New diagnosis and treatment marker DAO of chronic-acute hepatic failure and application

Abstract

The invention relates to application of a biomarker diamine oxidase (DAO) in prediction, diagnosis and treatment of chronic plus acute liver failure (ACLF). The invention also relates to correlation analysis of DAO and ACLF diagnostic markers such as TBIL, INR and the like. On the basis of existing TBIL and INR index models, the DAO index is added, early warning can be conducted on ACLF, complex instruments, equipment and schemes are not needed for DAO concentration detection, the detection period is short, and therefore a new marker is provided for clinical diagnosis and treatment of ACLF, and important clinical significance is achieved.

Description

Technical Field

[0001] The present invention specifically relates to a new diagnostic and therapeutic marker for acute-on-chronic liver failure and its application. Background Art

[0002] Acute-on-chronic liver failure (ACLF) is a potentially reversible condition that occurs in patients with chronic liver disease, with or without underlying cirrhosis. In the absence of treatment for the underlying liver disease, liver supportive care, or liver transplantation, it can lead to multi-organ failure and death within three months. Currently, there is no effective treatment for ACLF. Liver transplantation is the only potentially effective treatment for severe ACLF, but only a very small number of patients are eligible. Early identification of ACLF enables some patients to receive timely treatment, and accurate prognosis of ACLF patients can more rationally allocate medical resources. Although recent studies have shown that changes in gut microbial composition and function are associated with ACLF, and that microbial components and their metabolites may serve as biomarkers for the development and prognosis of ACLF, the precise mechanisms remain unclear.

[0003] Diamine oxidase (DAO) is a highly active intracellular enzyme found in the villi of the small intestinal mucosa of humans and mammals. Its activity can reflect the integrity and degree of damage to the intestinal mechanical barrier. Studies have shown that patients with liver failure experience disruption of the gut-liver axis, impaired intestinal microcirculation, and varying degrees of damage to the four major intestinal barriers. Through in-depth research into the mechanisms of ACLF, this study identifies DAO as a potential new diagnostic and therapeutic marker that can improve the efficiency and accuracy of ACLF diagnosis and treatment, possessing significant clinical value. Summary of the Invention

[0004] One object of the present invention is to provide the use of DAO in preparing ACLF diagnosis or prognosis prediction products.

[0005] To achieve the above technical purpose, the present invention first studies the serum content of DAO in ACLF population, chronic liver disease population and healthy control population, and finds that the serum content of DAO in healthy population and chronic liver disease population is not much different, and the serum content in ACLF population is reduced to almost undetectable. So we collected clinical biochemistry and other detection indicators of chronic liver disease (including ACLF) population, and did correlation analysis with DAO respectively. The results showed that the diagnostic indicators of ACLF, such as INR, TBIL, liver enzymes, were negatively correlated with DAO, while indicators such as PLT and PTA were positively correlated with DAO; the ACLF population was divided according to whether the DAO content in serum was greater than 0 U / L, and a survival curve was drawn. The results showed that the ACLF population with high DAO content in serum had a better prognosis. The above results show that DAO has a significant correlation with the clinical indicators of ACLF population. The higher the serum DAO content, the better the prognosis of ACLF patients, that is, the serum DAO content can prompt the occurrence and development of ACLF. According to the above research results, DAO can be preliminarily used as a marker of ACLF for the diagnosis or prognosis prediction of ACLF.

[0006] Furthermore, the present invention provides use of a reagent for detecting DAO expression in the preparation of a product for ACLF diagnosis or prognosis prediction.

[0007] The reagent includes a reagent for detecting the expression level or activity of DAO, the reagent includes a nucleic acid that binds to DAO or its DNA sequence, and the nucleic acid includes a PCR amplification primer used when using SYBRGreen, TaqMan probe, molecular beacon, double hybridization probe, or composite probe to detect the expression level of DAO.

[0008] Furthermore, the ACLF diagnosis or prognosis prediction product includes a chip, a test paper or a kit.

[0009] The chip includes a reagent for detecting the expression level or activity of DAO, wherein the reagent includes a nucleic acid that binds to DAO or its DNA sequence, and the nucleic acid includes a probe that can detect the expression level of DAO.

[0010] The kit includes an antibody against a certain peptide segment when detecting the expression amount or activity of DAO using Western blot, ELISA, chemiluminescence or immunofluorescence.

[0011] The present invention also provides the use of DAO in screening and preparing a pharmaceutical composition for treating ACLF.

[0012] The invention also provides a pharmaceutical composition for treating ACLF, wherein the pharmaceutical composition comprises an agent or compound that increases the DAO content.

[0013] The pharmaceutical composition of the present invention can be administered as a medicine alone or together with other drugs.

[0014] The pharmaceutical composition of the present invention can be prepared into various formulations as needed, including but not limited to tablets, solutions, granules, patches, ointments, capsules, aerosols, or suppositories for transdermal, mucosal, nasal, buccal, sublingual, or oral administration.

[0015] The administration route of the pharmaceutical composition of the present invention is not limited, as long as it can exert the desired therapeutic effect or preventive effect. In some cases, it can be administered systemically, and in some cases it can be administered locally.

[0016] The dosage of the pharmaceutical composition of the present invention is not limited as long as the desired therapeutic effect or preventive effect is achieved, and can be appropriately determined based on symptoms, gender, age, etc.

[0017] The dosage of the therapeutic pharmaceutical composition or preventive pharmaceutical composition of the present invention can be determined using, for example, the therapeutic effect or preventive effect on a disease as an indicator.

[0018] The present invention has discovered a marker for the diagnosis and treatment of ACLF, and provides a new use of DAO in the preparation of a method or product for the diagnosis or prognosis prediction of ACLF, thereby providing a new target for the diagnosis and treatment of clinical ACLF. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Analysis of serum DAO content in ACLF patients, *P<0.05.

[0020] Figure 2 Correlation analysis between serum DAO levels and clinical indicators of chronic liver disease and ACLF population, *P<0.05.

[0021] Figure 3 This is the survival curve of the effect of serum DAO on the prognosis of ALCF population, *P<0.05. DETAILED DESCRIPTION

[0022] The present invention is further described below by way of specific embodiments. The following examples are specific embodiments of the present invention, but the embodiments of the present invention are not limited to the following examples. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0023] Example 1: The ELISA method was used to detect the expression of DAO in the serum of ACLF patients, chronic liver disease patients and control patients.

[0024] Detection was performed according to the instructions of the Wuhan Cloud-Clone diamine oxidase (DAO) colorimetric test kit.

[0025] (1) Prepare all reagents, samples, and standards.

[0026] (2) Add 100 μl of standard or sample to each well and incubate at 37°C for 1 hour.

[0027] (3) Aspirate and add 100 μl of the prepared detection reagent A, and incubate at 37°C for 1 hour.

[0028] (4) Aspirate and wash 3 times.

[0029] (5) Add 100 μl of the prepared detection reagent B and incubate at 37°C for 30 minutes.

[0030] (6) Aspirate and wash 5 times.

[0031] (7) Add 90 μl of substrate solution and incubate at 37°C for 10-20 minutes.

[0032] (8) Add 50 μl of stop solution and read immediately at 460 nm.

[0033] Example 2: SPSS software was used to analyze the relationship between clinical biochemical indicators and DAO in patients with chronic liver disease and ACLF.

[0034] Methods Serum was collected from 37 patients with chronic liver disease and 49 patients with acute-on-chronic liver failure in the Department of Infectious Diseases, the Second Affiliated Hospital of Chongqing Medical University. SPSS Statistics 27 statistical analysis software was used to analyze the correlation between clinical indicators and serum DAO levels.

[0035] Example 3: Graphpad Prism software was used to analyze the survival curve of ACLF patients and the relationship between serum DAO levels, and to analyze the percentage accumulation graph of the detectable serum DAO levels in different populations.

[0036] The relationship between the 6-month survival rate of ACLF patients and their serum DAO levels was statistically analyzed. The ACLF population was divided according to whether the serum DAO level was greater than 0 U / L, and survival curves were drawn for each group. Statistical methods

[0037] The results were analyzed using Graphpad Prism 9.5.1 statistical analysis software, and the t-test was used for comparison between samples.

[0038] SPSS Statistics 27 statistical analysis software was used to analyze data correlation and non-parametric tests were used. result

[0039] Analysis of serum DAO content in ACLF population.

[0040] The experimental results showed that the expression of DAO in the serum of ACLF patients was higher than that in the normal control group and chronic liver disease group. Figure 1 .

[0041] Correlation analysis between serum DAO levels and clinical indicators in patients with chronic liver disease and ACLF.

[0042] The experimental results showed that the diagnostic indicators of ACLF, such as INR, TBIL, and liver enzymes, were negatively correlated with DAO, while PLT, PTA and other indicators were positively correlated with DAO. That is, DAO was significantly correlated with the clinical indicators of chronic liver disease and ACLF. The higher the serum DAO content, the better the patient's clinical indicators. Figure 2 .

[0043] Survival curves of the impact of serum DAO on the prognosis of ALCF population and the percentage accumulation diagram of whether serum DAO can be detected in different populations.

[0044] The experimental results showed that the higher the serum DAO content, the smoother the curve, the longer the median survival time of ACLF patients, the higher the survival rate, and the better the prognosis. Figure 3 .

Claims

1. Use of reagents for detecting DAO expression or activity in the preparation of products for the diagnosis, treatment, or prognosis of ACLF.

2. The product for ACLF diagnosis or prognosis prediction according to claim 1, characterized in that: The ACLF diagnosis or prognosis prediction product includes a chip, a test paper or a kit.

3. Application of DAO in screening drug compositions for the treatment of ACLF.

4. Application of DAO in the preparation of pharmaceutical compositions for the treatment of ACLF.

5. A pharmaceutical composition for treating ACLF, characterized in that: The pharmaceutical composition includes an agent or compound that inhibits the expression or activity of DAO.

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