] After cell-specific transfection of SIRT3 siRNA, the increase in cell survival rate and its inhibitory effect on cell apoptosis caused by the addition of idebenone in the above results were blocked by the loss of SIRT3 ( Figure 6 ).

[0087] In summary, idebenone can alleviate neuronal apoptosis in prion disease in a SIRT3-dependent manner.

[0088] 3. Conclusion

[0089] Neurons, as cells with high metabolism and high energy demand, are particularly sensitive to changes in mitochondrial dynamic balance. In diseases, mitochondria are the main site for the production of reactive oxygen species (ROS) in cells. When ROS exceeds the basal state, cells / organs will be in a state of oxidative stress. Mitochondrial damage and oxidative stress are early and key signs of neuronal damage in prion disease. As the site of aerobic respiration in cells, mitochondria produce a certain amount of reactive oxygen species during aerobic respiration. In prion disease, excessive production of reactive oxygen species damages biological macromolecules and organelles, leading to the fate of cell apoptosis. Idebenone is a multi-target quinone compound with pleiotropic effects, and SIRT3 is its important drug effect target. The present invention found that the SIRT3-SOD2 pathway of the prion disease cell model was impaired, which was specifically manifested as reduced activity of antioxidant enzymes represented by SOD2 and accumulation of mitochondrial ROS. The addition of idebenone can alleviate mitochondrial damage, thereby protecting N2a cells from PrP. 106-126The protective effects of idebenone in prion disease cell models were blocked by SIRT3 interference, indicating that SIRT3 is an important target of idebenone to affect mitochondrial quality control in prion disease.

[0090] Oxidative stress plays an important role in the development of various diseases. Compared with other organs, the brain has a lower cell regeneration capacity, so oxidation has a significant impact on the nervous system. Oxidative stress plays an important role in prion disease. c Combined with copper and iron, which have redox effects, it can scavenge free radicals, and PrP sc This may lead to metal imbalance in prion diseases. sc Binding to copper and iron makes the complex continuously redox active, further exacerbating oxidative stress and neurodegeneration. Idebenone can alleviate prion-induced oxidative stress, intervene in the progression of prion disease, and alleviate prion-induced neuronal apoptosis.

[0091] In summary, the significant downregulation of SIRT3 in the prion disease cell model and the positive regulation of SIRT3 on mitochondrial quality control make it a potential target for the treatment of prion disease. In the prion disease cell model, idebenone can regulate the deacetylation of SOD2 and its activity by increasing the content of SIRT3, an important mitochondrial deacetylase, thereby alleviating oxidative damage, mitochondrial damage and cell apoptosis, and has the potential to become a drug for alleviating and treating prion disease.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of idebenone in the preparation of drugs for treating prion diseases.

2. Use of an idebenone derivative or a composition comprising idebenone in the preparation of a medicament for treating prion disease.

3. The use according to claim 1 or 2, characterized in that: The prion disease is manifested by neuronal apoptosis, neuronal mitochondrial morphological damage and / or neuronal mitochondrial dysfunction.

4. The use according to claim 1 or 2, characterized in that: Idebenone treats prion diseases by alleviating neuronal apoptosis, neuronal mitochondrial morphological damage and / or neuronal mitochondrial dysfunction caused by prions.

5. Idebenone, its derivatives or compositions containing idebenone for the preparation of a method for treating prion protein PrP Sc Application of drugs in treating diseases of the nervous system.

6. The use according to claim 5, characterized in that: The pathogenic prion protein PrP Sc PrP 106 -126 .

7. The use according to claim 5 or 6, characterized in that: The nervous system disease is manifested by neuronal apoptosis, neuronal mitochondrial morphological damage and / or neuronal mitochondrial dysfunction.

8. Use of idebenone, its derivatives or a composition comprising idebenone in preventing or alleviating neuronal damage or apoptosis caused by prions.

9. Application of SIRT3 and / or SOD2 as drug targets for prion diseases in screening drugs for the treatment of prion diseases.

10. The use according to claim 9, characterized in that: The medicine includes an expression promoter of SIRT3 and / or SOD2.

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