Use of NPC1 protein or an agent that promotes expression thereof in the manufacture of a medicament for treating MASLD

Abstract

The application discloses application of NPC1 protein or an agent for promoting expression thereof in preparation of a medicine for treating MASLD, belongs to the technical field of biological medicines, and discloses that NPC1 is a key target point of autophagic flow downstream blockage in MASLD for the first time, discovers a brand-new functional domain, breaks through the traditional cognition that NPC1 is only a cholesterol transport protein, and proposes a synergistic treatment scheme of 'upstream activation + downstream dredging'. In-vivo experiments prove that the combined strategy or TFEB agonist monotherapy can significantly reduce liver steatosis, and the TFEB agonist can also effectively improve MASLD related impaired glucose tolerance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of NPC1 protein or reagents that promote its expression in the preparation of drugs for treating MASLD. Background Technology

[0002] Metabolic dysfunction-associated fatty liver disease (MASLD) is the most common chronic liver disease worldwide, characterized by excessive fat accumulation in the liver, which can progress to hepatitis, liver fibrosis, cirrhosis, and even liver cancer. Currently, clinical treatment mainly focuses on lifestyle interventions, but patient adherence is poor, resulting in limited efficacy. In 2024, the FDA approved the first commercially available drug for the treatment of MASH—the THR-β agonist Resmetirom. However, the variability in clinical manifestations of MASLD patients reflects the complexity of the disease, and further exploration of specific treatment options is needed to enrich clinical treatment choices.

[0003] Autophagy is a core process for maintaining cellular homeostasis, and its dysfunction is considered a key factor in the development and progression of MASLD. However, current therapeutic strategies targeting autophagy in MASLD are still in the research stage. Numerous studies have revealed that MASLD involves not only insufficient "upstream" activation but also "downstream" degradation barriers, particularly regarding the mechanisms of abnormal downstream autophagy degradation. Given this situation, there is currently a lack of effective treatments that simultaneously address both "upstream activation" and "downstream degradation" barriers. Therefore, there is an urgent need in this field to identify key targets leading to downstream autophagy blockage in MASLD, and to combine these targets with currently developed autophagy agonists to develop safe and effective therapeutic strategies that restore autophagy and promote the improvement of MASLD disease. Summary of the Invention

[0004] The purpose of this invention is to provide the application of NPC1 protein or reagents that promote its expression in the preparation of drugs for treating MASLD, drug combinations and strategies for treating MASLD by targeting NPC1 protein to restore autophagy function and by activating TFEB to synergistically upregulate autophagy activity and NPC1 expression.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] In a first aspect, the present invention provides the use of NPC1 protein or reagents that promote its expression in the preparation of medicaments for treating MASLD.

[0007] In the above technical solutions, the NPC1 protein is a biological agent containing the NPC1 protein or its functional domains.

[0008] In the above technical solution, the reagent is a TFEB agonist, which is an agonist that does not depend on the mTOR pathway.

[0009] In the above technical solutions, the reagent is a compound that can upregulate NPC1 expression or promote its function.

[0010] In the above technical solutions, the reagent is one of Thioperamide, DOPG, Alexidine, Voronostat, GEX1A, and Niacin.

[0011] In the above technical solution, the reagent is used in combination with an autophagy upstream activator to prepare a drug for treating MASLD.

[0012] In the above technical solutions, the autophagy upstream activator is an mTOR inhibitor or an AMPK agonist.

[0013] In a second aspect, the present invention provides a polypeptide comprising an amino acid sequence as shown in SEQ ID NO.1 or a functional variant thereof, said functional variant having more than 80% homology with the amino acid sequence shown in SEQ ID NO.1.

[0014] Thirdly, the present invention provides the use of the above-mentioned polypeptide, its fusion protein, or the nucleic acid molecule encoding it in the preparation of a medicament for treating MASLD.

[0015] Fourthly, the present invention provides a medicament comprising: a) an autophagy upstream activator; and b) an NPC1 function enhancer;

[0016] The autophagy upstream activator is an mTOR inhibitor or an AMPK agonist;

[0017] The NPC1 function enhancer is a compound that can upregulate NPC1 expression or promote its function, or a biological agent containing NPC1 protein or its functional domain.

[0018] The beneficial effects of this invention are as follows:

[0019] (1) Novel target: For the first time, it was revealed that NPC1 is a key target for downstream blockade of autophagy in MASLD, and its novel functional domain was discovered, breaking through the traditional understanding that it is only a cholesterol transporter.

[0020] (2) Advanced strategy: a synergistic treatment plan of "upstream activation + downstream unblocking" was proposed.

[0021] (3) Significant effects: In vivo experiments have confirmed that combination strategies or TFEB agonist monotherapy can significantly reduce hepatic steatosis, and TFEB agonists can also effectively improve MASLD-related glucose intolerance.

[0022] (4) High safety: The preferred regimen uses TFEB agonists, which avoids the side effects of rapamycin and has greater potential for clinical application.

[0023] (5) Strong translational potential: The changes in NPC1 and autophagy-related changes in the liver of MASLD mouse model and patients are relatively consistent, which has clinical translational potential. Attached Figure Description

[0024] Figure 1 Results of disease phenotype-related detection 16 weeks after the establishment of the MASLD mouse model. After 16 weeks of HFD feeding, (AE) MASLD mice showed significant increases in body weight, liver weight, and adipose tissue weight; (FG) fasting blood glucose and glucose tolerance also showed significant changes; (H) significant lipid deposition was formed in the liver, but no obvious fibrosis was formed; (I) the expression pattern of NPC1 in liver tissue with a centripetal distribution of central veins was significantly changed, and the expression level was significantly reduced.

[0025] Figure 2 This study investigated the effects of RAPA, an autophagy agonist, on MASLD mice. RAPA treatment showed a decreasing trend in body weight and adipose tissue weight in (AD) MASLD mice, but without statistical significance. Fasting blood glucose and impaired glucose tolerance were not significantly improved in (EF) mice; in contrast, RAPA treatment in the control group resulted in increased fasting blood glucose and impaired glucose tolerance. Hepatic steatosis was improved in (G) MASLD mice.

[0026] Figure 3 The effects of Thioperamide or DOPG monotherapy targeting NPC1 on the in vivo efficacy of MASLD mice were investigated. After Thioperamide treatment, (AE) there were no significant changes in body weight, liver weight, and adipose tissue weight in MASLD mice; (FG) there was a trend towards improvement in fasting blood glucose levels and impaired glucose tolerance; (H) there was no significant improvement in hepatic lipid deposition; (I) NPC1 expression levels in the liver of MASLD mice were significantly increased. After DOPG treatment, (JN) there were no significant changes in body weight, liver weight, and adipose tissue weight in MASLD mice; (OP) there was no significant improvement in fasting blood glucose levels and impaired glucose tolerance; (Q) there was no significant improvement in hepatic lipid deposition; and (R) NPC1 expression levels in the liver of MASLD mice were significantly increased.

[0027] Figure 4The combined intervention of Thioperamide and RAPA in MASLD mice was investigated. After the combined intervention, (AE) significantly reduced body weight, liver weight, and adipose tissue weight in MASLD mice, but body weight remained at a high level after a period of time and was still significantly different from the control group at the endpoint; (FG) fasting glucose elevation and glucose tolerance impairment tended to worsen; (H) liver lipid deposition level was significantly reduced, but significant lipid deposition was still present.

[0028] Figure 5 The effects of the TFEB agonist Tfa and Tfa combined with Thioperamide on MASLD mice in vivo were investigated. After Tfa administration alone or in combination, (AE) body weight, liver weight, and adipose tissue weight in MASLD mice were significantly reduced, with no significant difference between the two groups; (FG) fasting blood glucose levels and impaired glucose tolerance were significantly improved; (H) hepatic lipid deposition was significantly reduced; (I) NPC1 expression levels in the liver of MASLD mice were significantly increased; and (J) Tfa stimulation alone promoted NPC1 expression and activated autophagy in HepG2 cells in vitro.

[0029] Figure 6 To validate the key functional domains of NPC1: (AB) Based on the cryo-electron microscopy structure of the NPC1 protein, it was truncated into 5 large truncated variants; (CD) The interaction between NPC1 and the SNARE complex was predicted using AlphaFOLD3, and key sites were predicted as the basis for further truncation design; (E) The N-terminal truncated variants of the NPC1 protein were designed based on the prediction results; (F) Each truncated variant of NPC1 was expressed in NPC1 knockout HepG2 cells, and changes in autophagic flux were monitored using RFP-GFP-LC3, identifying the N-terminus of the NPC1 protein as the main functional region that regulates autophagic degradation; (G) NPC1 knockout led to abnormal co-localization of STX17 and VAMP8; (H) The selected functional truncated variants of NPC1 were co-expressed with STX17 and VAMP8 plasmids in NPC1 knockout cells, and it was found that the N-terminus of the NPC1 protein could significantly improve the abnormal co-localization of STX17 and VAMP8 caused by NPC1 knockout. Detailed Implementation

[0030] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below in conjunction with specific embodiments. This invention can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art. This invention will be defined only by the claims.

[0031] 1. Discovery and application of new targets

[0032] This invention is the first to discover that NPC1 protein (Niemann-Pick C1 type protein) is a key molecule for maintaining hepatic autophagy, and its expression is significantly downregulated in MASLD tissues. This invention clarifies that NPC1 deficiency is a significant cause of impaired autophagosome-lysosome fusion in MASLD. Based on this, this invention proposes the application of NPC1 protein or agents that promote its expression in the preparation of drugs for treating MASLD.

[0033] 2. New treatment strategies and drug combinations

[0034] This invention proposes a synergistic treatment strategy: combining "upstream autophagy activators" with "NPC1 expression promoters" to simultaneously overcome both "insufficient upstream activation" and "downstream degradation blockage" in autophagy. "Upstream autophagy activators" include, but are not limited to: mTOR inhibitors (such as rapamycin and its derivatives (Temsirolimus, Everolimus, Ridaforolimus), AZD8055, Torin 1, Torkinib, Sapanisertib), AMPK agonists (AICAR, A-769662, salicylates, resveratrol, metformin), etc. "NPC1 function enhancers" include, but are not limited to: compounds that can upregulate NPC1 expression or promote its function, such as Thioperamide, DOPG, Alexidine, Voronostat, GEX1A, Niacin; or biological agents containing NPC1 protein or its functional domains.

[0035] 3. Optimized treatment plan

[0036] This invention proposes a single-molecule synergistic treatment for MASLD: using transcription factor EB (TFEB) agonists, particularly mTOR-independent TFEB agonists (such as TFEB Activator 1 and other curcumin derivatives or analogs (Curcumin 5-8, Bisdemethoxycurcumin, Dimethoxycurcumin, Tetrahydrocurcumin, L48H37), Sulforaphane, trifluoperazine hydrochloride, desloratadine, digoxin, Ikarugamycin, and alexiconine). This approach simultaneously achieves: a) promoting upstream autophagy activation and increasing overall autophagy levels; b) promoting NPC1 protein expression and improving downstream autophagy degradation; thereby comprehensively restoring overall autophagic flux function, effectively improving MASLD, and avoiding the glucose metabolism side effects of mTOR inhibitors such as rapamycin.

[0037] 4. Key functional domains and biological agents

[0038] This invention is the first to discover and validate a novel functional domain in the NPC1 protein: the amino acid sequence located at the N-terminus from amino acid position 85 to 96. This functional domain has the function of directly regulating the autophagy fusion SNARE complex (STX17-SNAP29-VAMP8) independent of cholesterol transport function.

[0039] Therefore, this invention protects the use of polypeptides containing the amino acid sequence of the N-terminus of the NPC1 protein (85-96), their fusion proteins, or nucleic acid molecules encoding them in the preparation of medicaments for the treatment of MASLD.

[0040] 5. Experimental Design:

[0041] The reagents used in the following technical solutions are as follows: Thioperamide was purchased from abcam; dioleoylphosphatidylglycerol (DOPG) was purchased from Sigma; rapamycin (RAPA) and TFEB Acaivator1 (TFa) were purchased from MCE; NPC1, VAMP8, and STX17 expression plasmids were constructed in the laboratory; and GFP-RFP-LC3 lentivirus was purchased from Jikai Gene.

[0042] (1) MASLD animal model construction: 6-8 week old wild-type (WT) mice were fed a high-fat diet (HFD, containing 60% fat) for 12-20 weeks.

[0043] (2) Dosing regimen design:

[0044] 1) Rapamycin (RAPA) monotherapy: MASLD mice were injected intraperitoneally with RAPA (4ug / g / 3 days) for 8 weeks after 4 weeks of modeling, under continuous HFD feeding conditions.

[0045] 2) Thioperamide or DOPG monotherapy: After 10 weeks of MASLD modeling, mice were administered Thioperamide (8ug / g / 3 days) or DOPG (0.1 μmol / g / 3 days) via intraperitoneal injection for 6 weeks under continuous HFD feeding conditions.

[0046] 3) Thioperamide combined with rapamycin (RAPA): After 10 weeks of modeling in MASLD mice, a mixture of Thioperamide (8ug / g / 3 days) and RAPA (4ug / g / 3 days) was administered intraperitoneally for 6 weeks under continuous HFD feeding conditions.

[0047] 4) TFEB activator (TFa): MASLD mice were modeled 14 weeks after birth and were continuously fed HFD while receiving intraperitoneal injection of a mixture of TFEB Activator1 (20ug / g / 3 days) for 6 weeks.

[0048] 5) TFEB activator (TFa) combined with Thioperamide: MASLD mice were fed HFD for 6 weeks after 14 weeks of modeling, and were intraperitoneally injected with a mixture of TFEB Activator1 (20ug / g / 3 days) and Thioperamide (8ug / g / 3 days).

[0049] (3) Mouse model detection method: The weight of experimental mice was measured weekly. Fasting blood glucose and intraperitoneal glucose tolerance were measured 12-16 hours before sacrifice. After the test, mice were restored to a normal diet and sacrificed 24 hours later. Blood from the eyeballs, subcutaneous fat, epididymal fat and liver tissue were collected for subsequent detection. The liver tissue was divided into small pieces for paraformaldehyde fixation, frozen sectioning and cryopreservation for subsequent pathological staining and fluorescent staining.

[0050] (4) Identification of key functional domains of NPC1: Based on the resolved cryo-electron microscopy structure of NPC1, five large truncated variants containing the main functional domains of NPC1 were constructed and transfected into NPC1 knockout cells expressing FP-RFP-LC3 for salvage experiments. Autophagic flux changes were detected using GFP-RFP-LC3, referring to NPC1 knockout control cells (negative control) and full-length NPC1 expression (positive control), to identify functional segments of NPC1. Subsequently, based on the selected functional segments, their plasmids were co-transfected with VAMP8-GFP and mCherry-STX17 into NPC1 knockout cells to clarify the effect of these functional segments on the SNARE complex.

[0051] 6. Specific experimental methods

[0052] (1) In vivo drug administration preparation procedure in mice:

[0053] 1) Rapamycin (RAPA) monotherapy: Add 1.25 mL of Tween 80 to 20 mL of double-distilled water and heat to dissolve in a preheated 65°C water bath. After complete dissolution, cool in a refrigerator. Dissolve 10 mg of rapamycin in 1.25 mL of DMSO. After complete dissolution, add the DMSO to 2.5 mL of PEG400 and mix well. Mix the solutions obtained from the above two steps and shake well to obtain a 0.4 mg / mL RAPA solution. Filter through a 0.22 μm filter and store at -80°C.

[0054] 2) Thioperamide monotherapy: 50 mg of Thioperamide is completely dissolved in 62.5 mL of sterile physiological saline to obtain a Thioperamide solution of 0.8 mg / mL. After filtration through a 0.22 μm filter, it is stored at -80 °C.

[0055] 3) DOPG monotherapy: Dissolve 1.25 mL of Tween 80 in 20 mL of double-distilled water in a preheated 65°C water bath. After complete dissolution, cool in a refrigerator. Add 1.25 mL of anhydrous ethanol and 2.5 mL of PEG400, mix well, and centrifuge briefly. Filter sterilize using a 20 mL syringe with a 0.22 μm filter. Weigh 0.1594 g of DOPG and add it to 20 mL of the above solvent. Vortex until the powder is completely dissolved. Aliquot into 2 mL sterile tubes and store at -80°C.

[0056] 4) Thioperamide combined with rapamycin (RAPA): 50 mg of Thioperamide was completely dissolved in 62.5 mL of RAPA single-drug solution, filtered through a 0.22 μm filter, and stored at -80 °C.

[0057] 5) TFEB agonist: Add 2 mL of Tween 80 to 18 mL of double-distilled water and heat to dissolve in a preheated 65°C water bath. After complete dissolution, cool in a refrigerator. Mix 2 mL of PEG400, 1.6 mL of DMSO, and 16.4 mL of double-distilled water and shake well. Mix the above solution thoroughly. Add 10 mg of TFEB activator 1 (TFa) to 5 mL of the above mixture and shake to dissolve, obtaining a 2 mg / mL TFEB agonist solution. Filter through a 0.22 μm filter and store at -80°C.

[0058] 6) TFEB agonist combined with Thioperamide: 50 mg of Thioperamide was completely dissolved in 62.5 mL of TFEB agonist solution, filtered through a 0.22 μm filter, and stored at -80 °C.

[0059] (2) Intraperitoneal injection of drugs in mice: 10 μL of the above-mentioned drugs were added per gram of body weight for intraperitoneal injection. First, the skin of the back of the neck and tail of the mouse was fixed with the left hand, and the injection area was exposed with the abdomen facing upward. After disinfecting the skin of the mouse abdomen with 75% alcohol cotton balls, the drug solution corresponding to the mouse body weight was drawn with 1 mL of insulin needle. The needle was inserted into the skin at an angle of 30°-45° to the abdominal wall, 0.5 cm to the left or right of the midline of the mouse abdomen (avoiding the bladder and important organs). After aspirating and finding no blood or urine, the drug was slowly pushed in. After the injection was completed, the needle was quickly pulled out and the injection point was gently pressed with a dry cotton ball for a moment to prevent the drug solution from leaking out.

[0060] (3) Fasting blood glucose test in mice: After fasting for 12-16 hours, gently remove the mice from their cages and place them in a special mouse restraint, ensuring that the tail is stably exposed. Wipe the tip of the tail with an alcohol swab, and after the alcohol has completely evaporated and dried, quickly cut off about 1-2 mm from the tip of the tail with clean surgical scissors. Gently massage from the base of the tail to the tip, align the sampling groove of the blood glucose test strip with the edge of the blood drop, and allow the blood to be automatically drawn into the sampling area of ​​the test strip. Wait for the reading and record.

[0061] (4) Intraperitoneal glucose tolerance test in mice: After measuring fasting blood glucose in mice, the fasting blood glucose value was recorded as the baseline fasting blood glucose at 0 minutes. A 20% glucose solution was injected intraperitoneally at a ratio of 10 μL per 10 g body weight, using the same injection method as for drug injection. Timing began immediately after the injection, and blood glucose was measured at predetermined time points of 15, 30, 60, 90, and 120 minutes. Each blood collection procedure was performed gently and quickly to minimize stress on the mice. After completing all time point measurements, the mice were immediately given food and water, and all data were recorded.

[0062] (5) Animal tissue collection: After measuring glucose tolerance, mice were given food and water for 24 hours. The final weight of the mice at the end of the model was recorded using an electronic balance. Mice were anesthetized with ether, and blood was collected from the mice using the ocular blood collection method into sterile EP tubes. The blood was left at room temperature for 2 hours to allow it to coagulate. The mice were centrifuged at 3500 rpm for 10 minutes to collect the serum and stored in a -80°C freezer. Mice were euthanized by cervical dislocation. The mice were disinfected by immersing them in 75% alcohol and the limbs were fixed in the killing table. The abdominal skin and subcutaneous fat of the mice were separated using ophthalmic scissors and ophthalmic forceps. The subcutaneous and epididymal fat were separated and weighed. The liver was fully exposed, the hepatic ligaments were carefully separated, the gallbladder was removed, and the removed liver was washed in PBS, blotted dry with filter paper, and weighed. Two pieces of tissue the size of mung beans were fixed in 4% paraformaldehyde and OCT embedding medium, respectively, for subsequent experiments. The remaining liver tissue was cut into small pieces, placed in sterile cryovials, flash-frozen in liquid nitrogen, and finally transferred to a -80°C freezer for storage.

[0063] (6) Immunofluorescence assay: After routine dewaxing and hydration, the paraffin-embedded tissue sections were subjected to antigen retrieval using the EDTA high-pressure retrieval method. After histochemical circling, the sections were permeabilized with 0.1% Triton X-100 in PBS solution at room temperature for 30 minutes, and then 10% serum (identical to the secondary antibody source) was added for blocking at 37°C for 30 minutes. The serum was discarded, and the primary antigen solution was diluted with 10% serum to prepare the primary antibody working solution. 100 μl of the primary antibody working solution was added to each section, and the sections were incubated overnight at 4°C. The next day, the sections were removed from the refrigerator, placed at room temperature for 15 minutes to warm up, and washed three times with TBST. The secondary antigen solution was diluted with TBST to prepare the secondary antibody working solution. 100 μl of the secondary antibody working solution was added to each section, and the sections were incubated at 37°C for 45 minutes. The sections were washed three times with TBST. The TBST was discarded, and 100 μl of tyramine working solution was added to each section, and the sections were incubated at room temperature in the dark for 10 minutes. The sections were washed three times with TBST. After removing TBST, add 100 μl of DAPI working solution to each slide, stain the nuclei for 5 minutes in the dark, and then rinse with TBST. Mount the slides with fluorescent mounting medium and store at 4°C in the dark. Finally, acquire and analyze images using a fluorescence microscope or confocal microscope.

[0064] (7) Plasmid construction: HepG2 cells were collected, RNA was extracted using an RNA extraction kit, and after measuring the concentration, it was reverse transcribed into cDNA using a reverse transcription kit. SnapGene was used to design upstream and downstream primers for the corresponding gene fragments, and the corresponding gene fragments were amplified from the cDNA. The DNA fragments were separated and purified by agarose gel electrophoresis, ligated to the plasmid backbone, and transformed into competent cells. Single colonies were selected for sequencing. The correctly sequenced bacterial cultures were inoculated into 25 mL of liquid culture medium containing the same antibiotic and cultured at 37°C and 200 rpm for 16-18 hours. Plasmids were extracted using a plasmid drawer kit, and the concentration was measured for subsequent transfection experiments.

[0065] (8) Plasmid transfection: One day before transfection, seed an appropriate amount of cells into a confocal dish and add sufficient culture medium. During transfection, take a sterile EP tube, add 100 µL of Opti-MEM and the plasmid to be transfected (calculate the required volume in advance based on the plasmid concentration). Take another sterile EP tube, add 100 µL of Opti-MEM and the corresponding volume of transfection reagent, let stand at room temperature for 5 minutes, then slowly and gently add the culture medium containing the transfection reagent to the culture medium containing the plasmid, mix gently, and let stand for 20 minutes. Remove the cell culture dish from the incubator, discard the culture medium, wash the cells once with PBS, and add 800 µL of LDM medium. Slowly add the plasmid mixture into the culture dish, and replace with complete culture medium (containing 10% FBS) after 5-6 hours. Incubate overnight, and use for subsequent confocal imaging after 24 hours.

[0066] (9) Lentiviral transfection: One day before transfection, seed an appropriate amount of cells into a 12-well plate. During transfection, replace the medium with fresh medium, add the calculated volume of virus solution and transfection reagent according to the MOI determined in the preliminary experiment, mix gently, and incubate in an incubator. About 16 hours after transfection, aspirate the medium containing virus and transfection reagent, add fresh complete medium, and continue culturing. 48-72 hours after transfection, observe the expression of reporter genes (such as GFP) using a fluorescence microscope to determine the transfection status. When the cell confluence reaches 80%-90%, digest and resuspend the cells, transfer them to a 25T culture flask, add purine enzyme for selection, and perform Western blot (WB) detection on the selected cells for verification. Successfully verified cells are cryopreserved and passaged for subsequent experiments.

[0067] 7. Experimental Results:

[0068] like Figure 1-6 As shown, the details are as follows:

[0069] (1) Rapamycin (RAPA) monotherapy: It can improve hepatic steatosis to a certain extent, but it impairs glucose tolerance.

[0070] (2) Thioperamide monotherapy: It did not significantly improve hepatic steatosis, but it did improve fasting blood glucose and glucose tolerance.

[0071] (3) DOPG monotherapy: There was no significant improvement in hepatic steatosis, fasting blood glucose and glucose tolerance.

[0072] (4) Thioperamide combined with rapamycin (RAPA): significantly improved fatty degeneration, but still had an adverse effect on glucose tolerance.

[0073] Thioperamide targets NPC1, and RAPA activates autophagy. The combination of the two drugs can be used to treat MASLD. However, because RAPA alone has side effects, the combination therapy still has the side effects of RAPA.

[0074] (5) TFEB agonist (TFa): significantly improved fatty degeneration and improved impaired glucose tolerance.

[0075] TFEB agonist alone can achieve the effect of the two drugs in (4) without the side effects of RAPA. TFEB agonist is a single-target dual-action drug that can both promote NPC1 expression and activate autophagy.

[0076] (6) TFEB agonist (TFa) combined with Thioperamide: There is no significant difference compared with TFEB agonist (TFa) alone.

[0077] (7) Determination of key functional domains of NPC1: The N-terminus of NPC1 protein has the function of regulating the SNARE complex, which can significantly improve the abnormal autophagy degradation caused by NPC1 knockout.

[0078] In addition, it should be noted that the above experiment was conducted using Thioperamide and DOPG as examples. Other reagents that can promote NPC1 expression, such as Alexidine (1), Voronostat (2), GEX1A (3), Niacin (4), etc., can all achieve the same effect.

[0079] Alexidine, Voronostat, GEX1A, and Niacin are Thioperamide analogs that possess similar medicinal effects to Thioperamide and can promote NPC1 expression. Related reports on these four compounds are as follows:

[0080] (1) Pugach EK, Feltes M, Kaufman RJ, Ory DS, Bang AG. High-contentscreen for modifiers of Niemann-Pick type C disease in patient cells. Hum MolGenet 2018;27:2101-2112.

[0081] (2) Munkacsi AB, Hammond N, Schneider RT, Senanayake DS, Higaki K, Lagutin K, Bloor SJ, et al. Normalization of Hepatic Homeostasis in the Npc1(nmf164) Mouse Model of Niemann-Pick Type C Disease Treated with the HistoneDeacetylase Inhibitor Vorinostat. J Biol Chem 2017;292:4395-4410.

[0082] (3) Granatosky EA, DiPrimio N, Pickering JRE, Stevens DC, PerlsteinEO, Taylor RE. GEX1A, a Polyketide from Streptomyces chromofuscus, Correctsthe Cellular Defects Associated with Niemann-Pick Type C1 in HumanFibroblasts. J Nat Prod 2018;81:2018-2025.

[0083] (4) Yang Ping, Zhang Hanbin, Liu Yulin, Fang Shuxiang, Li Ping, Xu Xiaoyang; Niacin promotes the efflux of macrophage lysosome cholesterol via the LXRα / NPC1 pathway; Chinese Journal of Pathophysiology, 2020, No. 8.

[0084] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Application of TFEB activator1 in the preparation of drugs for treating MASLD.

2. Application of Thioperamide in combination with rapamycin in the preparation of drugs for the treatment of MASLD.

3. A drug, characterized in that: It contains: a) an autophagy upstream activator; and b) an NPC1 function enhancer; The autophagy upstream activator is rapamycin, and the NPC1 function enhancer is Thioperamide.

Images