Application of ANGPTL3 in the prognosis of hepatitis B-related acute-on-chronic liver failure

Abstract

The present invention discloses the use of ANGPTL3 in the prognosis of hepatitis B-related acute-on-chronic liver failure, relating to the fields of disease prognosis and biomedicine technology. The present invention discloses the use of ANGPTL3 as a biomarker in the preparation of products for the diagnosis and / or course diagnosis and / or prognosis assessment of hepatitis B-related acute-on-chronic liver failure. The beneficial effect of the present invention is that ANGPTL3 can be used as a biomarker for the prognosis of hepatitis B-related acute-on-chronic liver failure. By using ELISA technology to absolutely quantify the level of ANGPTL3 in blood samples of patients in different groups of HBV-ACLF patients and to absolutely quantify the dynamic level of ANGPTL3, the phenomenon and course of hepatitis B-related acute-on-chronic liver failure can be timely discovered, and other related indicators can be detected, thereby using ANGPTL3 to provide scientific guidance for the management and decision-making of hepatitis B-related acute-on-chronic liver failure.

Description

Technical Field

[0001] The present invention belongs to the field of disease prognosis and biomedicine technology, and specifically relates to the application of ANGPTL3 in the prognosis of hepatitis B-related acute-on-chronic liver failure. Background Art

[0002] Acute-on-chronic liver failure (ACLF) is a complex syndrome that develops in patients with chronic liver disease (CLD) during acute liver injury. It is characterized by intense systemic inflammation, organ failure, and high short-term mortality. Hepatitis B virus-associated acute-on-chronic liver failure (HBV-ACLF) presents with multi-organ failure within four weeks, high mortality, and a poor prognosis. Timely clinical intervention can halt the progression of HBV-ACLF and improve patient survival.

[0003] Currently, commonly used prognostic methods for liver failure, including the model for end-stage liver disease (MELD) and the CLIF-C ACLF model developed by the European Association for the Study of Chronic Liver Failure (CLIF), still have limitations. Many new prognostic markers and assessment systems for liver failure are under investigation. However, due to the complex mechanisms of liver failure, rapid disease progression, and numerous factors influencing prognosis, accurate prediction of liver failure is difficult. Developing more effective and sensitive tests to predict HBV-ACLF progression and grading is crucial to the treatment of HBV-ACLF. Summary of the Invention

[0004] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide an ANGPTL3 as a biomarker and its application in the prognosis of hepatitis B related acute-on-chronic liver failure.

[0005] The applicant used allele-based quantitative proteomics to evaluate HBV-ACLF disease-related proteomic changes and found that angiopoietin-like 3 (ANGPTL3) could be used as a candidate biological prognostic marker based on differentially expressed proteins (DEPs) in HBV-ACLF patients.

[0006] ANGPTL3 is a member of the angiopoietin-like protein family involved in regulating lipid metabolism. The protein encoded by the ANGPTL3 gene is produced exclusively in the liver and secreted into the circulation. In the liver, ANGPTL3 is produced exclusively by hepatocytes, and its expression is regulated by hydroxysterol-activated liver X receptors (LXRs). Previous studies have shown that the biological effects of ANGPTL3 are manifested in two aspects: regulating angiogenesis and lipid metabolism. To date, there have been no reports linking ANGPTL3 to HBV-ACLF. This study discovered this association and found that ANGPTL3 levels are closely associated with the course of HBV-ACLF.

[0007] The present invention provides ANGPTL3 as a biomarker for predicting the prognosis of hepatitis B acute-on-chronic liver failure. It can be used in an enzyme-linked immunosorbent assay (ELISA) product for testing patient plasma. It can efficiently, simply, and accurately predict the disease progression, survival time, and specific classification of HBV-ACLF patients, thereby better guiding clinical medication.

[0008] The technical solutions of the present invention are as follows:

[0009] A first aspect of the present invention provides a use of ANGPTL3 as a biomarker in the preparation of a product for diagnosing hepatitis B-related acute-on-chronic liver failure and / or diagnosing the course of disease and / or evaluating prognosis.

[0010] Optionally, the hepatitis B related acute-on-chronic liver failure includes hepatitis B related acute-on-chronic liver failure with underlying hepatitis B cirrhosis.

[0011] Optionally, the product includes a kit for detecting the content of the biomarker.

[0012] Optionally, the kit contains a reagent for detecting the content of the biomarker, and the reagent includes one of the reagents required for immunohistochemistry, Western-Blot, protein immunoblotting, and enzyme-linked immunosorbent assay.

[0013] Specifically, the kit is an ELISA kit, which includes a capture antibody, a detection antibody, a recombinant human ANGPTL3 protein standard, a sample processing solution, a color development solution, and a stop solution. More specifically, the detection antibody is a detection antibody labeled with horseradish peroxidase.

[0014] Optionally, the disease course diagnosis includes using the product to detect the patient's ANGPTL3 level before and after treatment, and judging whether the disease course is improving, fluctuating, or worsening based on the changes in ANGPTL3 levels.

[0015] Specifically, the course of disease diagnosis includes improvement, fluctuation and worsening of the disease, wherein improvement of the disease means that the patient's ANGPTL3 level increases during treatment, fluctuation of the disease means that the patient's ANGPTL3 level does not change significantly during treatment, and worsening of the disease means that the patient's ANGPTL3 level decreases during treatment.

[0016] A second aspect of the present invention provides a kit comprising a reagent for detecting the content of the biomarker, wherein the biomarker is ANGPTL3, and the reagent comprises one of the reagents required for detection by immunohistochemistry, Western-Blot, protein immunoblotting, and enzyme-linked immunosorbent assay;

[0017] When the kit is used to detect the content of the biomarker in the test sample, when the detection concentration of the biomarker in the test sample is lower than 64.87 ng / mL, the source of the test sample is determined to be a high-risk patient for HBV-ACLF.

[0018] Optionally, the kit is an ELISA kit, which includes a capture antibody, a detection antibody, a recombinant human ANGPTL3 protein standard, a sample processing solution, a color development solution and a stop solution, and the detection antibody is a detection antibody labeled with horseradish peroxidase.

[0019] A third aspect of the present invention provides the use of ANGPTL3 in constructing a prognosis prediction model for hepatitis B-related acute-on-chronic liver failure.

[0020] Specifically, the molecular marker ANGPTL3 was made into a prognostic prediction model for predicting the prognosis of hepatitis B-related acute-on-chronic liver failure.

[0021] A fourth aspect of the present invention provides the use of ANGPTL3 as a target in screening drugs for treating hepatitis B-related acute-on-chronic liver failure.

[0022] A fifth aspect of the present invention provides use of ANGPTL3 in the preparation of an inhibitor of hepatitis B-related acute-on-chronic liver failure.

[0023] Specifically, targeted therapeutic drugs or inhibitors could be designed for ANGPTL3 to treat hepatitis B-related acute-on-chronic liver failure. By designing an mRNA vaccine targeting ANGPTL3, the patient's immune system could be stimulated to produce ANGPTL3, thereby increasing its expression. Research on the regulation of ANGPTL3 expression could identify small molecules that can induce ANGPTL3 expression. Through drug or inhibitor screening and optimization, drugs or inhibitors that can activate ANGPTL3 expression could be designed.

[0024] A sixth aspect of the present invention provides a method for screening drugs, comprising the following steps:

[0025] Detecting the level of ANGPTL3 in patient samples before and after administration;

[0026] If the content of ANGPTL3 increases, the drug to be screened has the effect of treating HBV-ACLF;

[0027] If the ANGPTL3 content remains unchanged or decreases, the drug to be screened has no effect in treating HBV-ACLF;

[0028] The sample comprises blood.

[0029] The present invention has at least one of the following beneficial effects:

[0030] The applicant screened a biomarker for the prognosis of hepatitis B-related acute-on-chronic liver failure through extensive proteomics, and found that ANGPTL3 can be used as a biomarker for the diagnosis and / or course diagnosis and / or prognosis of hepatitis B-related acute-on-chronic liver failure. By using ELISA technology to absolutely quantify the levels of ANGPTL3 in blood samples of patients in different groups of HBV-ACLF and the absolute quantitative dynamic levels of ANGPTL3, the phenomenon and course of hepatitis B-related acute-on-chronic liver failure can be timely discovered, and other related indicators can be detected, thereby using ANGPTL3 to provide scientific guidance for the management and decision-making of hepatitis B-related acute-on-chronic liver failure.

[0031] ANGPTL3 can also be used to construct a prognosis prediction model for hepatitis B related acute-on-chronic liver failure, which is used to predict the prognosis of hepatitis B related acute-on-chronic liver failure.

[0032] ANGPTL3 can also be used as a target for screening drugs for treating hepatitis B-related acute-on-chronic liver failure and for preparing hepatitis B-related acute-on-chronic liver failure inhibitors, and for screening drugs and inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Figure 1 shows the correlation between admission ANGPTL3 levels and HBV-ACLF patients. Figure A shows the distribution of ANGPTL3 in the survival and non-survival groups of HBV-ACLF patients. Figure B shows the receiver operating characteristic curve for ANGPTL3 in predicting 30-day mortality in HBV-ACLF patients. Figure C shows the 30-day survival rates of patients in the high and low ANGPTL3 groups. Figure D shows the correlation between ANGPTL3 levels and mortality. * P < 0.001.

[0034] Figure 2Changes in ANGPTL3 levels between the initial (at admission) and final (14 days after admission or at discharge) assessments were performed for the improvement, fluctuation, and exacerbation groups.

[0035] Figure 3 is the standard curve equation in Example 1.

[0036] Figure 4 This is the sensitivity detection data in Example 3. DETAILED DESCRIPTION

[0037] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0038] Example 1

[0039] 1) Collect blood specimens, including HBV-ACLF blood specimens;

[0040] 2) Before testing ANGPTL3 levels in blood using ELISA, centrifuge the collected blood sample at 4000 rpm for 20 minutes to separate the serum. If the entire test cannot be completed in one sitting, aliquot and freeze at or below -20°C to avoid repeated freeze-thaw cycles. Thaw at room temperature before use to ensure that the sample is evenly and thoroughly thawed.

[0041] 3) The blood sample and all test kit components must be rewarmed for at least 120 minutes to ensure they are fully warmed to room temperature. Perform the test according to the following steps:

[0042] Among them, step 3) specifically includes:

[0043] (1) Set up standard wells, zero-value wells, blank wells, and sample wells. Add 50 μL of ANGPTL3 standard at different concentrations (80 ng / ml, 40 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, 2.5 ng / ml) to the standard wells, add 50 μL of sample diluent to the zero-value wells, leave the blank wells empty, and add 50 μL of the sample to be tested to the sample wells.

[0044] (ii) Add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to the standard wells, 0 value wells, and sample wells, except for the blank wells.

[0045] (3) Cover the reaction plate with sealing film and incubate in a 37°C water bath or incubator in the dark for 60 minutes.

[0046] (4) Remove the sealing film, discard the liquid, and pat dry on absorbent paper. Fill each well with wash solution, let it sit for 20 seconds, shake off the wash solution, and pat dry on absorbent paper. Repeat this process five times. If using an automated plate washer, wash the plate according to the machine's operating procedures. Adding a 30-second soaking period can improve assay accuracy. After washing, pat the plate dry on clean, lint-free paper before adding substrate.

[0047] (5) Mix substrate A (hydrogen peroxide) and substrate B (TMB) in the ELISA kit at a 1:1 volume ratio to obtain a substrate mixture; add 100 μL of the substrate mixture to all wells.

[0048] (6) Cover the reaction plate with sealing film and incubate in a 37°C water bath or incubator in the dark for 15 minutes.

[0049] (7) Add 50 μl of stop solution to all wells and read the absorbance (OD value) of each well on a microplate reader at a wavelength of 450 nm.

[0050] (VIII) Using the standard concentration as the horizontal axis (6 standard wells plus 1 zero well, a total of 7 concentration points) and the corresponding absorbance (OD value) as the vertical axis, use computer software and four-parameter logistic curve fitting (4-pl) to create the standard curve equation, see Figure 3 , the standard curve is y=(3.08485-0.03122) / [1+(x / 57.01298) -1.57071 ]+0.03122,r 2 =0.99951. Calculate the sample concentration using the equation based on the sample's absorbance (OD value). If the sample is diluted, the concentration measured using the above method must be multiplied by the dilution factor to obtain the final concentration.

[0051] 4) Validate the prognostic value of ANGPTL3 in patients with HBV-ACLF;

[0052] Among them, step 4) specifically includes:

[0053] (I) Clinical data of HBV-ACLF patients were collected, including admission test and examination data and complications. Patients were divided into the cirrhosis group and the non-cirrhosis group based on the presence or absence of hepatitis B cirrhosis. Patients were divided into the survival group and the non-survival group based on whether they survived for 30 days.

[0054] (II) SPSS 26.0 software was used to analyze univariate and multivariate Cox regression analysis, combined with clinical data, to determine whether ANGPTL3 could serve as an independent prognostic biomarker (Tables 1 and 2).

[0055] (III) Based on the results of step (II), ANGPTL3 was confirmed to be an independent prognostic biomarker. SPSS 26.0 software was used to divide HBV-ACLF into high ANGPTL3 group and low ANGPTL3 group using the receiver operating characteristic curve (ROC curve) and the area under the curve. The cumulative survival rates were compared. Figure 1 .in, Figure 1 The correlation between ANGPTL3 levels at admission and HBV-ACLF patients. Figure 1 Figure A represents the distribution of ANGPTL3 in the survival and non-survival groups of HBV-ACLF patients. It can be observed that during the 30-day follow-up period, the ANGPTL3 level of the non-survival group at admission was significantly lower than that of the survival group. Figure 1 B in the figure is the receiver operating characteristic curve for ANGPTL3 in predicting 30-day mortality in HBV-ACLF patients, where the red line represents the reference line with no diagnostic value, and the blue line is the ROC curve. The more the ROC curve deviates from the red line, the more meaningful it is. Figure 1 As shown in Figure B, the area under the ROC curve (AUC) of ANGPTL3 level for 30-day mortality was 0.77 (0.70-0.84), with a sensitivity of 0.74, a specificity of 0.68, and an optimal cut-off value of 64.87 ng / mL. Figure 1 C in the figure represents the survival rate of patients in the high ANGPTL3 group and the low ANGPTL3 group after 30 days. It can be seen that the cumulative survival rate of the low ANGPTL3 group was significantly lower than that of the high ANGPTL3 group. Figure 1 D in the figure is the correlation between ANGPTL3 level and mortality, where one blue point represents one sample and the red line is the trend line. Figure 1 As shown in Figure D, lower ANGPTL3 levels were associated with higher mortality rates, while increasing ANGPTL3 concentrations led to a decreasing mortality rate. *P < 0.001.

[0056] Step (1) Blood samples included 219 patients with HBV-ACLF; 51 patients were grouped as nonsurvivors and 168 patients were grouped as survivors; and 88 patients without HBV cirrhosis and 131 patients with HBV cirrhosis were grouped as cirrhosis (30 nonsurvivors, accounting for 58.8% of the nonsurvivors; 101 survivors, accounting for 60.1% of the survivors). See Tables 1 and 2 for details.

[0057] Step (2) Data analysis suggested that ANGPTL3 could still serve as an independent prognostic biomarker for HBV-ACLF in patients with underlying hepatitis B cirrhosis, as shown in Table 2.

[0058] Step (3) Data analysis results suggest that the worse the outcome of HBV-ACLF patients, the higher the ANGPTL3 level.

[0059] Table 1

[0060]

[0061] Table 2

[0062]

[0063] 5) determining from the results of step 4) that ANGPTL3 levels are associated with the course of hepatitis B-related acute-on-chronic liver failure;

[0064] Wherein, step 5) specifically includes:

[0065] (I) Clinical data of HBV-ACLF patients were collected and followed up, including test and examination results at admission and regular follow-up, and complications; patients were divided into survival group and non-survival group based on whether they survived for 30 days; patients were divided into improvement group, fluctuation group and deterioration group based on CLIF-SOF score (Chronic Liver Failure-Sequential Organ Failure Assessment, also known as chronic liver failure sequential organ failure assessment).

[0066] Improvement group: CLIF-SOFA score decreased by ≥2 points;

[0067] Fluctuation group: CLIF-SOFA score change (increase or decrease) <2 points;

[0068] The deterioration group was defined as an increase of ≥2 points in the CLIF-SOFA score.

[0069] (II) SPSS 26.0 software was used to analyze the Mann-Whitney U test and clinical data to determine whether there were significant differences in ANGPTL3 levels among the groups.

[0070] Step (1) Blood samples were collected from 84 patients with HBV-ACLF who were followed up. Among them, 36 patients were divided into the improvement group, 30 patients were divided into the fluctuation group, and 18 patients were divided into the deterioration group according to the course of disease. Blood samples were collected from 35 patients in the non-survival group and 49 patients in the survival group according to the non-survival and survival groups.

[0071] The results of step (2) suggest that ANGPTL3 levels are significantly correlated with the disease, as shown in Figure 2 .in Figure 2The middle shows the changes in ANGPTL3 levels between the initial (at admission) and final observation endpoint (14 days after admission or discharge) assessments in the improvement group, fluctuation group, and exacerbation group. It can be observed that there was no significant change between the initial and observation endpoints in the fluctuation group; the ANGPTL3 level in the exacerbation group was significantly lower at the observation endpoint than that at admission; and the ANGPTL3 level in the improvement group was significantly higher at the observation endpoint than that at admission.

[0072] Example 2

[0073] This embodiment provides a detection kit for use in the prognosis assessment of acute-on-chronic liver failure.

[0074] (1) Kit composition:

[0075] ANGPTL3-specific antibodies: including capture antibody (coated on microtiter plates) and human ANGPTL3 conjugate (coupled with horseradish peroxidase in the presence of preservatives), purchased from R&D Systems.

[0076] Standard: Recombinant human ANGPTL3 protein (concentration after reconstitution: 100 ng / mL) purchased from R&D Systems.

[0077] Standard diluent: Dilution buffer suitable for serum / plasma samples, animal serum with preservatives, purchased from R&D Systems.

[0078] Assay diluent: Buffered protein matrix with preservatives, purchased from R&D Systems.

[0079] Color development substrate: Color development reagent A (HRP) and color development reagent B (TMB) were evenly mixed in a volume ratio of 1:1 and purchased from R&D Systems.

[0080] Stop solution: 2N sulfuric acid stop solution.

[0081] (2) Detection method

[0082] step:

[0083] (A) Sample collection: Collect venous blood from the patient, centrifuge to separate serum / plasma (must be completed within 2 hours and stored at -80°C), collect serum, and obtain the sample to be tested.

[0084] (B) Testing process:

[0085] The standards and test samples were added to the microplate pre-coated with capture antibody, and 100 μl of assay diluent was added to each well and incubated at 37°C for 2 hours.

[0086] After washing, 200 μl of human ANGPTL3 conjugate was added to each well and incubated at room temperature for 1 hour.

[0087] After repeated washing steps, 200 μl of chromogenic substrate was added to each well and incubated at room temperature in the dark for 30 minutes.

[0088] Add 50 μl of stop solution to each well and measure the absorbance at 450 nm within 30 minutes after termination. The absorbance at 540 nm or 570 nm can be set for wavelength correction.

[0089] (C) Result analysis:

[0090] ANGPTL3 concentration was calculated using a standard curve.

[0091] The above-mentioned kit and detection method were used to test 219 HBV-ACLF patients in a tertiary hospital. The patients were divided into high-risk and low-risk groups based on their ANGPTL3 concentrations. The ANGPTL3 concentration in the high-risk group was lower than 64.87 ng / ml, while that in the low-risk group was higher than or equal to 64.87 ng / ml. The results showed:

[0092] The 28-day mortality rate was highly correlated with ANGPTL3 levels: 13 of 128 patients (10.1%) in the high-risk group died within 30 days, while 37 of 91 patients (40.6%) in the low-risk group died within 30 days.

[0093] Example 3

[0094] The specificity, sensitivity and precision of the kit in Example 2 were tested.

[0095] Specificity of the kit in Example 2: The kit in Example 2 was used to detect natural and recombinant human angiopoietin-like protein 3 (ANGPTL3) and structural analogs (purchased from Jianglai Biotechnology, model name JL19947-48T). The results showed that this kit recognized natural and recombinant human angiopoietin-like protein 3 (ANGPTL3) and had no cross-reactivity with structural analogs.

[0096] The sensitivity and precision of the kit in Example 2 were tested, as shown in Tables 3 to 5:

[0097] Sensitivity: The kit and method of Example 2 were used to detect standard samples with ANGPTL3 concentrations of 0.156 ng / ml, 0.313 ng / ml, 0.625 ng / ml, 1.25 ng / ml, 2.5 ng / ml, 5 ng / ml, and 10 ng / ml, respectively. The absorbance at 450 nm was measured. A standard curve was constructed with ANGPTL3 concentration as the abscissa and absorbance at 450 nm as the ordinate. The results are shown in FIG. Figure 4; The kit of Example 2 was used to detect 10 zero standards (blank controls without the substance to be tested), and the absorbance at 450 nm was measured. The average value of the OD of the 10 zero standards was added with twice the SD (standard deviation). The detection limit of the OD value (450 nm) was calculated to be 0.0562, corresponding to a minimum detection concentration of 0.006719 ng / ml. As shown in Tables 3 and Figure 4 .

[0098] Intra-assay precision: Three samples of known concentration were randomly selected and each sample was tested 20 times on the same plate to assess intra-assay precision. The intra-assay precision data are as follows: for all three samples, the number of tests, n, was 20. Sample 1 had a mean of 1.05 ng / mL, a standard deviation of 0.04, and a coefficient of variation of 3.8%; Sample 2 had a mean of 3.36 ng / mL, a standard deviation of 0.09, and a coefficient of variation of 2.7%; and Sample 3 had a mean of 6.80 ng / mL, a standard deviation of 0.18, and a coefficient of variation of 2.6%.

[0099] Interassay precision: Three samples of known concentration were tested in 20 independent analyses by at least three technicians using two batches of the ingredients to assess interassay precision. Interassay precision data showed that the number of analyses, n, for all three samples was 20. Sample 1 had a mean of 1.02 ng / mL, a standard deviation of 0.07, and a coefficient of variation of 6.9%; Sample 2 had a mean of 3.18 ng / mL, a standard deviation of 0.20, and a coefficient of variation of 6.3%; and Sample 3 had a mean of 6.74 ng / mL, a standard deviation of 0.60, and a coefficient of variation of 8.9%.

[0100] Table 3

[0101]

[0102] Table 4

[0103]

[0104] Table 5

[0105]

[0106] Therefore, it can be seen from Tables 4 and 5 that the intra-assay coefficient of variation is <10% and the inter-assay coefficient of variation is <15%. The above results indicate that the detection kit of the present invention has good precision and meets the requirements of clinical testing.

[0107] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. Use of reagents for detecting ANGPTL3 in the preparation of diagnostic or prognostic assessment kits for hepatitis B-related acute-on-chronic liver failure.

2. The use according to claim 1, characterized in that The hepatitis B related acute-on-chronic liver failure includes hepatitis B related acute-on-chronic liver failure with underlying hepatitis B cirrhosis.

3. The use according to claim 1, characterized in that The reagents include reagents required for enzyme-linked immunosorbent assay (ELISA).

4. The use according to claim 1, characterized in that The reagents include reagents required for immunohistochemistry or western blotting.

5. The use according to any one of claims 1 to 3, characterized in that: The diagnosis includes disease course diagnosis, which includes using the kit to detect the patient's ANGPTL3 level before and after treatment, and judging whether the disease course is improved, fluctuated, or worsened based on the changes in ANGPTL3 levels.

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