Application of attapulgite in recovery of lysosome acidity and autophagic flux

Abstract

The invention discloses application of attapulgite in recovery of lysosome acidity and autophagy flux, and belongs to the field of nano biotechnology medicine. The invention finds that attapulgite can recover lysosome acidity decrease caused by lipotoxicity, chloroquine, lysosome inhibitors and the like and recover blocked autophagy flux. The invention finds that ATT can effectively regulate cytotoxicity of chloroquine and recover lysosome acidity decrease and blocked autophagy flow caused by chloroquine or lipotoxicity. Besides, ATT shows a remarkable treatment effect on lysosomal dysfunction related diseases such as NAFLD, lysosomal acidity and autophagy flow in a high-fat feeding induced NAFLD model can be recovered, lipid accumulation and liver biochemical indexes can be effectively reduced, and the tolerance of glucose can be improved. In addition, the activity of lysosome hydrolase can be increased, and degradation of mutant huntingtin is accelerated.

Description

Technical Field

[0001] The present invention belongs to the field of nanobiotechnology medicine, and particularly relates to the use of attapulgite in restoring lysosomal acidity and autophagic flux. Background Art

[0002] Lysosomes are acidic organelles enclosed by a single membrane within cells, containing a variety of acid hydrolases (such as proteases, nucleases, and glycosidases), which can efficiently decompose biological macromolecules such as proteins, lipids, carbohydrates, and nucleic acids. Its membrane embeds key membrane proteins, such as lysosome-associated membrane protein 1 (LAMP1 / CD107a) and 2 (LAMP2 / CD107b), which maintain the homeostasis and functional integrity of lysosomes. In addition, the vacuolar ATPase (V-ATPase) on the lysosomal membrane pumps protons (H + ) into the lysosome to maintain its acidic environment and ensure the optimal activity of hydrolases. The lysosomal membrane also contains a variety of important proteins, including lysosomal membrane proteins (such as LIMP2), ion channels (such as TMEM175), receptor proteins (such as M6PR and LAMP2A), fusion proteins (such as Rab7 and SNARE proteins), and signaling proteins (such as mTORC1). These proteins work together to maintain the acidic environment, material degradation, ion balance, membrane fusion, and signal transduction of lysosomes, enabling them to play a central role in intracellular digestion, material recycling, and stress response. Through processes such as intracellular digestion, material recycling, nutrient sensing, receptor regulation, and exocytosis, lysosomes participate in cell metabolism, waste clearance, and stress response.

[0003] Lysosomes are closely related to the autophagy process. Autophagosomes fuse with lysosomes to form autolysosomes, which degrade damaged organelles and misfolded proteins through acidic hydrolases in lysosomes, maintaining the stability of the intracellular environment. Dysfunction of lysosomes and the autophagy-lysosome system is closely related to various diseases, such as neurodegenerative diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease), metabolic diseases (non-alcoholic fatty liver disease, diabetes), inflammation-related diseases (pancreatitis, atherosclerosis), and lysosomal storage diseases (Gaucher's disease, Niemann-Pick disease). In these diseases, lysosomal and autophagic dysfunction leads to the accumulation of intracellular waste, metabolic disorders, and cell damage, highlighting their central role in cell health and disease. For example, the pathological mechanism of Huntington's disease (HD) is closely related to the dysfunction of the autophagy-lysosome pathway (ALP). Aggregation of mutant huntingtin protein (mHTT) impairs autophagy and hinders the effective clearance of toxic proteins, while the decline in lysosomal function further hinders the degradation process, exacerbating neuronal damage. Autophagy can clear lipid droplets, damaged mitochondria, and toxic proteins in hepatocytes, maintaining liver homeostasis. However, in NAFLD, autophagic dysfunction leads to lipid accumulation and increased inflammation. Factors such as high-fat diet and oxidative stress inhibit autophagy and promote disease progression. Therefore, restoring lysosomal acidification and autophagic flux has the potential to reverse NAFLD and represents a new therapeutic strategy.

[0004] In addition, chloroquine (CQ) is a drug widely used in clinical treatment of malaria, autoimmune diseases (such as rheumatoid arthritis and systemic lupus erythematosus), and antiviral therapy. However, long-term or high-dose use of chloroquine may cause significant toxic side effects, including retinopathy, arrhythmia, neuromuscular toxicity, gastrointestinal reactions, blood abnormalities, and skin reactions. These side effects mainly result from its mechanism of action of inhibiting lysosomal function, disrupting mitochondrial metabolism, and affecting ion channels. Therefore, the development of novel nanomaterials with strong lysosomal regulatory ability and superior biosafety has become an important research direction in the biomedical field.

[0005] Attapulgite (ATT), a naturally occurring one-dimensional nanosilicate material, has significant ion exchange properties and biocompatibility due to its unique layered chain structure, controllable surface charge, and abundant hydroxyl groups. In 2012, ATT was approved as a food additive for processing aids (GB 29225-2012) due to its stability and safety. Recent studies have highlighted its unique advantages in biomedical applications, including drug delivery and antibacterial dressings. In addition, recent studies have also discovered the independent anti-inflammatory properties of ATT, expanding its clinical application prospects. However, its potential regulatory effects on the organelle microenvironment and autophagy pathway still need to be further explored. Summary of the Invention

[0006] In view of the above deficiencies in the prior art, the present invention provides the use of attapulgite in restoring lysosomal acidity and autophagic flux.

[0007] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is:

[0008] The object of the present invention is to provide the use of attapulgite in restoring lysosomal acidity and autophagic flux.

[0009] Another object of the present invention is to provide the use of attapulgite in the preparation of a preparation for restoring lysosomal acidity and autophagic flux.

[0010] Further, attapulgite restores the decrease in lysosomal acidity caused by lipotoxicity.

[0011] Further, attapulgite restores the decrease in lysosomal acidity caused by chloroquine.

[0012] Further, attapulgite can also inhibit chloroquine-induced cytotoxicity.

[0013] Further, attapulgite can restore the autophagic flux blocked by lysosomal inhibitors.

[0014] Another object of the present invention is to provide a preparation for restoring lysosomal acidity and autophagic flux, which comprises the above-mentioned attapulgite and a pharmaceutically acceptable carrier.

[0015] Another object of the present invention is to provide a preparation for inhibiting chloroquine cytotoxicity, which comprises the above-mentioned attapulgite and a pharmaceutically acceptable carrier.

[0016] Another object of the present invention is to provide the use of the above-mentioned attapulgite or preparation in the preparation of a drug for treating lysosomal decline and / or autophagy inhibition-related diseases.

[0017] Further, the diseases include obesity, metabolic syndrome, diabetes, non-alcoholic fatty liver disease and neurodegenerative diseases caused by lipid accumulation.

[0018] The beneficial effects of the present invention:

[0019] The present invention discovers that ATT can restore the lysosomal pH value in chloroquine-treated cells, activate cathepsin D, relieve autophagy blockage, reduce chloroquine-induced cell death. ATT can also promote the clearance of lipid droplets in palmitic acid-induced hepatocyte steatosis, reduce liver lipid accumulation in NAFLD mice induced by a high-fat diet, and improve fasting blood glucose levels. In addition, ATT shows significant therapeutic effects on lysosomal dysfunction-related diseases such as NAFLD, can restore lysosomal acidity and autophagic flux in the NAFLD model, and effectively reduce lipid accumulation. These findings establish the status of ATT as a lysosomal regulator and provide a basis for its role in alleviating the adverse effects of long-term chloroquine use, especially in improving neurodegenerative and metabolic disorders. Brief Description of the Drawings

[0020] Figure 1 It is a figure showing the identification result of ATT;

[0021] Figure 2 It is the effect of ATT on the decrease in lysosomal acidity and CTSD maturation caused by CQ;

[0022] Figure 3 It is the effect of ATT on the lysosomal autophagy inhibition caused by CQ;

[0023] Figure 4 It is the effect of ATT on the lysosomal autophagy inhibition caused by different lysosomal inhibitors;

[0024] Figure 5 It is the effect of ATT on lysosomal acidity and the clearance of mutant huntingtin;

[0025] Figure 6 It is the effect of ATT on lysosomal acidity and autophagic flux under lipotoxicity;

[0026] Figure 7 It is the effect of ATT on the cytotoxicity caused by CQ in HeLa cells;

[0027] Figure 8 It is the effect of ATT on the cytotoxicity caused by CQ in EA.hy926 and HaCaT cells;

[0028] Figure 9 It is the detection of the therapeutic effect of ATT on NAFLD. Detailed Description of the Invention

[0029] The specific embodiments of the present invention will be described below to facilitate the understanding of those skilled in the art of the present technology. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art of the present technology, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are within the scope of protection.

[0030] The experimental materials and equipment used in the present invention are as follows:

[0031] 1. Phosphate Buffered Saline (P1003), Penicillin-Streptomycin (P1400), Hoechst33342 (C0031), Proteasome Inhibitor (IKM1010), 5× Loading Buffer (20317ES05), 1.5M Tris-HCl Buffer (pH 8.8; T1010), 1M Tris-HCl Buffer (pH 6.8; T1020), Bovine Serum Albumin (A8010), Oil Red O Staining Kit (G1261) were purchased from Beijing Solarbio Science & Technology Co., Ltd. DMEM (PM150225) and RPMI-1640 (PM150110) media were purchased from Wuhan Punuo Life Science & Technology Co., Ltd. Lysosome Red Fluorescent Probe (C1046), Western and IP Cell Lysis Buffer (P0013), CCK-8 Cell Counting Kit (C0037), Hydrochloric Acid Ethanol Rapid Differentiation Solution (C0163) were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Chlorodiphosphate (S6999) and wortmannin (S2758) were purchased from Sigma-Aldrich. Palmitic Acid (57-10-3) was purchased from Shanghai Macklin Biochemical Co., Ltd. Bafilomycin A1 (HY-100558) was purchased from MedChemExpress, USA. Experimental rat maintenance feed (for normal fat diet) was purchased from Jiangsu Xianzheng Biotechnology Co., Ltd. mRFP-GFP-LC3 expression virus was purchased from Applied Biological Materials Inc. (ABM), Canada. High-fat rat feed was purchased from ResearchDiets, USA. Attapulgite was purchased from Mingguang Feizhou New Materials Co., Ltd. Alanine Aminotransferase (ALT / GPT) Detection Kit (Reitman-Frankel method, microplate method) (C-009-2-1) was purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd. GOT / AST Kit (enzymatic assay method) (C010-2-1) was purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd. Lysosome Yellow / Blue Fluorescent Probe (40768ES50) was purchased from Shanghai Yisheng Biotechnology Co., Ltd.

[0032] 2. The cathepsin D antibody (21327-1-AP) was purchased from Proteintech Group. The GAPDH antibody (AF7021) and SQSTM1 / p62 antibody (AF5384) were purchased from Affinity Biosciences. The LC3B antibody (NB100-2220) was purchased from NovusBiologicals. The GFP antibody (AE078) was purchased from ABclonal Technology.

[0033] 3. Preparation of ATT: Weigh 100 mg of ATT powder, add three times the amount of distilled water to dissolve it, and make up the volume to 10 mL to prepare a 10 mg / mL solution. The ultrasonic solution of ATT nanorods was ultrasonically treated in an ultrasonic cleaner for 2 hours and then stored at 4 °C for subsequent use. The following instruments were used for the characterization of ATT: Zetasizer Nano ZS-90 from Malvern Panalytical Ltd., UK; transmission electron microscope JEM-1400 from JEOL Ltd., Tokyo, Japan; and XRD instrument Rigaku Smartlab SE from Rigaku Corporation, Japan.

[0034] 4. Preparation of FITC-ATT: Weigh 10 mg of fluorescein isothiocyanate powder and dissolve it in 10 mL of dimethyl sulfoxide to prepare a 1 mg / mL solution. Prepare a 10 mg / mL ATT solution and mix it with the 1 mg / mL FITC solution at a mass ratio of 1:1. Stir the mixture overnight at room temperature and then centrifuge it three times at 10,000 rpm for 10 minutes each time. After centrifugation, redisperse the mixture in three portions of distilled water to obtain FITC-labeled ATT, which should be stored at 4 °C for later use. The successful labeling was confirmed by fluorescence imaging using a multifunctional fluorescence imaging system (Azure 600, Azure Biosystems).

[0035] 5. Construction of mRFP-GFP-LC3 / HeLa cells: Seed HeLa cells in a 24-well plate and add mRFP-GFP-LC3 lentivirus (Applied Biological Materials Inc, CS5005498) and polybrene to the cell culture medium. After incubating for 24 hours, replace the fresh medium and continue incubating for 48 hours. Subsequently, evaluate the virus infection efficiency under a fluorescence microscope. After successful infection, add 1 μg / mL of puromycin for screening.

[0036] 6. Cell culture: GFP-LC3 / HeLa cells and GFP-Htt(Q74) / PC12 cells were provided by Professor Wen Longping. HeLa cells, EA.hy926 cells, HaCaT cells, HepG2 cells, GFP-Htt(Q74) / PC12 cells, GFP-LC3 / HeLa cells and mRFP-GFP-LC3 / HeLa cells were all generously provided. All cell lines were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. All cell lines were maintained in an incubator at 37 °C and 5% carbon dioxide.

[0037] 7. Palmitate: BSA preparation: First, palmitate was dissolved in DMSO and then dissolved in fatty acid-free DMEM medium (without glucose) at 45 °C to prepare a 4 mM (10×) stock solution. As a control condition, a 10× DMEM medium stock solution containing 5% BSA and 1% DMSO was used. For the treatment condition, the 10× stock solution was added to DMEM medium containing 5% FBS, 50 U / mL penicillin, and 50 μg / mL streptomycin. The adjusted medium was sterile filtered using a 0.45 μm syringe filter.

[0038] The detection methods are as follows:

[0039] 1. Flow cytometry analysis of internalized FITC-labeled ATT: Hela cells were placed in a six-well plate and 500 μg / ml of FITC-labeled ATT was added. After incubation for different times, the cells were digested with trypsin and centrifuged, and then placed on ice for flow cytometry detection.

[0040] 2. Transmission electron microscopy observation of ATT: HeLa cells were seeded in cell culture dishes and allowed to adhere for 24 hours. Subsequently, the cells were exposed to 500 μg / mL of ATT nanorods for 6 hours. After treatment, the cells were gently rinsed with phosphate buffer (PBS) and fixed with 2.5% glutaraldehyde at 4 °C for 2 hours. After fixation, the cells were dehydrated with gradient ethanol and embedded in epoxy resin. The embedded samples were then cut into ultra-thin sections and stained for observation. Finally, all samples were examined using a JEM-1400 transmission electron microscope (JEOL Ltd., Japan).

[0041] 3. Western Blot: Cells were collected and lysed with RIPA lysis buffer containing protease inhibitors or WB / IP lysis buffer (Beyotime, P0013). Protein concentration was determined by Bradford method and mixed with 5× protein loading buffer. The cells were immersed in a metal bath at 99 °C for 10 minutes. Protein transfer was performed using a methanol-activated PVDF membrane (Millipore, ISEQ00010). The membrane was incubated with 5% non-fat milk at room temperature for 1 hour, then incubated with the pre-prepared primary antibody overnight at 4 °C, and then incubated with the secondary antibody for 1 hour at room temperature. The protein of interest was developed using an enhanced chemiluminescence system (SCG-W2000, Servicebio).

[0042] 4. Cell viability: Cells were seeded in 96-well plates at a density of approximately 10,000 cells per well. After the specified treatment, CCK-8: cell culture medium (1:9, 100 μL) was added and incubated for 45 min. Cell viability was measured using a microplate reader and the signal was detected at 450 nm.

[0043] 5. Nile red staining: To observe lipid droplets by immunofluorescence, cell samples treated differently were fixed with 4% paraformaldehyde for 30 minutes and stained with Nile red for 15 minutes in the dark at room temperature. Then, after washing the medium, images were observed and taken under a fluorescence microscope.

[0044] 6. Construction and treatment of NAFLD model: Four-week-old male C57BL / 6J mice were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. After one week of adaptive feeding, the C57BL / 6J mice were randomly divided into four groups. Two groups were fed a normal diet (ND, 1010088, Jiangsu Serui Bioengineering Co., Ltd.) for 16 weeks; the other two groups were fed a high-fat diet (HFD, D12492, 60% calories from fat, Research Diets, Inc.) for 16 weeks as well. Subsequently, the mice in the ND group were intravenously injected with 10 mg / kg ATT or PBS every other day for a total of four times; the mice in the HFD group were also intravenously injected with 10 mg / kg ATT or PBS every other day for the same four times. That is, the ND-PBS group (n = 3), ND-ATT group (n = 3), HFD-PBS group (n = 5), and HFD-ATT group (n = 7). Finally, blood samples of the mice were collected under anesthesia, and then euthanasia was performed to facilitate the collection of tissue samples. Subsequently, liver weight was measured and biochemical indexes were evaluated. There was no exclusion.

[0045] 7. Oral Glucose Tolerance Test (OGTT): The OGTT was performed after the mice had fasted for 12 hours. After determining the fasting blood glucose level, each mouse was administered 2 g / kg body weight of glucose via intragastric administration. Blood glucose levels were measured from the tail vein at 15, 30, 60, 90, and 120 min, respectively. The blood glucose concentrations at different time points were plotted, and the area under the curve (AUC) was calculated.

[0046] 8. Detection of Serum Biochemical Indexes: After anesthesia, venous blood was collected from the inner eye. The whole blood was placed at 4 °C for more than 2 hours, centrifuged at 3000 rpm for 20 min, the supernatant was aspirated, and the levels of ALT and AST in the blood were detected using alanine aminotransferase and aspartate aminotransferase (AST) test kits.

[0047] 9. Hematoxylin-Eosin Staining: The heart, liver, spleen, lung, and kidney were quickly removed, washed with blood, placed in a tissue embedding frame, and fixed with 4% paraformaldehyde for 48 hours. After fixation, the tissue blocks were embedded using an automatic paraffin embedding machine. The embedded tissue was cut into 5-μm-thick sections using an automatic paraffin slicer and attached to pathological glass slides. Before staining, the slides were baked at 65 °C for 2 hours, and then dewaxed, hydrated, stained, and sealed in sequence. Fluorescence microscopy was used for observation and photography.

[0048] 10. Oil Red O Staining of Liver: Fresh liver tissue was taken, placed in OCT embedding medium, and frozen on the surface of liquid nitrogen. The tissue was cut into 10-μm-thick sections using a cryostat and dried at room temperature for 20 minutes. After staining, the tissue was stained using an Oil Red O staining kit. After staining, the sections were sealed, and fluorescence microscopy was used for observation and photography.

[0049] 11. Detection of Cellular Lysosomal Acid: The cells were seeded in confocal culture dishes (cell density per well was 1×10^5), and after 24 hours of cell attachment, they were treated with different materials. The culture medium was removed, the cells were washed with PBS, and incubated with 75 nM Lysotracker Red dye preheated to 37 °C for 30 min. After washing again with PBS, fresh complete medium was added. Laser scanning confocal microscopy was used to observe the cells and capture images.

[0050] Preparation and Identification of ATT in Example 1

[0051] 1. Attapulgite was sourced from Mingguang Feizhou New Materials Co., Ltd., and its preparation process was as follows:

[0052] Weighed 100 mg of ATT powder, added three times the amount of distilled water to dissolve it, and made up the volume to 10 mL to prepare a 10 mg / mL solution. The ultrasonic solution of ATT nanorods was ultrasonically treated in an ultrasonic cleaner for 2 hours and then stored at 4 °C for subsequent use.

[0053] 2. The prepared ATT was characterized, and the results are shown in Figure 1。

[0054] As Figure 1 shown in A, the transmission electron microscopy image shows that ATT presents a good dispersion morphology, characterized by a needle-like rod structure. The results measured by the Malvin nanoparticle size analyzer indicate that the hydrated particle size of ATT is 396.00 ± 11.00 nm( Figure 1 B), and the Zeta potential is -16.60 ± 0.30 mV.

[0055] X-ray diffraction (XRD) analysis( Figure 1 C) shows that the characteristic peaks of ATT at 2θ angles show peaks corresponding to the basic framework of ATT, located at 8.2°, as well as internal silicon-oxygen absorption peaks, located at 13.6°, 19.8°, and 20.9° respectively. The characteristic absorption peaks of quartz at 26.7° and montmorillonite at 35.2° further indicate the presence of these mineral impurities. Fourier transform infrared spectroscopy( Figure 1 D) identifies the characteristic peaks related to the hexagonal unit cell of magnesium oxide in ATT, located at 459.2 cm -1 , 511.8 cm -1 and 883.4 cm -1 ]>. In addition, the peaks at 986.7 cm -1 and 1033.5 cm -1 correspond to the stretching vibrations caused by the asymmetry of the silicon-oxygen structure, while the peak at 1211.0 cm -1 is attributed to the stretching vibration of the silicon-oxygen-silicon structure. In summary, these physical and chemical analyses show that ATT exhibits good dispersibility, and its crystal structure and composition are consistent with the standard reference materials.

[0056] Example 2 Effects of ATT on Lysosomal Acidity and Autophagic Flux

[0057] 1. ATT restores the acidity decrease caused by CQ and promotes the maturation of CTSD

[0058] The CQ-treated HeLa cell model was used to study whether ATT can restore lysosomal acidity and the maturation of acid hydrolases. Lysotraker Red was used to detect the organelles related to acidic lysosomes, and the effect of ATT on lysosomal acidity was detected as follows:

[0059] HeLa cells were seeded in confocal culture dishes (cell density per well was 1 × 10^5), and after 24 hours of adhesion, they were treated with different materials. The medium was removed, washed with PBS, and incubated with 75 nM lysosome-tracking red dye preheated to 37 °C for 30 min. After washing again with PBS, fresh complete medium was added. The cells were observed using a laser scanning confocal microscope and images were taken, and the results are shown in Figure 2 .

[0060] in, Figure 2 A is a fluorescence image of Lyso-Tracker Red (75 nM; 30 minutes) staining in HeLa cells after treatment with ATT (500 μg / mL) and CQ (50 μM) for 24 hours; Figure 2 B is the immunoblot analysis of feline heparin D in HeLa cells under different treatment conditions, with treatment time of 24 h. ATT, 500 μg / mL; CQ, 50 μM; Figure 2 C shows immunoblot analysis of feline heparin D expression in HeLa cells following treatment with various concentrations of ATT for 24 hours in the presence of CQ. The experiment was repeated three times, and the right panel was quantified using Image J and GraphPad. NS: not significant; *p < 0.05, ***p < 0.001.

[0061] like Figure 2 As shown in A, CQ-reduced Lysotraker Red staining can be reduced by ATT treatment, demonstrating that ATT has the ability to restore lysosomal acidification. Furthermore, cells treated with ATT alone also showed increased Lysotraker Red staining, indicating that ATT can also induce lysosomal acidification in normally cultured cells. This suggests that ATT can restore the acidity of lysosomes that have been alkalinized by CQ.

[0062] In addition, the present study found that the use of ATT alone did not significantly change the maturation process of CTSD in HeLa cells ( Figure 2 B). However, it effectively counteracted the inhibitory effect of CQ on this process. Although CQ significantly hindered the maturation of CTSD, ATT successfully restored the expression level of mature CTSD, indicating that they are able to re-acidify CQ-alkalinized lysosomes and promote the maturation of lysosomal enzymes ( Figure 2 B). Meanwhile, the enhancement of CTSD maturation by ATT follows a clear dose-response relationship. With the increase of concentration, the maturation level gradually increases and reaches a peak at an ATT concentration of 500 μg / mL ( Figure 2 C) ATT can alleviate the CQ-induced maturation disorder of lysosomal acid hydrolase CTSD, and the effect is significant in a dose-dependent manner.

[0063] 2. ATT restores CQ-blocked autophagic flux

[0064] (1) After 24 hours of treatment in the above-mentioned HeLa cells, the expression of autophagy substrates was analyzed by immunoblotting. The results are shown in Figure 3 A.

[0065] like Figure 3As shown in Figure A, it can be seen from the expression levels of LC3-II and p62 proteins that CQ treatment significantly blocked the autophagic flux of lysosomes, while the addition of ATT effectively attenuated the autophagy inhibition caused by CQ. However, when ATT was used alone, it had no significant effect on the basal autophagic level of lysosomes.

[0066] (2) To further investigate autophagic activity, GFP-LC3 / HeLa cells were treated with ATT (500 μg / mL) and CQ (50 μM) in the presence of CQ (50 μM) for 24 hours to detect their effects on autophagic flux. The results are shown in Figure 3 Figures B - D.

[0067] As Figure 3 shown in Figure B, ATT significantly reduced the accumulation of GFP-LC3 puncta caused by CQ, indicating that they may play a role in restoring the degradation of autophagic substrates blocked by CQ. Meanwhile, western blot analysis showed that after co-treatment with CQ and ATT, the accumulation of LC3-II and p62 proteins decreased in a dose-dependent manner, and higher concentrations of ATT led to more obvious reduction of these autophagy markers ( Figure 3 Figures C and 3D). This indicates that ATT can effectively restore the autophagic flux blocked by CQ.

[0068] (3) Given the abundant presence of magnesium silicate, aluminum, and other components in ATT, further studies were conducted on whether silicates including magnesium silicate, aluminum silicate, and sodium silicate could similarly restore the CQ-inhibited autophagic flux. The results are shown in Figure 3 Figure E.

[0069] As Figure 3 shown in Figure E, silicates including magnesium silicate, aluminum silicate, and sodium silicate did not alleviate the accumulation of autophagic substrate proteins inhibited by CQ, indicating that the above-mentioned silicates do not have the ability to restore the blocked autophagic flux of lysosomes.

[0070] Subsequently, we used bafilomycin A1 and ammonium chloride to evaluate the general applicability of ATT in restoring autophagic flux. The Western blot results ( Figure 4 ) showed that both bafilomycin A1 and ammonium chloride led to the accumulation of autophagic substrate proteins and blocked autophagic flux. However, after the addition of ATT, the accumulation of these substrate proteins decreased. This indicates that ATT can relieve the autophagy block induced by bafilomycin A1 and ammonium chloride, thereby restoring autophagic flux and promoting the degradation of autophagic substrate proteins. In summary, ATT has the ability to restore the autophagic flux blocked by various lysosomal inhibitors.

[0071] (4) To further investigate the effect of ATT on autophagic flux, the mRFP-GFP-LC3 / HeLa cell line was used as a sensitive reporter system to detect the effect of ATT on autophagic flux. The results are shown in Figure 3 Figures F, G.

[0072] As Figure 3 shown in F and G, CQ treatment significantly inhibited the fusion of autophagosomes with lysosomes, which was confirmed by the persistence of GFP signal and the increase in yellow fluorescence. Notably, the simultaneous use of ATT effectively restored the acidity of lysosomes and promoted the fusion of autophagosomes with lysosomes, resulting in GFP quenching and a decrease in yellow fluorescence. Quantitative analysis of yellow spots showed that although CQ treatment significantly increased yellow fluorescence due to impaired autophagic flux, the simultaneous administration of ATT greatly reduced the yellow fluorescence intensity. Collectively, the above results indicate that ATT can counteract CQ-induced autophagy inhibition by restoring lysosomal function and promoting autophagosome-lysosome fusion, thereby re-establishing normal autophagic flux.

[0073] 3. ATT increases lysosomal acidity and accelerates the clearance of mutant huntingtin

[0074] In GFP-Htt(Q74) / PC12 cells, the effects of ATT on lysosomal acidity and mHTT degradation were investigated, and the results are shown in Figure 5 . Among them, A is the immunoblot analysis and quantitative analysis of the CTSD level in GFP-Htt(Q74) / PC12 cells after treatment with different concentrations of ATT for 72 hours. Three experiments were performed. B is the immunoblot analysis and quantitative analysis of the LC3 and p62 levels in GFP-Htt(Q74) / PC12 cells after treatment with different concentrations of ATT for 72 hours. C-D are the immunoblot analysis and quantitative analysis of the mutant huntingtin level in GFP-Htt(Q74) / PC12 cells after treatment with different concentrations of ATT for 72 hours. Three experiments were performed. E is the immunoblot analysis and quantitative analysis of the expression of mutant huntingtin in GFP-Htt(Q74) / PC12 cells under different treatment conditions. ATT (500 μg / mL), 72 hours; Wort (500 nM), 24 hours. Three experiments were performed. (F) Cell viability analysis of GFP-Htt(Q74) / PC12 cells after treatment with different doses of ATT for 72 hours. NS: no significant difference; **p < 0.01, ***p < 0.001.

[0075] As Figure 5 shown in A, after the addition of ATT, the lysosomal activity in GFP-Htt(Q74) / PC12 cells increased in a concentration-dependent manner. Western blot analysis showed that ATT treatment promoted the maturation of CTSD, manifested as the conversion of immature CTSD to its mature form, thus indicating that these nanorods enhanced the maturation of lysosomal enzymes. In addition, ATT was found to induce autophagy, which was demonstrated by the conversion of LC3 protein from its LC3-I form to LC3-II and the degradation of p62 protein ( Figure 5 B).

[0076] As Figure 5 shown in C and D, co-incubation of GFP-Htt(Q74) / PC12 cells with ATT significantly promoted the degradation of mHTT, and this effect became more obvious with the increase of ATT concentration. To further clarify the mechanism by which ATT promotes the degradation of mHTT, we used the autophagy inhibitor wortmannin. The results showed that wortmannin effectively inhibited the ATT-mediated degradation of mHTT, thus confirming that the degradation process occurred through the autophagy pathway( Figure 5 E). In addition, we also observed that ATT enhanced the survival rate of GFP-Htt(Q74) / PC12 cells( Figure 5 F), indicating its protective effect against the accumulation of toxic proteins. Based on the above results, it can be known that ATT can restore lysosomal acidity, induce autophagy, accelerate the lysosomal degradation of mHTT, and improve cell viability.

[0077] 4. ATT re-acidifies lysosomes and restores autophagic flux under lipotoxicity

[0078] High levels of free fatty acids in the liver can damage lysosomal acidification and inhibit autophagic flux. Based on this, it was considered to first evaluate in vitro whether ATT could alleviate the lysosomal acidity decrease and autophagic flux inhibition caused by palmitic acid (PA), and the results are shown in Figure 6 . Figure 6 A in shows a schematic diagram of the experimental protocol for treating cells 24 hours before detecting lysosomal acidity, autophagic flux, or lipid droplet accumulation. The experiment was performed with HepG2 cells treated with bovine serum albumin (BSA) as a control, PA (complexed with BSA at a ratio of 4:1), ATT + BSA, or a combination of PA and ATT.

[0079] B-C show LysoTracker Red (75 nM; 30 min) staining (B) and corresponding quantitative (C) analysis of HepG2 cells after exposure to different treatments for 24 hours. PA, 400 μM; ATT, 250 μg / mL. Quantitative analysis was performed using Image J and GraphPad. n = 3.

[0080] D-E show immunoblotting (D) and corresponding quantitative (E) analysis for detecting the expression of autophagy substrates in HepG2 cells after exposure to different treatments for 24 hours. PA, 400 μM; ATT, 250 μg / mL. Three experiments were performed.

[0081] F-G show immunoblotting (F) and corresponding quantitative (G) analysis for detecting the expression of autophagy substrates in HepG2 cells after treatment with different concentrations of ATT for 24 hours in the presence of PA (400 μM). Three experiments were performed.

[0082] Representative confocal images showing HepG2 cells stained with Nile Red dye (0.5 μg / mL) for 15 minutes and imaged using a fluorescence microscope. Nile Red dye rapidly accumulates in lipid droplets.

[0083] I shows the quantification results of the number of lipid droplets. After exposure to palmitic acid, ATT treatment significantly reduced the lipid droplet density in HepG2 cells. Addition of ATT in the control group did not reduce lipid droplets (n = 20 cells were analyzed under each condition). HepG2 cells after 24 hours of exposure to different treatments. PA, 400 μM; ATT, 250 μg / mL. Quantitative analysis was performed using Image J and Graph Pad. NS: No significant difference; **p < 0.01, ***p < 0.001.

[0084] As Figure 6 As shown in B and C, it was observed by confocal laser scanning microscopy (CLSM) that compared with the cells treated with BSA, the fluorescence intensity of the cells treated with PA was significantly reduced, indicating a decrease in lysosomal acidity. However, after adding ATT, the intracellular fluorescence intensity was restored, indicating that these nanoparticles effectively counteracted the lysosomal acidity decrease caused by PA.

[0085] The expression levels of autophagy substrate proteins in HepG2 cells were measured using western blot analysis, specifically targeting LC3-II and p62 ( Figure 6 D). The results showed that PA treatment significantly inhibited autophagic flux in HepG2 cells, resulting in blocked degradation of autophagy substrate proteins and accumulation of these proteins ( Figure 6 E). In contrast, addition of ATT promoted the recovery of autophagic flux and enhanced the degradation of autophagy substrate proteins ( Figure 6 D and E), and this effect was dose-dependent ( Figure 6 F and G). The above results further demonstrated that ATT could effectively reverse the autophagic flux inhibition caused by PA by restoring lysosomal acidification.

[0086] Using CLSM for observation, we found that after PA exposure, the number of lipid droplets in HepG2 cells increased significantly. In contrast, HepG2 cells treated with ATT showed a significant reduction in lipid droplet accumulation ( Figure 6 H and I). These results indicate that ATT can reverse the lysosomal acidity decrease caused by lipotoxicity, restore normal autophagic flux, and effectively reduce lipid accumulation.

[0087] Example 3 Effect of ATT on CQ-induced cytotoxicity

[0088] Chloroquine treatment may lead to lysosomal alkalinization, which subsequently increases lysosomal membrane permeability (LMP) and induces cytotoxic effects. Here, we also verified the cytotoxic potential of chloroquine. Experimental data showed that in HeLa cells, 50μM chloroquine caused only mild cytotoxicity, while 100μM treatment led to significant cytotoxic effects. Based on these findings, a concentration of 100μM was selected for subsequent evaluation of the potential cytoprotective properties of ATT. In addition, we also investigated the potential of ATT to restore cell viability in different cell types, and the results are shown in Figure 7 and Figure 8 .

[0089] like Figure 7 As shown, regardless of concentration, ATT alone had minimal effect on the viability of the above cell lines. However, when various concentrations of ATT were co-incubated with CQ for 24 hours, a reduction in CQ-induced HeLa cytotoxicity was observed. Subsequently, we evaluated the effect of ATT on the viability of EA.hy926 and HaCaT cells in the presence of CQ. The results showed that ATT could alleviate the accumulation of the autophagy substrate protein LC3-II and the cytotoxicity caused by CQ in EA.hy926 and HaCaT cells ( Figure 8 AD).

[0090] Example 4 ATT alleviates the condition of NAFLD mice

[0091] C57BL / 6 mice were treated with HFD for 16 weeks to establish an HFD-induced NAFLD mouse model. The in vivo therapeutic potential of ATT was then investigated based on the model. Figure 9 . Among them, A is the animal experiment schedule, and the mice were fed a normal diet (ND) or HFD for 16 weeks. Then, every other day, different groups of mice were injected with ATT (10 mg / kg) or PBS through the tail vein four times. B, C are the body weight and liver weight of mice under different treatments. D, E are the serum AST levels and serum ALT levels of mice under different treatments. F, G are oral glucose tolerance tests (OGTT) performed on mice that had fasted overnight for 12 hours. After measuring the blood glucose level after fasting, each mouse was given 2 g / kg body weight of glucose by intragastrically administering. Blood glucose levels were detected from the tail vein after 15, 30, 60, 90 and 120 minutes, respectively. H is Oil Red O staining and H&E staining of mouse liver. n≥3; NS: no significant difference; *p<0.05, **p<0.01, ***p<0.001.

[0092] like Figure 9As shown in Figures B and C, intravenous injection of ATT significantly reduced the weight gain and liver weight increase in HFD-fed mice. In addition, we evaluated the alanine aminotransferase (ALT) level and aspartate aminotransferase (AST) level in the liver tissues of HFD-fed mice ( Figure 9 Figures D and E), and found that ATT treatment significantly improved the ALT and AST levels, approaching the normal physiological range. These results indicate that ATT is effective in reducing mouse body weight and liver weight, and can normalize liver function indicators such as ALT and AST.

[0093] As Figure 9 shown in Figure F, by performing an oral glucose tolerance test (OGTT), it was confirmed that after treatment with ATT, the glucose tolerance of mice receiving HFD was enhanced, and their blood glucose levels decreased to levels comparable to those of normal mice. To further clarify the changes in the baseline fasting blood glucose levels among different treatment groups, we calculated the area under the OGTT curve. Compared with the HFD control group, administration of ATT significantly reduced the total blood glucose fluctuations ( Figure 9 Figure G), indicating a significant improvement in glucose responsiveness.

[0094] As Figure 9 shown in Figure H, significant lipid accumulation occurred in the livers of mice fed with HFD. In contrast, after treatment with ATT, the lipid accumulation in the liver tissues of these mice was significantly reduced. In addition, we also evaluated the degree of hepatic steatosis by hematoxylin and eosin staining (H&E). In the HFD-fed control group, liver tissue sections showed obvious regions of hepatocyte hypertrophy, indicating severe hepatic steatosis. However, after treatment with ATT, this condition was reversed ( Figure 9 Figure H).

[0095] Based on the above detections, the present invention found that ATT can effectively regulate the cytotoxicity of chloroquine, restore lysosomal acidity and autophagic flux. In addition, ATT shows significant therapeutic effects on lysosomal dysfunction-related diseases such as NAFLD, can restore lysosomal acidity and autophagic flux in the NAFLD model, and effectively reduce lipid accumulation.

[0096] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. Use of attapulgite in restoring lysosomal acidity and autophagic flux.

2. Use of attapulgite in the preparation of a preparation for restoring lysosomal acidity and autophagic flux.

3. The use according to claim 1 or 2, characterized in that, Attapulgite can restore the decrease in lysosomal acidity caused by lipotoxicity and the autophagic flux blocked thereby.

4. The use according to claim 1 or 2, characterized in that, Attapulgite can restore the decrease in lysosomal acidity caused by chloroquine and the autophagic flux blocked thereby.

5. The use according to claim 4, characterized in that, Attapulgite can also inhibit the cytotoxicity induced by chloroquine.

6. The use according to claim 1 or 2, characterized in that, Attapulgite can restore the autophagic flux blocked by lysosomal inhibitors.

7. A preparation for restoring lysosomal acidity and autophagic flux, characterized in that, Comprising the attapulgite described in claim 1, and a pharmaceutically acceptable carrier.

8. A preparation for inhibiting the cytotoxicity of chloroquine, characterized in that, Comprising the attapulgite described in claim 1, and a pharmaceutically acceptable carrier.

9. Use of attapulgite or the preparation described in claim 7 in the preparation of a drug for treating diseases related to lysosomal decline and / or autophagy inhibition.

10. The use according to claim 9, characterized in that, The diseases include obesity, metabolic syndrome, diabetes, non-alcoholic fatty liver disease and neurodegenerative diseases caused by lipid accumulation.

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