Application of artemisinene with double functions in promoting liver cell maturation and relieving liver lipid metabolism dysfunction

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of artemisinene with double functions in promoting liver cell maturation and relieving liver lipid metabolism dysfunction. It is found for the first time that ATT can specifically and efficiently promote human hepatic progenitor cell organoids to differentiate into functional mature hepatic cells, the effect of ATT depends on activation of a cAMP-EPAC signal channel, and the action mechanism and effect are remarkably different from those of a DHA-based scheme in the prior art. Meanwhile, the ATT can obviously relieve lipid deposition of liver cells, improve lipid metabolism disorder, effectively relieve liver cell oxidative stress and lipid peroxidation damage induced by lipid deposition, and prevent the cells from triggered apoptosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of artemisinin with dual functions in promoting hepatocyte maturation and alleviating hepatic lipid metabolism dysfunction. Background Technology

[0002] Metabolic dysfunction-related fatty liver disease (MASLD, formerly known as non-alcoholic fatty liver disease, NAFLD) is one of the most common chronic liver diseases worldwide, characterized by excessive lipid accumulation in hepatocytes, which can further develop into steatohepatitis (fatty liver), liver fibrosis, and even liver cancer. Currently, apart from a very few drugs such as recently approved resmetirom, there is still a lack of highly effective and safe treatments in clinical practice. Moreover, existing drugs or candidate compounds mostly target single pathways or phenotypes, and substances with multiple protective effects such as improving lipid metabolism, anti-oxidative stress, and anti-apoptosis are limited. Research on its mechanisms of action is insufficient, focusing mostly on phenomenological descriptions and lacking in-depth exploration of core signaling pathways. While cell therapy is effective, challenges exist in preparation, storage, safety, and cost. Therefore, developing new intervention strategies and drugs is an urgent priority.

[0003] In basic research and drug development, in vitro models that can accurately simulate the physiological and pathological state of the human liver are crucial. Traditional hepatocellular carcinoma cell lines (such as HepG2) have limitations in maintaining long-term hepatocyte function and complexity (Cottier KE, Bhalerao D, Lewis C, et al. Micropatterned Primary Hepatocyte Co-Culture (HEPATOPAC) for Fatty Liver Disease Modeling and Drug Screening. PREPRINT (Version 1) available at Research Square, 2022 Dec 20.). In recent years, hepatocyte organoids, as a three-dimensional culture model derived from stem cell differentiation, have been considered a more promising platform for disease modeling and drug screening due to their ability to better preserve the liver's tissue structure, cell types, and key functions. Their culture and differentiation techniques are currently a research hotspot. A common approach is to use a series of growth factors and small molecule compounds (such as FGF, BMP, HGF, Oncostatin M, etc.) to simulate the liver development process in stages, inducing functional hepatocytes or organoids from pluripotent stem cells.

[0004] Cells generated by existing organoid differentiation protocols often differ from adult primary hepatocytes in terms of drug-metabolizing enzyme activity, transporter function, and urea cycling, limiting their application value as high-fidelity disease models and transplant sources. This is because hepatocyte functional maturation involves an extremely complex transcriptional network, and existing factor targets are singular and cannot be globally regulated.

[0005] Artemisinin has shown therapeutic potential in many diseases, but it has some limitations in pharmacokinetics, such as low oral solubility, poor bioavailability, and short in vivo half-life. To address these issues, semi-synthetic and fully synthetic artemisinin and its derivatives have been developed, including artemisinin ester (ART), artemisinin ether, and its common active metabolite dihydroartemisinin (DHA). DHA, as a reduction product of artemisinin (hemiacetal structure), is formed by the reduction of the lactone in the molecule to a hydroxyl group under the action of an artemisinin re-reducing agent, generating dihydroartemisinin (Acton N, Klayman DL. Artemisitene, a newsesquiterpene lactone endoperoxide from Artemisia annua. Planta Medica. 1985;51(5):441-442.). However, another structurally different member exists within the artemisinin derivative family, such as artemisitene (ATT), a functionalized derivative of artemisinin at the C-15 position. It retains the carbonyl structure at the C-10 position and is fundamentally different from DHA in chemical properties. ATT is derived from the plant Artemisia annua, and its molecular formula is C0. 15 H 20 O5, with a molecular weight of 280.32 and CAS number 101020-89-7, is currently mainly derived from Artemisia annua ( Artemisia annuaArtemisinin was isolated from the extract of artemisinin, which is a direct derivative and chemical precursor of artemisinin. Artemisinin can be converted into artemisinin in microorganisms through specific treatments (Ma J, Weiss E, Kyle DE, Ziffer H. Acid catalyzed Michael additions to artemisitene. Bioorganic & Medicinal Chemistry Letters. 2000;10(14):1601-1603.), and artemisinin can also be converted into artemisinin through chemical reduction and deoxygenation reactions (El-Feraly FS, Ayalp A, Al Yahya A. Conversion of artemisinin to artemisitene. Journal of Natural Products. 1990;53(1):66.). Artemisinin belongs to the terpenoid class of compounds and is a compound with an isoprene basic unit structure, whose structural skeleton is composed of multiple isoprene units linked together. It possesses important functional groups such as carbon-carbon double bonds, which are unsaturated, endowing it with specific chemical reactivity (Hua L, Liang S, Zhou Y, et al. Artemisinin-derived artemisitene blocks ROS-mediated NLRP3 inflammasome and alleviates ulcerative colitis. International Immunopharmacology. 2022;113:109431.). Artemisinene obtained through structural modification removes the influence of the lactone ring carbonyl / hydroxyl groups on its activity, toxicity, and metabolic stability. The core of this structural modification process lies in the selective reduction of the target functional group while protecting the fragile peroxy bridge structure, which can lead to the development of more advantageous new drugs and thus achieve a better structure-pharmacodynamic relationship. Therefore, this medicinal chemistry structural modification process aims to alter the physicochemical properties of the parent compound.

[0006] Previous research on artemisinin and its derivatives has primarily focused on antimalarial and antitumor effects (through inducing oxidative damage), completely neglecting its potential in protecting normal hepatocyte function and promoting cell maturation, and its application in the cutting-edge field of hepatocyte organoids has been largely overlooked. Furthermore, different artemisinin derivatives exhibit variations in chemical structure and biological activity. Currently, research on the biological activity of ATT is relatively limited, particularly in the areas of liver regeneration and cell fate regulation, where no systematic reports have been found, and its specific efficacy and unique mechanisms remain completely undiscovered. Summary of the Invention

[0007] The technical problem this invention aims to solve is to improve hepatocyte function by optimizing the culture process of liver organoids derived from human embryonic stem cells, and to successfully simulate hepatocyte lipid accumulation when constructing metabolic liver disease models such as metabolic-associated fatty liver disease (MASLD) by combining existing modeling methods (such as free fatty acid (FFA) induction). On the other hand, there is currently a lack of effective compounds that can simultaneously reverse lipotoxicity, oxidative stress, and apoptosis while enhancing hepatocyte function. This invention proposes a technique that promotes the differentiation of hepatocyte progenitor cell organoids into mature hepatocyte organoids by adding the small chemical molecule artemisitene (ATT) during the organoid expansion stage. ATT significantly improves the maturity and functional stability of hepatocyte organoids, while alleviating FFA-induced lipid metabolism disorders, oxidative stress, and cell damage. This provides a more reliable model for liver disease research, drug screening, and liver regeneration therapy. Furthermore, ATT can also be used to prepare drugs, health products, and foods for the prevention or treatment of liver diseases.

[0008] The first aspect of this invention aims to provide applications of artemisinin.

[0009] A second aspect of the present invention is to provide a culture medium.

[0010] A third aspect of the present invention aims to provide a method for promoting the differentiation of hepatic progenitor cell organoids into hepatic cell organoids.

[0011] The fourth aspect of this invention aims to provide a method for improving the maturity and function of hepatocyte organoids.

[0012] The fifth aspect of this invention aims to provide a method.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows: ATT represents a novel class of compounds used to improve the maturity and function of hepatocyte organoids, providing a new technical means to address the problem of insufficient maturity in existing organoid models. On the other hand, ATT can be used as an active ingredient to improve or treat hepatocyte steatosis and related damage, and is suitable for preparing drugs, foods and health products for the prevention or treatment of liver diseases.

[0014] A first aspect of the invention provides the use of artemisinin (ATT) in any one of (1)-(11): (1) Promote the differentiation of hepatic progenitor cells into hepatocytes; (2) Prepare products that promote the differentiation of hepatic progenitor cells into hepatocytes; (3) Improve the maturity and function of hepatocytes in hepatocyte organoids; (4) Prepare products that improve the maturity and function of hepatocytes in hepatocyte organoids; (5) Construct liver organoids; (6) Prepare drugs for the treatment and / or prevention of diseases caused by lipid metabolism disorders; (7) Inhibit oxidative stress and lipid peroxidation damage of hepatocytes; (8) Prepare products that inhibit oxidative stress and lipid peroxidation damage of hepatocytes; (9) Inhibit apoptosis of hepatocytes; (10) Prepare products that inhibit apoptosis of hepatocytes; (11) Prepare health products that help with antioxidation, help control body fat, help maintain healthy blood lipid levels, or have an auxiliary protective effect against chemical liver damage.

[0015] In some embodiments of the present invention, the hepatic progenitor cells described in (1)-(2) include hepatic progenitor cells of hepatic progenitor cell organoids, and the hepatocytes include hepatocytes of hepatocyte organoids. In some embodiments of the present invention, the diseases caused by lipid metabolism disorders described in (6) include metabolic dysfunction-related fatty liver disease.

[0016] In some embodiments of the present invention, the drug prevents or treats fatty liver disease associated with metabolic dysfunction by regulating lipid metabolism disorders.

[0017] In some embodiments of the present invention, the regulation of lipid metabolism disorder includes regulating the expression of lipid metabolism-related genes (such as fatty acid storage genes FABP4 and DGAT2, lipid droplet coating protein gene PLIN2, and stress-related genes ASGR1 and TRIB3), inhibiting lipid droplet accumulation, reducing oxidative stress response, and inhibiting cell apoptosis.

[0018] In some embodiments of the present invention, the hepatocytes described in (7)-(10) include hepatocytes of hepatocyte organoids.

[0019] In some embodiments of the present invention, the effective concentration of artemisinin is 0.5-5 μM; further, it is 0.8-2 μM, such as any value or a range formed by any two of 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 μM.

[0020] In some embodiments of the present invention, the product includes reagents and pharmaceuticals.

[0021] In some embodiments of the present invention, the liver organoids are liver organoids derived from human pluripotent stem cells. Pluripotent stem cells refer to stem cells that possess the pluripotency to differentiate into all cells present in an organism, namely the three germ layers (endoderm, mesoderm, and ectoderm), and also have proliferative capacity. There are no particular limitations on the aforementioned pluripotent stem cells; examples include embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained through nuclear transfer (ntES cells), spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem cells (iPS cells), and pluripotent cells (Muse cells) derived from cultured fibroblasts or bone marrow stem cells. The human embryonic stem cells described in this invention are not human embryonic stem cells isolated or obtained from human embryos that have undergone in vivo development, nor are they stem cells isolated or obtained from human embryos that have not undergone in vivo development and have been fertilized for more than 14 days.

[0022] This invention provides a novel application of the natural compound artemisinin (ATT). ATT promotes the transformation of hepatic progenitor organoids into functionally mature hepatic organoids, demonstrating its ability to promote hepatocyte regeneration. Simultaneously, ATT can alleviate FFA-induced lipid metabolism disorders, oxidative stress, and apoptosis in hepatic organoids. These effects suggest that ATT has potential development value in the prevention or treatment of hepatocyte steatosis and degeneration (such as MASLD / NAFLD) and the repair and regeneration of related liver damage.

[0023] A second aspect of the present invention provides a culture medium comprising artemisinin and a liver progenitor cell organoid culture medium.

[0024] In some embodiments of the present invention, the final concentration of artemisinin in the culture medium is 0.5-5 μM; further, it is 0.8-2 μM, such as any value or a range formed by any two of 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 μM.

[0025] In some embodiments of the present invention, the liver progenitor cell organoid culture medium includes at least one of Matrigel, ALK5 inhibitor, GSK-3 inhibitor, FSK, growth factor and BMP signaling pathway activator.

[0026] In some embodiments of the present invention, the ALK5 inhibitor includes at least one selected from SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761; further comprising SB431542.

[0027] In some embodiments of the present invention, the concentration of the ALK5 inhibitor in the culture medium is 5-20 μM; more specifically 8-15 μM, such as any value or a range formed by any combination of 8, 9, 10, 11, 12, 13, 14 or 15 μM.

[0028] In some embodiments of the present invention, the GSK-3 inhibitor includes at least one of SB216763, GSK3β inhibitor VII, L803-mts, 6-bromo-indirubin-3'-oxime, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-Azakenpaullone, Indirubin, and CHIR99021; more specifically, CHIR99021.

[0029] In some embodiments of the present invention, the concentration of the GSK-3 inhibitor in the culture medium is 1-10 μM; further, it is 2-8 μM, such as any value of 2, 3, 4, 5, 6, 7 or 8 μM or a range formed by any two of them.

[0030] In some embodiments of the present invention, the BMP signaling pathway activator includes at least one of BMP2, BMP4, SB4, SJ000291942, SJ000063181, SJ000370178, isoliquiritin, geraniol, apigenin and chickpea sproutin; further comprising BMP4.

[0031] In some embodiments of the present invention, the concentration of the BMP signaling pathway activator in the culture medium is 10-40 ng / mL; more specifically, 10-30 ng / mL, such as any value or a range formed by any combination of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 ng / mL.

[0032] In some embodiments of the present invention, the growth factor comprises at least one of epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor-I, IGF-II, leukemia inhibitory factor, nerve growth factor, tumor suppressor M, platelet-derived endothelial growth factor, transforming growth factor-α, and vascular endothelial growth factor; and further comprises EGF and FGF (preferably FGF-4).

[0033] In some embodiments of the present invention, the concentration of EGF in the culture medium is 10-50 ng / mL; more specifically, 10-30 ng / mL, such as any value or a range formed by any combination of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 ng / mL.

[0034] In some embodiments of the present invention, the concentration of FGF-4 in the culture medium is 10-50 ng / mL; more specifically, 10-30 ng / mL, such as any value or a range formed by any combination of 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 ng / mL.

[0035] In some embodiments of the present invention, the concentration of Matrigel (Growth-factor reduced Matrigel) in the culture medium is 3v / v%-10v / v; further, it is 3v / v%-8v / v, such as any value or a range formed by any combination of 3v / v%, 4v / v%, 5v / v%, 6v / v%, 7v / v% or 8v / v%.

[0036] In some embodiments of the present invention, the concentration of FSK in the culture medium is 5-30 μM; more specifically, 5-15 μM, such as any value or a range formed by any combination of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 μM.

[0037] In some embodiments of the present invention, the liver progenitor cell organoid culture medium further includes FBS, ITS, NEAA, GlutaMAX, and nicotinamide.

[0038] In some embodiments of the present invention, the concentration of FBS in the culture medium is 5 w / w %-30 w / w %; more specifically, 5 w / w %-15 w / w %, such as any value or a range formed by any combination of 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15 w / w %.

[0039] In some embodiments of the present invention, the concentration of the ITS in the culture medium is 1×.

[0040] In some embodiments of the present invention, the concentration of NEAA in the culture medium is 1×.

[0041] In some embodiments of the present invention, the concentration of GlutaMAX in the culture medium is 0.5 w / w%-2 w / w; more specifically, 0.8 w / w%-1.5 w / w, such as any value or a range formed by any combination of 0.8 w / w%, 0.9 w / w%, 1 w / w%, 1.1 w / w%, 1.2 w / w%, 1.3 w / w%, 1.4 w / w%, or 1.5 w / w%.

[0042] In some embodiments of the present invention, the concentration of nicotinamide in the culture medium is 5-20 mM; more specifically, 5-15 mM, such as any value or a range formed by any combination of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mM.

[0043] In some embodiments of the present invention, the basal culture medium of the liver progenitor cell organoid culture medium is at least one of RPMI1640 and IMDM medium.

[0044] Compared with existing technologies, the culture medium provided by this invention can significantly improve the maturity of hepatocyte organoids, shorten the maturation cycle, and enhance the liver function characteristics of organoids (such as the maturity of metabolic function, protein expression, and gene expression). This culture medium not only provides a more accurate in vitro model for the study of liver-related diseases.

[0045] A third aspect of the present invention provides a method for promoting the differentiation of hepatic progenitor cell organoids into hepatic cell organoids, comprising culturing hepatic progenitor cell organoids using the culture medium of the second aspect of the present invention.

[0046] In some embodiments of the present invention, the liver progenitor cell organoids include liver progenitor cell organoids derived from human embryonic stem cells.

[0047] In some embodiments of the present invention, the culture time is 1-10 days, such as any value or a range formed by any two of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0048] In some embodiments of the present invention, the method involves culturing liver progenitor cell organoids on the first day of culture using the culture medium of the second aspect of the present invention, and subsequently adding the liver progenitor cell organoid culture medium of the second aspect of the present invention to the culture volume during subsequent culture processes.

[0049] In the differentiation culture system of hepatic progenitor cell organoids (HB-orgs) into mature hepatic cell organoids (P-hep-orgs), the introduction of a certain concentration of ATT for the first time can promote the directed differentiation of hepatic progenitor cell organoids into hepatocytes and improve the maturity and function of hepatic cell organoids.

[0050] A fourth aspect of the present invention provides a method for improving the maturity and function of hepatocyte organoids, comprising culturing hepatocyte progenitor organoids using the culture medium of the second aspect of the present invention.

[0051] In some embodiments of the present invention, the liver progenitor cell organoids include liver progenitor cell organoids derived from human embryonic stem cells.

[0052] In some embodiments of the present invention, the culture time is 1-10 days, such as any value or a range formed by any two of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0053] In some embodiments of the present invention, the method involves culturing liver progenitor cell organoids on the first day of culture using the culture medium of the second aspect of the present invention, and subsequently adding the liver progenitor cell organoid culture medium of the second aspect of the present invention to the culture volume during subsequent culture processes.

[0054] In some embodiments of the present invention, the method further includes the step of culturing the cells using a hepatocyte maturation culture medium.

[0055] In some embodiments of the present invention, the liver cell maturation culture medium is used to culture the cells for 7-10 days.

[0056] In some embodiments of the present invention, the hepatocyte maturation culture medium includes Growth-factor-reduced Matrigel, growth factors, tumor suppressor M, dimethyl sulfoxide, dexamethasone, and a first additive; the first additive includes ascorbic acid, BSA-FAF, hydrocortisone, transferrin, insulin, recombinant human epidermal growth factor, and GA-1000.

[0057] In some embodiments of the present invention, the final concentration of Matrigel in the hepatocyte maturation culture medium is 3-10 v / v; more specifically, 4-8 v / v.

[0058] In some embodiments of the present invention, the growth factors in the hepatocyte maturation culture medium include at least one of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor-I (IGF-1), IGF-II, leukemia inhibitory factor (LIF), nerve growth factor (NGF), oncogene M (OSM), platelet-derived endothelial growth factor (PDECGF), transforming growth factor-α (TGF-α), and vascular endothelial growth factor (VEGF).

[0059] In some embodiments of the present invention, the final concentration of HGF in the hepatocyte maturation culture medium is 10-30 ng / mL; more specifically, it is 15-25 ng / mL.

[0060] In some embodiments of the present invention, the final concentration of FGF-4 in the hepatocyte maturation culture medium is 10-30 ng / mL; more specifically, it is 15-25 ng / mL.

[0061] In some embodiments of the present invention, the final concentration of oncostatin M in the hepatocyte maturation culture medium is 30-70 ng / mL; more specifically, it is 40-60 ng / mL.

[0062] In some embodiments of the present invention, the final concentration of B27 in the hepatocyte maturation culture medium is 1 w / w %-5 w / w %; more specifically, it is 1 w / w %-3 w / w %.

[0063] In some embodiments of the present invention, the final concentration of dexamethasone in the hepatocyte maturation culture medium is 80-120 nM; more specifically, it is 90-110 nM.

[0064] In some embodiments of the present invention, the first additive consists of seven components from the Single Quots kit: 0.5 mL ascorbic acid, 5 mL BSA FAF, 0.5 mL hydrocortisone, 0.5 mL transferrin, 0.5 mL insulin, 0.5 mL recombinant human epidermal growth factor, and 0.5 mL GA 1000, Single Quots kit purchased from Lonza, item number CC 4182.

[0065] In some embodiments of the present invention, the basal culture medium of the hepatocyte maturation culture medium includes hepatocyte basal culture medium, such as HCM.

[0066] ATT treatment comprehensively enhances the functions of hepatocyte organoids in multiple dimensions, including gene expression, protein synthesis, metabolism, and drug response. It has the dual function of promoting hepatocyte differentiation and maturation and resisting hepatocellular lesions. It is suitable for constructing and optimizing liver disease models and for research on the mechanisms of liver diseases and for drug screening and development for the treatment of liver diseases.

[0067] A fifth aspect of the invention provides a method comprising the step of treating hepatocyte organoids with artemisinin; The method includes any one of 1)-3): 1) a method for inhibiting lipid deposition in hepatocyte organoids; 2) a method for inhibiting apoptosis of hepatocytes in hepatocyte organoids; 3) a method for improving the oxidative stress capacity of hepatocyte organoids.

[0068] In some embodiments of the present invention, the effective treatment concentration of artemisinin is 1-5 μM, such as any value or a range formed by any combination of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 μM.

[0069] In some embodiments of the present invention, the inhibition of hepatocyte organoid lipid deposition includes the inhibition of hepatocyte organoid lipid deposition induced by free fatty acids.

[0070] In some embodiments of the present invention, the apoptosis includes apoptosis of hepatocytes in hepatocyte organoids induced by free fatty acids.

[0071] The method provided by this invention can effectively reverse FFA-induced lipid accumulation, significantly reduce oxidative stress, and effectively inhibit apoptosis, providing a new platform for the development of liver regeneration and treatment methods. This suggests that ATT may be a candidate compound for the prevention or treatment of MASLD and other liver disease pathological manifestations, and has significant application value.

[0072] The beneficial effects of this invention are: This invention is the first to discover that ATT can specifically and efficiently promote the differentiation of human hepatic progenitor cell organoids (HB-orgs) into functionally mature hepatocytes. Its action depends on activating the cAMP-EPAC signaling pathway, a mechanism and effect significantly different from existing DHA-based approaches. Simultaneously, ATT can significantly alleviate lipid deposition in hepatocytes, improve lipid metabolism disorders, effectively reduce lipid deposition-induced oxidative stress and lipid peroxidation damage in hepatocytes, and prevent cells from triggering apoptosis. Mechanistic studies show that ATT reverses the abnormal activation of the MASLD and cholesterol metabolism pathways caused by lipid deposition by activating the AMPK signaling pathway and inhibiting the expression of key downstream lipid synthesis genes, thereby regulating lipid metabolism and alleviating hepatocyte steatosis.

[0073] Specifically, (1) it significantly and comprehensively improved the functional maturity of hepatocyte organoids.

[0074] Existing technology: Although some methods have been used for the maturation of hepatocyte organoids, the improvement in functional performance (such as ammonia metabolism, albumin secretion, drug metabolism, etc.) is not significant, and their stability in long-term culture is poor.

[0075] This invention significantly improves organoid maturity and function by adding ATT to the culture medium and treating it continuously for 4-6 days. It enhances the drug responsiveness of hepatocyte organoids, upregulates key drug-metabolizing enzymes (such as CYP3A4 and CYP1A2), and enables hepatocyte organoids to produce a more significant induction effect that is closer to the physiological state in vivo.

[0076] (2) It can improve lipid metabolism disorder, effectively reverse hepatocyte steatosis, and at the same time alleviate the accompanying oxidative stress and cell apoptosis, and has a multi-target protective effect.

[0077] While FFA induces pathological damage, it directly co-treats cells with specific concentrations of ATT (e.g., 1 or 2 μM). This process rapidly activates core protective pathways such as AMPK through ATT, inhibiting new lipid synthesis at its source (downregulating SREBP1 and FASN) on the one hand, and enhancing the cells' antioxidant capacity (restoring GSH-Px activity, reducing ROS and MDA) and anti-apoptotic capacity (regulating genes such as BIRC3 and BAD) on the other hand. This achieves synergistic intervention in steatosis and its key complications (many existing drugs may only focus on either lipid reduction or antioxidant effects). Attached Figure Description

[0078] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1Artemisinin treatment significantly promoted the differentiation of HB-orgs into mature hepatocytes and the maturation of P-hep-orgs. A shows the chemical structure of artemisinin (ATT); B shows the cell differentiation stages and cell images at each stage; C shows the changes in albumin secretion levels in HB-orgs after ATT treatment; D shows the changes in ALB and A1AT protein expression levels in HB-orgs after ATT treatment; E shows the expression of hepatocyte-specific marker genes in HB-orgs and the comparison between the ATT-treated group and the control group; F shows the changes in P-hep-orgs after ATT treatment. Changes in liver function-related genes in P-hep-orgs; G represents changes in urea cycle-related genes in P-hep-orgs after ATT treatment; H represents changes in albumin secretion levels in P-hep-orgs after ATT treatment; I represents functional changes in urea synthesis and secretion in P-hep-orgs after ATT treatment; J represents a comparison of the inducible activity levels of drug-metabolizing enzymes in P-hep-orgs after rifampin, omeprazole, and ATT treatment; K represents changes in the expression levels of ALB and A1AT proteins in P-hep-orgs after ATT treatment. In the figures, data are mean ± SEM; ns: no statistically significant difference. .

[0079] Figure 2 This section presents a comparative analysis of HB-orgs transcriptome sequencing with and without ATT treatment. A shows a heatmap illustrating differentially expressed genes with and without ATT treatment; B shows a volcano plot of differentially expressed genes after ATT treatment based on transcriptome sequencing; C shows KEGG pathway enrichment analysis of differentially expressed genes; and D shows GO pathway enrichment analysis of differentially expressed genes.

[0080] Figure 3 The figures show changes in the cAMP signaling pathway after ATT treatment. A represents intracellular cAMP levels after ATT treatment; B shows cell images on day 6 after ATT and inhibitor treatment of HB-orgs (scale bar = 100 μm); C shows changes in hepatocyte marker gene levels detected by qPCR after ATT and inhibitor treatment; D shows changes in albumin secretion levels detected by ELISA after ATT and ESI09 inhibitor treatment; E shows changes in ALB and A1AT protein expression levels detected by Western blotting after ATT and ESI09 inhibitor treatment, followed by quantitative analysis. Data in the figures are mean ± SEM; ns: no statistically significant difference. .

[0081] Figure 4Transcriptomic comparison of P-heporgs between the ATT-treated group and the control group; where A is a heatmap showing differential gene expression between ATT-treated and untreated P-heporgs; B is a volcano plot of DEGs after ATT treatment based on transcriptome sequencing; C is GO (gene ontology) enrichment analysis of DEGs; and D is KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of DEGs.

[0082] Figure 5 Changes in P-hep-orgs liver function-related genes after treatment with different concentrations of ATT. In the figure, data are mean ± SEM; ns: no statistically significant difference. .

[0083] Figure 6 The effect of ATT on FFA-induced lipid accumulation in P-hep-orgs is shown in the figure. A represents Oil Red O staining (scale bar = 20 μm); B represents BODIPY staining of neutral lipid droplets (scale bar = 20 μm); C represents quantitative analysis of lipid droplet area (percentage of BODIPY positive area); D represents the effect of ATT on intracellular triglyceride (TG) levels; E represents the effect of ATT on intracellular total cholesterol (TC) levels; and F represents the effect of ATT on lipid metabolism-related genes. Data in the figure are mean ± SEM values. ns: no statistically significant difference. .

[0084] Figure 7 The effects of ATT on FFA-induced oxidative stress and hepatocyte damage in organoids are shown. A represents the immunofluorescence detection of the effects of FFA and ATT treatments on cellular ROS; B represents the quantitative analysis of ROS levels; C represents the measurement of lipid peroxidation (MDA content); D represents the measurement of cellular glutathione (GSH) levels; E represents the TUNEL-stained immunofluorescence image (scale bar = 20 μm); and F represents the quantitative analysis of the fluorescence image results in E. In the figures, data are mean ± SEM; ns: no statistically significant difference. .

[0085] Figure 8 RNA-seq analysis showed that ATT regulates hepatic lipid metabolism; A is a heatmap of differentially expressed genes in the ATT-treated group, FFA model group, and control group; B is a volcano plot of differentially expressed genes in the ATT-treated group and FFA model group based on transcriptome sequencing; C is KEGG enrichment analysis of differentially expressed genes in the ATT-treated group and FFA model group; D is GO enrichment analysis of differentially expressed genes in the ATT-treated group and FFA model group; E is a heatmap of key genes related to MASLD among differentially expressed genes in the ATT-treated group and FFA model group; F is a heatmap enrichment analysis of apoptosis-related genes among the control group, ATT-treated group, and FFA model group.

[0086] Figure 9 This study aimed to demonstrate that ATT enhances liver function and reduces lipid deposition through the AMPK signaling pathway. Figure A shows the GSEA enrichment analysis of differentially expressed genes between the FFA model group and the control group, and between the ATT-treated group and the FFA model group. Figure B shows a heatmap of key genes enriched in the AMPK signaling pathway between the ATT-treated group and the FFA model group. Figure C shows the expression of AMPK and p-AMPK proteins detected by Western blotting, and the quantification of the p-AMPK to AMPK ratio. Figure D shows the expression changes of SREBP1, FASN, and SCD1 genes in P-hep-orgs detected by RT-qPCR. Figure E shows the expression changes and quantification of FASN and SCD1 proteins in P-hep-orgs detected by Western blotting. Data in the figures are mean ± SEM; ns: no statistically significant difference. . Detailed Implementation

[0087] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0088] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0089] The primer sequences used for RT-qPCR detection of target gene expression levels in this invention are shown in Table 1. The antibodies used for Western blotting detection of target protein expression and their dilution factors are shown in Table 2.

[0090] Table 1 Primer sequence information used for RT-qPCR

[0091] Table 2. Antibodies used in Western Blot assays and their dilution ratios.

[0092] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0093] Example 1: Artemisinin promotes the functional maturation and differentiation of hepatocyte organoids. Preparation of ATT solution: Artemisitene (CAS No.: 101020-89-7) was dissolved in DMSO (dimethyl sulfoxide, MP Biomedical, 196055) to prepare a 1 mM stock solution, which was stored at -20°C protected from light.

[0094] 1. Hepatocyte organoid expansion and differentiation (1) Preparation of hepatic progenitor cell organoids (HB-orgs) Human embryonic stem cell-derived liver progenitor cell organoids (HB-orgs, the preparation method of which is disclosed in Chinese Invention Patent 2025115803881) were digested into a single-cell suspension using TrypLE™ and seeded into low-adhesion 6-well plates (Corning, 3471) at a seeding density of 5 × 10⁶ cells / well. 5 Cells / well; add liver progenitor cell organoid culture medium (culture medium formulation: IMDM (Gibco, 31980030) + 10% w / w% FBS (fetal bovine serum, VISTECH, SE100-B7953) + 1×ITS (biogems, 00-101) + 1×NEAA (Gibco, 11140050) + 1×GlutaMAX (Gibco, 35050061) + 10 mM nicotinamide (Sigma, N0636) + 5 μM CHIR99021 (Selleck, CT99021) + 10 μM SB431542 (Selleckchem, S1067) + 10 μM MFSK (MCE, HY-15371) + 20 ng / mL FGF-4 (Peprotech, 100-18B) + 20 ng / mL BMP4 (Peprotech, 120-05) + 20 ng / mL EGF (Peprotech, AF-100-15) + 5v / v% Growth-factor reduced Matrigel (Corning, 354230) were cultured for 2-4 days to obtain liver progenitor cell organoids (HB-orgs).

[0095] (2) ATT treatment for hepatocyte organoid maturation The HB-orgs culture system (hepatic progenitor cell organoid medium) obtained above was treated with an effective concentration (final concentration of 1 μM) of ATT. ATT was added to the hepatic progenitor cell organoid medium only on Day 0, and no further additions were required to promote the differentiation and maturation of hepatic progenitor cell organoids. 2 mL of fresh hepatic progenitor cell organoid medium (without ATT) was added every 48 hours, and the culture was continued for 6 days.

[0096] (3) Induced into mature hepatocyte organoids (P-hep-orgs) After 6 days of culture, the cells were induced into mature hepatocyte organoids (P-hep-orgs). Specifically, the hepatocyte progenitor cell organoid culture medium was replaced with hepatocyte maturation culture medium (the culture medium formula was Hepatocyte basal medium (HCM) (Lonza, CC-3911) + Single Quots kit (Lonza, CC-4182) + 20 ng / mL HGF + 50 ng / mL oncostatin M (Peprotech, 300-10) + 100 nM dexamethasone (Sigma, D4902) + 20 ng / mL FGF-4 + 1×B27 (Gibco, 17504044) + 5 v / v% Growth-factor reduced Matrigel). Cultured under this system for 8 days, highly mature hepatocyte organoids were obtained.

[0097] After step (2) of the culture, hepatic progenitor cell organoids and their culture supernatant were collected for testing. A control group without ATT was used (i.e., cultured using only hepatic progenitor cell organoid culture medium). The detection methods included: ELISA assay: The amount of ALB secreted in the culture supernatant was detected using a human albumin (ALB) ELISA kit (Bethyl, E80-129).

[0098] Western Blot Analysis: Total organoid protein was extracted, and the protein expression levels of albumin (ALB) and α1-antitrypsin (A1AT) were detected by Western Blot. Specifically, after extracting and quantifying total organoid protein, equal amounts of protein were subjected to SDS-PAGE electrophoresis and transferred to a membrane. After blocking, the membrane was incubated sequentially with specific primary and secondary antibodies, and finally, ECL chemiluminescence detection was performed. GAPDH was used as an internal control, and the grayscale values ​​of the target protein bands were quantitatively analyzed. Specific antibody information is shown in Table 2.

[0099] RT-qPCR detection: After centrifugation and discarding the supernatant, the cell pellet was collected. Total RNA was extracted from the cells using TRIzol reagent (Invitrogen, 15596026). After determining the concentration and purity using Nanodrop, an equal volume of RNA was reverse transcribed into cDNA using a reverse transcription kit (Vazyme, R323). Using the cDNA as a template, amplification was performed on a real-time quantitative PCR instrument using the SYBR Green method. The relative expression levels of target genes (mature hepatocyte marker genes ALB and A1AT, nuclear transcription factors HNF4α and FOXA2, drug-metabolizing enzyme genes CYP3A4, CYP1A2, CYP1B1, and CYP2C19, and ammonia metabolism gene CPS1) were calculated using the 2^-ΔΔCt method, and normalized using GAPDH as an internal control. The primer sequences used are shown in Table 1 below.

[0100] The results are as follows Figure 1 ELISA results showed that, compared with the control group, ATT-treated HB-orgs exhibited a significant increase in ALB secretion. Figure 1 The results (C) suggest that ATT promotes hepatic differentiation of hepatic progenitor cells. Western blot analysis showed that the protein expression of ALB and A1AT in ATT-treated HB-orgs was significantly higher than that in the control group (C). Figure 1 (D). RT-qPCR results showed that ATT treatment significantly upregulated the expression of mature hepatocyte marker genes (ALB, A1AT, HNF4α, FOXA2), drug-metabolizing enzyme genes (CYP3A4, CYP1A2, CYP1B1, CYP2C19), and ammonia metabolism gene CPS1. Figure 1 (E).

[0101] After 6 days of treatment, the two groups of HB-orgs were transferred to HCM complete medium (hepatocyte maturation medium), supplemented with 5% Growth-factor reduced Matrigel, and cultured for 8 days until differentiation (step 3) into mature hepatocyte organoids (P-hep-orgs). After treatment, the hepatocyte organoids and their culture supernatant were collected for analysis (analysis method as above). The results showed that in P-hep-orgs, ATT significantly upregulated the expression of drug transporter genes (MDR1, MRP2, BSEP, NTCP), UGT enzyme gene (UGT1A1), and key urea cycle enzyme genes (CPS1, OTC, ASS1, ASL, NAGS). Figure 1 (F and G). ELISA was used to detect ALB secretion levels. The results showed that, compared with the control group, ATT-treated P-hep-orgs had significantly increased ALB secretion levels (F and G). Figure 1 (H).

[0102] Further functional assays were performed, and the urea yield in the culture supernatant was detected using a urea nitrogen assay kit (Beyotime, S0602). The results showed that the urea yield in ATT-treated P-hep-orgs was significantly higher than that in the control group (…). Figure 1 Middle I).

[0103] Furthermore, to systematically evaluate the regulatory effect of ATT on drug metabolism in liver organoids, this invention employs a classic chemical inducing agent treatment model. P-hep-orgs were exposed to the inducer (P-hep-orgs in the control group and ATT-treated group were exposed to 25 μM rifampicin or 100 μM omeprazole, respectively, for 48 h (37℃, 5% CO2)). After induction, organoid samples were collected, and total RNA was extracted using Trizol reagent. Subsequently, qRT-PCR was performed according to the above experimental steps to detect changes in the expression levels of genes such as CYP3A4, CYP2C9, CYP1A1, CYP1A2, and CYP1B1.

[0104] Exposure of P-hep-orgs to the inducer rifampin resulted in upregulation of the expression of liver metabolic enzymes CYP3A4, CYP2C9, and CYP2C19, with further enhanced induced expression of these enzymes in the ATT treatment group. Figure 1 Similarly, treatment with the inducer omeprazole also increased the expression of CYP1A1 and CYP1A2, with the increase being particularly pronounced in the ATT-treated cohort. Western blot analysis further confirmed these findings, showing increased protein levels of the functional proteins ALB and A1AT in P-hep-orgs after ATT treatment. Figure 1 (Middle K). These results indicate that ATT promotes hepatocyte maturation.

[0105] To further investigate the effects of ATT on hepatic progenitor cell organoids (HB-orgs) at the transcriptomic level, the inventors performed RNA-seq analysis. The results are as follows: Figure 2 Figures A and B show that, compared with the control group, the ATT-treated group had a large number of differentially expressed genes (DEGs). Functional enrichment analysis of these differentially expressed genes revealed that biological processes related to liver function were significantly enriched, suggesting that ATT may promote the differentiation of HB-orgs into hepatocytes by regulating the cellular microenvironment, extracellular matrix, and metabolic processes. Figure 2 (C and D). Notably, the cAMP signaling pathway was significantly observed in the enrichment analysis ( Figure 2 (C)

[0106] To investigate whether the cAMP signaling pathway mediates the differentiation-promoting effect of ATT, the inventors first examined the changes in intracellular cAMP levels after ATT treatment. The experiments confirmed that the addition of ATT significantly increased intracellular cAMP concentration, indicating that this signaling pathway was activated. Figure 3 (Type A). The main downstream effector targets of cAMP include PKA (protein kinase A) and EPAC (a cAMP-activated exchanger). To distinguish between the two, the invention used ESI-09, a specific inhibitor of EPAC, and H-89, a specific inhibitor of PKA, for intervention experiments (specifically, HB-org was treated with 1 μM ATT, along with ESI-09 (10 μM) and H89 (10 μM), and the supernatant and cell pellet were collected for detection after 6 days of continuous culture). Morphological observation showed that in the presence of ESI-09, the morphology and structure of organoids could not be maintained normally, and the differentiation-promoting effect of ATT was inhibited; while no similar inhibitory effect was observed with the addition of H-89. Figure 3 (B) Further RT-qPCR analysis revealed that ATT treatment upregulated the expression of differentiation genes related to the hepatic progenitor cell stage, while the co-addition of ESI-09 blocked this promoting effect of ATT; the co-addition of H-89 did not have this blocking effect. Figure 3 (C). The above results indicate that the role of ATT in promoting liver differentiation is mainly mediated through the cAMP-EPAC signaling pathway, rather than the cAMP-PKA pathway.

[0107] To verify the role of the cAMP-EPAC pathway in ATT-promoted hepatocyte functional maturation, the inventors examined albumin secretion and the expression of key functional proteins in hepatocyte progenitor cells. The results showed that ATT treatment promoted albumin secretion and ALB and A1AT protein expression in hepatocyte progenitor cells. When the EPAC inhibitor ESI-09 was added simultaneously, the promoting effect of ATT on ALB secretion was weakened. Western blot analysis further confirmed that ATT treatment upregulated the protein levels of ALB and A1AT in HB-orgs, while ESI-09 inhibited the ATT-induced upregulation of these two functional proteins. Figure 3 (D and E). The above results demonstrate that the inventors have confirmed that ATT promotes the differentiation of hepatic progenitor cells into functionally mature hepatocytes by activating the cAMP-EPAC signaling pathway and upregulates the expression of characteristic functional proteins of mature hepatocytes.

[0108] To further characterize the promoting effect of ATT on the functional maturation of P-hep-orgs at the transcriptomic level, the inventors performed RNA-seq analysis. The results showed that, compared with the control group, the ATT-treated group had a large number of differentially expressed genes (DEGs), among which genes related to liver function and hepatocyte maturation, such as CYP4F12, CYP26A1, SOX17, TGFBR3, and LRP2, were significantly upregulated. Figure 4 Volcano plot analysis showed that 277 genes were upregulated and 519 genes were downregulated in the ATT-treated group, indicating that ATT has a broad regulatory effect on gene expression. Figure 4 (B). Gene ontology (GO) enrichment analysis showed that ATT treatment enriched biological processes related to fatty acid transport, carboxylic acid transport, vitamin transport, organic acid transport, proteoglycan metabolism, and extracellular matrix structure, which are intrinsically linked to hepatocyte metabolism and functional maturation. Figure 4 (C). Furthermore, pathway analysis using the Kyoto Encyclopedia of Genetics and Genomes (KEGG) indicated that ATT treatment enriched pathways involved in bile secretion, drug-metabolizing enzymes (cytochrome P450 family), and nitrogen metabolism, thereby enhancing its role in promoting hepatocyte maturation and improving liver function. Figure 4 (D).

[0109] To determine the optimal concentration of ATT for promoting hepatic progenitor cell differentiation, the inventors tested the effects of different concentrations of ATT (0.5 μM, 1 μM, 2 μM, and 5 μM) on P-Hep-orgs (experimental procedures as described above). The results showed that 1 μM ATT significantly promoted the differentiation of HB-orgs into mature hepatocytes, specifically through the upregulation of expression of mature hepatocyte marker genes (such as ALB, CPS1, BSEP, and NTCP). Figure 5 In contrast, treatment with 0.5 μM ATT did not show a significant promoting effect, indicating that concentrations below this level may not be sufficient to effectively activate the relevant signaling pathways; similarly, treatment with 2 μM ATT did not show a significant differentiation-promoting effect, suggesting that excessively high concentrations may not produce the expected biological effects or that other regulatory mechanisms exist. The effect of ATT in promoting hepatic progenitor cell differentiation exhibits a clear effect window around a concentration of 1 μM, and its effect is concentration-specific.

[0110] The above results indicate that treatment with 1 μM ATT can effectively promote the hepatic differentiation of hepatic progenitor organoids HB-org and improve the maturity and function of hepatocytes in hepatocyte organoids by activating the cAMP-EPAC signaling pathway. Specifically, this is manifested in increased secretion of key functional proteins, upregulation of gene expression in mature hepatocytes, and enhanced metabolic function.

[0111] Subsequent experiments all used 1 μM ATT concentration for treatment.

[0112] Example 2: Artemisinin alleviates FFA-induced lipid accumulation in hepatocyte organoids To simulate the pathological environment of MASLD, free fatty acids (FFA, i.e., a mixture of palmitic acid and oleic acid = 2:1) were used to induce mature P-hep-orgs obtained in Example 1. At the same time or after FFA induction, an effective concentration of ATT was added to the culture system for co-treatment or intervention treatment to investigate the effect of artemisinin on lipid accumulation of hepatocyte organoids induced by FFA. The specific experimental procedure is as follows: The mature P-hep-orgs cultured in Example 1 (i.e., P-hep-orgs cultured without ATT treatment) were randomly divided into four groups: (1) Control group: cultured in normal culture medium (i.e., the hepatocyte maturation culture medium in Example 1); (2) FFA model group: cultured in culture medium containing 450 μM FFA (palmitic acid: oleic acid = 1:2); (3) FFA+ATT 1μM group: cultured in culture medium containing 450 μM FFA and 1 μM ATT; (4) FFA+ATT 2μM group: cultured in culture medium containing 450 μM FFA and 2 μM ATT. Each group was treated for 24 hours.

[0113] After treatment, lipid deposition phenotype detection was performed on the organoids, including: Oil Red O staining: After fixation with 4% paraformaldehyde, hepatocyte organoids were stained with Oil Red O (solarbio, 1320-06-5) and intracellular lipid droplet accumulation was observed under a microscope. BODIPY staining: Intracellular lipid droplet accumulation was observed using BODIPY (Beyotime Biotechnology, C2053S). Specifically, cells were first fixed, permeabilized with 0.5% Triton X-100 (Solarbio, T8200), and stained with BODIPY 493 / 503 (Beyotime Biotechnology, C2053S) for 20 min. After DAPI staining (1 μg / mL), cells were analyzed using confocal microscopy.

[0114] Biochemical assays: The levels of triglycerides (TG) (Nanjing Jiancheng Bioengineering Institute, A110-1) and total cholesterol (TC) (Nanjing Jiancheng Bioengineering Institute, A111-1-1) in the hepatocyte organoid lysates of each group were determined using a kit. The preparation method of the hepatocyte organoid lysate was as follows: First, the drug-treated cell suspension was removed and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, and the cell pellet was retained. The cells were washed 1-2 times with PBS, and centrifuged at 1000 rpm for 10 minutes. The supernatant was discarded, and the cell pellet was retained. Then, cell lysis was performed: the cells were lysed with lysis buffer (1-2% Triton X-100) for 30-40 minutes. The lysed liquid was the hepatocyte organoid lysate, which could be directly used for subsequent assays.

[0115] RT-qPCR detection: Detection of lipid metabolism-related gene expression (FABP4, DGAT2, PLIN2, ASGR1, TRIB3) (detection process is the same as in Example 1).

[0116] Test results as follows Figure 6 Oil Red O staining results showed that the FFA model group had a large number of red lipid droplets, while the area and number of lipid droplets in both ATT treatment groups were significantly reduced. Figure 6 (A). BODIPY staining results showed that FFA induced significant intracellular lipid droplet accumulation, while ATT treatment significantly reduced this accumulation. Figure 6 (B and C in the middle).

[0117] The levels of triglycerides (TG) and total cholesterol (TC) in the lysates of hepatocyte organoids in each group were determined using a triglyceride (TG) and total cholesterol (TC) assay kit. Results showed that TG and TC levels in the FFA model group were significantly higher than those in the control group; while compared with the FFA model group, TG and TC levels in the ATT treatment group decreased in a concentration-dependent manner. Figure 6 (D and E in the middle).

[0118] RT-qPCR results showed that FFA treatment significantly upregulated the expression of fatty acid storage genes (FABP4 and DGAT2), lipid droplet coating protein gene (PLIN2), and stress-related genes (ASGR1 and TRIB3). ATT treatment effectively reversed the abnormal expression of these genes. Figure 6 (Middle F).

[0119] The above results indicate that ATT can significantly alleviate lipid deposition in hepatocytes induced by FFA and regulate the expression of lipid metabolism-related genes, thereby improving lipid metabolism disorders.

[0120] Example 3: Artemisinin alleviates FFA-induced oxidative stress and apoptosis. 1. Oxidative stress level detection Hepatocyte organoids from each group treated in Example 2 were subjected to the following assays to assess lipid peroxidation and antioxidant capacity: ROS assay: Intracellular reactive oxygen species (ROS) levels were detected using a fluorescent probe (DCFH-DA), performed according to the kit (Nanjing Jiancheng Bioengineering Institute, E004-1-1). MDA assay: Intracellular malondialdehyde (MDA) content was detected using the thiobarbituric acid method, performed according to the kit (Beyotime Biotechnology, S0131S). GSH-Px assay: Glutathione peroxidase (GSH-Px) activity was detected using a kit (Beyotime Biotechnology, S0053), performed according to the kit.

[0121] The results are as follows Figure 7 As shown. Fluorescence microscopy and fluorescence intensity quantification revealed that ROS levels were significantly increased in the FFA model group, while ROS levels were significantly decreased in the ATT treatment group. Figure 7 (A and B); MDA detection results showed that the MDA content in the FFA model group was significantly increased, and ATT treatment effectively inhibited the increase of MDA ( Figure 7 (C); GSH-Px detection results showed that GSH-Px activity was reduced in the FFA model group, while ATT treatment significantly restored its activity (C). Figure 7 (D).

[0122] 2. Apoptosis detection TUNEL staining was used to detect and quantify FFA-induced apoptosis. Organoids were collected, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 (containing 0.1% sodium citrate), and then processed according to the instructions of the TUNEL assay kit (Beyotime Biotechnology, C1086).

[0123] The results showed that the TUNEL-positive cell rate in the FFA model group was significantly higher than that in the control group, while ATT treatment (especially 2 μM) significantly reduced the proportion of apoptotic cells. Figure 7 (E and F in the middle).

[0124] The above results indicate that ATT can effectively alleviate FFA-induced oxidative stress and lipid peroxidation damage in hepatocytes and protect cells from FFA-triggered apoptosis.

[0125] Example 4: Study on the mechanism by which artemisinin regulates lipid metabolism through the AMPK signaling pathway 1. Transcriptomics analysis RNA-seq transcriptome sequencing was performed on P-hep-orgs from the control group, FFA model group, and FFA+ATT (2μM) group in Example 2, and key signaling pathways regulated by ATT were screened using bioinformatics methods (such as GO, KEGG, and GSEA enrichment analysis).

[0126] The results are as follows Figure 8 Gene cluster heatmaps showed significant expression differences among the groups, with FFA treatment producing different gene expression profiles compared to the ATT-treated and control groups. Figure 8 Volcano plot analysis showed that, compared with the FFA model group, the ATT treatment group had 3,411 genes significantly upregulated and 2,469 genes downregulated ( ). Figure 8 KEGG enrichment analysis showed that differentially expressed genes were significantly enriched in the AMPK signaling pathway, fatty acid degradation, and bile secretion pathways after ATT treatment. Figure 8 (C). GO enrichment analysis showed that differentially expressed genes were mainly enriched in biological processes such as oxidative phosphorylation, fatty acid metabolism, and intracellular lipid catabolism; in addition, ATT treatment was associated with the enrichment of molecular functions such as NADH dehydrogenase activity, antioxidant activity, and lipid transport activity. Figure 8 (D).

[0127] Under MASLD pathway enrichment, ATT significantly suppressed the expression of genes involved in lipogenesis (such as PROX1 and BMP6) while promoting the expression of genes involved in lipolysis and fatty acid oxidation (including UCP2 and ALDH1A2). Furthermore, ATT treatment led to significant downregulation of key genes involved in lipid synthesis, cholesterol metabolism, and fatty acid uptake, including MLXIPL, ACAT1, SCARB1, SOAT2, CD36, and SLC27A2. Figure 8 (E).

[0128] Furthermore, apoptosis pathway analysis showed that FFA treatment induced apoptosis in P-Hep-orgs cells, while ATT treatment effectively alleviated this response. In the ATT-treated group, upregulation of anti-apoptotic genes, including IAP family members BIRC3, BIRC7, and BIRC6, and downregulation of pro-apoptotic genes, such as BAD, TNFRSF1A, TNFSF10, and SEPTIN4, were observed. Figure 8 (Middle F).

[0129] Gene set enrichment analysis (GSEA) further confirmed that ATT treatment reversed the abnormal activation of FFA-induced non-alcoholic fatty liver disease (NAFLD) pathways and cholesterol metabolism pathways. Figure 9 (A)

[0130] 2. Mechanism Validation The expression of key molecules in the core pathways of each group of organoids was verified by RT-qPCR and Western Blot (the experimental procedure is described in Example 1), such as detecting the protein levels of phosphorylated AMPK (p-AMPK) and its downstream lipid synthesis-related factors (such as SREBP1, FASN, and SCD-1).

[0131] Test results as follows Figure 9 ATT treatment significantly increased the protein level of phosphorylated AMPK (p-AMPK). Figure 9 (B and C). The study examined key factors in lipid synthesis, and the results showed that ATT treatment significantly inhibited the gene and protein expression of fatty acid synthase (FASN), sterol regulatory element-binding protein 1 (SREBP1), and stearoyl-CoA desaturase-1 (SCD-1). Figure 9 (D and E in the middle).

[0132] The above results indicate that ATT exerts its role in regulating lipid metabolism and alleviating hepatocyte steatosis by activating the AMPK signaling pathway and inhibiting the expression of key downstream lipid synthesis genes.

[0133] The experimental results of the above embodiments demonstrate that the present invention successfully improves the functionality of hepatocyte organoids by adding ATT, including enhancing ammonia metabolism, increasing albumin secretion, promoting the activity of hepatic drug-metabolizing enzymes, and inducing the expression of hepatocyte markers, significantly improving the maturity and physiological function of organoids. Simultaneously, ATT activates the AMPK signaling pathway and inhibits the expression of key downstream lipid synthesis genes, thereby regulating lipid metabolism, improving lipid metabolism disorders, significantly reducing lipid deposition in hepatocytes induced by FFA, and thus alleviating hepatocyte steatosis. Furthermore, ATT can effectively reduce FFA-induced oxidative stress and lipid peroxidation damage in hepatocytes and protect cells from FFA-triggered apoptosis.

[0134] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of artemisinin in any one of (1)-(11): (1) Promotes the differentiation of hepatic progenitor cells into hepatocytes; (2) Prepare products that promote the differentiation of hepatic progenitor cells into hepatocytes; (3) Improve the maturity and function of hepatocytes in hepatocyte organoids; (4) Prepare products that improve the maturity and function of hepatocytes in hepatocyte organoids; (5) Constructing liver organoids; (6) To prepare drugs for the treatment and / or prevention of diseases caused by lipid metabolism disorders; (7) Inhibits oxidative stress and lipid peroxidation damage in hepatocytes; (8) Prepare products that inhibit oxidative stress and lipid peroxidation damage in hepatocytes; (9) Inhibits hepatocyte apoptosis; (10) Prepare products that inhibit hepatocyte apoptosis; (11) Prepare health products that help with anti-oxidation, help control body fat, help maintain healthy blood lipid levels, or have an auxiliary protective effect against chemical liver damage.

2. The application according to claim 1, characterized in that, The liver progenitor cells mentioned in (1)-(2) include liver progenitor cells of liver progenitor cell organoids, and the liver cells include liver cells of liver cell organoids; And / or, the hepatocytes described in (7)-(10) include hepatocytes of hepatocyte organoids; And / or, the apoptosis described in (9)-(10) includes free fatty acid-induced apoptosis.

3. The application according to claim 1, characterized in that, (6) The diseases caused by lipid metabolism disorders include fatty liver disease related to metabolic dysfunction; And / or, the effective concentration of artemisinin is 0.5-5 μM.

4. A culture medium comprising artemisinin and a liver progenitor cell organoid culture medium; Preferably, the final concentration of artemisinin in the culture medium is 0.5-5 μM.

5. The culture medium according to claim 4, characterized in that, The liver progenitor cell organoid culture medium includes at least one of Matrigel, ALK5 inhibitor, GSK-3 inhibitor, FSK, growth factor, and BMP signaling pathway activator.

6. The culture medium according to claim 5, characterized in that, The ALK5 inhibitors include at least one of SB431542, SB-505, A-83-01, GW6604, IN-1130, Ki26894, LY2157299, LY364947, LY550410, LY573636, LY580276, NPC-30345, SB-505124, SD-093, Sm16, SM305, SX-007, Antp-Sm2A, and LY2109761; And / or, the GSK-3 inhibitor includes at least one of SB216763, GSK3β inhibitor VII, L803-mts, 6-bromo-indirubin-3'-oxime, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-Azakenpaullone, Indirubin, and CHIR99021; And / or, the BMP signaling pathway activator includes at least one of BMP2, BMP4, SB4, SJ000291942, SJ000063181, SJ000370178, isoliquiritin, geraniol, apigenin and chickpea sproutin; And / or, the growth factor comprises at least one of epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor-I, IGF-II, leukemia inhibitory factor, nerve growth factor, tumor suppressor M, platelet-derived endothelial growth factor, transforming growth factor-α, and vascular endothelial growth factor.

7. A method for promoting the differentiation of hepatic progenitor cell organoids into hepatocyte organoids or a method for improving the maturity and function of hepatocyte organoids, comprising culturing hepatic progenitor cell organoids using the culture medium of any one of claims 4-6.

8. The method according to claim 7, characterized in that, The culture time is 1-10 days.

9. A method comprising the step of treating hepatocyte organoids with artemisinin; The method includes any one of 1)-3): 1) A method for inhibiting lipid deposition in hepatocyte organoids; 2) A method for inhibiting apoptosis of hepatocytes in hepatocyte organoids; 3) A method to improve the oxidative stress capacity of hepatocyte organoids.

10. The method according to claim 9, characterized in that, The effective treatment concentration of artemisinin is 1-5 μM.

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