Application of cinnamoylglycine in preparing products for predicting or preventing metabolism-related fatty liver disease

Abstract

The present application relates to the field of biomedicine technology, and in particular to the use of cinnamoylglycine in the preparation of products for predicting or preventing metabolic-related fatty liver disease. The present invention is based on the use of cinnamoylglycine to prevent and / or treat metabolic-related fatty liver disease and its related conditions. Experimental results show that: cinnamoylglycine can effectively predict the risk of metabolic-related fatty liver disease and high-risk metabolic-related fatty liver hepatitis in community populations; cinnamoylglycine intervention can effectively reduce the body weight, triglyceride and cholesterol levels of mice induced by a high-fat and high-cholesterol diet, and improve liver lipid deposition, inflammatory cell infiltration and hepatocyte ballooning; cinnamoylglycine improves metabolic-related fatty liver disease by inhibiting the expression and activity of FDPS. The present invention provides new and effective intervention ideas and means for metabolic-related fatty liver disease and its related conditions.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to the use of cinnamoylglycine in preparing products for predicting, preventing or improving metabolism-related fatty liver disease and related disorders. Background Art

[0002] Metabolic fatty liver disease (MAFLD), previously known as non-alcoholic fatty liver disease (NAFLD), has become the most common cause of chronic liver disease, affecting approximately one-third of the global population and carrying a significant burden. The core pathological basis of MAFLD is excessive accumulation of triglycerides within hepatocytes, known as hepatic steatosis. During disease progression, persistent hepatic inflammatory infiltration and hepatic stellate cell activation can lead to MAFLD progression in 10% to 40% of patients, characterized by hepatocellular ballooning and lobular inflammation, with or without fibrosis. This progression can progress to end-stage liver disease such as cirrhosis and hepatocellular carcinoma. Because MAFLD is a complex metabolic disease caused by multiple factors, including genetic susceptibility, epigenetic regulation, diet, and lifestyle, its pathogenesis, clinical manifestations, and pathological features vary significantly between individuals. Only one drug, resmetirom, is approved for the treatment of patients with high-risk MAFLD. However, data are currently lacking on whether remediazole can provide sustained histological benefits, predict individual responses, outcomes related to metabolic fatty liver disease, and long-term safety in patients with metabolic fatty liver disease. Unlike drug treatments, which are often accompanied by side effects, small molecule metabolites, as endogenous substances, have natural biocompatibility and advantages such as wide tissue distribution, strong barrier-crossing ability, high toxicity threshold, and clear metabolic clearance pathways. Therefore, exploring small molecule metabolites that can effectively and safely improve liver lipid deposition, inhibit liver inflammation and hepatocyte damage, and clarifying their mechanisms of action are of great value in the prevention and clinical treatment of metabolic fatty liver disease and its progression.

[0003] Cinnamoylglycine is a product of the gut microbiota-mediated phenylalanine / cinnamic acid metabolic pathway and can be detected in the blood and urine of humans and mice. In recent years, several large human cohort studies using untargeted metabolomics data have identified cinnamoylglycine as a biomarker of gut health. However, no studies have investigated the effects of cinnamoylglycine on metabolic-related fatty liver disease and its associated conditions, nor have any studies experimentally demonstrated its metabolic health-improving effects or explored its underlying mechanisms.

[0004] Currently, most studies focusing on cinnamoylglycine and metabolic-related health outcomes are based on population epidemiological studies and are mainly non-targeted metabolomics. The specific concentration range of cinnamoylglycine is still unclear. No experimental studies have demonstrated the improvement effect of cinnamoylglycine on metabolic health, nor have studies revealed the potential molecular mechanism by which cinnamoylglycine improves host metabolic disorders. Summary of the Invention

[0005] In view of this, the present application provides the use of cinnamoylglycine in the preparation of products for predicting, preventing or improving metabolism-related fatty liver disease and its related conditions, aiming to provide the use of cinnamoylglycine in preventing and / or treating metabolism-related fatty liver disease and its related conditions.

[0006] In order to achieve the above-mentioned invention objectives, this application provides the following technical solutions:

[0007] The present application provides the use of cinnamoylglycine as a marker in the preparation of a detection product for predicting the risk of metabolism-related fatty liver disease.

[0008] In some specific embodiments of the present application, the prediction of the above application includes the following judgment rules: when the individual's plasma cinnamoylglycine concentration reaches or exceeds 8 ng / mL, it is considered low risk, and when it is lower than 8 ng / mL, it is considered a high-risk group.

[0009] The present application also provides the use of cinnamoylglycine in inhibiting the expression of liver FDPS and / or reducing the expression level of liver interleukin-1β.

[0010] The present application also provides the use of cinnamoylglycine in reducing the biosynthesis of liver cholesterol, improving liver steatosis, improving liver lipid deposition, reducing hepatocyte ballooning and / or improving liver inflammation.

[0011] The present application also provides the use of cinnamoylglycine in preventing and / or treating metabolism-related fatty liver disease.

[0012] The present application also provides the use of cinnamoylglycine in the preparation of a medicament for preventing and / or treating metabolism-related fatty liver disease.

[0013] In some specific embodiments of the present application, the cinnamoyl glycine used above prevents and / or treats metabolism-related fatty liver disease by improving liver lipid deposition.

[0014] In some specific embodiments of the present application, the cinnamoylglycine used above prevents and / or treats metabolism-related fatty liver disease by improving hepatic steatosis and hepatocyte ballooning.

[0015] In some specific embodiments of the present application, the cinnamoylglycine used above prevents and / or treats metabolism-related fatty liver disease by reducing the expression level of interleukin-1β in the liver and improving liver inflammation.

[0016] In some specific embodiments of the present application, the cinnamoylglycine used above prevents and / or treats metabolism-related fatty liver disease by inhibiting the expression of liver FDPS and reducing the biosynthesis of liver cholesterol.

[0017] In some specific embodiments of the present application, the metabolism-related fatty liver disease mentioned above includes metabolism-related fatty liver hepatitis.

[0018] The present application also provides a method for predicting, assisting in the diagnosis or diagnosing metabolism-related fatty liver disease based on cinnamoylglycine;

[0019] The cinnamoylglycine may refer to serum or plasma cinnamoylglycine concentration;

[0020] The metabolism-related fatty liver disease includes simple metabolism-related fatty liver and metabolism-related steatohepatitis.

[0021] The present application also provides a method for preventing and / or treating metabolism-related fatty liver disease based on cinnamoylglycine.

[0022] The present application also provides a method for preventing and / or treating metabolism-related fatty liver disease based on a composition containing cinnamoylglycine.

[0023] In some specific embodiments of the present application, the metabolism-related fatty liver disease in the above method includes simple metabolism-related fatty liver and metabolism-related steatohepatitis.

[0024] The present application also provides a detection product for predicting, assisting in the diagnosis or diagnosing metabolism-related fatty liver disease using cinnamoylglycine as a marker, wherein the detection product includes a chip, a device or a system;

[0025] The metabolism-related fatty liver disease includes simple metabolism-related fatty liver and metabolism-related steatohepatitis.

[0026] The present application also provides a drug for treating metabolic-related fatty liver disease containing cinnamoylglycine, wherein the drug includes an acceptable excipient or auxiliary agent;

[0027] The metabolism-related fatty liver disease includes simple metabolism-related fatty liver and metabolism-related steatohepatitis.

[0028] The present application also provides the use of cinnamoylglycine in preparing a product for reducing body fat and / or lowering blood lipids.

[0029] In some specific embodiments of the present application, the reducing body fat in the above application includes reducing liver fat.

[0030] In some specific embodiments of the present application, the lowering of blood lipids in the above application includes lowering blood triglycerides and / or lowering blood cholesterol.

[0031] In some specific embodiments of the present application, the product of the above application includes a medicine or a health product.

[0032] The present invention has the following beneficial effects:

[0033] (1) In a community-based natural population cohort, the application of the intestinal flora metabolite cinnamoylglycine as a biomarker for metabolic-related fatty liver disease and metabolic-related steatohepatitis was evaluated. Plasma cinnamoylglycine concentration can effectively predict the risk of metabolic-related fatty liver disease and high-risk metabolic-related steatohepatitis.

[0034] (2) Cinnamoylglycine helps lower blood lipids (cholesterol / triglycerides) and body fat, and can effectively improve lipid metabolism disorders and metabolic-related fatty liver disease in mice fed a high-fat, high-cholesterol diet, reduce the accumulation of triglycerides and cholesterol in the liver, and improve inflammation and hepatocyte ballooning.

[0035] (3) Cinnamoylglycine inhibits the expression and activity of farnesyl diphosphate synthase (FDPS) in the liver, reduces liver cholesterol biosynthesis, and thus improves metabolic-related fatty liver disease and its related conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art.

[0037] Figure 1 To show the risk stratification of plasma cinnamoylglycine concentration for metabolic-related fatty liver disease in the discovery cohort;

[0038] Figure 2 Figure 3 shows the prediction of plasma cinnamoylglycine concentration for the risk of metabolic-related fatty liver disease and metabolic-related steatohepatitis in an internal prospective validation cohort, where A shows the controlled attenuation parameter (CAP) and B shows the FibroScan-AST score (FAST);

[0039] Figure 3 To demonstrate the ability of plasma cinnamoylglycine concentration to stratify the risk of metabolic-related fatty liver disease in an external prospective validation cohort;

[0040] Figure 4Figure 2 shows the metabolic-related fatty liver disease mouse model induced by a high-fat and high-cholesterol diet and the intervention groups, where STC refers to standard control diet, HFHC refers to high-fat and high-cholesterol diet, PBS refers to phosphate-buffered saline, and CMG refers to cinnamoylglycine;

[0041] Figure 5 Comparison of body weight and blood glucose levels of mice in each group, where A shows body weight, B shows fasting blood glucose, and C shows postprandial blood glucose;

[0042] Figure 6 Comparison of blood lipid levels of mice in each group, where A shows serum triglyceride (TG) level, B shows serum total cholesterol (TC) level, C shows serum high-density lipoprotein cholesterol (HDL-c) level, and D shows serum low-density lipoprotein cholesterol (LDL-c) level;

[0043] Figure 7 Comparison of liver lipid levels in mice of each group, where A shows liver triglyceride content and B shows liver total cholesterol content;

[0044] Figure 8 The liver morphology and serum alanine aminotransferase levels of mice in each group are shown, where A shows the liver morphology and B shows the serum alanine aminotransferase levels;

[0045] Figure 9 Shown are the liver tissue sections of mice in each group (H&E staining);

[0046] Figure 10 The semi-quantitative evaluation of H&E staining results of the livers of mice in each group is shown, where A shows the liver steatosis score, B shows the inflammatory infiltration score, C shows the ballooning score, and D shows the total NAS score;

[0047] Figure 11 Comparison of liver Filipin staining and liver cholesterol content in each group of mice, where A shows the Filipin staining results and B shows the relative intensity of Filipin;

[0048] Figure 12 Comparison of FDPS expression and activity in mice of each group, where A shows the mRNA expression level of Fdps, B shows the protein expression level of FDPS, and C shows the enzyme activity of FDPS;

[0049] Figure 13 Comparison of interleukin-1β expression in the liver of mice in each group, where A shows the mRNA expression level of interleukin-1β, B shows the immunohistochemical image, and C shows the interleukin-1β-positive area. DETAILED DESCRIPTION

[0050] The present application discloses the use of cinnamoylglycine in the preparation of products for predicting, preventing or improving metabolic-related fatty liver disease and its related conditions. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of the present invention.

[0051] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0052] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0053] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0054] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and does not limit the scope of the present application. No language in this specification should be construed as indicating any non-claimed element is essential to the practice of the present application.

[0055] In addition, the numerical ranges and parameters used to define this application are approximate values. The relevant numerical values ​​in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, amounts, values, and percentages used in this disclosure are modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0056] Based on the limitations of research progress described in the background technology, the purpose of the present invention is to specifically address the current situation where there is no effective treatment for metabolic-related fatty liver disease, and to provide an application of cinnamoylglycine in the prevention and / or treatment of metabolic-related fatty liver disease and related conditions. Cinnamoylglycine can effectively predict the risk of metabolic-related fatty liver disease and high-risk metabolic-related steatohepatitis in community populations; provide an intervention regimen of cinnamoylglycine, which can effectively reduce the body weight, triglyceride and cholesterol levels of mice induced by a high-fat and high-cholesterol diet, and improve liver lipid deposition, inflammatory cell infiltration and hepatocyte ballooning; provide an application of inhibiting FDPS expression and activity in the cinnamoylglycine-mediated improvement effect of metabolic-related fatty liver disease, providing new and effective intervention ideas and means for metabolic-related fatty liver disease and related conditions.

[0057] Some of the terms involved in this application are explained as follows:

[0058] Metabolic-associated fatty liver disease (MAFLD): Previously known as non-alcoholic fatty liver disease (NAFLD), MAFLD is a multisystem metabolic disorder of the liver. The term MAFLD was proposed by an international expert panel in 2020 and is endorsed and supported by the Asia-Pacific Association for the Study of the Liver and the Chinese Medical Association's Society of Hepatology. MAFLD is defined as the presence of hepatic steatosis accompanied by overweight / obesity, type 2 diabetes, or metabolic dysfunction.

[0059] Hepatic steatosis: Hepatic steatosis is the core pathological feature of metabolic-related fatty liver disease. It is defined as the abnormal accumulation of lipids such as triglycerides in hepatocytes, with the content exceeding 5% of the liver weight, or the presence of lipid droplets in more than 5% of hepatocytes histologically.

[0060] Metabolic-associated steatohepatitis: Metabolic-associated steatohepatitis, formerly known as nonalcoholic steatohepatitis, is an advanced stage of metabolic-associated fatty liver disease. It is characterized by hepatic steatosis, combined with lobular inflammation and hepatocellular ballooning, with or without fibrosis. Metabolic-associated steatohepatitis can progress to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma.

[0061] High-risk metabolic-related steatohepatitis: High-risk metabolic-related steatohepatitis refers to a clinical subtype of patients with metabolic-related steatohepatitis with a high risk of rapid progression to advanced fibrosis, cirrhosis or hepatocellular carcinoma. It is characterized by the combination of grade 2 or higher fibrosis on the basis of the pathological characteristics of metabolic-related steatohepatitis.

[0062] Hepatic lobular inflammation: Hepatic lobular inflammation is one of the key histological features of metabolic-related fatty liver disease, manifested as inflammatory cell infiltration, mainly lymphocytes, in the hepatic lobular parenchyma, often accompanied by hepatocellular ballooning and punctate necrosis and other hepatocellular damaging changes.

[0063] Hepatocellular ballooning: Hepatocellular ballooning is a specific morphological manifestation of hepatocellular injury, characterized by a significant increase in hepatocyte size, rarefied cytoplasm, and a "balloon-like" transparent appearance under light microscopy. This is often accompanied by the formation of Mallory-Denk bodies within the cytoplasm. Hepatocellular ballooning is a necessary histological criterion for the diagnosis of metabolic-related steatohepatitis and is used to assess MASH disease activity.

[0064] Cinnamoylglycine: Cinnamoylglycine is a derivative of cinnamic acid and glycine, produced by the phenylalanine / cinnamic acid metabolic pathway mediated by intestinal flora. As an endogenous metabolite in humans, it can be detected in the blood circulation and urine. In this application, cinnamoylglycine was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (S83815).

[0065] Farnesyl diphosphate synthase: Farnesyl diphosphate synthase (FDPS; also known as farnesyl pyrophosphate synthase, FPPS) is an enzyme encoded by the FDPS gene (EC 2.5.1.10). As a key enzyme in the cholesterol biosynthesis pathway, FDPS catalyzes the production of farnesyl pyrophosphate, an important cholesterol precursor.

[0066] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this application are all common commercial products and can be purchased from the market.

[0067] The present invention will be further described below with reference to the embodiments.

[0068] Example 1

[0069] This example quantitatively detected the plasma cinnamoylglycine content in community populations and determined that cinnamoylglycine can effectively predict the future risk of metabolic-related fatty liver disease and high-risk metabolic-related steatohepatitis in community populations. Specifically, the following steps were included:

[0070] (1) Community population cohort data collection and diagnosis of metabolic-related fatty liver disease

[0071] The discovery cohort was a prospective cohort study of chronic non-communicable diseases in a natural population in a certain region, including 1,381 subjects who underwent baseline questionnaires, physical examinations, and biochemical tests. Hepatic steatosis was assessed using the fatty liver index. A higher fatty liver index indicates a greater likelihood of hepatic steatosis. A further 1,014 subjects who underwent vibration-controlled transient elastography during the follow-up phase, with a mean follow-up of 3.65 years, were included as an internal prospective validation cohort. Hepatic lipid content was assessed using the controlled attenuation parameter (CAP); higher values ​​indicate greater lipid accumulation. The FibroScan-AST score (FAST score) was used to assess the risk of high-risk metabolic-related steatohepatitis; higher scores indicate a greater risk.

[0072] A subcohort of a community-based population-based cohort served as a validation cohort, including 386 participants who were negative for metabolic fatty liver disease on abdominal ultrasound at baseline and who underwent repeat abdominal ultrasound during follow-up for a mean follow-up of 2.25 years. Abdominal ultrasound was performed using a color Doppler ultrasound system to assess hepatic steatosis, defined as a diffuse increase in fine echogenicity in the liver parenchyma compared with the kidney or spleen parenchyma.

[0073] A standardized questionnaire was used to collect basic information, such as gender, age, smoking, alcohol consumption, diet, physical activity, past medical history, and medication history. Trained staff or nurses collected a fasting blood sample and completed a physical examination, including height, weight, waist circumference, and blood pressure. Biochemical tests, such as liver and kidney function, and blood lipid and glucose levels, were performed by laboratory technicians. Baseline hypertension and current diabetes were diagnosed based on self-reported medical history, medication history, or baseline measurements.

[0074] (2) Targeted detection of plasma cinnamoylglycine concentration

[0075] 100 μL of human plasma was taken for sample pretreatment, [2,2- 2 H2]-N-trans-Cinnamoylglycine (IsoReag) was used as an internal standard, and the plasma cinnamoylglycine content of the human population was quantitatively detected by liquid chromatography-mass spectrometry (LC-MS, Agilent 6495, Agilent).

[0076] (3) Prediction of the risk of metabolic-related fatty liver disease and metabolic-related steatohepatitis by plasma cinnamoylglycine concentration

[0077] ① Determine the risk stratification of plasma cinnamoylglycine for metabolic-related fatty liver disease

[0078] The association between plasma cinnamoylglycine concentration and fatty liver index was assessed in 1,381 subjects in the discovery cohort. Restricted cubic spline regression models revealed a nonlinear relationship between plasma cinnamoylglycine concentration and fatty liver index (nonlinearity test P = 0.003), with 8 ng / mL being the critical threshold concentration. Before this threshold, cinnamoylglycine had a significant regulatory effect on the fatty liver index, but above this concentration, the effect leveled off, as shown in Table 1. Figure 1 shown.

[0079] Taking 8 ng / mL as the risk judgment standard, when the individual's plasma cinnamoylglycine concentration reaches or exceeds 8 ng / mL, it is considered low risk, and when it is lower than 8 ng / mL, it is considered a high-risk group.

[0080] ② Prediction of the risk of metabolic-related fatty liver disease and metabolic-related steatohepatitis by plasma cinnamoylglycine concentration

[0081] The association between baseline plasma cinnamoylglycine concentration and liver fat content and high-risk metabolic steatohepatitis during follow-up was assessed in 1,014 subjects in an internal prospective validation cohort. After adjusting for potential confounding effects such as age, sex, smoking, alcohol consumption, dietary diversity, physical activity, medication history, diabetes, hypertension, obesity, and estimated glomerular filtration rate, baseline plasma cinnamoylglycine concentration was significantly inversely correlated with the degree of liver lipid accumulation during follow-up (r = -0.125, P < 0.001). adj =0.001), such as Figure 2 Further analysis found that after adjusting for potential confounding factors, baseline plasma cinnamoylglycine concentration was significantly negatively correlated with the risk of high-risk metabolic-related steatohepatitis (r=-0.063, P adj =0.013), such as Figure 2 As shown in B.

[0082] The ability of baseline plasma cinnamoylglycine concentration to stratify the risk of metabolic-related fatty liver disease was further evaluated in 386 subjects in an external prospective validation cohort. The subjects were divided into two groups according to whether the baseline plasma cinnamoylglycine concentration was ≥8 ng / mL. Kaplan-Meier survival analysis was used to compare the cumulative risk of metabolic-related fatty liver disease in the groups with higher or lower plasma cinnamoylglycine concentrations during an average follow-up of 2.25 years. The log-rank test determined that people with higher plasma cinnamoylglycine concentrations had a lower risk of metabolic-related fatty liver disease (P=0.007). See Figure 3From day 500 of follow-up, the two risk curves and their 95% confidence intervals separated and did not overlap. The curve for the lower-concentration group was higher than that for the higher-concentration group at all time points, indicating that those with higher baseline plasma cinnamoylglycine concentrations had a lower risk of developing metabolic-related fatty liver disease. Compared with those with lower baseline plasma cinnamoylglycine concentrations, participants with baseline plasma cinnamoylglycine concentrations ≥8 ng / mL had a 59% reduced risk of developing metabolic-related fatty liver disease (hazard ratio (HR) = 0.41, 95% CI: 0.18-0.94).

[0083] Example 2: Application of Inhibiting FDPS Expression and Activity in Cinnamoylglycine-Mediated Improvement of Metabolism-Related Fatty Liver Disease

[0084] This example uses in vivo animal experiments to verify the ameliorative effects of cinnamoylglycine on metabolic-related fatty liver disease and related conditions. This study further reveals the potential molecular mechanism by which cinnamoylglycine reduces liver cholesterol biosynthesis by inhibiting hepatic FDPS expression and activity. All experiments were conducted under the premise of ensuring animal welfare and experimental guidelines. The specific experimental steps included:

[0085] (1) Establishment of a high-fat, high-cholesterol diet-induced metabolic-related fatty liver disease mouse model and intervention groups

[0086] 8-week-old SPF male C57BL / 6J mice were randomly divided into 3 groups, 8 mice in each group, and housed in an SPF-grade experimental animal center. They were allowed to eat and drink freely, with a 12-hour light / dark cycle, and the room temperature was controlled at 23℃±2℃. One group was fed with a standard control diet (STC) and gavaged with sterile PBS every day, namely the Ctrl group; the other two groups were fed with a high-fat and high-cholesterol diet (HFHC), and gavaged with sterile PBS and cinnamoylglycine (CMG, 0.2 mg / kg body weight, simulating a serum concentration of 8 ng / mL), respectively, namely the HFHC group and the CMG group. The formula of the high-fat and high-cholesterol diet is 16.9% protein, 40.3% fat (0.2% of which is cholesterol), and 42.8% carbohydrates. The above intervention lasted for 12 weeks. Figure 4 shown.

[0087] Food intake and body weight of the mice were recorded weekly. After the intervention, serum and liver tissue were collected to measure blood glucose and lipid levels. Hematoxylin and eosin (H&E) staining was used to evaluate liver pathology, including steatosis, inflammation, and ballooning. Filipin staining was used to measure liver cholesterol levels. Gene expression was assessed by real-time quantitative PCR, FDPS protein expression by Western blot, and interleukin-1β levels by immunohistochemistry.

[0088] (2) Cinnamoylglycine alleviates weight gain in mice with metabolic-related fatty liver disease and has no significant effect on glucose metabolism

[0089] After 12 weeks of intervention, the body weight of mice in the HFHC group increased significantly compared with the Ctrl group (P<0.001), indicating that cinnamoylglycine intervention can alleviate the weight gain induced by high-fat and high-cholesterol diet. Figure 5 A. Cinnamoylglycine intervention had no significant effect on glucose metabolism in mice with metabolic-related fatty liver disease (P>0.05). Figure 5 As shown in B and C.

[0090] (3) Cinnamoylglycine improves lipid metabolism disorders in mice with metabolic-related fatty liver disease

[0091] Lipid metabolism disorder is the core pathogenesis of metabolic-related fatty liver disease. After the intervention, the serum of each group of mice was collected after fasting for 14 hours, and the levels of four blood lipids were tested. Figure 6 As shown, the serum triglyceride and total cholesterol levels of mice treated with cinnamoylglycine for 12 weeks (CMG group) were significantly reduced by 26% and 9% compared with those of the HFHC group (see Figure 6 A and B in (P < 0.05), but there was no significant difference in the levels of high-density lipoprotein cholesterol and low-density lipoprotein cholesterol (see Figure 6 C, D in the figure).

[0092] The liver tissue homogenates of mice in each group were further collected to detect the liver lipid levels. Figure 7 As shown in Figures A and B, after 12 weeks of high-fat, high-cholesterol diet feeding, liver triglyceride and total cholesterol levels in the HFHC group were significantly higher than those in the Ctrl group (P<0.05), indicating hepatic lipid deposition. Compared with the HFHC group, mice treated with cinnamoylglycine (CMG) showed a significant decrease in liver triglyceride and total cholesterol levels by 20% and 24% (P<0.05).

[0093] The above results show that cinnamoylglycine intervention helps lower blood lipids (cholesterol / triglycerides), can effectively improve hypertriglyceridemia and hypercholesterolemia induced by a high-fat and high-cholesterol diet, reduce liver lipid deposition, and help reduce body fat.

[0094] (4) Cinnamoylglycine safely and effectively improves hepatic steatosis, inflammation, and hepatocellular ballooning in mice with metabolic-associated fatty liver disease

[0095] like Figure 8 As shown, the livers of mice fed a high-fat, high-cholesterol diet for 12 weeks showed typical metabolic-related fatty liver disease features, including yellow-white and enlarged livers in the HFHC group (see Figure 8A), serum alanine aminotransferase was significantly higher than that in the Ctrl group (see Figure 8 Compared with the HFHC group, cinnamoylglycine intervention for 12 weeks significantly improved the gross morphology of the liver in mice (see Figure 8 A, CMG group), significantly reduced serum alanine aminotransferase levels (see Figure 8 The difference was statistically significant (P<0.05).

[0096] The pathological characteristics of mouse liver tissue sections were further observed by H&E staining. Figure 9 As shown, compared with the typical pathological characteristics of metabolic-related fatty liver disease in HFHC group mice, the range of hepatic steatosis, the number of lobular inflammatory foci, and the number of ballooning hepatocytes in mice treated with cinnamoylglycine (CMG group) were reduced.

[0097] The H&E staining results of the livers of mice in each group were semi-quantitatively evaluated using the NAS scoring system. Figure 10 As shown, compared with the Ctrl group, the HFHC group mice had hepatic steatosis (see Figure 10 A in), inflammatory infiltration (see Figure 10 B) and ballooning (see Figure 10 C) scores of each item and the total NAS score (see Figure 10 The D in the NAS score (P<0.001) was significantly increased, indicating that the high-fat, high-cholesterol diet successfully induced a metabolic-associated fatty liver disease model in mice. Compared with the HFHC group, the 12-week cinnamoylglycine intervention (CMG) group significantly reduced the NAS total score and the hepatocyte steatosis, lobular inflammation, and ballooning degeneration subscores (P<0.05).

[0098] The above results show that cinnamoylglycine can effectively improve metabolism-related fatty liver disease and its related symptoms, has a certain protective effect on liver function, and has good safety.

[0099] (5) Cinnamoylglycine inhibits FDPS expression and activity, reducing hepatic cholesterol biosynthesis

[0100] To further confirm the effect of cinnamoylglycine on hepatic cholesterol synthesis, Filipin staining was performed on the liver tissue sections of mice in each group. Figure 11 As shown in Figures A and B, compared with the Ctrl group, the free cholesterol content in the liver of mice in the HFHC group was significantly increased (P<0.001), indicating that the high-fat, high-cholesterol diet induced enhanced liver cholesterol synthesis. Compared with the HFHC group, the 12-week cinnamoylglycine treatment group significantly improved the liver cholesterol accumulation in mice, with the content decreasing by 15% (P<0.05), indicating that cinnamoylglycine inhibits liver cholesterol biosynthesis in mice with metabolic-associated fatty liver disease.

[0101] At the transcriptional level, real-time fluorescence quantitative PCR revealed that cinnamoylglycine significantly downregulated the mRNA expression level of Fdps, a key gene involved in cholesterol biosynthesis (P<0.05). Figure 12 A. The expression level of FDPS protein in mouse liver was detected by Western Blot. Figure 12 As shown in Figure B, after 12 weeks of high-fat, high-cholesterol diet intervention, the expression level of FDPS in the liver of mice in the HFHC group was significantly increased by 53% compared with the Ctrl group (P < 0.05). Compared with the HFHC group, 12 weeks of cinnamoylglycine intervention reduced the expression level of FDPS in the liver of mice by 47%, with a statistically significant difference (P < 0.05). The FDPS enzyme activity in the liver of mice in each group was measured by the phosphorus colorimetric method. Figure 12 As shown in Figure C, 12 weeks of cinnamoylglycine intervention can reduce the liver FDPS enzyme activity promoted by a high-fat, high-cholesterol diet. Compared with the HFHC group mice, the liver FDPS enzyme activity of the mice in the cinnamoylglycine intervention group was significantly decreased by 72% (P<0.05).

[0102] The above results further demonstrate that cinnamoylglycine improves metabolism-related fatty liver disease by inhibiting hepatic FDPS expression and reducing hepatic cholesterol biosynthesis.

[0103] (6) Cinnamoylglycine improves hepatic inflammatory response in mice with metabolic-related fatty liver disease

[0104] In Example 1, cinnamoylglycine can effectively predict the risk of high-risk metabolic-related fatty liver disease. In this example, H&E staining found that cinnamoylglycine can improve liver lobule inflammation in mice with metabolic-related fatty liver disease. Real-time fluorescence quantitative PCR was further used to detect the transcriptional expression level of interleukin-1β (Il-1β) in the liver of mice with metabolic-related fatty liver disease. It was found that cinnamoylglycine significantly reduced the mRNA expression level of Il-1β in the liver of mice with metabolic-related fatty liver disease (P<0.05). Figure 13 As shown in Figure A. Immunohistochemical analysis showed that compared with the HFHC group, 12 weeks of cinnamoylglycine intervention significantly reduced the expression level of IL-1β in the liver of mice. Figure 13 As shown in Figures B and C, it further demonstrated that cinnamoylglycine could improve the hepatic inflammatory response in mice with metabolic-associated fatty liver disease.

[0105] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of this application.

Claims

1. Application of cinnamoylglycine as a biomarker in the preparation of a test product for predicting the risk of metabolic-related fatty liver disease.

2. Use of cinnamoylglycine in the preparation of a medicament for preventing and / or treating metabolism-related fatty liver disease.

3. The use according to claim 2, characterized in that The cinnamoyl glycine prevents and / or treats metabolism-related fatty liver disease by improving liver lipid deposition.

4. The use according to claim 2, characterized in that The cinnamoyl glycine prevents and / or treats metabolism-related fatty liver disease by improving liver steatosis and hepatocyte ballooning.

5. The use according to claim 2, characterized in that The cinnamoyl glycine prevents and / or treats metabolism-related fatty liver disease by reducing the expression level of liver interleukin-1β and improving liver inflammation.

6. The use according to claim 2, characterized in that The cinnamoylglycine prevents and / or treats metabolism-related fatty liver disease by inhibiting the expression of liver FDPS and reducing the biosynthesis of liver cholesterol.

7. The use according to any one of claims 1 to 6, characterized in that The metabolism-related fatty liver disease includes simple metabolism-related fatty liver and / or metabolism-related steatohepatitis.

8. Use of cinnamoylglycine in preparing a product for reducing body fat and / or lowering blood lipids, wherein the reducing body fat is reducing liver fat, and the lowering blood lipids is reducing blood triglycerides and / or blood cholesterol.

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