Endogenous substances serving as biomarkers of alcohol-related liver diseases and prevention and treatment application of endogenous substances

Abstract

The invention belongs to the technical field of biological medicine, and relates to a novel application of endogenous substances in alcohol related liver disease (ALD). The invention proposes for the first time that an endogenous substance with regular concentration change in the ALD process can be used as a biomarker for auxiliary diagnosis and / or stage evaluation of ALD, the exogenous supplement of the endogenous substance has a prevention and treatment effect on ALD, and the endogenous substance has double values of being used as an excellent biomarker and an effective therapeutic agent. The substances include but are not limited to taurochenodeoxycholic acid (TCDCA), dehydroepiandrosterone sulfate (DHEA-S) and the like. Wherein the serum concentration of TCDCA is obviously increased along with disease progression, and the concentration of DHEA-S is obviously reduced. The diagnostic value of the substance on ALD is remarkably superior to that of a traditional liver injury marker, and ethanol-induced liver cell injury and mouse liver pathological change can be remarkably improved through exogenous supplementation. The invention provides a brand new strategy for accurate diagnosis and prevention of ALD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. It relates to a class of endogenous substances that exhibit regular concentration changes during the progression of alcohol-related liver disease, which can serve as biomarkers for the auxiliary diagnosis and / or staging assessment of alcohol-related liver disease. Exogenous supplementation of these substances has a preventive and therapeutic effect on alcohol-related liver disease. Background Technology

[0002] Alcoholic Liver Disease (ALD) is a group of chronic liver diseases caused by long-term excessive alcohol consumption. Its spectrum includes alcoholic fatty liver, alcoholic hepatitis, liver fibrosis, cirrhosis, and even hepatocellular carcinoma, and it has become a significant global public health problem. In China, the disease burden caused by ALD is particularly prominent, making it imperative to address this challenge.

[0003] However, the current clinical management of ALD still faces two major challenges and unmet needs: First, there is a lack of highly sensitive and specific biomarkers for early diagnosis and accurate staging. Clinically, apart from detailed questioning about alcohol consumption history, there is a lack of objective and specific biomarkers to effectively determine whether liver damage is caused by alcohol. Currently, the diagnosis of ALD primarily relies on confirming a long history of excessive alcohol consumption. However, patients often conceal or underestimate their alcohol intake due to psychosocial factors, leading to inaccurate history taking. More importantly, there is currently no blood test indicator that can serve as a specific serological marker to definitively indicate that liver damage originates from alcohol. This makes it necessary for doctors to rule out other liver diseases (such as non-alcoholic fatty liver disease, viral hepatitis, etc.) during etiological differentiation, making the diagnostic process complex and uncertain. Currently, clinically relied-upon liver function biochemical indicators (such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) are not sensitive to early ALD and lack specificity, making it difficult to accurately distinguish between simple fatty liver and high-risk states that are about to progress to hepatitis and cirrhosis. Imaging examinations have limited effectiveness in assessing early fibrosis and inflammation. This directly leads to a large number of subclinical or early-stage ALD patients being missed, thus missing the optimal intervention window. Second, targeted therapies are extremely scarce. Although abstinence from alcohol is the cornerstone of ALD treatment, patient compliance is poor, and for significant liver damage already established, abstinence alone has limited effectiveness. Currently, there are no approved targeted therapies specifically targeting the key pathogenesis of ALD worldwide. Clinical treatment options are very limited, mainly relying on glucocorticoids (for severe alcoholic hepatitis) and symptomatic supportive care, with unsatisfactory efficacy and significant side effects. Therefore, developing novel biomarkers for the early diagnosis / staging of ALD, as well as safe and effective preventive and therapeutic drugs, is a major challenge and urgent need in clinical practice.

[0004] Existing research largely focuses on exogenous compounds or single known pathways, lacking the ability to discover targets with both diagnostic and therapeutic value from the perspective of systemic changes in endogenous substances. In particular, there are no systematic reports on using the dynamic changes of endogenous substances in ALD as both biomarkers and preventive drugs. Summary of the Invention

[0005] In view of this, the present invention provides a new use for a class of endogenous substances. The core of the invention lies in proposing and validating an innovative concept: endogenous substances that exhibit significant and regular concentration changes during the progression of ALD represent a valuable resource for discovering "dual-function" molecules with both excellent diagnostic / staging value and effective therapeutic potential. Based on this, the present invention specifically provides the application of these substances as ALD biomarkers and preventative drugs, offering a novel strategy and material basis for the accurate diagnosis and effective prevention of ALD.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of a class of endogenous substances that undergo regular changes in the progression of alcoholic liver disease (ALD) as biomarkers for the auxiliary diagnosis and / or staging assessment of ALD.

[0008] In some embodiments of the present invention, the above application is achieved by detecting the concentration of the endogenous substance in a subject's blood sample.

[0009] In some embodiments of the present invention, the above-described application, the regular changes include a significant increase or decrease in concentration as the severity of the disease increases.

[0010] In some embodiments of the present invention, the endogenous substances described above include bile acids and steroid hormones.

[0011] In some embodiments of the present invention, the bile acid described above is taurine chenodeoxycholic acid (TCDCA).

[0012] In some embodiments of the present invention, the steroid hormone described above is dehydroepiandrosterone sulfate (DHEA-S).

[0013] This invention also provides the use of a class of endogenous substances that undergo regular changes in the ALD process in the preparation of medicaments for the prevention and / or treatment of ALD.

[0014] In some embodiments of the present invention, the endogenous substances described above include bile acids and steroid hormones.

[0015] In some embodiments of the present invention, the bile acid described above is taurine chenodeoxycholic acid (TCDCA).

[0016] In some embodiments of the present invention, the steroid hormone described above is dehydroepiandrosterone sulfate (DHEA-S).

[0017] The beneficial effects of this invention include:

[0018] This invention is the first to propose and verify that a class of endogenous substances can serve as a common source of ALD biomarkers and preventive drugs, opening up new research and development directions. The discovered biomarkers (such as TCDCA and DHEA-S) have the characteristics of high diagnostic accuracy, strong correlation with disease severity, and the ability to assist in disease staging. It is the first to demonstrate that exogenous supplementation of such substances can exert a clear hepatoprotective effect through a multi-target mechanism. The active ingredients are endogenous substances with wide availability and good safety, and have great potential to be developed into diagnostic reagents and therapeutic drugs. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0020] Figure 1 Example 1 illustrates the correlation between human serum TCDCA levels and clinical indicators, as well as their diagnostic and staging value. In the figure: A represents the TCDCA content in human serum; B represents the Spearman correlation analysis between human TCDCA levels and ALD severity; CH represents the correlation analysis between human TCDCA levels and traditional clinical liver injury indicators ALT, AST, AST / ALT, TBIL, ALB, and PLT; I represents the ROC curve of TCDCA distinguishing between healthy individuals and ALD patients (hepatitis, cirrhosis, and liver cancer); J represents the comparison of ROC curves of TCDCA and traditional liver injury indicators AST, AST / ALT, TBIL, ALB, and PLT distinguishing between healthy individuals and ALD patients (cirrhosis and liver cancer) under the same conditions; K represents the ROC curve of TCDCA distinguishing between patients with alcoholic hepatitis and alcoholic cirrhosis; L represents the ROC curve of TCDCA distinguishing between patients with alcoholic liver cancer. In the figure, * indicates significance analysis with the healthy group, * represents p<0.05, and ** represents p<0.01.

[0021] Figure 2Example 1 illustrates the correlation between human serum DHEA-S levels and clinical indicators, as well as their diagnostic and staging value. In this example: A represents the DHEA-S content in human serum; B represents the Spearman correlation analysis between DHEA-S levels and ALD severity; CD represents the correlation analysis between DHEA-S levels and traditional clinical liver injury indicators ALB and PLT; E represents the ROC curve of DHEA-S distinguishing between healthy individuals and ALD patients (hepatitis, cirrhosis, liver cancer); F represents the comparison of ROC curves of DHEA-S and traditional liver injury indicators AST, AST / ALT, TBIL, ALB, and PLT distinguishing between healthy individuals and ALD patients (cirrhosis, liver cancer) under the same conditions; G represents the ROC curve of DHEA-S distinguishing between patients with alcoholic hepatitis and alcoholic cirrhosis; H represents the ROC curve of DHEA-S distinguishing between patients with alcoholic liver cancer. *** in the figure represents p<0.001.

[0022] Figure 3 This figure shows the cell viability and related gene expression of AML12 hepatocytes with acute ethanol injury in Example 2, where: A represents the cell viability of AML12 hepatocytes with acute ethanol injury after TCDCA intervention; BD represents the mRNA expression levels of Nrf2, Tnf-α, and Fas in AML12 hepatocytes with acute ethanol injury after TCDCA intervention. In the figure, # represents the comparison between the control group and the model group; * represents the comparison between the TCDCA intervention group and the model group; # represents p < 0.05, ### represents p < 0.001, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0023] Figure 4 This figure shows the cell viability and related gene expression of AML12 hepatocytes with acute ethanol injury in Example 2. A represents the cell viability of AML12 hepatocytes with acute ethanol injury treated with DHEA-S; BC represents the mRNA expression levels of Tnf-α and Fas in AML12 hepatocytes with acute ethanol injury treated with DHEA-S. In the figure, # indicates the comparison between the control group and the model group; * indicates the comparison between the DHEA-S intervention group and the model group; # represents p < 0.05, ### represents p < 0.001, * represents p < 0.05, and ** represents p < 0.01.

[0024] Figure 5 The image shows serum biochemical indicators and liver section staining images of mice with acute alcohol-related liver injury in Example 3. AC represents the serum ALT, AST, and TG levels of Gao-binge mice; D represents the Oil Red O staining image of mouse liver; * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0025] Figure 6The image shows serum biochemical indicators and liver section staining images of mice with acute alcohol-related liver injury in Example 3. AC represents the serum ALT, AST, and TG levels of Gao-binge mice; D represents the Oil Red O staining image of mouse liver; * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[0026] All data are expressed as mean ± SEM. One-way ANOVA was performed on the experimental data, and p < 0.05 was considered statistically significant. Detailed Implementation

[0027] This invention discloses a class of endogenous substances that exhibit regular concentration changes during the progression of alcohol-related liver disease.

[0028] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0029] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0030] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0031] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0032] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0033] Firstly, this invention provides a class of endogenous substances that exhibit regular changes during the progression of ALD, which can be used as biomarkers in the auxiliary diagnosis and / or staging assessment of ALD. The "regular changes" encompass various patterns, such as significant increases or decreases in concentration as the disease progresses. These substances include, but are not limited to, taurine chenodeoxycholic acid (TCDCA) and dehydroepiandrosterone sulfate (DHEA-S). While their trends are opposite, both are closely related to disease severity, demonstrating the diversity and application potential of this class of biomarkers.

[0034] As a representative of this class of substances, TCDCA shows a significant positive correlation in the progression of ALD, demonstrating excellent diagnostic and staging potential. This invention, for the first time through clinical sample analysis, reveals a significant positive correlation between serum TCDCA levels and the severity of ALD. Further evaluation confirms that TCDCA has excellent diagnostic value for ALD, with diagnostic efficacy significantly superior to commonly used traditional liver injury markers. Simultaneously, TCDCA demonstrates good ability to distinguish early stages of the disease. Therefore, detecting the concentration of TCDCA in the blood of subjects can be used to assess an individual's risk of developing ALD, provide high-precision diagnosis, or assist in assessing the stage of disease progression.

[0035] As another representative of this class of substances, DHEA-S shows a significant negative correlation in the progression of ALD and demonstrates unique value in assessing liver function. Clinical studies have found a significant negative correlation between serum DHEA-S levels and ALD severity. Importantly, DHEA-S levels are significantly positively correlated with key clinical indicators reflecting liver synthetic function, suggesting it is a biomarker closely related to liver functional reserve. DHEA-S has good diagnostic efficacy for ALD, and its diagnostic value is superior to many traditional liver injury markers. Particularly noteworthy is the extremely high accuracy of DHEA-S in distinguishing early stages of the disease, showing great potential for predicting early disease progression. Therefore, detecting serum DHEA-S concentration, in addition to its use in auxiliary diagnosis, is particularly suitable for assessing patients' liver function and the risk of early disease progression.

[0036] The above findings reflect the pathophysiological changes of ALD from different perspectives. They not only verify the universality of "regularly changing endogenous substances" as biomarkers, but also provide a combination of indicators that can be used in combination and complement each other. Therefore, detecting the concentration of such endogenous substances in the blood of subjects can be used to assess an individual's risk of having ALD, diagnose patients, or assist in assessing their disease stage and liver function status.

[0037] Secondly, the present invention provides the use of such endogenous substances in the preparation of medicaments for the prevention and / or treatment of ALD.

[0038] Based on the above clinical findings, this invention further confirms through in vitro and in vivo experiments that exogenous supplementation with such substances (such as TCDCA or DHEA-S) has a clear ameliorative effect on ALD. Specifically: In vitro, both TCDCA and DHEA-S can significantly rescue ethanol-induced decline in hepatocyte viability and regulate the expression of genes related to inflammation and lipid metabolism. In vivo, both can significantly reduce liver pathological indicators (such as serum AST, ALT, and TG levels) in a mouse model of alcohol-related liver injury and effectively improve pathological damage and lipid deposition in liver tissue.

[0039] Therefore, this invention not only provides the application of TCDCA and DHEA-S as specific drugs, but more importantly, establishes a new drug discovery strategy: supplementing depleted beneficial endogenous substances in the disease (such as DHEA-S), or regulatoryally supplementing compensatorily elevated beneficial substances to activate protective mechanisms (such as TCDCA), can both become effective treatments for ALD. These endogenous substances can be used to prepare pharmaceutical compositions for preventing the occurrence of ALD, delaying its progression, or treating ALD.

[0040] Based on the second aspect of application, the present invention provides a pharmaceutical composition for the prevention and / or treatment of ALD, comprising an effective dose of the endogenous substance and a pharmaceutically acceptable carrier. The pharmaceutical composition can be formulated into dosage forms such as oral preparations using conventional techniques in the art. The effective dose can be adjusted according to the subject's condition and disease severity; for example, the effective dose of TCDCA can be 20-100 mg per kilogram of body weight per day, and the effective dose of DHEA-S can be 5-10 mg per kilogram of body weight per day.

[0041] These endogenous substances are naturally occurring components with high biocompatibility. This invention reveals for the first time their dual value as both excellent biomarkers and effective preventative and therapeutic components, providing a novel approach for the precise diagnosis and nutritional intervention of ALD.

[0042] Terminology Explanation:

[0043] The term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention, but does not exclude other aspects.

[0044] The term "prevention and / or treatment" in some embodiments refers to improving a disease or condition (i.e., slowing or stopping or alleviating the development of a disease or at least one of its clinical symptoms). In other embodiments, it refers to mitigating or improving at least one bodily parameter, including bodily parameters that may not be perceptible to the subject. In still other embodiments, it refers to regulating a disease or condition physically (e.g., stabilizing perceptible symptoms) or physiologically (e.g., stabilizing bodily parameters) or both. In still other embodiments, it refers to preventing or delaying the onset, flare-up, or worsening of a disease or condition.

[0045] The term "effective dose" refers to the amount of a compound that is sufficient to treat a disease when administered to a subject. The effective dose can vary depending on the severity of the disease and the physical condition, age, weight, and sex of the subject to be treated.

[0046] This invention provides novel uses for a class of endogenous substances, specifically including: first, their application as biomarkers for the auxiliary diagnosis and / or staging assessment of ALD; and second, their application in the preparation of drugs for the prevention and / or treatment of ALD. The serum concentrations of these endogenous substances exhibit regular changes during the progression of ALD. This invention, using TCDCA and DHEA-S as representative examples, jointly reveals and verifies this innovative discovery.

[0047] Regarding the first aspect, clinical studies have found a significant association between these endogenous substances and the severity of ALD, but their patterns of change are diverse. Specifically, TCDCA shows a significant positive correlation in the progression of ALD, with its serum concentration increasing sharply as the disease progresses, peaking in the alcoholic cirrhosis stage (approximately 184 times that of the healthy group). It exhibits near-perfect efficacy in diagnosing ALD (AUC > 0.99) and can effectively differentiate early lesions (hepatitis vs. cirrhosis). Conversely, DHEA-S shows a significant negative correlation in the progression of ALD, with its serum concentration decreasing significantly as the disease progresses, reaching its lowest point in the alcoholic cirrhosis stage (approximately 19% of that in the healthy group). Importantly, DHEA-S levels are strongly positively correlated with albumin, a key indicator reflecting liver synthetic function, suggesting it is a biomarker closely related to liver functional reserve. DHEA-S also shows good efficacy in diagnosing ALD (AUC = 0.9054) and demonstrates extremely high accuracy in differentiating early lesions (AUC = 0.9700). TCDCA and DHEA-S show opposite trends, but both are closely related to disease severity, demonstrating the application potential and complementary value of these biomarkers in disease assessment.

[0048] Regarding the second aspect, in vitro and in vivo experiments have confirmed that exogenous supplementation of these endogenous substances has a clear ameliorative effect on ALD. In vitro, both TCDCA and DHEA-S can significantly rescue ethanol-induced decline in hepatocyte viability and regulate the expression of inflammatory factors such as TNF-α and adipogenesis gene Fas. TCDCA can also upregulate the antioxidant factor Nrf2. In vivo, both TCDCA (20-100 mg / kg) and DHEA-S (5-10 mg / kg) can significantly reduce serum ALT, AST, and TG levels in a mouse model of acute alcoholic liver injury and effectively improve hepatic lipid deposition and pathological damage. This indicates that although their endogenous levels change in different directions, exogenous supplementation can exert a hepatoprotective effect by intervening in key pathological processes of the disease.

[0049] The human serum samples involved in the embodiments of the present invention were obtained from Beijing Ditan Hospital affiliated to Capital Medical University.

[0050] The TCDCA and DHEA-S used in the embodiments of this invention were purchased from Nanjing Bencao Yikang Biotechnology Co., Ltd., with a purity of ≥ 98%.

[0051] The mouse hepatocyte cell line AML12 used in the embodiments of this invention was purchased from the Institute of Zoology, Chinese Academy of Sciences.

[0052] The C57BL6 / J mice used in the embodiments of this invention were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0053] In Examples 1 to 3 of this invention, all raw materials and reagents used can be purchased from the market.

[0054] The present invention will be further illustrated below with reference to the embodiments:

[0055] Example 1: Correlation, diagnostic, and staging value analysis of serum TCDCA and DHEA-S levels in human ALD.

[0056] Serum samples were collected from healthy volunteers (20 cases), patients with alcoholic hepatitis (5 cases), patients with alcoholic cirrhosis (20 cases), and patients with alcoholic liver cancer (31 cases). The levels of TCDCA and DHEA-S in all samples were determined using liquid chromatography-tandem mass spectrometry.

[0057] Results regarding TCDCA showed that serum TCDCA levels changed significantly during the progression of ALD: the serum TCDCA concentration in the healthy group was 61.31±18.04 ng / mL. Compared with the healthy group, the serum TCDCA concentration in ALD patients increased sharply. Specifically, the concentration in the alcoholic hepatitis group (3531.36±3232.52 ng / mL) was approximately 58 times that of the healthy group; the peak concentration in the alcoholic cirrhosis group (11274.74±2464.09 ng / mL) was approximately 184 times that of the healthy group; and the concentration in the alcoholic liver cancer group (9211.24±2610.95 ng / mL) was approximately 150 times that of the healthy group. The TCDCA levels in both the alcoholic cirrhosis group (**p < 0.01) and the alcoholic liver cancer group (*p < 0.05) were significantly higher than those in the healthy group. Figure 1 A).

[0058] To systematically evaluate the clinical significance of serum TCDCA in ALD, we assessed the correlation between serum TCDCA levels and clinical indicators, as well as its diagnostic and staging value. First, we analyzed its correlation with disease progression and traditional liver injury markers. The results are as follows: Figure 1 As shown in Figure B, serum TCDCA levels were significantly positively correlated with disease severity (healthy controls = 0, alcoholic hepatitis = 1, alcoholic cirrhosis = 2, alcoholic liver cancer = 3) (Spearman ρ = 0.60, 95% CI 0.43–0.73, p < 0.0001), suggesting it may be a potential biomarker for ALD progression. Further analysis revealed that TCDCA levels were significantly positively correlated with key indicators reflecting hepatocellular damage (…). Figure 1 The levels of DF (diethyltoluene, hydroxychloroquine), including AST (ρ = 0.6212, P < 0.0001), AST / ALT ratio (ρ = 0.5353, P < 0.0001), and total bilirubin (TBIL) (ρ = 0.7677, P < 0.0001). Meanwhile, TCDCA levels showed a significant negative correlation with indicators reflecting liver synthetic function and disease severity. Figure 1 The levels of GH (glucose), such as albumin (ALB) (ρ = -0.7271, P < 0.0001) and platelet count (PLT) (ρ = -0.5942, P < 0.0001). Notably, TCDCA was not significantly correlated with ALT (ρ = 0.1086, P = 0.4047). Figure 1 C), which is consistent with the characteristic of alcoholic liver injury being mainly characterized by elevated AST.

[0059] To quantify the diagnostic value of TCDCA, its ability to distinguish between healthy individuals and ALD patients was evaluated using ROC curves. The results are as follows: Figure 1 As shown in Figure I, in the comparison of "healthy controls (n=20) vs. all ALD patients (n=56)," the area under the curve (AUC) of TCDCA was as high as 0.9911 (95% CI: 0.9763-1.000), indicating that it has near-perfect efficacy for screening and diagnosing ALD. To fairly compare the diagnostic efficacy of TCDCA with commonly used clinical indicators, a subset of samples with data for all indicators to be evaluated (i.e., healthy controls and patients with alcoholic cirrhosis and liver cancer) was further analyzed. Results ( Figure 1 The results showed that in this cohort, TCDCA had a significantly higher diagnostic efficacy (AUC=0.9961) than traditional liver injury biomarkers such as PLT (AUC=0.9180), AST (AUC=0.9160), AST / ALT ratio (AUC=0.9070), and TBIL (AUC=0.9005). These results collectively confirm that TCDCA is a superior ALD diagnostic biomarker compared to most traditional liver injury indicators.

[0060] To explore whether TCDCA can be used for disease severity staging, we conducted an analysis within ALD patients. TCDCA demonstrated good discriminatory power in differentiating between early-stage alcoholic hepatitis and late-stage alcoholic cirrhosis (AUC=0.8100, 95% CI: 0.5133-1.000, P=0.0352). Figure 1 The presence of K suggests that it may help identify early disease progression. Although the limited sample size (n=5) in the alcoholic hepatitis group resulted in a wide confidence interval, the AUC=0.81 and P<0.05 were statistically significant, preliminarily indicating that TCDCA can not only be used for disease diagnosis, but may also show significant changes in the early stages of the disease (progression from hepatitis to cirrhosis), showing the potential to predict disease progression. As a promising finding, it deserves further validation in a larger cohort. However, TCDCA's discriminatory power was limited in distinguishing precancerous lesions (hepatitis + cirrhosis) from hepatocellular carcinoma (AUC=0.5548) and cirrhosis from liver cancer (AUC=0.6387). Figure 1 This indicates that although TCDCA levels significantly increase with ALD progression, its specificity in distinguishing advanced lesions (cirrhosis and hepatocellular carcinoma) is relatively insufficient. This result reveals the clear scope of TCDCA's application in disease staging: its main value lies in assisting diagnosis and early warning of progression, rather than specifically differentiating advanced cirrhosis from hepatocellular carcinoma.

[0061] Results regarding DHEA-S showed that serum DHEA-S levels changed significantly during the progression of ALD: the serum DHEA-S level in the healthy group was 1866.31±18.04 ng / mL. Compared with the healthy group, the serum DHEA-S concentration in ALD patients showed a progressive decrease. The alcoholic hepatitis group (1756.20±307.89 ng / mL) showed a slight decrease, the alcoholic cirrhosis group (358.27±82.63 ng / mL) reached the lowest level, approximately 19% of the healthy group, and the alcoholic liver cancer group (478.78±67.53 ng / mL) showed a slight increase, but remained at extremely low levels. The DHEA-S levels in the alcoholic cirrhosis group (*** p < 0.001) and the alcoholic liver cancer group (*** p < 0.001) were significantly lower than those in the healthy group, and there were also highly significant differences between the alcoholic hepatitis group and the cirrhosis group, and between the liver cancer group (*** p < 0.001). Figure 2 A).

[0062] To systematically evaluate the clinical significance of serum DHEA-S in ALD, we assessed the correlation between serum DHEA-S levels and clinical indicators, as well as their diagnostic and staging value. The results are as follows: Figure 2 As shown in Figure B, serum DHEA-S levels were significantly negatively correlated with disease severity (healthy controls = 0, alcoholic hepatitis = 1, alcoholic cirrhosis = 2, alcoholic liver cancer = 3) (Spearman ρ = -0.56, 95% CI -0.70 to -0.38, P < 0.0001), suggesting it may be a potential protective indicator for the progression of alcoholic liver disease. Further analysis revealed a strong positive correlation between DHEA-S levels and ALB, a key indicator reflecting hepatic synthetic function (ρ = 0.62, P < 0.0001). Figure 2 The C value was positively correlated with PLT (ρ = 0.53, P < 0.0001). Figure 2 (D). This indicates that DHEA-S is a biomarker highly correlated with liver functional status.

[0063] To quantify the diagnostic value of DHEA-S, its ability to distinguish between healthy individuals and ALD patients was assessed using ROC curves. The results are as follows: Figure 2 As shown in Figure E, in the comparison of "healthy controls (n=20) vs. all ALD patients (n=56)," the AUC of DHEA-S was 0.9054 (95% CI: 0.8038-1.000), indicating that DHEA-S has good diagnostic efficacy for ALD. To fairly compare the diagnostic efficacy of DHEA-S with commonly used clinical indicators, a subset of samples with data for all assessed indicators (i.e., healthy controls and patients with alcoholic cirrhosis and liver cancer) was further analyzed. Results ( Figure 2 The results (F) showed that in this cohort, DHEA-S had a superior diagnostic efficacy (AUC=0.9251) compared to traditional liver injury biomarkers such as PLT (AUC=0.9180), AST (AUC=0.9160), AST / ALT ratio (AUC=0.9070), and TBIL (AUC=0.9005). These results collectively confirm that DHEA-S is a good biomarker for ALD diagnosis, superior to most traditional liver injury indicators.

[0064] To explore whether DHEA-S can be used for disease severity staging, we conducted an analysis within ALD patients. DHEA-S demonstrated superior discriminatory power in differentiating between early-stage alcoholic hepatitis and late-stage alcoholic cirrhosis (AUC=0.9700, 95% CI: 0.9053-1.000, P=0.0014). Figure 2 The presence of G in the DHEA-S suggests that it may help identify early disease progression. Although the limited sample size (n=5) in the alcoholic hepatitis group resulted in a wide confidence interval, the AUC=0.97 and P<0.01 were statistically significant, preliminarily indicating that DHEA-S can not only be used for disease diagnosis, but may also show significant changes in the early stages of the disease (progression from hepatitis to cirrhosis), showing the potential to predict disease progression. As a promising finding, it deserves further validation in a larger cohort. However, DHEA-S had limited discriminatory power in distinguishing precancerous lesions (hepatitis + cirrhosis) from hepatocellular carcinoma (AUC=0.5084) and cirrhosis from liver cancer (AUC=0.6306). Figure 2 This indicates that although DHEA-S levels decrease significantly with ALD progression, its specificity in distinguishing advanced lesions (cirrhosis and hepatocellular carcinoma) is relatively insufficient. This result reveals the clear applicability of DHEA-S in disease staging: its main value lies in assisting diagnosis and early warning of progression, rather than specifically distinguishing advanced cirrhosis from hepatocellular carcinoma.

[0065] Example 2: Protective effect of TCDCA and DHEA-S against ethanol-induced acute hepatocellular injury

[0066] Mouse AML12 hepatocytes were cultured in DMEM medium containing 10% fetal bovine serum and routinely cultured in an incubator at 37℃ and 5% CO2. Four groups were established: a normal control group, an ethanol model group, a TCDCA intervention group, and a DHEA-S intervention group. An acute ethanol injury model was established by treating AML12 cells with 500 mM ethanol for 24 hours. The TCDCA intervention groups were co-treated with 1 μM, 10 μM, and 100 μM TCDCA, respectively, concurrently with ethanol treatment. The DHEA-S intervention groups were co-treated with 0.1 μM and 1 μM DHEA-S, respectively, concurrently with ethanol treatment.

[0067] Regarding the TCDCA component, cell viability in each group was assessed using the CCK-8 assay to evaluate the protective effect of TCDCA against ethanol-induced hepatocyte damage. Results are as follows: Figure 3 As shown in Figure A, compared with the control group, treatment with 500 mM ethanol significantly reduced AML12 cell viability (p < 0.001), decreasing it to 81.72% of the control group. Compared with the model group, all TCDCA concentration groups increased cell viability in a concentration-dependent manner, with the 100 μM TCDCA treatment group showing the most significant effect (p < 0.05), restoring cell viability to 89.74% of the control group.

[0068] The mRNA expression levels of Nrf2, Tnf-α, and Fas in cells were further detected using qRT-PCR. The results are as follows: Figure 3 As shown in the BD diagram, compared with the model group, 10 μM and 100 μM TCDCA significantly upregulated the expression of the antioxidant Nrf2 (**p < 0.01, ***p < 0.001) and significantly downregulated the expression levels of the inflammatory cytokine TNF-α and the adipogenesis gene Fas (*p < 0.05, ***p < 0.001). These results indicate that TCDCA has a clear protective effect against ethanol-induced acute hepatocellular injury.

[0069] Regarding the DHEA-S component, cell viability in each group was assessed using the CCK-8 assay to evaluate the protective effect of DHEA-S against ethanol-induced hepatocyte damage. Results are as follows: Figure 4 As shown in Figure A, compared with the control group, treatment with 500 mM ethanol significantly reduced AML12 cell viability (p < 0.001), decreasing it to 81.72% of the control group. In contrast, compared with the model group, 0.1 μM and 1 μM DHEA-S significantly increased cell viability (p < 0.05), raising it to 88.83%–89.31%.

[0070] The mRNA expression levels of Tnf-α and Fas in cells were further detected using qRT-PCR. The results are as follows: Figure 4As shown in the BC diagram, compared with the model group, both 0.1 μM and 1 μM DHEA-S significantly downregulated the expression levels of the inflammatory cytokine TNF-α and the adipogenic gene Fas (*p < 0.05, **p < 0.01). These results indicate that DHEA-S has a clear protective effect against ethanol-induced acute hepatocellular injury.

[0071] Example 3: Protective effects of TCDCA and DHEA-S on a mouse model of acute alcohol-related liver injury.

[0072] Seventy-two 7-week-old male C57BL / 6J mice were housed in an SPF-grade environment and, after one week of acclimatization, were randomly divided into six groups (n=12) based on body weight: a paired feeding group, an ethanol model group, a low-dose TCDCA (20 mg / kg) group, a high-dose TCDCA (100 mg / kg) group, a low-dose DHEA-S (5 mg / kg) group, and a high-dose DHEA-S (10 mg / kg) group. From day 1 to day 10, each intervention group was administered the corresponding dose of TCDCA solution by gavage in the morning, while the control and model groups were administered an equal volume of water. In the afternoon, except for the paired feeding group which was administered an equal amount of glucose by gavage, all other groups were administered 5 g / kg ethanol by gavage. Nine hours after ethanol gavage on day 11, all mice were sacrificed, and serum and various tissues and organs were collected and cryopreserved at -80℃. Serum ALT, AST, and TG levels were detected using kits, and liver tissue was stained with Oil Red O.

[0073] Regarding the results of TCDCA, as follows: Figure 5 As shown in the AC results, compared with the paired feeding group, the serum ALT, AST, and TG levels in the ethanol model group mice were significantly increased (**p < 0.01, ***p < 0.001), indicating that short-term high-intensity alcohol exposure successfully induced hepatocellular damage and lipid metabolism disorders, and the model was successfully established. In the intervention group, TCDCA supplementation (20 mg / kg and 100 mg / kg) significantly reversed the increase in the above indicators induced by ethanol. Among them, the intervention effect of high-dose TCDCA was the most significant, bringing serum ALT, AST, and TG levels back to near the control group level, with statistically significant differences from the model group (*p < 0.05, **p < 0.01, ***p < 0.001). In addition, liver Oil Red O staining also showed that TCDCA intervention significantly reduced hepatic lipid deposition ( Figure 5 The results (D) fully demonstrate that TCDCA has a clear protective effect in acute alcoholic liver injury.

[0074] Regarding the results of DHEA-S, as follows: Figure 6As shown in the AC results, compared with the paired feeding group, the serum ALT, AST, and TG levels in the ethanol model group mice were significantly increased (**p < 0.01, ***p < 0.001), indicating that short-term high-intensity alcohol exposure successfully induced hepatocellular damage and lipid metabolism disorders, and the model was successfully established. In the intervention group, DHEA-S supplementation (5 mg / kg and 10 mg / kg) significantly reversed the elevation of the above indicators induced by ethanol, showing statistically significant differences compared with the model group (*p < 0.05, **p < 0.01, ***p < 0.001). Furthermore, Oil Red O staining of the liver also showed that DHEA-S intervention significantly reduced hepatic lipid deposition (…). Figure 6 (D), which fully demonstrates that DHEA-S has a clear protective effect in acute alcohol-related liver injury.

[0075] In summary, Examples 1 to 3 collectively demonstrate that in the field of alcohol-related liver disease, there exists a class of endogenous substances (represented by TCDCA and DHEA-S) whose serum concentration changes are systematically correlated with disease progression, serving as excellent biomarkers; simultaneously, exogenous supplementation of these substances has a clear preventive and therapeutic effect on the disease. Although the endogenous changes of TCDCA and DHEA-S occur in opposite directions, both successfully reveal the core principle that "changes in the level of endogenous substances indicate pathological states and can themselves serve as therapeutic agents." Therefore, this invention not only provides two specific application schemes for TCDCA and DHEA-S, but more importantly, it reveals a new source of targets with both diagnostic and therapeutic value, providing a novel approach and solid scientific basis for the development of precision diagnosis and prevention strategies for ALD.

[0076] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a class of endogenous substances that undergo regular changes in the progression of alcoholic liver disease (ALD) as biomarkers for the auxiliary diagnosis and / or staging assessment of ALD.

2. The application according to claim 1, characterized in that, The application is achieved by detecting the concentration of the endogenous substance in a subject's blood sample.

3. The application according to claim 1 or 2, characterized in that, The regular changes include a significant increase or decrease in concentration as the severity of the disease increases.

4. The application according to claim 1, characterized in that, The endogenous substances include bile acids and steroid hormones.

5. The application according to claim 4, characterized in that, The bile acid is taurine chenodeoxycholic acid (TCDCA).

6. The application according to claim 4, characterized in that, The steroid hormone is dehydroepiandrosterone sulfate (DHEA-S).

7. The application of a class of endogenous substances that undergo regular changes in the ALD process in the preparation of drugs for the prevention and / or treatment of ALD.

8. The application according to claim 7, characterized in that, The endogenous substances include bile acids and steroid hormones.

9. The application according to claim 8, characterized in that, The bile acid is taurine chenodeoxycholic acid (TCDCA).

10. The application according to claim 8, characterized in that, The steroid hormone is dehydroepiandrosterone sulfate (DHEA-S).

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