Application of cinnamyl glycine in preparation of product for predicting or preventing and treating metabolism-related fatty liver disease

Abstract

The invention relates to the technical field of biological medicines, in particular to application of cinnamyl glycine in preparation of products for predicting or preventing and treating metabolism-related fatty liver diseases. The present invention is based on the prevention and / or treatment of metabolism-related fatty liver diseases and related conditions thereof on the basis of cinnamoylglycine. Experimental results show that the cinnamyl glycine can effectively predict the risk of metabolism-related fatty liver diseases and high-risk metabolism-related fatty hepatitis of community population; cinnamoylglycine intervention can effectively reduce the weight, the triglyceride level and the cholesterol level of mice induced by high-fat and high-cholesterol diet, and improve liver lipid deposition, inflammatory cell infiltration and liver cell ballooning. Cinnamoylglycine can improve metabolism-related fatty liver diseases by inhibiting FDPS expression and activity. The invention provides a new effective intervention thought and means for metabolism-related fatty liver diseases and related diseases thereof.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and particularly to the application of cinnamoyl glycine in the preparation of products for predicting, preventing or improving metabolic associated fatty liver disease and its related disorders. Background Art

[0002] Metabolic associated fatty liver disease, formerly known as non-alcoholic fatty liver disease, has become the most common cause of chronic liver disease, affecting approximately one-third of the global population and imposing a significant disease and economic burden. The core pathological basis of metabolic associated fatty liver disease is the excessive accumulation of triglycerides in hepatocytes, i.e., hepatic steatosis. During the progression of the disease, persistent hepatic inflammatory infiltration and hepatic stellate cell activation can lead to 10% - 40% of patients with metabolic associated fatty liver disease progressing to metabolic associated steatohepatitis, presenting with hepatocyte ballooning degeneration, lobular inflammation, with or without fibrosis, and can further progress to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma. Since metabolic associated fatty liver disease is a complex metabolic disease caused by the combined action of multiple factors such as genetic susceptibility, epigenetic regulation, diet, and lifestyle, its pathogenesis, clinical manifestations, and pathological features all show significant individual differences. Only one drug, Resmetirom, has been approved for the treatment of patients with high-risk metabolic associated steatohepatitis. However, there is currently a lack of data on whether Resmetirom can provide sustained histological benefits, individual response prediction, metabolic associated fatty liver disease-related outcomes, and long-term safety when treating patients with metabolic associated fatty liver disease. Different from the common side effects of drug treatment, small molecule metabolites, as endogenous substances, have natural biocompatibility and possess advantages such as wide tissue distribution, strong cross-barrier ability, high toxicity threshold, and clear metabolic clearance pathways. Therefore, exploring small molecule metabolites that can effectively and safely improve hepatic lipid deposition, inhibit hepatic inflammation, and hepatocyte injury, and clarifying their mechanisms of action, is of great value for the prevention and clinical treatment of metabolic associated fatty liver disease and its progressive diseases.

[0003] Cinnamoyl glycine is a product of the phenylalanine / cinnamic acid metabolic pathway mediated by gut microbiota and can be detected in the blood and urine of humans and mice. In recent years, several large population cohorts have used non-target metabolomics data to find that cinnamoyl glycine is a biomarker of gut health. However, there is currently no study exploring the effect of cinnamoyl glycine on metabolic associated fatty liver disease and its related disorders, nor is there any study experimentally demonstrating the improvement effect of cinnamoyl glycine on metabolic health or exploring its potential mechanism of action.

[0004] Current research on cinnamoyl glycine and its metabolic-related health outcomes is mostly based on population-based epidemiological studies, and mainly non-targeted metabolomics. The specific concentration range of cinnamoyl glycine is still unclear. There is no experimental study to prove the improvement effect of cinnamoyl glycine on metabolic health, nor any study to reveal the potential molecular mechanism by which cinnamoyl glycine improves host metabolic disorders. Summary of the Invention

[0005] In view of this, the present application provides the use of cinnamoyl glycine in the preparation of products for predicting, preventing or improving metabolic-related fatty liver disease and its related disorders, aiming to provide the use of cinnamoyl glycine in the prevention and / or treatment of metabolic-related fatty liver disease and its related disorders.

[0006] To achieve the above-mentioned invention purpose, the present application provides the following technical solutions: The present application provides the use of cinnamoyl glycine as a biomarker in the preparation of detection products for predicting the risk of metabolic-related fatty liver disease.

[0007] In some specific embodiments of the present application, for the above-mentioned prediction, the judgment rule includes: when the plasma cinnamoyl glycine concentration of an individual reaches or exceeds 8 ng / mL, it is regarded as a low-risk group, and when it is lower than 8 ng / mL, it is regarded as a high-risk group.

[0008] The present application also provides the use of cinnamoyl glycine in inhibiting the expression of liver FDPS and / or reducing the expression level of liver interleukin-1β.

[0009] The present application also provides the use of cinnamoyl glycine in reducing liver cholesterol, improving hepatic steatosis, improving liver lipid deposition, reducing hepatocyte ballooning and / or improving the biosynthesis of liver inflammation.

[0010] The present application also provides the use of cinnamoyl glycine in the prevention and / or treatment of metabolic-related fatty liver disease.

[0011] The present application also provides the use of cinnamoyl glycine in the preparation of drugs for preventing and / or treating metabolic-related fatty liver disease.

[0012] In some specific embodiments of the present application, the cinnamoyl glycine in the above-mentioned application prevents and / or treats metabolic-related fatty liver disease by improving liver lipid deposition.

[0013] In some specific embodiments of the present application, the cinnamoyl glycine in the above-mentioned application prevents and / or treats metabolic-related fatty liver disease by improving hepatic steatosis and hepatocyte ballooning.

[0014] In some specific embodiments of the present application, the cinnamoyl glycine in the above application prevents and / or treats metabolic associated fatty liver disease by reducing the expression level of interleukin-1β in the liver and improving liver inflammation.

[0015] In some specific embodiments of the present application, the cinnamoyl glycine in the above application prevents and / or treats metabolic associated fatty liver disease by inhibiting the expression of FDPS in the liver and reducing the biosynthesis of cholesterol in the liver.

[0016] In some specific embodiments of the present application, the metabolic associated fatty liver disease in the above application includes metabolic associated steatohepatitis.

[0017] The present application also provides a method for predicting, assisting in diagnosing, or diagnosing metabolic associated fatty liver disease based on cinnamoyl glycine; The cinnamoyl glycine may refer to the concentration of cinnamoyl glycine in serum or plasma; The metabolic associated fatty liver disease includes simple metabolic associated fatty liver and metabolic associated steatohepatitis.

[0018] The present application also provides a method for preventing and / or treating metabolic associated fatty liver disease based on cinnamoyl glycine.

[0019] The present application also provides a method for preventing and / or treating metabolic associated fatty liver disease based on a composition containing cinnamoyl glycine.

[0020] In some specific embodiments of the present application, the metabolic associated fatty liver disease in the above method includes simple metabolic associated fatty liver and metabolic associated steatohepatitis.

[0021] The present application also provides a detection product for predicting, assisting in diagnosing, or diagnosing metabolic associated fatty liver disease using cinnamoyl glycine as a marker, and the detection product includes a chip, a device, or a system; The metabolic associated fatty liver disease includes simple metabolic associated fatty liver and metabolic associated steatohepatitis.

[0022] The present application also provides a drug for treating metabolic associated fatty liver disease containing cinnamoyl glycine, and the drug includes acceptable excipients or adjuvants; The metabolic associated fatty liver disease includes simple metabolic associated fatty liver and metabolic associated steatohepatitis.

[0023] The present application also provides the use of cinnamoyl glycine in the preparation of a product for reducing body fat and / or reducing blood lipid.

[0024] In some specific embodiments of the present application, the reduction of body fat in the above application includes the reduction of liver fat.

[0025] In some specific embodiments of the present application, the reduction of blood lipids in the above application includes reducing blood triglycerides and / or reducing blood cholesterol.

[0026] In some specific embodiments of the present application, the product in the above application includes drugs or health products.

[0027] The present invention has the following beneficial effects: (1) In a community natural population cohort, the application of the gut microbiota metabolite cinnamoyl glycine as a biomarker for metabolic associated fatty liver disease and metabolic associated steatohepatitis was evaluated, and the plasma cinnamoyl glycine concentration can effectively predict the risk of metabolic associated fatty liver disease and high-risk metabolic associated steatohepatitis.

[0028] (2) Cinnamoyl glycine helps to reduce blood lipids (cholesterol / triglycerides) and body fat, can effectively improve lipid metabolism disorders and metabolic associated fatty liver disease in mice fed with high-fat and high-cholesterol diets, and reduce liver triglyceride and cholesterol accumulation, improve inflammation and hepatocyte ballooning degeneration.

[0029] (3) Cinnamoyl glycine improves metabolic associated fatty liver disease and its related conditions by inhibiting the expression and activity of liver farnesyl diphosphate synthase (FDPS), and reducing liver cholesterol biosynthesis. Description of the Drawings

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0031] Figure 1 Shows the risk stratification concentration of plasma cinnamoyl glycine for metabolic associated fatty liver disease in the discovery cohort; Figure 2 Shows the prediction of plasma cinnamoyl glycine concentration for the risk of metabolic associated fatty liver disease and metabolic associated steatohepatitis in the internal prospective validation cohort, where A shows the controlled attenuation parameter (CAP), and B shows the FibroScan-AST score (FAST); Figure 3 Shows the stratification ability of plasma cinnamoyl glycine concentration for the incidence risk of metabolic associated fatty liver disease in the external prospective validation cohort; Figure 4 Shows the metabolic associated fatty liver disease mouse model induced by high-fat and high-cholesterol diet and the intervention grouping, where STC refers to standard control diet, HFHC refers to high-fat and high-cholesterol diet, PBS refers to phosphate buffer solution, and CMG refers to cinnamoyl glycine; Figure 5Comparison of body weights and blood glucose levels of each group of mice. Among them, A represents body weight, B represents fasting blood glucose, and C represents postprandial blood glucose; Figure 6 Comparison of blood lipid levels of each group of mice. Among them, A represents serum triglyceride (TG) level, B represents serum total cholesterol (TC) level, C represents serum high-density lipoprotein cholesterol (HDL-c) level, and D represents serum low-density lipoprotein cholesterol (LDL-c) level; Figure 7 Comparison of liver lipid levels of each group of mice. Among them, A represents liver triglyceride content and B represents liver total cholesterol content; Figure 8 Showing the liver morphology and serum alanine aminotransferase level of each group of mice. Among them, A represents liver morphology and B represents serum alanine aminotransferase level; Figure 9 Showing liver tissue sections (H&E staining) of each group of mice; Figure 10 Showing the semi-quantitative evaluation of the H&E staining results of the liver of each group of mice. Among them, A represents the liver steatosis score, B represents the inflammatory infiltration score, C represents the ballooning degeneration score, and D represents the total NAS score; Figure 11 Showing the Filipin staining of the liver and the comparison of liver cholesterol content of each group of mice. Among them, A represents the Filipin staining result and B represents the relative intensity of Filipin; Figure 12 Comparison of FDPS expression and activity in each group of mice. Among them, A represents the mRNA expression level of Fdps, B represents the FDPS protein expression level, and C represents the FDPS enzyme activity; Figure 13 Showing the comparison of interleukin-1β expression in the liver of each group of mice. Among them, A represents the mRNA expression level of interleukin-1β, B represents the immunohistochemical image, and C represents the positive area of interleukin-1β; Detailed implementation mode

[0032] The present application discloses the use of cinnamoyl glycine in the preparation of products for predicting, preventing or improving metabolic associated fatty liver disease and its related disorders. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those related can obviously make changes or appropriate alterations and combinations to 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.

[0033] It should be understood that the expression "one or more of..." individually includes each of the recited objects following said expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0034] The terms "comprising", "having", or "including", including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unrecited elements or steps, unless specifically stated otherwise or otherwise understood from the context.

[0035] It should be understood that as long as the present application remains operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be performed simultaneously.

[0036] The use of any and all examples or exemplary language in this document, such as "for example" or "including", is merely intended 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 that any unclaimed element is essential for the practice of the present application.

[0037] In addition, the numerical ranges and parameters used to define the present application are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any numerical value inherently and inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, numerical values, and percentages used in this disclosure are modified by "about". Herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific numerical value or range.

[0038] Based on the limitations of the research progress described in the background art, the object of the present invention is to specifically address the current situation that there is no effective treatment drug for metabolic associated fatty liver disease, and to provide an application of cinnamoyl glycine in the prevention and / or treatment of metabolic associated fatty liver disease and its related disorders. Cinnamoyl glycine can effectively predict the risks of metabolic associated fatty liver disease and high-risk metabolic associated steatohepatitis in the community population; to provide an intervention plan for cinnamoyl glycine, which can effectively reduce the body weight, triglyceride, and cholesterol levels of mice induced by high-fat and high-cholesterol diet, and improve liver lipid deposition, inflammatory cell infiltration, and hepatocyte ballooning; to provide an application of inhibiting the expression and activity of FDPS in the improvement effect of cinnamoyl glycine-mediated metabolic associated fatty liver disease, which provides new effective intervention ideas and means for metabolic associated fatty liver disease and its related disorders.

[0039] Some terms involved in this application are explained as follows: Metabolic associated fatty liver disease: Metabolic associated fatty liver disease, formerly known as non-alcoholic fatty liver disease, is a liver disease with multi-system metabolic disorders. Metabolic associated fatty liver disease was proposed by an international expert panel in 2020 and has been recognized and supported by the Asian-Pacific Association for the Study of the Liver and the Chinese Liver Disease Society of the Chinese Medical Association. The definition of metabolic associated fatty liver disease is the presence of hepatic steatosis, accompanied by overweight / obesity, type 2 diabetes, or metabolic dysfunction.

[0040] Hepatic steatosis: Hepatic steatosis is the core pathological feature of metabolic associated fatty liver disease. It is defined as the abnormal accumulation of lipids such as triglycerides in hepatocytes, with a content exceeding 5% of the liver weight, or more than 5% of hepatocytes showing lipid droplets histologically.

[0041] Metabolic associated fatty hepatitis: Metabolic associated fatty hepatitis, formerly known as non-alcoholic fatty hepatitis, is the progressive stage of metabolic associated fatty liver disease. It is characterized by the combination of lobular inflammation and hepatocyte ballooning on the basis of hepatic steatosis, with or without fibrosis. Metabolic associated fatty hepatitis can further progress to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma.

[0042] High-risk metabolic associated fatty hepatitis: High-risk metabolic associated fatty hepatitis refers to a clinical subtype among patients with metabolic associated fatty hepatitis who have a high risk of rapidly progressing 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 features of metabolic associated fatty hepatitis.

[0043] Hepatic lobular inflammation: Hepatic lobular inflammation is one of the key histological features of metabolic associated fatty hepatitis, manifested as the infiltration of inflammatory cells mainly composed of lymphocytes in the hepatic lobular parenchyma, often accompanied by hepatocyte ballooning and focal necrosis and other hepatocyte injury changes.

[0044] Hepatocyte ballooning: Hepatocyte ballooning is a special morphological manifestation of hepatocyte injury. It is characterized by a significant increase in the volume of hepatocytes, cytoplasmic rarefaction, and a "balloon-like" transparency under light microscopy, often accompanied by the formation of Mallory-Denk bodies in the cytoplasm. Hepatocyte ballooning is a necessary histological criterion for the diagnosis of metabolic associated fatty hepatitis and is used to evaluate the disease activity of MASH.

[0045] Cinnamoyl glycine: Cinnamoyl glycine is a derivative formed by the combination of cinnamic acid and glycine, and is produced by the phenylalanine / cinnamic acid metabolic pathway mediated by gut microbiota. As an endogenous metabolite in the human body, it can be detected in blood circulation and urine. In this application, the cinnamoyl glycine used is from Shanghai Yuanye Bio-Technology Co., Ltd. (S83815).

[0046] Farnesyl diphosphate synthase: Farnesyl diphosphate synthase (FDPS; also known as farnesyl pyrophosphate synthase, FPPS) is an enzyme (EC 2.5.1.10) encoded by the FDPS gene. As a key enzyme in the cholesterol biosynthesis pathway, FDPS catalyzes the formation of farnesyl diphosphate, an important precursor of cholesterol.

[0047] Unless otherwise specified, the raw materials, reagents, consumables, and instruments involved in this application are all ordinary commercially available products and can be purchased from the market.

[0048] The present invention will be further described below in conjunction with embodiments.

[0049] Example 1 In this example, the plasma cinnamoyl glycine content of the community population was quantitatively detected, and it was determined that cinnamoyl glycine can effectively predict the risk of future metabolic associated fatty liver disease and high-risk metabolic associated steatohepatitis in the community population. Specifically, it includes the following steps: (1) Collection of community population cohort data and diagnosis of metabolic associated fatty liver disease Using a prospective cohort study of chronic non-communicable diseases - omics sub-cohort of the natural population in a certain region as the discovery cohort, 1,381 research subjects who received questionnaire surveys, physical examinations, and biochemical tests at baseline were included. The fatty liver index was used to evaluate hepatic steatosis. The higher the fatty liver index, the greater the likelihood of an individual having hepatic steatosis. Further, 1,014 research subjects who received vibration-controlled transient elastography examinations during the follow-up period with an average follow-up time of 3.65 years were included as the internal prospective validation cohort. The controlled attenuation parameter (CAP) was used to evaluate liver lipid content, and the higher it was, the higher the degree of lipid accumulation; the FibroScan-AST score (FAST score) was used to evaluate the risk of high-risk metabolic associated steatohepatitis, and the higher the score, the greater the risk.

[0050] Using a community population cohort - sub-cohort as the validation cohort, 386 research subjects who did not have metabolic associated fatty liver disease by abdominal ultrasound examination at baseline and underwent abdominal ultrasound examination again during the follow-up period with an average follow-up time of 2.25 years were included. Abdominal ultrasound examination was completed using a color Doppler ultrasound diagnostic system to evaluate hepatic steatosis, which was defined as a diffuse increase in fine echoes in the liver parenchyma compared to the renal or splenic parenchyma.

[0051] Basic information such as gender, age, smoking, drinking, diet, physical activity, past medical history, and medication history was collected using a standardized questionnaire. Fasting blood samples were collected by trained staff or nurses, and physical examinations were completed, including height, weight, waist circumference, blood pressure, etc. Biochemical tests such as liver function, kidney function, blood lipids, and blood glucose were completed by laboratory technicians. Incident cases of baseline hypertension and diabetes were diagnosed based on self-reported medical history, medication history, or baseline measurements.

[0052] (2)Targeted detection of plasma cinnamoyl glycine concentration Take 100 µL of population plasma for sample pretreatment. [2,2- 2 H2]-N-trans-Cinnamoylglycine (IsoReag) was used as the internal standard, and the content of cinnamoyl glycine in population plasma was quantitatively detected by liquid chromatography-mass spectrometry (LC-MS, Agilent 6495, Agilent).

[0053] (3)Prediction of the risk of metabolic associated fatty liver disease and metabolic associated steatohepatitis by plasma cinnamoyl glycine concentration ① Determine the risk stratification concentration of plasma cinnamoyl glycine for metabolic associated fatty liver disease Among the 1,381 study subjects in the discovery cohort, the association between plasma cinnamoyl glycine concentration and fatty liver index was evaluated. The restricted cubic spline regression model found that the plasma cinnamoyl glycine concentration was nonlinearly associated with the fatty liver index (nonlinear test P = 0.003), and 8 ng / mL was the critical threshold concentration. Before this threshold point, the regulatory effect of cinnamoyl glycine on the fatty liver index was relatively significant, while after exceeding this concentration, the effect tended to level off, as Figure 1 shown.

[0054] Taking 8 ng / mL as the risk judgment criterion, when the plasma cinnamoyl glycine concentration of an individual reaches or exceeds 8 ng / mL, it is regarded as low risk, and when it is lower than 8 ng / mL, it is regarded as a high-risk population.

[0055] ② Prediction of the risk of metabolic associated fatty liver disease and metabolic associated steatohepatitis by plasma cinnamoyl glycine concentration Among the 1,014 study subjects in the internal prospective validation cohort, the association between baseline plasma cinnamoyl glycine concentration and follow-up liver fat content and high-risk metabolic associated steatohepatitis was evaluated. After adjusting for potential confounding effects such as age, gender, smoking, drinking, diet diversity, physical activity, medication history, diabetes, hypertension, obesity, and estimated glomerular filtration rate, the baseline plasma cinnamoyl glycine concentration was significantly negatively correlated with the degree of liver lipid accumulation during the follow-up period (r = -0.125, P adj = 0.001), as Figure 2as shown in A of adj = 0.013), as shown in Figure 2 B of

[0056] Furthermore, in 386 subjects of an external prospective validation cohort, the ability of baseline plasma cinnamoyl glycine concentration to stratify the risk of developing metabolic associated fatty liver disease was evaluated. The subjects were divided into two groups according to whether the baseline plasma cinnamoyl glycine concentration was ≥ 8 ng / mL. Kaplan-Meier survival analysis was used to compare the cumulative incidence risks of metabolic associated fatty liver disease in the groups with higher or lower plasma cinnamoyl glycine concentration during an average follow-up period of 2.25 years, and the log-rank test was used to determine that the subjects with higher plasma cinnamoyl glycine concentration had a lower risk of developing metabolic associated fatty liver disease (P = 0.007). See Figure 3 , starting from 500 days of follow-up, the two risk curves and their 95% confidence interval bands separated from each other and did not overlap, and the curve of the lower concentration group was higher than that of the higher concentration group at each time point, indicating that the group with a higher baseline plasma cinnamoyl glycine concentration had a lower risk of developing metabolic associated fatty liver disease. Compared with the group with a lower baseline plasma cinnamoyl glycine concentration, the risk of developing metabolic associated fatty liver disease in the subjects with a baseline plasma cinnamoyl glycine concentration ≥ 8 ng / mL was reduced by 59% (hazard ratio HR = 0.41, 95% CI: 0.18 - 0.94).

[0057] Example 2: Application of inhibiting FDPS expression and activity in the improvement effect of cinnamoyl glycine-mediated metabolic associated fatty liver disease In this example, in vivo animal experiments were used to verify the improvement effect of cinnamoyl glycine intervention on metabolic associated fatty liver disease and its related conditions. The potential molecular mechanism by which cinnamoyl glycine reduces hepatic cholesterol biosynthesis by inhibiting hepatic FDPS expression and activity was further revealed. All the above experiments were carried out on the premise of ensuring animal welfare and experimental guidelines. Specifically, the experimental steps include: (1) Construction of a mouse model of metabolic associated fatty liver disease induced by a high-fat and high-cholesterol diet and intervention grouping Eight-week-old SPF male C57BL / 6J mice were randomly divided into three groups of eight mice each and housed in an SPF-class experimental animal center. They were allowed free access to food and water, with a 12-hour light / dark cycle, and the room temperature was controlled at 23°C ± 2°C. One group was fed a standard control diet (STC) and gavaged with sterile PBS daily, designated as the Ctrl group; the other two groups were fed a high-fat and high-cholesterol diet (HFHC) and gavaged with sterile PBS and cinnamoyl glycine (CMG, 0.2 mg / kg body weight, simulating a concentration of 8 ng / mL in serum) daily, designated as the HFHC group and the CMG group, respectively. The high-fat and high-cholesterol diet formula consisted of 16.9% protein, 40.3% fat (including 0.2% cholesterol), and 42.8% carbohydrates. The above interventions continued for 12 weeks, as Figure 4 shown.

[0058] The food intake and body weight of the mice were regularly recorded every week. After the intervention ended, the mouse serum and liver tissues were collected to detect the blood glucose and blood lipid levels. Hematoxylin-eosin (H&E) staining was used to evaluate the pathological manifestations of the mouse liver, including hepatic steatosis, inflammation, and ballooning degeneration. Filipin staining was used to detect the cholesterol content in the liver. Real-time fluorescence quantitative PCR was used to detect gene expression, Western Blot was used to detect the FDPS protein expression level, and immunohistochemistry was used to detect the interleukin-1β level.

[0059] (2)Cinnamoyl glycine alleviates weight gain in mice with metabolic-associated fatty liver disease and has no significant effect on glucose metabolism After 12 weeks of intervention, compared with the Ctrl group, the body weight of the mice in the HFHC group increased significantly (P < 0.001), indicating that cinnamoyl glycine intervention could alleviate the weight gain induced by a high-fat and high-cholesterol diet, as shown in Figure 5 A. Cinnamoyl glycine intervention had no significant effect on glucose metabolism in mice with metabolic-associated fatty liver disease (P > 0.05), as shown in Figure 5 B and C.

[0060] (3)Cinnamoyl glycine improves lipid metabolism disorders in mice with metabolic-associated fatty liver disease Lipid metabolism disorder is the core pathogenesis of metabolic-associated fatty liver disease. After the intervention ended, the serum of each group of mice after 14 hours of fasting was collected and the levels of four blood lipids were detected. As Figure 6 shown, compared with the mice in the HFHC group, the serum triglyceride and total cholesterol levels of the mice intervened with cinnamoyl glycine for 12 weeks (CMG group) were significantly reduced by 26% and 9% (see Figure 6 A, B, 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).

[0061] Further collect the liver tissue homogenates of each group of mice and detect the liver lipid levels. As Figure 7 shown in A and B of Figure 7 , after 12 weeks of high-fat and high-cholesterol diet feeding, the contents of triglyceride and total cholesterol in the livers of mice in the HFHC group were significantly higher than those in the Ctrl group (P<0.05), indicating liver lipid deposition. Compared with the mice in the HFHC group, the contents of triglyceride and total cholesterol in the livers of mice intervened with cinnamoyl glycine (CMG group) were significantly reduced by 20% and 24% respectively, and the difference was statistically significant (P<0.05).

[0062] The above results indicate that cinnamoyl glycine intervention helps to reduce blood lipids (cholesterol / triglyceride), can effectively improve hypertriglyceridemia and hypercholesterolemia induced by high-fat and high-cholesterol diet, and reduce liver lipid deposition, which helps to reduce body fat.

[0063] (4) Cinnamoyl glycine safely and effectively improves hepatic steatosis, inflammation and hepatocyte ballooning in mice with metabolic associated fatty liver disease As Figure 8 shown, after 12 weeks of high-fat and high-cholesterol diet feeding, the livers of mice showed typical characteristics of metabolic associated fatty liver disease, including that the gross morphology of the livers of mice in the HFHC group presented yellowish-white and enlarged (see A in Figure 8 ), and the serum alanine aminotransferase was significantly higher than that in the Ctrl group (see B in Figure 8 , P<0.05). Compared with the mice in the HFHC group, cinnamoyl glycine intervention for 12 weeks significantly improved the gross morphology of the livers of mice (see A in Figure 8 , CMG group), and significantly reduced the level of serum alanine aminotransferase (see B in Figure 8 ), and the difference was statistically significant (P<0.05).

[0064] Further observe the pathological characteristics of mouse liver tissue sections by H&E staining. As Figure 9 shown, compared with the pathological characteristics of typical metabolic associated fatty liver disease in the HFHC group, the range of hepatic steatosis in the mice intervened with cinnamoyl glycine (CMG group) was reduced, the number of lobular inflammatory foci was decreased, and the number of ballooned hepatocytes was reduced.

[0065] Semi-quantitatively evaluate the H&E staining results of the livers of each group of mice by NAS scoring system. As Figure 10 shown, compared with the Ctrl group, the scores of hepatic steatosis (see A in Figure 10 ), inflammatory infiltration (see B in Figure 10 ) and ballooning (see C in Figure 10 ) in the livers of mice in the HFHC group, and the total NAS score (see Figure 10In D), they were all significantly increased (P<0.001), indicating that the high-fat and high-cholesterol diet successfully induced a mouse model of metabolic associated fatty liver disease. Compared with the HFHC group, the total NAS score and the sub-items of hepatocyte steatosis, lobular inflammation and ballooning degeneration in the 12-week cinnamoyl glycine intervention (CMG group) were all significantly decreased, and the differences were statistically significant (P<0.05).

[0066] The above results indicate that cinnamoyl glycine can effectively improve metabolic associated fatty liver disease and its related diseases, has a certain protective effect on liver function, and has good safety.

[0067] (5) Cinnamoyl glycine inhibits FDPS expression and activity, reducing hepatic cholesterol biosynthesis To further confirm the effect of cinnamoyl glycine on hepatic cholesterol synthesis, Filipin staining was performed on liver tissue sections of mice in each group. As Figure 11 shown in A and B, compared with the Ctrl group, the content of free cholesterol in the liver of HFHC group mice was significantly increased (P<0.001), suggesting enhanced hepatic cholesterol synthesis induced by the high-fat and high-cholesterol diet. Compared with the HFHC group mice, the degree of hepatic cholesterol accumulation in the mice in the 12-week cinnamoyl glycine intervention group was significantly improved, and the content decreased by 15% (P<0.05), that is, cinnamoyl glycine inhibits hepatic cholesterol biosynthesis in mice with metabolic associated fatty liver disease.

[0068] At the transcriptional level, real-time fluorescence quantitative PCR found that cinnamoyl glycine significantly down-regulated the mRNA expression level of the key gene Fdps involved in cholesterol biosynthesis (P<0.05), as shown in Figure 12 A. The protein expression level of FDPS in the liver of mice was detected by Western Blot. As Figure 12 shown in B, after 12 weeks of high-fat and high-cholesterol diet intervention, compared with the Ctrl group, the expression level of FDPS in the liver of HFHC group mice was significantly increased by 53% (P<0.05). Compared with the HFHC group mice, 12-week cinnamoyl glycine intervention could reduce the expression level of FDPS in the liver of mice by 47%, and the difference was statistically significant (P<0.05). The FDPS enzyme activity in the liver of mice in each group was detected by the phosphorus determination colorimetric method. As Figure 12 shown in C, 12-week cinnamoyl glycine intervention could reduce the hepatic FDPS enzyme activity promoted by the high-fat and high-cholesterol diet. Compared with the HFHC group mice, the hepatic FDPS enzyme activity in the cinnamoyl glycine intervention group mice was significantly decreased by 72% (P<0.05).

[0069] The above results further prove that cinnamoyl glycine improves metabolic associated fatty liver disease by inhibiting hepatic FDPS expression and reducing hepatic cholesterol biosynthesis.

[0070] (6)Cinnamoyl glycine improves liver inflammatory response in mice with metabolic associated fatty liver disease In Example 1, cinnamoyl glycine could effectively predict the risk of high-risk metabolic associated steatohepatitis. In this example, H&E staining found that cinnamoyl glycine could improve the lobular inflammation of the liver in mice with metabolic associated fatty liver disease. Further, the transcriptional expression level of interleukin-1β (Il-1β) in the liver of mice was detected by real-time fluorescence quantitative PCR, and it was found that cinnamoyl glycine significantly reduced the mRNA expression level of Il-1β in the liver of mice with metabolic associated fatty liver disease (P<0.05), as shown in A of Figure 13 . Immunohistochemical analysis found that compared with the HFHC group, 12-week cinnamoyl glycine intervention significantly reduced the expression level of IL-1β in the liver of mice, as shown in B and C of Figure 13 , further indicating that cinnamoyl glycine can improve the liver inflammatory response in mice with metabolic associated fatty liver disease.

[0071] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of this application.

Claims

1. Use of cinnamoyl glycine as a biomarker in the preparation of a detection product for predicting the risk of metabolic associated fatty liver disease.

2. Use of cinnamoyl glycine in the preparation of a drug for preventing and / or treating metabolic associated fatty liver disease.

3. The application according to claim 2, wherein The cinnamoyl glycine prevents and / or treats metabolic associated fatty liver disease by improving liver lipid deposition.

4. The application according to claim 2, characterized in that The cinnamoyl glycine prevents and / or treats metabolic associated fatty liver disease by improving hepatic steatosis and ballooning degeneration of hepatocytes.

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

6. The application according to claim 2, characterized in that, The cinnamoyl glycine prevents and / or treats metabolic associated fatty liver disease by inhibiting the expression of FDPS in the liver and reducing the biosynthesis of liver cholesterol.

7. The application according to any one of claims 1 to 6, characterized in that, The metabolic associated fatty liver disease includes simple metabolic associated fatty liver and / or metabolic associated steatohepatitis.

8. Use of cinnamoyl glycine in the preparation of a product for reducing body fat and / or reducing blood lipid.

9. The application according to claim 8, characterized in that, The reduction of body fat includes the reduction of liver fat.

10. The application according to claim 8, wherein The reduction of blood lipid includes the reduction of blood triglyceride and / or the reduction of blood cholesterol.

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