Application of aframycin in preparation of products for treating autophagy related diseases

Abstract

This application discloses the use of tetrandrine in the preparation of products for treating autophagy-related diseases. Through research, this application found that tetrandrine can significantly induce autophagy and promote TFEB nuclear translocation, making it suitable as an autophagy inducer and TFEB activator. Furthermore, it is naturally derived, has low toxicity, and high activity, providing a safe and effective new strategy for treating autophagy-related diseases, especially Gaucher disease, a lysosomal storage disorder.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to the application of tebufenozide in the preparation of products for treating autophagy-related diseases. Background Technology

[0002] Autophagy is a normal physiological process by which cells degrade and recycle intracellular components. When cells face nutrient deprivation or pathogen infection, they activate the autophagy mechanism, encapsulating substrates to be degraded (such as misfolded toxic proteins, aging organelles, or invading microorganisms) into a double-membrane structure called an "autophagosome." Subsequently, the autophagosome fuses with lysosomes to form an autolysosome, using hydrolytic enzymes to degrade the contents into reusable small molecules (such as amino acids and fatty acids). This process is crucial for maintaining cellular homeostasis and self-renewal under stress. Therefore, autophagy inducers can initiate or enhance this process, promoting efficient cellular self-cleaning and resource recycling.

[0003] Autophagy is regulated at multiple levels, with transcription factor TFEB considered a core regulator of this pathway. Its activity is closely related to phosphorylation and nucleoplasmic shuttle. Under adequate nutrient levels and other physiological conditions, TFEB is phosphorylated at the Ser211 site. Phosphorylated TFEB is recognized and bound by the 14-3-3 adaptor protein, thus becoming "confined" to the cytoplasm and unable to enter the nucleus. Under stress conditions (such as viral infection or toxic protein aggregation), TFEB is dephosphorylated, releasing it from the 14-3-3 protein's binding and translocating into the nucleus. This initiates the transcription of autophagy and lysosome-related genes, driving the autophagy process. Therefore, TFEB activators can enhance autophagy flux by promoting its nuclear translocation, thereby achieving effective cellular cleanup and homeostasis maintenance.

[0004] Insufficient or decreased autophagy mediated by TFEB can induce various autophagy-related diseases, including lysosomal storage diseases (LSD), cardiovascular diseases, and infectious diseases, with a particularly close relationship to LSD. LSD is a group of inherited metabolic diseases caused by mutations in lysosome-related genes, leading to the abnormal accumulation of large amounts of undegraded products in lysosomes. Based on the type of primary stored substance, LSD can be mainly classified into lipid storage diseases (such as Gaucher disease, Fabry disease, and Niemann-Pick disease), glycoprotein storage diseases (such as fucoside storage diseases), mucolipid storage diseases (such as mucolipid storage disease type II and III), and glycogen storage diseases (such as Pompe disease). Studies have found that TFEB agonists, by enhancing the nuclear translocation of TFEB and activating the autophagy process, accelerate the degradation of stored substances and are an important means of treating LSD. Developing safe and highly specific novel small molecule autophagy inducers and TFEB agonists will undoubtedly open up new treatment avenues for a range of diseases caused by the accumulation of "cellular waste".

[0005] However, currently reported drug-based treatments that induce autophagy or enhance TFEB transcriptional activity generally suffer from significant toxic side effects, limiting their clinical translation. Therefore, identifying highly effective and low-toxicity dual-activators of autophagy and TFEB from natural plant resources has become an important and urgent research task in this field. Summary of the Invention

[0006] Therefore, it is necessary to provide information on the application of tebufenozide in the preparation of products for treating autophagy-related diseases.

[0007] The first aspect of this application provides the use of tetrandrine in the preparation of products for treating autophagy-related diseases.

[0008] In some embodiments, the product includes a drug.

[0009] In some embodiments, the drug includes tetrandrine and pharmaceutically acceptable excipients;

[0010] Optionally, the excipients include pharmaceutically acceptable excipients such as one or more of diluents, binders, wetting agents, disintegrants, lubricants, solubilizers, pH adjusters, and osmotic pressure adjusters.

[0011] In some embodiments, the dosage form of the product includes one or more of tablets, granules, capsules, oral preparations, and injections.

[0012] In some embodiments, the autophagy-related diseases include one or more of lysosomal storage diseases, cardiovascular diseases, and infectious diseases.

[0013] In some embodiments, the autophagy-related disease specifically refers to Gaucher disease, a type of lysosomal storage disease.

[0014] The second aspect of this application provides the use of tebufenozide in the preparation of autophagy inducers.

[0015] In some embodiments, the tebufenozide enhances autophagy by promoting the nuclear translocation of the transcription factor TFEB.

[0016] In some embodiments, the cells in the autophagy inducer include at least one of human embryonic kidney cells and neuroma cells.

[0017] The third aspect of this application provides the use of tebufenozide in the preparation of transcription factor TFEB activators.

[0018] In some embodiments, the terpinene significantly promotes the protein expression of the transcription factor TFEB in the cell nucleus.

[0019] In some embodiments, the African tetrandrine promotes TFEB activation by binding to the Ser211 phosphorylation site of TFEB.

[0020] This application reveals that tetrandrine can significantly induce autophagy and promote TFEB nuclear translocation. It can be used as an autophagy inducer and TFEB activator. It is derived from nature, has low toxicity and high activity, and provides a safe and effective new strategy for the treatment of autophagy-related diseases, especially Gaucher disease, a lysosomal storage disease. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0022] Figure 1 This image shows the results of a significant increase in the expression of autophagy marker proteins in HEK293T / APPswe cells by tebufenozide in one embodiment of this application.

[0023] Figure 2 This is a diagram showing the results of a significant increase in the number of autophagosomes in HEK293T / APPswe cells by tebufenozide in one embodiment of this application, where the arrows indicate autophagosomes;

[0024] Figure 3This is a diagram showing the results of tebufenozide promoting TFEB nuclear translocation in HEK293T / APPswe cells according to one embodiment of this application;

[0025] Figure 4 This is a diagram showing the results of tebufenozide promoting TFEB nuclear translocation in N2a cells according to one embodiment of this application. In this diagram, A is an immunofluorescence staining image of TFEB subcellular localization in N2a cells, and B is a quantitative analysis diagram of the ratio of TFEB fluorescence in the nucleus to the cytoplasm of N2a cells.

[0026] Figure 5 This is a diagram showing the results of tebufenozide promoting TEEB protein expression in a Gaucher disease model according to one embodiment of this application. In this diagram, A is a Western blot diagram of TFEB protein expression in the nucleus and cytoplasm of Huh7 cells, and B is a quantitative analysis diagram of TFEB protein expression.

[0027] Figure 6 This is a schematic diagram illustrating the structure by which tebufenozide blocks the recognition of the TFEB Ser211 phosphorylation site by the 14-3-3 protein in one embodiment of this application. Detailed Implementation

[0028] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0030] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0031] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0032] The terms “having,” “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0033] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0034] In this application, if the unit of a data range is only followed by the right endpoint, it means that the units of the left and right endpoints are the same.

[0035] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0036] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0037] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0038] In this application, when numerical intervals (i.e., numerical ranges) are mentioned, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0039] In this application, the terms "room temperature" or "normal temperature" generally refer to 4°C to 35°C, for example, 20°C ± 5°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 10°C to 30°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 20°C to 30°C.

[0040] Currently, most autophagy inducers and TFEB activators in the preclinical research stage are chemically synthesized compounds, all of which exhibit significant toxicity, hindering their further clinical development. For example, rapamycin (an autophagy inducer) can cause infection, oral ulcers, and severely impair wound healing. Torin1 (a TFEB activator) shows significant cytotoxicity to various cell lines at high concentrations or with prolonged treatment; this toxicity stems not only from its induced cell cycle arrest but may also from its inhibition of other kinases. Therefore, discovering highly effective and low-toxicity naturally derived autophagy inducers and TFEB activators has become an urgent research task.

[0041] African tetrandrine is a natural, small-molecule isoquinoline alkaloid derived from the rhizomes of plants such as Coptis chinensis (Ranunculaceae) and Menispermum erythrorhizon (Menispermaceae), possessing advantages such as low toxicity and high activity. However, its role in autophagy and TFEB regulation has not been reported.

[0042] Based on this, the embodiments of this application at least provide the use of tebufenozide in the preparation of products for treating autophagy-related diseases.

[0043] In a first aspect of this application, the use of tetrandrine in the preparation of products for treating autophagy-related diseases is provided.

[0044] In this application, "autophagy-related diseases" refers to diseases that are caused, developed, or aggravated directly or indirectly by functional abnormalities in the autophagy process.

[0045] In some implementations, autophagy-related diseases include one or more of cardiovascular diseases, infectious diseases, and lysosomal storage diseases. Cardiovascular diseases include, but are not limited to, myocardial hypertrophy and heart failure; infectious diseases include, but are not limited to, tuberculosis, bacterial or viral infections; and lysosomal storage diseases are primarily Gaucher disease.

[0046] In some implementations, the autophagy-related disease is Gaucher disease.

[0047] In some implementations, the product includes a drug.

[0048] In this application, "drug" includes any agent, compound, composition, or mixture that provides physiological and / or pharmacological effects in vivo or in vitro, and often provides beneficial effects. The scope of the physiological and / or pharmacological effects produced by a "drug" in vivo is not particularly limited; it may have systemic effects or only local effects. The activity of the "drug" is not particularly limited; it may be an active substance that can interact with other substances or an inert substance that does not interact with other substances.

[0049] In some implementations, the drug includes tetrandrine and pharmaceutically acceptable excipients.

[0050] In this application, "pharmaceutically acceptable" means those ligands, materials, compositions, and / or dosage forms that are suitable for administration to patients within the bounds of reasonable medical judgment and that are commensurate with a reasonable benefit / risk ratio.

[0051] In some embodiments, the excipients include, but are not limited to, one or more of the following: diluents, binders, humectants, disintegrants, lubricants, solubilizers, pH adjusters, and osmotic pressure regulators. Diluents may include, but are not limited to: starches, sugars, cellulose compounds, and inorganic salts. Binders may include, but are not limited to: starch paste and polyvinylpyrrolidone. Humectants may include, but are not limited to: water and ethanol. Disintegrants may include, but are not limited to: starch. Lubricants may include, but are not limited to: talc, calcium stearate, and magnesium stearate. Solubilizers may include, but are not limited to: Tween compounds. pH adjusters may include, but are not limited to: hydrochloric acid, sodium hydroxide, and sodium bicarbonate. Osmotic pressure regulators may include, but are not limited to: glucose, sodium chloride, sodium citrate, sorbitol, and xylitol.

[0052] This application does not specifically limit the dosage form of the drug, which may be a liquid or solid dosage form. Liquid dosage forms refer to dosage forms containing a liquid phase, such as, but not limited to, oral dosage forms and injections; solid dosage forms may be, but are not limited to, tablets, granules, and capsules.

[0053] In some implementations, the drug may be administered via a suitable route of administration, which may include, but is not limited to, oral administration, enema administration, or parenteral administration, depending on clinical needs.

[0054] In this application, "subject" is an animal, preferably a mammal, more preferably a human, and the subject includes, but is not limited to, patients with diseases, conditions and / or symptoms.

[0055] In a second aspect of this application, a method for treating autophagy-related diseases is provided, comprising administering to a subject a therapeutically effective amount of a drug as defined in the first aspect.

[0056] In some implementations, the autophagy-related disease is Gaucher disease.

[0057] In this application, "therapeutic effective amount" refers to the amount of a pharmaceutical active ingredient that will elicit a biological or medical response in an individual in response to a disease, condition, and / or symptom, such as the amount of the active ingredient of this application that brings positive physiological and / or pharmacological effects to an individual. These positive physiological and / or pharmacological effects include, but are not limited to, reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating conditions, slowing or delaying disease progression, or preventing disease.

[0058] In a third aspect of this application, the use of tetrandrine in the preparation of autophagy inducers is provided.

[0059] In some implementations, tebufenozide enhances autophagy by promoting the nuclear translocation of the transcription factor TFEB.

[0060] In some embodiments, the cells in the autophagy inducer include at least one of human embryonic kidney cells and neuroma cells.

[0061] In some implementations, autophagy inducers can be used to prepare drugs for treating diseases related to insufficient autophagy function.

[0062] In a fourth aspect of this application, the use of tetrandrine in the preparation of transcription factor TFEB activators is provided.

[0063] In some implementations, tebufenozide promotes TFEB activation by binding to the Ser211 phosphorylation site of TFEB.

[0064] Compared to synthetic drugs, the core advantage of African tetrandrine lies in its combination of "clear dual pharmacological activity" and "high safety of natural drugs." This component is extracted directly from the roots and rhizomes of traditional Chinese medicines such as Coptis chinensis and Scutellaria baicalensis, possessing a profound pharmacological basis and avoiding the high toxicity and side effects commonly found in synthetic drugs. It provides a highly promising lead compound for the development of novel, safe, and effective drugs to treat related diseases.

[0065] The following are some examples.

[0066] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0067] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0068] Example 1

[0069] 1. Experimental materials:

[0070] African tetrandrine standard (catalog number: 3621-36-1) was provided by Baoji Chenguang Biotechnology Co., Ltd. HEK293T / APPswe (human embryonic kidney cells) was provided by Heyuan Biotechnology (Shanghai) Co., Ltd., and N2a (mouse neuroma cells) and Huh7 (human liver cancer cells) were obtained from Shenzhen Second People's Hospital. DMEM high-glucose medium (catalog number: SH30243.01) was purchased from HyClone. LC3B antibody (catalog number: ab192890) and Beclin1 antibody (catalog number: ab207612) were purchased from Abcam. Histone H3 (catalog number: 9715S) and β-actin antibody (catalog number: 3700S) were purchased from Cell Signaling Technology. TFEB antibody (catalog number: PA5-96632) was purchased from Invitrogen. The nuclear and cytoplasmic protein extraction kit (catalog number: P0028) and BeyoGold™ 35mm glass-bottom confocal culture dishes (catalog number: FCFC016-150pcs) were purchased from Beyotime Biotechnology Co., Ltd. The Pierce™ ECL (catalog number: 32106) chemiluminescent substrate for Western blot was purchased from Thermo Fisher Scientific. Conduritol-β-epoxide (CβE, catalog number: HY-100944) was purchased from MCE Biotechnology Co., Ltd.

[0071] 2. Experimental methods:

[0072] 2.1 Cell Culture

[0073] All three cell types were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1× penicillin-streptomycin antibiotics at 37°C and 5% CO2. When the cell confluence reached approximately 90%, the cells were passaged at a ratio of 1:3.

[0074] 2.2 Construction of Gaucher disease cell model and drug administration

[0075] CβE is a specific, irreversible inhibitor of glucocerebrosidase and is widely used to construct cell and animal models of Gaucher disease. Huh7 cells were seeded in 10 cm culture dishes, and control, model, and treatment groups were established. The model and treatment groups were treated with 150 mM CβE for 5 days to establish the Gaucher disease model. The treatment group was then treated with 10 μM tetrandrine in addition to the existing model. The model and control groups received an equal volume of sterile PBS.

[0076] 2.3 Extraction of whole-cell proteins and nuclear proteins

[0077] 1) Whole-cell protein extraction:

[0078] HEK293T / APPswe cells were seeded at a density of 150,000 per well in 6-well plates. After cell attachment, different concentrations of tetrandrine (0.5, 1, 5, 10, 25 μM) were added and incubated with the cells for 24 hours. The culture medium was then discarded, cell lysis buffer was added, and the cells were lysed on ice for 30 minutes. The cells were then centrifuged (4°C, 12,000 rpm, 10 minutes) and the supernatant (whole cell protein) was transferred to a new EP tube.

[0079] 2) Extraction of nuclear proteins:

[0080] HEK293T / APPswe cells were seeded at a density of 2 million per dish in 10cm culture dishes. After cell attachment, different concentrations of tetrandrine (10, 25 μM) were added and incubated with the cells for 24 hours. The cells were then gently rinsed once with PBS. Cells were scraped off using a cell scraper, centrifuged, and the cell pellet was collected into 1.5 mL centrifuge tubes. The supernatant was discarded, and 100 μL of cytoplasmic protein extraction reagent A was added to each pellet. The cells were vortexed vigorously for 5 seconds to fully resuspend them. After incubating on ice for 15 minutes, 5 μL of cytoplasmic protein extraction reagent B was added, and the mixture was vortexed again and incubated on ice for another minute. The cells were then centrifuged (4°C, 12,000 rpm, 10 minutes), and the supernatant was transferred to pre-chilled EP tubes, which contained the cytoplasmic protein fraction. 25 μL of nuclear protein extraction reagent was added to the remaining pellet, and the mixture was vortexed vigorously for 30 seconds, then placed on ice. This vortex-ice incubation process was repeated 10 times. Centrifuge again (4℃, 12,000 rpm, 10 minutes), and collect the supernatant to obtain the nucleoprotein component.

[0081] 2.4 Western blot

[0082] Protein samples obtained according to method 2.3 were quantified using the BCA method, and then loaded with loading buffer at a ratio of 1:4. The mixture was heated in a 100°C water bath for 5 minutes. SDS-PAGE gel electrophoresis was then performed, and the proteins in the gel were transferred to a PVDF membrane using a wet transfer method. The membrane was blocked with 5% skim milk at room temperature for 2 hours, and washed three times with TBST washing buffer for 5 minutes each time. Then, LC3B, Beclin 1, and β-actin antibodies (for whole-cell proteins) or TFEB and Histone H3 antibodies (for nuclear proteins) were added, and the membrane was incubated overnight at 4°C. The next day, the corresponding HRP-labeled secondary antibody was added, and the membrane was incubated at room temperature for 45 minutes. After washing, the membrane was developed and exposed using ECL chemiluminescent substrate, and the images were finally analyzed using ImageJ software.

[0083] 2.5 Observation of intracellular autophagosomes using transmission electron microscopy

[0084] 1) Cell sample preparation: HEK293T / APPswe cells were seeded at a density of 2 million per dish in 10cm culture dishes, and a control group and a tetrandrine group were set up. After the cells adhered, tetrandrine was added to the experimental group to a final concentration of 10 μM, and an equal volume of sterile PBS was added to the control group.

[0085] 2) Cell collection and fixation: 24 hours after drug intervention, the cells were collected by centrifugation (1,500 rpm, 5 minutes) and fixed with 2.5% glutaraldehyde fixative at 4°C in the dark for more than 24 hours.

[0086] 3) Agar pre-embedding: Collect the fixed cell suspension by centrifugation (1,500 rpm, 10 minutes), wash with PBS for 5 minutes, centrifuge again, and repeat the washing three times. After discarding the supernatant, add preheated 1% agarose solution (slightly cooled) to the cell pellet. Before the agar solidifies, use a toothpick to suspend and encapsulate the cell pellet. Cut a 1 mm³ piece from the cell-rich area.

[0087] 4) Post-fixation: Fix with 1% osmium tetroxide at room temperature in the dark for 2 hours, then rinse with PBS 3 times, 15 minutes each time.

[0088] 5) Room temperature dehydration: The sample was sequentially dehydrated by ascending dehydration with 30%, 50%, 70%, 80%, 95%, 100% alcohol and 100% acetone, 10 minutes per step.

[0089] 6) Infiltration and embedding: Infiltrate the sample sequentially using acetone:812 embedding medium at a ratio of 1:1 (37°C, 2–4 hours), 1:2 (37°C, overnight), and pure 812 embedding medium (37°C, 5–8 hours). Then place the sample into the embedding plate and incubate overnight at 37°C.

[0090] 7) Polymerization: Place the embedding plate in a 60℃ oven for 48 hours to polymerize and make resin blocks for later use.

[0091] 8) Ultrathin sectioning: The resin block was cut into 70 nm sections using an ultrathin slicer and retrieved using a 200-mesh copper wire mesh.

[0092] 9) Staining: The copper sheet was stained successively with 2% uranium acetate saturated alcohol solution (protected from light, 10 minutes) and lead citrate solution (protected from CO2, 10 minutes). After each staining, it was washed 3 times with ultrapure water, blotted dry with filter paper, and dried overnight at room temperature.

[0093] 10) Observation: Observe the sample under a transmission electron microscope (HITACHI, HT7800).

[0094] 2.6 TFEB cell immunofluorescence staining

[0095] N2a cells were seeded at a density of 100,000 per dish in confocal culture dishes. After cell adhesion, the cells were co-incubated with 10 μM tetrandrine for 24 hours. The culture medium was then aspirated, and the cells were fixed with 4% paraformaldehyde solution for 15 minutes, followed by permeabilization with 0.1% Triton X-100. After blocking with normal goat serum for 1 hour at room temperature, the cells were incubated overnight at 4°C with the corresponding TFEB primary antibody. After washing with PBS, the cells were incubated for 1 hour with Alexa Fluor 488-labeled goat anti-rabbit / mouse IgG secondary antibody under light-protected conditions. Cell nuclei were counterstained with DAPI, and finally, fluorescence images were acquired and analyzed using a confocal microscope (Olympus FV101).

[0096] 2.7 Molecular docking simulation of the interaction between tetrandrine and the TFEB-14-3-3 complex

[0097] Molecular docking simulations of tetrandrine and TFEB were performed using AutoDock Vina v1.2.0 software. The crystal structure of human TFEB (6a5q) was obtained from the RCSB PDB database (https: / / www.rcsb.org / ), and the chemical structure of tetrandrine was obtained from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). Based on the docking free energy (kcal / mol), the optimal docking conformation was screened, and the complex structure with the lowest docking free energy was extracted. Finally, the binding interaction between the complex and the protein active site residues was visualized and analyzed using PyMOL software in a three-dimensional model.

[0098] 2.8 Statistics and Analysis

[0099] Data were obtained from at least three independent biological replicates and are expressed as mean ± standard error (mean ± SEM). Statistical analysis was performed using IBM SPSS 25.0, with a significance threshold of p < 0.05. Differences between groups were tested using the following methods: (i) Student's t-test for comparisons between two groups; (ii) one-way ANOVA for comparisons between multiple groups, with appropriate post-hoc tests selected based on homogeneity of variance (LSD test for homogeneity of variance, Dunnett's T3 test for unequal variances).

[0100] 3. Experimental Results

[0101] 3.1 Stephania tetrandra increases the expression of autophagy marker proteins in HEK293 cells.

[0102] like Figure 1 As shown, after 24 hours of treatment with different concentrations of tetrandrine, the expression levels of autophagy-related proteins LC3B-II and Beclin 1 in HEK293T / APPswe cells significantly increased. At concentrations of 0.5, 1, 5, 10, and 25 μM, the expression level of LC3B-II increased from 1.00 ± 0.10 in the control group to 1.31 ± 0.16, 1.45 ± 0.26, 1.55 ± 0.13, 2.54 ± 0.64, and 1.91 ± 0.20, respectively; while the expression level of Beclin 1 increased from 1.00 ± 0.20 in the control group to 1.27 ± 0.19, 1.78 ± 0.30, 1.94 ± 0.19, 2.04 ± 0.12, and 1.67 ± 0.11, respectively.

[0103] 3.2 African tetrandrine significantly increased the number of autophagosomes in HEK293 cells.

[0104] like Figure 2 As shown, transmission electron microscopy revealed that treatment with 10 μM tetrandrine significantly increased the number of autophagosomes in HEK293 cells. Specifically, the number of autophagosomes in the control group was 3.13 ± 0.90, while the number of autophagosomes in the tetrandrine-treated group increased to 13.50 ± 1.32.

[0105] 3.3 Tetrandrine promotes TFEB nuclear translocation in different cell models.

[0106] like Figure 3 As shown in the figure, Western blot analysis revealed that treatment with 10 μM and 25 μM tetrandrine significantly increased the expression level of TFEB protein in the nucleus of HEK293T / APPswe cells, with the relative expression levels increasing from 1.00 ± 0.07 in the control group to 2.22 ± 0.43 and 2.05 ± 0.50, respectively. Subcellular localization of TFEB was further observed in N2a cells using immunofluorescence assays. Figure 4 In the control group, TFEB (red fluorescence) was mainly distributed in the cytoplasm, with less distribution in the nuclei labeled with DAPI (blue fluorescence). However, after treatment with 10 μM tetrandrine, the red TFEB signal and the blue DAPI signal showed significant overlap, exhibiting a purplish-red fluorescence, indicating a significantly enhanced translocation of TFEB into the nucleus. Quantitative analysis showed that tetrandrine treatment increased the fluorescence intensity of TFEB in the nucleus from 1.00 ± 0.09 in the control group to 1.26 ± 0.08, a statistically significant difference.

[0107] 3.4 In the Gaucher disease model, tetrandrine significantly restored the nuclear TFEB protein expression level that was reduced due to CβE treatment.

[0108] like Figure 5 As shown, compared with the control group (1.00 ± 0.27), CβE treatment for 5 days significantly reduced the expression level of TFEB protein in the nucleus of Huh7 cells to 0.17 ± 0.03; while after intervention with 10 μM tetrandrine, the expression level of this protein significantly increased to 0.54 ± 0.15, and the differences were statistically significant.

[0109] 3.5 Tetrandrine blocks the binding of the 14-3-3 adaptor protein to TFEB, promoting TFEB nuclear translocation.

[0110] like Figure 6As shown, molecular docking results indicate that tetrandrine can directly interact with the Ser211 phosphorylation site of TFEB, forming three stable hydrogen bonds at its Lys-11, Lys-87, and Asp-138 residues, with a binding energy of -6.7 kcal / mol. This interaction directly and competitively interferes with the recognition and binding of the 14-3-3 adaptor protein to the TFEB phosphorylation site, thereby releasing TFEB from the cytoplasmic anchorage of the 14-3-3 mediator and allowing it to enter the nucleus in free form to exert its transcriptional regulatory function.

[0111] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0112] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Application of tebufenozide in the preparation of products for treating autophagy-related diseases.

2. Use according to claim 1, wherein The products include pharmaceuticals.

3. Use according to claim 2, wherein the compound is ###0002### The drug includes tetrandrine and pharmaceutically acceptable excipients; Optionally, the pharmaceutically acceptable excipients include one or more of diluents, binders, wetting agents, disintegrants, lubricants, solubilizers, pH adjusters, and osmotic pressure adjusters.

4. The use according to claim 3, wherein the compound is ###0002### The dosage form of the product includes one or more of tablets, granules, capsules, oral preparations, and injections.

5. The application as described in any one of claims 1 to 4, characterized in that, The autophagy-related diseases include one or more of lysosomal storage diseases, cardiovascular diseases, and infectious diseases.

6. Application of African tetrandrine in the preparation of autophagy inducers.

7. The application as described in claim 6, characterized in that, The African tetrandrine enhances autophagy by promoting the nuclear translocation of the transcription factor TFEB.

8. The application as described in claim 7, characterized in that, The cells in the autophagy inducer include at least one of human embryonic kidney cells and neuroma cells.

9. Application of African tetrandrine in the preparation of transcription factor TFEB activator.

10. The application as described in claim 9, characterized in that, The African tetrandrine promotes TFEB activation by binding to the Ser211 phosphorylation site of TFEB.

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