Agents targeting akr1b10 and use of akr1b10 in treating and detecting mash ballooning

Abstract

The present application relates to the field of diagnosis of ballooning hepatocytes of metabolic-associated steatohepatitis (MASH), and application of spatial transcriptome combined with single-cell sequencing technology to detect liver tissue of MASH patients, and it is found that AKR1B10 is specifically highly expressed in ballooning degeneration area. Database analysis shows that AKR1B10 is positively correlated with the degree of MASH ballooning degeneration, and in-vivo experiments also verify that AKR1B10 is only expressed in MASH ballooning degeneration hepatocytes, thereby providing a new potential biological marker for diagnosis of MASH hepatocyte ballooning degeneration.

Description

Technical Field

[0001] This invention relates to the field of detection of ballooning degeneration hepatocytes in metabolic-associated steatohepatitis, and particularly to the use of spatial transcriptomics combined with single-cell sequencing technology to accurately identify potential biomarkers specifically expressed in ballooning degeneration hepatocytes. Background Technology

[0002] With the prevalence of obesity and type 2 diabetes, metabolic-associated fatty liver disease (MASLD) has become the leading chronic liver disease. Its pathological scope includes simple metabolic-associated fatty liver (MASL) and metabolic-associated steatohepatitis (MASH). MASL has a good prognosis, but 25% of patients can progress to MASH, which presents with inflammation, hepatocyte ballooning degeneration and necrosis, gradually developing into fibrosis with excessive accumulation of extracellular matrix (ECM) proteins dominated by hepatic stellate cells (HSCs), and even evolving into cirrhosis and liver cancer. Mitochondrial dysfunction in MASH leads to a decrease in intracellular ATP and a decline in the activity of the membrane Na+ / K+ pump, resulting in water and sodium retention and increased cell volume, and loose and transparent cytoplasm; endoplasmic reticulum stress leads to endoplasmic reticulum expansion and breakage into filaments, and overload of unfolded protein response; at the same time, the cytoskeleton is damaged, and abnormal cytoskeleton keratin 8 / 18 (CK8 / 18), molecular chaperone heat shock proteins, proteasome subunits, and autophagy mutations cause p62 to aggregate together to form Mallory bodies. The above-mentioned cellular damage is MASH ballooning degeneration. This pathological feature is not only an important basis for the diagnosis of MASH, but also a landmark event in the progression of liver disease. Studies have shown that the appearance of ballooning degeneration marks the transformation from simple fatty liver to inflammatory liver disease and is significantly associated with the occurrence and progression of fibrosis. Furthermore, the degree of ballooning degeneration is positively correlated with the risk of cirrhosis and hepatocellular carcinoma, and is therefore considered an important indicator for prognostic assessment of MASH patients. However, although the importance of ballooning degeneration in MASH is widely recognized, its molecular mechanisms remain not fully elucidated.

[0003] While ballooning degeneration is an important basis for the diagnosis and grading of MASH (Mandatory Action of Ash), there are currently no non-invasive biomarkers, requiring invasive pathological examination for identification. Studies have shown that the breakage and disappearance of keratin 8 / 18 (CK8 / 18) is an important marker of ballooning degeneration, which can be identified through immunohistochemical detection of liver tissue. Furthermore, abnormal aggregation of p62 / SQSTM1 protein is also considered one of the molecular characteristics of ballooning degeneration, and its accumulation in hepatocytes is closely related to autophagy dysfunction. Due to the subjective nature of the morphological characteristics of ballooning degeneration and the low diagnostic consistency among pathologists, its application in clinical diagnosis is limited, necessitating the exploration of new biomarkers.

[0004] Given that hepatocellular ballooning degeneration is a significant pathological change in MASH (Maleather's Asthenic Atrophy), elucidating the molecular mechanisms underlying its development and identifying potential biomarkers is crucial. However, current research on ballooning degeneration is limited and lacks systematic and integrative analysis. Previous molecular biology methods, including immunohistochemistry and in situ hybridization, can only analyze specific proteins or genes in a single study; while detection techniques such as genome sequencing and proteomics require tissue homogenization, and the output information often represents the average level of tissue cells, leading to the loss of heterogeneity and spatial morphological information. Therefore, traditional detection techniques cannot provide precise studies of hepatocellular ballooning degeneration in MASH.

[0005] Aldehyde reductase family 1 member B10 (AKR1B10) is an NADPH-dependent enzyme located in the cytoplasm. Its main functions are to reduce aldehyde and ketone carbonyl compounds, promote lipid synthesis, and regulate vitamin A metabolism. Recent studies have found that endoplasmic reticulum stress and oxidative stress activate transcription factors such as NRF2 to regulate AKR1B10 expression; free fatty acid palmitic acid does not increase AKR1B10 levels in hepatocytes, while the endoplasmic reticulum stress inducer tunicamycin does. The mechanism by which it exerts a protective or pathogenic effect on MASH remains controversial, but it may be related to changes in the disease-specific microenvironment and target cells. Yongzhen Liu et al. suggested that compensatory upregulation of AKR1B10 can protect hepatocytes from oxidative stress during hepatocellular carcinoma development. Other studies have shown that AKR1B10 can exacerbate steatosis by enhancing de novo lipogenesis catalyzed by acetyl-CoA carboxylase, while bis(demethoxycurcumin) can significantly reduce AKR1B10 expression in a diet-induced MASH mouse model, alleviating steatosis, inflammation, and fibrosis. Regarding its potential link to ballooning degeneration, AKR1B10 may participate in its development by regulating the stability of cytoskeletal proteins and the balance of lipid metabolism. However, the specific mechanism of action of AKR1B10 in MASH remains unclear, especially regarding its role in ballooning degeneration. Therefore, further exploration of the mechanism of action of AKR1B10 in MASH ballooning degeneration will not only help elucidate the pathogenesis of MASH but may also provide new targets for its diagnosis and treatment. Summary of the Invention

[0006] To address the shortcomings and deficiencies of existing technologies and to elucidate the role of AKR1B10 in MASH ballooning degeneration, this invention, for the first time, combines spatial transcriptomics and single-cell sequencing technologies to systematically analyze the role of AKR1B10 in MASH ballooning degeneration. It innovatively utilizes spatial transcriptomics to reveal the spatial distribution characteristics of MASH ballooning degeneration regions, and combines single-cell sequencing technology to analyze the expression patterns and dynamic changes of AKR1B10 in different cell types. Through a multi-level research perspective, from molecules to cells and from cells to tissues, it comprehensively reveals the mechanism of action of AKR1B10 in ballooning degeneration.

[0007] This invention provides the use of an AKR1B10-targeting agent in the preparation of a drug for treating MASH ballooning degeneration.

[0008] In some cases, the reagent inhibits and / or reduces the expression level of the AKR1B10 gene and / or protein.

[0009] In some cases, the reagent is selected from antisense nucleic acid molecules complementary to the AKR1B10 gene sequence, interfering RNA that knocks down the expression level of AKR1B10 protein, antagonist antibodies that antagonize the function of AKR1B10 protein, and small molecule compounds that selectively inhibit AKR1B10 protein.

[0010] In some cases, the interfering RNA used to knock down AKR1B10 protein expression levels is selected from siRNA, shRNA, single-stranded interfering RNA, and microRNA.

[0011] This invention also provides the application of AKR1B10 as a target in the preparation of drugs for treating MASH ballooning degeneration.

[0012] In a preferred embodiment of the invention, inhibition and / or reduction of AKR1B10 protein expression levels is achieved by knocking down AKR1B10 protein expression levels with interfering RNA. When introduced in vivo, the interfering RNA forms an RNA-induced silencing complex (“RISC”) with other proteins and initiates a process called RNA interference. During RNA interference, the RISC incorporates one strand of a single-stranded or double-stranded interfering RNA. The incorporated strand serves as a template for the RISC to recognize a complementary mRNA transcript. Once the complementary mRNA is identified, the protein component in the RISC is activated and cleaves the mRNA, resulting in knockdown of the target gene expression. Non-limiting examples of interfering RNA molecules used for knocking down target gene expression include small interfering RNA (siRNA), short hairpin RNA (shRNA), single-stranded interfering RNA, and microRNA (miRNA). Methods of using these interfering RNAs are well known to those skilled in the art.

[0013] In this invention, interfering RNA refers to an RNA nucleic acid molecule that is double-stranded or single-stranded and capable of inducing an RNA interference mechanism that targets the expression of knockdown gene knockout.

[0014] In this invention, siRNA is a double-stranded RNA, typically less than 30 nucleotides in length. Gene silencing via siRNA begins with one strand of the siRNA incorporating into a ribonucleoprotein complex called the RNA-induced silencing complex (RISC). The strand incorporating into the RISC recognizes mRNA molecules that are at least partially complementary to the incorporated siRNA strand, and the RISC then cleaves these target mRNAs or inhibits their translation.

[0015] In this invention, miRNA is a small non-coding RNA molecule that can hybridize with complementary sequences within an mRNA molecule, thereby causing mRNA cleavage or destabilizing the mRNA by shortening its poly(A) tail.

[0016] In this invention, single-stranded interfering RNA (SRNA) can achieve mRNA silencing in a similar manner to double-stranded siRNA, although with lower efficiency. Single-stranded SRNA typically has a length of approximately 19 to approximately 49 nucleotides.

[0017] In this invention, shRNA is an artificial RNA molecule with a tight hairpin turn, which can be used to silence target gene expression via siRNA produced in cells. shRNA expression in cells is typically achieved via plasmid vectors or via viral or bacterial vectors.

[0018] In this invention, antagonist antibodies are used in the broadest sense and include antibodies that inhibit or reduce the biological activity of the anti-AKR1B10 to which the antibody binds. Therefore, AKR1B10 antagonist antibodies encompass antibodies that bind to AKR1B10 and block, inhibit, counteract, antagonize, or reduce AKR1B10 agonist activity to any meaningful extent (including significantly).

[0019] This invention also provides the application of AKR1B10 in the preparation of a kit for detecting MASH balloon-like degeneration.

[0020] The present invention also provides the use of AKR1B10 with at least one of S100A10, CD74, VIM, CXCL12 and LYZ in the preparation of a kit for detecting MASH balloon-like degeneration. Preferably, the use of AKR1B10 with S100A10, CD74, VIM, CXCL12 and LYZ in the preparation of a kit for detecting MASH balloon-like degeneration.

[0021] In this invention, the kit for detecting MASH ballooning degeneration can be an immunoassay-based kit, such as an immunochromatographic kit, an immunoturbidimetric kit, or an ELISA kit, or a molecular detection-based kit, such as a kit containing amplification primers and probes for detecting genes such as AKR1B10 or their transcripts.

[0022] This invention reveals for the first time the specific high expression of AKR1B10 in the MASH ballooning degeneration region and its potential regulatory mechanism: the study is the first to confirm that AKR1B10 is a highly specific molecular marker of hepatocyte ballooning degeneration. Its expression is specifically localized in ballooning degeneration hepatocytes and is absent in the surrounding interstitial and inflammatory cells, thus overcoming the inherent limitations of traditional transcriptomics methods in elucidating cell origin.

[0023] This invention is the first to verify the key role of AKR1B10 in MASH ballooning degeneration through in vivo experiments: database analysis showed that the expression level of AKR1B10 increased with the degree of ballooning degeneration; verification experiments in both clinical liver tissue and peripheral blood consistently showed that serum AKR1B10 levels were independently and positively correlated with the severity of hepatocellular ballooning degeneration, and demonstrated excellent diagnostic performance for the presence of MASH and ballooning degeneration.

[0024] This invention proposes for the first time AKR1B10 as a novel target for early diagnosis and targeted therapy of MASH: Through integrated analysis of spatial transcriptomics and single-cell sequencing technologies, the functional positioning of AKR1B10 in MASH ballooning degeneration is clarified, providing a solid theoretical basis for its use as a diagnostic biomarker and therapeutic target; in vivo experiments showed that tail vein injection of AAV-packaged liver-targeting shRNA-AKR1B10 into diet-induced MASH mice significantly improved liver inflammation and fibrosis, suggesting that targeted regulation of AKR1B10 may effectively intervene in the pathological process of MASH, providing an important direction for the development of novel treatment strategies.

[0025] This invention is the first to utilize multi-omics technology to reveal the molecular mechanism of MASH ballooning degeneration: by constructing cell pseudo-time trajectories through visualization techniques such as tSNE and UMAP, the differentiation pathway of AKR1B10+ cells in ballooning degeneration and their interaction with other cell populations are revealed.

[0026] This invention provides new tools and ideas for precision medicine research on MASH for the first time: by utilizing spatial transcriptomics and single-cell sequencing technologies, it breaks through the limitations of traditional molecular biology techniques, captures the spatial heterogeneity of tissues and the dynamic changes at the single-cell level, and provides new methods for the precise diagnosis and personalized treatment of MASH; it provides a complete technical path from basic research to clinical translation, laying the foundation for future research and treatment of related diseases.

[0027] The beneficial effects of this invention are: (1) Innovation in systematic and integrative analysis: Existing technologies mostly focus on the study of single molecules or pathways, lacking systematic and integrative analysis of ballooning degeneration. This invention, for the first time, combines spatial transcriptomics and single-cell sequencing technology to comprehensively analyze the role mechanism of AKR1B10 in MASH ballooning degeneration from a multi-level perspective from molecules, cells to tissues, overcoming the limitations of traditional research methods in revealing tissue spatial heterogeneity and cell functional relationships; (2) Revealing tissue spatial heterogeneity and single-cell dynamic changes: Traditional molecular biology techniques are usually based on the analysis of whole tissues or cell populations, making it difficult to By capturing the significant differences in MASH pathological features across different liver regions and cell types, this invention reveals the spatial distribution characteristics of MASH ballooning degeneration regions through spatial transcriptomics technology. Combined with single-cell sequencing technology, it analyzes the expression patterns and dynamic changes of AKR1B10 in different cell types, filling a gap in existing technologies. (3) Clinical translation potential: This invention proposes AKR1B10 as a new target for early diagnosis and targeted therapy of MASH. The research results combined with spatial transcriptomics and single-cell sequencing technologies provide a solid theoretical foundation and technical support for developing novel diagnostic biomarkers and treatment strategies. This advantage significantly enhances the clinical translation value of the research and represents a significant breakthrough that existing technologies have failed to achieve. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0029] Figure 1 A. The violin diagram shows the UMI detection statistics for each spot on each spatial transcriptome sequencing chip; the heatmap shows the UMI expression statistics of each spot mapped to the H&E staining map on each chip, with red indicating high UMI expression in the sample and blue indicating low expression; B. Low-power images of whole liver sections are shown to provide spatial background information, along with high-power magnified images of the same section, clearly presenting the histological characteristics of ballooning hepatocytes.

[0030] Figure 2Multidimensional characterization of hepatocyte ballooning degeneration: from pathological mapping to key gene expression. A. Stacked bar charts show the percentage distribution of pathological components (normal, fatty degeneration, ballooning degeneration, inflammation, fibrosis) in 5 MASLD patient samples (numbers: 20201869, 20200714, 20201896, 20232357, 181394); B. Venn diagrams show the intersection and union of differentially expressed genes (DEGs) screened from ballooning degeneration hepatocyte regions compared with normal and fatty degeneration regions in 4 MASH patient samples (numbers: 20200714, 20201896, 20232357, 181394); C. Violin diagrams show the distribution and statistical significance of AKR1B10 expression levels after log2 transformation in the above 4 MASH samples (ballooning degeneration region vs. normal / fatty degeneration region).

[0031] Figure 3 Explanation of GO and KEGG enrichment analysis results based on differentially expressed gene sets in ballooning hepatocytes. Y-axis: Represents specific biological processes or molecular functions from the Gene Ontology (GO) database. The GO database is an internationally recognized standardized classification system for gene function, annotating gene function from three independent dimensions: biological process (e.g., cell cycle regulation, inflammatory response), molecular function (e.g., ATP binding, kinase activity), and cellular component (e.g., mitochondria, cell membrane). Z-score: A directional indicator. A positive Z-score indicates that upregulated genes dominate the gene set, directly reflecting the overall expression trend of differentially expressed genes in the corresponding function / pathway, helping to determine whether the pathway is in an activated or inhibited state; -log 10 (Corrected P-value, P_adj): The height of the bar chart represents the statistical significance of enrichment. P_adj is the P-value after correction for multiple hypothesis testing.

[0032] Figure 4 The crucial role of immune cells in MASH ballooning degeneration. A. UMAP plot showing 4114 cells isolated from MASL and MASH patients, with major cell groups labeled; B. AKR1B10 expression in different cell types: AKR1B10 high expression regions are specifically enriched in hepatocyte clusters (red area overlaps with the green hepatocyte area in Figure A), while expression is almost undetectable in other cell types; C. Box plot comparing the difference in the proportion of AKR1B10-positive cells between different cell types in the ballooning degeneration and fatty degeneration regions (***p<0.001). Figure 5A. Overall pseudo-time distribution map colored by pseudo-time values, showing a continuous developmental process with a clear branching pattern; B & C. Cell type-specific trajectories. The figures show different developmental paths of seven major cell populations: each cell type exhibits a unique trajectory pattern with clear starting nodes and subsequent branching structures, suggesting that it is in a different developmental state and that there is a potential fate-determining process.

[0033] Figure 6 Representative images (10x magnification) of liver tissue sections from healthy controls (Ctrl), MASL patients, and MASH patients were obtained. The sections were stained with H&E, and immunohistochemically / immunofluorescently stained with AKR1B10 and the ballooning degeneration marker CK8 / 18.

[0034] Figure 7 A. AKR1B10 expression levels in liver tissues of healthy controls, overweight individuals, MASL patients, and MASH patients in the GSE126848 database; B. AKR1B10 expression levels in patients with different degrees of ballooning degeneration in MASLD patients in the GSE130970 database.

[0035] Figure 8 Diagnostic efficacy of AKR1B10 in hepatocellular ballooning degeneration in MASH patients. A. Bar chart showing the expression level of the AKR1B10 gene in seven groups: healthy controls, and patients with alcoholic liver disease (ALD), drug-induced liver injury (DILI), chronic hepatitis B (CHB), chronic hepatitis C (CHC), MASL, and MASH. Intergroup comparisons were performed using a combination of the overall test (Kruskal–Wallis test) and a post-hoc test with multiple comparisons (Dunn test) (*** indicates p<0.001, ** indicates p<0.01). Abbreviations: ALD, alcoholic liver disease; DILI, drug-induced liver injury; CHB, chronic hepatitis B; CHC, chronic hepatitis C; MASL, metabolic dysfunction-associated fatty liver disease; MASH, metabolic dysfunction-associated fatty liver disease; B. Quantitative relationship between AKR1B10 gene expression level and liver pathological severity. Y-axis: Liver AKR1B10 expression level (transformed by natural logarithm for easy comparison of differences in expression levels); X-axis: Grading based on pathological features.

[0036] Figure 9 Diagnostic efficacy of AKR1B10 in healthy controls, patients with metabolic dysfunction-associated fatty liver disease (MASL), and patients with metabolic dysfunction-associated steatohepatitis (MASH).

[0037] Figure 10Honeycomb plot. This plot shows the expression levels of AKR1B10 in different groups: the horizontal axis is divided into three groups, namely the non-MASLD control group, the MASLD group, and the MASH group; the vertical axis represents the expression level of AKR1B10 (unit: pg / ml).

[0038] Figure 11 ROC curves. A comprehensive comparison of the diagnostic efficacy of the ballooning lesion diagnostic model with that of single biomarkers AKR1B10 and FIB-4 for ballooning lesions. The X-axis represents 1 - specificity (i.e., false positive rate), with values ​​closer to 0 indicating higher accuracy; the Y-axis represents sensitivity (i.e., true positive rate), with higher values ​​indicating better accuracy.

[0039] Figure 12 Tail vein injection of adeno-associated virus-packaged shRNA-AKR1B10 can improve liver fibrosis in MASH mice. A, B. Cell experiments: qPCR and WB selection of the siRNA with the best effect; C. Mouse experiment: feeding and operation procedures; D. Mouse shRNA-AKR1B10 transfection effect and pathological manifestations (200-fold); E. AKR1B10 expression in mouse liver tissue; F. Comparison of NAS pathological scores of liver tissue in the two groups of mice; G. Comparison of peripheral blood AKR1B10, AST, TG, UA and Glu levels in the two groups of mice. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0041] Example 1: Spatial transcriptomics reveals the spatial distribution characteristics of ballooning degeneration in MASLD The raw data were quantitatively analyzed using Space Ranger 1.3.1 software to obtain the gene expression profiles corresponding to the diseased cells. Figure 1 A). Based on this expression profile and combined with histopathological features, we performed regional annotation on liver tissue sections ( Figure 1 B). The analysis results showed that as MASLD progressed, the histopathological features exhibited a dynamic evolution trend: the area of ​​fatty degeneration gradually decreased with disease progression, the area of ​​ballooning hepatocytes peaked in the middle of the disease and then began to decline, while the area of ​​fibrosis progressively expanded (B). Figure 2 A).

[0042] To screen for novel biomarkers defining ballooning hepatocyte regions, we selected four samples at the MASH stage and performed differential expression analysis on ballooning hepatocyte regions compared to normal liver tissue and steatotic hepatocyte regions. Through cross-matching of differentially expressed genes across all samples, we identified a core gene, AKR1B10, which showed persistent and significant differential expression in all MASH samples. Figure 2 B). Quantitative analysis showed that in all four MASH samples, the mean expression level of the AKR1B10 gene in the ballooning hepatocyte region was significantly higher than that in the paired normal liver tissue and steatotic hepatocyte regions. Figure 2 C).

[0043] Furthermore, functional enrichment analysis of differentially expressed gene sets in each sample revealed unique molecular characteristics at different disease stages: the molecular phenotype in the mid-stage of MASH was mainly associated with cytoskeleton dynamics, energy metabolism disorders, and mitochondrial stress; while as the disease progressed to the late stage, the molecular characteristics underwent a fundamental transformation, showing significant enrichment in the tissue and assembly-related functions of the extracellular matrix (ECM), suggesting that fibrosis has become the dominant pathological process at this stage. Figure 3 ).

[0044] Example 2: Integrating single-cell sequencing data to reveal the key role of immune cells in MASH ballooning degeneration By integrating spatial transcriptome data with the single-cell sequencing dataset (GSE189175), we identified seven cell types in the GSE189175 dataset. Figure 4 A). Compared to fatty degeneration areas, the proportion of T cells and fibroblasts was significantly increased in ballooning degeneration areas ( Figure 4 C). To investigate the evolutionary relationships of cells during the progression of MASLD, we used Monocle2 software to analyze the developmental trajectories of these cells: In the initial stage of the pseudo-timeline, most cells were typical hepatocytes; as the pseudo-timeline progressed, the proportion of T lymphocytes and Kupffer cells gradually increased; subsequently, influenced by T lymphocytes and Kupffer cells, the proportion of hepatic stellate cells (HSCs) and fibroblasts gradually increased (C). Figure 5 ).

[0045] Example 3: Metabolic enrichment of AKR1B10-positive hepatocytes and activity of endoplasmic reticulum stress-related transcription factors By integrating previously obtained single-cell RNA sequencing data, we found that AKR1B10 expression was mainly enriched in hepatocytes, rather than in other non-parenchymal cell types. Figure 4B). To investigate the potential role of AKR1B10+ cells in hepatocyte ballooning degeneration, we used SCENIC analysis to divide cells into AKR1B10+ and AKR1B10- groups based on AKR1B10 expression levels. We then screened for the most activated transcription factors (TFs) and their associated signaling pathways by predicting transcription factor (TF) activity. The results showed that these TFs were significantly enriched in metabolic regulation and endoplasmic reticulum (ER) stress pathways: In metabolic regulation, PPARA, PPARG, PPARG coactivator 1α (PPARGC1A), sterol regulatory element binding protein 1 / 2 (SREBF1, SREBF2), and MLXIPL (ChREBP) regulate fatty acid oxidation and lipid synthesis through the PPAR signaling pathway. In ER stress, XBP1, ATF4, ATF6, ATF3, CREB3L1, CREB3L2, and HSF1 regulate protein processing and cell survival through the ER stress response pathway.

[0046] Example 4: High expression of AKR1B10 in ballooning degeneration regions of MASH tissue To evaluate the expression specificity and diagnostic value of AKR1B10 in hepatocellular ballooning degeneration, we performed H&E staining and immunofluorescence (IF) analysis on 20 samples from 5 groups (healthy controls (Gilbert's syndrome), patients with alcoholic liver disease (ALD), patients with drug-induced liver injury (DILI), MASL, and MASH patients). Histological analysis revealed the following patterns: AKR1B10 expression in disease stage and cell localization: AKR1B10 expression was almost undetectable in the healthy control group and the MASL group; however, in the MASH group, AKR1B10 was specifically highly expressed in the cytoplasm and cell membrane of ballooning degeneration hepatocytes. Figure 6 This characteristic is consistent with existing research findings: AKR1B10 is not expressed or only expressed in small amounts locally in MASL, but is significantly positive in ballooning hepatocytes in the MASH stage, and its expression level increases with the increase of MASH fibrosis stage (F stage).

[0047] Complementary expression pattern of AKR1B10 and the classic marker CK8 / 18: In the same lesion area, there is significant spatial overlap between areas of AKR1B10 expression gain and areas of CK8 / 18 expression loss, jointly constituting the dual molecular characteristics of ballooning hepatocytes. CK8 / 18 is a core protein of the hepatocyte cytoskeleton. When ballooning occurs, the cytoskeleton is damaged, and CK8 / 18 is no longer evenly distributed throughout the cytoplasm, but dispersed to the periphery of the cell. Therefore, in immunohistochemical detection, ballooning hepatocytes are CK8 / 18 negative, while normal hepatocytes are positive, which is an objective indicator for identifying ballooning. AKR1B10 expression in other liver diseases with ballooning: AKR1B10 is also expressed in the tissues of patients with ALD and DILI with ballooning. This complementary pattern of "AKR1B10 positive - CK8 / 18 negative" provides robust internal validation for pathological interpretation: when AKR1B10 positivity and CK8 / 18 loss occur simultaneously, its positive predictive value for hepatocyte ballooning is significantly increased. Figure 6 Based on the above findings, we reassessed the ballooning degeneration score by combining AKR1B10 or CK8 / 18 immunostaining with H&E staining. The results showed that in 10 out of 20 samples (50%), the ballooning degeneration score was improved due to additional evidence provided by immunohistochemistry, while only 1 case (5%) showed a decrease in score. This result directly addresses a current pain point in MASH diagnosis: the observer concordance Kappa value for identifying ballooning degeneration using traditional H&E staining is only 0.54–0.56, far lower than that for fatty degeneration (0.77–0.79) and fibrosis (0.75–0.84). However, combining immunohistochemical markers such as AKR1B10 and CK8 / 18 can improve diagnostic concordance to 85%–92%, which is of significant value for assessing the pathological endpoints in clinical trials of new MASH drugs.

[0048] To investigate the changes in AKR1B10 expression during the progression of MASLD, this study integrated and analyzed two GEO public datasets: the analysis based on the GSE126848 dataset (which includes liver tissue RNA sequencing data from patients with normal weight, obesity, MASL, and MASH) showed that the AKR1B10 mRNA expression level in the MASH group was significantly higher than that in the other three groups, while there was no significant difference among the normal weight, obesity, and MASL groups. Figure 7 A). Further stratified analysis using the GSE130970 dataset (covering histologically normal to full-lineage MASLD samples) showed that in MASH patients, AKR1B10 expression levels were positively correlated with the severity of hepatocellular ballooning degeneration. Figure 7B). The above results collectively suggest that the upregulation of AKR1B10 expression is closely related to the progression of MASLD to MASH and can directly reflect the severity of hepatocellular damage. It can serve as an auxiliary marker for identifying ballooning degeneration at the histopathological level and as a potential indicator for non-invasive assessment of MASH progression and prognosis at the serum level.

[0049] Example 5: Expression characteristics of AKR1B10 in different liver diseases Based on tissue-level evidence, AKR1B10 is specifically upregulated in hepatocellular ballooning degeneration in MASH patients, compensating for the loss of CK8 / 18 expression and thus improving the objectivity of hepatocellular ballooning degeneration detection. Next, we investigated whether this molecular signature is universal across different etiologies and circulatory systems. To systematically elucidate the characteristic expression patterns of AKR1B10 under different etiologies, we measured peripheral blood AKR1B10 levels from seven cohorts: healthy controls and patients with ALD, DILI, CHB, CHC, MASL, and MASH (…). Figure 8 A). Serum AKR1B10 measurements showed stable expression in normal liver tissue (180.32±223.15 pg / mL), while the disease group showed a significant difference. Compared with all other groups, the MASH group had significantly higher AKR1B10 expression (4874.12±11872.15 pg / mL, P<0.01), and this difference persisted after excluding outliers. Compared with the control group, serum AKR1B10 in MASL patients was only slightly elevated (161.43±190.92 pg / mL), and intragroup heterogeneity may indicate a risk of progression. In the viral hepatitis group, the level in the CHB group (482.36±731.45 pg / mL) exceeded that in the CHC group (338.12±428.03 pg / mL), while the levels in the ALD and DILI groups covered a wider range (1119.0±2585.1 and 223.56±341.72 pg / mL, respectively).

[0050] ROC curve analysis was used to evaluate the diagnostic performance of AKR1B10 in distinguishing between healthy individuals and patients with MASLD. The results showed that it had high value in distinguishing between MASL and MASH patients (AUC = 0.841; 95% CI: 0.758–0.924), the best value in distinguishing between MASH patients and healthy controls (AUC = 0.862; 95% CI: 0.789–0.935), and moderate value in distinguishing between healthy controls and MASLD patients (AUC = 0.757; 95% CI: 0.669–0.845). Figure 9In the external validation cohort, the AKR1B10 expression level in the MASH group was significantly higher than that in the non-MASLD control group (P<0.001) and the MASL group (P<0.05), while there was no statistically significant difference between the non-MASLD control group and the MASL group. Figure 10 The ROC curve results show that AKR1B10 performs well in distinguishing between MASL and MASH (AUC=0.891); specifically, it is even more effective in distinguishing between MASH and healthy individuals (AUC=0.906).

[0051] Example 6: Development of a non-invasive diagnostic model for hepatocellular ballooning degeneration After detecting significantly elevated AKR1B10 expression in MASH patients and its strong discriminative power across different etiologies, we further focused on pathologically confirmed MASLD patients to correlate these epidemiological findings with histological mechanisms and phenotypic manifestations. This study included 77 pathologically confirmed MASLD patients, comprising 23 MASL patients (29.9%) and 54 MASH patients (70.1%). Demographic analysis showed that MASH patients were slightly older (mean age 44.2 years) compared to MASL patients (mean age 43.0 years). Sex distribution showed a male predominance (65.2%) in MASL patients and a female predominance (59.3%) in MASH patients. BMI stratification revealed a disease severity gradient: the prevalence of overweight / obesity was 73.9% in MASL patients (47.8% overweight, 26.1% obese) and 87.0% in MASH patients (63.0% overweight, 24.1% obese). Comorbidity analysis showed that the prevalence of diabetes mellitus (27.8%) was significantly higher in MASH patients than in MASL patients (13.0%), and hypertension was also more common in MASH patients (22.2% vs. 17.4%). Notably, two MASH patients (3.7%) had cardiovascular disease, while none of the MASL patients had this condition. Liver function tests showed significantly elevated AST and ALP levels in MASH patients (p<0.001 and p=0.001, respectively). No significant differences were found in metabolic parameters (UA, CHOL, TG, HDL-C, LDL-C). The core histological features (ballooning degeneration, lobular inflammation, and fibrosis stage) were significantly more severe in MASH patients (p<0.001), while there was no significant difference in the prevalence of steatosis between the two groups. Ordinal logistic regression analysis showed a strong correlation between lnAKR1B10 expression and the severity of ballooning degeneration (OR=1.541 per unit, p=0.003), with diabetes increasing the risk of progression by 1.483-fold (95% CI: 1.109–1.982) (SDC). In terms of feature importance, the liver injury composite index (lnAKR1B10 × AST / ALT × FIB-4) (22.3%) and FIB-4 (18.7%) contributed the most, followed by lnAKR1B10 (16.5%), diabetes status (14.2%), ALT level (12.8%), age category (9.6%), and AST / ALT ratio (6.1%). The final model contains six significant predictors, described by the following multivariate logistic equation: Risk score = 0.43×lnAKR1B10 + 0.29×FIB-4 + 0.22×ALT − 0.19×AST / ALT + 0.39×diabetes + 0.32×age category.The model achieved an accuracy of 78.2% and an AUC of 0.863 (95% CI: 0.812–0.914). In terms of classification performance, the model showed sensitivities of 82.1%, 74.5%, and 73.8% for grades 0, 1, and 2, respectively, with specificities of 92.3%, 89.7%, and 93.5%. Patients were categorized into three risk groups based on a risk stratification system: low risk (<1.2 points), intermediate risk (1.2–2.8 points), and high risk (≥2.8 points). These stratifications correspond to histological grades 0, 1, and 2 of hepatocellular ballooning degeneration, respectively. The clinical significance of each category varied—low-risk patients were recommended for annual follow-up, intermediate-risk patients required lifestyle interventions and reassessment every 3–6 months, while high-risk patients required specialist treatment, with or without liver biopsy evaluation. Furthermore, the diagnostic performance of AKR1B10, FIB-4, and ballooning degeneration models for hepatocellular ballooning degeneration was systematically evaluated. The results showed that the ballooning lesion model exhibited the best diagnostic performance in distinguishing the presence of ballooning lesions (ballooning lesion 0 vs. 1–2), with an AUC of 0.958. AKR1B10 ranked second in diagnostic efficiency (AUC = 0.874), while FIB-4 had relatively limited diagnostic efficiency (AUC = 0.670). Further analysis showed that in identifying high-risk ballooning lesions (ballooning lesion 0–1 vs. 2), the ballooning lesion model again demonstrated significantly superior diagnostic performance (AUC = 0.976), significantly outperforming individual indicators AKR1B10 (AUC = 0.863) and FIB-4 (AUC = 0.689). Figure 11 ).

[0052] Example 7: Correlation analysis between AKR1B10 expression and the severity of MASLD This study systematically evaluated the association between AKR1B10 gene expression and core pathological features of MASLD. Results ( Figure 8B) shows that although AKR1B10 expression levels increased with the severity of hepatic steatosis (ρ = 0.215), the difference was not statistically significant (P = 0.062). Furthermore, no statistically significant differences in AKR1B10 expression levels were observed among different steatosis grades (P = 0.115). However, AKR1B10 expression was significantly positively correlated with hepatocellular ballooning degeneration and hepatic inflammatory activity. Ballooning degeneration grade was strongly correlated with AKR1B10 expression levels (Spearman ρ = 0.624; P < 0.001). When the ballooning degeneration grade progressed from 0 to 2, the median AKR1B10 expression significantly increased from 4.94 to 7.50, and pairwise comparisons between groups showed significant differences between grades (all P < 0.01). Similarly, inflammation grade was significantly positively correlated with AKR1B10 expression levels (ρ = 0.57, P < 0.001). When the inflammation grade increased from 0 to 2, the median expression level rose from 4.82 to 7.06, with a statistically significant difference between groups. Furthermore, AKR1B10 expression levels were positively correlated with the degree of liver fibrosis (ρ = 0.456, P < 0.001). The median expression level increased from 5.40 in stage 0 of fibrosis to 7.28 in stages 3–4, showing a clear dose-dependent relationship. Notably, no significant difference in AKR1B10 expression levels was found between stage 0 and stage 1 of fibrosis (P > 0.05).

[0053] Example 8: Knockdown of liver AKR1B10 improves liver fibrosis in MASH mice Primary mouse hepatocytes were isolated and extracted, and transiently transfected with siRNA-NC and siRNA-AKR1B10, respectively. The siRNA-AKR1B10-1 with the best interference effect was selected to construct an adeno-associated virus vector. Figure 12 A, 12B).

[0054] Six-week-old male C57BL / 6J mice were fed an AMLN diet. After 12 weeks, they were divided into two groups: the shRNA-AKR1B10 group received tail vein injection of HBAAV-TBG-mir30-AKR1B10-ZsGreen to target and knock down AKR1B10 in the liver; the shRNA-NC group received tail vein injection of HBAAV-TBG-ZsGreen. Six mice were in each group. Serum and liver samples were collected 12 weeks after tail vein injection. Figure 12 C). Six 30-week-old male C57BL / 6J mice fed a normal diet were set up as a negative control group (HC).

[0055] Frozen sections of liver tissue from mice in the shRNA-AKR1B10 and shRNA-NC groups showed abundant expression of green fluorescent protein, while the HC group showed only a small amount of autofluorescence. Figure 12D). For example Figure 12 As shown in E and 12G, Western blot results indicated a significant decrease in AKR1B10 expression in the liver tissue of mice in the shRNA-AKR1B10 group, and a significant decrease in the quantitative AKR1B10 level in peripheral blood. Compared with the shRNA-NC group, the shRNA-AKR1B10 group showed a decreasing trend in serum aspartate aminotransferase (AST), triglycerides (TG), uric acid (UA), and blood glucose levels, while pathological results indicated a significant improvement in inflammation and fibrosis (p<0.05). Figure 12 D, 12F).

[0056] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of an AKR1B10-targeting agent in the preparation of a medicament for treating MASH ballooning degeneration.

2. The application as described in claim 1, characterized in that, The reagents inhibit and / or reduce the expression levels of the AKR1B10 gene and / or protein.

3. The use as described in claim 2, characterized in that, The reagents are selected from antisense nucleic acid molecules complementary to the AKR1B10 gene sequence, interfering RNA that knocks down the expression level of AKR1B10 protein, antagonist antibodies that antagonize the function of AKR1B10 protein, and small molecule compounds that selectively inhibit AKR1B10 protein.

4. The use according to claim 3, characterized in that, The interfering RNAs used to knock down AKR1B10 protein expression levels were selected from siRNA, shRNA, single-stranded interfering RNA, and microRNA.

5. Application of AKR1B10 as a target in the preparation of drugs for the treatment of MASH ballooning degeneration.

6. Application of AKR1B10 in the preparation of a kit for detecting MASH balloon-like degeneration.

7. The use of AKR1B10 in the preparation of a kit for detecting MASH balloon-like degeneration, along with at least one of S100A10, CD74, VIM, CXCL12 and LYZ.

8. Application of AKR1B10 in the preparation of a kit for detecting MASH balloon-like degeneration, along with S100A10, CD74, VIM, CXCL12 and LYZ.

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