Application of PYGL as auxiliary diagnosis marker of hepatitis E related acute hepatic failure

Abstract

The invention discloses application of PYGL as an auxiliary diagnosis marker for hepatitis E related acute hepatic failure, and belongs to the technical field of biomedical detection. By detecting the expression level of PYGL in serum of a subject, hepatitis E related acute hepatic failure patients (HEV-ALF), acute hepatitis E patients (AHE) and healthy control people (HCs) can be effectively distinguished. The result shows that the serum PYGL expression level of HEV-ALF patients is obviously higher than that of acute hepatitis E patients and healthy control people; the serum PYGL expression level has significant correlation with the clinical outcome of patients with hepatitis E related acute hepatic failure, and the serum PYGL expression level of patients in a death group is significantly higher than that of patients in a survival group; the expression level of the serum PYGL is also associated with the number of organ failure of the patient, and can reflect the dynamic change of the condition of the patient. Therefore, the serum PYGL can be used as an effective biomarker for auxiliary diagnosis of the hepatitis E related acute hepatic failure and prognosis evaluation of the hepatitis E related acute hepatic failure.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to the application of PYGL as an auxiliary diagnostic marker for hepatitis E-related acute liver failure. Background Technology

[0002] Hepatitis E virus (HEV) is a single-stranded, positive-sense RNA virus and one of the leading causes of acute viral hepatitis worldwide. Its genome typically encodes three open reading frames (ORF1, ORF2, and ORF3). According to the World Health Organization, approximately 20 million people are infected with hepatitis E globally each year, with 3.4 million developing clinical symptoms, resulting in about 70,000 deaths and 3,000 stillbirths, placing a heavy burden on global public health. Hepatitis E virus is primarily transmitted through the fecal-oral route. Eight different genotypes have been identified, with genotypes 1 and 2 infecting only humans, and genotypes 3 and 4 being zoonotic. In addition, one case of infection with genotype 7 due to consumption of contaminated camel meat and milk has been reported.

[0003] Although hepatitis E infection is usually asymptomatic or self-limiting in immunocompetent individuals, it can lead to acute hepatitis and even liver failure in pregnant women, the elderly, or patients with underlying liver disease, resulting in a high mortality rate. However, the diagnosis of hepatitis E-associated acute liver failure (HEV-ALF) currently relies primarily on a comprehensive assessment of virological tests (anti-HEV antibodies, HEV RNA) and traditional liver function and coagulation indicators (such as ALT, AST, TBIL, and INR). These indicators have significant limitations in early disease warning, risk stratification for severe illness, and prognostic assessment, lacking highly sensitive and specific biomarkers.

[0004] Glycogen phosphorylase (GP) is a key rate-limiting enzyme in glycogenolysis, catalyzing the conversion of glycogen into glucose-1-phosphate, which is involved in the body's energy supply. Human GP has three main tissue-specific subtypes: PYGL (liver type, 97 kDa), PYGM (muscle type, 97 kDa), and PYGB (brain type, 96.6 kDa). Recent studies have shown that GP not only participates in physiological metabolic regulation, but its abnormal expression is also associated with disease progression. Among them, the role of liver-type glycogen phosphorylase (PYGL) in the development and progression of hepatocellular carcinoma (HCC) has attracted attention. Studies have shown that increased PYGL expression in HCC tissues promotes tumor cell proliferation, migration, and invasion; while downregulation of PYGL expression inhibits HCC progression. Further mechanistic studies have revealed that hepatitis B virus DNA polymerase can upregulate PYGL expression, reprogram glucose metabolism pathways, enhance glycolysis, and promote liver cancer progression. Although the role of PYGL in chronic liver diseases such as hepatocellular carcinoma has been studied, its expression level and clinical value in acute liver injury, especially acute liver failure caused by hepatitis E infection, remain unclear. Currently, there are no studies reporting on the diagnosis and prognostic assessment of PYGL in hepatitis E-related acute liver failure.

[0005] Therefore, there is an urgent need in this field for a novel biomarker that can identify patients with hepatitis E-related acute liver failure early and specifically, and can effectively predict disease diagnosis and prognosis. Summary of the Invention

[0006] Technical issues The technical problem to be solved by this invention is the urgent need in the field for an objective and reliable biomarker that can identify patients with hepatitis E-related acute liver failure early and specifically, and differentiate them from acute hepatitis E (AHE), and can effectively predict the auxiliary diagnosis and prognosis of the disease.

[0007] Technical solution This invention provides a biomarker for the early auxiliary diagnosis of hepatitis E-associated acute liver failure (HEV-ALF) and its differentiation from acute hepatitis E (AHE), wherein the biomarker is serum liver glycogen phosphorylase (PYGL).

[0008] The auxiliary diagnosis refers to distinguishing between patients with hepatitis E-related acute liver failure, patients with acute hepatitis E, and relatively healthy individuals. The relatively healthy individuals refer to those who do not have AHE or HEV-ALF.

[0009] The biomarkers can provide early and accurate prediction of clinical outcomes (such as survival / death) in HEV-ALF patients.

[0010] The biomarkers can dynamically reflect the number of organ failures and disease progression in HEV-ALF patients, and are used for risk assessment and disease monitoring.

[0011] This invention provides the use of the biomarker in the preparation of products for the diagnosis and / or prognostic assessment of hepatitis E-related acute liver failure.

[0012] The product can be a diagnostic kit that detects the concentration of glycogen phosphorylase L in a subject's serum and compares it to a reference value, wherein the reference value is selected from the average serum glycogen phosphorylase L concentration of patients with acute hepatitis E. If the subject's serum glycogen phosphorylase L concentration is significantly higher than the reference value, it indicates an increased risk of the subject progressing to hepatitis E-related acute liver failure. This test can serve as an auxiliary diagnostic tool to differentiate between ordinary acute hepatitis E and hepatitis E-related acute liver failure. The diagnostic kit can be a quantitative assay kit, an ELISA diagnostic kit, or a colloidal gold kit.

[0013] The product can be a prognostic kit capable of detecting the expression level of glycogen phosphorylase L in a patient's serum sample at at least one time point during diagnosis or treatment; wherein the expression level is significantly correlated with the patient's clinical outcome; specifically, higher serum PYGL expression levels are associated with poor prognosis (such as death) and the number of organ failures; dynamic monitoring of changes in the expression level can be used to assess the evolution trend of the patient's condition. The kit can be a quantitative detection kit, an ELISA diagnostic kit, or a colloidal gold kit.

[0014] The product can also be a disease discrimination or prognostic prediction model built based on serum PYGL expression levels. This model is capable of solving binary classification problems, such as an elastic regression network model established using the biomarker. The model can also be built using multivariate statistical analysis algorithms such as orthogonal partial least squares discriminant analysis (OPLS-DA).

[0015] The product can also be a system for assisted diagnosis and prognosis of HEV-ALF, the system comprising: (1) Data acquisition module, used to collect the PYGL content in the serum sample to be tested; (2) Diagnostic module, used to input the serum sample PYGL content data collected by the data acquisition module into the orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate statistical model based on serum PYGL expression and clinical indicators, and output whether the sample to be tested may belong to HEV-ALF; (3) Prediction module, which is used to input the serum sample PYGL content data collected by the data acquisition module into the orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate statistical model based on serum PYGL expression and clinical indicators, and predict whether the sample belongs to the category of improvement, fluctuation or deterioration of the condition.

[0016] The system also includes necessary hardware such as a data processor, a keyboard or barcode reader, and a display.

[0017] Beneficial effects Compared with the prior art, the present invention has the following significant advantages and beneficial effects: 1. High diagnostic efficacy: Serum PYGL shows excellent discriminative ability in differentiating between hepatitis E-related acute liver failure and acute hepatitis E. Its area under the receiver operating characteristic curve (AUC) can reach 0.911, and its sensitivity and specificity are both higher than 85%, which are significantly better than traditional liver function indicators.

[0018] 2. Strong prognostic predictive value: Serum PYGL levels are closely associated with mortality in HEV-ALF patients and can effectively distinguish between the survival and death groups (AUC up to 0.859). High serum PYGL levels are an independent predictor of poor prognosis in patients.

[0019] 3. Reflects disease severity: Serum PYGL expression levels are associated with the number of organ failures in patients and can dynamically reflect the deterioration or improvement of the condition, providing a quantitative tool for clinical risk stratification and real-time monitoring.

[0020] 4. Providing clinical decision support: Decision curve analysis shows that the prediction model based on serum PYGL can provide positive net benefits for clinical decision-making over a wide range of threshold probabilities, and has good clinical applicability and translational potential.

[0021] 5. Innovation and Specificity: This invention reveals for the first time the clinical application value of serum PYGL in acute liver failure, especially hepatitis E-related acute liver failure, providing a novel and effective biomarker for precision medicine of hepatitis E-related acute liver failure. Attached Figure Description

[0022] Figure 1 Standard curve for serum PYGL detection.

[0023] Figure 2 A risk assessment model for the progression of acute hepatitis E to severe illness based on serum PYGL.

[0024] Figure 3 Predictive efficacy of serum PYGL in assessing the risk of severe progression of acute hepatitis E and its clinical decision-making effects.

[0025] Figure 4 A prognostic stratification model for hepatitis E-related acute liver failure based on serum PYGL.

[0026] Figure 5 Predictive efficacy of serum PYGL for prognosis of hepatitis E-related acute liver failure and clinical decision analysis.

[0027] Figure 6 Association analysis of serum PYGL expression levels with disease severity and disease progression. Detailed Implementation

[0028] The following detailed description of the technical solution, implementation process, and technical effects of the present invention, in conjunction with specific embodiments, will fully elucidate the innovativeness, practicality, and technical features of the present invention. It should be noted that the embodiments described herein are merely illustrative examples, intended to clearly demonstrate the implementation process of the present invention and assist in understanding its core content, and are not intended to limit the scope of protection of the present invention. Based on the technical principles and design concepts disclosed in this invention, any other implementation schemes achieved by those skilled in the art using conventional experimental methods or equivalent techniques without departing from the essence of the present invention should be considered to fall within the scope defined by the claims of this invention. Unless otherwise specified, the experimental methods used in the embodiments of this invention refer to standardized operating procedures in the field; all reagents, calibrators, consumables, and testing instruments and equipment involved, unless otherwise stated, are registered or filed commercially available products that can be obtained through public commercial channels.

[0029] Example 1: Differences in serum PYGL expression levels among healthy controls, patients with acute hepatitis E, and patients with hepatitis E-related acute liver failure. Ⅰ Research Subjects A total of 280 serum samples were included, comprising 80 healthy controls (HCs), 100 patients with acute hepatitis E (AHE), and 100 patients with hepatitis E-related acute liver failure (HEV-ALF). All AHE and HEV-ALF samples and their corresponding clinical information were obtained from the First Affiliated Hospital of Zhejiang University School of Medicine, and were collected before patients underwent treatment or surgery to reflect the levels of biomarkers in the natural state of the disease. The healthy control samples were obtained from Suzhou Hospital Affiliated to Nanjing Medical University, and were confirmed by routine examinations to have normal liver and kidney function and no active viral hepatitis infection.

[0030] II. Detection Procedure for Serum PYGL Serum PYGL was detected using a kit. The kit used in the following experiment was the Glycogen Phosphorylase L (PYGL) Detection Kit (96 samples / kit) manufactured by Abmart Pharmaceutical Technology (Shanghai) Co., Ltd., batch number: AB-K184901A.

[0031] 1. Sample Addition: Set up standard wells, sample wells, and blank wells. Set up 7 standard wells, adding 100 μL of standard solution of different concentrations sequentially. The standard solution concentration is 20 ng / mL (reconstitute with 1 mL of standard diluent before use, let stand at room temperature for about 10 minutes, gently stirring to avoid air bubbles). Prepare 7 EP tubes, each pre-filled with 500 μL of standard diluent. Serially dilute the reconstituted standards to obtain a series of standard concentrations of 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, and 0.312 ng / mL. Use the standard diluent (0 ng / mL) directly as a blank control, adding 100 μL to the blank wells. Add 100 μL of the sample to each of the remaining sample wells. After adding samples to all wells, cover the ELISA plate with sealing film and incubate at 37°C for 1 hour.

[0032] 2. Discard the liquid, shake dry, no washing required.

[0033] 3. Add 100 μL of working solution A (prepare immediately before use) to each well, cover the microplate with a membrane, and incubate at 37°C for 1 hour.

[0034] 4. Discard the liquid in each well. Wash each well with 350 μL of washing buffer, soaking for 1-2 minutes. Gently tap the plate on absorbent paper to remove all liquid from the wells. Repeat the washing process 3 times. After the final wash, aspirate or pour out the remaining washing buffer, invert the plate onto absorbent paper, and blot away any remaining liquid from the wells. This process can also be performed using a spray buffer, a multi-channel pipette, or an automated plate washer.

[0035] 5. Add 100 μL of detection solution B working solution (prepare immediately before use) to each well, cover the microplate with a membrane, and incubate at 37°C for 30 minutes.

[0036] 6. Discard the liquid in the well, shake dry, and wash the plate 5 times, following the same method as step 4.

[0037] 7. Add 90 μL of TMB substrate solution to each well, cover the microplate with a membrane, and incubate at 37°C in the dark (control the reaction time to 10-20 minutes, do not exceed 30 minutes. Stop the reaction when there is a clear gradient of blue in the first 3-4 standard wells and the gradient is not obvious in the last 3-4 wells).

[0038] 8. Add 50 μL of stop solution to each well to terminate the reaction. The blue color will immediately turn yellow. The stop solution should be added in the same order as the substrate solution. If uneven coloring occurs, gently shake the plate to mix the solutions thoroughly.

[0039] 9. After ensuring that there are no water droplets at the bottom of the ELISA plate and no air bubbles in the wells, immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an ELISA reader.

[0040] 10. Plot a standard curve using the measured absorbance values ​​(OD values) of the standard wells and blank wells. The results are as follows: Figure 1 As shown. The goodness of fit of this standard curve is R² > 0.99. p <0.001 indicates that the curve has a good linear relationship and significance.

[0041] III. Analysis of serum PYGL expression levels in the three groups As shown in Table 1, there were no statistically significant differences in age and sex among hepatitis E-associated acute liver failure (HEV-ALF), acute hepatitis E (AHE), and healthy controls (HCs). p >0.05). Significant differences were observed in erythrocyte distribution width (RDW), platelet count (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), total protein (TP), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), creatinine (CR), prothrombin time (PT), international normalized ratio (INR), and alpha-fetoprotein (AFP) among the three groups. p <0.05); there was no significant difference in blood urea nitrogen (UREA) among the three groups. p >0.05). Notably, serum PYGL expression levels were significantly elevated in HEV-ALF patients compared to AHE and HCs. p <0.001).

[0042] Table 1: Clinical baseline characteristics of the included subjects

[0043] Example 2: Evaluation of the predictive ability of serum PYGL for severe hepatitis E I. Clinical baseline characteristics of patients with acute hepatitis E and hepatitis E-related acute liver failure As shown in Table 2, there were no statistically significant differences in AHE and HEV-ALF between age and sex (p>0.05). RDW, PLT, ALT, AST, TP, ALB, TBIL, DBIL, CR, PT, INR, and AFP were statistically significant. p <0.05); GGT and UREA showed no statistically significant difference between the two groups ( p >0.05). Notably, serum PYGL was significantly elevated in HEV-ALF patients compared to AHE patients (p <0.001).

[0044] Table 2: Clinical baseline characteristics of patients with acute hepatitis E and hepatitis E-related acute liver failure

[0045] II. Establishing an auxiliary diagnostic model for the progression of acute hepatitis E to severe illness based on serum PYGL levels. To effectively distinguish between acute hepatitis E (AHE) and hepatitis E-associated acute liver failure (HEV-ALF), we constructed a multivariate statistical model based on serum PYGL expression and clinical indicators using orthogonal partial least squares discriminant analysis (OPLS-DA).

[0046] like Figure 2 As shown, the model exhibits excellent discriminative power, with an area under the ROC curve (AUC) as high as 0.9822. The 3D score plot visually demonstrates a clear separation between AHE and HEV-ALF patients in 3D space. Further analysis using loading plots and variable importance projection (VIP) reveals that serum PYGL has the largest loading value on the first predictive component among all variables, and its VIP value is higher than other clinical indicators. These results consistently indicate that serum PYGL is the key variable contributing the most to this discriminative model and has significant predictive value in distinguishing between AHE and HEV-ALF.

[0047] III. Evaluation of the auxiliary diagnostic efficacy of serum PYGL in patients with severe acute hepatitis E The ability of serum PYGL to distinguish between AHE and HEV-ALF was assessed using receiver operating characteristic (ROC) curve analysis. Figure 3 As shown, the area under the curve (AUC) was 0.911 (95% confidence interval: 0.869–0.953), the optimal cutoff value was 12.13 ng / ml, the diagnostic sensitivity was 89%, and the specificity was 85%. This result indicates that serum PYGL has excellent discriminatory power in distinguishing between hepatitis E progression to acute liver failure. Furthermore, its clinical applicability was evaluated through decision curve analysis. Within a wider risk threshold range, clinical decision-making based on serum PYGL to predict HEV-ALF risk can bring higher net clinical benefits to the patient population, demonstrating its significant value in assisting clinical decision-making.

[0048] Example 3: Evaluation of the predictive value of serum PYGL for the prognosis of patients with hepatitis E-associated acute liver failure (HEV-ALF) I. Clinical baseline characteristics of the survival and death groups To assess the predictive value of serum PYGL concentration for the prognosis of HEV-ALF patients, we defined HEV-ALF patients into survival and death groups based on clinical prognostic outcomes, and then measured the patients' serum PYGL concentration levels. As shown in Table 3, there was no significant difference in gender between the death and survival groups (p>0.05), but age showed a significant difference between the two groups (p<0.001). Significant differences were found in RDW, ALT, TP, ALB, TBIL, DBIL, UREA, PT, INR, and AFP. p <0.05); PLT, AST, GGT, and CR showed no statistical significance. p >0.05). Notably, serum PYGL levels were significantly higher in the death group compared to the survival group ( p <0.001).

[0049] Table 3: Clinical baseline characteristics of patients in the survival and death groups

[0050] II. Establishment of a prognostic prediction model for hepatitis E-related acute liver failure based on serum PYGL One hundred HEV-ALF patients were divided into a survival group (n = 78) and a death group (n = 22) based on their clinical outcomes. We constructed a prognostic model using orthogonal partial least squares discriminant analysis (OPLS-DA) with serum PYGL expression levels at admission as the key variable, combined with routine clinical indicators. Figure 4 As shown, the model exhibits excellent discriminative power, with an area under the ROC curve (AUC) as high as 0.950. The 3D score plot visually demonstrates a clear separation between the survival and death groups in 3D space. Further analysis using loading plots and variable importance projection (VIP) reveals that serum PYGL has the largest loading value on the first predictive component among all variables, and its VIP value is significantly higher than other clinical indicators. These results consistently indicate that serum PYGL is the key variable contributing the most to this discriminative model and has significant predictive value in distinguishing between the survival and death groups.

[0051] III. Independent predictive value of serum PYGL in assessing mortality risk in patients with hepatitis E-related acute liver failure To quantitatively assess the predictive ability of serum PYGL for mortality in HEV-ALF patients, receiver operating characteristic (ROC) curve analysis was performed. Figure 5As shown, the area under the curve (AUC) for serum PYGL in predicting mortality in HEV-ALF patients was 0.859 (95% confidence interval: 0.747–0.971), with a sensitivity of 77.3% and a specificity of 92.3%. Further decision curve analysis was used to evaluate its clinical applicability. Within a wide risk threshold range, clinical decision-making based on serum PYGL prediction of HEV-ALF mortality risk can bring higher net clinical benefits to the patient population, demonstrating its significant value in assisting clinical decision-making.

[0052] IV. Correlation between serum PYGL levels and organ failure severity and disease progression HEV-ALF patients were divided into different subgroups based on the number of organ failures (2, 3, >3 organ failures). For example... Figure 6 As shown, serum PYGL expression levels increased significantly with the number of organ failures. Specifically, the serum PYGL levels in patients with >3 organ failures were significantly higher than those in the 3-organ failure and 2-organ failure groups. This result indicates that serum PYGL levels can reflect the severity of multi-organ dysfunction in HEV-ALF patients.

[0053] To evaluate the application value of serum PYGL in disease monitoring, serum PYGL was measured in HEV-ALF patients. Based on the overall clinical progression during this period, patients were divided into improvement, fluctuation, and deterioration groups. Data analysis showed that patients in the deterioration group experienced a sustained increase or maintenance of high serum PYGL levels throughout the disease course; while patients in the improvement group showed a significant decreasing trend in serum PYGL levels; and patients in the fluctuation group experienced changes in serum PYGL levels between the two groups. This indicates that the dynamic pattern of serum PYGL changes can reflect the direction of disease progression in HEV-ALF patients.

[0054] Example 4: System for HEV-ALF Diagnosis and Prognosis The system includes: (1) Data acquisition module, used to collect the PYGL content in the serum sample to be tested; (2) Diagnostic module, used to input the serum sample PYGL content data collected by the data acquisition module into the orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate statistical model based on serum PYGL expression and clinical indicators, and output whether the sample to be tested belongs to HEV-ALF; (3) Prediction module, which is used to input the serum sample PYGL content data collected by the data acquisition module into the orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate statistical model based on serum PYGL expression and clinical indicators, and predict whether the sample belongs to the category of improvement, fluctuation or deterioration of the condition.

[0055] The system also includes necessary hardware such as a data processor, a keyboard or barcode reader, and a display.

[0056] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Application of serum glycogen phosphorylase L as a biomarker for the auxiliary diagnosis and / or prognostic assessment of hepatitis E-related acute liver failure.

2. The application according to claim 1, characterized in that, The auxiliary diagnosis refers to distinguishing between patients with hepatitis E-related acute liver failure, patients with acute hepatitis E, and relatively healthy individuals.

3. The application according to claim 2, characterized in that, The auxiliary diagnosis includes detecting the concentration of liver glycogen phosphorylase in the serum of the subject and comparing it with the concentration in patients with acute hepatitis E and healthy controls, wherein the concentration of liver glycogen phosphorylase in the serum of patients with hepatitis E-related acute liver failure is significantly higher than that in patients with acute hepatitis E and healthy controls.

4. The application according to claim 1, characterized in that, The prognostic assessment includes predicting the clinical outcome of patients with hepatitis E-related acute liver failure based on serum glycogen phosphorylase expression levels.

5. The application according to claim 4, characterized in that, The expression level of serum liver glycogen phosphorylase was significantly correlated with the number of patients with organ failure.

6. The application according to claim 4, characterized in that, The dynamic changes in serum liver glycogen phosphorylase expression levels are correlated with the severity of the patient's condition.

7. The application according to claim 1, characterized in that, A kit for assisting in the diagnosis and / or prognostic assessment of hepatitis E-related acute liver failure was prepared using the biomarkers.

8. The application according to claim 7, characterized in that, The kits are quantitative detection kits, ELISA diagnostic kits, and colloidal gold kits.

9. Products prepared using serum glycogen phosphorylase L as a biomarker for the auxiliary diagnosis and / or prognostic assessment of hepatitis E-related acute liver failure.

10. The product according to claim 9, characterized in that, include: Reagent kits, models, and diagnostic systems.

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