Application of ergothioneine in preparation of medicine for improving metabolism-related fatty liver disease

Abstract

The invention provides application of ergothioneine in preparation of a medicine for improving metabolism-related fatty liver diseases. In the disclosure, it is verified through cell and animal models that the ergothioneine can improve the metabolism-related fatty liver diseases, and the ergothioneine can be used for preparing the medicine for improving the metabolism-related fatty liver diseases.

Description

Technical Field

[0001] This disclosure relates to the field of biomedical technology, specifically to the use of ergothionein in the preparation of a drug for improving metabolic-related fatty liver disease. Background Technology

[0002] Metabolic associated fatty liver disease (MAFLD) is a prevalent chronic disease worldwide, with a global prevalence of 24%, which continues to rise with increasing global obesity rates. It is typically associated with lifestyle factors, obesity, hypertension, diabetes, high cholesterol, and metabolic syndrome. Metabolic factors play a significant role in the development of MAFLD, including lipid metabolism disorders, inflammation and oxidative stress, gut microbiota dysbiosis, insulin resistance and glucose metabolism disorders, and genetic factors. Most cases of MAFLD are chronic and progressive, characterized primarily by fat accumulation in the liver (fatty liver). Early stages are often asymptomatic, but mild abnormalities may be observed during liver function tests. However, without timely intervention and treatment, serious complications can develop. In some patients, MAFLD may progress to hepatitis, leading to impaired liver function, liver fibrosis, cirrhosis, and even liver cancer. MAFLD is currently a leading cause of cirrhosis and hepatocellular carcinoma worldwide.

[0003] Currently, the main treatment for metabolic-related fatty liver disease is to reduce fat accumulation in the liver and lower the patient's weight, cholesterol, and blood sugar levels by changing lifestyle and dietary habits.

[0004] However, due to the complex and unclear pathophysiological mechanisms of metabolic-associated fatty liver disease, there are currently no approved drugs for its treatment. Therefore, there is an urgent need to provide new drugs that can be used to improve metabolic-associated fatty liver disease. Summary of the Invention

[0005] This disclosure was made in view of the above-mentioned state of the prior art, and its purpose is to provide the use of ergothionein in the preparation of a medicament for improving metabolic-related fatty liver disease.

[0006] Therefore, the first aspect of this disclosure provides the use of ergothioneine in the preparation of a medicament for improving metabolic-associated fatty liver disease (MAFLD). Ergothioneine (EGT) is a naturally occurring betaine amino acid, a natural thiourea derivative of histidine (2-mercaptohistidine trimethyl betaine), currently commonly used as an antioxidant and cosmetic additive. Ergothioneine has been proven safe under the intended conditions of use by the European Food Safety Authority's Food Products, Nutrition and Allergy Panel and is recommended as a dietary supplement. In this disclosure, ergothioneine's ability to improve MAFLD has been validated using cell and animal models, providing a solid foundation for the application of ergothioneine in MAFLD and facilitating its use in the preparation of medicaments for improving MAFLD.

[0007] In the applications covered by the first aspect of this disclosure, optionally, ergothioneine improves metabolic-associated fatty liver disease by reducing lipid accumulation, promoting lipid metabolism and transport, enhancing antioxidant enzyme activity, reducing oxidative damage, promoting autophagy, and improving apoptosis. This disclosure investigates and validates the regulatory mechanism by which ergothioneine improves metabolic-associated fatty liver disease.

[0008] In the applications covered by the first aspect of this disclosure, optionally, ergothioneine improves metabolic-associated fatty liver disease by alleviating insulin resistance and impaired hepatic lipid metabolism. This disclosure investigates and validates the regulatory mechanism by which ergothioneine improves metabolic-associated fatty liver disease.

[0009] In the applications covered by the first aspect of this disclosure, optionally, the medicament is a pharmaceutical composition comprising ergothioneine, wherein the pharmaceutical composition refers to a pharmaceutical composition consisting of ergothioneine and pharmaceutically permissible excipients.

[0010] In the applications covered by the first aspect of this disclosure, the excipient may optionally be at least one selected from diluents, excipients, binders, fillers, solubilizers, sustained-release agents, flavoring agents, and sweeteners. This can improve the performance of the pharmaceutical composition and make it more suitable for clinical needs.

[0011] In the applications covered by the first aspect of this disclosure, the drug may optionally be administered orally.

[0012] In the applications covered by the first aspect of this disclosure, optionally, the dosage of the drug is not less than 25 mg / kg / day.

[0013] A second aspect of this disclosure provides an formulation for improving metabolic-associated fatty liver disease, characterized by comprising ergothioneine. In this disclosure, ergothioneine has been validated in cell and animal models to improve metabolic-associated fatty liver disease, and a formulation containing ergothioneine is provided for use in improving metabolic-associated fatty liver disease.

[0014] Optionally, the formulations involved in the second aspect of this disclosure are pharmaceuticals, food products, or health supplements. This allows for the selection of application scenarios for the formulations as needed.

[0015] In the formulations disclosed in the second aspect of this invention, optionally, the formulation comprises ergothioneine and excipients, wherein the excipients are permitted in pharmaceutical, general food, health food, or special medical food applications. This can improve the performance of the formulation and make it more suitable for clinical needs.

[0016] According to this disclosure, it is possible to provide the use of ergothionein in the preparation of a medicament for improving metabolic-related fatty liver disease. Attached Figure Description

[0017] Figure 1 This is a schematic diagram showing the relevant results of mouse body weight, intraperitoneal glucose tolerance test, and intraperitoneal insulin sensitivity test involved in the embodiments of this disclosure.

[0018] Figure 2 This is a schematic diagram illustrating experimental results related to liver lipid metabolism function in mice as described in the embodiments of this disclosure.

[0019] Figure 3 This is a schematic diagram illustrating experimental results related to the liver structure of mice in embodiments of this disclosure.

[0020] Figure 4 This is a schematic diagram illustrating experimental results related to the level of liver inflammation in mice according to embodiments of this disclosure.

[0021] Figure 5 This is a schematic diagram illustrating the results of the selection of intervention concentration and time in the cell experiments involved in the embodiments of this disclosure.

[0022] Figure 6 This is a schematic diagram showing the results of liver lipid metabolism deposition and cell viability of AML12 according to embodiments of this disclosure.

[0023] Figure 7 This is a schematic diagram illustrating the results related to the apoptosis level of AML12 in the embodiments of this disclosure.

[0024] Figure 8 This is a schematic diagram illustrating the results related to oxidative damage of AML12 according to an embodiment of this disclosure.

[0025] Figure 9 This is a schematic diagram illustrating the results related to the autophagy level of AML12 cells involved in the embodiments of this disclosure. Detailed Implementation

[0026] Hereinafter, preferred embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. In the following description, the same reference numerals are used for the same components, and repeated descriptions are omitted. Furthermore, the drawings are merely schematic diagrams, and the proportions of the components or the shapes of the components may differ from actual figures.

[0027] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0028] It should also be understood that terms such as those defined in general dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless defined as here.

[0029] This disclosure relates to the following aspects:

[0030] The use of ergothionein in the preparation of a drug to improve metabolic-related fatty liver disease;

[0031] The application of ergothionein in the preparation of a formulation that improves metabolic-related fatty liver disease;

[0032] An agent for improving metabolic-associated fatty liver disease, comprising ergothioneine;

[0033] One method for improving metabolic-associated fatty liver disease involves administering ergothioneine to a subject.

[0034] The ergothioneine (EGT) disclosed herein has the molecular formula C9H. 15 N3O2S is a naturally occurring betaine amino acid, a natural thiourea derivative of histidine (2-mercaptohistidine trimethyl betaine), currently used as an antioxidant and cosmetic additive. Ergothioneine has been proven safe under intended use by the European Food Safety Authority's Food Products, Nutrition and Allergy Panel and is recommended as a dietary supplement; however, there are currently no reports of its application in metabolically related fatty liver disease (MAFLD). In this disclosure, ergothioneine was demonstrated to improve MAFLD using cell and animal models, providing a solid foundation for its application in MAFLD and facilitating its use in the preparation of drugs to improve MAFLD.

[0035] In this disclosure, ergothioneine can be used to improve metabolic-associated fatty liver disease.

[0036] In some examples, ergothioneine can be used to prepare drugs that improve metabolic-associated fatty liver disease. Ergothioneine may include ergothioneine, and / or its isomers, and / or its pharmaceutically acceptable salts, and / or its prodrugs.

[0037] In some cases, ergothioneine can improve metabolic-associated fatty liver disease by reducing lipid accumulation, promoting lipid metabolism and transport, enhancing antioxidant enzyme activity, reducing oxidative damage, promoting autophagy, and improving apoptosis. This publication investigates and validates the regulatory mechanism by which ergothioneine improves metabolic-associated fatty liver disease.

[0038] In some cases, ergothioneine can improve metabolic-associated fatty liver disease by alleviating insulin resistance and impaired hepatic lipid metabolism. This publication investigates and validates the regulatory mechanism by which ergothioneine improves metabolic-associated fatty liver disease.

[0039] As previously stated, this disclosure provides a formulation for improving metabolic-related fatty liver disease, including ergothioneine. In some examples, the formulation may be a drug, food, or health supplement. This allows for selection of the application scenario of the formulation as needed. The following detailed description will use a drug formulation as an example.

[0040] In some examples, ergothioneine can be used as the sole active ingredient in the preparation of a medicament for improving metabolic-related fatty liver disease. In other words, ergothioneine can be the sole active ingredient in the medicament. A sole active ingredient is a single ingredient that has a significant impact on the technical efficacy of the medicament. Therefore, this disclosure also provides the use of ergothioneine as the sole active substance in the preparation of a medicament for improving metabolic-related fatty liver disease. In some examples, the medicament may also include excipients permitted in pharmaceutical, general food, health food, or special medical food formulations. Excipients do not have a significant impact on the technical efficacy of the medicament. Thus, adding excipients can help improve the performance of the medicament, making it more suitable for clinical needs.

[0041] In some examples, the formulation may consist of ergothioneine and excipients. In some examples, a drug for improving metabolic-related fatty liver disease may be a pharmaceutical composition comprising ergothioneine. Here, a pharmaceutical composition refers to a pharmaceutical composition consisting of ergothioneine and pharmaceutically permissible excipients.

[0042] In some examples, the excipients may be at least one of diluents, excipients, binders, fillers, solubilizers, sustained-release agents, flavoring agents, and sweeteners. This can help improve the performance of the pharmaceutical composition and make it better meet clinical needs.

[0043] In this disclosure, "administration" and "dosage" can refer to providing a drug to a subject via a known route. In some examples, the route of administration of the drug may include oral, intravenous, parenteral, local, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal administration. In some examples, preferably, the drug may be administered orally.

[0044] In some examples, the drug may be in liquid dosage form (e.g., suspension, gel, and paste), solid dosage form (e.g., tablet, pill, powder, and packaged powder), or gaseous dosage form.

[0045] In some examples, the dosage of the drug can range from 10 mg / kg / day to 200 mg / kg / day. For example, the dosage of the drug can be 10 mg / kg / day, 15 mg / kg / day, 20 mg / kg / day, 25 mg / kg / day, 30 mg / kg / day, 35 mg / kg / day, 40 mg / kg / day, 50 mg / kg / day, 60 mg / kg / day, 70 mg / kg / day, 80 mg / kg / day, 90 mg / kg / day, 100 mg / kg / day, 110 mg / kg / day, 120 mg / kg / day, 130 mg / kg / day, 140 mg / kg / day, 150 mg / kg / day, 160 mg / kg / day, 170 mg / kg / day, 180 mg / kg / day, 190 mg / kg / day, or 200 mg / kg / day. In some examples, preferably, the dosage of the drug may be no less than 25 mg / kg / day.

[0046] In this disclosure, the dosage of the drug can be adjusted based on different subject types. Specifically, regarding the administration of EGT, in mice, a dose of 35 mg / kg / day is sufficient to saturate the intracellular accumulation and metabolism of EGT in mice; however, in humans, existing studies have shown that administering a dose of 25 mg / kg / day of EGT to humans for 7 days (the mouse equivalent dose is 307.5 mg / kg / day) results in a proportional increase in EGT levels in both blood and urine, indicating that EGT metabolism in humans requires a higher dose than in mice to reach saturation.

[0047] To further illustrate this disclosure, the following detailed description, in conjunction with embodiments, illustrates the application of ergothioneine provided in this disclosure in improving metabolic-related fatty liver disease.

[0048] In the embodiments disclosed herein, unless otherwise specified, all materials, reagents, instruments and software used are commercially available products, and the operating procedures are performed in accordance with the instructions for the reagents, instruments or software.

[0049] [Example]

[0050] 1.1 Experimental Cells

[0051] AML12 cells were purchased from ATCC.

[0052] 1.2 Laboratory Animals

[0053] Twenty male C57 mice (strain: C57BL / 6J) were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.

[0054] 2 Experimental Methods

[0055] 2.1 AML12 cell culture

[0056] (1) Preparation of complete culture medium: DMEM / F-12 basal medium + 10% FBS + 1% 100X penicillin / streptomycin mixture + 10μg / mL Insulin + 5.5μg / mL Transferrin + 5ng / mL Selenium + 40ng / mL Dexamethasone;

[0057] (2) External culture environment: 37℃ constant temperature incubator with 5% CO2 saturated humidity.

[0058] 2.2 Handling and Sampling of Laboratory Animals

[0059] To investigate the dynamic pathological characteristics of the effects of ergothioneine (EGT) on the liver of high-fat mice, we divided C57 mice into four groups by feeding them a normal diet, a high-fat diet, and a diet with or without ergothioneine: the normal diet group (NCD), the normal diet + ergothioneine group (NCD+EGT), the high-fat diet group (HFD), and the high-fat diet + ergothioneine group (HFD+EGT). The dose of ergothioneine was 35 mg / kg / day.

[0060] Mouse body weight was monitored weekly. After 16 weeks of feeding, intraperitoneal glucose tolerance test (IPGTT), intraperitoneal insulin tolerance test (IPITT), dual-energy X-ray absorptiometry were performed to measure mouse body fat content, and serum and liver tissue were collected.

[0061] 2.3 Measurement of liver function indicators and blood lipids

[0062] Serum alanine aminotransferase (ALT), aspartate transaminase (AST), triglycerides (TG), total cholesterol (TC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) levels in mice of each group were measured using an automated biochemical analyzer.

[0063] 2.4 In vivo experimental index detection

[0064] IL-6 and IL-10 were detected by hematoxylin-eosin staining, Oil Red O staining of frozen sections, PAS staining, immunohistochemical staining for the inflammatory marker F4 / 80, and ELISA kit. Antioxidant enzyme activity in mouse serum was measured according to the kit instructions, detecting serum catalase (CAT), glutathione peroxidase (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) levels in each group of mice.

[0065] 2.5 In vitro experimental index detection

[0066] In vitro experiments used palmitic acid (PA) to stimulate AML12 cells to simulate liver lipotoxicity. The mRNA and protein expression levels of lipid synthesis, transport, oxidation, autophagy, and apoptosis-related molecules were detected by qPCR and Western blotting. Cell proliferation-toxicity was assessed using CCK8 assay, apoptosis was detected by Annexin V-FITC / PI double staining, Oil Red O staining was performed, and reactive oxygen species (ROS) were detected.

[0067] Experimental results

[0068] Ergothioneine can significantly improve liver metabolic dysfunction in mice fed a high-fat diet.

[0069] 1.1 Ergothionein reduced the body weight of mice fed a high-fat diet and improved their glucose tolerance and insulin sensitivity.

[0070] Figure 1 This is a schematic diagram showing the relevant results of mouse body weight, intraperitoneal glucose tolerance test, and intraperitoneal insulin sensitivity test involved in the embodiments of this disclosure. Figure 1 Part a is a graph showing the change in mouse body weight. Figure 1 Part b shows the statistical analysis of the intraperitoneal glucose tolerance test (GTT) and the area under the curve (AUC) in mice. Figure 1Part c shows the statistical analysis of the intraperitoneal insulin sensitivity test (ITT) and area under the curve (AUC) in mice. Mice's tolerance to changes in blood glucose were monitored at week 22. n = 5. HFD vs. NCD: *P<0.05, **P<0.01, ****P<0.0001; HFD+EGT vs. HFD: #P<0.05, ###P<0.001, ####P<0.0001.

[0071] Mice were grouped weekly after week 6. After 12 weeks of feeding, using NCD as a control, there were no statistically significant differences in body weight, glucose tolerance, and insulin sensitivity between the NCD+EGT group and the control group. The HFD group had significantly higher body weight, area under the glucose tolerance test curve (AUC), and area under the insulin sensitivity curve (AUC) than the control group, indicating significant liver metabolic dysfunction. However, compared to the HFD group, the HFD+EGT group had significantly lower body weight, AUC, and AUC, suggesting that ergothioneine can reduce body weight and insulin resistance in HFD mice.

[0072] 1.2 Ergothionein improves body fat, liver function and blood lipid levels in mice fed a high-fat diet.

[0073] Figure 2 This is a schematic diagram illustrating experimental results related to liver lipid metabolism function in mice as described in the embodiments of this disclosure. Figure 2 Part a of the diagram shows the results of dual-energy X-ray ablation (DXA) in mice. Mice underwent DXA anesthesia at week 23 for body fat imaging. Figure 2 Statistical analysis of body fat percentage in mice in part b. Analysis of body fat percentage after dual-energy X-ray diffraction (DXD) detection. Figure 2 The middle section (ch) is a statistical analysis graph of automated biochemical detection of liver metabolic function in mouse serum. Section c represents mouse serum ALT level, section d represents mouse serum AST level, section e represents mouse serum TG level, section f represents mouse serum TC level, section g represents mouse serum HDL level, and section h represents mouse serum LDL level.

[0074] Using NCD as a control, no statistically significant differences were found between the NCD+EGT group and the control group in terms of body fat content, liver function, and blood lipid levels. The HFD group showed higher body fat content, liver function, and blood lipid levels than the control group, indicating significant lipid metabolism disorder in the HFD group. However, compared with the HFD group, the HFD+EGT group showed significantly better body fat content, liver function, and blood lipid levels. In conclusion, ergothioneine can improve liver lipid metabolism dysfunction in HFD mice.

[0075] 1.3 Ergothionein improves liver structural damage, lipid and glycogen deposition in high-fat fed mice.

[0076] Figure 3 This is a schematic diagram illustrating experimental results related to the liver structure of mice involved in the embodiments of this disclosure. Specifically, (HE) mouse liver hematoxylin-eosin staining: In the NCD group, the livers of normal mice showed clear lobular structures; in the HFD group, the livers of mice showed increased steatosis and vacuolation.

[0077] HFD+EGT group mice showed reduced hepatic steatosis. (PAS) mouse liver glycogen staining: HFD group mice showed increased hepatic glycogen deposition; HFD+EGT group mice showed decreased hepatic glycogen deposition. (Oil Red) mouse liver Oil Red O staining: NCD group mice showed no orange-red lipid droplet deposition; HFD group mice showed steatosis with obvious orange-red lipid droplet deposition; HFD+EGT group mice showed reduced hepatic steatosis, with a significant decrease in the number and size of lipid droplets. HE, PAS, Oil Red, bar = 100 μm. n = 5.

[0078] Using NCD as a control, no significant differences were found in liver structure, lipid, glycogen deposition, and fibrosis levels between the NCD+EGT group and the control group. The HFD group showed significant vacuolar degeneration and lipid deposition in the liver, with increased glycogen accumulation. However, compared to the HFD group, the HFD+EGT group exhibited reduced vacuolar degeneration and lipid deposition, and decreased glycogen accumulation. In conclusion, ergothioneine can improve liver structural damage in HFD mice.

[0079] 1.4 Ergothionein improves oxidative stress and inflammatory response levels in high-fat fed mice.

[0080] Figure 4 This is a schematic diagram illustrating experimental results related to the level of liver inflammation in mice according to embodiments of this disclosure. Figure 4 Part a is immunohistochemical staining F4 / 80b. Figure 4 The bg portion is a mouse serum ELISA, where part b is the mouse serum IL-1β level, part c is the mouse serum IL-6 level, part d is the mouse serum SOD level, part e is the mouse serum CAT level, part f is the mouse serum GSH level, and part g is the mouse serum MDA level. n = 5. HFD vs. NCD: *P<0.05, **P<0.01, ****P<0.0001; HFD+EGT vs. HFD: #P<0.05, ###P<0.001, ####P<0.0001.

[0081] Using NCD as a control, no significant differences were found in inflammation levels and antioxidant enzyme activity between the NCD+EGT group and the control group. The HFD group showed significantly increased liver inflammation and decreased antioxidant enzyme activity. However, compared to the HFD group, the HFD+EGT group exhibited reduced inflammation levels and increased antioxidant enzyme activity. In conclusion, ergothioneine can improve liver inflammation levels and enhance antioxidant enzyme activity in HFD mice.

[0082] 2PA stimulus condition exploration

[0083] Palmitic acid stimulation of cells is a classic method for in vitro lipotoxicity modeling. Mouse hepatocyte line AML12 was stimulated with palmitic acid (PA) at different concentrations (0, 0.1, 0.2, 0.3, 0.4, 0.5 mmol / L) and for different durations (6, 12, 24, 48 h) to select the optimal stimulation concentration and duration.

[0084] Figure 5 This is a schematic diagram illustrating the results of the selection of intervention concentration and time in the cell experiments involved in the embodiments of this disclosure. Figure 5 Part a shows the survival status of AML12 cells stimulated with different concentrations of PA for 48 hours. Figure 5 Part b shows the percentage of AML12 cell viability as detected by CCK8. Figure 5 Part c shows the lipid deposition status of AML12 cells as detected by Oil Red O, after PA intervention for 48 hours. Bar: 20µm.

[0085] AML12 cells were stimulated with PA, and a PA concentration of 0.2 mmol / L was selected as the optimal condition for simulating lipotoxicity in vitro, considering cell survival, cell viability, and lipid deposition status.

[0086] 3EGT improves lipid deposition and lipotoxic damage induced by PA in mouse AML12 cells.

[0087] Based on the presence or absence of PA and ergothioneine intervention, the in vitro experiments were divided into four groups: control group (no intervention, Ctrl), ergothioneine-only intervention group (PA+EGT), PA-only intervention group (PA), and PA and ergothioneine combined intervention group (PA+EGT). We used different concentrations of ergothioneine (0-2 mmol / L) to intervene in AML12 cells. Based on the improvement in lipid deposition and cell viability, 0.5 mmol / L ergothioneine was ultimately selected for intervention.

[0088] 3.1 EGT improves PA-induced lipid deposition and impaired lipid metabolism in AML12.

[0089] Figure 6 This is a schematic diagram showing the results of liver lipid metabolism deposition and cell viability of AML12 according to embodiments of this disclosure. Figure 6 Part a shows the detection of lipid deposition status of AML12 by different concentrations of ergothionein using Oil Red O. Bar: 20 μm. Figure 6 Part b shows the percentage of cell viability detected by CCK8 assay at different concentrations of ergothioneine. Figure 6 Part c represents the ApoB mRNA expression level. Figure 6 The d-part represents the FASN mRNA expression level. Figure 6 The e part represents the CPT1α mRNA expression level. Figure 6 Part f in the table represents the expression level of PPARα mRNA. n = 3. PA vs. Ctrl: *P < 0.05, **P < 0.01, ****P < 0.0001; PA+EGT vs. PA: #P < 0.05, ###P < 0.001, ####P < 0.0001.

[0090] Compared with the Ctrl group, there was no significant difference in the mRNA transcription levels of AML12 lipid transport (ApoB), fatty acid synthesis (FASN), and fatty acid oxidation-related genes (CPT1α and PPARα) in the Ctrl+EGT group. In the PA group, the mRNA transcription level of AML12 fatty acid synthesis (FASN) genes was increased, while the mRNA transcription levels of lipid transport (ApoB) and fatty acid oxidation-related genes (CPT1α and PPARα) were inhibited. However, compared with the PA group, the mRNA transcription levels of the aforementioned lipid metabolism-related genes were improved and restored in the PA+EGT group. In conclusion, ergothioneine can alleviate PA-induced hepatic lipid deposition in AML12 and improve cell viability.

[0091] 3.2 EGT alleviates PA-induced AML12 apoptosis

[0092] Figure 7 This is a schematic diagram illustrating the results related to the apoptosis level of AML12 in the embodiments of this disclosure. Figure 7 Part a shows the flow cytometry-based detection of apoptosis levels and statistical analysis. Figure 7 Part b represents the Bax mRNA expression level. Figure 7 Part c represents the expression level of Bcl2 mRNA. Figure 7 The d-part represents the Bax / Bcl2 mRNA expression level ratio. Figure 7 The e part represents the caspase 7 mRNA expression level. Figure 7 Part f in the table represents the caspase 9 mRNA expression level. n = 3. PA vs. Ctrl: *P < 0.05, **P < 0.01, ****P < 0.0001; PA+EGT vs. PA: #P < 0.05, ###P < 0.001, ####P < 0.0001.

[0093] Compared with the Ctrl group, there was no significant difference in AML12 apoptosis levels and apoptosis-related gene mRNA expression levels in the Ctrl+EGT group. In the PA group, AML12 apoptosis increased, the Bax / Bcl2 ratio of apoptosis-related gene mRNA expression levels increased, and the mRNA expression levels of caspase 7 and caspase 9 were enhanced. However, compared with the PA group, the PA+EGT group showed significantly improved apoptosis levels and apoptosis-related gene mRNA levels. In conclusion, ergothioneine can improve PA-induced AML12 apoptosis levels.

[0094] 3.3 EGT alleviates PA-induced AML12 oxidative stress damage and enhances antioxidant activity

[0095] Figure 8 This is a schematic diagram illustrating the results related to oxidative damage of AML12 according to an embodiment of this disclosure. Figure 8 Part a of the diagram shows the detection of reactive oxygen species (ROS) in AML12. Bar: 100µm. Figure 8 Part b represents the SOD3 mRNA expression level. Figure 8 Part c represents the CAT mRNA expression level. Figure 8 Part d represents the GPX4 mRNA expression level. n = 3. *P < 0.05, **P < 0.01, ****P < 0.0001 PA vs. Ctrl; #P < 0.05, ###P < 0.001, ####P < 0.0001 PA+EGT vs. PA.

[0096] Compared with the Ctrl group, there was no significant difference in ROS levels and antioxidant enzyme activities in AML12 in the Ctrl+EGT group; in the PA group, ROS levels of AML12 increased, and the mRNA expression levels of antioxidant enzyme genes CAT, SOD3, and GPX4 were inhibited; however, compared with the PA group, ROS levels of the PA+EGT group decreased, and the mRNA expression levels of antioxidant enzyme genes CAT, SOD3, and GPX4 were improved. In conclusion, ergothioneine can alleviate PA-induced oxidative damage to AML12.

[0097] 3.4 EGT enhances AML12 autophagy and improves PA-induced AML12 lipotoxicity damage.

[0098] Figure 9 This is a schematic diagram illustrating the results related to the autophagy level of AML12 cells involved in the embodiments of this disclosure. Figure 9 Part a represents the p62 mRNA expression level. Figure 9 Part b represents the Beclin1 mRNA expression level. Figure 9Part c represents the Atg5 mRNA expression level. Figure 9 The d-part represents the mTOR mRNA expression level. Figure 9 Part e represents the serum TG level in mice. Figure 9 The middle part (f) represents the serum TC level in mice. Figure 9 The g portion represents the mouse serum HDL level. Figure 9 The h-value represents the serum LDL level in mice. n = 3. *P < 0.05, **P < 0.01, ****P < 0.0001 PA vs. Ctrl; #P < 0.05, ###P < 0.001, ####P < 0.0001 PA+EGT vs. PA.

[0099] Compared to the Ctrl group, there was no significant difference in autophagy levels in AML12 cells between the Ctrl+EGT group and the PA group. In the PA group, autophagy levels were suppressed, with increased mRNA transcription levels of autophagy-related repressor genes p62 and mTOR, and decreased mRNA transcription levels of autophagy-related promoter genes Beclin1, Atg5, and ULK1. Protein expression levels of the autophagy-related repressor gene p62 were increased, while protein expression levels of the autophagy-related promoter genes Beclin1 and Atg5 were decreased. However, compared to the PA group, autophagy levels were significantly improved in the PA+EGT group. In conclusion, ergothionein can enhance autophagy levels in AML12 cells.

[0100] In this disclosure, a mouse MAFLD model was established by feeding C57 mice a high-fat diet. The successful establishment of the MAFLD model was confirmed by experiments including mouse weight monitoring, insulin resistance-related tests, blood lipids, body fat, and liver tissue staining. Figure 1-4 As shown in the diagram. Simultaneously, we used ergothioneine to intervene in EGT in mice fed a water-based diet. Since metabolic-associated fatty liver disease is a chronic metabolic condition, we began high-fat diets and ergothioneine (35 mg / kg / day) in mice from week 6 for 4 months. A dose of 35 mg / kg / day was sufficient to saturate intracellular EGT accumulation and metabolism in mice; its safety has been thoroughly studied and approved by regulatory agencies such as the FDA and EFSA. Furthermore, weekly monitoring revealed no developmental abnormalities or deaths in the EGT intervention group, thus ensuring and confirming the safety of EGT.

[0101] No abnormalities were found in mouse weight, insulin resistance-related tests, blood lipids, body fat, oxidative stress, inflammatory response, or liver tissue staining between the NCD control group and the EGT intervention group alone. Figure 1-4 As shown in the figure. This indicates that EGT itself has no other adverse effects on mice. However, in the HFD+EGT group, a significant reduction in body weight and insulin resistance was observed in mice, as shown in the figure. Figure 1As shown. Furthermore, body fat percentage, liver function, and blood lipid levels were all lower in the EGT group than in the HFD group, indicating that EGT can significantly improve lipid metabolism disorders; such as Figure 2 As shown. It was also found that the livers of HFD mice treated with EGT showed significant improvement in vacuolar degeneration and lipid deposition, and reduced glycogen accumulation; as shown. Figure 3 As shown. Furthermore, the HFD+EGT group mice exhibited reduced liver inflammation and enhanced antioxidant enzyme activity; such as... Figure 4 As shown in the figure. These results demonstrate the protective effect of EGT against liver damage induced by a high-fat diet in mice.

[0102] Excessive lipid accumulation in hepatocytes associated with MAFLD poses a significant challenge to human health. Therefore, as a saturated fatty acid, PA has been widely used in in vitro cell line lipotoxicity studies to induce hepatic lipid metabolism disorders. Based on cell state and viability, our study established a hyperlipidemic model of AML-12 cells using 0.2 mM PA intervention for 48 h, which significantly induced lipid deposition, such as... Figure 5 As shown.

[0103] Lipid metabolism in hepatocytes is mainly determined by the uptake and oxidation of free fatty acids and lipid synthesis. Here, our study demonstrated decreased fatty acid β-oxidation and fatty acid transport levels, and increased lipogenesis and lipid deposition levels in PA-treated AML12 cells. Similar to these results, previous studies reported low protein expression levels of CPT1α and phosphorylated ACC, which are involved in regulating fatty acid β-oxidation and lipogenesis in PA-treated HepG2 cells and increasing lipid droplet content; similarly, phosphorylated ACC and CPT1α levels were significantly lower in PA-treated rat myoblasts. After EGT intervention, these levels changed in the opposite direction, indicating improved lipid metabolism function. Figure 6 As shown.

[0104] Excessive lipid deposition induces lipid peroxidation, leading to ROS accumulation and reduced antioxidant capacity, causing oxidative stress damage to hepatocytes. Studies have found that PA treatment significantly increases MDA and ROS levels in mouse spermatogonia, reduces mitochondrial membrane potential, and impairs mitochondrial function. Similarly, our study found that PA significantly downregulated the expression levels of SOD3, CAT, and GPX4 while increasing ROS levels. However, EGT, as a natural antioxidant, can significantly upregulate the activity of these antioxidant enzymes, reduce ROS levels, and mitigate oxidative stress-induced cell damage, thereby exerting a protective effect against AML12. Figure 8 As shown.

[0105] Autophagy and apoptosis are mutually regulated, jointly regulating homeostasis and cell death in the body. In most cases, autophagy can inhibit the activation of apoptosis-related proteins, promoting cell survival. Autophagy is involved in regulating cellular lipid homeostasis because autophagy activation can reduce lipid content by regulating the expression of proteins involved in lipid metabolism, while autophagy inhibition can lead to excessive lipid accumulation. Furthermore, restoring autophagy flux can attenuate or prevent the progression of NAFLD. Autophagy dysfunction leads to excessive lipid accumulation in hepatocytes, resulting in fatty liver disease. Beclin-1 is a substrate of caspase 3, which has two cleavage sites at positions 124 and 149. This cleavage can induce apoptosis by inhibiting autophagy. Studies have found PA-induced apoptosis, such as the increased percentage of apoptosis and BAX / BCL 2 expression levels in PA-treated AML-12. Similar to our results, PA treatment significantly increased the level of apoptosis in human hepatocytes, upregulated the BAX / BCL 2 ratio, and upregulated the mRNA expression levels of caspase 7 and caspase 9. Figure 7 As shown. Meanwhile, in AML12 cells, we detected suppressed autophagy levels in the PA group, with increased expression levels of autophagy-related inhibitory genes p62 and mTOR, and decreased expression levels of autophagy-related promoting genes Beclin1, Atg5, and ULK1; however, compared to the PA group, we found significantly improved autophagy levels in the PA+EGT group, as shown. Figure 9 As shown. Therefore, EGT intervention can prevent lipid metabolism disorders by promoting autophagy and inhibiting apoptosis in PA-treated hepatocytes.

[0106] Steroid degeneration leads to increased signaling of the transcription factor NF-κB via upstream activation of IKKβ (an inhibitor of nuclear factor-κB). NF-κB activation induces the production of pro-inflammatory mediators such as TNF-α (tumor necrosis factor-α), IL-6 (interleukin-6), and IL-1β (interleukin-1β). These cytokines contribute to the recruitment and activation of Kupffer cells (resident hepatic macrophages).

[0107] In summary, this disclosure demonstrates that EGT intervention can improve MAFLD by reducing lipid accumulation, promoting lipid metabolism and transport, enhancing antioxidant enzyme activity, reducing oxidative damage, promoting autophagy, and improving apoptosis, providing a new approach for the clinical treatment of MAFLD.

[0108] In summary, this disclosure provides the use of ergothioneine in the preparation of a medicament for improving metabolic-related fatty liver disease.

[0109] While the present disclosure has been specifically described above in conjunction with the accompanying drawings and examples, it is to be understood that the foregoing description does not limit the present disclosure in any way. Those skilled in the art can make modifications and variations to the present disclosure as needed without departing from its essential spirit and scope, and all such modifications and variations shall fall within the scope of the present disclosure.

Claims

1. The use of ergothionein in the preparation of a drug for improving metabolic-related fatty liver disease.

2. The application according to claim 1, characterized in that, Ergothioneine improves metabolic-associated fatty liver disease by reducing lipid accumulation, promoting lipid metabolism and transport, enhancing antioxidant enzyme activity, reducing oxidative damage, promoting autophagy, and improving apoptosis.

3. The application according to claim 1, characterized in that, Ergothioneine improves metabolic-associated fatty liver disease by reducing insulin resistance and impaired liver lipid metabolism.

4. The application according to claim 1, characterized in that, The drug is a pharmaceutical composition comprising ergothioneine, wherein the pharmaceutical composition refers to a pharmaceutical composition consisting of ergothioneine and pharmaceutically permissible excipients.

5. The application according to claim 4, characterized in that, The excipients are at least one of diluents, excipients, binders, fillers, cosolvents, sustained-release agents, flavoring agents, and sweeteners.

6. The application according to claim 1, characterized in that, The drug is administered orally.

7. The application according to claim 1 or 6, characterized in that, The dosage of the drug shall not be less than 25 mg / kg / day.

8. A formulation for improving metabolic-related fatty liver disease, characterized in that, Including ergothioneine.

9. The formulation according to claim 8, characterized in that, The preparation is a drug, food, or health product.

10. The formulation according to claim 8, characterized in that, The formulation consists of ergothioneine and excipients, wherein the excipients are permitted in pharmaceutical, general food, health food, or special medical food applications.

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