Application of inhibitor of cathepsin D or coding gene thereof in preparation of product for preventing and / or treating type 2 diabetes

Abstract

The invention provides application of an inhibitor of cathepsin D or a coding gene thereof in preparation of a product for preventing and / or treating type 2 diabetes mellitus. The inhibitor realizes prevention and / or treatment of the type 2 diabetes mellitus by inhibiting expression of the cathepsin D or the coding gene thereof or inhibiting activity of the cathepsin D; applications are achieved by administering an inhibitor of cathepsin D expression, an inhibitor of cathepsin D coding gene expression, or an inhibitor of cathepsin D activity. The inhibitor inhibits the activation of a JAK2-STAT3 signal channel by inhibiting the expression of cathepsin D or a coding gene thereof, or inhibiting the activity of cathepsin D, so as to prevent and / or treat type 2 diabetes mellitus. The cathepsin D disclosed by the invention can be used as a key molecular target for intervening liver insulin resistance for the first time, glycolipid metabolism disorder of a high-fat induced mouse model is remarkably improved by specifically inhibiting the activity of the cathepsin D, and a new strategy is provided for treating liver insulin resistance related diseases T2DM.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to new uses of cathepsin D (CTSD), specifically, to the use of cathepsin D (CTSD) and its inhibitors in the preparation of products for the prevention and / or treatment of type 2 diabetes mellitus (T2DM). Background Technology

[0002] Diabetes is a major global public health problem, affecting more than 425 million people. Type 2 diabetes, accounting for 90% of cases, is characterized by insulin resistance—that is, insufficient cellular response to insulin, leading to impaired glucose uptake and utilization. Although some metabolic mechanisms are known, the complete metabolic profile of insulin resistance and the reasons for individual susceptibility differences remain unclear. A deeper understanding of its metabolic mechanisms is crucial for identifying high-risk groups for type 2 diabetes.

[0003] A key mechanism in the development of insulin resistance is lipotoxicity—the harmful effects of lipid accumulation in non-adipose tissues. Lysosomes, as crucial organelles, are responsible for lipid degradation. Under lipotoxic conditions (such as increased intracellular lipid influx), lysosomal function is impaired. In fact, lipid-induced lysosomal membrane damage can lead to the abnormal release of lysosomal enzymes into the cytoplasm and / or extracellular environment.

[0004] Cathepsin D (CTSD) is a widely distributed lysosomal aspartic protease. The liver is rich in CTSD, having one of the highest protein levels among organs, and also possesses the largest population of macrophages containing high levels of lysosomal enzymes, including CTSD. Although previous studies have found a positive correlation between plasma CTSD activity and glucose metabolism parameters in patients with type 2 diabetes, its relationship with hepatic insulin sensitivity remains unknown.

[0005] Currently, there are no studies on the application of cathepsin D or its encoding gene inhibitors in the preparation of products for the prevention and / or treatment of type 2 diabetes. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide an application of cathepsin D (CTSD) or an inhibitor of its encoding gene in the preparation of products for the prevention and / or treatment of type 2 diabetes mellitus (T2DM).

[0007] The present invention achieves the above-mentioned technical objectives through the following technical means.

[0008] The use of an inhibitor of cathepsin D or its encoding gene in the preparation of products for the prevention and / or treatment of type 2 diabetes.

[0009] In the above scheme, the inhibitor achieves prevention and / or treatment of type 2 diabetes by inhibiting the expression of cathepsin D or its encoding gene, or by inhibiting the activity of cathepsin D.

[0010] In the above scheme, the cathepsin D or its encoding gene is derived from mammals. Preferably, it is derived from humans, mice, rats, or rabbits; more preferably, it is derived from humans.

[0011] In the above scheme, the amino acid sequence of cathepsin D is shown in SEQ ID NO.1.

[0012] In the above scheme, the application is achieved by administering an inhibitor of cathepsin D expression, an inhibitor of the expression of the cathepsin D encoding gene, or an inhibitor of cathepsin D activity.

[0013] Preferably, the inhibitors of cathepsin D or its encoding gene include: inhibitors of cathepsin D activity and inhibitors of the expression of cathepsin D or its encoding gene.

[0014] Furthermore, the inhibitor of cathepsin D activity includes a small molecule inhibitor or a cathepsin D antibody; preferably, the small molecule inhibitor includes Pepstatin A.

[0015] Preferably, the Pepstatin A is purchased from Sigma-Aldrich, with product number P-5138.

[0016] Furthermore, the inhibitor of cathepsin D or its encoding gene expression is an antisense RNA, siRNA, shRNA, or miRNA that inhibits the expression of cathepsin D or its encoding gene.

[0017] Furthermore, the target sequence of the shRNA is shown in SEQ ID NO.2. The oligonucleotide sequence of the shRNA is shown in SEQ ID NO.3.

[0018] Furthermore, the shRNA is PL-Ctsd-RNAi.

[0019] Preferably, the PL-Ctsd-RNAi is purchased from Jikai Gene, with the catalog number P24L0390.

[0020] Preferably, the product includes a drug.

[0021] In the above scheme, the inhibitor inhibits the expression of cathepsin D or its encoding gene, or inhibits the activity of cathepsin D, thereby inhibiting the activation of the JAK2-STAT3 signaling pathway, thereby preventing and / or treating type 2 diabetes.

[0022] JAK2, or Janus kinase 2, is abbreviated as JAK2. The amino acid sequence of JAK2 is shown in SEQ ID NO. 5.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] This invention reveals for the first time that cathepsin D (CTSD) can serve as a key molecular target for intervening in hepatic insulin resistance. Specific inhibition of CTSD activity significantly improves glucose and lipid metabolism disorders in a high-fat induced mouse model, and elucidates that this effect is closely related to the functional remodeling of the lysosome-insulin signaling axis. This discovery not only provides a new perspective for elucidating the pathological mechanisms of lysosomal dysfunction in the progression of metabolic diseases, but also lays a theoretical foundation for developing multi-organ metabolic homeostasis remodeling strategies based on cathepsin D (CTSD) activity regulation, possessing significant translational value for achieving precise intervention in type 2 diabetes mellitus (T2DM).

[0025] This invention uses RNAi (shRNA) to knock down cathepsin D (CTSD) at the cellular level and uses Western blotting (WB) to detect insulin signaling-related proteins (PS6, PAKT, PGSK-3β). The results show that knocking down CTSD can reverse hyperlipidemia-induced hepatic insulin resistance and alleviate glucose and lipid metabolism disorders.

[0026] This invention discovers that JAK2 can bind to and interact with CTSD, and can be activated by CTSD. To elucidate the mechanism, this invention demonstrates that blocking the JAK2-STAT3 signaling pathway can improve insulin signaling. This discovery reveals the molecular mechanism by which CTSD induces insulin resistance by activating the JAK2-STAT3 pathway, thus confirming that inhibiting CTSD is an effective strategy for treating T2DM, providing a new strategy for the treatment of liver insulin resistance-related diseases (T2DM). Attached Figure Description

[0027] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0028] Figure 1 CTSD expression and activity were increased in T2DM mice; among them, Figure 1(A) Serum CTSD enzyme activity was detected using a fluorescent substrate method to obtain serum samples by blood collection from the posterior orbital venous plexus. Figure 1 (B) Western blot detection of CTSD protein in mouse liver; Figure 1 (C) represents the activity of liver CTSD enzyme.

[0029] Figure 2 Inhibiting CTSD activity improves serum biochemical levels in T2DM mice; among which, Figure 2 (A) is serum TG in T2DM mice; Figure 2 (B) Serum TC in T2DM mice; Figure 2 (C) represents serum AST from T2DM mice; Figure 2 (D) represents serum ALT in T2DM mice; Figure 2 (E) represents serum ALP in T2DM mice.

[0030] Figure 3 Inhibiting CTSD activity improves glucose tolerance and insulin tolerance in T2DM mice; among which, Figure 3 (A) Glucose tolerance test performed during week 4 of PepA treatment; Figure 3 (B) is the area under the curve (AUC), used to quantify the GTT results.

[0031] Figure 4 Inhibiting CTSD activity improved body weight and blood glucose levels in T2DM mice; among which, Figure 4 (A) represents the body weight levels of mice from week 5 to week 8; Figure 4 (B) represents the fasting blood glucose levels of mice from week 5 to week 8.

[0032] Figure 5 Inhibiting CTSD activity improves insulin signaling in T2DM mice; among which, Figure 5 (A) ELISA detection of insulin levels in mice; Figure 5 (B) is the insulin resistance index.

[0033] Figure 6 Inhibition of CTSD activity improves hepatic glucose and lipid metabolism in T2DM mice; among which, Figure 6 (A) H&E, PAS, ORO, and immunohistochemical staining of mouse liver; Figure 6 (B) Detection of G6pc gene in mouse liver; Figure 6 (C) Detection of Pck1 gene in mouse liver.

[0034] Figure 7 High-fat and high-glucose diets induce increased expression of AML-12 and HepG2 CTSD genes; among them, Figure 7(A) qPCR detection of CTSD expression in HepG2 cells under PA conditions; Figure 7 (B) qPCR detection of Ctsd expression in AML-12 cells under PA conditions; Figure 7 (C) qPCR detection of CTSD expression in HepG2 cells under PA+HG conditions; Figure 7 (D) qPCR detection of Ctsd expression in AML-12 cells under PA+HG conditions.

[0035] Figure 8 High-fat and high-glucose diets induce increased expression and activity of CTSD protein in AML-12 and HepG2 cells; among them, Figure 8 (A) Western blot analysis of CTSD expression in HepG2 cells under PA conditions; Figure 8 (B) Western blot detection of CTSD expression in AML-12 cells under PA conditions; Figure 8 (C) Western blot detection of CTSD expression in HepG2 cells under PA+HG conditions; Figure 8 (D) Western blot detection of CTSD expression in AML-12 cells under PA+HG conditions; Figure 8 (E) Gray-scale scanning detection of CTSD protein bands in HepG2 cells under PA conditions; Figure 8 (F) Gray-scale scanning detection of CTSD protein bands in AML-12 cells under PA conditions; Figure 8 (G) PA+HG conditions for grayscale scanning detection of CTSD protein bands in HepG2 cells; Figure 8 (H) PA+HG conditions for grayscale scanning detection of CTSD protein bands in AML-12 cells; Figure 8 (I) CTSD activity of HepG2 cell culture supernatant under PA conditions; Figure 8 (J) CTSD activity of AML-12 cell culture supernatant under PA conditions; Figure 8 CTSD activity of AML-12 cell culture supernatant under (K)PA+HG conditions.

[0036] Figure 9 High-fat and high-glucose diets induce increased CTSD protein levels in AML-12 and HepG2 cells; among them, Figure 9 (A) IF detection of CTSD expression in HepG2 cells under PA conditions; Figure 9 (B) IF detection of CTSD expression in AML-12 cells under PA conditions; Figure 9 (C) IF detection of CTSD expression in HepG2 cells under PA+HG conditions; Figure 9 (D) IF detection of CTSD expression in AML-12 cells under PA+HG conditions.

[0037] Figure 10 Inhibition of cathepsin D activity improves insulin signaling in AML-12 cells; among which, Figure 10 (A) Western blot analysis of β-actin, CTSD, p-S6, p-GSK-3β, T-GSK-3β, p-AKT, and T-AKT levels in AML-12 cells; Figure 10 (B) represents the normalization of p-GSK-3β levels and T-GSK-3β levels; Figure 10 (C) represents the normalization of p-AKT and T-AKT levels.

[0038] Figure 11 Inhibition of cathepsin D activity improves insulin signaling in HepG2 cells; among which, Figure 11 (A) Western blot analysis of β-actin, CTSD, p-S6, p-GSK-3β, T-GSK-3β, p-AKT, and T-AKT levels in HepG2 cells; Figure 11 (B) represents the normalization of p-GSK-3β levels and T-GSK-3β levels; Figure 11 (C) represents the normalization of p-AKT and T-AKT levels.

[0039] Figure 12 : An investigation into the mechanism by which CTSD damages hepatic insulin signaling; among which, Figure 12 (A) Control vs recombinant CTSD protein treatment group: differentially expressed genes, |LOGFC|>1, P-value<0.05 (TOP20 markers); Figure 12 (B) Control vs. recombinant CTSD protein treatment group: KEGG enrichment analysis P-value < 0.05 (TOP20 Findings); Figure 12 (C) Insulin resistance induction group vs. PepA intervention group: GO analysis; Figure 12 (D) KEGG enrichment analysis of insulin resistance induction group vs. PepA intervention group.

[0040] Figure 13 CTSD and JAK2 have protein-level interactions; among them, Figure 13 (A) Predicts the binding of JAK2 to CTSD using molecular docking; Figure 13 (B) To verify the binding of JAK2 to CTSD in 293T cells (overexpressing CTSD) for CO-IP; Figure 13(C) Validation of the binding of JAK2 to CTSD in 293T cells (treated with 200 μM PA for 24 h) using fluorescence colocalization. Scale bar, 50 μm.

[0041] Figure 14 High-fat or high-fat-high-glucose diets induce CTSD and increase p-JAK2, while decreasing p-AKT; among them, Figure 14 (A) PA induces an increase in CTSD and a decrease in p-AKT in AML-12 cells; Figure 14 (B) PA induces an increase in CTSD and p-JAK2 in AML-12 cells; Figure 14 (C) PA+HG induces an increase in CTSD and p-JAK2 and a decrease in p-AKT in AML-12 cells.

[0042] Figure 15 CTSD affects the activation of JAK2 and AKT; among them, Figure 15 (A) Western Blot analysis of the expression levels of p-JAK2, T-JAK2, p-STAT3, STAT3, p-AKT, and T-AKT after CTSD overexpression; Figure 15 (B) Western Blot analysis of the expression levels of p-JAK2, T-JAK2, p-STAT3, STAT3, p-AKT, and T-AKT after CTSD knockdown.

[0043] Figure 16 Phosphorylation of JAK2 affects the phosphorylation levels of AKT and STAT3. Detailed Implementation

[0044] This invention provides the use of cathepsin D (CTSD) inhibitors in products for the prevention and / or treatment of type 2 diabetes mellitus (T2DM). The invention is described in detail below with reference to specific examples to facilitate further understanding by those skilled in the art. However, the examples described below are only a part of the embodiments of this invention and should not be considered as any form of limitation on this invention. It should be noted that adjustments and improvements made by those skilled in the art based on the concept of this invention should be considered within the scope of protection of this invention. Specific technical operation steps and operators not specified in the examples are all performed according to the general technical conditions described in the literature or relevant product instructions. Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the reagents and materials used in the following examples are commercially available unless otherwise specified.

[0045] Example 1: Construction of a Type 2DM animal model and evaluation of the therapeutic effect of CTSD inhibitors

[0046] 1 Experimental Methods

[0047] 1.1 T2DM mouse model

[0048] Six- to seven-week-old male C57BL / 6 mice (purchased from the Experimental Animal Research Center of Jiangsu University, housed in an SPF-grade environment with conditions of 22°C, 45-55% humidity, and a 12-hour light / 12-hour dark cycle. This experiment was approved by the Animal Ethics Committee of Jiangsu University, approval number: UJS-IACUC-AP-2023022702, and followed relevant animal use guidelines) were randomly divided into a Control group, a T2DM group, and a T2DM+PepA (Pepstatin A) group. After one week of acclimatization, the Control group maintained a normal diet, while the T2DM and T2DM+PepA groups were given a high-fat diet (45% fat for energy) for four weeks. At the end of the fourth week, all mice were administered STZ intraperitoneally (50 mg / kg) under a fasting state. Fasting blood glucose was measured 24 hours after injection to assess the effectiveness of the model construction. From the fifth week onwards, the T2DM+PepA group received weekly intraperitoneal injections of PepA (50 mg / kg), while the Control group received an equal volume of physiological saline. Before the experimental endpoint, glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were completed. The following day, blood samples were collected from the eye, heart, liver, spleen, lungs, kidneys, fat, muscle, pancreas, colon, and feces. Serum was centrifuged and liver function indicators were measured using a Mindray BS-200 fully automated biochemical analyzer. Liver tissue was stained with H&E, PAS, ORO, and CTSD immunohistochemically. The mice's mental state and behavioral activity were observed daily.

[0049] 1.2 Detection of GTT and ITT in mice

[0050] GTT: After fasting for 12 h (with free access to water), fasting blood glucose (0 min baseline value) was measured using a portable blood glucose meter in each group of mice. 50% D-glucose solution (2 g / kg) was injected intraperitoneally (ip). Tail vein blood was collected at 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min after administration. Blood glucose changes were dynamically monitored using a glucose oxidase test strip method (commercial blood glucose test strips were used).

[0051] ITT: Mice were fasted for 6 h (with free access to water) and then injected intraperitoneally with recombinant human insulin (2 IU / kg) (Beyotime, Shanghai, China, #P3376-100 IU). Blood glucose levels in the tail vein were measured before injection (0 min) and at 15 min, 30 min, 60 min, 90 min, and 120 min after injection.

[0052] 1.3 HOMA-IR Calculation

[0053] Calculated based on the last fasting blood glucose and insulin levels:

[0054] HOMA-IR = Fasting blood glucose × Fasting insulin / 22.5

[0055] 1.4 Detection of CTSD activity in mouse serum and liver

[0056] The cathepsin D activity assay kit (Abcam, Cambridge, UK, #ab65302) was used to detect CTSD enzyme activity in mice and liver.

[0057] (1) Reagent preparation: Thaw the reagents from -20 ℃ to -4 ℃ in advance, and bring the reagents to room temperature 30 min before the experiment; buffer solution; specific substrate: detach briefly before use.

[0058] (2) Sample preparation: Mouse ocular serum was taken out from the -80°C freezer in advance, thawed on ice and diluted 10 times; Mouse liver protein extraction: 50 mg sample + 500 μL RIPA + 5 μL PMSF was ground until there were no large tissue blocks visible to the naked eye, and lysed on ice for 15 min; centrifuged at 4 ℃, 14000 rpm / min for 15 min, and the supernatant was collected; the protein concentration was measured by BCA method, and the protein supernatant was diluted and adjusted to the same concentration for later use; the buffer and substrate were mixed at a ratio of 50 μL + 2 μL per well, and 10% more was prepared.

[0059] (3) Sample addition: Add 5 μL of 1×PBS + 52 μL of detection buffer to the blank well; add 5 μL of diluted sample + detection buffer to the sample well.

[0060] (4) Incubation: Incubate at 37 ℃ in the dark for 2 h.

[0061] (5) Detection reading: Read the data on the microplate reader within 30 min (Ex / Em = 328 / 460 nm).

[0062] 1.5 Western Blot Detection of Liver CTSD Protein

[0063] BCA method (Sangon Biotech, Shanghai, China, # C503021) for protein concentration determination:

[0064] (1) Reagent preparation: The reagents were brought to room temperature in advance from -20 ℃ and -4 ℃ to allow them to recover to room temperature; the total working solution was prepared according to 200 μL of working solution per well (A solution: B solution = 50: 1), and 10% more was prepared.

[0065] (2) Sample preparation: The protein supernatant obtained in the method of 2.2.4 was diluted 10 times with PBS for later use; the standard solution was diluted to a gradient concentration (0 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml).

[0066] (3) Adding samples: Add 20 μL of standard solution + 200 μL of working solution to the standard sample; add 20 μL of diluted sample solution + 200 μL of working solution to the sample well.

[0067] (4) Incubation: Incubate at 37 ℃ in the dark for 30 min.

[0068] (5) Reading: After cooling to room temperature, measure well A on the microplate reader. 562 The value of is determined, and a standard curve is plotted. The results are then calculated.

[0069] Western blot detection of CTSD protein in the liver:

[0070] (1) Prepare protein samples according to the BCA results (calculated sample volume + 20 μL 5×Loading Buffer + 1×Loading Buffer to make up to 100 μL).

[0071] (2) Sample loading: Add 10 μL of sample to the sample loading well of the pre-cast gel (FuturePAGE™ 4-12% 15 Wells); and apply 5 μL of pre-stained protein marker (NCM, Suzhou, China, # P9006) to both sides of the sample.

[0072] (3) Electrophoresis: Constant voltage electrophoresis (80 V for 20 min, 135 V for 45 min) was performed in electrophoresis buffer (MOPS-SDS Running Buffer).

[0073] (4) Transfer: Cut the PVDF membrane according to the position and size of the protein on the gel, pre-wet it in methanol for 30 s, and then equilibrate it in 1× rapid transfer solution; assemble the wet transfer "sandwich" (foam pad + filter paper + gel + membrane), clamp it tightly, and then immerse it in the transfer tank containing 1× rapid transfer solution, and perform constant flow (400mA, 40 min) transfer at room temperature.

[0074] (5) Blocking: Take out the PVDF membrane from (4), cut the membrane according to the location of the protein and refer to the marker, and then immerse the membrane in 5% skim milk powder (1.25 g dissolved in 25 mL 1×TBST), and incubate on a shaker (room temperature, medium speed) for 2 h. After the incubation, rinse with 1×TBST to remove excess milk powder.

[0075] (6) Primary antibody binding: Place the membrane in a sufficient amount of primary antibody solution (anti-CTSD antibody (Abcam, #ab75852) diluted at 1:2000, anti-β-Actin antibody (Proteintech, #66009-1-Ig) diluted at 1:20000 in universal antibody diluent, antibody information is detailed in Table 4), ensuring that the protein surface on the membrane can fully contact the antibody, and incubate overnight at 4 ℃ on a shaker (medium speed).

[0076] (7) Washing the membrane: Use a 1×TBST shaker to wash the membrane 4 times at medium speed, 5 min each time.

[0077] (8) Secondary antibody binding: Place the membrane in a sufficient amount of HRP secondary antibody (HRP Goat Anti-Rabbit IgG (H+L);

[0078] In HRP Goat Anti-Mouse IgG (H+L), ensure the protein side of the membrane is in complete contact with the antibody, and incubate at room temperature on a shaker (medium speed) for 1 h. (Antibodies used in the experiment are listed in Table 4.)

[0079] (9) Washing the membrane: Use a 1×TBST shaker to wash the membrane 4 times at medium speed, 5 min each time.

[0080] (10) Exposure: Prepare a sufficient amount of working solution of chemiluminescent substrate; place the protein-binding side of the membrane facing up, add the working solution of substrate, and use an imaging system to perform blotting imaging.

[0081] 1.6 Detection of Insulin in Mouse Serum

[0082] (1) Preparation of reagents (BYabscience, Nanjing, China, #BY-EM220778): The reagents and strips were brought from -4 ℃ to room temperature in advance to allow them to recover to room temperature; the concentrated washing solution was diluted with distilled water at a ratio of 1:20 to prepare the working washing solution; substrate A and substrate B were mixed at a ratio of 1:1 and used within 15 min (prepare the solution when needed).

[0083] (2) Sample preparation: The serum sample was thawed on ice in advance and diluted.

[0084] (3) Sample addition: Add 50 μL of different concentrations of standard to the standard wells, add 50 μL of diluted sample to the sample wells; add 50 μL of sample diluent to the 0 value wells, and do not add to the blank wells; except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to the standard wells, 0 value wells and sample wells.

[0085] (4) Incubation: Seal the reaction plate with a sealing film and incubate at 37 °C in the dark for 60 min.

[0086] (5) Washing: Open the sealing film, discard the liquid, pat dry with absorbent paper, fill each well with washing liquid, let stand for 20 seconds, shake off the washing liquid, pat dry with absorbent paper, and repeat 5 times.

[0087] (6) Adding substrate: Add 100 μL of the pre-mixed substrate mixture to all wells, seal the reaction plate with the sealing film, and incubate at 37 °C in the dark for 15 min.

[0088] (7) Reading: Add 50 μL of stop solution to all wells and measure the A value of each well on a microplate reader. 450 The value of is determined, and a standard curve is plotted. The results are then calculated.

[0089] 1.7 HE staining of mouse liver

[0090] (1) Fixation: After heart perfusion with PBS, mouse livers were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for H&E staining.

[0091] (2) Dewaxing: The slices are laid flat in a 70 ℃ oven and heated for 30 min.

[0092] (3) Hydration: The wax was washed with xylene (2×20 min) in sequence, followed by xylene removal with anhydrous ethanol (2×10 min), then hydration with gradient ethanol (95%, 85%, 70%) for 10 min each, followed by PBS rinsing for 5 min.

[0093] (4) Nuclear staining: stain with hematoxylin and eosin for 5 min → rinse with running water for 5 min → differentiate with 1% hydrochloric acid and ethanol for 30 s → return to blue with running water.

[0094] (5) Staining: Counterstain with eosin solution for 1-3 min → remove excess stain with running water.

[0095] (6) Dehydration: rapid dehydration with gradient ethanol (70%, 85%, 95%, 100%) for 10 s each → xylene clearing for 10 min → air drying.

[0096] (7) Mounting: Seal with neutral resin and observe under a microscope or store the sample at 4 ℃ in the dark.

[0097] 1.8 Mouse liver glycogen staining

[0098] (1) Preparation of reagents (Beyotime, Shanghai, China, # C0142S): periodic acid solution, Schiff reagent, sodium bisulfite solution, hematoxylin stain (for counterstaining).

[0099] (2) Slice preparation: Cut the embedded tissue into thin slices (about 4-6 μm), place them on a glass slide, and bake at 60 ℃ for 1-2 hours.

[0100] (3) Dewaxing and hydration: The sections were placed in xylene I and II for 10 minutes each, then subjected to a gradient of ethanol (100%, 95%, 80%, 70%) for 1-2 minutes each, and finally rinsed with distilled water.

[0101] (4) Oxidation: Soak in periodic acid solution for 5–10 min → rinse with distilled water.

[0102] (5) Staining: Immerse with Schiff's reagent for 10–30 min (the polysaccharide structure turns purple-red).

[0103] (6) Differentiation and rinsing: Rinse the sections three times with sodium bisulfite solution for 1-2 minutes each time to remove non-specific staining.

[0104] (7) Counterstaining: Counterstain the cell nuclei with hematoxylin solution for 2-5 min until the cell nuclei turn blue.

[0105] (8) Dehydration, clearing and sealing: Gradient ethanol dehydration → xylene clearing → neutral resin sealing.

[0106] 1.9 Oil Red Staining of Mouse Liver

[0107] (1) Fixation: The sections were fixed in 4% paraformaldehyde for 10 min and then rinsed slightly with distilled water.

[0108] (2) Immersion: Immerse the slices in 60% isopropanol for 20 seconds.

[0109] (3) Staining: Immerse the sections in Oil Red O staining solution for 10 min.

[0110] (4) Color separation: Wash away excess dye with 60% isopropanol and rinse with distilled water.

[0111] (5) Counterstaining cell nuclei: Counterstain in hematoxylin for 2 min, then rinse with distilled water.

[0112] (6) Mounting: Glycerin gelatin mounting tablets are used for mounting.

[0113] 1.10 Histochemical staining of mouse liver

[0114] (1) Dewaxing and hydration: Before dewaxing, the paraffin slices were first baked in a 60 ℃ oven for 60 min, soaked in xylene for 15 min, and then soaked for 15 min after replacing the xylene; then they were subjected to a gradient of ethanol (100%, 95%, 75%) for 5 min each.

[0115] (2) Antigen retrieval: After microwaving sodium citrate buffer (pH 6.0), place the slice in the solution and let it stand for 1-2 cycles (5-10 minutes apart).

[0116] (3) Permeabilization: After natural cooling, the sections were soaked in PBS for 5 min, then in 0.5% Triton X-100 at room temperature for 10 min, and washed with PBS 2-3 times for 5 min each time.

[0117] (4) Blocking: 5% BSA for 1 h.

[0118] (5) Incubation of primary antibody: Dilute with antibody diluent, incubate overnight at 4 ℃, and rewarm at 37 ℃ for 1 h. Wash 3 times with PBS, 5 min each time.

[0119] (6) Incubation of secondary antibody: Dilute with antibody diluent and incubate at 37 °C for 45 min. Wash 3 times with PBS, 5 min each time.

[0120] (7) DAB staining: Add DAB staining solution, let stand for 2 minutes, and check the staining degree under a microscope. Rinse with running tap water for 3 minutes.

[0121] (8) Counterstaining: Hematoxylin counterstaining for 2 min, then rinse with running tap water for 5 min.

[0122] (9) Dehydration and clearing: After 2 min each of gradient ethanol (75%, 95%, 100%), xylene was used to clear the product for 2 min.

[0123] (10) Mounting: Seal with neutral resin, air dry in a fume hood, and examine under a microscope.

[0124] 1.11 Cellular Insulin Resistance Model

[0125] AML-12 and HepG2 cells were treated with 200 μM palmitic acid (PA) or 200 μM PA combined with 30 mM glucose for 24 h to induce lipid accumulation. To activate the insulin signaling pathway, hepatogenic cells were stimulated with 100 nM insulin within 30 min before the treatment was terminated.

[0126] 1.12 Cell Culture

[0127] AML-12 mouse liver cells (cultured in DMEM / F12 medium containing 10% FBS in a 37 ℃ CO2 incubator, with 1% ITS and 40 ng / ml dexamethasone added to the medium) and HepG2 human liver cancer cells (cultured in DMEM high-glucose medium containing 10% FBS in a 37 ℃ CO2 incubator).

[0128] 1.13 Cell resuscitation and culture

[0129] Cell resuscitation procedure:

[0130] (1) Equipment pre-balancing: Preheat in a 37 ℃ constant temperature water bath for 30 min.

[0131] (2) Environmental sterilization: The biosafety cabinet is irradiated with ultraviolet light for 30 min, and 75% ethanol is sprayed and ventilated for 15 min before operation.

[0132] (3) Reagent preparation: Preheat the complete culture medium to 37 ℃ (containing double antibiotics and 10% FBS).

[0133] (4) Thawing: Quickly remove the cryovial from -80 ℃ and immediately immerse it in a 37 ℃ water bath. Shake it horizontally continuously until the contents are about to turn into liquid.

[0134] (5) After sterilizing the surface of the cryovials with ethanol, transfer them to a biosafety cabinet. Slowly inject the suspension into a 15 mL centrifuge tube, add pre-warmed culture medium at a ratio of 1:3, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of complete culture medium, gently pipette to mix, and then transfer to a 60 mm culture dish. Add another 3-5 mL of complete culture medium, mix using the cross-mixing method, and transfer to a cell culture incubator. Observe the cell status one day later.

[0135] Cellular digestion:

[0136] (1) Reagent rewarming: Place PBS, trypsin digestion solution and complete culture medium in a 37 ℃ water bath for equilibration for 15 min.

[0137] (2) Environmental sterilization: Turn on the biosafety cabinet for 30 minutes in advance, ventilate for 10 minutes before operation and spray with 75% ethanol for disinfection.

[0138] (3) Aseptic transfer: After wiping the outer wall of the culture dish with alcohol, quickly transfer it into the biosafety cabinet.

[0139] (4) Discard the old culture medium (avoid touching the cell layer), slowly inject 2 mL of PBS (pre-warmed to 37℃) along the wall of the dish, gently shake the culture dish twice using the cross-shaking method, add 0.5-1 mL of trypsin, digest at 37℃ for 90-120 s, confirm under the microscope that more than 80% of the cells show edge shrinkage and cell body retraction characteristics, quickly inject 2 mL of stop solution (volume ratio greater than or equal to 1:3), use a pipette to gently pipette 8-10 times and then transfer to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add 1 mL of complete culture medium, gently pipette to mix, and then proceed to the next step.

[0140] Cell count:

[0141] After digestion, take approximately 10 µL of cell suspension (if there are many cells, dilute appropriately in an EP tube) and drop it into a prepared counting chamber (covered with a coverslip). Use a 10× objective to locate the central 25-square area, and a 40× objective to confirm the morphology of individual cells. Count the cells according to a specific rule (cells with double-line contact: count the left / top boundary, exclude the right / bottom boundary). Finally, calculate: Cell density = (Total number of cells in the four large squares / 4) × 10⁻⁶ 4 ×Dilution factor / mL

[0142] Cell passage:

[0143] Microscopic examination requirements: Cells can be passaged when the confluence reaches 80-90%. After digestion, aspirate culture medium at an appropriate ratio, seed into new culture dishes, and add an appropriate amount of complete culture medium. Transfer to a cell culture incubator and observe the cell status after one day.

[0144] Cell cryopreservation:

[0145] Cells were cryopreserved when they reached passage 4-5 and entered the logarithmic growth phase (confluence 80%-90%). The cells were digested and centrifuged to obtain a precipitate. The cells were then gently resuspended in cryopreservation buffer containing 10% DMSO (freshly prepared with 900 μL fetal bovine serum and 100 μL DMSO; serum-free cryopreservation buffer can also be used). The cells were aliquoted into cryovials (1 mL / tube) and labeled with the cell line name and passage number. The cryovials were placed in a programmed cooling box and cooled at a gradient rate of -1 °C / min before being transferred to a -80 °C freezer for temporary storage (preferably, long-term storage in liquid nitrogen within 24 hours).

[0146] 1.14 Protein extraction and Western blot technology

[0147] Extracting cell proteins:

[0148] (1) Reagent preparation: Ice up in advance, place all reagents and consumables such as EP tubes on ice, and prepare RIPA lysis buffer (containing 1% PMSF, prepare fresh and use immediately) and label it.

[0149] (2) Cell lysis: Remove cells from the CO2 incubator, discard the cell culture supernatant, wash twice with ice-cold PBS, add the appropriate RIPA lysis buffer to each well to lyse the cells (e.g., 150 μL for a 6-well plate), lyse on ice for 5 min, scrape off cells with a cell scraper or pipette tip, and collect them into EP tubes as soon as possible. Maintain low temperature throughout the process. Shake the EP tubes on a shaker (3 times, 1 min each time).

[0150] (3) Centrifugation: Centrifuge at 14000 rpm / min for 15 min, take 80 µL of supernatant and add 20 µL of 5× Loading Buffer.

[0151] (4) Denaturation: Boil in a water bath at 100 °C for 10 min, cool to room temperature, and store at -20 °C. (Alternatively, protein concentration can be determined by the BCA method first, and then the mixture can be prepared and boiled.)

[0152] (5) Sample loading: Add 10 μL of sample to the sample loading well of the pre-cast gel (FuturePAGE™ 4-12% 15 Wells); and apply 5 μL of pre-stained protein marker to both sides of the sample (Sunbio Biotechnology, Suzhou, China, #P9006).

[0153] (6) Electrophoresis: Perform constant voltage electrophoresis in electrophoresis buffer (MOPS-SDS Running Buffer) (80 V for 20 min, 135 V for 45 min).

[0154] (7) Transfer: Cut the PVDF membrane according to the position and size of the protein on the gel, pre-wet it in methanol for 30 s, and then equilibrate it in 1× rapid transfer solution; assemble the wet transfer "sandwich" (foam pad + filter paper + gel + membrane), clamp it tightly, and then immerse it in the transfer tank containing 1× rapid transfer solution, and perform constant current (400mA, 40 min) transfer at room temperature.

[0155] (8) Blocking: Take out the PVDF membrane from (4), cut the membrane according to the location of the protein and refer to the marker, and then immerse the membrane in 5% skim milk powder (1.25 g dissolved in 25 mL 1×TBST), and incubate on a shaker (room temperature, medium speed) for 2 h. After the incubation, rinse with 1×TBST to remove excess milk powder.

[0156] (9) Primary antibody binding: Place the membrane in a sufficient amount of primary antibody solution (anti-CTSD antibody diluted at 1:2000 and anti-β-Actin antibody diluted at 1:20000 in universal antibody diluent), ensuring that the protein surface on the membrane can fully contact the antibody, and incubate overnight at 4 ℃ on a shaker (medium speed).

[0157] (10) Washing the membrane: Use a 1×TBST shaker to wash the membrane 4 times at medium speed, 5 min each time.

[0158] (11) Secondary antibody binding: Place the membrane in a sufficient amount of HRP secondary antibody, ensuring that the protein surface on the membrane can fully contact the antibody, and incubate at room temperature on a shaker (medium speed) for 1 h.

[0159] (12) Washing the membrane: Use a 1×TBST shaker to wash the membrane 4 times at medium speed, 5 min each time.

[0160] (13) Exposure: Prepare a sufficient amount of working solution of chemiluminescent substrate; place the protein-binding side of the membrane facing up, add the working solution of substrate, and use an imaging system to perform blotting imaging.

[0161] 1.15 RNA extraction and qPCR technology

[0162] Cellular RNA extraction:

[0163] (1) Reagent preparation: Pre-ice the reagents and consumables such as EP tubes, place them on ice or at room temperature, and label them.

[0164] (2) Cell lysis: Remove the cells from the CO2 incubator, discard the cell culture supernatant, wash twice with pre-cooled PBS on ice (make sure to remove the PBS completely), add the appropriate RIPA lysis buffer to each well to lyse the cells (e.g., add 500 μL Trizol lysis buffer to a 12-well plate), scrape off the cells with a cell scraper or pipette tip, collect them into a labeled EP tube, and lyse at room temperature for 5-10 min.

[0165] (3) Then add one-fifth of the lysis buffer volume of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 3 minutes, and centrifuge at 12,000 rpm and 4 °C for 10 minutes.

[0166] (4) Transfer the upper aqueous phase (three layers in total) to a clean, labeled EP tube, add an equal volume of isopropanol, mix well, and let stand at room temperature for 20 min.

[0167] (5) Centrifuge at 12000 rpm and 4 ℃ for 10 min, then discard the supernatant.

[0168] (6) Add 500µL of 75% ethanol (prepared with DEPC water) to wash the precipitate (wash once or twice). Centrifuge at 12000rpm and 4℃ for 3 min, discard the supernatant, and dry at room temperature for 5-10 min.

[0169] (7) Add about 30 µL of RNase-free ddH2O to fully dissolve the RNA, and store the resulting RNA solution at -80°C or use it for subsequent experiments.

[0170] Reverse transcription:

[0171] (1) Reagent preparation: Ice the container in advance, place the reagents and consumables such as EP tubes on the ice and mark them; first measure the concentration and purity of RNA, and use DEPC water to prepare the RNA to the same appropriate concentration, preferably 200 ng / μL.

[0172] (2) Reaction system: A commercial reverse transcription kit (Thermo Fisher Scientific, Shanghai, China, #4374967) was used. Following the instructions, 2.0 μL of 10×RT Buffer, 0.8 μL of 25×dNTP Mix (100 mM), 2.0 μL of 10×RT Random Rrimers, and 1.0 μL of MultiScribe were added sequentially to a 200 μL EP tube. TM The system consisted of 1.0 μL Reverse Transcriptyase, 1.0 μL LRNase Inhibitor, 3.2 μL Nuclease-Free H2O, and 10.0 μL of the corresponding RNA sample, for a total volume of 20 μL.

[0173] (3) Reverse transcription reaction conditions: 25 ℃ for 10 min, 37 ℃ for 120 min, 85 ℃ for 5 min. After reverse transcription, it can be used for subsequent experiments or stored at -80 ℃.

[0174] The primers used in the experiment were designed based on the mRNA sequences of mouse and human CTSD, G6pc, Pck1, and the internal control β-actin from NCBI, and synthesized by a biotechnology company after being submitted to PrimerBank. The specific primer sequences are shown below:

[0175] Table 1. Primer Design and Synthesis

[0176]

[0177] Note: β-Actin, Ctsd, G6pc, and Pck1 are mouse gene primers; β-ACTIN and CTSD are human gene primers. β-Actin and β-ACTIN are internal reference genes from mice and humans, respectively.

[0178] The above experiment used a qRT-PCR reaction system (ChamQ Blue Universal SYBR qPCR Master Mix: Vazyme Biotech, Nanjing, China, #Q312-02).

[0179] Table 2 qRT-PCR reaction system

[0180]

[0181] 40 cycles: 95 ℃ (15 s), 60 ℃ (60 s). Data analysis was performed using 2... -△△Ct The relative expression level of the target gene mRNA was analyzed using a method.

[0182] 1.16 Immunofluorescence detection of CTSD and JAK2 proteins (antibodies used in the experiment are listed in Table 4)

[0183] (1) Before seeding cells into the plate, put a drop of PBS on the bottom, cover the bottom with the burnt smear, dry in an oven, seed 10,000-30,000 cells per well (12-well plate), and perform cell treatment one day later.

[0184] (2) One hour before the sample collection time, prepare the reagents and consumables such as EP tubes to be used and mark them.

[0185] (3) Wash twice with PBS

[0186] (4) Fix with 4% paraformaldehyde (Biosharp, Hefei, China, #BL539A) (pre-cooled methanol) for 15 min (or overnight at 4 ℃); after fixation, wash 4 times with PBS for 5 min each time.

[0187] (5) Punching: 0.1% Triton X-100 (Sigma–Aldrich, St. Louis, MO, USA, #T9284) (prepared with PBS, 500µL per well) incubated at room temperature for 30 min, washed twice with PBS, 5 min each time.

[0188] (6) Blocking: Block with blocking solution (5% BSA, prepared in PBS) at room temperature for 2 h. (400 μL per well)

[0189] (7) Dilute the corresponding antibody with universal antibody diluent (the antibody is the same as the antibody used for WB, and the dilution ratio is 1:100), add 50 μL to the slide, and ensure complete coverage (incubate in a humidified chamber) and place at 4 °C overnight.

[0190] (8) Take it out the next day and let it stand at room temperature for 1 h (to make the antibody binding stronger). Recover the primary antibody (which can be diluted for Western Blot). Place the smear in the unused wells of the plate and wash with PBS 3 times for 5 min each time.

[0191] (9) Add fluorescent secondary antibody (CoraLite594 – conjugated Goat Anti-RabbitIgG(H+L)) prepared in PBS at a ratio of 1:500 (500 μL per well) and incubate at room temperature in the dark for 2 h (the fluorescent secondary antibody used for JAK2 antibody in fluorescent colocalization is CoraLite488-conjugated Goat Anti-Mouse IgG(H+L)).

[0192] (10) Wash with PBS 3 times, 10 min each time.

[0193] (11) Counterstain the cell nuclei with 500µl Hoechest (10 μg / ml) (Sigma–Aldrich, St. Louis, MO, USA, #B2261) for about 5 min.

[0194] (12) Wash three times with PBS, 10 min each time. After washing, mount with anti-fluorescence quenching mounting medium (5 µL).

[0195] (13) Take 5 random photos of each cell slide using a fluorescence microscope and save them.

[0196] (14) Image J was used to count the number of cells (based on the number of cell nuclei) and fluorescence intensity (integrated density), and the fluorescence intensity / cell number value was used for statistical analysis.

[0197] 1.17 Plasmid transfection of 293T cells

[0198] (1) Seed 293T cells into DMEM wells or culture dishes containing 10% FBS and cultured in a CO2 cell incubator. Transfection was performed when the cell density in the plate or dish reached 50-70%.

[0199] (2) Preparation of transfection reagent (jetPRIME® transfection reagent: Polyplus, Illkirch, France, #101000046): Taking a 12-well plate as an example, each well contains 100 μL buffer + 1 μg cDNA + 2 μL jetPRIME. ® Mix the transfection reagent thoroughly and incubate at room temperature for 10-15 minutes.

[0200] (3) Slowly add 100 μL of transfection mixture to each well and incubate at 37 °C and 5% CO2 for 36 to 48 h.

[0201] (4) Real-time quantitative PCR (qRT-PCR) and Western blotting were used to detect the knockdown efficiency.

[0202] To investigate the gene function of CTSD, its expression was knocked down using RNA interference technology. Specifically, a shRNA expression plasmid targeting the mouse Ctsd gene (product number: PL-Ctsd-RNAi (P24L0390)) was used for transfection. The target sequence of the shRNA (SEQ ID NO.2) is GCACTGACTCCAAGTACTACC, and the complete oligonucleotide sequence encoding this shRNA is shown in SEQ ID NO.3 and SEQ ID NO.4. It consists of a double-stranded DNA formed by annealing a pair of complementary single strands (P24L0390a and P24L0390b) listed in Table 3. This plasmid can mediate the intracellular expression of specific shRNA. Through this method, the specific knockdown of the CTSD gene was successfully achieved in the target cells.

[0203] Table 3 Single-stranded sequences used to constitute the complete DNA oligonucleotide sequences (SEQ ID NO.3 and SEQ ID NO.4) of shRNA

[0204]

[0205] Note: In this table, P24L0390a and P24L0390b are a pair of complementary single-stranded DNA oligonucleotides. The double-stranded DNA molecule formed after annealing is the complete DNA oligonucleotide sequence of shRNA (SEQ ID NO.3 and SEQ ID NO.4).

[0206] 1.18 Co-IP detection of protein-protein binding

[0207] (1) Reagent preparation: Ice up in advance, place all reagents and consumables such as EP tubes on ice, label them, and prepare CO-IP lysis buffer (Proteintech, Rosemont, IL, USA, #PR20037) (containing 1% PMSF, prepare fresh before use) (PMSF: Sangon Biotech, Shanghai, China, #C510006-0100).

[0208] (2) Sample preparation: Taking adherent cells in a 10 cm culture dish as an example (preferably, all cell lines used are adherent cells), aspirate the cell culture medium, wash once with PBS, and then add the prepared lysis buffer for lysis.

[0209] (3) Lyse on ice for 5 min, scrape off the cells with a cell scraper or pipette tip, and collect them into EP tubes as soon as possible. Perform the entire process at low temperature. Shake the EP tubes on a shaker (3 times, 1 min each time).

[0210] (4) Centrifugation: Centrifuge at 14000 rpm / min for 15 min, and the supernatant is the protein lysis buffer.

[0211] (5) Remove non-specific binding: Take the appropriate volume of protein lysis buffer (200 μL to 1 mL), add about 1 μg of ordinary IgG of the same species as the IgG used in immunoprecipitation and 20 μL of fully resuspended Protein A+G Agarose (Beyotime, Shanghai, China, #P2055-2 ml), shake slowly at 4 ℃ for 30 min to 2 h, centrifuge at 2500 rpm for 5 min, and take the supernatant for subsequent immunoprecipitation.

[0212] (6) Add 0.2-2 μg of primary anti-CTSD antibody (Abcam, #ab75852, see Table 4) for immunoprecipitation, and shake slowly overnight at 4 ℃.

[0213] (7) Add 30 μL of fully resuspended Protein A+G Agarose and incubate slowly at 4 °C for 1-3 h.

[0214] (8) Centrifuge at 2500 rpm for 5 min, carefully remove the supernatant, and wash the precipitate 5 times with CO-IP lysis buffer (centrifugation conditions: 2500 rpm for 5 min).

[0215] (9) After the last wash, remove the supernatant, add 30 μL of 1×Loading Buffer protein loading buffer to resuspend the precipitate, and centrifuge at high speed for a short time to centrifuge the sample to the bottom of the tube.

[0216] (10) Boil in a water bath at 100 ℃ for 10 min, cool to room temperature, and then perform electrophoresis directly or store at -20 ℃.

[0217] Table 4. Antibody information used in the experiment

[0218]

[0219] 1.19 Molecular docking predicts protein-protein binding

[0220] To predict the potential interaction between CTSD and JAK2, molecular docking simulations were performed, with the following steps:

[0221] (a) Molecular docking via AlphaFold Fold3

[0222] (1) Log in to the AlphaFold Fold Server official website.

[0223] (2) Enter the sequences of two or more proteins that you want to predict (search on the UniProt website), and then click Prediction (Continue and preview job).

[0224] (3) Download the prediction results and select model 0 (highest confidence level).

[0225] (4) Visualize using PyMOL.

[0226] (ii) Molecular docking via HDOCK

[0227] (1) RCSB Protein Database ( RCSB PDB - 6QCB: Crystal structure of human cathepsin D in complex with macrocyclic inhibitor 9 ; RCSB PDB - 8C08: Crystal Structure of JAK2 JH2-K539L Select a suitable protein crystal (CTSD protein crystal PDB is 6QCB) and save it as a .Pdb file. Perform preliminary processing using PyMOL (based on the RCSB crystal structure, chain A is the desired protein; extract it, remove water molecules and other non-protein structures, and save as a .Pdb file). Perform the same processing on JAK2 (8C08) (based on the RCSB crystal structure, chains A and B are the desired proteins; extract them, remove water molecules and other non-protein structures, and save as a .Pdb file).

[0228] (2) Log in to the HDOCK official website.

[0229] (3) Input the two protein crystals processed above, and then click docking. Select active regions that cover the entire chain as much as possible.

[0230] CTSD Sequence (SEQ ID NO.1):

[0231] MQPSSLLPLALCLLAAPASALVRIPLHKFTSIRRTMSEVGGSVEDLIAKGPVSKYSQAVPAVTEGPIPEVLKNYMDAQYYGEIGIGTPPQCFTVVFDTGSSNLWVPSIHCKLLDIACWIHHKYNSDKSSTYVKNGTSFDIHYGSGSLSGYLSQDTVSVPCQSASSASALGGVKVERQVFGEATKQPGITFIAAKFDGILGMAYPRISVNNVLPVFDNLMQQKLVDQNIFSFYLSRDPDAQPGGELMLGGTDSKYYKGSLSYLNVTRKAYWQVHLDQVEVASGLTLCKEGCEAIVDTGTSLMVGPVDEVRELQKAIGAVPLIQGEYMIPCEKVSTLPAITLKLGGKGYKLSPEDYTLKVSQAGKTLCLSGFMGMDIPPPSGPLWILGDVFIGRYYTVFDRDNNRVGFAEAARL

[0232] JAK2 Sequence(SEQ ID NO.5):

[0233]

[0234] 2. Experimental Results

[0235] 2.1 Increased serum CTSD activity and liver CTSD expression and activity in T2DM mice

[0236] Previous studies have suggested that plasma cathepsin D activity is closely related to metabolic parameters in men with type 2 diabetes. . To verify the causal relationship, this study induced a type 2 diabetic mouse model using a high-fat diet combined with streptozotocin injection. At the experimental endpoint, blood was collected via the orbital sinus, and serum was separated by centrifugation. CTSD enzyme activity was detected using a fluorescent substrate method. Simultaneously, liver tissue proteins were extracted, and CTSD protein expression was detected by Western blot. Results are as follows: Figure 1 As shown: Compared with the Control group, the serum CTSD activity of mice in the T2DM group was significantly increased (CTSD activity increased from 259817±12819 RFU / μL to 299310±3396 RFU / μL) (**p<0.01). Figure 1 As shown in (A), the expression level of hepatic CTSD protein also showed a significant increase, and the hepatic CSTD activity increased from (13632±309.6) RFU / μg to (22580±515.6) RFU / μg (**p<0.01). Figure 1 (B)-(C).

[0237] 2.2 Inhibition of CTSD activity improves serum biochemical levels in T2DM mice

[0238] To investigate the regulatory effect of CTSD activity inhibition on metabolic function in T2DM mice, blood was collected via the orbital venous plexus at the experimental endpoint. Serum was separated and the levels of TG, TC, AST, ALT, and ALP were measured using a Mindray BS-200 fully automated biochemical analyzer. Results showed that compared to the Control group, serum TG, TC, AST, ALT, and ALP levels were significantly increased in the T2DM group; however, after T2DM+PepA intervention, these indicators showed a decreasing trend compared to the T2DM group. Figure 2 As shown in the figure. These results confirm that inhibition of CTSD activity can effectively improve lipid metabolism disorders and liver function damage in T2DM mice. Among them, Figure 2 (A) is serum TG in T2DM mice; Figure 2 (B) Serum TC in T2DM mice; Figure 2 (C) represents serum AST from T2DM mice; Figure 2 (D) represents serum ALT in T2DM mice; Figure 2(E) Serum ALP in T2DM mice. Mean ± SEM, n=5. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns showed no statistical significance.

[0239] 2.3 Inhibition of CTSD activity improves glucose tolerance and insulin tolerance in T2DM mice

[0240] To systematically evaluate the regulatory effect of inhibiting CTSD activity on glucose metabolism homeostasis, this invention used glucose tolerance test (GTT) to detect insulin sensitivity and glucose metabolism capacity. Mice were fasted for 12 h before the experiment and then injected intraperitoneally with 2 g / kg glucose solution. Blood glucose levels were measured at 0, 15, 30, 60, and 120 min. Results showed that compared with the Control group, the area under the glucose curve (AUC) of mice in the T2DM group was significantly increased (AUC increased from 38.066±2.659 to 123.567±3.184) (****p<0.0001), indicating impaired glucose tolerance. However, the AUC value of the T2DM+PepA group was significantly lower than that of the T2DM group, decreasing from 123.567±3.184 to 81.383±5.883 (***p<0.001). Figure 3 As shown, inhibition of CTSD activity can significantly improve insulin signaling. Among them, Figure 3 (A) Glucose tolerance test performed during week 4 of PepA treatment; Figure 3 (B) is the area under the curve (AUC), used to quantify the GTT results. Mean ± SEM, n=5. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0241] 2.4 Inhibition of CTSD activity improves body weight and blood glucose levels in T2DM mice

[0242] like Figure 4 As shown, weight and blood glucose were monitored weekly during the administration period. Results showed that, with prolonged administration, the T2DM+PepA group experienced a certain degree of weight reduction compared to the T2DM group. Figure 4 As shown in (A), the mouse body weight level from week 5 to week 8; blood glucose levels also showed some improvement. Figure 4 (B) shows the fasting blood glucose levels in mice from week 5 to week 8. Mean ± SEM, n=5.

[0243] 2.5 Inhibition of CTSD activity improves insulin resistance in T2DM mice

[0244] Further testing revealed that serum insulin levels and HOMA-IR were significantly higher in the T2DM group than in the Control group. Insulin increased from 41.29±2.889 mIU / L to 58.78±4.3 mIU / L; HOMA-IR increased from 12.68±1.078 mM*mIU / L to 30.15±3.531 mM*mIU / L (*p<0.05, **p<0.01). Figure 5 As shown; the above indicators in the T2DM+PepA group were significantly improved compared with those in the T2DM group, with insulin decreasing from 58.78±4.3 mIU / L to 39.69±3.342 mIU / L; and HOMA-IR decreasing from 30.15±3.531 mM*mIU / L to 20.67±1.528 mM*mIU / L (*p<0.05). Figure 5 (A) ELISA detection of insulin levels in mice; Figure 5 (B) represents the insulin resistance index. Mean ± SEM, n=5. *p<0.05, **p<0.01.

[0245] 2.6 Inhibition of CTSD activity improves hepatic glucose metabolism in T2DM mice

[0246] The livers of the three groups of mice were also subjected to H&E, PAS, ORO, and immunohistochemical staining, and mouse liver RNA was extracted to detect gluconeogenesis-related genes G6pc and Pck1. Figure 6 As shown, the staining results indicate that, Figure 6 (A) H&E, PAS, ORO, and immunohistochemical staining of mouse liver; Figure 6 (B) Detection of G6pc gene in mouse liver; Figure 6 (C) Detection of Pck1 gene in mouse liver. Scale bar, 100 μm. Mean ± SEM, n=3. *p<0.05, **p<0.01, ***p<0.001. Compared with the control group, the liver of the T2DM group showed obvious fatty vacuoles, while the T2DM+PepA group showed significant improvement. Figure 6 As shown in (A). Glycogen staining results showed that glycogen synthesis was reduced in the T2DM group, and glycogen synthesis recovered after CTSD activity was inhibited (the intensity of purple was positively correlated with the amount of glycogen). Glycognate gene qPCR results showed the opposite. Figure 6 (B) and Figure 6As shown in (C), the T2DM group exhibits increased gluconeogenesis gene expression, leading to increased glucose production and decreased glycogen synthesis, resulting in elevated blood glucose levels. The PepA group shows decreased gluconeogenesis gene expression, reduced glucose production, and enhanced glycogen synthesis, leading to decreased blood glucose levels. The lipid droplet staining results are consistent with glycogen staining (lipid droplets stain orange-red, with intensity and quantity representing lipid droplet content). Furthermore, CTSD immunohistochemistry shows a significant increase in CTSD formation in the T2DM group, consistent with previous results. Figure 6 (A).

[0247] 2.7 High-fat, high-fat, and high-glucose treatments increased CTSD gene expression in AML-12 and HepG2 cells.

[0248] The in vivo experiments in the T2DM mouse model indicated that the expression and enzyme activity of CTSD were significantly upregulated in the insulin-resistant mouse model. Given the crucial role of the liver in the development of insulin resistance, this invention selected mouse liver-derived AML-12 cell lines and human hepatocellular carcinoma HepG2 cell lines to construct in vitro models for mechanism verification. Experimental data showed that under high-fat (PA) or high-fat-high-glucose (PA+HG) stimulation conditions, the expression levels of CTSD mRNA in both cell models were significantly increased, and the response trend of AML-12 cells was consistent with that of the human HepG2 cell model (*p<0.05, **p<0.01, ***p<0.001). Figure 7 As shown in (A)-(D), Figure 7 (A) qPCR detection of CTSD expression in HepG2 cells under PA conditions; Figure 7 (B) qPCR detection of Ctsd expression in AML-12 cells under PA conditions; Figure 7 (C) qPCR detection of CTSD expression in HepG2 cells under PA+HG conditions; Figure 7 (D) qPCR detection of Ctsd expression in AML-12 cells under PA+HG conditions. Mean ± SEM, n=4. *p<0.05, **p<0.01, ***p<0.001.

[0249] 2.8 High-fat, high-fat, and high-glucose treatments increased CTSD protein expression and activity in AML-12 and Hepg2 cells.

[0250] To further verify the multidimensional regulatory characteristics of CTSD, this study employed Western blot quantitative analysis and immunofluorescence microscopy for detection. Figure 8 As shown, where, Figure 8 (A) Western blot analysis of CTSD expression in HepG2 cells under PA conditions; Figure 8(B) Western blot detection of CTSD expression in AML-12 cells under PA conditions; Figure 8 (C) Western blot detection of CTSD expression in HepG2 cells under PA+HG conditions; Figure 8 (D) Western blot detection of CTSD expression in AML-12 cells under PA+HG conditions; Figure 8 (E) Gray-scale scanning detection of CTSD protein bands in HepG2 cells under PA conditions; Figure 8 (F) Gray-scale scanning detection of CTSD protein bands in AML-12 cells under PA conditions; Figure 8 (G) PA+HG conditions for grayscale scanning detection of CTSD protein bands in HepG2 cells; Figure 8 (H) PA+HG conditions for grayscale scanning detection of CTSD protein bands in AML-12 cells; Figure 8 (I) HepG2 cells on culture medium under PA conditions

[0251] Clear CTSD activity; Figure 8 (J) CTSD activity of AML-12 cell culture supernatant under PA conditions; Figure 8 (K) CTSD activity in AML-12 cell culture supernatant under PA+HG conditions; Mean ± SEM, n=3. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns showed no statistical significance. Results showed that, consistent with the trend in mRNA levels, CTSD protein abundance significantly increased in AML-12 and HepG2 cells after 24 hours of PA / PA+HG treatment (*p<0.05, **p<0.01, ***p<0.001). Figure 8 (A)-(H), and CTSD activity increased synchronously (*p<0.05, **p<0.01, ****p<0.0001) as shown. Figure 8 (I)-(K). Immunofluorescence assays also showed a significant increase in fluorescence intensity of CTSD after 24 hours of PA / PA+HG treatment (*p<0.05, **p<0.01, ***p<0.001). Figure 9 As shown in (A)-(D), high-fat and high-glucose diets induced increased CTSD protein levels in AML-12 and HepG2 cells, suggesting that the insulin-resistant microenvironment may promote CTSD synthesis and synergistically regulate its function. Figure 9 (A) IF detection of CTSD expression in HepG2 cells under PA conditions; Figure 9 (B) IF detection of CTSD expression in AML-12 cells under PA conditions; Figure 9 (C) IF detection of CTSD expression in HepG2 cells under PA+HG conditions; Figure 9 (D) IF detection of CTSD expression in AML-12 cells under PA+HG conditions. Scale bar, 50 μm. Mean ± SEM, n=3. *p<0.05, **p<0.01, ***p<0.001.

[0252] 2.9 Inhibition of cathepsin D activity improves insulin signaling in AML-12 cells

[0253] Based on previous animal experiments demonstrating that inhibiting CTSD activity can improve hepatic glucose metabolism and enhance insulin signaling pathways, this invention further constructs an in vitro cell model to verify the molecular mechanism by which elevated CTSD activity leads to hepatic insulin resistance. First, a lipid accumulation model was established by inducing mouse hepatic AML-12 cells with palmitic acid (PA). Western blot analysis showed that PA treatment significantly downregulated the phosphorylation levels of key effector molecules in the insulin signaling pathway (including S6, AKT, and GSK-3β) (**p<0.01). Figure 10 As shown, inhibition of CTSD enzyme activity significantly increased the phosphorylation levels of the aforementioned signaling molecules (*p<0.05), indicating that inhibition of CTSD activity restored insulin signaling in liver cells. This result is effectively corroborated by animal experimental data. Figure 10 (A) Western blot analysis of β-actin, CTSD, p-S6, p-GSK-3β, T-GSK-3β, p-AKT, and T-AKT levels in AML-12 cells; Figure 10 (B) Normalization of p-GSK-3β levels and T-GSK-3β levels; Figure 10 (C) Normalization of p-AKT and T-AKT levels. Mean ± SEM, n=3. *p<0.05, **p<0.01, ns showed no statistical significance.

[0254] 2.10 Inhibition of cathepsin D activity improves insulin signaling in HepG2 cells

[0255] The same results were verified in the mouse hepatocellular carcinoma cell line HepG2, and this invention further verified the above findings in the human hepatocellular carcinoma cell line (HepG2). Figure 11 As shown, PA intervention significantly inhibited the phosphorylation activation of key molecules in the insulin signaling pathway in HepG2 cells (*p<0.05). Figure 11 As shown, in the CTSD inhibitor treatment group, the phosphorylation levels of related signaling molecules were restored (*p<0.05, **p<0.01), further confirming that CTSD can regulate the insulin signaling pathway. Among these, Figure 11(A) Western blot analysis of β-actin, CTSD, p-S6, p-GSK-3β, T-GSK-3β, p-AKT, and T-AKT levels in HepG2 cells; Figure 11 (B) Normalization of p-GSK-3β levels and T-GSK-3β levels; Figure 11 (C) Normalization of p-AKT and T-AKT levels. Mean ± SEM, n=3, * p<0.05， **p<0.01, ns indicates no statistical significance.

[0256] 2.11 CTSD may affect insulin signaling through the JAK2-STAT3 signaling pathway.

[0257] Based on the aforementioned finding that inhibiting CTSD can improve insulin signaling in hepatocytes, this invention further focuses on elucidating its molecular regulatory network. By establishing a dual-model system for hepatocytes: ① a basic model (Control vs. recombinant CTSD protein treatment group); ② a pathological model (insulin resistance induction group vs. PepA intervention group), combined with whole transcriptome sequencing and KEGG pathway enrichment analysis, it was found that the JAK2-STAT3 signaling pathway showed significant activation characteristics in both models, suggesting that CTSD may participate in the insulin resistance process by positively regulating the JAK-STAT signaling cascade. Figure 12 As shown. This discovery provides an important entry point for elucidating the multidimensional regulatory mechanism of "ligand-receptor-kinase" in CTSD-mediated glucose metabolism disorders. Among them, Figure 12 (A) Control vs recombinant CTSD protein treatment group: differentially expressed genes, |LOGFC|>1, P-value<0.05 (TOP20 markers); Figure 12 (B) Control vs. recombinant CTSD protein treatment group: KEGG enrichment analysis P-value < 0.05 (TOP20 Findings); Figure 12 (C) Insulin resistance induction group vs. PepA intervention group: GO analysis; Figure 12 (D) KEGG enrichment analysis of the insulin resistance induction group vs. the PepA intervention group. (LOGFC: Log Fold Change; KEGG: Kyoto Encyclopedia of Genes and Genomes; GO: Gene Ontology)

[0258] 2.12 CTSD and JAK2 have protein-level interactions.

[0259] Previous studies have confirmed that abnormal activation of the STAT3 signaling pathway can lead to pancreatic islet cell dysfunction (Khodarahmi, A., et al., Quercetin Mitigates Hepatic Insulin Resistance in Rats with BileDuct Ligation Through Modulation of the STAT3 / SOCS3 / IRS1 Signaling Pathway. JFood Sci, 2019. 84(10): p. 3045-3053). http: / / doi.org / 10.1111 / 1750-3841.14793 Based on this, CTSD in this invention inhibits insulin signaling by regulating the JAK2-STAT3 pathway. Figure 13 To clarify the interaction between JAK2 and CTSD, the following experiments were conducted: Molecular docking analysis showed that CTSD (sky blue) and JAK2 (green) have potential binding sites, such as... Figure 13 As shown in (A); CTSD was overexpressed in 293T cells (36 h), and then Co-IP experiments confirmed that the two physically bind at the endogenous level. Figure 13 As shown in (B); PA treatment of 293T cells for 24 h induced insulin resistance, and then fluorescence colocalization experiments revealed that CTSD and JAK2 had significant spatial colocalization in the cytoplasm, as shown in Figure (B). Figure 13 As shown in (C). The above results consistently demonstrate that CTSD can bind to JAK2, providing crucial molecular interaction evidence for subsequent mechanism studies.

[0260] 2.13 Under high-fat or high-fat-high-glucose conditions, CTSD increases, p-JAK2 increases, and p-AKT decreases.

[0261] Experimental results based on the interaction between JAK2 and CTSD ( Figure 14 As shown in the figure, further investigation was conducted to determine whether JAK2 signaling pathway activation was related to CTSD expression levels. In AML-12 cells, insulin resistance was induced by high-fat or high-fat + high-glucose treatment, and CTSD activity was inhibited using PepA. The results showed that p-JAK2 expression levels were upregulated with increasing CTSD expression; p-AKT (a key effector protein in insulin signaling) expression levels decreased with increasing CTSD expression.

[0262] Adjust as Figure 14 As shown in (A)-(C), where, Figure 14 (A) PA induces an increase in CTSD and a decrease in p-AKT in AML-12 cells; Figure 14 (B) PA induces an increase in CTSD and p-JAK2 in AML-12 cells; Figure 14(C) PA+HG induced an increase in CTSD and p-JAK2, and a decrease in p-AKT in AML-12 cells. These results indicate that under high-fat or high-fat + high-glucose conditions, high expression of CTSD may inhibit hepatic insulin signaling by activating the JAK2-STAT3 signaling pathway.

[0263] 2.14 Effects of CTSD overexpression or knockdown on JAK2-STAT3 and insulin signaling pathways

[0264] To verify this mechanism, CTSD was overexpressed in 293T cells. For example... Figure 15 As shown, the results indicate that p-JAK2 levels are increased and p-AKT levels are decreased in the CTSD overexpression group. Figure 15 As shown in (A), the phenotype is consistent with the elevated CTSD induced by high-fat / high-glucose diets; in the CTSD knockdown group, p-JAK2 levels are decreased, and p-AKT levels are increased as shown in (A). Figure 15 As shown in (B). The above results indicate that the expression level of CTSD is positively correlated with JAK2 phosphorylation and negatively correlated with AKT phosphorylation, suggesting that CTSD inhibits hepatic insulin signaling by activating the JAK2-STAT3 pathway. Figures 12 to 15 ). Figure 15 (A) After overexpression of CTSD, the expression levels of p-JAK2, T-JAK2, p-STAT3, STAT3, p-AKT, and T-AKT were detected by Western Blot; Figure 15 (B) After knocking down CTSD, Western Blot was used to detect the expression levels of p-JAK2, T-JAK2, p-STAT3, STAT3, p-AKT, and T-AKT.

[0265] 2.15 Inhibition of p-JAK2 improves insulin signaling.

[0266] Based on the established regulatory role of CTSD in the JAK2-STAT3 signaling pathway, this study further elucidated its mechanistic association with insulin sensitivity through pharmacological inhibition. Treatment of AML-12 cells with the JAK2 phosphorylation inhibitor WP1066 (2.5 μM, 24 hours) (WP1066 purchased from MedChemExpress, catalog number HY-15312) resulted in upregulation of p-AKT levels and a decrease in CTSD protein abundance. Figure 16As shown, JAK2 phosphorylation affects the phosphorylation levels of AKT and STAT3. After inhibiting JAK2 phosphorylation, Western blotting was used to detect the expression levels of p-JAK2, T-JAK2, p-STAT3, STAT3, p-AKT, and T-AKT. This may be the reason for the relief of insulin resistance. The results again showed a strong negative correlation between CTSD levels and p-AKT activation, suggesting that CTSD inhibits hepatic insulin signaling by overactivating the JAK2-STAT3 pathway. This invention establishes the JAK2-STAT3 pathway as the core regulatory axis for CTSD-induced damage to hepatic cell insulin sensitivity.

[0267] It should be understood that although this specification is described according to various embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0268] The detailed descriptions listed above are merely specific illustrations of feasible embodiments of the present invention and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of an inhibitor of cathepsin D or its encoding gene in the preparation of products for the prevention and / or treatment of type 2 diabetes.

2. The application according to claim 1, characterized in that, The inhibitors prevent and / or treat type 2 diabetes by inhibiting the expression of cathepsin D or its encoding gene, or by inhibiting the activity of cathepsin D.

3. The application according to claim 1, characterized in that, The inhibitors prevent and / or treat type 2 diabetes by inhibiting the expression of cathepsin D or its encoding gene, or by inhibiting the activity of cathepsin D, thereby inhibiting the activation of the JAK2-STAT3 signaling pathway.

4. The application according to claim 1, characterized in that, The cathepsin D or its encoding gene is derived from mammals.

5. The application according to claim 1, characterized in that, The amino acid sequence of the cathepsin D is shown in SEQ ID NO.

1.

6. The application according to claim 1, characterized in that, The application is achieved by administering an inhibitor of cathepsin D expression, an inhibitor of the expression of the cathepsin D encoding gene, or an inhibitor of cathepsin D activity.

7. The application according to claim 6, characterized in that, The inhibitor of cathepsin D activity includes a small molecule inhibitor or a cathepsin D antibody; preferably, the small molecule inhibitor includes Pepstatin A.

8. The application according to claim 6, characterized in that, The inhibitor of cathepsin D or its encoding gene expression is an antisense RNA, siRNA, shRNA or miRNA that inhibits the expression of cathepsin D or its encoding gene.

9. The application according to claim 8, characterized in that, The target sequence of the shRNA is shown in SEQ ID NO.

2.

10. The application according to claim 9, characterized in that, The shRNA is PL-Ctsd-RNAi.

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