Application of dehydrocostus lactone and medicine for treating steatohepatitis

Abstract

The invention discloses application of dehydrocostus lactone and a medicine for treating steatohepatitis, relates to the technical field of biological medicines, and solves the problems that in the prior art, the application scene of the dehydrocostus lactone is limited, and the application of the dehydrocostus lactone in a complex pathological environment of metabolic dysfunction related steatohepatitis has a technical blank. Dehydrocostus lactone can reduce lipid accumulation, inflammation and fibrosis in liver cells; the weight, liver weight, serum triglyceride content, serum total cholesterol content and insulin resistance level of animals with steatohepatitis are reduced; the content of serum glutamic oxalacetic transaminase, glutamic-pyruvic transaminase, tumor necrosis factor alpha, interleukin 1beta, malondialdehyde and bivalent ferrous ions is reduced, and the content of serum glutathione is increased; the compound can regulate and control lipid metabolism and mitochondrial energy metabolism pathways, reprograms fatty degeneration liver cell energy metabolism phenotypes, and is applied to preparation of drugs for treating steatohepatitis, ferroptosis inhibitors and glutamic oxalacetic transaminase and / or glutamic-pyruvic transaminase inhibitors.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of dehydroauracetam and a drug for treating fatty liver disease. Background Technology

[0002] Metabolic dysfunction-associated steatotic liver disease (MASLD) is inflammatoryly subtyped as metabolic dysfunction-associated steatohepatitis (MASH). Clinically, it manifests as hepatic steatosis, damage, and inflammation in non-alcoholic individuals, often accompanied by varying degrees of liver fibrosis, and some patients may progress to cirrhosis. With the continued prevalence of metabolic diseases such as obesity and type 2 diabetes, MASLD has become the most prevalent chronic progressive liver disease globally, particularly prominent in my country. Unlike malignant liver diseases such as liver cancer, which are characterized by abnormal cell proliferation, MASLD is essentially a chronic inflammatory liver injury driven by metabolic disorders, primarily caused by lipid deposition, insulin resistance, and oxidative stress imbalance. In terms of treatment philosophy, liver cancer focuses on tumor cell clearance and proliferation inhibition, while MASH emphasizes metabolic remodeling, inflammation control, and reversible regulation of cell damage. It requires intervention in lipid metabolism homeostasis, oxidative stress pathways, and programmed cell death processes to slow disease progression and promote liver function recovery. MASH has a bidirectional promoting relationship with diseases such as metabolic syndrome, and can accelerate complications such as atherosclerosis, chronic kidney disease, liver decompensation, and hepatocellular carcinoma. MASH has become an increasingly serious and important public health challenge facing my country now and in the future.

[0003] Ferroprelation is an iron-dependent cell death pathway characterized by intracellular iron overload leading to abnormal accumulation of reactive oxygen species and a cascade amplification of lipid peroxidation, accompanied by glutathione depletion and loss of GPX4 activity, ultimately resulting in cell membrane rupture and death. Studies have shown that ferroptosis plays a crucial role in the occurrence and progression of macrosomia schizophrenia (MASH). MASH patients often experience hepatic iron homeostasis imbalance and elevated oxidative stress, which exacerbates insulin resistance and metabolic disorders. Given the central role of the liver in iron homeostasis and lipid metabolism, targeting and inhibiting ferroptosis is considered a potential therapeutic strategy for MASH. However, current interventions targeting the ferroptosis pathway remain limited, and there is an urgent need to develop safe and effective treatment strategies to meet the unmet clinical needs of MASH patients.

[0004] Dehydrocostus lactone (Dehy) is a natural sesquiterpene lactone compound extracted from the traditional Chinese medicine *Aucklandia lappa*. Studies have confirmed its broad biological activities, including anti-ulcer, anti-inflammatory, antitumor, antioxidant, immunomodulatory, smooth muscle relaxant, and antispasmodic effects. Structurally, it exhibits good stability, maintaining its active conformation and function under various physiological conditions. Its moderate lipophilicity facilitates penetration of cell membrane barriers, enabling more efficient delivery to intracellular targets and the exertment of its intended biological activities. Current research largely focuses on the pro-ferroptosis effect of dehydrocostus lactone in tumor treatment (such as liver cancer), primarily based on its single application pathway of inducing tumor cell damage through regulation of oxidative stress pathways. However, these studies have not systematically elucidated the mechanism of action of dehydrocostus lactone in non-tumor chronic liver diseases, particularly lacking a clear understanding of its regulatory direction on ferroptosis under conditions of coexisting inflammation and metabolic disorders. This results in a significant disease-context-dependent biological effect, limiting its potential application in the field of metabolically related liver injury. Furthermore, current research on the application of dehydroauracetam in non-neoplastic chronic liver diseases is mainly limited to in vitro steatosis cell models, demonstrating only its local ameliorative effects on lipid accumulation and oxidative stress. A holistic disease model reflecting the complex pathological environment involving the intertwining of inflammatory responses, metabolic state changes, and cell death pathways has not yet been established. Therefore, the application of dehydroauracetam in metabolic-associated steatohepatitis still faces significant technical bottlenecks, and its potential therapeutic value urgently requires further systematic evaluation. Summary of the Invention

[0005] To address the limited application scenarios of dehydroauracetam in existing technologies and the technological gap in its application within the complex pathological context of metabolic dysfunction-related steatohepatitis, this invention proposes the application of dehydroauracetam and a drug for treating steatohepatitis. The technical solution of this invention is as follows: The application of dehydroauracetam lactone is in the preparation of drugs for treating fatty liver disease. The structural formula of dehydroauracetam lactone is shown in Formula I: Formula I; The aforementioned fatty liver disease refers to fatty liver disease associated with metabolic dysfunction caused by a high-fat, high-cholesterol, and high-fructose diet.

[0006] Furthermore, the drug for treating steatohepatitis is a ferroptosis inhibitor or an aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) inhibitor.

[0007] The dehydroauracetam lactone reduces lipid accumulation, inflammation, and fibrosis in hepatocytes; The dehydroauracetam lactone reduced body weight and liver weight in animals with fatty liver disease. The dehydroauracetam lactone reduces serum triglyceride levels, serum total cholesterol levels, and insulin resistance levels; The dehydroausyl lactone reduces serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The dehydrocostunolide reduces serum tumor necrosis factor α and interleukin-1β levels. The dehydrocostunolide improves metabolic disorders by regulating lipid metabolism and mitochondrial energy metabolism pathways and reprogramming the energy metabolism phenotype of steatotic hepatocytes. The dehydrocostunolide increases serum glutathione levels and decreases malondialdehyde and ferrous ion levels.

[0008] A drug for treating steatohepatitis, prepared using the above-mentioned dehydroauracetam, comprising dehydroauracetam and pharmaceutically acceptable excipients.

[0009] Furthermore, the dosage form of the drug for treating fatty liver disease includes any one of the following: injection, tablet, powder, suspension, capsule, pill, or syrup.

[0010] Compared with existing technologies, this invention solves the problems of limited application scenarios for dehydroauracetam and the technological gap in its application in the complex pathological environment of metabolic dysfunction-related steatohepatitis. Specifically, the beneficial effects are as follows: 1. This invention specifically applies dehydroauracetam to the preparation of drugs for treating metabolic dysfunction-related steatohepatitis, significantly expanding the application scenarios of dehydroauracetam in the field of metabolic liver diseases. It specifically demonstrates the comprehensive intervention effect of dehydroauracetam on metabolic dysfunction-related steatohepatitis, a complex disease characterized by lipid deposition, inflammatory damage, and fibrotic progression. Dehydroauracetam not only effectively inhibits abnormal weight gain and pathological hepatic weight gain, but also significantly reduces hepatic lipid deposition, improves histological changes such as steatosis and ballooning degeneration, reduces the degree of inflammatory cell infiltration, and inhibits the progression of liver fibrosis, thereby achieving overall regulation of the entire process of lipid deposition-inflammatory damage-fibrotic evolution. This invention effectively demonstrates that dehydroauracetam is no longer limited to improving a single metabolic indicator or a single pathological link, but has the ability to exert a comprehensive therapeutic effect in a complex pathological environment driven by multiple factors, significantly enhancing its application value in the field of metabolic liver diseases.

[0011] 2. This invention clarifies that dehydroauracetam possesses multi-target synergistic regulatory characteristics, exerting an overall improving effect on obesity, lipid metabolism disorders, and related metabolic abnormalities, significantly broadening its application scenarios. It can also be used to prepare aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) inhibitors. Dehydroauracetam can reduce serum triglyceride and total cholesterol levels, improve insulin sensitivity, reduce abnormally elevated transaminases, alleviate hepatocyte damage, and simultaneously downregulate the expression of inflammatory factors, improving the imbalance of the liver microenvironment. Furthermore, dehydroauracetam can optimize the energy metabolism structure of hepatocytes, enhance mitochondrial oxidative metabolism, and inhibit abnormally enhanced glycolytic compensatory processes, thereby achieving enhanced lipid oxidation capacity and restoration of energy metabolism homeostasis.

[0012] 3. This invention clarifies that dehydroauracetam can exert a specific protective effect under pathological conditions involving significant iron-dependent lipid peroxidation and oxidative stress, further establishing its application as an inhibitor of ferroptosis. Dehydroauracetam can enhance antioxidant reserves, reduce the accumulation of lipid peroxidation products and free ferrous ions, inhibit iron-dependent cell damage, and alleviate ferroptosis-related oxidative stress at the mechanistic level. Simultaneously, dehydroauracetam exhibits stable and significant functional characteristics in regulating iron homeostasis, enhancing antioxidant defense, and blocking the lipid peroxidation chain reaction. This makes it not only suitable for intervention in metabolic dysfunction-related steatohepatitis but also has potential value for extended applications to other ferroptosis-related diseases, filling the application gap of dehydroauracetam in the regulation of ferroptosis. Attached Figure Description

[0013] Figure 1 The results show the changes in body weight and liver function in mice after 8 weeks of intervention in each group induced by the HFHC diet; among them, Figure 1 (a) is a graph showing the changes in body weight of mice in each group; Figure 1 (b) is a graph showing the changes in liver weight in each group of mice; Figure 1 (c) is a graph showing the liver weight / body weight results for each group of mice; Figure 2 Images show the results of hematoxylin-eosin, Oil Red O, Masson's red, and Sirius red staining of the livers of HFHC-induced mice after 8 weeks of intervention in various groups, along with pathological analysis results. Figure 2 (a) The results of hematoxylin-eosin, oil red O, masson and Sirius red staining of the liver of HFHC diet-induced mice after 8 weeks of intervention in each group; Figure 2 (b) A graph showing the non-alcoholic fatty liver disease activity (NAS) score of liver sections stained with hematoxylin and eosin; Figure 2 (c) A graph showing the quantitative analysis of the lipid-positive area in liver sections stained with Oil Red O; Figure 2 (d) is a graph showing the quantitative analysis of fibrosis area in liver sections stained with Masson's disease. Figure 2 (e) is a graph showing the quantitative analysis of collagen area in liver sections stained with Sirius red. Figure 3 The graph shows the levels of biochemical indicators in HFHC-induced mice after 8 weeks of intervention in each group; among them, Figure 3 (ag) are graphs showing the changes in serum triglycerides, serum total cholesterol, insulin resistance index, serum aspartate aminotransferase, serum alanine aminotransferase, serum tumor necrosis factor α, and serum interleukin 1β levels in HFHC diet-induced mice after 8 weeks of intervention in each group. Figure 4 The graph shows the changes in serum glutathione, malondialdehyde, and ferrous ion levels in HFHC-induced mice after 8 weeks of intervention in various groups; among them... Figure 4 (a) is a graph showing the changes in serum glutathione levels in HFHC-induced mice after 8 weeks of intervention in each group; Figure 4 (b) is a graph showing the changes in malondialdehyde (MDA) content in mice after 8 weeks of intervention in each group induced by the HFHC diet. Figure 4 (c) is a graph showing the changes in ferrous ion content in mice after 8 weeks of intervention in each group induced by HFHC diet. Figure 5 The figure shows the results of in vitro flow analysis in a mouse primary hepatocyte model induced by oleic acid / palmitic acid; among them, Figure 5 (a) is a graph showing the change in mitochondrial oxygen consumption rate of hepatocytes over time in each group; Figure 5 (b) Quantitative analysis results of basal respiration, ATP production, and maximum respiratory capacity; Figure 5 (c) is a graph showing the dynamic change of extracellular acidification rate; Figure 5 (d) is a statistical analysis chart of glycolysis and glycolytic capacity; Figure 6 The graph shows the changes in key ferroptosis indicators in an oleic acid / palmitic acid-induced mouse hepatocyte in vitro model after different interventions; among them, Figure 6 (a) is a graph showing the changes in glutathione content in cells of each group; Figure 6 (b) Fe in the cells of each group 2+ Content change results graph; Figure 6 (c) is a flow cytometry distribution curve of lipid peroxidation detected by Liperfluo fluorescent probe; Figure 6 (d) is a graph showing the statistical results of the proportion of lipid peroxidation-positive cells; Figure 7 The image shows the expression results of key ferroptosis proteins in an in vitro mouse hepatocyte model induced by oleic acid / palmitic acid after treatment with dehydroauracetam and ferroptosis inhibitors, respectively; among them, Figure 7 (a) is a Western blot image of the proteins in each group; Figure 7 (b) is a graph showing the results of the corresponding protein grayscale quantitative analysis. Detailed Implementation

[0014] To make the technical solutions of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be noted that the following embodiments are only used to better understand the technical solutions of the present invention and should not be construed as limiting the present invention.

[0015] Example 1. A mouse model of metabolic dysfunction-related steatohepatitis induced by a high-fat, high-cholesterol, and high-fructose (HFHC) diet was established. C57BL / 6 mice were fed an HFHC diet consisting of 40 kcal% fat, 2% cholesterol, and 22% fructose. HFHC-induced steatohepatitis is widely recognized as a classic model of NASH (Neuro-Associated Spondylitis). Rodents fed HFHC for a long period (≥16 weeks) will naturally progress to hepatic steatosis, accompanied by inflammation and fibrosis. Male C57BL / 6 mice were acclimatized in a standard experimental environment for 7 days and then randomly divided into a model group, a dehydroausinolone intervention group, and a positive control group. The dehydrocostunolide intervention groups were administered dehydrocostunolide at doses of 15 mg / kg and 30 mg / kg daily via gavage, designated as the Dehy 15 mg / kg group and the Dehy 30 mg / kg group, respectively. The positive control group was administered obeticholic acid (OCA) at doses of 10 mg / kg daily via gavage, designated as the OCA 10 mg / kg group. Each group contained 6 mice, and the experiment lasted for 24 weeks. The model group was fed a HFHC diet for 24 weeks. The remaining groups underwent HFHC feeding for 16 weeks to establish the model, followed by an 8-week intervention under a continuous HFHC diet. During the intervention phase, the Dehy 15 mg / kg group, the Dehy 30 mg / kg group, and the OCA 10 mg / kg group were administered dehydrocostunolide at doses of 15 mg / kg / 2 days, 30 mg / kg / 2 days, and 10 mg / kg / 2 days, respectively, every two days via gavage.

[0016] Near the end of the experiment, mice were fasted for 12 hours before sampling (with free access to water). After final body weight measurement, blood samples were collected using the retroorbital venous plexus method, followed by cervical dislocation as euthanasia. Liver tissue was rapidly separated after euthanasia and sampled from different areas according to their intended use. 300 mg of tissue from the largest lobe of the liver was collected as a liver sample, placed in RNase-free cryovials, and immersed in liquid nitrogen before being transferred to [other storage facilities]. Store at 80 ℃ for later use. Take another 400 mg tissue sample (1 cm × 1 cm × 0.5 cm) from the middle region of the same site, fix it in 10% formaldehyde solution, and place it in a container... Store at 80℃ for later use. Take several pieces of right liver lobe tissue, 300 mg per piece, and place them in separate 15 mL centrifuge tubes. Store at 80℃ for later use.

[0017] I. Test results of liver weight and body weight in patients with metabolic dysfunction-related steatohepatitis, improved by dehydroaurateurin: like Figure 1 The figure shows the changes in body weight and liver function in mice induced by the HFHC diet in Example 1 after 8 weeks of intervention in each group. Among them, Figure 1 (a) is a graph showing the changes in body weight of mice in each group. As can be seen from the graph, the body weight of mice in the model group gradually increased under continuous HFHC diet induction. However, after intervention with dehydroausendin, the trend of body weight increase in mice was significantly inhibited. In particular, the body weight of the Dehy 30 mg / kg group was significantly lower than that of the model group, showing a clear dose-dependent decreasing trend. Moreover, the overall decrease was better than that of the positive control OCA group, which proves that dehydroausendin can effectively improve the abnormal body weight increase induced by HFHC. Figure 1 (b) is a graph showing the changes in liver weight in each group of mice. The graph shows that the liver weight of the model group mice was significantly increased, indicating that the HFHC diet successfully induced hepatic lipid deposition and hepatic hypertrophy. After intervention with dehydroauracetam, the liver weight of all treatment groups decreased significantly, with the Dehy 30 mg / kg group showing the largest decrease and exhibiting a better improvement effect than the OCA group, demonstrating that dehydroauracetam can significantly alleviate HFHC-induced hepatomegaly. Figure 1 (c) shows the results of liver weight / body weight in each group of mice. As can be seen from the figure, compared with the model group, the liver weight / body weight ratio of each dose group of dehydroauracetin was significantly reduced, and the improvement was more significant in the high dose group, which proves that dehydroauracetin can effectively alleviate liver lipid deposition and pathological liver weight gain induced by HFHC diet.

[0018] II. Test results of dehydroauracetamole in improving pathological tissues of metabolic dysfunction-related steatohepatitis: Hematoxylin-eosin staining: Tissue from the middle two-thirds of the liver lobe of mice in each group of the study (Example 1) was uniformly collected, placed in an embedding cassette, and fixed overnight in 10% neutral buffered formalin solution. Subsequently, dehydration and clearing were performed to prepare paraffin-embedded liver tissue blocks. Paraffin sections with a thickness of 5 μm were cut and baked in a 75 ℃ incubator for 1 h to obtain liver paraffin sections, which were then stored at room temperature for later use. For staining, the liver paraffin sections were immersed in hematoxylin staining solution for 5 min, rinsed with tap water, differentiated with differentiation solution, rinsed with tap water, blued with blue solution, and rinsed with running water. The sections were then sequentially immersed in 85% and 95% graded ethanol solutions for 5 minutes each for dehydration, followed by staining with eosin for 5 minutes each. After staining, the sections were sequentially immersed in anhydrous ethanol I, anhydrous ethanol II, and anhydrous ethanol III for 5 minutes each for dehydration, and then cleared with xylene I and xylene II for 5 minutes each to obtain neutral resin mountings. These were then examined under an optical microscope, and images were acquired for pathological analysis. Figure 2 (a) shows the results of hematoxylin-eosin staining of the liver of mice induced by HFHC diet in Example 1 after 8 weeks of intervention in each group. As can be seen from the figure, after hematoxylin-eosin staining, the hepatocytes of the model group mice showed more obvious fatty degeneration, with round vacuoles of different sizes in the cytoplasm, as well as ballooning degeneration, cell swelling, cytoplasmic vacuolation, inflammatory cell infiltration, and a noticeably greasy feel when touched. After 8 weeks of intervention with dehydroausinolone, the pathological damage of liver tissue was significantly improved, the hepatocyte structure gradually recovered its integrity, the liver plates tended to be arranged regularly, and the inflammatory infiltration was significantly reduced. Moreover, the overall improvement of the high-dose group was better than that of the OCA positive control group. Figure 2 (b) is a graph showing the NAS score results of liver sections stained with hematoxylin and eosin. As can be seen from the graph, compared with the model group, the NAS scores of each dose group of dehydroauracetin were significantly reduced, proving that dehydroauracetin can effectively alleviate the overall pathological damage of HFHC-induced fatty liver disease.

[0019] Oil Red O staining: Tissue from the lower third of the middle lobe of the liver of mice in each group as described in Example 1 was uniformly taken and completely immersed in embedding medium, then stained in liquid nitrogen. Rapid freezing of the air interface until complete OCT solidification was performed to prepare frozen liver tissue embedding blocks. Store at 20 ℃. Liver sections were prepared using a cryostat at 20 ℃, with a section thickness of 8 μm. The frozen liver sections were then placed in... Store at 80 ℃ for later use. For staining, thaw frozen sections, fix in fixative for 5 min, rinse with tap water, and air dry. Immerse sections in Oil Red O staining solution for 10 min, cover, and protect from light. After staining, remove sections and hold for 3 s, then immerse in two separate tanks of 60% isopropanol for differentiation (3 s, 5 s each), followed by immersion in two separate tanks of pure water for 10 s each. After rinsing, remove sections and hold for 3 s, then counterstain in hematoxylin for 5 min, rinse in three separate tanks of pure water for 5 s, 10 s, and 30 s each, differentiate in differentiation solution (60% alcohol as solvent) for 5 s, rinse in two separate tanks of distilled water for 10 s each, and then in blue solution for 1 s. Gently immerse sections in two separate tanks of tap water for 5 s and 10 s each. Examine staining results under an optical microscope. Mount with glycerin-gelatin mounting medium. Observe and photograph under a white light microscope, randomly acquiring fields of view. Figure 2 (a) shows the Oil Red O staining results of HFHC diet-induced mice after 8 weeks of intervention in each group in Example 1. The figure shows that a large amount of red lipid deposition was observed in the liver tissue of the model group, with widespread and obvious lipid droplet distribution. After intervention with dehydroauscin, the number and area of ​​lipid droplets in hepatocytes were significantly reduced, and the degree of lipid deposition was significantly decreased. Figure 2 (c) shows the quantitative analysis of the lipid-positive area of ​​liver sections stained with Oil Red O. The Oil Red O positive area in the dehydrocostunolide treatment group was significantly lower than that in the model group, proving that dehydrocostunolide can effectively inhibit HFHC-induced liver lipid deposition.

[0020] Masson staining: Tissue sections from the lower 1 / 3 of the middle lobe of the liver of mice in each group of Example 1 were uniformly collected. The sections were treated sequentially with xylene I, xylene II, and xylene III for 5 min each; then treated with anhydrous ethanol, 95% ethanol, and 75% ethanol for 1 min each; rinsed with tap water for a few seconds to dewax to water; stained with Weigert iron hematoxylin staining solution for 8 min; differentiated in acidic ethanol differentiation solution for 15 s, and washed with water; blued with Masson blue solution for 5 min, and washed with water. Subsequently, the sections were washed with distilled water for 1 min, stained with Ponceau S and fuchsin staining solution for 5 min, washed with weak acid working solution for 1 min, washed with phosphomolybdic acid solution for 1 min, washed with weak acid working solution for 1 min, stained with aniline blue staining solution for 2 min, and washed with weak acid for 1 min; then rapidly dehydrated with 95% ethanol for 2-3 s, dehydrated with anhydrous ethanol 3 times (5-10 s each time), cleared with xylene 3 times (1-2 min each time), and mounted with neutral resin. Then, the sections were examined under an optical microscope, and images were acquired for pathological analysis. like Figure 2(a) shows the results of marsonod staining of the liver of mice induced by the HFHC diet in Example 1 after 8 weeks of intervention in each group. The figure shows that obvious collagen fiber deposition was observed in the liver tissue of the model group, indicating early fibrosis. After intervention with dehydroausendin, the blue-stained collagen fiber deposition was significantly reduced, and the degree of fibrosis was significantly alleviated. Figure 2 (d) shows a quantitative analysis of the fibrosis area in a liver section stained with massonite. Dehydrocostunolide can significantly reduce the level of liver tissue fibrosis.

[0021] Sirius red staining: Tissue sections from the lower 1 / 3 of the liver of mice in each group of Example 1 were uniformly collected. The sections were sequentially treated with xylene I and xylene II for 20 min each, followed by treatment with anhydrous ethanol I and anhydrous ethanol II for 10 min each, and then with 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 5 min each. After washing with distilled water to dewax to water, the sections were stained with Sirius red staining solution for 1 h, rinsed with running water for 10 s, and immersed in 0.5% acetic acid for 20 s. The sections were then directly immersed in anhydrous ethanol for differentiation and dehydration. Clearing and mounting: The sections were dehydrated and cleared with xylene I for 5 min and xylene II for 5 min, and then mounted with neutral resin. The sections were then examined under an optical microscope, and images were acquired for pathological analysis. Figure 2 (a) shows the Sirius red staining results of the livers of mice induced by the HFHC diet in Example 1 after 8 weeks of intervention in each group. The figure shows that the liver tissue of the model group exhibits significant fibrosis and collagen fiber hyperplasia. In contrast, after 8 weeks of treatment with dehydroauscin, the liver cells gradually became rounder and fuller, the liver plates became more regularly and neatly arranged, there was no significant dilation or compression of the hepatic sinusoids, the area of ​​red lipid droplets on hepatocytes was significantly reduced, and the ballooning degeneration and fibrosis of hepatocytes were weakened, with no greasy feel upon touch. Figure 2 (e) shows the quantitative analysis of collagen area in liver slices stained with Sirius red. The proportion of collagen deposition was significantly reduced in each dose group of dehydrocostunolide, demonstrating that dehydrocostunolide has a significant inhibitory effect on liver fibrosis.

[0022] III. Results of tests on serum triglyceride, total cholesterol, insulin resistance, aspartate aminotransferase (AST), alanine aminotransferase (ALT), tumor necrosis factor-α, and interleukin-1β levels in patients with metabolic dysfunction-related steatohepatitis regulated by dehydrocostunolide: Following the instructions of the commercial kits for measuring serum triglycerides, total cholesterol, insulin resistance, aspartate aminotransferase (AST), alanine aminotransferase (ALT), tumor necrosis factor-α (TNF-α), and interleukin-1β, the levels of these biochemical indicators in the serum of mice in Example 1 were measured in each group. The results are as follows: Figure 3 As shown, where, Figure 3(ag) shows the changes in serum triglycerides, total cholesterol, insulin resistance index, serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), serum tumor necrosis factor-α (TNF-α), and serum interleukin-1β (IL-1β) levels in mice after 8 weeks of intervention induced by the HFHC diet. The figures show that dehydroauracetam significantly reduced HFHC-induced dyslipidemia, serum triglycerides, and total cholesterol levels, demonstrating its excellent interventional effect on lipid metabolism disorders. Simultaneously, dehydroauracetam effectively reduced the insulin resistance index, proving its effectiveness in... It has significant potential for systemic metabolic regulation in improving metabolic dysfunction; intervention with dehydrocostunolide can significantly reduce serum aspartate aminotransferase and alanine aminotransferase levels, proving that dehydrocostunolide can effectively alleviate abnormal hepatocyte membrane permeability and cell damage, and has a clear protective effect on liver function; in addition, dehydrocostunolide can significantly reduce the expression levels of pro-inflammatory factors tumor necrosis factor α and interleukin 1β, indicating that dehydrocostunolide can effectively inhibit the chronic low-grade inflammatory response induced by HFHC diet, further confirming its anti-inflammatory potential in metabolic dysfunction-related steatohepatitis.

[0023] In summary, dehydroauracetam not only improves lipid metabolism abnormalities and insulin resistance, but also simultaneously alleviates liver function damage and inflammatory microenvironment dysregulation, demonstrating a holistic biological effect of multi-target synergistic regulation. Based on the above experimental results, dehydroauracetam has a clear functional role in reducing serum transaminase levels and can be used to prepare aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) inhibitors. By inhibiting abnormally elevated transaminase levels, it can reduce the degree of hepatocyte damage, thereby intervening in and improving liver function disorders caused by metabolic dysfunction-related steatohepatitis. Furthermore, from the perspective of mechanism of action, the downregulation of transaminases by dehydroauracetam may be related to multiple mechanisms, including alleviating lipid deposition-induced mitochondrial stress, inhibiting inflammatory factor-mediated cell damage, and improving hepatocyte energy metabolism homeostasis, demonstrating obvious systemic regulatory characteristics.

[0024] IV. Test results of serum glutathione, malondialdehyde, and ferrous ion biochemical indicators in fatty liver disease associated with metabolic dysfunction regulated by dehydrocostunolide: The levels of biochemical indicators such as glutathione, malondialdehyde, and ferrous ions in the serum of mice in Example 1 were determined according to the instructions of the commercial kit for the determination of serum glutathione, malondialdehyde, and ferrous ions.

[0025] The measurement results are as follows Figure 4 As shown, where, Figure 4(ac) shows the changes in serum glutathione, malondialdehyde, and ferrous ion levels in mice after 8 weeks of intervention with HFHC diet. The figures show that after 8 weeks of intervention with dehydrocostunolide, serum glutathione levels in mice significantly increased, while malondialdehyde and ferrous ion levels significantly decreased, demonstrating that dehydrocostunolide can effectively improve lipid peroxidation and oxidative stress related to ferrous ion accumulation. Simultaneously, dehydrocostunolide inhibits iron-dependent lipid peroxidation by regulating key biochemical indicators of ferroptosis, thereby alleviating oxidative stress damage in ferroptosis-mediated metabolic dysfunction-related steatohepatitis at the mechanistic level, demonstrating its potential as a ferroptosis inhibitor.

[0026] V. Test results of dehydroauracetamide improving hepatocyte energy metabolism phenotype: (1) The effect of dehydroauracetam on the energy metabolism phenotype in a mouse primary hepatocyte model was determined by an in vitro flow analysis system. The specific steps for the mitochondrial aerobic respiration pressure (oxygen consumption rate) test are as follows: S1: Primary mouse hepatocytes were isolated, and the cell suspension was seeded in a special culture medium for primary mouse cells, with the cell number adjusted to a suitable level. Using a pipette tip, 50 μL of cell suspension was slowly added to the wells of a microplate (blank background wells were only added with culture medium). 400 μL of phosphate-buffered saline (PBS, to prevent evaporation) was added to each well on the side. The cells were then cultured overnight at 37 ℃ in a 5% CO2 incubator. Subsequently, a special culture medium containing 200 μM oleic acid and 100 μM palmitic acid was added to the cell suspension, and the volume was adjusted to 50 μL. The cells were cultured for another 24 h. After successfully establishing the in vitro cell model, dehydroauric acid lactone (10 μM, 15 μM) and obeticholic acid (5 μM) were added for 24 h, respectively. A control model group was also set up.

[0027] S2: Hydration probe: Add 200 μL of hydration equilibration solution to each well of the probe plate and 400 μL of hydration equilibration solution to each side well. Then place it in a 37 ℃ CO2-free constant temperature incubator for overnight hydration. The probe plate needs to be placed in a 37 ℃ incubator for more than 12 hours. S3: Prepare the culture medium for analysis: Weigh 0.45 g glucose, add 2 mL ultrapure water, mix thoroughly to dissolve, and bring the final concentration to 2.5 M; weigh 0.1461 g glutamine, add 5 mL ultrapure water, mix thoroughly to dissolve, and bring the final concentration to 0.2 M; weigh 0.11 g sodium pyruvate, add 10 mL ultrapure water, mix thoroughly to dissolve, and bring the final concentration to 0.1 M; add 200 μL glucose, 200 μL glutamine, and 100 μL sodium pyruvate to 9.5 mL of serum- and bicarbonate-free basal cell culture medium. When the temperature of the analysis medium reaches 37 ℃, adjust the pH to 7.3 with 1 mM NaOH solution. S4: Preparation of metabolic regulation drugs: Oligomycin was added to 252 μL of analytical medium to a final concentration of 1 μM; oxidative phosphorylation uncoupling agent was added to 288 μL of analytical medium to a final concentration of 0.5 μM; antimycin A was added to 216 μL of analytical medium to a final concentration of 0.5 μM. S5: Take the cells cultured in S1, and use a pipette to aspirate the culture medium to maintain a volume of 20 μL; add 200 μL of the prepared analytical medium to each well for washing and then aspirate it all, repeating this step once; add 160 μL of analytical medium to each well, making the final volume of analytical medium in each well 180 μL, and place in a CO2-free incubator for later use; remove the hydrated probe plate, add 20 μL of oligomycin to each well in the first drug injection well (AH well), 22 μL of oxidative phosphorylation uncoupling agent to each well in the second drug injection well (AH well), and 25 μL of antimycin A to each well in the third drug injection well (AH well). S6: Power on the Seahorse extracorporeal flow analyzer and preheat it to 37 ℃. Select the mitochondrial program. The probe plate will automatically enter the program for calibration. After calibration, the probe plate base plate will exit the program automatically. Remove the probe plate base plate and replace it with the prepared sample cell culture plate. Confirm the program is running. After the analysis is completed, use Wave software to open the raw data for data processing.

[0028] (2) The specific steps for the glycolysis (extracellular acidification rate) test are as follows: S1: The procedure is the same as the oxygen consumption rate test S1. S2: The procedure is the same as the oxygen consumption rate test S2. S3: Prepare the culture medium for analysis: Weigh 0.1461 g of glutamine, add 5 mL of ultrapure water, mix thoroughly to dissolve, and make the final concentration 0.2 M; take 200 μL of glutamine and add it to 9.8 mL of serum- and bicarbonate-free cell basal culture medium. When the temperature of the analysis medium reaches 37 ℃, adjust the pH to 7.4 with 1 mM NaOH solution. S4: Preparation of metabolic regulation drugs: Add glucose to 300 μL of analytical medium to a final concentration of 10 mM; add oligomycin to 288 μL of analytical medium to a final concentration of 10 mM; add 2-deoxy-D-glucose to 300 μL of analytical medium to a final concentration of 50 mM. S5: Take the cells cultured in S11 and use a pipette to aspirate the culture medium to a volume of 20 μL; add 200 μL of the prepared analytical medium to each well for washing and then aspirate it all, repeating this step once; add 160 μL of analytical medium to each well, making the final volume of analytical medium in each well 180 μL, and place in a CO2-free incubator for later use; remove the hydrated probe plate, add 20 μL of glucose to each well in the first drug injection well (AH well), 22 μL of oligomycin to each well in the second drug injection well (AH well), and 25 μL of 2-deoxy-D-glucose to each well in the third drug injection well (AH well). S6: The Seahorse extracorporeal flow analyzer should be preheated to 37°C before use. Select the glycolysis program, and the probe plate will automatically calibrate. After calibration, the probe plate base plate will exit the program automatically. Remove the probe plate base plate and replace it with the prepared sample cell culture plate. Confirm the program is running. After the analysis is complete, use Wave software to open the raw data for processing.

[0029] The results of the in vitro flow analysis system for the above-mentioned oleic acid / palmitic acid-induced mouse primary hepatocyte in vitro model are as follows: Figure 5 As shown, where, Figure 5 (a) is a graph showing the change in mitochondrial oxygen consumption rate of hepatocytes over time in each group; Figure 5 (b) Quantitative analysis results of basal respiration, ATP production, and maximum respiratory capacity; Figure 5 (c) is a graph showing the dynamic change of extracellular acidification rate; Figure 5 (d) is a statistical analysis chart of glycolysis and glycolytic capacity. From Figure 5 As shown in (a–b), compared with the model group, the basal respiratory level and the maximum respiratory capacity induced by oxidative phosphorylation uncoupling agent in hepatocytes were significantly increased after treatment with dehydrocostunolide, demonstrating that dehydrocostunolide can effectively restore mitochondrial oxidative phosphorylation function and improve fatty acid oxidation capacity. Meanwhile, there was no significant difference in non-mitochondrial respiration after antimycin A inhibition, indicating that dehydrocostunolide mainly acts on mitochondrial function improvement rather than non-specific oxygen consumption processes. Figure 5(c–d) It can be seen that the extracellular acidification rate of hepatocytes was significantly lower after dehydroauracetam intervention compared with the model group. The glycolytic capacity after glucose stimulation and the glycolytic reserve induced by oligomycin both showed a decreasing trend, indicating that dehydroauracetam can inhibit the compensatory glycolytic enhancement phenomenon. After treatment with dehydroauracetam, the primary hepatocyte model was reprogrammed to improve the energy metabolism phenotype of hepatocytes and accelerated lipid oxidation to improve metabolic disorders.

[0030] VI. Results of the application of dehydroauracetam as an inhibitor of ferroptosis in an oleic acid / palmitic acid-induced mouse hepatocyte in vitro model: (1) Biochemical indicators such as glutathione and ferrous ions in an in vitro mouse hepatocyte model induced by oleic acid / palmitic acid were determined using a commercially available biochemical indicator kit: Mouse hepatocytes were seeded in 96-well plates and cultured overnight at 37 °C in a 5% CO2 incubator; free fatty acid induction medium containing 200 μM oleic acid and 100 μM palmitic acid was added to the cell suspension and cultured for 24 h; after successfully establishing the in vitro cell model, dehydroaurolactone (15 μM) and the classical ferroptosis inhibitor Ferrostatin-1 (Fer-1, 1 μM) were added and treated for 24 h. The contents of biochemical indicators such as glutathione and ferrous ions in the cells of each group of the in vitro mouse hepatocyte model induced by oleic acid / palmitic acid were determined according to the instructions of the commercially available kit.

[0031] like Figure 6 The figure shows the changes in key indicators of ferroptosis in an in vitro mouse hepatocyte model induced by oleic acid / palmitic acid after different interventions. Figure 6 (a) is a graph showing the changes in glutathione content in cells of each group. As can be seen from the graph, compared with the model group, the intracellular glutathione levels were significantly increased after treatment with dehydroausinol and Fer-1. Figure 6 (b) Fe in the cells of each group 2+ The content change results are shown in the figure. The figure shows that the treatment with dehydroauracetam lactone can significantly reduce the level of free divalent ferrous ions in cells, and the trend is similar to that of Fer-1. Figure 6 (c) is a flow cytometry distribution curve of lipid peroxidation detected by Liperfluo fluorescent probe. The figure shows that the fluorescence signal shifted to the left after treatment with dehydroausinol, which proves that the lipid peroxidation level decreased. Figure 6 (d) is a statistical result of the proportion of lipid peroxidation positive cells. As can be seen from the figure, both dehydroauracetin and Fer-1 can significantly reduce the level of lipid peroxidation. This proves that dehydroauracetin exhibits similar effects to classic ferroptosis inhibitors in terms of enhancing antioxidant reserves, inhibiting ferrous ion accumulation and reducing lipid peroxidation.

[0032] (2) Detection of lipid peroxides in an in vitro mouse hepatocyte model induced by oleic acid / palmitic acid using Liperfluo dye: Mouse hepatocytes were seeded in 6-well plates and cultured overnight at 37 °C in a 5% CO2 incubator. Free fatty acid induction medium containing 200 μM oleic acid and 100 μM palmitic acid was added to the cell suspension and cultured for 24 h. After successful establishment of the in vitro cell model, dehydroauronin (15 μM) and the classical ferroptosis inhibitor Fer-1 (1 μM) were added and incubated for 24 h. The supernatant was removed, and the cells were washed once with serum-free medium. 1 μM Liperfluo working solution was added, and the cells were incubated at 37 °C for 30 min. The supernatant was removed, and the cells were washed twice with serum-free medium. Cell analysis was performed using flow cytometry.

[0033] (3) The expression of key proteins of ferroptosis in an in vitro mouse hepatocyte model induced by oleic acid / palmitic acid was determined by Western blot, specifically including the following steps: P1: Primary mouse hepatocytes were isolated, and the cell suspension was seeded in a special culture medium for primary mouse cells. An in vitro mouse hepatocyte model was established by oleic acid / palmitic acid induction. After treatment with dehydroauronin (15 μM) and the classic ferroptosis inhibitor Fer-1 (1 μM) for 24 h, the cell pellet was collected. An appropriate amount of IP lysis buffer (containing phosphatase inhibitor, protease inhibitor, and PMSF) was added, and the cells were lysed on ice for 30 min. After sonication, the cells were centrifuged at 12000 rpm for 8 min at 4 ℃, and the supernatant was transferred to a new EP tube; this completed the total protein extraction step. P2: Prepare a 25 mg / mL protein standard solution (30 mg BSA); take 40 μL of the protein standard solution, add 1960 μL of deionized water, and dilute to a final concentration of 0.5 mg / mL. It can be used immediately after preparation, or left to stand. Store at 20℃ for later use; BCA working solution preparation and protein concentration determination: Prepare an appropriate amount of BCA working solution by mixing BCA reagents A and B in a 50:1 ratio. Dilute the protein standard solution to concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. Add 2 μL of each sample to a labeled 96-well plate, add 18 μL of deionized water to a final volume of 20 μL, and then add 200 μL / well of the prepared BCA working solution to each sample. Incubate in a CO2-free 37℃ incubator in the dark for 30 min. Measure the absorbance at 562 nm using a microplate reader. Construct a standard curve based on the relationship between standard protein concentration and absorbance, and use this curve to calculate the protein concentration of the sample. P3: Preparation method of PAGE gel rapid preparation kit: Take equal volumes of 4.0 mL of lower gel buffer and lower gel solution, and mix thoroughly by pipetting to obtain a lower gel mixture; add 80 μL of modified coagulant to the lower gel mixture and mix thoroughly by pipetting to obtain a mixed solution; pour the mixed solution into the thick and thin glass plates, making the distance between the liquid surface of the mixed solution and the upper edge of the thin glass plate 50 mm longer than the comb teeth, and then pour 1 mL of deionized water to cover the lower gel solution; at room temperature, the lower gel will solidify after 10 min, remove the deionized water covering the upper layer; take equal volumes of 1.0 mL of colored upper gel buffer and upper gel solution, and mix thoroughly by pipetting to obtain a mixed solution; add 20 μL of modified coagulant to the upper gel mixture and mix thoroughly by pipetting to obtain a mixed solution; pour the upper gel mixture obtained in the above steps into the thick and thin glass plates, and insert the gel casting comb; at room temperature, 15 min... After min, once the upper gel has solidified, remove the comb to begin protein electrophoresis. Add 25 μg / well of protein sample to each gel well to start electrophoresis. Use 80 V for the upper gel and switch to 120 V for the lower gel. Stop electrophoresis when the bromocyanine blue at the tip of the sample reaches the lower edge of the lower gel. P4: Soak the PVDF membrane in methanol solution beforehand, then transfer it to membrane transfer buffer; lay moistened transfer filter paper on the transfer apparatus beforehand, then place the PVDF membrane on the transfer filter paper, remove the gel between the gel casting plates, place it on the laid PVDF membrane, and finally cover the gel with another transfer filter paper; perform electrotransfer at 15 V for 40 min. P5: Use 1×TBST blocking solution with a mass concentration of 5% (W / V) skim milk powder to block at room temperature for no less than 3 h, and use a horizontal shaker at 50 rpm; P6: The primary antibodies against anti-glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), nuclear factor-E2-associated factor 2 (Nrf2), transferrin receptor 1 (Trf1), and β-actin were diluted to 1:1000 using blocking buffer. The antibodies were then incubated with the blocked PVDF membrane overnight at 4 °C. After antibody incubation, the PVDF membrane was washed 5 times with 1×TBST at 100 rpm on a horizontal shaker for 6 min each time. The secondary antibodies of the corresponding species were diluted with blocking buffer (1:1000), and the PVDF membrane was placed at room temperature and incubated for 40 min. After secondary antibody incubation, the PVDF membrane was washed 5 times with 1×TBST at 100 rpm on a horizontal shaker for 6 min each time. P7: Development was performed using an ultrasensitive ECL chemiluminescent solution, and exposure was performed using a DNR Bio-Imaging Systems chemiluminescence imager.

[0034] like Figure 7The image shows the expression results of key ferroptosis proteins in an in vitro mouse hepatocyte model induced by oleic acid / palmitic acid, after treatment with dehydroauracetam and the ferroptosis inhibitor Fer-1, respectively. Figure 7 (a) is a Western blot image of the proteins in each group. Figure 7 (b) shows the results of the corresponding protein grayscale quantitative analysis. From Figure 7 As shown in (ab), compared with the model group, dehydroauracetam treatment significantly upregulated the antiferroptosis protein GPX4 and systemic Xc. - The expression of the key subunit SLC7A11 induces the activation of the Nrf2 antioxidant pathway; while the expression of the iron uptake-related protein Trf1 is significantly downregulated. The above trends are basically consistent with those of the Fer-1 group, which proves that dehydroauracetin can exert an anti-ferroptosis effect by regulating iron homeostasis and the lipid peroxidation defense system.

[0035] In summary, dehydroauracetamide exhibited clear inhibitory activity against ferroptosis in an oleic acid / palmitic acid-induced mouse hepatocyte in vitro model. Dehydroauracetamide also showed activity against glutathione recovery and Fe... 2+ In terms of multiple key ferroptosis indicators, such as accumulation inhibition, reduction of lipid peroxidation, and regulation of ferroptosis core proteins, dehydroauracetam showed a consistent trend with the classic ferroptosis inhibitor Fer-1, with no significant differences. This systematically demonstrates that dehydroauracetam possesses the functional properties of a typical ferroptosis inhibitor, providing direct experimental evidence for its application in ferroptosis-related diseases such as metabolic dysfunction-associated steatohepatitis, and strengthening its drug development value in the treatment of ferroptosis-related diseases such as metabolic dysfunction-associated steatohepatitis.

[0036] In summary, this invention explicitly applies dehydroauracetam to the preparation of drugs for treating metabolic dysfunction-related steatohepatitis, significantly expanding the application scenarios of dehydroauracetam in the field of metabolic liver diseases and achieving holistic regulation of the entire process of fat deposition-inflammatory damage-fibrosis progression. It also clarifies that dehydroauracetam possesses multi-target synergistic regulatory characteristics and can exert specific protective effects under pathological conditions involving significant iron-dependent lipid peroxidation and oxidative stress. Furthermore, it can be applied to the preparation of aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) inhibitors and ferroptosis inhibitors, effectively broadening the application scenarios of dehydroauracetam in the field of metabolic dysfunction-related steatohepatitis.

[0037] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0038] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of dehydroauracetam, characterized in that, For use in the preparation of drugs for treating fatty liver disease, the structural formula of the dehydroauracetam lactone is shown in Formula I: Equation I; The aforementioned fatty liver disease refers to fatty liver disease associated with metabolic dysfunction caused by a high-fat, high-cholesterol, and high-fructose diet.

2. The application of the dehydroaurate lactone according to claim 1, characterized in that, The medication for treating fatty liver disease is a ferroptosis inhibitor or an aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) inhibitor.

3. A drug for treating fatty liver disease as described in claim 1, characterized in that, The drug for treating steatohepatitis is prepared from dehydroauracetin and includes dehydroauracetin and pharmaceutically acceptable excipients.

4. The drug for treating fatty liver disease according to claim 3, characterized in that, The dosage form of the drug for treating fatty liver disease includes any one of the following: injection, tablet, powder, suspension, capsule, pill, or syrup.

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