Application of reagent for detecting expression level of ALOX15 in preparation of product for predicting or diagnosing reverse liver fibrosis

Abstract

The invention provides application of a reagent for detecting the expression level of ALOX15 in preparation of products for predicting or diagnosing reverse liver fibrosis, and belongs to the technical field of preparation of disease diagnosis and treatment reagents. Research finds that compared with a mouse with hepatic fibrosis, the expression level of ALOX15 in a mouse with reverse hepatic fibrosis is remarkably reduced, and it is indicated that the reverse hepatic fibrosis can be accurately predicted or diagnosed by detecting the expression level of ALOX15. Besides, compared with activated hepatic stellate cells cultured by a macrophage culture medium knocking down ALOX15, the expression level of hepatic fibrosis related proteins alpha-SMA and COL1A1 is remarkably reduced, and it is indicated that inhibition of the expression level of ALOX15 can be used for preventing and / or treating hepatic fibrosis. The ALOX15 provides more choices for treating or diagnosing the hepatic fibrosis and predicting or diagnosing the reversal of the hepatic fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of preparation technology of disease diagnosis and treatment reagents, and particularly relates to the application of a reagent for detecting ALOX15 expression level in the preparation of products for predicting or diagnosing liver fibrosis reversal. Background Technology

[0002] Liver fibrosis is a pathophysiological process of the liver caused by a variety of factors such as viral infection and metabolic abnormalities. Its core feature is the imbalance between the synthesis and degradation of extracellular matrix (ECM) in the liver, which leads to the accumulation of a large amount of abnormal scar tissue (Roehlen N, Crouchet E, Baumert TF. Liver Fibrosis: Mechanistic Concepts and Therapeutic Perspectives. Cells. 2020 Apr 3;9(4):875.). Liver fibrosis is a key pathological link in the progression of chronic liver disease. When the liver is continuously damaged, the fibrosis process will continue to worsen. If it is not effectively intervened, it can eventually progress to cirrhosis and even induce liver cancer (Bataller R, Brenner DA. Liverfibrosis. J Clin Invest. 2005 Feb;115(2):209-18. doi: 10.1172 / JCI24282.Erratum in: J Clin Invest. 2005 Apr;115(4):1100.).

[0003] With the rapid development of technology and the deepening of liver research, more and more research data show that if the pathogenic factors of chronic liver injury can be effectively eliminated, liver fibrosis can be reversed to varying degrees. This phenomenon is particularly significant in patients with HBV and HCV (Li Manbiao, Li Jinyu, Feng Duiping. Research progress on reversal of liver fibrosis [J]. Journal of Clinical Hepatology, 2023, 39 (01): 193-198.). The progression and reversal of liver fibrosis show significant differentiation in pathophysiological mechanisms. The former is dominated by excessive ECM deposition and continuous activation of hepatic stellate cells (HSCs), while the latter depends on repair processes such as fibrosis degradation and cell function remodeling. This difference in mechanism is directly reflected in the molecular characteristics of diagnostic markers. That is, markers for the progression stage focus more on indicators related to fibrosis accumulation and inflammatory damage, while markers for the reversal process focus more on specific molecular changes that reflect tissue repair and metabolic improvement. Therefore, systematically elucidating the molecular mechanisms of liver fibrosis reversal and identifying diagnostic biomarkers and therapeutic targets with clinical translational potential are of significant theoretical and clinical value for promoting the construction of a precision medicine system for liver fibrosis.

[0004] Macrophages play a crucial role in the complex cellular interaction network of liver fibrosis. Under different microenvironmental stimuli, macrophages can polarize into either the M1 type, which promotes inflammation and fibrosis, or the M2 type, which participates in tissue repair and matrix degradation. In the early stages of injury, activated M1 macrophages secrete large amounts of pro-inflammatory factors, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), activating hepatic stellate cells and driving their transformation into myofibroblasts, thus accelerating the fibrotic process. Therefore, a thorough understanding of the regulation of macrophage function is essential for comprehending the mechanisms underlying the development and progression of liver fibrosis.

[0005] 15-Lipoxygenase (ALOX15), an important member of the lipoxygenase family, primarily functions by catalyzing the production of various bioactive lipid mediators from arachidonic acid, including the metabolism of arachidonic acid to 15-hydroxyeicosatetraenoic acid (15-hydroxyeicosatetraenoic acid) and 12-hydroxyeicosatetraenoic acid (12-hydroxyeicosatetraenoic acid), and the metabolism of linoleic acid to 13(S)-hydroxyoctadecadienoic acid (13-hydroxyoctadecadienoic acid). However, no research reports have yet been published regarding whether ALOX15 participates in the reversal of liver fibrosis and its specific molecular regulatory mechanisms. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a reagent for detecting ALOX15 expression levels in the preparation of products for predicting or diagnosing liver fibrosis reversal.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of a reagent for detecting ALOX15 expression levels in the preparation of products for predicting or diagnosing liver fibrosis reversal.

[0008] This invention provides the application of a reagent for detecting ALOX15 expression levels in the preparation of products for predicting or diagnosing liver fibrosis or grading the progression of liver fibrosis.

[0009] Preferably, the reagent for detecting ALOX15 expression levels includes ALOX15 antibody or primer pair.

[0010] Preferably, the nucleotide sequences of the primer pair are shown in SEQ ID NO.3~SEQ ID NO.4.

[0011] This invention provides the use of a reagent that inhibits ALOX15 expression levels in the preparation of drugs for the prevention and / or treatment of liver fibrosis.

[0012] Preferably, the reagent for inhibiting ALOX15 expression includes a reagent for knocking down or silencing ALOX15; the reagent for knocking down or silencing ALOX15 includes siRNA, shRNA, or miRNA.

[0013] Preferably, the siRNA consists of a sense strand and an antisense strand, the sequence of which is shown in SEQ ID NO.1 and the sequence of which is shown in SEQ ID NO.2.

[0014] This invention provides a system for predicting or diagnosing reversal of liver fibrosis, comprising a data processing device and a detection device. The detection device detects the expression level of ALOX15 protein in a test sample and a liver fibrosis patient sample. The data processing device includes a conclusion output module. If the expression level of ALOX15 protein in the test sample is significantly lower than that in the liver fibrosis patient sample, then the test sample is predicted or diagnosed as having undergone fibrosis reversal.

[0015] Preferably, the sample to be tested is a liver fibrosis patient who is receiving drug treatment.

[0016] This invention provides a system for predicting or diagnosing liver fibrosis or the grade of liver fibrosis progression, including a data processing device and a detection device, wherein the detection device is used to detect the expression level of ALOX15 protein in a sample to be tested. The liver fibrosis progression was graded according to the modified Scheuer scoring system, with liver histology scores divided into S1-S2 and S3-S4 grades. The data processing device includes a conclusion output module. If the ALOX15 expression level in the sample to be tested is ≤0.6623, it indicates that the sample does not have liver fibrosis; if the ALOX15 expression level in the sample to be tested is >0.6623, it indicates that the sample has liver fibrosis. If the ALOX15 expression level in the sample to be tested is >1.724, it indicates that the degree of liver fibrosis in the sample is S3-S4. If the ALOX15 expression level in the sample to be tested is ≤1.724 and >0.6623, it indicates that the degree of liver fibrosis in the sample is S1-S2.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of a reagent for detecting ALOX15 expression levels in the preparation of products for predicting or diagnosing liver fibrosis reversal. The study found that ALOX15 expression levels were significantly lower in mice with liver fibrosis compared to mice with liver fibrosis, indicating that detecting ALOX15 expression levels can accurately predict or diagnose liver fibrosis reversal. Furthermore, the study found that the expression levels of liver fibrosis-related proteins α-SMA and COL1A1 were significantly reduced when activated hepatic stellate cells cultured in a medium with knockdown of ALOX15, indicating that inhibiting ALOX15 expression levels can be used for the prevention and / or treatment of liver fibrosis. ALOX15 provides a novel target for the treatment or diagnosis of liver fibrosis and for predicting or diagnosing liver fibrosis reversal. Attached Figure Description

[0018] Figure 1 The results of the liver fibrosis reversal mouse model and proteomics analysis are as follows: A: Heatmap of differentially expressed proteins in liver tissue (left) and serum (right) of mice in the liver fibrosis model group, the 4-week reversal group, and the 12-week reversal group; B: Immunohistochemical staining of liver tissue with H&E, Masson, and ALOX15 (left) and statistical results of ALOX15 expression level detected by immunohistochemical staining (right) in mice in the control group, CCl4 model group, the 4-week reversal group, and the 12-week reversal group; C: ALOX15 protein level in liver tissue of the control group, the CCl4 model group, the 4-week reversal group, and the 12-week reversal group detected by Western blot (left) and statistical results (right). Figure 2 Immunohistochemical staining results of H&E, Masson, and ALOX15 in liver tissues of groups S0, S1-2, and S3-4 (left side) and differential analysis results of ALOX15 protein levels among different groups (right side); Figure 3 ROC curve for detecting liver fibrosis by ALOX15 expression level; Figure 4ROC curves for detecting liver fibrosis grade S3-S4 by ALOX15 expression level; Figure 5 ROC curves for detecting liver fibrosis grade S1-S2 by ALOX15 expression level; Figure 6 The results of detecting ALOX15 expression levels in Raw264.7 cells with ALOX15 knockout are shown in Figure A: qRT-PCR detection of ALOX15 mRNA levels in ALOX15 knockout cells; and Western blot detection of ALOX15 protein expression in ALOX15 knockout cells (left side) and statistical results of expression levels (right side). Figure 7 The effect of ALOX15 knockout on the mRNA levels of hepatic stellate cell fibrosis markers is shown in the left figure, which shows the COL1A1 mRNA level detected by qRT-PCR; the right figure shows the α-SMA mRNA level detected by qRT-PCR. Figure 8 The effect of ALOX15 on the levels of COL1A1 and α-SMA protein, markers of hepatic stellate cell fibrosis. Detailed Implementation

[0019] This invention provides the application of a reagent for detecting ALOX15 expression levels in the preparation of products for predicting or diagnosing liver fibrosis reversal.

[0020] This invention provides the application of a reagent for detecting ALOX15 expression levels in the preparation of products for predicting or diagnosing liver fibrosis or grading the progression of liver fibrosis.

[0021] In this invention, the liver fibrosis progression grading refers to the differentiation of the severity of liver fibrosis.

[0022] In this invention, the reagent for detecting ALOX15 expression levels includes ALOX15 antibodies or primer pairs. This invention does not specifically limit the source of the ALOX15 antibody; commercially available antibody products in the art can be used. The primer pair is a primer pair for detecting ALOX15 expression levels, and the preferred nucleotide sequences of the primer pair are shown in SEQ ID NO. 3~SEQ ID NO. 4.

[0023] In this invention, the product includes a kit. When using a reagent to detect ALOX15 expression levels for predicting or diagnosing liver fibrosis reversal, the kit also includes a positive sample of liver fibrosis, such as serum or liver tissue. A significantly decreased ALOX15 expression level in the test sample compared to a positive sample of liver fibrosis indicates that the test sample has undergone liver fibrosis reversal.

[0024] In this invention, when using a reagent to detect ALOX15 expression levels to predict or diagnose liver fibrosis, the sample may be serum or liver tissue. If the ALOX15 expression level in the sample is ≤0.6623, it indicates that the sample does not have liver fibrosis; conversely, if the ALOX15 expression level in the sample is >0.6623, it indicates that the sample has liver fibrosis.

[0025] In this invention, when using reagents for detecting ALOX15 expression levels to predict or diagnose the progression of liver fibrosis, the invention utilizes a modified Scheuer scoring system to classify liver fibrosis patients into grades S0, S1-S2, or S3-S4. If the ALOX15 expression level in the tested tissue sample is >1.724, it indicates that the liver fibrosis level of the sample is grade S3-S4; if the ALOX15 expression level in the tested tissue sample is ≤1.724 and >0.6623, it indicates that the liver fibrosis level of the sample is grade S1-S2.

[0026] This invention provides the use of a reagent that inhibits ALOX15 expression levels in the preparation of drugs for the prevention and / or treatment of liver fibrosis.

[0027] In this invention, the ALOX15 inhibitor is a reagent that reduces ALOX15 expression, including reagents that knock down or silence ALOX15. The reagents that knock down or silence ALOX15 include siRNA, shRNA, or miRNA. The siRNA consists of a sense strand and an antisense strand, the sequence of which is shown in SEQ ID NO.1, and the sequence of which is shown in SEQ ID NO.2. This invention has found that this siRNA can significantly reduce the expression level of ALOX15 in cells.

[0028] This invention provides a system for predicting or diagnosing reversal of liver fibrosis, comprising a data processing device and a detection device. The detection device detects the expression level of ALOX15 protein in a test sample and a tissue sample from a liver fibrosis patient. The data processing device includes a conclusion output module. If the expression level of ALOX15 protein in the test sample is significantly lower than that in the tissue sample from a liver fibrosis patient, then the test sample is predicted or diagnosed as having undergone fibrosis reversal.

[0029] In this invention, the test sample is a liver fibrosis patient undergoing drug treatment. The data processing device further includes a data input module, a data recording module, and a data comparison module; the data input module is configured to input the ALOX15 expression levels of the test sample and the liver fibrosis patient sample; the data recording module is configured to store data on the ALOX15 expression levels of the test sample and the liver fibrosis patient sample; the data comparison module is configured to receive the ALOX15 expression levels of the test sample and the liver fibrosis patient sample sent by the data input module, and compare the ALOX15 expression levels of the liver fibrosis patient sample stored in the data recording module with the ALOX15 expression levels of the test sample; the data comparison can be performed using conventional difference significance analysis methods in the art; the conclusion output module is configured to receive the comparison results sent by the data comparison module and determine the comparison results according to predetermined judgment conditions. The predetermined judgment condition is that if the expression level of ALOX15 in the test sample is significantly lower than that in the liver fibrosis patient sample, then the test sample has undergone fibrosis reversal. The test sample is a liver fibrosis patient who is receiving drug treatment.

[0030] This invention provides a system for predicting or diagnosing liver fibrosis or the grade of liver fibrosis progression, including a data processing device and a detection device, wherein the detection device is used to detect the expression level of ALOX15 protein in a sample to be tested. The liver fibrosis progression was graded according to the modified Scheuer scoring system, with liver histology scores divided into S1-S2 and S3-S4 grades. The data processing device includes a conclusion output module. If the ALOX15 expression level in the sample to be tested is ≤0.6623, it indicates that the sample does not have liver fibrosis; if the ALOX15 expression level in the sample to be tested is >0.6623, it indicates that the sample has liver fibrosis. If the ALOX15 expression level in the sample to be tested is >1.724, it indicates that the degree of liver fibrosis in the sample is S3-S4. If the ALOX15 expression level in the sample to be tested is ≤1.724 and >0.6623, it indicates that the degree of liver fibrosis in the sample is S1-S2.

[0031] The data processing device further includes a data input module, a data recording module, and a data comparison module; the data input module is configured to input the ALOX15 expression level of the sample to be tested; the data recording module is configured to store data on the ALOX15 expression level of the sample to be tested; the data comparison module is configured to receive the ALOX15 expression level of the sample to be tested sent by the data input module, and compare the data with the ALOX15 expression level of the sample to be tested based on predetermined judgment conditions stored in the data recording module; and make a judgment on the comparison result according to the predetermined judgment conditions. The predetermined judgment criteria are as follows: if the ALOX15 expression level in the sample to be tested is ≤0.6623, it indicates that the sample does not have liver fibrosis; if the ALOX15 expression level in the sample to be tested is >0.6623, it indicates that the sample has liver fibrosis; if the ALOX15 expression level in the sample to be tested is >1.724, it indicates that the degree of liver fibrosis in the sample is S3-S4; if the ALOX15 expression level in the sample to be tested is ≤1.724 and >0.6623, it indicates that the degree of liver fibrosis in the sample is S1-S2.

[0032] In this invention, the modified Scheuer scoring system is used to score liver histology, specifically including four aspects: no fibrosis score S0, portal enlargement score S1, portal fibrosis with a small amount of fibrous septum formation score S2, large amount of fibrous septum formation with lobule formation and structural disorder score S3, and possible or certain cirrhosis score S4.

[0033] In this invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] In the following examples, the mice used in this invention were male C57BL / 6J mice, purchased from Beijing Cyagen Biotech Co., Ltd. They were housed in the animal facility of the Beijing Academy of Science and Technology Analysis and Testing Institute, SPF grade, with the temperature maintained at 22±2℃ and humidity maintained at 50%~60%. The housing process followed the mice's natural living habits, with a 12-hour light cycle followed by 12 hours of darkness.

[0036] Preparation method of 12.5% ​​carbon tetrachloride: Mix 12.5 mL of carbon tetrachloride with 87.5 mL of olive oil until homogeneous to obtain 12.5% ​​carbon tetrachloride.

[0037] Example 1 Examples of the application of ALOX15 as a marker for reversing liver fibrosis or for diagnosis 1. Model construction and ALOX15 expression 1.1 Construction of mouse liver fibrosis model and mouse liver fibrosis reversal model Mice were randomly divided into four groups: control group (Ctrl), CCl4 model group (CCl4), 4-week reversal group (4W), and 12-week reversal group (12W).

[0038] The control group consisted of healthy mice, i.e. mice that were not treated with 12.5% ​​carbon tetrachloride.

[0039] I. Construction of a mouse liver fibrosis model: Mice were injected intraperitoneally with 12.5% ​​carbon tetrachloride at a rate of 0.01 mL / g twice a week for 8 consecutive weeks to obtain a mouse model of liver fibrosis (referred to as the CCl4 model group or model group).

[0040] II. Construction of a mouse model for reversing liver fibrosis: (1) Establishment of a model of spontaneous reversal of liver fibrosis in mice for 4 weeks (referred to as the 4-week reversal group). Mice were intraperitoneally injected with 12.5% ​​carbon tetrachloride at a concentration of 0.01 mL / g twice a week for eight weeks. The intraperitoneal injection of 12.5% ​​carbon tetrachloride was then stopped, and the spontaneous reversal occurred for four weeks.

[0041] (2) Model construction of spontaneous reversal of liver fibrosis in mice for 12 weeks (hereinafter referred to as the 12-week reversal group). Mice were injected intraperitoneally with 12.5% ​​carbon tetrachloride at a concentration of 0.01 mL / g twice a week for eight weeks. The intraperitoneal injection of 12.5% ​​carbon tetrachloride was then stopped, and the spontaneous reversal occurred for 12 weeks.

[0042] Liver tissues were collected from the control group, CCl4 model group, 4-week reversal group, and 12-week reversal group, and subjected to HE staining and Masson staining. The inflammation level and liver fibrosis level of each group were also detected.

[0043] Figure 1 The results from the B study showed that mice in the CCl4 model group exhibited significant collagen fiber deposition and inflammatory cell infiltration, with some areas showing the formation of pathological pseudolobular structures. In contrast, the liver tissue in the control group appeared normal, indicating that the liver fibrosis mouse model was successfully established. In the liver fibrosis reversal model mice, collagen fiber deposition and inflammatory cell infiltration were significantly reduced in the 4-week and 12-week reversal groups, and were positively correlated with the reversal period.

[0044] 1.2 Changes in ALOX15 expression levels during liver fibrosis reversal 1.2.1 Serum and liver samples were randomly collected from mice in the model group, the 4-week reversal group, and the 12-week reversal group for proteomics analysis.

[0045] Figure 1 The results from the study showed that, compared with the model group, the expression of ALOX15 protein was significantly downregulated in the serum and liver of mice in the 4-week and 12-week reversal groups, and decreased with the reversal of liver fibrosis.

[0046] 1.2.2 Liver proteins were extracted from mice in the successfully constructed control group, CCl4 model group, 4-week reversal group, and 12-week reversal group. ALOX15 expression levels were detected by Western blot. Primary antibody information is shown in Table 1. Simultaneously, liver tissue sections were prepared from the above mice, and ALOX15 protein expression was analyzed using immunohistochemistry.

[0047] Table 1 Primary Antibody Information

[0048] Figure 1 The results of the B-test showed that ALOX15 expression was lowest in the control group, but significantly increased in the liver tissue of mice with liver fibrosis; after reversal of liver fibrosis, its expression level decreased significantly again. Western blot results were consistent with immunohistochemical results, showing that ALOX15 expression in mouse livers decreased with reversal of fibrosis. (See...) Figure 1 (C in the middle).

[0049] Example 2 Applications of detecting ALOX15 expression levels in predicting or diagnosing liver fibrosis or the grading of liver fibrosis progression. 2.1 Expression of ALOX15 in liver tissue of patients with liver fibrosis Liver tissue biopsy samples were collected from 55 patients with liver fibrosis and 9 healthy individuals from Beijing Ditan Hospital, affiliated with Capital Medical University. Among the 55 patients, 21 were male (38.18%) and 34 were female (61.82%), with a mean age of 46 years (range 30-62 years).

[0050] Patients with liver fibrosis were diagnosed via liver biopsy and scored using the modified Scheme scoring system. The scoring system included four aspects: no fibrosis (S0), portal enlargement (S1), portal fibrosis with minor fibrotic septum formation (S2), extensive fibrotic septum formation with lobule formation and structural disorder (S3), and possible or confirmed cirrhosis (S4). The samples were then divided into three groups based on the modified Scheme scoring system.

[0051] In this embodiment, liver tissues from the above groups were stained with HE and Masson staining, and the inflammation level and liver fibrosis level of each group were analyzed.

[0052] Figure 2The results showed that, according to HE staining and Masson staining, compared with the healthy control group, the S1-S2 group and the S3-S4 group had significantly increased collagen fiber deposition and inflammatory cell infiltration. Among them, the S3-S4 group had more severe collagen fiber deposition and inflammatory cell infiltration than the S1-S2 group, indicating that the S1-S2 group and the S3-S4 group were patients with liver fibrosis, and the degree of liver fibrosis was different between the two groups.

[0053] Furthermore, in this embodiment, 9 healthy control samples, 17 S0 samples (S0 group), 19 S1-S2 samples (S1-S2 group), and 19 S3-S4 samples (S3-S4 group) were selected as the validation sample set. The expression level of ALOX15 was detected by immunohistochemical analysis (ALOX15 antibody information is shown in Table 1) to predict or diagnose liver fibrosis or liver fibrosis progression. At the same time, healthy samples were used as the control group (denoted as HC) to diagnose whether the test samples had the risk of liver fibrosis.

[0054] If the ALOX15 expression level in the sample is ≤0.6623, it indicates that the sample does not have liver fibrosis; if the ALOX15 expression level in the sample is >0.6623, it indicates that the sample has liver fibrosis. If the ALOX15 expression level in the sample is >1.724, it indicates that the liver fibrosis level of the sample is S3-S4. If the ALOX15 expression level in the sample is ≤1.724 and >0.6623, it indicates that the liver fibrosis level of the sample is S1-S2.

[0055] Immunohistochemical analysis of ALOX15 protein expression levels was performed on each group using the method described in Example 1.

[0056] according to Figure 2 The results showed that, based on immunohistochemical expression levels of ALOX15, compared with the HC control group, the expression levels of ALOX15 were significantly increased in all tissue samples from groups S1-S2 and S3-S4, while there was no significant difference in ALOX15 expression levels in all tissue samples from group S0. Furthermore, the disease status of samples from groups S0, S1-S2, and S3-S4 was consistent with the results of HE and Masson staining. Therefore, detecting ALOX15 protein expression levels can be used to determine whether a sample has fibrosis.

[0057] based on Figure 2 The results of detecting ALOX15 protein expression levels in different groups were analyzed for sensitivity and specificity in patients with liver fibrosis or different degrees of liver fibrosis, and ROC curves were plotted.

[0058] Specifically, if the ALOX15 expression level in the sample is ≤0.6623, it indicates that the sample does not have liver fibrosis; conversely, if the ALOX15 expression level in the sample is >0.6623, it indicates that the sample has liver fibrosis. Figure 3 ROC curves for detecting liver fibrosis.

[0059] Figure 3 The results showed that the AUC value of detecting ALOX15 expression level was 0.9954, indicating that the detection of ALOX15 expression level has high specificity and sensitivity for the diagnosis of liver fibrosis, and the detection sample results showed a diagnostic efficacy of 99.54%.

[0060] If the ALOX15 expression level in the sample is >1.724, it indicates that the liver fibrosis level of the sample is S3-S4. Figure 4 ROC curves were used to detect liver fibrosis at levels S3-S4.

[0061] Figure 4 The results showed that the AUC value of detecting ALOX15 expression level was 0.8450, indicating that the detection of ALOX15 expression level showed a diagnostic efficacy of 84.50% for the test samples with liver fibrosis grade S3-S4.

[0062] If the ALOX15 expression level in the sample is ≤1.724 and >0.6623, it indicates that the liver fibrosis level of the sample is S1-S2.

[0063] Figure 5 The results showed that the AUC value of detecting ALOX15 expression level was 0.9907, indicating that the diagnostic efficacy of detecting ALOX15 expression level in diagnosing liver fibrosis grade S1-S2 was 99.07%.

[0064] Figures 4-5 The results showed that detecting the expression level of ALOX15 can efficiently detect the severity of liver fibrosis in liver fibrosis samples.

[0065] Example 3 Application of reagents that inhibit ALOX15 expression levels in the preparation of products for treating liver fibrosis 3.1 Raw264.7 cells were transfected with siRNA-ALOX15, and the steps are as follows: (1) Raw264.7 cells in the logarithmic growth phase were taken, digested, and prepared into a single-cell suspension, and then mixed at 5×10⁻⁶ cells / cells. 5 The cells were seeded at high density into 6-well plates containing complete culture medium and incubated overnight at 37°C in a 5% CO2 incubator, so that the cell density reached about 50% on the day of transfection the next day. (2) For each well of cells, siRNA was added for transfection. After being starved of serum for 12 hours, the cells were cultured for 12 hours to obtain ALOX15 knockout Raw264.7 cell culture. The cells were centrifuged at 3000 rpm for 5 min, the ALOX15 knockout Raw264.7 cells were discarded, and the culture medium of ALOX15 knockout Raw264.7 cells was collected. The siRNA sequence is as follows: sense strand, 5'-GCUGUGCUGAAGAAGUUCATT-3' (SEQ ID NO.1); antisense strand, 5'-UGAACUUCUUCAGCACAGCTT-3' (SEQ ID NO.2).

[0066] RNA and protein were extracted and detected by qRT-PCR and Western blot, respectively.

[0067] For qRT-PCR detection, the qRT-PCR primer sequences are shown in Table 2. After preparing the qRT-PCR reaction system according to Table 3, the amplification is carried out according to the qRT-PCR reaction procedure in Table 4.

[0068] Table 2 qRT-PCR primer sequences

[0069] Table 3 qRT-PCR reaction system

[0070] Table 4 SYBR ® Green qRT-PCR reaction program

[0071] 3.2 Raw264.7 cells were pretreated with hepatic stellate cells (LX2 cells) as follows: (A1) Take LX2 cells in the logarithmic growth phase, digest them, prepare a single-cell suspension, and then mix them at 5 × 10⁻⁶ cells / cells. 5 The cells were seeded at a density into 6-well plates containing complete culture medium and incubated overnight at 37°C with 5% CO2, so that the cell density reached about 50% the next day when the knockout medium was obtained. (A2) The experiment was divided into three groups: the Ctrl group, the TGF group, and the TGF+si-ALOX15 group. For each well of LX2 cells, the culture medium for knocking out Raw264.7 cells with ALOX15 was added, and then the cultured cells were stimulated with 10 ng / mL TGF-β1 for 24 hours to obtain activated LX2 cells (denoted as TGF+si-ALOX15 group).

[0072] For each LX2 cell well, add culture medium that has not been treated with ALOX15 knockout Raw264.7 cells, and then stimulate the cultured cells with 10 ng / mL TGF-β1 for 24 hours to obtain activated LX2 cells 1 (denoted as TGF group).

[0073] For each well of LX2 cells, add the culture medium of Raw264.7 cells that have not been knocked out by ALOX15, and then culture the resulting cells without stimulation with 10 ng / mL TGF-β1 for 24 hours to obtain unactivated LX2 cells 2 (denoted as Ctrl).

[0074] Figure 6 The results in A-B showed that, compared with the control group, the expression levels of ALOX15 mRNA and protein in ALOX15 knockout Raw264.7 cells were significantly reduced, indicating that the ALOX15 knockout Raw264.7 cell model was successfully constructed.

[0075] RNA and protein were extracted from LX2 cells in the Ctrl, TGF, and TGF+si-ALOX15 groups. The expression levels of COL1A1 and α-SMA were detected by RT-PCR or Western blot. The primary antibody information for COL1A1 and α-SMA is shown in Table 1.

[0076] Figure 7 The results showed that, compared with the Ctrl group, the TGF group had significantly increased mRNA levels of fibrosis markers α-SMA and COL1A1 induced by TGF-β1. Conversely, compared with the TGF group, ALOX15 knockout in the TGF+si-ALOX15 group significantly inhibited TGF-β1-induced mRNA levels of α-SMA and COL1A1.

[0077] Figure 8 The results confirmed that ALOX15 knockout significantly inhibited TGF-β1-induced α-SMA and COL1A1 expression levels.

[0078] In summary, ALOX15 knockout can inhibit the activation of hepatic stellate cells, thereby achieving the purpose of preventing and / or treating liver fibrosis.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a reagent for detecting ALOX15 expression levels in the preparation of products for predicting or diagnosing liver fibrosis reversal.

2. The application of a reagent for detecting ALOX15 expression levels in the preparation of products for predicting or diagnosing liver fibrosis or grading the progression of liver fibrosis.

3. The application according to claim 1 or 2, characterized in that, The reagents used to detect ALOX15 expression levels include ALOX15 antibodies or primer pairs.

4. The application according to claim 1 or 2, characterized in that, The nucleotide sequences of the primer pairs are shown in SEQ ID NO.3~SEQ ID NO.

4.

5. The use of a reagent that inhibits ALOX15 expression levels in the preparation of drugs for the prevention and / or treatment of liver fibrosis.

6. The application according to claim 5, characterized in that, The reagents for inhibiting ALOX15 expression include reagents that knock down or silence ALOX15; the reagents that knock down or silence ALOX15 include siRNA, shRNA, or miRNA.

7. The application according to claim 6, characterized in that, The siRNA consists of a sense strand and an antisense strand, the sequence of which is shown in SEQ ID NO.1 and the sequence of which is shown in SEQ ID NO.

2.

8. A system for predicting or diagnosing the reversal of liver fibrosis, characterized in that, The device includes a data processing unit and a detection unit. The detection unit is used to detect the expression level of ALOX15 protein in the test sample and the liver fibrosis patient sample. The data processing unit includes a conclusion output module. If the expression level of ALOX15 protein in the test sample is significantly reduced compared with that in the liver fibrosis patient sample, the test sample is predicted or diagnosed to have undergone fibrosis reversal.

9. The system according to claim 8, characterized in that, The samples to be tested were liver fibrosis patients who were receiving drug treatment.

10. A system for predicting or diagnosing liver fibrosis or the grading of liver fibrosis progression, characterized in that, It includes a data processing device and a detection device, wherein the detection device is used to detect the expression level of ALOX15 protein in the sample to be tested; The liver fibrosis progression was graded according to the modified Scheuer scoring system, with liver histology scores divided into S1-S2 and S3-S4 grades. The data processing device includes a conclusion output module. If the ALOX15 expression level in the sample to be tested is ≤0.6623, it indicates that the sample does not have liver fibrosis; if the ALOX15 expression level in the sample to be tested is >0.6623, it indicates that the sample has liver fibrosis. If the ALOX15 expression level in the sample to be tested is >1.724, it indicates that the degree of liver fibrosis in the sample is S3-S4. If the ALOX15 expression level in the sample to be tested is ≤1.724 and >0.6623, it indicates that the degree of liver fibrosis in the sample is S1-S2.

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