Use of chrysin in the preparation of drugs and health foods for preventing or treating high-fat diet related liver injury

Abstract

The application discloses application of fisetin in preparation of medicines and health foods for preventing or treating high-fat diet related liver injury. The fisetin promotes BNIP3 related mitochondrial autophagy, reduces accumulation of mitochondrial reactive oxygen species, and thus simultaneously inhibits hepatocyte apoptosis, necrotic apoptosis and pyroptosis. Through a mouse liver injury model induced by a high-fat diet and a palmitic acid induced AML12 hepatocyte injury model, it is proved that the fisetin can improve liver tissue and hepatocyte injury, reduce mitochondrial dysfunction and reduce accumulation of mitochondrial reactive oxygen species. Further research shows that after knocking down BNIP3, the effect of the fisetin on promoting mitochondrial autophagy and reducing mitochondrial reactive oxygen species is obviously weakened; after knocking down BMAL1, the above regulation effect is obviously weakened. In addition, the application also relates to application of the fisetin in preparation of health foods for assisting in protecting liver function.

Description

Technical Field

[0001] This invention belongs to the fields of medicine and food technology, specifically relating to the application of flavonoids in the preparation of drugs and health foods for the prevention or treatment of liver damage related to a high-fat diet. Background Technology

[0002] High-fat diets (HFD) refer to dietary patterns where fat constitutes a relatively high proportion of energy. Long-term high-fat diets can cause energy metabolism imbalances and lipid metabolism disorders, promote lipid accumulation in the liver, and further induce oxidative stress, mitochondrial dysfunction, and hepatocyte damage. Therefore, identifying bioactive substances that can effectively intervene in high-fat diet-related liver damage is of great significance.

[0003] Programmed hepatocyte death is a crucial pathological process in the development of high-fat diet-related liver injury. Apoptosis, necroptosis, and pyroptosis involve different molecular regulatory mechanisms, but they can occur simultaneously and jointly contribute to hepatocyte injury under metabolic stress. Therefore, intervening in the common upstream regulatory links of various programmed cell death mechanisms may provide a new approach to improving high-fat diet-related hepatocyte injury.

[0004] Mitochondrial dysfunction is a crucial factor in hepatocyte damage induced by high-lipid conditions. Damaged mitochondria can lead to abnormal accumulation of mitochondrial reactive oxygen species (mtROS), and persistently elevated mtROS further promotes various programmed cell death processes, including apoptosis, necroptosis, and pyroptosis. Mitophagy can selectively clear damaged mitochondria and maintain mitochondrial homeostasis, with BCL2-interacting protein 3 (BNIP3) being a key receptor protein involved in the regulation of mitophagy. Previous studies have shown that BNIP3-related mitophagy is involved in the quality control and damage regulation of hepatocyte mitochondria during non-alcoholic fatty liver disease and hepatic steatosis (e.g., Li et al., Therapeutic effect of Sirtuin 3 on ameliorating nonalcoholic fatty liver disease: The role of the ERK-CREB pathway and Bnip3-mediated mitophagy, Redox Biology, 2018; Chen et al., RNF31 alleviates liver steatosis by promoting p53 / BNIP3-related mitophagy in hepatocytes, Free Radical Biology and Medicine, 2024).

[0005] BMAL1, an ARNT-like protein 1 in the brain and muscle, is a crucial core protein in the circadian rhythm system and participates in the regulation of cellular metabolism and mitochondrial homeostasis. Previous studies have shown that BMAL1 can regulate hepatic mitochondrial dynamics, mitophagy, and oxidative metabolism. Restoring hepatic BMAL1 activity under high-fat dietary conditions can improve metabolic abnormalities (e.g., Wang et al., Polydatin improves mitochondrial dynamics dysrhythmia by regulating BMAL1 to prevent and treat metabolic dysfunction-associated fatty liver disease, Journal of Ethnopharmacology, 2027). Therefore, BMAL1-mediated mitochondrial quality control may be closely related to hepatocyte damage and programmed cell death under high-fat conditions.

[0006] Fisetin (FIS) is a natural flavonoid compound with various biological activities, including antioxidant, anti-inflammatory, and metabolic regulation. Studies have reported that Fisetin can improve phenotypes such as hepatic lipid metabolism disorders, liver fibrosis, and insulin resistance in models of high-fat diets or metabolic abnormalities (e.g., Choi et al., Fisetin Alleviates Hepatic and Adipocyte Fibrosis and Insulin Resistance in Diet-Induced Obese Mice, Journal of Medicinal Food, 2020; Guo et al., Fisetin Ameliorates atherosclerosis through activating FXR-mediated hepatic cholesterol metabolism and transintestinal cholesterol excretion, Food Research International, 2025). Xu et al. (Activated TNF-α / RIPK3 signaling is involved in prolonged high-fat diet-stimulated hepatic inflammation and lipid accumulation: inhibition by dietary fisetin intervention, Food & Function, 2019) further reported that fuzzin improves high-fat diet-induced liver inflammation and lipid accumulation by inhibiting the TNF-α / RIPK3 signaling pathway. This study shows that fuzzin can regulate the necroptosis pathway. However, this study only involves necroptosis, a programmed cell death pathway, and does not involve the regulatory mechanism of mitophagy or the regulation of mtROS levels. Currently, there are no reports of fuzzin simultaneously inhibiting hepatocyte apoptosis, necroptosis, and pyroptosis under high-fat conditions, nor has it revealed the key role of mtROS in the above-mentioned synergistic regulation, and it has not addressed the regulatory relationship between BNIP3-related mitophagy and BMAL1 on this process.

[0007] To address the shortcomings of the prior art, the technical problem to be solved by this invention is to provide a new use of flavin in the preparation of drugs and health foods for the prevention or treatment of liver damage related to a high-fat diet. Specifically, it provides a new use of flavin that simultaneously inhibits hepatocyte apoptosis, necrotizing apoptosis, and pyroptosis by promoting BNIP3-related mitophagy to reduce mtROS accumulation, as well as a new mechanism by which BMAL1 participates in regulating the above processes. Summary of the Invention

[0008] The purpose of this invention is to provide a new use for flavin in the preparation of drugs and health foods for the prevention or treatment of liver damage related to a high-fat diet.

[0009] To achieve the above objectives, the present invention provides the use of flavin in the preparation of a medicament for the prevention and / or treatment of high-fat diet-related liver injury, wherein the flavin reduces the accumulation of mitochondrial reactive oxygen species by promoting BNIP3-associated mitophagy, thereby simultaneously inhibiting hepatocyte apoptosis, necrotizing apoptosis, and pyroptosis.

[0010] Furthermore, the high-fat diet-related liver injury includes pathological liver tissue damage and / or abnormal liver weight gain caused by a high-fat diet, as well as hepatocyte oxidative stress and / or mitochondrial dysfunction caused by fatty acid overload. In animal experiments, flavonoids at concentrations of 10-40 mg / kg body weight / day showed protective effects, with 20-40 mg / kg body weight / day showing better efficacy; in AML12 cell experiments, the effective working concentration of flavonoids was 5-20 μM.

[0011] Furthermore, fuzzin can promote BNIP3-related mitophagy and reduce mtROS accumulation; after BNIP3 knockdown, the regulatory effect of fuzzin on LC3, P62 expression and mtROS level is significantly weakened.

[0012] Furthermore, BMAL1 is involved in the regulation of BNIP3-related mitophagy by fuzzin. Knockdown of BMAL1 significantly weakened the regulatory effect of fuzzin on the expression of BNIP3, LC3, and P62, while mtROS levels increased again, and its regulatory effect on apoptosis, necroptosis, and pyroptosis-related proteins was weakened.

[0013] Furthermore, the drug may be an oral formulation, which may be a tablet, capsule, granule, or oral liquid. The drug may have flavonoids as the sole active ingredient, or it may be used in combination with other active ingredients, and may include pharmaceutically acceptable carriers or excipients.

[0014] On the other hand, the present invention also provides the application of flavin in the preparation of health food for the auxiliary protection of liver function. The health food can be used for liver health maintenance in people with high-fat diets. The product form can be powder, tablet, capsule or beverage, and includes excipients or auxiliary ingredients acceptable in food.

[0015] The present invention has the following beneficial effects: (1) The present invention provides a new use of lacquer for intervening in liver injury related to high-fat diet. It has been found that lacquer can reduce mtROS accumulation and simultaneously inhibit hepatocyte apoptosis, necrotizing apoptosis and pyroptosis, providing a new technical solution for intervention against common upstream events of multiple programmed cell death; (2) Lacquer can improve liver tissue and hepatocyte damage caused by high-fat diet or fatty acid overload, reduce oxidative stress and mitochondrial structural and functional abnormalities, and reduce mtROS accumulation, thereby playing a protective role for hepatocytes; (3) The present invention further shows that lacquer can promote BNIP3-related mitophagy; after knocking down BNIP3, its effect of promoting mitophagy and reducing mtROS is significantly weakened; after knocking down BMAL1, the regulatory effect of lacquer on BNIP3-related mitophagy, mtROS, apoptosis, necrotizing apoptosis and pyroptosis is significantly weakened. The above results support that BMAL1 participates in the regulation of BNIP3-related mitophagy and multiple programmed cell death processes by lacquer. Attached Figure Description

[0016] Figure 1 The results show the effects of flavonoids on alleviating high-fat diet-induced liver and hepatocyte damage. A shows H&E staining of mouse liver tissue (scale bar: 200 μm); B shows F4 / 80 immunohistochemical staining of mouse liver tissue (scale bar: 200 μm); C and D show mouse body weight and liver-to-body ratio; E shows PI staining results for AML12 cells (scale bar: 50 μm). An asterisk (*) indicates a comparison with the control group, and a hash (#) indicates a comparison with the HFD or PA group. Single-symbol P < 0.05, double-symbol P < 0.01.

[0017] Figure 2 The results show the effects of rosin on the improvement of high-fat diet-induced mitochondrial damage and oxidative stress in hepatocytes. A shows the ultrastructure of mouse hepatocyte mitochondria observed by transmission electron microscopy (TEM) (scale bar: 1, 2 μm, or 500 nm); B shows the fluorescence staining of reactive oxygen species (ROS) in mouse liver tissue (scale bar: 5 μm); C shows the results of mitochondrial permeability transition pore (mPTP) detection in AML12 cells (scale bar: 200 μm); D shows the mitochondrial membrane potential of AML12 cells detected by flow cytometry; and E shows the results of mtROS fluorescence staining in AML12 cells (scale bar: 200 μm).

[0018] Figure 3 This study investigated the experimental results of using rosin to reduce mtROS accumulation and alleviate high-fat diet-induced apoptosis, necrotizing apoptosis, and pyroptosis in mouse hepatocytes. In the figures, A represents the expression of apoptosis-related proteins in mouse liver tissue; B represents the expression of necrotizing apoptosis-related proteins; C represents the expression of pyroptosis-related proteins; D represents the expression of apoptosis-related proteins after Mito-TEMPO intervention; E represents the expression of necrotizing apoptosis-related proteins after Mito-TEMPO intervention; and F represents the expression of pyroptosis-related proteins after Mito-TEMPO intervention. An asterisk (*) indicates a comparison with the control group; a hash (#) indicates a comparison with the HFD group, with single-symbol P < 0.05 and double-symbol P < 0.01.

[0019] Figure 4 Experimental results showing that rutin reduced mtROS accumulation and alleviated palmitic acid-induced apoptosis, necroptosis, and pyroptosis in AML12 cells; where A represents the expression of apoptosis-related proteins in AML12 cells; B represents the expression of necroptosis-related proteins; C represents the expression of pyroptosis-related proteins; D represents the expression of apoptosis-related proteins after Mito-TEMPO intervention; E represents the expression of necroptosis-related proteins after Mito-TEMPO intervention; and F represents the expression of pyroptosis-related proteins after Mito-TEMPO intervention. An asterisk (*) indicates a comparison with the control group; a hash (#) indicates a comparison with the PA group, with single-symbol P < 0.05 and double-symbol P < 0.01.

[0020] Figure 5 The results show the experimental effects of flavonoids reducing mtROS accumulation through BNIP3-related mitophagy. A represents the expression of BNIP3, LC3, and P62 proteins in mouse liver tissue; B represents the expression of BNIP3, LC3, and P62 proteins in AML12 cells; C represents the knockdown effect of si-BNIP3 on BNIP3 as verified by Western blot; D represents the expression of LC3 and P62 proteins in AML12 cells after si-BNIP3 intervention; and E represents the mtROS fluorescence staining results in AML12 cells after si-BNIP3 intervention (scale bar: 10 μm). An asterisk (*) indicates a comparison with the control group; a hash (#) indicates a comparison with the PA group; and a sum (&) indicates a comparison with the PA + 20 μM FIS group. Single-symbol P < 0.05, double-symbol P < 0.01.

[0021] Figure 6This study presents the experimental results of how flavin improves the rhythmic oscillation disorder of BMAL1 and CLOCK proteins induced by a high-fat diet and palmitic acid. In this study, A shows the changes in BMAL1 and CLOCK protein expression in mouse liver tissue at different time points; B shows the changes in BMAL1 and CLOCK protein expression in AML12 cells at different time points. An asterisk (*) indicates a comparison with the control group; a hash (#) indicates a comparison with the HFD or PA group. Single-symbol P < 0.05, double-symbol P < 0.01.

[0022] Figure 7 The results show the experimental findings of BMAL1's involvement in promoting mitophagy and reducing mtROS accumulation through urushiol. A represents the Western blot validation of the BMAL1 knockdown effect of si-BMAL1; B shows the protein expression of BNIP3, LC3, and P62 in AML12 cells after si-BMAL1 intervention; C shows the mtROS fluorescence staining results in AML12 cells after si-BMAL1 intervention (scale bar: 10 μm). An asterisk (*) indicates a comparison with the control group; a hash (#) indicates a comparison with the PA group; and a sum (&) indicates a comparison with the PA + 20 μM FIS group. Single-symbol P < 0.05, double-symbol P < 0.01.

[0023] Figure 8 The results show the effects of BMAL1 on the inhibition of palmitic acid-induced apoptosis, necrotizing apoptosis, and pyroptosis in AML12 cells by fuzzycin. In this study, A represents the expression of apoptosis-related proteins after si-BMAL1 intervention; B represents the expression of necrotizing apoptosis-related proteins after si-BMAL1 intervention; and C represents the expression of pyroptosis-related proteins after si-BMAL1 intervention. An asterisk (*) indicates a comparison with the control group; a hash (#) indicates a comparison with the PA group; and a sum (&) indicates a comparison with the PA + 20 μM FIS group. Single-symbol P < 0.05, double-symbol P < 0.01. Detailed Implementation

[0024] To further illustrate the technical solution and effects of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0025] Unless otherwise stated, the experimental methods used in the embodiments of the present invention are all conventional experimental methods in the art, and the materials and reagents used are all commercially available.

[0026] I. Materials and Methods

[0027] 1. Main experimental materials and reagents

[0028] The experimental animals were SPF-grade healthy male C57BL / 6 mice, 6-8 weeks old, weighing 20-22 g, purchased from Liaoning Changsheng Biotechnology Co., Ltd.; normal mouse hepatocyte cell line (AML12) was purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; rosin (purity ≥98%, catalog number: B20953) was purchased from Shanghai Yuanye Co., Ltd.; palmitic acid (purity ≥99%, catalog number: P0500) was purchased from Sigma-Aldrich, Inc.; high-fat diet (catalog number: D12492) was purchased from Beijing Xiaoshu Youtai Co., Ltd.; fetal bovine serum (catalog number: CF-01S-02) was purchased from Changsha Cellbox Technology Co., Ltd.; PI staining kit (catalog number: C1008) was used. The following reagents were purchased from Shanghai Beyotime Biotechnology Co., Ltd.: Mitochondrial membrane potential (MMP) assay kit (C2003S), mPTP assay kit (C2009S), ROS assay kit (C0064S), and BCA protein concentration assay kit (P0009); MitoSOX (C36007) fluorescent probe was purchased from Thermo Fisher Scientific, Inc.; BMAL1, CLOCK, BNIP3, LC3, P62, and fluorescent secondary antibody were purchased from Wuhan Sanying Biotechnology Co., Ltd.

[0029] 2. AML12 cell culture

[0030] AML12 cells were cultured in DMEM / F-12 complete medium. This complete medium contained 10% fetal bovine serum, 1% penicillin-drug antibiotics, 1% ITS, and 40 ng / mL dexamethasone. Cells were cultured in a 37°C, 5% CO2 incubator. Routine passages were performed when cell confluence reached approximately 80%.

[0031] 3. Western blot detection

[0032] Total protein was extracted from mouse liver tissue or AML12 cells according to the experimental design, and the protein concentration was determined by the BCA method. Equal amounts of protein were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking, the membrane was incubated overnight at 4°C with the corresponding primary antibody. The next day, the membrane was incubated with the corresponding secondary antibody. Chemiluminescence was used for development, and band grayscale analysis was performed using ImageJ software.

[0033] 4. Data Processing and Statistical Analysis

[0034] Cell experiments were independently repeated at least three times; animal experiments were performed using the actual number of animals in each experimental group as biological replicates; experimental results were expressed as mean ± standard deviation; Western blot bands were quantified using ImageJ software, and statistical analysis and plotting were performed using GraphPad Prism 9.0 software; independent samples t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups, with Tukey's method used for post-hoc comparisons, and P < 0.05 was considered statistically significant.

[0035] Example 1: Rhubarb flavonoids alleviate liver and hepatocellular damage induced by a high-fat diet.

[0036] 1. Establishment of a high-fat diet mouse model and intervention with rosin.

[0037] Forty-eight healthy male SPF-grade C57BL / 6 mice were selected and randomly divided into six groups of eight mice each after one week of acclimatization: (1) Normal control group: fed with standard diet (SCD) for 16 weeks; (2) High-fat diet group: fed with high-fat diet (HFD) for 16 weeks; (3) HFD + low-dose FIS group: fed with high-fat diet and administered 10 mg / kg lacquer flavonoids by gavage daily; (4) HFD + medium-dose FIS group: fed with high-fat diet and administered 20 mg / kg lacquer flavonoids by gavage daily; (5) HFD + high-dose FIS group: fed with high-fat diet and administered 40 mg / kg lacquer flavonoids by gavage daily; (6) HFD + high-dose FIS + Mito-TEMPO group: fed with high-fat diet and 40 mg / kg lacquer flavonoids, and administered 1 mg / kg Mito-TEMPO intraperitoneally daily starting from week 13. The weight of the mice was measured and recorded weekly during the experiment. Animal experiments were conducted within the barrier facility of the Experimental Animal Center at Jilin University, and all animal procedures were approved by the Animal Ethics and Welfare Committee of Jilin University. Based on the intervention results of three dosage groups (10, 20, and 40 mg / kg body weight / day), the effective animal dosage of rosin in this invention is 10-40 mg / kg body weight / day, with 20-40 mg / kg body weight / day showing better efficacy.

[0038] 2. Animal sample collection

[0039] After the experiment, mice were anesthetized with sodium pentobarbital, and blood and liver tissue were collected. The liver tissue was processed differently according to the needs of subsequent experiments: some liver tissue was fixed with 10% neutral formalin for H&E staining and F4 / 80 immunohistochemical staining; some liver tissue was fixed with glutaraldehyde for transmission electron microscopy to observe the ultrastructure of mitochondria; the remaining liver tissue was rapidly cryopreserved for subsequent detection such as Western blot.

[0040] 3. Liver tissue H&E staining and F4 / 80 immunohistochemical staining

[0041] Liver tissue was fixed, embedded in paraffin, and sectioned. Hematoxylin and eosin (H&E) staining was used to observe liver tissue structure and pathological changes. For F4 / 80 immunohistochemical staining, 5 μm thick paraffin sections were dewaxed, rehydrated with graded ethanol, and then antigen retrieval was performed using sodium citrate buffer. After eliminating endogenous peroxidase activity, the sections were blocked with 5% BSA, incubated overnight at 4°C with F4 / 80 primary antibody, and then incubated the following day with the appropriate secondary antibody. After DAB staining and hematoxylin counterstaining, the sections were observed under a microscope.

[0042] 4. Palmitic acid-induced AML12 cell damage model and rosin treatment

[0043] A hyperlipidemic injury model was established in vitro using palmitic acid-induced AML12 cells. During the experiment, AML12 cells were treated with 200 μM palmitic acid for 48 h. Urushibrin was prepared as a 10 mM stock solution in DMSO, and diluted to working concentrations of 5 μM, 10 μM, and 20 μM using complete culture medium. These solutions were then co-treated with 200 μM palmitic acid for 48 h. Mito-TEMPO was prepared as a 50 mM stock solution in DMSO and diluted before use in cell experiments. In the AML12 cell experiments, the final concentration of Mito-TEMPO was 50 μM, and the treatment time was 48 h. Based on the results of 5, 10, and 20 μM ursicrin treatments, the effective working concentration of ursicrin for AML12 cells in this invention is 5-20 μM, with 10-20 μM showing better results.

[0044] 5. PI staining

[0045] After drug treatment, PI staining was used to observe the cell membrane integrity and cell death of AML12 cells, and images were acquired using a fluorescence microscope.

[0046] The results are as follows Figure 1 As shown in Figure A, compared with the normal control group, the liver tissue structure of mice in the high-fat diet group was significantly damaged, with disordered hepatocyte arrangement and inflammatory cell infiltration; Figure 1 As shown in Figure B, the F4 / 80 positive signal was significantly enhanced. After intervention with flavonoids, the liver tissue structure of mice was significantly improved, inflammatory cell infiltration was reduced, and the F4 / 80 positive signal was significantly decreased, indicating that flavonoids can alleviate liver tissue pathological damage and inflammatory response induced by a high-fat diet; Figure 1 As shown in C and 1D, the body weight and liver-to-body ratio of mice in the high-fat diet group were significantly increased. After intervention with rosin, the body weight gain of mice was inhibited, and the liver-to-body ratio decreased significantly, indicating that rosin can alleviate excessive body weight gain and abnormal liver enlargement in mice induced by a high-fat diet. Figure 1As shown in E, the proportion of PI-positive cells in AML12 cells increased significantly after palmitic acid treatment, while the proportion of PI-positive cells decreased significantly after laccase intervention.

[0047] The above results indicate that flavin can effectively alleviate liver and hepatocyte damage induced by high-fat diet and palmitic acid.

[0048] Example 2: Okratin improves high-fat diet-induced mitochondrial damage and reduces ROS and mtROS accumulation.

[0049] 1. Observation of mitochondrial ultrastructure using transmission electron microscopy

[0050] A high-fat diet mouse model was established according to the method described in Example 1. Fixed liver tissue samples were taken, and after routine fixation, dehydration, embedding, and ultrathin sectioning, TEM observation was performed, focusing on mitochondrial morphology, mitochondrial cristae integrity, and matrix structure changes.

[0051] 2. ROS and mtROS detection

[0052] ROS staining of mouse liver tissue was performed by incubation at 37°C for 30 min in 5 μM dihydroethidium (DHE) working solution in the dark. mtROS staining of AML12 cells was performed by incubation at 37°C for 40 min in 3 μM MitoSOX working solution in the dark. After washing with PBS, the cells were observed and images were acquired using a fluorescence microscope or a confocal microscope.

[0053] 3. mPTP and mitochondrial membrane potential detection

[0054] AML12 cells were treated with appropriate drugs according to the experimental design. mPTP detection was performed according to the kit instructions. After drug treatment, cells were collected, incubated with the appropriate working solution, and observed and images were acquired using a fluorescence microscope to evaluate the degree of mPTP opening. Mitochondrial membrane potential was detected using appropriate fluorescent probes, and changes in mitochondrial membrane potential in each group of AML12 cells were analyzed by flow cytometry.

[0055] The results are as follows Figure 2 As shown in Figure A, the high-fat diet group of mice showed significant swelling of mitochondria in their hepatocytes, decreased matrix density, and breakage or disappearance of mitochondrial cristae. However, after intervention with rosin, the aforementioned ultrastructural damage to mitochondria was significantly improved. Figure 2 As shown in Figure B, the ROS fluorescence signal in the liver tissue of mice in the high-fat diet group was significantly enhanced, and the ROS fluorescence intensity was significantly reduced after intervention with rosin. Figure 2 C and Figure 2 As shown in D, palmitic acid treatment led to abnormal mPTP opening and a decrease in mitochondrial membrane potential in AML12 cells, while rutin intervention significantly reduced abnormal mPTP opening and restored mitochondrial membrane potential; Figure 2As shown in E, palmitic acid treatment significantly increased mtROS in AML12 cells, while rutin intervention significantly reduced mtROS levels.

[0056] The above results indicate that flavonoids can improve the structural and functional damage of hepatocytes mitochondria induced by a high-fat diet and reduce the accumulation of ROS and mtROS.

[0057] Example 3: Rhubarb flavonoids alleviated high-fat diet-induced apoptosis, necrotizing apoptosis, and pyroptosis in mouse hepatocytes by reducing mtROS accumulation.

[0058] A high-fat diet mouse model was established according to the method described in Example 1, and urochrome intervention was performed. The Mito-TEMPO intervention group received intraperitoneal injections of 1 mg / kg Mito-TEMPO daily starting from week 13, in addition to high-dose urochrome treatment. After the experiment, liver tissue was collected, and Western blot was used to detect the expression of apoptosis-related proteins such as BAX, Bcl-2, Cyt-c, Caspase-3, and Caspase-8; necroptosis-related proteins such as RIPK1, RIPK3, MLKL, and p-MLKL; and pyroptosis-related proteins such as NLRP3, Caspase-1, GSDMD, and GSDMD-N. Results are as follows: Figure 3 As shown in AC, after high-fat diet treatment, the expression of apoptosis-related proteins such as BAX, Cyt-c, Caspase-3, and Caspase-8 increased, while the expression of Bcl-2 decreased in mouse liver tissue; the expression of necroptosis-related proteins such as RIPK1, RIPK3, MLKL, and p-MLKL increased; and the expression of pyroptosis-related proteins such as NLRP3, Caspase-1, GSDMD, and GSDMD-N increased. All of these changes were significantly improved after intervention with lacquer xanthophyll. Figure 3 As shown in DF, the expression of apoptosis, necroptosis, and pyroptosis-related proteins was further reduced after combining Mito-TEMPO with fuzzycin intervention.

[0059] The above results indicate that mtROS is involved in various programmed cell death processes induced by a high-fat diet, and that flavin inhibits mouse hepatocyte apoptosis, necrotizing apoptosis, and pyroptosis by reducing mtROS accumulation.

[0060] Example 4: Rhubarb alleviates palmitic acid-induced apoptosis, necroptosis, and pyroptosis in AML12 cells by reducing mtROS accumulation.

[0061] A palmitic acid-induced AML12 cell damage model was established according to the method described in Example 1, followed by intervention with urochrome. The final concentration of Mito-TEMPO was 50 μM. After treatment, Western blot was used to detect the expression of apoptosis, necroptosis, and pyroptosis-related proteins. The results are as follows: Figure 4As shown in AC, palmitic acid treatment significantly increased the expression of apoptosis, necroptosis, and pyroptosis-related proteins in AML12 cells, while laccase intervention significantly decreased the expression of these related proteins. Figure 4 As shown in the DF, after combining Mito-TEMPO with rutin intervention, the expression of three programmed cell death-related proteins was further reduced, including RIPK1, RIPK3, MLKL, p-MLKL, as well as NLRP3, Caspase-1, GSDMD and GSDMD-N.

[0062] The above results further indicate that rosin alleviates palmitic acid-induced apoptosis, necrotizing apoptosis, and pyroptosis in AML12 cells by inhibiting mtROS accumulation.

[0063] Example 5: Okratin reduces mtROS accumulation by promoting BNIP3-associated mitophagy

[0064] The si-BNIP3 sequence was designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.; the sense strand sequence of si-BNIP3 is 5′-GGACGAAGUAGCUCCAAGATT-3′, and the antisense strand sequence is 5′-UCUUGGAGCUACUUCGUCCTT-3′; the sense strand sequence of the negative control NC is 5′-UUCUCCGAACGUGUCACGUTT-3′, and the antisense strand sequence is 5′-ACGUGACACGUUCGGAGAATT-3′.

[0065] AML12 cells were seeded in 100 mm cell culture dishes and transfected when confluence reached 70%-80%. 600 pmol siRNA and 45 μL GP-transfect-Mate were each mixed with 500 μL Opti-MEM, incubated for 5 min, then mixed again and incubated for another 10 min. Cells were washed with D-Hanks buffer, then 8 mL of serum-free Opti-MEM was added, followed by the transfection complex. Cells were incubated at 37°C for 6 h, then replaced with fresh complete culture medium. Subsequently, palmitic acid and urochrome treatments were performed according to the experimental design.

[0066] The results are as follows Figure 5 A and Figure 5 As shown in Figure B, after a high-fat diet and palmitic acid treatment, BNIP3 expression decreased, P62 accumulated, and LC3-II showed abnormal changes. After intervention with rutin, BNIP3 expression increased, LC3-II further increased, and P62 expression decreased, indicating that rutin can improve mitophagy disorders caused by a high-fat diet. Figure 5 As shown in Figure C, si-BNIP3 treatment significantly reduced BNIP3 protein expression. Figure 5As shown in Figure D, knocking down BNIP3 significantly weakened the regulatory effect of rutin on LC3 and P62 expression. Figure 5 As shown in E, flavonoids can reduce palmitic acid-induced mtROS accumulation in AML12 cells, while this effect is significantly weakened after si-BNIP3 treatment.

[0067] The above results indicate that rosin reduces mtROS accumulation by promoting BNIP3-associated mitophagy.

[0068] Example 6: Rhubarb extract improves BMAL1 and CLOCK protein rhythmic oscillation disorder induced by high-fat diet and palmitic acid.

[0069] Another 45 healthy male C57BL / 6 mice of SPF grade, aged 6-8 weeks and weighing 20-22 g, were randomly divided into 3 groups of 15 mice each. Normal control group: fed with SCD for 16 weeks; HFD group: fed with a high-fat diet for 16 weeks; HFD+FIS group: fed with a high-fat diet for 16 weeks, and administered 40 mg / kg of flavonoids by gavage daily. 24 h after the last administration, samples were collected at five time points: ZT0, ZT6, ZT12, ZT18, and ZT24. Three mice were randomly selected from each group at each time point, and liver tissue was collected for the detection of BMAL1 and CLOCK protein rhythm expression. AML12 cells were treated with 50% horse serum for 2 h for rhythm synchronization. Cells were divided into a normal control group, a 200 μM PA group, and a 200 μM PA+20 μM FIS group. After 48 h of treatment, cells were collected at five time points: ZT0, ZT6, ZT12, ZT18, and ZT24 for the detection of BMAL1 and CLOCK protein expression.

[0070] The results are as follows Figure 6 As shown in Figure A, BMAL1 and CLOCK proteins in the liver tissue of normal mice exhibit distinct rhythmic oscillations. High-fat diet treatment significantly abnormalized the rhythmic oscillations of BMAL1 and CLOCK, while intervention with rosin significantly improved their rhythmic oscillation patterns. Figure 6 As shown in Figure B, BMAL1 and CLOCK also exhibit distinct rhythmic oscillations in normal AML12 cells. Palmitic acid treatment caused abnormalities in the rhythmic phase and amplitude of both proteins, while intervention with rutin improved these rhythmic disturbances.

[0071] The above results indicate that flavin can improve the rhythmic oscillation disorder of BMAL1 and CLOCK proteins induced by a high-fat diet and palmitic acid.

[0072] Example 7: BMAL1 participates in lacquinone's promotion of mitophagy and reduction of mtROS accumulation.

[0073] The si-BMAL1 sequence was designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.; the sense strand sequence of si-BMAL1 is 5′-GCAAACUACAAGCCAACAUTT-3′, and the antisense strand sequence is 5′-AUGUUGGCUUGUAGUUUGCTT-3′; the remaining transfection conditions and cell processing steps are the same as in Example 5.

[0074] The results are as follows Figure 7 As shown in Figure A, si-BMAL1 treatment significantly reduced BMAL1 protein expression, indicating successful BMAL1 knockdown. Figure 7 As shown in Figure B, knocking down BMAL1 significantly weakened the regulatory effect of rutin on the expression of BNIP3, LC3, and P62. Figure 7 As shown in C, rosin can significantly reduce palmitic acid-induced mtROS accumulation, while mtROS levels rise again after si-BMAL1 treatment.

[0075] The above results indicate that BMAL1 is involved in the process by which rosin promotes BNIP3-related mitophagy and reduces mtROS accumulation.

[0076] Example 8: BMAL1 participates in the mitigation of palmitic acid-induced apoptosis, necroptosis, and pyroptosis in AML12 cells by urochrome.

[0077] The si-BMAL1 transfection and palmitic acid and rosin treatment methods for AML12 cells were the same as in Example 7. After treatment, Western blot was used to detect the expression of apoptosis, necroptosis, and pyroptosis-related proteins. The results are as follows: Figure 8 As shown in Figure A, after si-BMAL1 treatment, the effect of rutin in reducing BAX, Caspase-3, and Caspase-8 expression and increasing Bcl-2 expression was significantly weakened. Figure 8 As shown in Figure B, after si-BMAL1 treatment, the inhibitory effect of rutin on the expression of RIPK1, RIPK3, MLKL, and p-MLKL was significantly weakened. Figure 8 As shown in Figure C, after treatment with si-BMAL1, the inhibitory effect of rosin on the expression of NLRP3, Caspase-1, GSDMD, and GSDMD-N was significantly weakened.

[0078] The above results indicate that BMAL1 is involved in the inhibitory effect of flavin on palmitic acid-induced apoptosis, necrotizing apoptosis, and pyroptosis in AML12 cells.

[0079] In summary, the results of this invention demonstrate that flavin can improve liver tissue and hepatocyte damage caused by a high-fat diet or fatty acid overload, promote BNIP3-related mitophagy and reduce mtROS accumulation, while inhibiting hepatocyte apoptosis, necrotizing apoptosis, and pyroptosis. BMAL1 participates in the regulation of BNIP3-related mitophagy and the above processes by flavin. Therefore, flavin can be used to prepare drugs for the prevention or treatment of high-fat diet-related liver injury, and can also be used to prepare health foods that help protect liver function.

Claims

1. The application of flavonoids in the preparation of drugs for the prevention or treatment of liver injury related to a high-fat diet, characterized in that, The flavin reduces mitochondrial reactive oxygen species accumulation by promoting BNIP3-associated mitophagy, thereby simultaneously inhibiting hepatocyte apoptosis, necroptosis, and pyroptosis.

2. The application according to claim 1, characterized in that, The high-fat diet-related liver injury includes pathological damage to liver tissue and / or abnormal liver weight gain caused by a high-fat diet, as well as hepatocyte oxidative stress and / or mitochondrial dysfunction caused by fatty acid overload.

3. The application according to claim 1, characterized in that, The dosage of the flavonoid is 10-40 mg / kg body weight / day.

4. The application according to any one of claims 1 to 3, characterized in that, The drug is an oral preparation, which includes tablets, capsules, granules or oral liquids, and includes a pharmaceutically acceptable carrier or excipient.

5. The application according to claim 1, characterized in that, The drug uses flavin as its sole active ingredient, or flavin is used in combination with other active ingredients.

6. The application according to claim 1, characterized in that, The promoting effect of laccasein on BNIP3-related mitophagy is regulated by BMAL1.

7. The application of flavonoids in the preparation of health food products for the auxiliary protection of liver function, characterized in that, The health food product is intended for liver health maintenance in people with a high-fat diet.

8. The application according to claim 7, characterized in that, The health food products may be in the form of powder, tablets, capsules or beverages, and may include excipients or auxiliary ingredients acceptable in food.

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