Use of adugamycin H for the preparation of a medicament for the prevention and / or treatment of metabolic-related fatty liver disease

Abstract

The present application provides the use of Adaglicin H in the preparation of a drug for preventing and / or treating metabolic-related fatty liver disease, wherein the Adaglicin H has a structure as shown in formula (I). The present application first discloses that Adaglicin H has a dual activity of simultaneously improving liver cell lipid deposition and inhibiting liver stellate cell activation, can significantly reduce the transaminase and NAS score of an animal model, reduce lipid accumulation and collagen deposition, reverse the pathological damage of MASH in an all-round way, and has low toxicity and wide safety window, thereby providing a new technical scheme for MASH treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. Specifically, this invention relates to the use of adegamycin H in the preparation of a medicament for the prevention and / or treatment of metabolic-related fatty liver disease. Background Technology

[0002] As one of the most important risk factors for cirrhosis and hepatocellular carcinoma, metabolic dysfunction-associated steatohepatitis (MASH) is a chronic liver disease characterized by abnormal lipid accumulation in hepatocytes, persistent inflammatory response, hepatocyte damage, and progressive fibrosis. Currently, the global prevalence of MASH is showing an increasing trend and a younger age of onset. It is estimated that by 2030, the number of MASH patients worldwide will exceed 490 million, becoming a serious public health problem that endangers human health. The pathological process of MASH mainly involves three key links: (1) lipid deposition caused by disordered lipid metabolism in hepatocytes; (2) persistent inflammatory response mediated by hepatic macrophages; and (3) abnormal activation and proliferation of hepatic stellate cells (HSCs). Among them, excessive lipid deposition in hepatocytes is the initiating factor for the occurrence of MASH. It induces excessive production of reactive oxygen species (ROS), leading to a series of cellular stress responses such as DNA damage, protein oxidation, and lipid peroxidation, ultimately causing hepatocyte damage and death. Liver fibrosis is a key marker of poor prognosis in patients with metastatic liver injury (MASH). Its prominent feature is the excessive deposition of the extracellular matrix (ECM), particularly collagen, leading to progressive damage to the normal physiological structure and function of the liver. Notably, when hepatocytes suffer damage such as lipid deposition, the intracellular NOD-like receptor protein 3 (NLRP3) inflammasome is activated, promoting HSC activation through the secretion of inflammatory factors such as TNFα and IL-1β. Activated HSCs proliferate extensively and secrete ECM, forming a positive feedback loop that exacerbates the pathological process of MASH. Therefore, intervention strategies that simultaneously improve lipid degeneration and liver fibrosis are a key focus of current anti-MASH drug development.

[0003] Nearly one hundred candidate drugs with different mechanisms of action have entered clinical trials to address the aforementioned pathological processes, such as the farnesoid X receptor (FXR) agonist obeticholic acid and the fatty acid synthase (FASN) inhibitor TVB-2640. However, these candidate drugs have generally shown limited efficacy or significant toxic side effects in clinical trials. Although semaglutide and resmetiro have recently been approved for adjuvant treatment in MASH patients, their benefits are essentially indirect, achieved by improving lipid metabolism, and they suffer from limitations such as restricted patient populations (semaglutide is limited to patients with F2-F3 stage fibrosis) and low clinical response rates (resmetiro's response rate is only 25%). Therefore, there is an urgent need to develop a novel therapeutic drug that can effectively intervene in the pathological process of MASH, especially one that combines the effects of improving lipid deposition and anti-liver fibrosis.

[0004] Aldgamycins are a class of natural sixteen-membered macrocyclic lactones produced by actinomycetes. Their parent nucleus is linked to β-D-mycinose at position C-20 via a glycosidic bond, and to position C-5 with a rare aldose branch, octose, namely D-aldgarose or decarboxylated D-aldgarose. Since adegamycin E was first isolated from *Streptomyces spp.* in 1964, fewer than 20 members of this family have been reported. Existing reports mainly focus on the antibacterial activity of these compounds. Studies have shown that most adegamycin compounds exhibit good inhibitory activity against Gram-positive bacteria. For example, adegamycin G, when administered orally to a mouse model of *Staphylococcus aureus* infection, showed efficacy comparable to josamycin and erythromycin, demonstrating the good in vivo safety and drug-like characteristics of this class of compounds.

[0005] As a member of the adegamycin family, Aldgamycin H (Ald-H) has received extremely limited research reports. Only US2007 / 0202574A1 describes the isolation of Aldgamycin H and its inhibitory activity against some Gram-positive bacteria (Micrococcus luteus, Bacillus subtilis, and Mycobacterium smegmatum). To date, research on adegamycin compounds has been limited to the antibacterial field. Summary of the Invention

[0006] The present invention aims to develop new pharmaceutical uses for adegamycin H, particularly its application in the preparation of drugs for the treatment and / or prevention of metabolic dysfunction-related steatohepatitis and / or liver fibrosis, in order to address the shortcomings of existing MASH treatment drugs, such as limited efficacy and significant side effects.

[0007] Therefore, the object of this invention is to provide a new treatment for metabolic-related fatty liver disease.

[0008] The above-mentioned objective of the present invention is achieved by providing the following technical solution:

[0009] This invention provides the use of adegamycin H in the preparation of medicaments for the prevention and / or treatment of metabolic-associated fatty liver disease.

[0010] Adegamycin H is a macrolide compound with the structure shown in formula (I):

[0011] .

[0013] According to some embodiments of the present invention, the metabolic-associated fatty liver disease is selected from one or more of metabolic dysfunction-associated steatohepatitis (MASH), liver injury, liver fibrosis, cirrhosis, hepatocellular carcinoma, and liver failure.

[0014] According to some embodiments of the present invention, the prevention and / or treatment are to block hepatic steatosis, inflammation and / or fibrosis, and / or to improve hepatic lipid accumulation, liver injury, inflammation and / or reverse liver fibrosis.

[0015] According to some embodiments of the present invention, the drug further comprises pharmaceutically acceptable excipients.

[0016] Preferably, the pharmaceutically acceptable excipient is selected from one or more of surfactants, preservatives, antioxidants, hardeners, thickeners, and absorption enhancers.

[0017] According to some embodiments of the present invention, the dosage form of the drug is selected from one or more of tablets, capsules, suspensions, syrups, granules, creams, ointments, pills, powders, elixirs, suppositories, creams, drop pills, gels, patches, sprays, aerosols, oral liquids, and injections.

[0018] According to some embodiments of the present invention, the adegamycin H is obtained by fermentation of Saccharothrix sp. strain 16Sb2-4. The preservation information of Saccharothrix sp. strain 16Sb2-4 is as follows: preservation number is CGMCC No. 39500, preservation date is June 29, 2026, and the depository institution is China General Microbiological Culture Collection Center.

[0019] Preferably, the preparation method of adegamycin H includes the following steps:

[0020] (1) The Saccharothrix sp. strain 16Sb2-4 was fermented and cultured, and the fermentation broth was collected;

[0021] (2) The fermentation broth is centrifuged, the supernatant is extracted with an organic solvent, the organic phase is collected, and the crude extract is obtained by dehydration and concentration.

[0022] (3) The crude extract was purified by gel chromatography, and the eluent containing adegamycin H was collected to obtain the first eluent.

[0023] (4) The first elution fraction is eluted by reverse phase chromatography gradient elution, and the elution fraction containing adegamycin H is collected to obtain the second elution fraction;

[0024] (5) The second elution fraction was purified by high performance liquid chromatography, and the adegamycin H fraction was collected, concentrated and dried to obtain adegamycin H.

[0025] According to some embodiments of the present invention, in step (1), the fermentation culture includes: inoculating the Saccharothrix sp. strain 16Sb2-4 into ISP 2 medium and culturing it at a constant temperature of 28°C for 7 days to prepare a plate culture; inoculating the plate culture into ISP 2 liquid medium and culturing it at 28°C and 180 rpm for 3 days to obtain a seed culture; transferring the seed culture into ISP 2 liquid medium and culturing it at 28°C and 180 rpm for 7 days to collect the fermentation broth.

[0026] According to some embodiments of the present invention, in step (2), the organic solvent is ethyl acetate, and the extraction is performed 2-4 times; preferably, the dehydration is performed using anhydrous sodium sulfate; preferably, the concentration is performed under reduced pressure.

[0027] According to some embodiments of the present invention, in step (3), the gel chromatography uses a Sephadex LH-20 gel chromatography column and methanol as the eluent.

[0028] Preferably, in step (4), the reversed-phase chromatography uses an ODS reversed-phase chromatography column, the elution solvent is a methanol-water mixed solution, and the elution gradient is successively 20%, 40%, 60%, 70% and 100% methanol aqueous solution by volume fraction.

[0029] According to some embodiments of the present invention, in step (5), the chromatographic column of the high performance liquid chromatography is an Agilent ZORBAX SB-C18 column with a specification of 9.4 mm × 250 mm and a packing particle size of 5 μm.

[0030] Preferably, the mobile phase is a 50% (v / v) methanol aqueous solution, and the flow rate is 2.0 mL / min.

[0031] Compared with the prior art, the present invention has at least the following beneficial effects:

[0032] This invention reveals for the first time the novel use of adgamycin H in the preparation of drugs for the prevention and / or treatment of metabolic-associated steatohepatitis (MASH) and its associated liver injury and fibrosis. Experimentally, this invention demonstrates that this compound can simultaneously act on two key pathological stages in the MASH disease progression in vitro: firstly, in an oleic acid-palmitic acid-induced human hepatocyte L02 lipid deposition model, adgamycin H significantly reduces intracellular neutral lipid accumulation and upregulates the expression of the lipid peroxidation gene PPARγ, effectively reversing hepatocyte steatosis; secondly, in a TGF-β1-induced human hepatic stellate cell LX2 activation model, this compound significantly downregulates the protein and mRNA expression levels of fibrosis markers such as COL1A1, α-SMA, and TGF-β1, directly inhibiting abnormal activation of hepatic stellate cells. This dual activity of simultaneously improving lipid metabolism disorders and blocking the initiation of fibrosis is difficult to achieve with existing candidate drugs that target only a single pathological stage, possessing unique technical advantages.

[0033] In terms of in vivo efficacy validation, this invention used a classic MASH model in C57BL / 6 mice induced by a high-fat, high-cholesterol diet. After oral administration of adegamycin H at a dose of 100 mg / kg, the compound demonstrated a comprehensive and definitive effect in reversing MASH pathological damage. The gross hepatic lesions induced by the high-fat diet in the treatment group were significantly improved, with marked relief of hepatomegaly and granular lesions, and a significant decrease in the abnormally elevated liver weight / body weight ratio. Simultaneously, serum ALT and AST transaminase levels in the model mice were significantly reduced, fully demonstrating the excellent in vivo hepatoprotective effect of this compound. Histopathological examination further corroborated this, showing that adegamycin H significantly reduced hepatocyte ballooning degeneration, significantly decreased NAS activity scores, and comprehensively improved the complex histological damage to the liver induced by MASH. Oil Red staining of liver tissue and quantitative detection of intrahepatic triglycerides demonstrated that this compound can effectively reverse the massive accumulation of lipids in the liver caused by a high-fat, high-cholesterol diet. Furthermore, collagen-specific Masson's staining and fibrosis marker detection results showed that adegamycin H can significantly reduce collagen fiber deposition in liver tissue, effectively inhibit the continued progression of liver fibrosis, and block the development of MASH into cirrhosis. Its oral administration mode of action is also more suitable for routine clinical drug use, and it has high potential for formulation conversion.

[0034] The safety evaluation results of this invention show that, at doses with significant therapeutic effects, adegamycin H does not have a significant negative impact on the body weight of mice, indicating that the compound has low in vivo toxicity and a wide oral safety window.

[0035] In summary, the novel pharmaceutical use of adegamycin H disclosed in this invention provides a new technical solution for developing MASH therapeutic drugs with multiple effects such as lipid-lowering, liver protection, anti-inflammatory, and anti-fibrotic properties. It effectively overcomes the shortcomings of existing MASH therapeutic drugs, which have a single target and cannot address multiple pathological damages simultaneously. It has outstanding clinical application value and promising prospects for drug industrialization. Attached Figure Description

[0036] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0037] Figure 1 The adegamycin H (compound shown in Formula I) of this invention 1 H NMR spectrum.

[0038] Figure 2 The adegamycin H (compound shown in Formula I) of this invention 13 C NMR spectrum.

[0039] Figure 3 This study demonstrated that adegamycin H inhibited lipid deposition activity in an in vitro L02 cell model. Figure 3 A shows the staining results of L02 cells induced with OA (0.1 mM) + PA (0.1 mM) and treated with adegamycin H (40 µM) for 24 hours, followed by Oil Red and Nile Red staining. Figure 3 B shows the statistical results of Oil Red staining; Figure 3 C shows that L02 cells were induced with OA (0.1 mM) + PA (0.1 mM) and simultaneously treated with adegamycin H (0 µM, 20 µM, 40 µM) for 24 hours, and the expression level of PPARγ was detected by Western blotting. Figure 3 D shows L02 cells induced with OA+PA and simultaneously treated with adegamycin H (40 µM) for 24 hours, with qRT-PCR used to detect PPARG mRNA levels. N=3, * p<0.05; # p<0.05.

[0040] Figure 4 This study demonstrated that adegamycin H inhibited hepatic stellate cell activation in an in vitro LX2 cell model. LX-2 cells were treated with serum-free starved adegamycin H (20 µM, 40 µM, 60 µM, and 80 µM) for 24 hours, followed by induction with TGF-β1 (2 ng / mL) for another 24 hours. Figure 4 A shows the expression levels of COL1A1, α-SMA, and TGF-β1 as detected by Western blot. Figure 4B shows the mRNA levels of TGFB1 and COL1A1 detected by qRT-PCR. N=3. * p<0.05 vs COL1A1 group; # p<0.05 vsTGF-β1 group.

[0041] Figure 5 The study showed that adegamycin H improved liver structure in HFHC-induced C57BL / 6 mice. Among other things, Figure 5 A shows a schematic diagram of the MASH mouse model constructed using the HFHC diet; Figure 5 B shows the body weight of mice in the NCD control group, HFHC model group, and HFHC+Ald-H treatment group (100 mg / kg); Figure 5 C shows the liver morphology of mice in the NCD control group, HFHC model group, and HFHC+Ald-H treatment group (100 mg / kg); Figure 5 D shows the liver weight / body weight of the mouse. N=6. ** p<0.01 vs NCD group; ## p<0.01 vs HFHC group.

[0042] Figure 6 The results showed that adegamycin H improved serum markers and liver pathology in HFHC-induced C57BL / 6 mice. Figure 6 A shows the levels of ALT and AST in the serum of MASH mice; Figure 6 B shows a representative schematic diagram of H&E staining in liver tissue; Figure 6 C shows the results of balloon-like deformation scoring based on H&E staining; Figure 6 D shows the NAS score, which integrates lipid degeneration, ballooning degeneration, and inflammation. N=6. ** p<0.01 vs NCD group; # p<0.05, ## p<0.01 vs HFHC group.

[0043] Figure 7 This study demonstrated that adegamycin H improved HFHC-induced lipid deposition in C57BL / 6 mice. Among other things, Figure 7 A shows a representative schematic diagram of Oil Red staining of liver tissue; Figure 7 B shows the results of lipid denaturation scoring based on Oil Red staining results; Figure 7 C shows the level of total triglycerides (TG) detected in liver tissue; Figure 7 D shows the total protein extracted from liver tissue, and the expression level of the lipid metabolism-related protein PPARγ was detected by Western blotting. N=6. * p<0.05,** p<0.01 vsNCD group; # p<0.05, ## p<0.01 vs HFHC group.

[0044] Figure 8 This study demonstrated that adegamycin H inhibited HFHC-induced fibrosis progression in C57BL / 6 mice. Figure 8 A shows a representative schematic diagram of Masson staining in liver tissue; Figure 8 B shows the total RNA extracted from liver tissue, and the expression of fibrosis-related genes (Col1a1, Tgfb1, and Acta2) detected by qRT-PCR; Figure 8 C shows the total protein extracted from liver tissue, and the expression levels of fibrosis-related proteins (COL1A1, α-SMA, and TGF-β1) were detected by Western blotting. N=6. * p<0.05 vs NCD group; # p<0.05 vs HFHC group. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0046] Example 1: Fermentation of Saccharothrix sp. strain 16Sb2-4

[0047] The strain used for fermentation in this embodiment is Saccharothrix sp. 16Sb2-4, and its preservation information is as follows: preservation number is CGMCC No. 39500, preservation date is June 29, 2026, and the depository is China General Microbiological Culture Collection Center.

[0048] Saccharothrix sp. 16Sb2-4 was inoculated onto ISP 2 agar plates. The agar medium was prepared by mixing 4.0 g glucose, 4.0 g yeast extract, 10.0 g malt extract, 15.0 g agar, and 1.0 L deionized water. After adjusting the pH to 7.2, the medium was sterilized at 121°C for 15 minutes. After inoculation, the plates were incubated at 28°C for 7 days. Under aseptic conditions, mycelia and spores were scraped from the surface of the plates using a sterile bamboo stick and inoculated into 500 mL Erlenmeyer flasks containing 100 mL of ISP 2 liquid medium. The flasks were then placed in a shaker and incubated at 28°C and 180 rpm for 3 days to prepare the seed culture.

[0049] Take 100 mL of the above seed culture and transfer it to a 5 L Erlenmeyer flask containing 1 L of ISP 2 liquid culture medium. The total volume of the entire fermentation system is 15 L. Place all Erlenmeyer flasks on a shaker and culture them at 28 ℃ and 180 rpm for 7 days. After the culture is completed, collect the fermentation broth.

[0050] Example 2: Extraction, purification and identification of adegamycin H

[0051] The fermentation broth prepared in Example 1 was centrifuged. The supernatant was extracted three times with equal volumes of ethyl acetate (15 L × 3). All organic phases were combined, dehydrated and dried with anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain 0.9 g of a brownish crude extract. The crude extract was thoroughly dissolved in a small amount of methanol and then loaded onto a Sephadex LH-20 gel chromatography column (2.5 cm × 120 cm). Elution was performed with pure methanol at a flow rate of approximately 1 mL / min, and the eluent was collected in 15 mL tubes. All eluted fractions were analyzed by LC-MS and combined to obtain four fractions, labeled Ga, Gb, Gc, and Gd, where Gb is the target fraction containing adegamycin H.

[0052] The Gb fraction was loaded onto a 120g Biotage SNAP Cartridge KP-C18-HS reversed-phase ODS column and subjected to gradient fractional elution on a medium-pressure Flash rapid purification system at a flow rate of 20 mL / min. The elution solvent was a methanol-water mixture with volume fraction gradients of 20%, 40%, 60%, 70%, and 100%, yielding 10 sub-fractions labeled Gb1-Gb10. All sub-fractions were screened by LC-MS, and fractions Gb5-Gb8 were found to contain the target product. These effective fractions were combined and prepared by HPLC. The preparative chromatographic conditions were as follows: an Agilent ZORBAX SB-C18 column (9.4 mm × 250 mm, 5 μm particle size) was used; the mobile phase was 50% methanol-water solution at a flow rate of 2.0 mL / min; and the UV detection wavelength was set to 254 nm. The UV characteristic absorption fraction corresponding to a retention time of 43 minutes was collected, and the collected liquids were combined and the methanol solvent was removed by rotary evaporation. The remaining aqueous solution was freeze-dried to finally obtain 7.8 mg of pure adegamycin H.

[0053] Measurements were performed using a 600 MHz nuclear magnetic resonance spectrometer. 1 H NMR and 13C13 NMR spectra were obtained using deuterated chloroform (CDCl3) as solvent and tetramethylsilane (TMS) as internal standard. The 1H NMR was measured at 600 MHz, and the 13C NMR was measured at 150 MHz (broadband proton decoupling). The results are as follows:

[0054] 1 H NMR (600 MHz, CDCl3) δ 6.62 (dd, J = 15.4, 10.7 Hz, 1H), 6.56 (d, J= 15.4 Hz, 1H), 6.41 (dd, J = 15.4, 9.3 Hz, 1H), 5.80 (d, J = 15.4 Hz, 1H), 5.34 (dq, J = 12.4, 6.2 Hz, 1H), 4.60 (d, J = 7.6 Hz, 1H), 4.56 (d, J = 7.7Hz, 1H), 4.16 (dd, J = 10.1, 2.9 Hz, 1H), 3.95 - 3.87 (m, 1H), 3.75 (t, J =2.8 Hz, 1H), 3.68 - 3.64 (m, 1H), 3.63 (dd, J = 10.3, 3.0 Hz, 1H), 3.60 (s,3H), 3.60 - 3.58 (m, 1H), 3.54 (s, 3H), 3.52 (dd, J = 9.1, 6.3 Hz, 1H), 3.33(d, J = 10.2 Hz, 1H), 3.26 (d, J = 9.3 Hz, 1H), 3.21 - 3.16 (m, 1H), 3.09 -3.05 (m, 2H), 2.83 (s, 1H), 2.74 - 2.66 (m, 1H), 2.62 - 2.55 (m, 1H), 2.37(s, 1H), 1.63 - 1.53 (m, 2H), 1.53 - 1.47 (m, 1H), 1.37 (dd, J = 10.5, 5.7Hz, 2H), 1.32 (d, J = 6.3 Hz, 3H), 1.29 - 1.23 (m, 6H), 1.23 - 1.17 (m, 4H), 1.16 (d, J = 6.2 Hz, 3H), 1.12 (d, J = 6.9 Hz, 3H), 0.97 (d, J = 6.8 Hz, 2H); 13C NMR (150 MHz, CDCl3) δ 200.97, 165.42, 151.16, 144.13, 125.63, 120.64,101.76, 101.02, 86.70, 81.98, 79.73, 74.10, 73.76, 72.79, 72.59, 70.82,68.79, 67.13, 66.97, 61.80, 59.78, 59.13, 59.00, 49.56, 44.72, 41.87, 39.38,34.18, 32.10, 20.88, 18.97, 18.51, 18.23, 17.88, 17.72, 17.08.

[0055] 1 H NMR spectrum and 13 The C NMR spectra are as follows: Figure 1 and Figure 2 As shown.

[0056] Example 3: Pharmacological Study

[0057] (I) Experimental Methods

[0058] 1. Oil Red O staining test

[0059] This experiment used oleic acid (OA) combined with palmitic acid (PA) to induce human hepatocyte L02 cells (purchased from the ATCC cell bank) to establish an in vitro model for evaluating the activity of compounds in improving cellular lipid deposition. This model is a classic cell model for screening lipid-lowering active compounds. Oil Red staining is a classic staining technique that can specifically label intracellular triglycerides, cholesterol esters, and other neutral lipids, and is also commonly used as a specific staining method for cellular steatosis. The experimental procedure was as follows: L02 cells were treated with a final concentration of 0.1 mMOA and 0.1 mM PA for 24 hours, followed by continuous intervention with adegamycin H (Ald-H) prepared in Example 2 for 24 hours. After drug treatment, Oil Red staining was performed using the following method:

[0060] (1) Fixation: Use 4% paraformaldehyde (PFA) solution to fix the cell samples for 10-30 minutes;

[0061] (2) Rinsing: After the fixation operation is completed, gently rinse the cell sample with distilled water 2-3 times to remove residual fixative;

[0062] (3) Pre-infiltration: The cells were then rapidly infiltrated with 60% isopropanol for 1-2 minutes.

[0063] (4) Staining: After soaking, add freshly filtered Oil Red O working solution and stain for 10-20 minutes at room temperature in the dark;

[0064] (5) Differentiation: After staining, rinse the sample quickly with 60% isopropanol for a few seconds until the stained background is clear and free of impurities;

[0065] (6) Rinsing: Gently rinse the cell sample 2-3 times with distilled water;

[0066] (7) Mounting and observation: Add water-based mounting medium to complete the mounting. Immediately after mounting, place the slide under a microscope to observe the cell staining state and take micrographs.

[0067] 2. Nile Red staining test

[0068] Nile red is a high-performance lipid fluorescent dye. When it binds to neutral lipids such as triglycerides, it exhibits bright golden-yellow or red fluorescence with low background interference. In this experiment, human hepatocytes L02 were induced with a final concentration of 0.1 mM oleic acid and 0.1 mM palmitic acid for 24 hours. Then, adegamycin H prepared in Example 2 was added to further intervene in the cells for another 24 hours. After drug treatment, Nile red staining was performed according to the following procedure:

[0069] (1) Remove the original culture medium from the cells and add preheated fresh culture medium with a final Nile Red concentration of 1-2 μg / mL to the cell culture system;

[0070] (2) Incubate the cells at 37°C in the dark for 10-30 minutes;

[0071] (3) After incubation, replace the culture medium with a fresh one, wash off any unbound free dye on the cell surface, and then place the cells under a microscope to observe the fluorescence staining and take microscopic images.

[0072] 3. Protein sample lysis treatment

[0073] (1) Cell sample lysis procedure

[0074] Remove the original culture medium from the cells. Gently wash the cells once with phosphate-buffered saline (PBS) in a culture plate or dish. Then add cell lysis buffer (purchased from Shanghai Beyotime Biotechnology Co., Ltd., P0013) containing protease inhibitors and phosphatase inhibitors, and incubate on ice for 30 minutes to fully lyse the cells. Use a cell scraper to scrape off the cells and collect them into 1.5 mL centrifuge tubes. Centrifuge the tubes at 4 ℃ and 12000 rpm for 20 minutes. After centrifugation, transfer the supernatant to a brand new EP tube for later use.

[0075] (2) Animal tissue sample lysis procedure

[0076] Remove the tissue block stored at -80 ℃, quickly weigh approximately 100 mg of tissue block and place it in a glass homogenizing tube. Add 1 mL of pre-chilled cell lysis buffer containing protease and phosphatase inhibitors to each tube. Use a handheld high-speed homogenizer to homogenize thoroughly for about 1 minute until no visible lumps of tissue remain in the tube, forming a homogenized tissue homogenate. Place the homogenate on ice and let it stand for 30 minutes to complete lysis. After lysis, centrifuge at 4 ℃ and 12000 rpm for 10 minutes, and transfer the supernatant obtained from centrifugation to a brand new EP tube for later use.

[0077] 4. Western blot analysis of proteins

[0078] This technology enables the specific analysis of target proteins through protein extraction, electrophoresis, membrane transfer, and antibody detection.

[0079] (1) First, the protein concentration was determined by the BCA method (BCA protein concentration assay kit, Beyotime). The protein concentration of the cells or animal tissue samples that had been lysed was then quantified.

[0080] (2) Mix equal amounts of protein samples with SDS-PAGE loading buffer and boil at 95-100 ℃ for 5 minutes to denature and reduce the protein.

[0081] (3) Add protein molecular weight marker and perform SDS-PAGE electrophoresis.

[0082] (4) Transfer the electrophoretically separated proteins onto a PVDF membrane. After the transfer is complete, block the non-specific binding sites with blocking buffer (PBS-T).

[0083] (5) The PVDF membrane was incubated overnight at 4°C with the specific primary antibody (as shown in Table 1) in PBS-T buffer. The membrane was washed 3 times with PBS-T, and HRP-labeled secondary antibody (as shown in Table 1) was added and incubated at room temperature for 1 hour to form a complex with the primary antibody.

[0084] (6) Use chemiluminescent substrate (ECL) to detect the secondary antibody labeling signal and obtain the target protein band.

[0085]

[0086] 5. Triglyceride (TG) level detection

[0087] The triglyceride content was determined using an enzymatic assay kit (Beijing Pulilai Gene Technology Co., Ltd., E1025-105). The kit was operated according to the instructions as follows:

[0088] (1) Accurately weigh the liver tissue sample, cut it into small pieces, add 500 μL of lysis buffer for every 10 mg of tissue, homogenize it using an electric high-speed homogenizer, let it stand at room temperature for 10 minutes, and then centrifuge it at 2000 rpm for 5 minutes. Take the supernatant to measure TG and protein concentration.

[0089] (2) Preparation of working solution for triglyceride detection: Calculate the volume of working solution required for the reaction of sample and standard. Mix the reagents evenly according to the ratio of R1:R2=4:1 to obtain the working solution. Use immediately or store at 4℃ for <1 day. Discard if discoloration occurs.

[0090] (3) Standard dilution: Dilute the 4 mM glycerol standard with distilled water or physiological saline to 1000, 500, 250, 125, 62.5, 31.25, 15.625 and 7.813 μmol / L. At the same time, set up a standard 0 concentration tube, i.e. a blank tube (distilled water + working solution).

[0091] (4) Triglyceride concentration determination:

[0092] Using a clean, transparent standard 96-well plate, add 10 μL of standard or sample to each well, along with 190 μL of triglyceride detection working solution. After thoroughly mixing the liquids in each well, incubate the plate in a 37°C water bath for 15 minutes. After the reaction is complete and the plate is cooled to room temperature, measure the OD value at 550 nm.

[0093] 6. LX2 cell model

[0094] This experiment used TGF-β1-induced human hepatic stellate cells (LX2, purchased from the ATCC cell bank) to establish an in vitro model for evaluating the inhibition of liver fibrosis progression by compounds. This model is a classic cell model for screening compounds with anti-fibrotic activity. The experimental procedure was as follows: LX2 cells were treated with TGF-β1 at a final concentration of 2 ng / mL for 24 hours, and then treated with adegamycin H prepared in Example 2 at concentrations of 20 µM, 40 µM, 60 µM, and 80 µM for another 24 hours. After drug treatment, protein samples were lysed and subsequent expression levels were detected.

[0095] 7. High-fat, high-cholesterol (HFHC) diet-induced MASH model in mice

[0096] Male C57BL / 6 mice aged 8-10 weeks (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used in the experiment. The animals were divided into three groups: a normal control diet (NCD) group, an HFHC model induction group, and an HFHC combined with adegamycin H (HFHC+Ald-H) treatment group. Mice in the NCD control group were fed a normal diet throughout the entire modeling period; mice in the HFHC model induction group and the HFHC+Ald-H treatment group were fed an HFHC diet (TD.12058) for 14 weeks. In the last four weeks of the modeling period, the HFHC+Ald-H treatment group received adegamycin H daily by gavage at a dose of 100 mg / kg.

[0097] On the day the modeling and drug administration cycle ended, all experimental mice were weighed, blood was collected and serum was separated for the detection of liver function biochemical indicators such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The liver tissue of the mice was dissected and removed. A portion of the liver tissue was fixed in 4% paraformaldehyde solution for subsequent pathological section preparation and histopathological observation. The remaining liver tissue was quickly frozen in liquid nitrogen and stored at -80℃ for subsequent related tests.

[0098] This animal testing protocol has been reviewed and approved by the Animal Ethics Committee of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences.

[0099] 8. Biochemical indicators and liver tissue pathological examination

[0100] The Mindray fully automated biochemical analyzer and biochemical indicator detection kits (purchased from Shenzhen Mindray Bio-Medical Electronics Co., Ltd.) were used to conduct various indicator tests. The specific test contents are as follows:

[0101] Mouse serum samples were collected to detect two liver function indicators: alanine aminotransferase and aspartate aminotransferase. If the initial test value of the sample exceeded the instrument's detection range, the serum was diluted 10 times with double-distilled water and tested again.

[0102] Liver histopathological sectioning and staining were performed by Wuhan Saiweier Biotechnology Co., Ltd., to detect pathological changes in the liver tissue. Hematoxylin and eosin (H&E) staining was used to evaluate basic liver lesions and inflammation; Oil Red staining was used to evaluate lipid degeneration; and Masson staining was used to detect the collagen fiber content within the liver tissue. All stained sections underwent high-resolution panoramic scanning and image acquisition using a Pannooramic Scan device, and NAS pathological scoring was performed based on the modified Ishak scoring system.

[0103] 9. Extraction and purification of total RNA

[0104] (1) Sample processing

[0105] (1.1) Cell sample processing: Aspirate the culture medium from the culture system and, at a ratio of 10 cm⁻¹, remove the culture medium from the culture medium. 2 Add Trizol reagent at a ratio of 1 mL to 1 mL of culture medium, let stand at room temperature for 5 minutes to fully lyse the cells, and then use a pipetting operation to completely detach the cells from the culture medium. Transfer the cell lysis mixture to a 1.5 mL EP tube.

[0106] (1.2) Animal tissue sample processing: Take out the animal tissue frozen at -80℃, weigh 50-100 mg of tissue block and pre-cool it in liquid nitrogen; add 1 mL of Trizol to each glass grinding tube (or a flow cytometer can be used instead), put the tissue block into the tube, and grind it rapidly with a handheld high-speed homogenizer under ice-water bath conditions until there are no visible tissue blocks in the tube. Place the resulting tissue homogenate on ice for 30 minutes to fully lyse, and then centrifuge at 4℃ and 12000 rpm for 15 minutes. After centrifugation, aspirate the supernatant and transfer it to a brand new EP tube for later use.

[0107] (2) Add chloroform reagent to the sample system obtained by step (1) at a ratio of 200 μL chloroform for every 1 mL Trizol reagent, tighten the cap of the EP tube, shake vigorously for 15 s, and let stand at room temperature for 3 minutes.

[0108] (3) Centrifuge the mixture at 4°C and 12,000 rpm for 15 minutes. After centrifugation, the sample is divided into three layers: the bottom layer is a yellow organic phase, the top layer is a colorless aqueous phase, and there is an intermediate layer between the two phases. RNA is mainly distributed in the upper aqueous phase, and the volume of the aqueous phase is about 60% of the initial volume of Trizol reagent added.

[0109] (4) Carefully transfer the upper layer of liquid to a new 1.5 mL EP tube. A single tube can aspirate approximately 400 μL of liquid. When aspirating the upper layer of liquid, be careful to avoid aspirating the middle layer.

[0110] (5) Use the Kangwei Century RNA Purification Kit to complete the RNA purification operation. Add an equal volume or slightly excess of 70% ethanol (prepared by mixing anhydrous ethanol and pure water in a volume ratio of 7:3) to the collected aqueous phase liquid. Gently invert the centrifuge tube to mix the liquid. After standing at room temperature for 2 minutes, transfer all the mixed liquid to the purification column of the kit.

[0111] (6) The purification process is carried out at room temperature. There is no need to cool the centrifuge to 4°C. Centrifuge at 12,000 rpm for 1 minute. After centrifugation, discard the waste liquid at the bottom of the collection tube and put the purification column back into the collection tube.

[0112] (7) Add 700 μL of RA1 reagent from the Kangwei Century RNA Purification Kit, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid in the collection tube.

[0113] (8) Add 500 μL of RA2 reagent from the Kangwei Century RNA Purification Kit. Before the first use of RA2 reagent, add 200 mL of anhydrous ethanol, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid in the collection tube; repeat the addition of RA2 reagent and centrifugation once.

[0114] (9) Centrifuge at 12000 rpm for 2 minutes to remove the residual liquid in the purification column.

[0115] (10) Transfer the purification column to a new 1.5 mL RNase-free EP tube, open the tube cap and let it stand for 10-15 minutes to allow the residual ethanol in the column to evaporate completely.

[0116] (11) Vertically suspend the adsorption membrane of the purification column and add RNase-free water preheated to 65℃ (if the RNA content of cells is low, add 20-30 μL of RNase-free water; if the RNA content of animal tissues is high, add 100-200 μL of RNase-free water). After adding, let stand at room temperature for 2 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the RNA solution obtained by elution in the tube; if the RNA concentration is low, the first elution buffer can be added back to the adsorption column for a second elution to increase the RNA concentration. Take 2 μL from each tube to determine the concentration and purity, and store the rest in a -80℃ freezer.

[0117] 10. Reverse transcription PCR to synthesize cDNA

[0118] Weigh 1 μg of total RNA and synthesize cDNA using the Roche Transcriptor first strand cDNA synthesis kit. The specific steps are as follows:

[0119] (1) Prepare the reaction system as shown in Table 2 in a 0.2 mL RNase-free PCR tube:

[0120]

[0121] After mixing the above components, centrifuge briefly, denature at 65°C for 10 minutes, and immediately place in an ice bath.

[0122] (2) Add the components shown in Table 3 to the above system to prepare a 20 μL reaction system:

[0123]

[0124] Mix the 7 μL mixture in all wells, then add 7 μL to each well, mix well, and centrifuge briefly. Incubate at 55°C for 30 minutes to synthesize cDNA.

[0125] (3) Heat at 85℃ for 5 minutes to inactivate Transcriptor Reverse Transcriptase. Add 100 μL of sterile water to the obtained cDNA, mix well, transfer to a 1.5 mL centrifuge tube and store at -20℃ or use directly for subsequent experiments.

[0126] 11. TaqMan real-time quantitative PCR

[0127] Real-time quantitative PCR using the Taqman probe (ThermoFisher) method was performed using the Roche FastStart Universal Probe Master (ROX) kit. The obtained cDNA was used as the amplification template, and the GAPDH gene was used as an internal control. The reaction system (20 μL) was prepared according to Table 4 below.

[0128]

[0129] Prepare a basic reaction mixture without cDNA template. Aliquot 18 μL of this mixture into each well of a 96-well Real-Time PCR plate, then add 2 μL of cDNA template to each well. Gently mix all reaction liquids in each well and centrifuge at 1000 rpm for 5 minutes to remove air bubbles. After centrifugation, place the 96-well plate in an ABI QuantStudio 3 Real-Time PCR instrument and set the amplification program parameters: 95℃ pre-denaturation for 5 minutes; then perform 45 amplification cycles, with single-cycle parameters of 95℃ denaturation for 15 seconds and 60℃ annealing extension for 30 seconds.

[0130] In this process, fluorescence signal values ​​are collected at the end of each cycle. After the reaction is completed, the expression levels of the target gene and the internal reference gene are analyzed using the relative CT value method based on the CT values ​​of the target gene and the internal reference gene given by the instrument for each sample, so as to determine the effect of the drug on the expression of related genes in cells or animal tissues.

[0131] 12. Statistical Analysis

[0132] Data are expressed as mean ± standard error (Mean ± SEM). Statistical analysis was performed using GraphPad Prism 9.0 software. For normally distributed data with homogeneous variance, one-way or two-way ANOVA was used for inter-group comparisons, followed by Dunnett's post-hoc test to further analyze differences between groups. For data that did not conform to a normal distribution or had unequal variances, nonparametric tests were used. P < 0.05 and P < 0.01 were considered statistically significant.

[0133] (II) Experimental Results

[0134] 1. Study on the regulation of lipid levels by adegamycin H in L02 cell model

[0135] L02 hepatocytes induced by OA+PA were treated with 40 μM adegamycin H for 24 hours. Oil Red and Nile Red fluorescence staining were performed to detect the expression levels of lipid metabolism-related genes within the cells. The results of Oil Red and Nile Red fluorescence staining showed that adegamycin H significantly inhibited OA+PA-induced lipid deposition in L02 cells. Figure 3 A and 3B); Western blot and quantitative real-time PCR (qRT-PCR) results confirmed that adegamycin H can upregulate the expression of the lipid peroxidation gene PPARγ. Figure 3 C and 3D).

[0136] The above experimental results demonstrate that adegamycin H exerts its lipid-lowering effect by regulating lipid metabolism-related pathways.

[0137] 2. Study on the inhibition of hepatic stellate cell activation by adegamycin H in the LX2 cell model

[0138] LX2 cells induced by TGF-β1 were treated with adegamycin H at gradient concentrations of 20 μM, 40 μM, 60 μM, and 80 μM for 24 hours. Protein analysis showed that adegamycin H at a concentration of 40 μM significantly downregulated the protein expression of fibrosis markers such as COL1A1, α-SMA, and TGF-β1. Figure 4 A); Real-time quantitative nucleic acid amplification results showed that 40 μM adegamycin H significantly reduced the mRNA levels of TGFB1 and COL1A1 genes ( Figure 4 B).

[0139] The above results confirm that adegamycin H can significantly inhibit hepatic stellate cell activation and possesses good in vitro anti-hepatic fibrosis activity. Combined with the aforementioned lipid-lowering activity of adegamycin H in improving hepatocyte lipid deposition, it can be concluded that adegamycin H can simultaneously regulate hepatocyte lipid metabolism pathways and inhibit the activation process of hepatic stellate cells, demonstrating outstanding potential for the prevention and treatment of metabolism-related steatohepatitis.

[0140] 3. Hepatoprotective effect of adegamycin H on C57BL / 6 mice induced by a high-fat, high-cholesterol diet.

[0141] The overall design scheme of this animal experiment is as follows: Figure 5 As shown in Figure A, C57BL / 6 mice were fed a high-fat, high-cholesterol (HFHC) diet for 10 weeks to establish the model. Subsequently, adegamycin H was administered by gavage at a dose of 100 mg / kg daily for 4 consecutive weeks to conduct intervention treatment. The in vivo efficacy of adegamycin H was evaluated using this animal model of metabolic-associated steatohepatitis induced by a simulated Western diet.

[0142] After the administration period, the body weight of mice in each group was measured. The results showed that adegamycin H had no significant effect on mouse body weight, suggesting that the compound has low in vivo toxicity. Figure 5 B). Gross observation of mouse liver showed that adegamycin H significantly improved hepatomegaly and granular lesions induced by HFHC (B). Figure 5 C). Meanwhile, adegamycin H significantly reversed the liver weight / body weight ratio after HFHC induction ( Figure 5 (D) The above results preliminarily confirm that adegamycin H has a protective effect against liver injury induced by HFHC.

[0143] 4. The effect of adegamycin H in improving HFHC-induced serum abnormalities and liver pathological damage in C57BL / 6 mice.

[0144] Biochemical indicators showed that, compared with the HFHC-induced group, the serum ALT and AST levels of mice treated with adegamycin H were significantly reduced. Figure 6 A) confirmed that adegamycin H has good in vivo liver protection effects.

[0145] Observation of liver tissue pathological morphology by hematoxylin-eosin (H&E) staining ( Figure 6 B), and a double-blind scoring method was used to pathologically score the degree of liver lesions in each group to systematically evaluate the effect of adegamycin H on improving liver pathological damage. The results showed that hepatic ballooning degeneration was significantly reduced in the adegamycin H treatment group (B). Figure 6 C), the NAS score also decreased significantly ( Figure 6 D).

[0146] The above experimental results fully demonstrate that adegamycin H can effectively improve pathological liver damage in C57BL / 6 mice caused by a high-fat, high-cholesterol diet and correct abnormal serum liver function indicators in mice.

[0147] 5. The effect of adegamycin H on improving hepatic lipid deposition in HFHC-induced C57BL / 6 mice.

[0148] The effect of adegamycin H on improving hepatic lipid deposition in C57BL / 6 mice induced by a high-fat, high-cholesterol (HFHC) diet was systematically analyzed using Oil Red staining combined with the detection of lipid metabolism-related indicators. Oil Red staining results showed a significant increase in the number of red lipid droplets in the liver tissue of mice in the HFHC-induced group. After treatment with adegamycin H, the accumulation of red lipid droplets in the liver of mice was significantly reduced. Figure 7 A), and the liver tissue lipid degeneration score was significantly reduced ( Figure 7 B).

[0149] The results of total triglyceride content detection in liver tissue showed that adegamycin H could significantly reverse HFHC-induced triglyceride deposition in the liver. Figure 7 C). Further extraction of total protein from mouse liver tissue and Western blot analysis showed that the expression level of the fatty acid oxidation gene PPARγ in the liver of mice treated with adegamycin H was significantly upregulated. Figure 7 D).

[0150] In summary, adegamycin H exhibits excellent ability to improve hepatic lipid deposition in vivo, demonstrating good potential for lipid-lowering applications.

[0151] 6. Adegamycin H inhibits the progression of HFHC-induced liver fibrosis in C57BL / 6 mice.

[0152] The intervention effect of adegamycin H on liver fibrosis was comprehensively analyzed by combining marson staining and detection of liver fibrosis-related markers (COL1A1, α-SMA, and TGF-β1). Marson staining results of liver tissue showed that the area of ​​positive fibrosis staining in the liver tissue of mice treated with adegamycin H was significantly lower than that in the HFHC-induced group (…). Figure 8 A). Meanwhile, the results of fibrosis marker detection showed that the mRNA levels and protein expression levels of COL1A1, α-SMA, and TGF-β1 were all decreased ( Figure 8 B and Figure 8 C).

[0153] The above results indicate that adegamycin H can effectively inhibit the progression of HFHC-induced liver fibrosis in C57BL / 6 mice.

[0154] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or related embodiments obtained by those skilled in the art through minor modifications or variations of the disclosed technical content without departing from the scope of the present invention fall within the scope of the present invention.

Claims

1. The use of adegamycin H in the preparation of medicaments for the prevention and / or treatment of metabolic-related fatty liver disease, wherein, The adegamycin H has the structure shown in formula (I): 。 2. The use according to claim 1, wherein, The metabolic-associated fatty liver disease is selected from one or more of the following: metabolic dysfunction-associated steatohepatitis (MASH), liver injury, liver fibrosis, cirrhosis, hepatocellular carcinoma, and liver failure.

3. The use according to claim 1, wherein, The prevention and / or treatment are to block hepatic steatosis, inflammation and / or fibrosis, and / or improve hepatic lipid accumulation, liver injury, inflammation and / or reverse liver fibrosis.

4. The use according to claim 1, wherein, The drug also contains pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipient is selected from one or more of surfactants, preservatives, antioxidants, hardeners, thickeners, and absorption enhancers.

5. The use according to claim 1, wherein, The dosage form of the drug is selected from one or more of the following: tablets, capsules, suspensions, syrups, granules, creams, ointments, pills, powders, elixirs, suppositories, creams, drop pills, gels, patches, sprays, aerosols, oral liquids, and injections.

6. The use according to claim 1, wherein, The adegamycin H was prepared by fermentation of Saccharothrix sp. strain 16Sb2-4, whose preservation number is CGMCC No. 39500; Preferably, the preparation method of adegamycin H includes the following steps: (1) The Saccharothrix sp. strain 16Sb2-4 was fermented and cultured, and the fermentation broth was collected; (2) The fermentation broth is centrifuged, the supernatant is extracted with an organic solvent, the organic phase is collected, and the crude extract is obtained by dehydration and concentration. (3) The crude extract was purified by gel chromatography, and the eluent containing adegamycin H was collected to obtain the first eluent. (4) The first elution fraction is eluted by reverse phase chromatography gradient elution, and the elution fraction containing adegamycin H is collected to obtain the second elution fraction; (5) The second elution fraction was purified by high performance liquid chromatography, and the adegamycin H fraction was collected, concentrated and dried to obtain adegamycin H.

7. The use according to claim 6, wherein, In step (1), the fermentation culture includes: inoculating the Saccharothrix sp. strain 16Sb2-4 into a culture medium and culturing it at a constant temperature of 28°C for 7 days to prepare a plate culture; inoculating the plate culture into a liquid culture medium and culturing it at 28°C and 180 rpm for 3 days to obtain a seed culture; transferring the seed culture into a liquid culture medium and culturing it at 28°C and 180 rpm for 7 days to collect the fermentation broth.

8. The use according to claim 6, wherein, In step (2), the organic solvent is ethyl acetate, and the extraction is performed 2-4 times; preferably, the dehydration is performed using anhydrous sodium sulfate; preferably, the concentration is performed under reduced pressure.

9. The use according to claim 6, wherein, In step (3), the gel chromatography uses a Sephadex LH-20 gel chromatography column with methanol as the eluent; Preferably, in step (4), the reversed-phase chromatography uses an ODS reversed-phase chromatography column, the elution solvent is a methanol-water mixed solution, and the elution gradient is successively 20%, 40%, 60%, 70% and 100% methanol aqueous solution by volume fraction.

10. The use according to claim 6, wherein, In step (5), the chromatographic column of the high performance liquid chromatography is an Agilent ZORBAX SB-C18 column with a specification of 9.4 mm × 250 mm and a packing particle size of 5 μm; Preferably, the mobile phase is a 50% (v / v) methanol aqueous solution, and the flow rate is 2.0 mL / min.

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