Application of brucein D in treating fatty liver disease related to metabolic dysfunction

Abstract

The invention is applicable to the technical field of biological medicines, and provides application of brucein D in treating fatty liver diseases related to metabolic dysfunction. The invention finds that the brucein D has potential advantages in lipid-lowering treatment such as multi-target regulation and control of lipid metabolism, anti-inflammatory / antioxidant synergistic effect and possible anti-fibrosis activity, and can make up for the problems of insufficient improvement of fibrosis, significant side effects and the like of existing drugs; the compound can be further applied to prevention and treatment of fatty liver diseases related to metabolic dysfunction through deep research.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an application of brucein D in treating fatty liver disease associated with metabolic dysfunction. Background Art

[0002] Metabolic dysfunction-associated steatotic liver disease (MASLD) is currently the most common chronic liver disease worldwide. 25% of patients with MASLD will develop metabolic dysfunction-associated steatohepatitis (MASH) and develop fibrosis within 3 years. The development of MASLD is associated with multiple factors, including metabolic syndrome such as obesity, type 2 diabetes, hypertension and dyslipidemia. MASLD can be roughly divided into two categories: metabolic dysfunction-associated fatty liver (MAFL), which is the non-progressive form of MASLD; and metabolic dysfunction-associated steatohepatitis (MASH), which is the progressive form of MASLD. MAFL is hepatic steatosis, which usually occurs in zone 3 of the liver lobule and may or may not be accompanied by mild inflammation. The histological characteristics of the progression of MAFL to MASH are the appearance of three main features: steatosis, lobular inflammation and hepatocyte ballooning, which may be accompanied by fibrosis. The process of reversal of MASH to MAFL can be highly dynamic and may even occur in a short period of time. Abnormal lipid metabolism is an important cause of MASLD, and the endoplasmic reticulum, as an important organelle for lipid metabolism and protein synthesis, plays a key role in the occurrence and regulation of MASLD. Inflammatory response is a key factor in the progression of MASLD to MASH. Endoplasmic reticulum stress promotes the release of inflammatory factors by activating signaling pathways such as NF-κB, which further aggravate the inflammatory response of the liver. At present, although the pathogenesis of MAFLD has been deeply studied at home and abroad, the exact pathogenesis and treatment of MASLD have not yet been fully clarified. Therefore, the search for highly effective and low-toxic small molecule drugs has become a new strategy for the clinical prevention and treatment of MASLD.

[0003] The treatment of metabolic dysfunction-associated fatty liver disease (MASLD, formerly known as non-alcoholic fatty liver disease / NAFLD) currently focuses on improving metabolic disorders and anti-inflammatory and anti-fibrosis. Approved or widely studied drugs include pioglitazone, vitamin E (α-tocopherol), semaglutide, tirzepatide, SGLT2 inhibitors (such as empagliflozin, dapagliflozin), resmetirom, obeticholic acid (OCA), etc.

[0004] As a representative of thiazolidinediones, pioglitazone specifically activates peroxisome proliferator-activated receptor γ (PPAR-γ), enhances insulin sensitivity, regulates adipocyte differentiation and lipid metabolism-related gene expression, and thus reduces ectopic lipid deposition in the liver. Its clinical efficacy has been verified in multiple randomized controlled trials. For example, the results of the PIVENS trial (Pioglitazone vs. Vitamin E vs. Placebo for NASH) showed that the pathological improvement rate of liver steatosis and lobular inflammation in patients treated with pioglitazone was significantly better than that in the placebo group (34% vs. 19%), but its inhibitory effect on the progression of liver fibrosis did not reach statistical significance (P=0.24). Based on this, the American Association for the Study of Liver Diseases (AASLD) guidelines recommend that this drug can be used preferentially in patients with MASLD with type 2 diabetes, but be alert to its potential adverse reactions, including peripheral edema (incidence of about 4-6%), weight gain (average increase of 2-4 kg), and the risk of decreased bone density that may result from long-term use.

[0005] As a fat-soluble antioxidant, vitamin E can reduce liver oxidative stress damage by scavenging free radicals and inhibiting lipid peroxidation. The PIVENS trial for non-diabetic NASH patients showed that after 96 weeks of treatment with 800 IU high-dose vitamin E daily, 36% of patients achieved a significant reduction in non-alcoholic steatohepatitis activity score (NAS), and the degree of liver steatosis and ballooning was significantly improved. However, the effect of this intervention on fibrosis reversal was limited (the fibrosis improvement rate was only 19%). It is worth noting that long-term high-dose application may cause dose-dependent adverse reactions, such as increased bleeding tendency (prolonged prothrombin time) and increased incidence of prostate cancer as suggested by epidemiological studies (SELECT trial hazard ratio HR=1.17). Therefore, clinical guidelines emphasize the need to strictly screen indications and regularly evaluate the risk-benefit ratio.

[0006] As a long-acting glucagon-like peptide-1 (GLP-1) receptor agonist, semaglutide achieves weight loss (average reduction of 10-15%) and liver fat content through central appetite suppression, delayed gastric emptying and direct regulation of the insulin signaling pathway of hepatocytes. The results of a Phase III clinical trial (NCT02970942) published in 2021 showed that after 72 weeks of subcutaneous injection of 2.4 mg semaglutide once a week, 39% of NASH patients achieved histologically defined inflammatory remission (NAS score decreased by ≥2 points and no fibrosis worsening), but the improvement rate of fibrosis stage was only 26%, indicating that its anti-fibrotic effect is still limited. The drug has not yet been approved by the FDA for the indication of NASH.

[0007] By activating GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) receptors simultaneously, tebuconazole synergistically enhances insulin secretion, inhibits glucagon release, and promotes browning of white adipose tissue. Data from its Phase II clinical trial (SURPASS-NASH) showed that the liver fat content of patients in the 15 mg dose group decreased by 8-10% compared with baseline, and 47% of patients achieved improvement in NAS scores at 52 weeks. Given its excellent weight loss effect (average body weight reduction of 20-25%), the drug is considered a potential preferred option for patients with NASH and obesity. Its Phase III clinical trial (SYNERGY-NASH) is underway to further verify its improvement effect on fibrosis endpoints.

[0008] SGLT2 inhibitors, represented by Empagliflozin and Dapagliflozin, improve systemic glucose and lipid metabolism disorders by inhibiting proximal renal tubular glucose reabsorption and promoting urinary glucose excretion. Observational cohort studies (such as the EMPA-REG OUTCOME subgroup analysis) have shown that SGLT2i treatment can reduce serum ALT levels (average decrease of 8-12 IU / L) and liver fat fraction (MRI-PDFF assessment decrease of 3-5%), but whether its mechanism of action directly targets the liver remains controversial. There is currently a lack of high-quality randomized controlled trial evidence for NASH. The European Association for the Study of the Liver (EASL) guidelines recommend that the use of this type of drug should focus on MASLD patients with atherosclerotic cardiovascular disease or chronic kidney disease.

[0009] Resmetirom, as a liver-selective thyroid hormone receptor β (THR-β) agonist, accelerates liver lipid metabolism and excretion by upregulating mitochondrial fatty acid oxidase and apolipoprotein B100 expression. The results of its key Phase III clinical trial MAESTRO-NASH (NCT03900429) showed that after 52 weeks of treatment with a 100 mg dose, 26% of patients achieved an improvement in fibrosis stage ≥1 and no worsening of NASH, meeting the FDA's accelerated approval criteria. Based on this, the drug was approved by the FDA in March 2024 for the treatment of adult patients with NASH with moderate to severe fibrosis (stage F2-F3), becoming the first approved NASH targeted drug. Common adverse reactions include transient diarrhea (incidence 24%) and nausea (18%), and thyroid hormone levels need to be monitored regularly to avoid the risk of exogenous hyperthyroidism.

[0010] Obeticholic acid inhibits hepatic neolipogenesis, enhances bile acid metabolism, and regulates fibrosis-related genes (such as TGF-β and COL1A1) by activating the farnesoid X receptor (FXR). The interim analysis of the Phase III REGENERATE trial (NCT02548351) showed that the proportion of patients in the 25 mg dose group with fibrosis improvement of ≥1 grade was 23% (vs. 12% in the placebo group, P=0.0002), but due to the high incidence of adverse reactions of itching (51%) (moderate to severe in 28%) and the potential risk of elevated low-density lipoprotein (LDL), the FDA did not approve its NASH indication.

[0011] Different drugs for treating fatty liver disease associated with metabolic dysfunction have different mechanisms of action and cause different side effects. Therefore, contraindications and precautions for use are also different. In general, hypoglycemic drugs should be used with extreme caution in patients with fatty liver disease associated with metabolic dysfunction who are at higher risk of liver damage and cardiovascular disease, and should be used with caution based on the patient's various clinical indicators. In addition, the use of drugs in obese patients and patients with various complications or comorbidities should also take into account drug toxicity, side effects, and interactions between drugs. Therefore, it is urgent to find a new drug for the treatment of fatty liver disease associated with metabolic dysfunction. Summary of the invention

[0012] The purpose of the present invention is to provide an application of brucein D in treating fatty liver disease associated with metabolic dysfunction, aiming to solve the problems raised in the background technology.

[0013] In view of the above problems, the present invention is achieved by providing a use of brucein D in the preparation of a drug for treating and / or preventing fatty liver disease associated with metabolic dysfunction and / or its complications.

[0014] Preferably, the complications of metabolic dysfunction-related fatty liver disease include metabolic dysfunction-related steatohepatitis and liver fibrosis.

[0015] Preferably, the brucein D acts through the following mechanism:

[0016] By reducing fatty acid synthesis, it reduces hepatic triglyceride synthesis, lowering hepatic triglycerides and low-density lipoprotein cholesterol;

[0017] Reduce the phosphorylation level of JNK / NF-kb, thereby reducing the expression of inflammatory factors and alleviating liver inflammation;

[0018] Enhance antioxidant capacity and alleviate oxidative stress damage.

[0019] Preferably, the drug is suitable for patients with metabolic dysfunction-related fatty liver disease who are also suffering from obesity, type 2 diabetes or cardiovascular disease.

[0020] Preferably, the drug comprises brucein D and a pharmaceutically acceptable carrier and / or excipient.

[0021] Preferably, the drug is in the form of an injection, oral tablet or capsule.

[0022] Preferably, the medicine further comprises other active ingredients; the other active ingredients comprise vitamin E.

[0023] The present invention finds that the potential advantage of brucein D in lipid-lowering therapy lies in multi-target regulation of lipid metabolism, anti-inflammatory / antioxidant synergistic effects and possible anti-fibrosis activity, which may make up for the problems of insufficient improvement of fibrosis and significant side effects of existing drugs; in-depth research can further apply it to the prevention and treatment of fatty liver disease associated with metabolic dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1The experimental results of the mouse model induced by MCD feeding are shown in the figure; in the figure, (A) the modeling method of metabolic dysfunction-related fatty hepatitis in MCD-induced mice; (B) changes in body weight of MCD-induced mice; (C) photos of MCD-induced mouse livers; (D) the ratio of liver weight to body weight of MCD-induced mice; (E) H&E and Oil Red-O-staining of MCD-induced mouse livers (×200, scale bar: 50μm); (F) TG content in MCD-induced mouse livers; (G) RT-qPCR detection of the expression of fatty acid synthesis genes in MCD-induced mouse livers; (H) Western-blot detection of the expression of fatty acid synthesis genes in MCD-induced mouse livers; (I) CD11b and F4 / 80 staining of MCD-induced mouse livers (×200, scale bar: 50μm); (J) RT-qPCR detection of the expression of inflammatory response genes in MCD-induced mouse livers; (K) Western-blot detection of the expression of inflammatory response genes in MCD-induced mouse livers.

[0025] Figure 2 The experimental results of the mouse model induced by HFHC feeding; in the figure, (A) the modeling method of HFHC diet-induced metabolic dysfunction-related fatty liver disease mouse; (B) the weight change of mice induced by HFHC diet; (C) the photo of HFHC diet-induced mouse liver; (D) the ratio of liver weight to body weight of HFHC diet-induced mice; (E) H&E and Oil Red-O-staining of HFHC diet-induced mouse liver (×200, scale bar: 50μm); (F) TG content in HFHC diet-induced mouse liver; (G) RT-qP CR was used to detect the expression of fatty acid synthesis genes in the liver of mice induced by HFHC diet; (H) Western-blot was used to detect the expression of fatty acid synthesis genes in the liver of mice induced by HFHC diet; (I) CD11b and F4 / 80 staining of the liver of mice induced by HFHC diet (×200, scale bar: 50 μm); (J) RT-qPCR was used to detect the expression of inflammatory response genes in the liver of mice induced by HFHC diet; (K) Western-blot was used to detect the expression of inflammatory response genes in the liver of mice induced by HFHC diet.

[0026] Figure 3 The figures show the experimental results of treating mouse liver primary cells with brucein D. In the figure, (A) brucein D was added to mouse liver primary cells after PA / OA induction, and the cell TG content was detected; (B) Oil red-O staining was used to observe the accumulation of lipid droplets (×200, scale bar: 50μm; ×400, scale bar: 20μm); (C) Western-blot was used to detect the expression of genes related to lipid synthesis; (D) Western-blot was used to detect the expression of genes related to inflammation. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] Bruceine D (BD) is a quasinoid compound purified from the seeds of Brucea brucea. As a traditional Chinese medicine that can be used in clinical practice, Brucea brucea has played its medicinal role in clearing away heat and detoxification, stopping malaria, and stopping dysentery. Bruceine D was first considered to be a Notch inhibitor, which means that it can interfere with the Notch signaling pathway, which plays a key role in processes such as cell differentiation, proliferation, and apoptosis. In anti-cancer research, Bruceine D showed significant activity and was able to induce apoptosis in a variety of human cancer cells, which is of great significance for the development of new anti-cancer drugs. In addition to its anti-cancer activity, Bruceine D is also an effective plant-based insect repellent. It has excellent systemic properties and can significantly inhibit the growth of pests. Experimental data show that it exhibits strong anthelmintic activity. This property makes Bruceine D have potential application value in the field of agricultural pest control. Recent studies have shown that brucein D has a variety of biological effects, including inhibiting Parkinson's disease, treating ulcerative colitis, and preventing the progression of multiple cancer types, such as lung cancer, hepatocellular carcinoma, pancreatic cancer, osteosarcoma, chronic myeloid leukemia, and breast cancer. Recent studies have reported that brucein D inhibits HIF-1α-mediated glucose metabolism in hepatocellular carcinoma by blocking ICAT / β-catenin interaction. It also suggests that brucein D may be related to regulating the body's glucose and lipid metabolism, and brucein D may be used as a potential drug for the treatment of MAFLD.

[0029] Pioglitazone has limited efficacy: although it can improve liver steatosis and inflammation, it has a weak effect on the reversal of liver fibrosis (e.g., the improvement of fibrosis in the PIVENS trial did not reach statistical significance). Significant side effects: Peripheral edema (4-6% of patients) and weight gain (average 2-4 kg) are common. Long-term use may increase the risk of osteoporosis, especially for postmenopausal women or elderly patients. Limited applicable population: The guidelines only recommend it for MASLD patients with type 2 diabetes, and there is insufficient evidence for non-diabetic patients. Vitamin E (α-tocopherol) has insufficient efficacy: although it can relieve inflammation in non-diabetic NASH patients, the improvement rate of fibrosis is only 19% (PIVENS trial), which cannot meet the needs of advanced liver disease. Long-term safety controversy: Long-term use of high doses (800 IU / day) may increase the risk of bleeding (abnormal coagulation function) and the incidence of prostate cancer (HR=1.17 in the SELECT trial). Population restrictions: It is not recommended for patients with diabetes or cirrhosis, and potential toxicity needs to be monitored regularly. Semaglutide has limited improvement in fibrosis: Only 26% of patients in the Phase III trial had improved fibrosis stages, suggesting that its core role is focused on metabolic regulation rather than direct anti-fibrosis. Dosage and tolerability: Weekly subcutaneous injections are required, and some patients terminate treatment due to gastrointestinal reactions (nausea, vomiting). Indications not approved: There are legal and reimbursement barriers to off-label use, and long-term cardiovascular safety data are lacking. Problems with Tirzepatide include weight loss and liver fat reduction in Phase II trials, but Phase III trials (SYNERGY-NASH) are still ongoing and the efficacy of fibrosis has not yet been determined. Side effect risk: Similar to GLP-1 drugs, the incidence of gastrointestinal reactions (diarrhea, vomiting) is high, and strong weight loss may lead to muscle loss. Cost and accessibility: As new drugs, they are expensive and have limited medical insurance coverage. Empagliflozin and dapagliflozin have low levels of evidence: Existing supporting data mostly come from observational studies or subgroup analyses of diabetes trials, and there is a lack of high-quality randomized controlled trials (RCTs) for NASH. Urogenital system risks: May increase the risk of urinary tract infection (more common in women) and diabetic ketoacidosis (rare but serious). Unclear mechanism: It is unclear whether the mechanism of improving liver fat is independent of systemic metabolic improvement, and its targeting is questionable. Resmetirom (Rezdiffra) has complex side effects that require management: diarrhea (24%) and nausea (18%) are common, requiring dose adjustment or symptomatic treatment; long-term use requires monitoring of thyroid dysfunction (such as TSH suppression). Narrow indications: It is only approved for use in patients with NASH with moderate to severe fibrosis (stage F2-F3), and its efficacy has not been verified for early or late stage patients. Long-term safety is unknown: As the first approved NASH targeted drug, there is a lack of long-term follow-up data of more than 52 weeks.51% of patients with obeticholic acid (OCA) experience pruritus (28% moderate to severe), resulting in a high rate of treatment discontinuation; it may increase LDL cholesterol (statins are required in combination). Due to safety issues, it has not been formally approved for NASH, and its off-label use is limited. It can be seen that the improvement rate of liver fibrosis by most drugs is less than 30%, which cannot meet the needs of patients in the advanced stage. Side effects include but are not limited to weight gain (pioglitazone), pruritus (OCA), gastrointestinal reactions (GLP-1 drugs), etc., which affect long-term compliance. Drugs such as SGLT2 inhibitors lack NASH-specific RCT data; the long-term safety of new drugs (such as Resmetirom) is unknown. Targeted drugs are expensive and have limited medical insurance coverage, which restricts their use by patients in low- and middle-income countries. Drugs need to be selected based on comorbidities (diabetes, cardiovascular disease), degree of fibrosis and patient preferences, and there is no unified best solution.

[0030] In contrast, the advantages of brucein D: brucein D (brucein D) is a quasinoid compound isolated from the traditional Chinese medicine Brucea javanica, which has been used as a Chinese medicine component for clinical treatment. In recent years, brucein D has shown potential value in the field of lipid-lowering and metabolic disease treatment. Although its clinical application is still in the preclinical research stage, based on existing experimental data, animal experiments have shown that brucein D can upregulate the expression of peroxisome proliferator-activated receptor α (PPARα), promote lipid decomposition and lipoprotein metabolism, and reduce serum triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C). By reducing the release of proinflammatory factors (such as TNF-α, IL-6), improving adipose tissue inflammation, it may delay the progression of metabolic dysfunction-associated steatohepatitis (MASH). Antioxidant activity can reduce liver oxidative stress damage, similar to vitamin E but may synergize through different pathways. In vitro studies suggest that it may have a direct intervention effect on liver fibrosis by blocking the TGF-β / Smad pathway and inhibiting collagen deposition, which is superior to drugs with limited efficacy in treating fibrosis, such as pioglitazone and semaglutide.

[0031] Specifically, in one embodiment of the present invention, there is provided a use of brucein D in the preparation of a drug for treating and / or preventing fatty liver disease associated with metabolic dysfunction and / or its complications.

[0032] Among them, the complications of metabolic dysfunction-related fatty liver disease include metabolic dysfunction-related steatohepatitis and liver fibrosis.

[0033] Brucein D works through the following mechanisms:

[0034] By reducing fatty acid synthesis, it reduces hepatic triglyceride synthesis, lowering hepatic triglycerides and low-density lipoprotein cholesterol;

[0035] Reduce the phosphorylation level of JNK / NF-kb, thereby reducing the expression of inflammatory factors and alleviating liver inflammation;

[0036] Enhance antioxidant capacity and alleviate oxidative stress damage.

[0037] In another embodiment of the present invention, a drug for treating and / or preventing metabolic dysfunction-related fatty liver disease and / or its complications is provided, and the drug is suitable for patients with metabolic dysfunction-related fatty liver disease who are concurrently obese, type 2 diabetes or cardiovascular disease.

[0038] In a preferred embodiment of the present invention, the above-mentioned drug includes brucein D and a pharmaceutically acceptable carrier and / or excipient; for example, the pharmaceutically acceptable carrier includes but is not limited to physiological saline, liposomes, polysaccharide solutions, capsules, etc.; pharmaceutically acceptable excipients include but are not limited to fillers, adhesives, disintegrants, lubricants, wetting agents, flavoring agents and coating materials, etc.

[0039] In a preferred embodiment of the present invention, the dosage form of the above-mentioned drug is injection, oral tablet or capsule, but not limited thereto.

[0040] In a preferred embodiment of the present invention, the above-mentioned medicine also includes other active ingredients; for example, it may also include other active ingredients such as vitamin E; brucein D can work synergistically with vitamin E.

[0041] In another embodiment of the present invention, relevant animal experiments are provided to verify the efficacy of brucein D, as follows:

[0042] 1. Construction of animal model of fatty liver:

[0043] (1) 8-week-old male C57BL / 6J mice were housed in an SPF animal room with a 12h:12h light / dark cycle. They were fed with a methionine and choline deficiency diet (MCD) (purchased from Changzhou Shuyi Shuer Co., Ltd.) for 3 weeks. The control group mice were fed with a normal diet (ND) (purchased from Changzhou Shuyi Shuer Co., Ltd.).

[0044] (2) 4-week-old male C57BL / 6J mice: They were housed in an SPF animal room with a light / dark cycle of 12h:12h. They were fed with a high fat and high cholesterol (HFHC) diet (purchased from Changzhou Shuyi Shuer Co., Ltd.) for 12 weeks. The control group mice were fed with a normal diet (ND) (purchased from Changzhou Shuyi Shuer Co., Ltd.).

[0045] 2. Dosage method:

[0046] (1) Three groups of 8-week-old male C57BL / 6 mice (n=8 / group) were set up in the experiment: the control group of mice was fed a normal diet (ND) for one week and then received intraperitoneal injections of dimethyl sulfoxide (DMSO) solution for two consecutive weeks; the second group of mice was fed MCD for three consecutive weeks and was injected with an equal volume of DMSO solution daily from the second week as the model group (MCD group); the third group (BD group) of mice was fed an MCD diet and was given intraperitoneal injections of brucein D solution (1 mg / kg, dissolved in the same concentration of DMSO) daily from the second week. All injections maintained the same dosage volume and time interval.

[0047] (2) Three groups of 4-week-old male C57BL / 6 mice (n=8 / group) were set up in the experiment: the control group of mice was fed a normal diet (ND) for ten weeks and then received intraperitoneal injections of DMSO solution for two consecutive weeks; the second group of mice was fed HFHC for twelve consecutive weeks and injected with an equal volume of DMSO solution daily from the tenth week as the model group (HFHC); the third group (BD group) of mice was fed HFHC diet and received intraperitoneal injections of brucein D solution (1 mg / kg, dissolved in the same concentration of DMSO) daily from the tenth week. All injections maintained the same dosage volume and time interval.

[0048] 3. The livers of mice in the drug-treated group and the control group were identified by RT-qPCR, Western-blot, H&E staining, Oil Red-O staining, immunohistochemistry and other experimental methods to observe whether brucein D can significantly improve the symptoms of diet-induced fatty liver.

[0049] The results are as follows Figure 1 and Figure 2 As shown; it can be seen from the figure that under the conditions of MCD feeding and HFHC feeding, the symptoms of fatty liver in the liver of mice injected with brucea bruxellinus D were alleviated, the lipid droplets in the liver decreased, the triglyceride content in the liver decreased, the expression of lipid synthesis-related genes was significantly reduced, the expression of inflammation-related genes in the liver was also reduced, and the macrophage infiltration in the liver was reduced.

[0050] 4. Isolation of primary liver cells: Animal preparation: 6-8 week old male C57BL / 6J. The solution is prepared as follows:

[0051] Solution C: potassium chloride (KCl, 480 mM), magnesium sulfate (MgSO 4 , 120 mM), potassium dihydrogen phosphate (KH 2 PO 4 , 120mM);

[0052] CaCl2 Solution: Anhydrous calcium chloride (CaCl 2 , 1M), calcium chloride dihydrate (CaCl 2 :2H 2 O, 1M);

[0053] Ethylene glycol ether diamine tetraacetic acid (EGTA) solution: weigh 190.175 mg of EGTA powder, add a small amount of pure water to dissolve the powder, if it is not completely dissolved, add an appropriate amount of sodium hydroxide (NAOH) solution to completely dissolve the EGTA, adjust the pH to neutral with hydrochloric acid (HCl) solution, and finally make up to 10 mL;

[0054] Krebs solution: sodium chloride (NaCl, 3.5 g), sodium bicarbonate (NaHCO 3 , 1.0g), glucose (1.8g), 1 mol / L HEPES (pH=7.45) 2.5mL, solution C 5mL, dilute to 500mL. All the above solutions are neutral solutions and are stored at 4℃;

[0055] Perfusate I: 90 μL EGTA solution added to 45 mL Krebs solution;

[0056] Perfusate II: 40 mL Krebs solution with 54.88 μL CaCl 2 solution, 0.024 g collagenase;

[0057] Aspirate perfusion fluid I into the syringe, remove bubbles, weigh 0.023g collagenase and dissolve it in perfusion fluid II. Mix well and put it in a 37℃ water bath. Anesthetize the mouse, wipe the skin with alcohol for disinfection, open the abdominal cavity, and separate the inferior vena cava. Use a surgical rope to pass through the inferior vena cava. Slowly push perfusion fluid I for a total of 45mL. After the liver becomes granular and the color changes, immediately cut the portal vein to allow the blood in the liver to flow out of the portal vein. The push speed should be slow in the first 3min. After 20min, perfusion fluid I is connected to perfusion fluid II (drop a few drops of perfusion fluid at the connection before connecting to the connecting tube to prevent bubbles from affecting the experiment during the connection process). Perfusion fluid II 50ml is perfused for about 9 minutes, and the perfusion is stopped. Remove the liver and transfer it to a 6cm culture dish (1640 culture medium is added to the dish in advance). Use tweezers to cut the liver to disperse the hepatocytes, filter it with a filter, and transfer it to a 50mL centrifuge tube. Centrifuge it at 4℃ and 500rpm for 3min. After centrifugation, discard the supernatant and add 1640 culture medium to the remaining cell suspension to 15mL. Place the centrifuge tube on ice and centrifuge at 4℃ for 3 minutes at 500rpm. After centrifugation, discard the supernatant, add 1640 culture medium to 15mL, centrifuge at 500rpm for 3 minutes, discard the supernatant after centrifugation, add 1640 culture medium to 15mL, and centrifuge at 700rpm for 3min. Mix the cell suspension and start plating. Add two milliliters of primary liver culture medium to each well of the six-well plate, and then add an equal volume (about 200μL) of cell suspension. The amount of cell suspension added is determined according to the total amount of cells extracted. You can add one well first and observe under the microscope; before each addition, mix the cell suspension to ensure that the amount of cells in each well is roughly the same; since primary liver cells cannot proliferate and divide, they should be spread as densely as possible when plating. After three to four hours of culture in the incubator, observe the cell adhesion under a microscope. After good adhesion, change the medium and conduct subsequent experiments.

[0058] 5. Palmitic acid PA & oleic acid OA were added to primary liver cells to simulate a high-fat environment. Brucein D was then added to treat primary mouse liver cells, and the effect of brucein D on the expression level of key genes involved in lipid metabolism and the changes in cellular lipid droplets were verified by Western-blot and Oil Red-O-staining.

[0059] The results are as follows Figure 3 As shown in the figure, it can be seen that after brucei bruxin D was used to treat mouse liver primary cells, the intracellular triglyceride content was reduced, the protein expression of lipid synthesis-related genes and inflammatory response-related genes was significantly reduced, and the number and size of intracellular lipid droplets were reduced.

[0060] In summary, in the MCD feeding-induced and HFHC feeding-induced mouse models, there was no significant difference in the body weight of mice in the brucein D-treated group and the ND group, suggesting that brucein D may have less side effects on the health of mice. Histochemical results showed that after brucein D treatment, brucein D improved macrophage infiltration in the liver of mice, suggesting that brucein D may have a more obvious effect on improving fibrosis. The potential advantages of brucein D in lipid-lowering therapy lie in multi-target regulation of lipid metabolism, anti-inflammatory / antioxidant synergistic effects, and possible anti-fibrotic activity, which may make up for the problems of insufficient improvement of fibrosis and significant side effects of existing drugs. In-depth research can be further applied to the prevention and treatment of fatty liver disease associated with metabolic dysfunction.

[0061] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of brucein D in the preparation of drugs for treating and / or preventing fatty liver disease associated with metabolic dysfunction and / or its complications.

2. The use according to claim 1, characterized in that: The complications of metabolic dysfunction-related fatty liver disease include metabolic dysfunction-related steatohepatitis and liver fibrosis.

3. The use according to claim 1, characterized in that: The brucein D acts through the following mechanism: By reducing fatty acid synthesis, it reduces hepatic triglyceride synthesis, lowering hepatic triglycerides and low-density lipoprotein cholesterol; Reduce the phosphorylation level of JNK / NF-kb, thereby reducing the expression of inflammatory factors and alleviating liver inflammation; Enhance antioxidant capacity and alleviate oxidative stress damage.

4. The use according to claim 1, characterized in that: The drug is suitable for patients with fatty liver disease associated with metabolic dysfunction combined with obesity, type 2 diabetes or cardiovascular disease.

5. The use according to claim 1 or 4, characterized in that: The drug comprises brucein D and a pharmaceutically acceptable carrier and / or excipient.

6. The use according to claim 5, characterized in that: The dosage form of the drug is injection, oral tablet or capsule.

7. The use according to claim 5, characterized in that: The medicine also includes other active ingredients; the other active ingredients include vitamin E.

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