Application of platycodin D2 in preparation of medicine for treating or relieving fatty liver

Abstract

The application discloses application of platycodin D2 in preparation of a medicine for treating or relieving fatty liver, and belongs to the field of biomedical technology. The application aims to provide a medicine for treating or relieving fatty liver. The application provides application of platycodin D2 in preparation of a medicine for treating or relieving fatty liver. The platycodin D2 can down-regulate FASN protein by inhibiting palmitoyl transferase ZDHHC23, so that the effect of treating non-alcoholic fatty liver is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of platycodon saponin D2 in the preparation of drugs for treating or alleviating fatty liver. Background Technology

[0002] Fatty liver disease refers to a condition characterized by excessive fat accumulation in liver cells. It is classified into alcoholic fatty liver disease and non-alcoholic fatty liver disease (NAFLD). Fatty liver disease is not only a manifestation of metabolic syndrome but also reflects a systemic chronic inflammatory state in the liver, and its harm increases as the disease progresses.

[0003] Harmful effects: Fatty liver can lead to liver cell damage, inflammation, and fibrosis, gradually progressing to non-alcoholic steatohepatitis (NASH), cirrhosis, and even liver cancer. It significantly increases the risk of cardiovascular disease, as it is often accompanied by obesity, insulin resistance, dyslipidemia, and hypertension. NAFLD is also closely associated with type 2 diabetes, chronic kidney disease, polycystic ovary syndrome, and increases all-cause mortality. Compared to alcoholic fatty liver disease, NAFLD has a larger patient population, insidious onset, and often no symptoms in the early stages, frequently being discovered during physical examinations, easily overlooked, and delaying intervention.

[0004] Current treatments and limitations: Current treatments primarily focus on lifestyle interventions, including weight loss, dietary control, and regular exercise, which can effectively improve mild to moderate NAFLD, but have limited effectiveness for severe cases or those with fibrosis. Regarding medications, very few drugs are approved for NASH (such as the thyroid hormone receptor agonist Resmetirom), and most are still in clinical trials. Commonly used insulin sensitizers (such as pioglitazone) and antioxidants (vitamin E) have some improvement effects, but have side effects (weight gain, cardiovascular risk, risk of hemorrhagic stroke, etc.). Metabolic regulators and gut microbiota regulators are still under investigation, lacking large-scale long-term efficacy evidence. Furthermore, current therapies have poor adherence, making sustained weight loss and lifestyle changes difficult, and there is a lack of effective means to reverse advanced fibrosis. In summary, fatty liver, especially NAFLD, has widespread harmful effects. Current treatments are mainly based on lifestyle interventions, with limited drug options and significant side effects, necessitating a safer and more effective targeted therapy. Summary of the Invention

[0005] The purpose of this invention is to provide the use of platycodon saponin D2 in the preparation of drugs for treating or alleviating fatty liver.

[0006] This invention provides the application of platycodon saponin D2 in the preparation of drugs for treating or alleviating fatty liver.

[0007] To further specify, fatty liver refers to non-alcoholic fatty liver.

[0008] Further specifying, the dosage form of the drug is liquid, suppository, tablet, powder, or ointment.

[0009] Furthermore, platycodon saponin D2 is identified as the active ingredient in palmitoyltransferase inhibitors.

[0010] Further specifying, palmitoyltransferase is palmitoyltransferase ZDHHC23.

[0011] Beneficial effects: Platycodon saponin D2 can downregulate FASN protein by inhibiting palmitoyltransferase ZDHHC23, thereby achieving the effect of treating non-alcoholic fatty liver disease. Attached Figure Description

[0012] Figure 1 The graph shows the changes in mouse body weight; the horizontal axis represents the number of weeks, and the vertical axis represents body weight (g). Figure 2 A graph showing the results of changes in liver indices; Figure 3 The graph shows the results of changes in blood parameters; A is ALT level; B is AST level; C is LDL-C level; D is TG level. Figure 4 This is a diagram showing the results of liver pathological changes. Figure 5 The results are shown in the transcriptomics diagram; A is a KEGG pathway enrichment map of transcriptomic cellular components; B is a KEGG pathway enrichment map of transcriptomic molecular functions; C is the palmitoylation level of the whole protein before and after PD2 addition as detected by Western blotting. Figure 6 Figure A shows the palmitoylation modification proteomics results; Figure B shows the differential ranking results of palmitoylated proteins; Figure C shows the volcano plot of changes in palmitoylated proteins. Figure 7 The graph shows the changes in palmitoylation transferase protein expression levels after drug administration. Figure 8 To illustrate how PD2 inhibits the interaction between endogenous ZDHHC23 and endogenous FASN proteins; A is the result of Co-IP detection of palmitoylated transferases interacting with FASN protein; B is the result of Western blotting detection of FASN, ZDHHC23, and ZDHHC5 protein expression levels before and after PD2 addition. Figure 9 Figure A shows the results of PD2 downregulating the palmitoylation level of FASN protein; Figure B shows the results of Co-IP and ABE detection of FASN protein palmitoylation level before and after PD2 addition; Figure C shows the results of WB detection of FASN protein expression level before and after PD2 addition. Figure 10 The image shows the molecular-protein docking results of PD2 and ZDHHC23. Figure 11 The image shows the protein-protein docking results of ZDHHC23 and FASN. Detailed Implementation

[0013] Platycodon saponin D2, CAS No.: 66663-90-9, chemical formula as follows: .

[0014] Example 1. The intervention effect of PD2 on non-alcoholic fatty liver disease in mice. Animal experiment: 24 C57 mice were randomly divided into 4 groups: blank control group, model group (non-alcoholic fatty liver model), positive drug group (simvastatin 10mg / kg / d), and PD2 group (platycodon saponin D2, 5mg / kg / d).

[0015] The control group was fed a normal diet, while the other groups were fed a high-fat diet for 8 weeks. Subsequently, the drug-treated groups were administered PD2 (solvents: 10% DMSO, 40% PEG300, 5% Tween-80, and 45% Saline) by gavage, and the positive control group (solvents: 10% DMSO, 40% PEG300, 5% Tween-80, and 45% Saline) for 6 consecutive weeks. Body weight was measured weekly. One hour after the first administration at the end of the 6th week, mice were anesthetized by intraperitoneal injection of urethane, and blood was collected from the orbital artery. After standing for 30 minutes, the blood was centrifuged (4°C, 3500 rpm, 15 minutes) to separate serum. Serum ALT, AST, LDL-C, and TG levels were measured using a kit. The liver was harvested, cleaned of surface blood with pre-cooled saline, weighed, and its liver index was calculated. Liver tissue was harvested, rinsed with physiological saline, soaked in 4% paraformaldehyde for 12 hours, dehydrated, embedded in paraffin, sectioned, spread, heated, stained with hematoxylin and eosin, dehydrated and sealed; then stained again with eosin. Images of the sections were acquired using a microscopic imaging system to observe specific lesions. Liver index = liver mass / body mass × 100%.

[0016] General feed: Spifor (Beijing) Biotechnology Co., Ltd., product number F002 High-fat feed: Xiaoshu Youtai (Beijing) Biotechnology Co., Ltd., Product No.: XSYT-ED-078 Results: A high-fat diet caused rapid weight gain in mice, while PD2 and the positive control drug reduced the rate of weight gain and the recovery of liver index to varying degrees. Figure 1-2 The results of PD2's effect on serum influencing factors in non-alcoholic fatty liver rats are shown in [the table below]. Figure 3 Compared with the control group, the serum ALT, AST, LDL-C and TG levels in the model group were significantly increased, while the serum ALT, AST, LDL-C and TG levels in the positive drug group and PD2 were decreased compared with the model group.

[0017] Pathological observation of the liver revealed that in the model group, extensive fatty degeneration of hepatocytes was observed in most of the liver parenchyma, with varying numbers and sizes of round vacuoles in the cytoplasm, and diffuse infiltration of numerous inflammatory cells in the lobules; while in the PD2 group, after PD2 intervention, both round vacuoles and diffuse infiltration of inflammatory cells were alleviated. Figure 4 Oil Red staining of the liver revealed a large area of ​​lipid droplets in the liver tissue of the model group, while the accumulation of lipid droplets in the PD2 intervention group was reduced compared with the blank control group, and the positive rate was significantly decreased. Figure 4 ).

[0018] Example 2. PD2 exhibits depalmitoylation activity and weakens the activity of palmitoylation transferase ZDHHC23. I. Transcriptomics analysis (RNA analysis of mouse liver tissue) 1. Total RNA quality testing (1) NanoDrop 2000 spectrophotometer: detects the purity and concentration of RNA; (2) Agient2100 / LabChip GX assay: accurately detects the integrity of RNA.

[0019] 2. After the sample passes the testing, library construction is performed. The main process is as follows: (1) Enrich eukaryotic mRNA with magnetic beads containing Oligo (dT); (2) Add Fragmentation Buffer to randomly fragment the mRNA; (3) Use mRNA as a template to synthesize the first and second strands of cDNA and purify the cDNA; (4) Repair the ends of the purified double-stranded cDNA, add A tails and ligate sequencing adapters, and then use AMPure XP beads to select fragment sizes; (5) Finally, obtain the cDNA library by PCR enrichment.

[0020] 3. After the library is constructed, a preliminary quantification is performed using a Qubit 3.0 fluorescence quantitative analyzer, with a concentration of at least 1 ng / ul. Then, the Qsep400 high-throughput analysis system is used to detect the inserted fragments in the library. If the inserted fragments meet the expectations, the effective concentration of the library (effective concentration of the library > 2 nM) is accurately quantified using the Q-PCR method to ensure the quality of the library.

[0021] 4. After the library passes quality control, use a high-throughput sequencing platform to perform PE150 mode sequencing.

[0022] 5. After sequencing data is processed, the bioinformatics analysis workflow provided by BMKCloud (www.biocloud.net) is used for data analysis. The processed data is filtered to obtain Clean Data, which is then aligned with a specified reference genome to obtain Mapped Data. Library quality assessment, structural analysis, differential expression analysis, gene function annotation, and functional enrichment are then performed.

[0023] II. Palmitoylation Modification Omics Analysis 1. Protein ABE treatment Tissue lysis was performed at 4°C, 12000 rpm for 10 min, and the supernatant was collected. A suitable amount of protein sample (liver tissue samples from the model group and PD2 group) was precipitated once with chloroform-methanol (CM) at room temperature, 6000 rpm for 10 min, and the precipitate was collected and air-dried in a fume hood for 2-3 min. 180 μL of 4SB (4-sulfobenzaldehyde) and 20 μL of TCEP (tris(2-carboxyethyl)phosphine) were added to the protein precipitate, and the mixture was incubated at 37°C for 10 min, occasionally shaking the centrifuge tube until the precipitate dissolved. Another chloroform-methanol (CM) precipitation was performed at room temperature, 6000 rpm for 10 min, and the precipitate was collected and air-dried in a fume hood for 2-3 min. 200 μL of 4SB and 10 mM NEM (N-ethylmaleimide) were added to the protein precipitate, and the mixture was incubated overnight at 4°C in the dark to block free sulfhydryl groups. Three CM precipitations were performed consecutively the following day to remove residual NEM. After the final CM precipitation, add 4SB to dissolve the precipitate. Perform one more CM precipitation to remove residual HA. Add 200 μL of 4SB and incubate at 37°C for 10 min to dissolve the precipitate. Then, add Low Biotin-HPDP buffer to the dissolved protein sample and incubate at room temperature for 1 h for labeling. Perform three more CM precipitations on the labeled protein sample to remove unreacted Biotin-HPDP. After each CM precipitation, add 200 μL of 4SB and shake the centrifuge tube until the precipitate dissolves. The dissolved protein solution is ready for use.

[0024] 2. Proteolytic enzyme digestion Add the prepared protein solution to a FASP ultrafiltration tube and wash three times with UA. After centrifugation, add an appropriate amount of trypsin and incubate at 37°C for 16 hours. The next day, centrifuge to collect the enzymatically digested peptides for later use.

[0025] 3. Peptide enrichment Add an appropriate amount of streptavidin agarose gel beads for washing and equilibration, then invert at room temperature for 5 min. Centrifuge at 2000g for 30 s at room temperature, and carefully discard the supernatant after the gel beads settle to the bottom of the tube. Add the collected peptide sample to the washed and equilibrated streptavidin agarose gel beads, and invert overnight at 4℃. The next day, centrifuge at 2000g for 30 s at room temperature, and carefully aspirate the supernatant. Wash the gel beads 4 times with LB elution buffer. Each time, invert at room temperature for 5 min, then centrifuge at 2000g for 30 s at room temperature, and carefully discard the supernatant after the gel beads settle to the bottom of the tube. Add 50-70 μL of elution buffer to the washed gel beads, incubate at 37℃ for 15 min, centrifuge at 2000g for 1 min at room temperature, collect the supernatant, repeat the elution once, combine the two eluates, add IAA (iodoacetamide), and incubate at room temperature in the dark for 1 h. After incubation, the sample is ready for desalting.

[0026] 4. Desalination and freeze-drying The sample peptides were desalted and purified using a C18 tip, pre-equilibrated with 50% ACN (acetonitrile), washed with 0.1% trifluoroacetic acid, and then slowly and repeatedly pipetted the C18 tip into the sample dissolved in 0.1% trifluoroacetic acid to ensure sufficient binding of the C18 peptides to the sample. After binding, the sample was washed with 0.1% trifluoroacetic acid / 5% ACN, and finally eluted with 100 μL of a solution containing 50% acetonitrile and 0.1% trifluoroacetic acid. The desalted and purified sample was concentrated under vacuum and evaporated to dryness.

[0027] 5. Liquid chromatography-tandem mass spectrometry and data analysis The peptides were analyzed using an Easy-nLC 1000 chromatograph and an LTQ Obitrap ETD mass spectrometer (Thermo Fisher Scientific). The relevant database (uniprot-human.fasta) was then searched using Maxquant 2.1 (Thermo) software to obtain the final protein identification and analysis results.

[0028] III. Western Blot After grinding, liver tissue samples from the model group and PD2 group were lysed in RIPA lysis buffer on ice for 10 min, then centrifuged at 14000 r / min for 5 min, the precipitate was discarded, and the supernatant protein was quantified using a BCA protein quantification kit. Then, 5× protein loading buffer was added according to the volume of supernatant protein, and the samples were boiled in boiling water for 10 min. The protein samples were then stored at -80℃.

[0029] Take 30 μg of each quantified protein sample for protein electrophoresis. Add the protein sample to the well of the pre-prepared electrophoresis gel and add the pre-stained protein marker. Adjust the voltage to 80 V. After the marker bands disperse, adjust the voltage to 120 V until the electrophoresis is complete. After electrophoresis, carefully remove the gel, set the voltage to 100 V, and the time to 90 min. Transfer the protein on the gel to an NC membrane. Then, block the protein on the membrane with 5% skim milk solution at room temperature for 2 h. Dilute the primary antibody to be incubated at a ratio of 1:1000 with primary antibody dilution buffer. Trim the protein on the membrane according to the size of the protein to be incubated with the primary antibody, and then place it in the diluted primary antibody solution. Incubate overnight at 4°C. The next day, aspirate the primary antibody and wash the membrane with TBST for 10 min each time, three times. Add the secondary antibody prepared at a ratio of 1:3000 and incubate at room temperature for 45 min. Then aspirate the secondary antibody and continue washing with TBST for 10 min each time, three times. Finally, a developer is dropped onto the film, and the film is exposed and developed under the developer.

[0030] IV. Immunoprecipitation The following experiments were performed using the Pierce™ cross-linked magnetic bead immunoprecipitation / co-immunoprecipitation kit.

[0031] 1. Bind antibodies to protein A / G magnetic beads. (1) For each immunoprecipitation reaction, prepare 2 ml of 1× modified European buffer, which is to dilute 0.1 ml of 20× coupling buffer and 0.1 ml of immunoprecipitation lysis / rinse buffer in 1.8 ml of ultrapure water.

[0032] (2) Vortex the Pierce protein A / G magnetic bead bottle to obtain a uniform suspension. Add 25 μL of magnetic beads to a centrifuge tube, place the centrifuge tube on a magnetic rack for 1 min, collect the magnetic beads, and remove the storage solution.

[0033] (3) Add 500 μL of the 1x modified coupling buffer prepared in step 1 to the centrifuge tube, mix gently, incubate at room temperature on a vortex mixer for 1 min, collect the magnetic beads using a magnetic rack, and remove the supernatant. Repeat this step once.

[0034] (4) Dilute the antibody with 20× conjugation buffer and immunoprecipitation lysis / wash buffer at a ratio of 1:20 to make the final antibody concentration 5ug per 100ul. For example, to prepare 100ul of antibody solution, dilute the antibody stock solution with 5ul of 20X conjugation buffer, 5ul of immunoprecipitation lysis / wash buffer, and then add ultrapure water to make the final volume 100ul.

[0035] (5) Add 100 μL of the prepared antibody solution to the magnetic beads, mix gently, and incubate on a rotator at room temperature for 3 hours.

[0036] (6) Collect the magnetic beads with a magnetic rack and remove the supernatant.

[0037] (7) Add 100 μL of 1× modified coupling buffer, gently vortex or invert to mix, collect the magnetic beads with a magnetic rack, and remove the supernatant.

[0038] (8) Add 300 μL of 1× modified coupling buffer, gently vortex or invert to mix, collect the magnetic beads with a magnetic rack, and remove the supernatant. Repeat this step once.

[0039] 2. Cross-linked antibodies (1) Pierce the aluminum bag of a tube of DSS with a pipette tip and add 217 μL of DMSO or DMF to prepare a 10× stock solution (25 mM). Mix the solution thoroughly with the pipette tip until the DSS dissolves.

[0040] (2) Dilute DSS at a ratio of 1:100 in DMSO or DMF (10 μL of 10×DSS is added to 990 μL of solvent) to make the DSS concentration 0.25 mM.

[0041] (3) Add 2.5 μL of 20× coupling buffer, 4 μL of 0.25 mM DSS and 43.5 μL of ultrapure water to the magnetic beads, making the final solution volume 50 μL. The added DSS is 10× in excess to the Pierce protein A / G magnetic beads, with a working concentration of 20 μM.

[0042] (4) At room temperature, incubate the cross-linking reaction for 30 min with a vortexer or stirrer. During the incubation process, gently vortex the magnetic beads every 5-10 min to keep the magnetic beads in a suspended state.

[0043] (5) Collect the magnetic beads with a magnetic rack, remove and retain the liquid to confirm antibody cross-linking.

[0044] (6) Add 100 μL of elution buffer to the magnetic beads and gently mix on a vortex mixer for 5 min at room temperature to remove uncrosslinked antibodies and terminate the crosslinking reaction. Collect the magnetic beads with a magnetic rack and remove the supernatant.

[0045] (7) Add 100 μL of elution buffer to the magnetic beads and gently vortex or invert to mix. Collect the magnetic beads using a magnetic rack and remove the supernatant. Repeat once.

[0046] (8) Add 200 μL of pre-chilled immunoprecipitation lysis / wash buffer to the magnetic beads and gently vortex or invert to mix. Collect the magnetic beads using a magnetic rack and remove the supernatant. Repeat once.

[0047] 3. Tissue lysis Add pre-chilled immunoprecipitation lysis / wash buffer to the homogenized tissue. Use 500 μL of immunoprecipitation lysis / wash buffer per 50 mg of tissue (i.e., a 10:1 volume / mass ratio), and incubate on ice for 10 min, mixing several times during incubation. Centrifuge at 13000 rpm for 10 min to remove tissue and cell debris, transfer the supernatant to a new tube, and determine the protein concentration for subsequent experiments.

[0048] 4. Manual antigen immunoprecipitation (1) Dilute the protein lysis buffer from the previous step to 500 μL with immunoprecipitation lysis / wash buffer.

[0049] (2) Add 500 μL of the diluted protein lysis buffer to a centrifuge tube containing antibody cross-linked magnetic beads. Incubate overnight at 4°C on a rotary oscillator or mixer.

[0050] (3) Collect magnetic beads with a magnetic rack, remove unbound samples, and store them for analysis.

[0051] (4) Add 500 μL of immunoprecipitation lysis / wash buffer to the centrifuge tube, mix gently, collect the magnetic beads, and discard the supernatant. Repeat this operation once.

[0052] (5) Add 500 μL of ultrapure water to the tube and mix gently. Collect the magnetic beads with a magnetic rack and discard the supernatant.

[0053] (6) Add 30-50 μL of 1× protein loading buffer to the magnetic beads, boil in boiling water for 10 min, and collect the supernatant with a magnetic rack. This step can be repeated once to obtain more samples.

[0054] (7) The following steps are the same as those for Western Blot.

[0055] Results: We found a significant enrichment of palmitoylation signals in the transcriptomic results of the livers of PD2 group mice. Figure 5 Furthermore, palmitoylation level detection revealed a significant decrease in palmitoylation levels in the PD2 group. Palmitoylation is a common post-translational modification of proteins, one of its functions being the regulation of protein stability: protecting proteins from proteasome degradation or accelerating degradation. We then performed palmitoylation modification proteomics (…). Figure 6 The study found that PD2 significantly downregulated palmitoylation of proteins in liver tissue, with notable downregulation observed in FASN, ALB, CAST, PITHD1, NES, STRN4, KHSRP, TSGA10, and TARDBP. Since FASN is a key protein related to lipid metabolism, the subsequent experiments focused on FASN to analyze the regulatory effect of PD2 on palmitoylation.

[0056] Palmitoylation modification relies on the action of palmitoylation transferases. We investigated the effects of PD2 on the protein expression of palmitoylation and depalmitoylation transferases. From transcriptomics results, we screened for relevant palmitoylation transferases: ZDHHC20, ZDHHC21, ZDHHC5, ZDHHC2, and ZDHHC23, as well as two depalmitoylation transferases, ABHD17A and PPT1. Western blotting analysis revealed that only palmitoylation transferases ZDHHC23 and ZDHHC5 showed a significant decrease in protein expression in the PD2 group. Figure 7 ).

[0057] Example 3. PD2 inhibits the interaction between ZDHHC23 and FASN proteins. I. Molecular-protein docking: The Autodock Vina software was used to search for possible interaction modes between the ligand and protein. First, the crystal structure of the target protein was obtained from the Uniprot database; simultaneously, the SMILES sequence of Platycodin D2 was obtained from the Pubchem database and converted into its spatial structure using RDKit. Then, the AutoDock Tools tool was used to add hydrogen atoms to the protein to refine its chemical structure and ensure the accuracy of subsequent docking. Next, a 100×100×100 Å grid was set to define the search area for molecular docking. Finally, Autodock Vina was run to perform docking calculations. After docking, the conformation with the lowest binding energy was selected from numerous docking results for in-depth analysis. This conformation was considered to be the most likely representative of the interaction mode between the ligand and protein under real physiological conditions, providing crucial evidence for further elucidating the interaction mechanism.

[0058] II. Acyl-Biotin Exchange (ABE) Method for Determining Palmitoylation Palmitoylation of FASN protein was determined using the Immuno-Precipitation-ABE Kit (catalog number AM10314, brand AIMSMASS) for Western blotting. The following steps were performed on the protein sample prepared by immunoprecipitation.

[0059] Collect at least 500 μg of total protein (liver tissue samples from the model group and PD2 group), grind the tissue, add RIPA lysis buffer, and lyse on ice for 10 min. Then centrifuge at 14000 rpm for 5 min, discard the precipitate, and quantify the supernatant protein using a BCA protein quantification kit. Perform immunoprecipitation of the target protein. After the IP process, place the EP tube on a magnetic rack, wait 10 s, discard the supernatant, and obtain the protein eluent according to the Immuno-Precipitation-ABE Kit procedure. Take an equal volume of the protein eluent for subsequent Western blotting experiments.

[0060] III. Protein-protein docking Protein-protein molecular docking was performed using the HDOCK program (http: / / hdock.phys.hust.edu.cn / ). The HDOCK server is a hybrid docking platform integrating template-based and ab initio computation methods. Prior to docking, the protein structure was processed by removing heteroatoms and optimizing the hydrogen bond network. Using default parameters and a rotation step size of 15°, 100 potential binding conformations were generated through global sampling based on Fast Fourier Transform. The resulting complexes were energy-ranked using ITScorePP statistical potential, and the conformation with the highest score was selected for subsequent analysis. Three-dimensional structural visualization was performed using Pymol, and LigPlot+ generated two-dimensional interaction maps detailing hydrogen bonds (<3.5 Å) and hydrophobic interactions.

[0061] Results: 1. In our Co-IP experiment, we first used FASN protein for IP incubation, and then incubated ZDHHC5 and ZDHHC23 antibodies respectively. The results showed that there was an interaction between ZDHHC23 protein and FASN protein, and PD2 could weaken the interaction between them, while there was no interaction between ZDHHC5 and FASN protein. Figure 8 ).

[0062] 2. PD2 downregulates palmitoylation levels in FASN protein We already know from palmitoylation transcriptomics results that PD2 downregulates the palmitoylation level of FASN protein. We then further validated this using IP-ABE experiments. We first enriched FASN protein with IP, and then measured the palmitoylation level of FASN protein. HAM, an abbreviation for hydroxylamine, plays a crucial role in specifically cleaving the thioester bond between palmitic acid and thiol groups on cysteine ​​residues, releasing free thiol groups for subsequent labeling and enrichment. HAM is weakly basic, only cleaving the thioester bond (a characteristic bond of palmitoylation), without affecting disulfide bonds or other amide bonds, ensuring the specificity of the palmitoylation signal. We first blocked free thiol groups in the sample with NEM, then treated it with HAM (+HAM group) to remove the palmitoyl group and expose the thiol group at the modification site; the untreated group (-HAM) served as a negative control to exclude non-specific signals. After HAM treatment, the newly released thiol groups were labeled with biotinylated reagents, enriched with streptavidin, and then detected by Western blotting or mass spectrometry. Experimental results showed that the palmitoylation level of FASN protein was significantly downregulated in the PD2 group. Figure 9 ).

[0063] 3. Results of molecular-protein docking between PD2-ZDHHC23 and ZDHHC23-FASN To investigate the direct effect of PD2 on ZDHHC23, PD2-ZDHHC23 molecular docking was performed using software. During the docking simulation, the software calculated the binding energy between the ligand molecule PD2 and the ZDHHC23 protein, with the lowest binding energy reaching -8.0 kcal / mol. Figure 10In the field of molecular docking, binding energy is an important indicator of the tightness of intermolecular binding. Generally, the lower the binding energy, the tighter the binding and the stronger the interaction. A binding energy value of -8.0 kcal / mol strongly indicates a tight binding relationship between the ligand molecule PD2 and the ZDHHC23 protein. This tight binding may be achieved through various intermolecular forces, such as hydrogen bonds, van der Waals forces, and hydrophobic interactions. These forces work together to allow the ligand molecule to stably attach to the surface of the ZDHHC23 protein. The 2D diagram visualizes the binding between the ligand molecule and the protein. Red eyelash-shaped amino acid residues indicate hydrophobic interactions with the ligand molecule, while green text showing the specific molecular structure of the amino acid indicates hydrogen bond interactions with the ligand molecule. Hydrogen bonds are represented by green dashed lines, and the numbers in the middle represent the hydrogen bond length in Å (10 Å = 1 nm, 10 angstroms = 1 nanometer). In exploring the interaction between PD2 and ZDHHC23 proteins, molecular docking simulations and subsequent structural analysis revealed that several key amino acid residues in the ZDHHC23 protein—Glu382, Tyr367, Gln386, Arg390, His132, His288, and His287—play a crucial role in their binding process.

[0064] Next, we used software to perform protein-protein docking between ZDHHC23 and FASN proteins, further demonstrating the interaction between the two proteins. The results are as follows... Figure 11 The interaction between the ZDHHC23 and FASN proteins is crucial for maintaining the stability of the protein-protein complex. Specifically, six key amino acid residues on the ZDHHC23 protein—His109, Tyr338, His104, Trp55, Leu42, and Asp49—form hydrogen bonds with amino acid residues on the FASN protein—Glu1525, Arg1552, Glu1526, Glu1130, Arg1171, Thr1150, and Gln1154, respectively. These hydrogen bonds play a key role in maintaining the structural stability of the protein-protein complex. In addition to hydrogen bonding, numerous hydrophobic interactions also provide important support for the stable binding of the ZDHHC23 and FASN proteins. Among these hydrophobic interactions, the Cys1186 residue of the FASN protein is particularly active, contributing to the protein-protein binding.

Claims

1. Application of Platycodon saponin D2 in the preparation of drugs for treating or alleviating fatty liver.

2. The application according to claim 1, characterized in that, Fatty liver is non-alcoholic fatty liver disease.

3. The application according to claim 1, characterized in that, The dosage forms of the drug are liquid, suppository, tablet, powder or ointment.

4. The application according to claim 1, characterized in that, Platycodon saponin D2 is the active ingredient of palmitoyltransferase inhibitor.

5. The application according to claim 4, characterized in that, Palmitoyltransferase is palmitoyltransferase ZDHHC23.

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