Marker, kit and prediction method for predicting treatment effect of autoimmune hepatitis

Abstract

The invention discloses a marker, a kit and a prediction method for predicting the treatment effect of AIH (autoimmune hepatitis), the marker is JUNB protein, and the identifier of the JUNB protein in a UniProt database is UniProtID: P17535; the kit is used for detecting the JUNB protein. According to the method, the expression level of JUNB in the PBMC is detected, under the determined cut-off value, a complete biochemical response group and an insufficient response group of an AIH standard treatment scheme are efficiently and accurately distinguished, and objective and accurate data support is provided for clinicians to early recognize refractory patients and timely adjust treatment strategies. According to the present invention, with the application of the kit to predict the refractory patient of AIH, the accuracy is high, the missed diagnosis rate is zero, the sensitivity of the prediction method is high, the clinical missed diagnosis risk on the refractory patient is eliminated, and the safety and the reliability are high.

Description

Technical Field

[0001] This invention relates to a biomarker, reagent kit, and prediction method for predicting the treatment efficacy of autoimmune hepatitis (AIH). Background Technology

[0002] Autoimmune hepatitis (AIH) is an immune-mediated, chronic, progressive inflammatory disease of the liver, clinically characterized by elevated serum transaminases, hyperimmunoglobulin G (IgG) hyperemia, and the presence of autoantibodies. Currently, the standard first-line treatment for AIH primarily relies on immunosuppressive therapy using corticosteroids (such as prednisone or prednisolone) alone or in combination with azathioprine.

[0003] The primary goal of clinical treatment is to achieve a "complete biochemical response," meaning that serum transaminase (ALT / AST) and IgG levels return to normal. Numerous studies have shown that patients achieving a complete biochemical response have significantly better outcomes in terms of liver inflammation resolution, fibrosis reversal, and long-term survival than those with an "inadequate response." Therefore, in-depth analysis of the patient's immune status using samples such as peripheral blood mononuclear cells (PBMCs) in the early stages of treatment or at the diagnostic stage is crucial for accurately differentiating the patient's potential response to treatment. This is of paramount clinical importance for developing individualized treatment plans and improving patient prognosis.

[0004] Currently, the diagnosis of AIH mainly relies on autoantibody testing, including: antinuclear antibodies (ANA) and anti-smooth muscle antibodies (ASMA): used for the diagnosis of type I AIH; anti-liver and kidney microsomal antibodies (LKM): the core marker of type II AIH; and anti-soluble liver antigen / hepatopancreatic antigen antibodies (SLA / LP): a highly specific marker of type III AIH. These antibodies have been clinically validated and are the main basis for diagnosing AIH.

[0005] Although existing standardized treatment regimens are effective for most AIH patients, in clinical practice, some patients still exhibit "incomplete response" or no response to treatment. Current techniques for assessing AIH treatment response mainly suffer from the following problems:

[0006] ① Lag in assessment indicators: Currently, clinical practice mainly relies on retrospectively assessing efficacy by monitoring the decline trend of serum biochemical indicators (such as ALT, AST, and IgG) after a period of treatment (usually 3-6 months or even longer). This "trial and error" method has a significant lag and cannot predict whether a patient belongs to the treatment-resistant (i.e., inadequate response) group at the initial stage of treatment or at the time of diagnosis. ② Lack of precise molecular markers: Existing assessment systems lack specific molecular markers based on pathogenesis (such as specific protein expression levels). Although some studies focus on cytokine levels, their short half-life and large fluctuations make them difficult to use as stable predictive indicators. ③ Limitations of invasive examinations: Although liver biopsy is the gold standard for assessing liver inflammation and fibrosis, it is invasive, carries the risk of complications such as bleeding, and makes it difficult to dynamically monitor treatment response through repeated sampling.

[0007] To overcome the aforementioned technical limitations, current methods primarily employ combined scoring systems based on clinical biochemical indicators (such as models based on pre-treatment bilirubin and transaminase levels) or detection based on single immune cell subsets (such as the proportion of Treg cells) to assess the effectiveness of treatment regimens for AIH patients. However, these techniques also have the following drawbacks:

[0008] ① Insufficient sensitivity and specificity: Relying solely on baseline levels of routine biochemical indicators makes it difficult to accurately distinguish between the "complete response" and "insufficient response" groups, as the biochemical manifestations of the two groups at the onset of the disease largely overlap, resulting in a typically low area under the ROC curve (AUC) of the predictive model and a high false positive rate. ② Inability to reflect core immune pathways: AIH is essentially a disruption of immune tolerance. Existing crude indicators cannot reflect the key differences of specific transcription factors (such as AP-1 family members) in the immune regulatory network, thus making it difficult to reveal the deep biological mechanisms underlying the differences in response. Summary of the Invention

[0009] The purpose of this invention is to address the problem that existing technologies cannot accurately distinguish between patients with "complete biochemical response" and "insufficient response" in the early stages of autoimmune hepatitis (AIH) treatment, resulting in some refractory patients not receiving timely intensive treatment and having poor prognosis. This invention provides a biomarker, a detection kit, and a prediction method for predicting the treatment effect of autoimmune hepatitis (AIH).

[0010] This invention detects the expression level of the biomarker JUNB protein in peripheral blood mononuclear cells (PBMCs) and establishes a highly sensitive and specific predictive model using a specific cutoff value. This model enables accurate prediction of treatment response in AIH patients and can be used to screen high-risk AIH patients (i.e., those with inadequate drug response) who require more aggressive immunosuppressive therapy. It also serves as a biological marker for patient stratification in drug clinical trials.

[0011] According to a first aspect of the present invention, a biomarker for predicting the therapeutic effect of treating AIH with a standard treatment regimen is provided, wherein the biomarker is JUNB protein in peripheral blood mononuclear cells of AIH patients before treatment, and the identifier of JUNB protein in the UniProt database is UniProtID:P17535; wherein AIH is autoimmune hepatitis.

[0012] Preferably, if the cutoff value of the relative expression level of JUNB protein in peripheral blood mononuclear cells of AIH patients before treatment is ≥13.715, then AIH patients are determined to achieve "complete biochemical response" if treated with the standard treatment regimen; if the cutoff value of the relative expression level of JUNB protein in peripheral blood mononuclear cells of AIH patients before treatment is <13.715, then AIH patients are determined to achieve "insufficient response" if treated with the standard treatment regimen; the standard treatment regimen is the standard glucocorticoid combined with azathioprine treatment regimen.

[0013] According to a second aspect of the present invention, a kit for predicting the therapeutic effect of treating AIH with a standard treatment regimen includes a substance for detecting the expression level of JUNB protein in peripheral blood mononuclear cells of AIH patients before treatment; the identifier of the JUNB protein in the UniProt database is UniProtID:P17535; and the AIH is autoimmune hepatitis.

[0014] Preferably, the substances include: lymphocyte separation solution, cell lysis buffer, PBS buffer, trypsin, urea, dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate buffer, formic acid, acetonitrile, and BCA protein concentration assay reagent.

[0015] Preferably, the detection of JUNB protein expression level includes:

[0016] Peripheral blood mononuclear cells (PBMCs) were obtained by separating and washing peripheral blood from AIH patients using lymphocyte separation medium and PBS buffer.

[0017] PBMCs were lysed using cell lysis buffer to obtain total protein extract;

[0018] Impurities in the total protein extract were removed by acetone precipitation, then urea was added to dissolve the protein precipitate, and BCA protein concentration was quantified using BCA protein assay reagent.

[0019] Dithiothreitol was added to the protein sample for BCA protein quantification to reduce disulfide bonds;

[0020] Then, after cooling, iodoacetamide is added to carry out the alkylation reaction;

[0021] Then add ammonium bicarbonate buffer to dilute the urea concentration to below 1M;

[0022] Next, sequencing-grade trypsin was added, and enzymatic hydrolysis was performed at an enzyme:protein ratio of 1:50 (w / w) to obtain a peptide mixture;

[0023] The reconstituted peptide mixture was detected using a liquid chromatography-tandem mass spectrometry system to obtain raw mass spectrometry data of the peptide mixture;

[0024] The relative expression level of JUNB protein in patients was obtained by processing the short mixed mass spectrometry data using MaxQuant software.

[0025] Preferably, if the relative expression level of JUNB protein in the patient is ≥ cutoff value 13.715, then the patient is predicted to have a “complete biochemical response” if treated with the standard treatment regimen; otherwise, the patient is predicted to have an “inadequate response” if treated with the standard treatment regimen; wherein the standard treatment regimen is the standard glucocorticoid combined with azathioprine treatment regimen.

[0026] Preferably, the lymphocyte separation solution is Ficoll separation solution.

[0027] Preferably, the cell lysis buffer is a RIPA lysis buffer containing a protease inhibitor.

[0028] Preferably, PBMCs are lysed using RIPA cell lysis buffer containing protease inhibitors under the following conditions: lysis on ice for 30 min, with vortexing every 10 min during the lysis; after lysis, centrifugation is performed, and the supernatant is collected to obtain the total protein extract.

[0029] Preferably, the protease inhibitor is one or more of benzyl sulfonyl fluoride, aprotinin, leucine, or a mixture of protease inhibitors.

[0030] According to a third aspect of the present invention, a method for predicting the therapeutic effect of treating AIH with a standard treatment regimen; comprising:

[0031] Detection of JUNB protein expression levels in peripheral blood mononuclear cells of AIH patients before treatment;

[0032] If the relative expression level of JUNB protein in a patient is ≥13.715, then the patient is predicted to have a “complete biochemical response” if treated with standard therapy for AIH; otherwise, the patient is predicted to have an “inadequate response” if treated with standard therapy for AIH.

[0033] The identifier of the JUNB protein in the UniProt database is UniProtID:P17535; the standard treatment regimen is the standard glucocorticoid combined with azathioprine treatment regimen.

[0034] Preferably, the detection of JUNB protein expression level includes:

[0035] Peripheral blood mononuclear cells (PBMCs) were obtained by separating and washing peripheral blood from AIH patients using lymphocyte separation medium and PBS buffer.

[0036] PBMCs were lysed using cell lysis buffer to obtain total protein extract;

[0037] Impurities in the total protein extract were removed by acetone precipitation, then urea was added to dissolve the protein precipitate, and BCA protein concentration was quantified using BCA protein assay reagent.

[0038] Dithiothreitol was added to the protein sample for BCA protein quantification to reduce disulfide bonds;

[0039] Then, after cooling, iodoacetamide is added to carry out the alkylation reaction;

[0040] Then add ammonium bicarbonate buffer to dilute the urea concentration to below 1M;

[0041] Next, sequencing-grade trypsin was added, and enzymatic hydrolysis was performed at an enzyme:protein ratio of 1:50 (w / w) to obtain a peptide mixture;

[0042] The reconstituted peptide mixture was detected using a liquid chromatography-tandem mass spectrometry system to obtain raw mass spectrometry data of the peptide mixture;

[0043] The relative expression level of JUNB protein in patients was obtained by processing the short mixed mass spectrometry data using MaxQuant software.

[0044] This invention detects the expression abundance of JUNB in ​​PBMCs and, at a defined cutoff value, efficiently distinguishes between groups with complete biochemical responses and those with inadequate responses, thereby providing objective and accurate data support for clinicians to identify refractory patients early and adjust treatment strategies in a timely manner.

[0045] This invention confirms that decreased JUNB protein expression levels in PBMCs are an independent risk factor for inadequate response to standard treatment in patients with autoimmune hepatitis. By setting a threshold of 13.715, this invention provides a non-invasive detection method that can effectively identify patients with refractory AIH, which has important clinical guiding significance.

[0046] The core of the standard treatment regimen for AIH (autoimmune hepatitis) is immunosuppressive therapy, which includes single-dose glucocorticoid therapy (such as prednisone) and glucocorticoid therapy (such as prednisone) combined with azathioprine. The standard for treatment efficacy is the return of serum transaminase (ALT / AST) and IgG levels to the normal range.

[0047] The JUNB protein biomarker of this invention is used to predict patients with AIH refractory. A precise quantitative cutoff value (Cut off = 13.715) transforms ambiguous clinical judgments into objective digital indicators. Standardized data results can be obtained through proteomics or subsequently developed immunological detection methods (such as ELISA and flow cytometry), reducing errors from human experience-based judgments. This facilitates widespread application in medical institutions at different levels, providing doctors with accurate judgment criteria and avoiding the difficulty doctors often face in determining whether escalation of treatment is necessary for patients in a "gray area" (such as those with mildly elevated but not yet normalized transaminase levels), as is often the case in existing technologies.

[0048] Compared with the prior art, the present invention has the following significant advantages and beneficial effects:

[0049] 1. The JUNB protein biomarker for predicting the treatment efficacy of autoimmune hepatitis (AIH) of this invention exhibits excellent diagnostic efficacy and extremely high sensitivity, solving the technical problem that existing methods relying solely on baseline biochemical indicators (such as ALT, AST, and IgG) are insufficient to accurately predict the treatment prognosis of AIH. The predictive accuracy is high. The predictive model constructed using the JUNB protein has a receiver operating characteristic (AUC) as high as 0.95, significantly superior to traditional clinical scoring models. The overall accuracy of the JUNB biomarker in identifying and distinguishing patients with complete biochemical responses from those with inadequate responses is demonstrated. In this invention, AUC = 0.95 (95% CI: 0.88–1.00).

[0050] 2. The kit for predicting the treatment effect of standard AIH treatment using the present invention has a zero false negative rate. At the cutoff value (13.715) for determining "high risk" and "low risk," the kit achieves 100% sensitivity, indicating that patients with JUNB expression levels below this threshold are accurately identified as high-risk individuals with "insufficient response." This characteristic significantly reduces the risk of missed diagnosis for refractory patients in clinical practice, demonstrating extremely high safety and reliability. Furthermore, it has a high exclusion rate for "complete response" patients, i.e., a low false positive rate, and a specificity as high as 0.80 (80%), effectively avoiding missed diagnoses of refractory cases in clinical applications.

[0051] 3. The use of biomarkers in this invention to predict the prognosis of standard treatments for AIH patients represents a breakthrough from "retrospective assessment" to "prospective prediction," solving the problem of "lag" in treatment effect evaluation. Treatment efficacy can be determined before treatment. If the patient is identified as having AIH refractory disease, the treatment plan can be adjusted in a timely manner to carry out intensive treatment, avoiding patients with poor response from missing the optimal window for early adjustment and improving efficacy.

[0052] Traditional AIH efficacy assessments typically require waiting 6 months or even longer after treatment before evaluation can be conducted. However, with the biomarkers and reagent kits of this invention, accurate judgment and assessment can be performed before treatment.

[0053] 4. By examining the expression level of the marker JUNB protein in peripheral blood PBMCs of AIH patients, the patient's response potential can be predicted immediately at the initial stage of treatment or even at the time of AIH diagnosis. For patients with low JUNB expression (<13.715), clinicians can provide early warning and directly initiate second-line treatment regimens (such as mycophenolate mofetil, tacrolimus, etc.) or increase the frequency of follow-up, thereby truly achieving "personalized precision treatment" for AIH and avoiding the waste of time and the risk of disease progression caused by ineffective treatment.

[0054] 5. The biomarkers and detection kits of the present invention also provide a non-invasive detection method based on the pathogenesis, which solves the problem of "difficulty in obtaining materials" in the existing gold standard for AIH detection (i.e., liver tissue biopsy).

[0055] 6. Compared with the traditional "gold standard" of liver biopsy, this invention has significant non-invasive advantages. The kit of this invention is used to detect the expression level of the biomarker JUNB protein. Only peripheral blood of the patient needs to be collected and PBMCs separated, avoiding the risks of bleeding, infection and pain caused by liver puncture, and the patient compliance is good.

[0056] 7. The biomarker JUNB protein of this invention directly addresses the immune cell level, which is more effective than simply detecting enzyme indicators in the blood in understanding the essence of AIH immune tolerance imbalance. Compared with non-specific inflammatory factors in serum, PBMCs (peripheral blood mononuclear cells) serve as the core carriers of the immune response, and the expression level of their internal transcription factor (JUNB) can more directly reflect the patient's overall immune regulation status. Attached Figure Description

[0057] Figure 1 A statistical analysis of JUNB protein expression levels in PBMC samples from patients with autoimmune hepatitis (AIH);

[0058] Figure 2Receiver operating characteristic (ROC) curves for predicting treatment response in AIH patients using JUNB protein expression levels. Detailed Implementation

[0059] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0060] This invention, through proteomics screening and validation in clinical samples, reveals for the first time that the expression level of JUNB protein in peripheral blood mononuclear cells (PBMCs) of patients with autoimmune hepatitis is closely related to treatment response. JUNB protein is a transcription factor encoded by the JUNB gene, a core component of the AP-1 complex, and participates in the regulation of cell cycle, proliferation, differentiation, and inflammatory responses. JUNB protein is a nuclear-localized protein with a molecular weight of approximately 35.03 kD, composed of 347 amino acids, possessing hydrophilicity and three potential glycosylation sites; its coding sequence (CDS region) is 1044 base pairs long and corresponds to the NCBI number NM_002229.

[0061] Through research, the inventors discovered that the JUNB protein, by regulating DOCK2 gene expression, affects the migration and infiltration of pathogenic CD4⁺ T cells, thereby participating in the progression of AIH. This mechanism provides a molecular basis for understanding the disease's pathogenesis.

[0062] Description of the main reagents and instruments used in this invention:

[0063] Lymphocyte separation medium (Ficoll-PaquePLUS, Ficoll separation medium): purchased from Cytiva.

[0064] Cell lysate (RIPA) and protease inhibitors were purchased from Thermo Fisher Scientific.

[0065] BCA protein concentration assay kit: purchased from Beyotime.

[0066] Sequencing-grade trypsin: purchased from Promega.

[0067] Liquid chromatography-tandem mass spectrometry (LC-MS / MS): ThermoFisher QExactive HF-X (or a similar high-resolution mass spectrometer).

[0068] Data analysis software: MaxQuant (v1.6) and GraphPadPrism 9.0.

[0069] Note: The reagents and instruments described in this embodiment are all commercially available products. The manufacturers listed are only the brands actually used in this embodiment and are not intended to limit the invention. Those skilled in the art can implement this invention using other brands of products with equivalent performance.

[0070] On the one hand, the present invention provides a biomarker for predicting the therapeutic effect of standard treatment for autoimmune hepatitis (AIH). The biomarker is JUNB protein in peripheral blood mononuclear cells of AIH patients before treatment. The identifier of JUNB protein in the UniProt database is UniProtID:P17535, corresponding to NCBI number NM_002229.

[0071] Specifically, if the cutoff value of the relative expression level of JUNB protein in peripheral blood mononuclear cells of AIH patients before treatment is ≥13.715, then AIH patients are considered to have achieved a "complete biochemical response" if treated with the standard treatment regimen; if the cutoff value of the relative expression level of JUNB protein in peripheral blood mononuclear cells of AIH patients before treatment is <13.715, then AIH patients are considered to have achieved an "inadequate response" if treated with the standard treatment regimen; the standard treatment regimen is the standard glucocorticoid combined with azathioprine treatment regimen.

[0072] On the other hand, the present invention provides a kit for predicting the therapeutic effect of treating AIH with standard treatment regimens, including a substance for detecting the expression level of JUNB protein in peripheral blood mononuclear cells of AIH patients before treatment; the identifier of JUNB protein in the UniProt database is UniProtID:P17535, corresponding to NCBI number NM_002229.

[0073] The substances used in this invention for detecting the expression level of JUNB protein in peripheral blood mononuclear cells of AIH patients before treatment include: lymphocyte separation medium, cell lysis buffer (RIPA lysis buffer), PBS buffer, trypsin, urea, dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate buffer, formic acid, acetonitrile, and BCA protein concentration assay reagent.

[0074] The present invention detects JUNB protein expression levels by including:

[0075] Peripheral blood mononuclear cells (PBMCs) were obtained by separating and washing peripheral blood from AIH patients using lymphocyte separation medium and PBS buffer.

[0076] PBMCs were lysed using cell lysis buffer to obtain total protein extract;

[0077] Impurities in the total protein extract were removed by acetone precipitation, then urea was added to dissolve the protein precipitate, and BCA protein concentration was quantified using BCA protein assay reagent.

[0078] Dithiothreitol was added to the protein sample for BCA protein quantification to reduce disulfide bonds;

[0079] Then, after cooling, iodoacetamide is added to carry out the alkylation reaction;

[0080] Then add ammonium bicarbonate buffer to dilute the urea concentration to below 1M;

[0081] Next, sequencing-grade trypsin was added, and enzymatic hydrolysis was performed at an enzyme:protein ratio of 1:50 (w / w) to obtain a peptide mixture;

[0082] The reconstituted peptide mixture was detected using a liquid chromatography-tandem mass spectrometry system to obtain raw mass spectrometry data of the peptide mixture;

[0083] The relative expression level of JUNB protein in patients was obtained by processing the short mixed mass spectrometry data using MaxQuant software.

[0084] If the relative expression level of JUNB protein in a patient is ≥ cutoff value 13.715, then the patient is predicted to have a “complete biochemical response” if treated with the standard treatment regimen; otherwise, the patient is predicted to have an “inadequate response” if treated with the standard treatment regimen. The standard treatment regimen is the standard glucocorticoid combined with azathioprine treatment regimen.

[0085] The lymphocyte separation medium was Ficoll separation medium. The cell lysis buffer was RIPA lysis buffer containing a protease inhibitor. In this invention, PBMCs were lysed using RIPA cell lysis buffer containing a protease inhibitor under the following conditions: lysis on ice for 30 min, with vortexing every 10 min; after lysis, centrifugation was performed, and the supernatant was collected to obtain the total protein extract. The protease inhibitor was one or more of benzyl sulfonyl fluoride, aprotinin, leucine, or a mixture of protease inhibitors.

[0086] Furthermore, this invention provides a method for predicting the therapeutic effect of treating AIH with a standard treatment regimen; comprising:

[0087] Detection of JUNB protein expression levels in peripheral blood mononuclear cells of AIH patients before treatment;

[0088] If the relative expression level of JUNB protein in a patient is ≥13.715, then the patient is predicted to have a “complete biochemical response” if treated with standard therapy for AIH; otherwise, the patient is predicted to have an “inadequate response” if treated with standard therapy for AIH.

[0089] The identifier of the JUNB protein in the UniProt database is UniProtID:P17535; the standard treatment regimen is the standard glucocorticoid combined with azathioprine treatment regimen.

[0090] Preferably, the detection of JUNB protein expression level includes:

[0091] Peripheral blood mononuclear cells (PBMCs) were obtained by separating and washing peripheral blood from AIH patients using lymphocyte separation medium and PBS buffer.

[0092] PBMCs were lysed using cell lysis buffer to obtain total protein extract;

[0093] Impurities in the total protein extract were removed by acetone precipitation, then urea was added to dissolve the protein precipitate, and BCA protein concentration was quantified using BCA protein assay reagent.

[0094] Dithiothreitol was added to the protein sample for BCA protein quantification to reduce disulfide bonds;

[0095] Then, after cooling, iodoacetamide is added to carry out the alkylation reaction;

[0096] Then add ammonium bicarbonate buffer to dilute the urea concentration to below 1M;

[0097] Next, sequencing-grade trypsin was added, and enzymatic hydrolysis was performed at an enzyme:protein ratio of 1:50 (w / w) to obtain a peptide mixture;

[0098] The reconstituted peptide mixture was detected using a liquid chromatography-tandem mass spectrometry system to obtain raw mass spectrometry data of the peptide mixture;

[0099] The relative expression level of JUNB protein in patients was obtained by processing the short mixed mass spectrometry data using MaxQuant software.

[0100] In a specific example, detecting the JUNB protein expression level in peripheral blood mononuclear cells of AIH patients before treatment includes the following steps:

[0101] 1) PBMC separation: Peripheral blood from AIH patients was stacked on Ficoll separation solution and centrifuged horizontally at room temperature; the intermediate white membrane layer after centrifugation was aspirated to obtain peripheral blood mononuclear cells (PBMCs); then the obtained PBMCs were washed with PBS buffer.

[0102] 2) Protein extraction: PBMCs were lysed using RIPA cell lysis buffer containing protease inhibitors. Lysis conditions: Lysis was performed on ice for 30 min, with vortexing every 10 min. After lysis, the cells were centrifuged (4℃, 14,000×g, 15 min), and the supernatant was collected to obtain the total protein extract.

[0103] 3) Proteolytic digestion and pretreatment (key step):

[0104] Impurities in the total protein extract were removed by acetone precipitation.

[0105] Add 8M urea to dissolve the protein precipitate and perform BCA protein quantification;

[0106] Dithiothreitol (DTT) was added to reduce disulfide bonds (56℃, 30 min).

[0107] After cooling, iodoacetamide (IAA) was added to carry out the alkylation reaction (room temperature, protected from light, 30 min);

[0108] Dilute the urea concentration to below 1M by adding ammonium bicarbonate buffer (NH4HCO3);

[0109] Add sequencing-grade trypsin at a ratio of enzyme:protein = 1:50 (w / w) and hydrolyze overnight (12-16 hours) at 37°C to obtain a peptide mixture.

[0110] 4) Desalting treatment: The enzymatically hydrolyzed peptide mixture was desalted using a C18 desalting column and then vacuum dried.

[0111] 5) LC-MS / MS Detection: The reconstituted peptide mixture was detected using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system. Chromatographic conditions: A Nano-LC system was used, with mobile phase A being 0.1% formic acid aqueous solution and mobile phase B being 0.1% formic acid acetonitrile solution. Linear gradient elution was used for 60 minutes (5% B phase to 35% B phase), at a flow rate of 300 nL / min. Mass spectrometry conditions: Data-dependent acquisition (DDA) mode was used. The primary mass spectrometry scan range was 350–1600 m / z with a resolution of 60,000; the secondary mass spectrometry resolution was 15,000.

[0112] 6) Data Analysis and Judgment: The raw mass spectrometry data were imported into MaxQuant software and searched using the Uniprot human proteome database. Parameter settings: The fixed modification was cysteine ​​carbamoyl methylation, and the variable modification was methionine oxidation. Quantitative calculation: A label-free algorithm was used to obtain the LFQ intensity value of JUNB protein in the sample, and the intensity value was log2 transformed to obtain the final relative expression level of JUNB protein. Result judgment: The obtained relative expression level of JUNB protein in this patient was compared with the cutoff value of 13.715: if ≥13.715, it was judged as "adequate response"; if <13.715, it was judged as "insufficient response".

[0113] Example 1: Screening and grouping of clinical research subjects

[0114] This embodiment describes the source and grouping criteria of the clinical samples required for this invention.

[0115] Inclusion criteria: Treatment-naïve patients diagnosed with autoimmune hepatitis (AIH) who were treated at Peking University First Hospital between January 2021 and January 2025 were included. All patients met the diagnostic criteria of the International Autoimmune Hepatitis Group (IAIHG). Exclusion criteria: Patients with concurrent viral hepatitis, drug-induced liver injury, alcoholic liver disease, primary biliary cholangitis (PBC) overlap syndrome, and other autoimmune diseases were excluded.

[0116] Grouping criteria: All patients received standard glucocorticoid combined with azathioprine (i.e., standard treatment regimen) and were followed up for 6 months. Based on the biochemical indicators at 6 months of treatment, patients were retrospectively divided into two groups:

[0117] Complete Biochemical Response (CBR) group: defined as serum transaminase (ALT and AST) and IgG levels returning to the normal reference range after 6 months of standard treatment, including 15 cases in the CBR group.

[0118] Incomplete Response (IR) group: defined as those whose serum transaminase or IgG levels failed to fully return to normal or did not meet the above CBR criteria after 6 months of standard treatment, including 15 cases in the IR group.

[0119] Sample collection: Before the patient receives treatment (i.e., at baseline), collect 5-10 mL of fasting peripheral venous blood in the morning and place it in a blood collection tube containing EDTA anticoagulant (to obtain anticoagulated peripheral blood). Store at 4°C and perform subsequent PBMC separation within 4 hours.

[0120] Six months after the patient received treatment, 5-10 mL of fasting peripheral venous blood was collected in the morning and placed in a blood collection tube containing EDTA anticoagulant (to obtain anticoagulated peripheral blood). The blood was stored at 4°C and PBMCs were separated within 4 hours.

[0121] Example 2: Isolation and protein extraction from peripheral blood mononuclear cells (PBMCs)

[0122] 1. PBMC separation:

[0123] The collected anticoagulated peripheral blood was diluted with sterile PBS buffer at a 1:1 volume ratio. 3 mL of Ficoll separation buffer was pre-added to a 15 mL centrifuge tube. The diluted blood sample was then slowly stacked along the tube wall onto the surface of the separation buffer, maintaining a clear interface.

[0124] Centrifuge at 400×g for 25 minutes at room temperature. After centrifugation, aspirate the white membrane layer (i.e., the PBMC cell layer) located between the plasma layer and the separation liquid layer.

[0125] Add 5 times the volume of PBS buffer to the PBMC cell layer, resuspend the PBMC cells, centrifuge at 300×g for 10 minutes, wash twice, discard the supernatant, and obtain the PBMC cell pellet.

[0126] 2. Protein extraction and quantification:

[0127] Add RIPA lysis buffer containing protease inhibitors to the PBMC cell pellet. Incubate on ice for 30 minutes, vortexing every 10 minutes. Centrifuge at 14,000×g, 4°C for 15 minutes, and collect the supernatant as the total protein extract.

[0128] Protein concentration was determined using a BCA kit. The protein concentration of each sample was adjusted to a uniform level, aliquoted, and stored at -80℃ for later use.

[0129] Example 3: Proteomics detection and JUNB identification based on LC-MS / MS

[0130] This embodiment describes the specific technical process for determining the expression level of JUNB protein.

[0131] 1. Protein hydrolysis:

[0132] Take an equal amount of protein (e.g., 100 μg) from each group of samples, remove impurities using acetone precipitation; add 8M urea to dissolve the protein precipitate, add dithiothreitol (DTT) to reduce disulfide bonds (56℃, 30 min); then add iodoacetamide (IAA) for alkylation reaction (room temperature, protected from light, 30 min); add sequencing-grade trypsin, and enzymatically hydrolyze overnight (12-16 hours) at 37℃ at an enzyme:protein ratio of 1:50 (w / w) to obtain a peptide mixture;

[0133] 2. Desalination treatment:

[0134] The enzymatically hydrolyzed peptide mixture was desalted using a C18 desalting column and then vacuum-dried to obtain desalted peptides.

[0135] 3. Liquid chromatography-mass spectrometry analysis:

[0136] The dried peptide fragments were reconstituted in 0.1% formic acid solution and then analyzed by liquid chromatography and mass spectrometry, wherein:

[0137] Liquid chromatography conditions: A Nano-LC system was used, with mobile phase A being 0.1% formic acid aqueous solution and mobile phase B being 0.1% formic acid acetonitrile solution. Linear gradient elution was performed for 60 minutes (from 5% B phase to 35% B phase) at a flow rate of 300 nL / min.

[0138] Mass spectrometry conditions: Mass spectrometry analysis was performed using data-dependent acquisition mode (DDA), with the first-stage mass spectrometry scanning range of 350-1600 m / z and a resolution of 60,000; and the second-stage mass spectrometry resolution of 15,000.

[0139] 4. Data retrieval and quantification:

[0140] Label-free quantitative analysis of processed peptides was performed using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system. The relative expression levels (intensities) of JUNB protein in each sample were obtained through database retrieval and bioinformatics analysis.

[0141] The raw mass spectrometry data were imported into MaxQuant software and searched using the Uniprot human proteome database. The fixed modification was set to cysteine ​​carbamoyl methylation, and the variable modification was set to methionine oxidation. Then, the relative abundance (intensity) of JUNB protein in each sample was calculated using the label-free quantification (LFQ) algorithm.

[0142] Example 4: Data Analysis

[0143] Differential expression analysis and results of JUNB protein (see appendix) Figure 1 Statistical analysis was performed on the data obtained in Example 3.

[0144] The quantitative data (relative expression level) of JUNB protein were compared between the two groups of patients after being grouped in Example 1 (CBR group n=15 cases, IR group n=15 cases).

[0145] Unpaired t-tests (or Mann-Whitney U tests) were performed using GraphPadPrism software to compare the expression differences of JUNB protein between the CBR group and the IR group.

[0146] Analysis of PBMC samples from both groups of patients revealed that the expression abundance of JUNB protein was significantly higher in the complete biochemical response group (CBR) than in the inadequate response group (IR).

[0147] Statistical analysis shows that, for example Figure 1 As shown, the expression level of JUNB protein in the CBR group was significantly higher than that in the IR group, and the relative expression level of JUNB protein in PBMCs of patients in the CBR group (mean after Log2 conversion was approximately 14.5) was significantly higher than that in the IR group (mean after Log2 conversion was approximately 13.0).

[0148] The median expression level of JUNB protein in the CBR group was approximately 14.5, while the median expression level in the IR group was significantly lower to approximately 13. The P-value between the two groups was less than 0.0001, indicating a highly statistically significant difference. This suggests that high expression of JUNB protein in PBMCs is associated with a good treatment response, while low expression of JUNB is significantly associated with an inadequate response to AIH treatment, indicating a poor response.

[0149] Figure 1 The horizontal axis represents different patient groups, where CBR represents the complete biochemical response group and IR represents the inadequate response group; the vertical axis represents the relative expression level of JUNB protein.

[0150] Example 5: Diagnostic efficacy assessment of JUNB in ​​predicting treatment response

[0151] JUNB Diagnostic Efficacy Assessment and Threshold Setting for Predicting Treatment Response

[0152] ROC curve construction: Statistical software (such as R or SPSS) was used to analyze the difference in JUNB expression between the two groups. Receiver operating characteristic (ROC) curves were constructed.

[0153] Using treatment response outcome (CBR group vs IR group) as the state variable and the relative expression level of JUNB protein as the test variable, a receiver operating characteristic (ROC) curve was plotted, as follows: Figure 2 As shown.

[0154] Calculate the area under the curve (AUC) and determine the optimal cutoff value based on the Youden Index.

[0155] Figure 2 The red curve represents the performance of the JUNB protein as a classifier. The horizontal axis represents the false positive rate, and the vertical axis represents the true positive rate.

[0156] Depend on Figure 2 The area under the curve (AUC) of JUNB protein in predicting AIH treatment response was 0.95 (95% CI: 0.88–1.00), indicating that this biomarker has extremely high discriminative power.

[0157] Cutoff value determination: Based on the principle of maximizing the Youden Index (sensitivity + specificity - 1), the optimal diagnostic cutoff value was calculated to be 13.715 (relative expression level units).

[0158] ROC curve analysis showed that JUNB protein, as a single biomarker, has excellent classification performance, and JUNB protein has extremely high accuracy in distinguishing between the CBR group and the IR group. The curve is close to the upper left corner, indicating that the indicator has both high sensitivity and high specificity; moreover, its area under the curve (AUC) reached 0.95 (95% CI: 0.88–1.00), which is much higher than conventional clinical indicators (usually AUC>0.9 is considered to have extremely high diagnostic value).

[0159] The optimal diagnostic cutoff value determined based on the Yoden Index is 13.715.

[0160] When the cutoff value was set to 13.715, the predictive sensitivity was 1.00 and the specificity was 0.80. These results indicate that JUNB expression levels below 13.715 can highly sensitively and specifically identify patients with inadequate responses.

[0161] When the expression level of JUNB protein in the PBMC of the test sample is <13.715, the patient is identified as a high-risk group with "insufficient response"; when the expression level is ≥13.715, the patient is identified as a high-probability group with "complete biochemical response".

[0162] At a cutoff value of 13.715, the predictive sensitivity of the markers of this invention was 1.00 (100%), and all patients with inadequate responses (15 cases) showed low expression, with no missed diagnoses.

[0163] This invention can determine the treatment plan for AIH patients based on whether the response is adequate or inadequate.

[0164] When the cutoff value of the relative expression level of JUNB protein (Log2 converted LFQ intensity value) in peripheral blood mononuclear cells (PBMCs) of AIH patients is ≥ 13.715, it is determined that the patient can achieve the standard of "complete biochemical response" with standard treatment, and it is recommended that the AIH patient continue to be treated with standard treatment.

[0165] The standard treatment regimen is immunosuppressive therapy using corticosteroids (such as prednisone or prednisolone) alone or in combination with azathioprine. The criteria for a complete biochemical response are: normalization of serum transaminases (ALT / AST) and IgG levels after 6 months of treatment.

[0166] When the cutoff value of the relative expression level of JUNB protein (Log2 converted LFQ intensity value) in peripheral blood mononuclear cells (PBMCs) of AIH patients is < 13.715, the patient is deemed to have failed to achieve a "complete biochemical response" (i.e., "inadequate response") with standard treatment. Therefore, intensive treatment is recommended for these AIH patients. The intensive treatment regimen consists of: replacing azathioprine with mycophenolate mofetil (MMF) in combination with corticosteroids; or replacing azathioprine with tacrolimus in combination with corticosteroids.

[0167] Meanwhile, the specificity is 0.80 (80%), indicating that the prediction method of the present invention can effectively avoid missed diagnosis of refractory cases in clinical applications.

[0168] Although the present invention has been described in detail above, it is not limited thereto, and those skilled in the art can make various modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the protection scope of the present invention.

Claims

1. A biomarker for predicting the treatment efficacy of standard treatment regimens for AIH, characterized in that, The biomarker is JUNB protein in peripheral blood mononuclear cells of AIH patients before treatment, and the identifier of JUNB protein in the UniProt database is UniProtID:P17535; AIH refers to autoimmune hepatitis.

2. The marker as described in claim 1, characterized in that, If the cutoff value of the relative expression level of JUNB protein in peripheral blood mononuclear cells of AIH patients before treatment is ≥13.715, then AIH patients are considered to have achieved a "complete biochemical response" when treated with the standard treatment regimen; if the cutoff value of the relative expression level of JUNB protein in peripheral blood mononuclear cells of AIH patients before treatment is <13.715, then AIH patients are considered to have achieved an "inadequate response" when treated with the standard treatment regimen. The standard treatment regimen is a combination of standard glucocorticoids and azathioprine.

3. A kit for predicting the treatment efficacy of standard treatment regimens for AIH, characterized in that, It includes substances for detecting the expression level of JUNB protein in peripheral blood mononuclear cells of AIH patients before treatment; the identifier of the JUNB protein in the UniProt database is UniProtID:P17535; the AIH is autoimmune hepatitis.

4. The reagent kit as described in claim 3, characterized in that, The substances include: lymphocyte separation medium, cell lysis buffer, PBS buffer, trypsin, urea, dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate buffer, formic acid, acetonitrile, and BCA protein concentration assay reagent.

5. The kit according to claim 4, characterized in that, The detection of JUNB protein expression levels includes: Peripheral blood mononuclear cells (PBMCs) were obtained by separating and washing peripheral blood from AIH patients using lymphocyte separation medium and PBS buffer. PBMCs were lysed using cell lysis buffer to obtain total protein extract; Impurities in the total protein extract were removed by acetone precipitation, then urea was added to dissolve the protein precipitate, and BCA protein concentration was quantified using BCA protein assay reagent. Dithiothreitol was added to the protein sample for BCA protein quantification to reduce disulfide bonds; Then, after cooling, iodoacetamide is added to carry out the alkylation reaction; Then add ammonium bicarbonate buffer to dilute the urea concentration to below 1M; Next, sequencing-grade trypsin was added, and enzymatic hydrolysis was performed at an enzyme:protein ratio of 1:50 (w / w) to obtain a peptide mixture; The reconstituted peptide mixture was detected using a liquid chromatography-tandem mass spectrometry system to obtain raw mass spectrometry data of the peptide mixture; The relative expression level of JUNB protein in patients was obtained by processing the short mixed mass spectrometry data using MaxQuant software.

6. The reagent kit as described in claim 5, characterized in that, If the relative expression level of JUNB protein in a patient is ≥ cutoff value 13.715, then the standard treatment regimen for AIH is predicted to be a "complete biochemical response"; otherwise, the standard treatment regimen for AIH is predicted to be an "inadequate response"; wherein the standard treatment regimen is the standard glucocorticoid combined with azathioprine treatment regimen.

7. The kit according to claim 5, characterized in that, The lymphocyte separation solution is Ficoll separation solution.

8. The reagent kit as described in claim 5, characterized in that, The cell lysis buffer is a RIPA lysis buffer containing a protease inhibitor.

9. A method for predicting the treatment effect of AIH using a standard treatment regimen; comprising: Detection of JUNB protein expression levels in peripheral blood mononuclear cells of AIH patients before treatment; If the relative expression level of JUNB protein in a patient is ≥13.715, then the patient is predicted to have a "complete biochemical response" if treated with standard treatment regimens for AIH; otherwise, the patient is predicted to have an "inadequate response" if treated with standard treatment regimens for AIH. The identifier of the JUNB protein in the UniProt database is UniProtID:P17535; the standard treatment regimen is the standard glucocorticoid combined with azathioprine treatment regimen.

10. The kit according to claim 9, characterized in that, The detection of JUNB protein expression levels includes: Peripheral blood mononuclear cells (PBMCs) were obtained by separating and washing peripheral blood from AIH patients using lymphocyte separation medium and PBS buffer. PBMCs were lysed using cell lysis buffer to obtain total protein extract; Impurities in the total protein extract were removed by acetone precipitation, then urea was added to dissolve the protein precipitate, and BCA protein concentration was quantified using BCA protein assay reagent. Dithiothreitol was added to the protein sample for BCA protein quantification to reduce disulfide bonds; Then, after cooling, iodoacetamide is added to carry out the alkylation reaction; Then add ammonium bicarbonate buffer to dilute the urea concentration to below 1M; Next, sequencing-grade trypsin was added, and enzymatic hydrolysis was performed at an enzyme:protein ratio of 1:50 (w / w) to obtain a peptide mixture; The reconstituted peptide mixture was detected using a liquid chromatography-tandem mass spectrometry system to obtain raw mass spectrometry data of the peptide mixture; The relative expression level of JUNB protein in patients was obtained by processing the short mixed mass spectrometry data using MaxQuant software.

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