Use of aloxe3 in promoting liver cell transplanted repopulation of the liver and methods

Abstract

The application provides an application and a method of Aloxe3 in promoting liver cell transplantation to repopulate a liver, relates to the field of medical bioengineering, and discloses that Aloxe3 is a negative regulation factor for transplanted liver cells to repopulate in a liver of a recipient through CRISPR screening for the first time, that knocking down Aloxe3 can significantly enhance the resistance of liver cells to ferroptosis, and that the liver cell transplantation is promoted, which is manifested by up-regulation of GPX4 protein expression and down-regulation of ACSL4 protein expression. The application provides an application of an Aloxe3 inhibitor in the preparation of a medicine for promoting liver cell transplantation to repopulate after transplantation, a medicine composition containing the Aloxe3 inhibitor, and a cell preparation containing liver cells with inhibited expression of an Aloxe3 gene. The application provides a new intervention target and strategy for breaking through a core bottleneck of low efficiency of liver cell transplantation to repopulate, and has a good clinical transformation prospect.

Description

Technical Field

[0001] This invention relates to the field of medical bioengineering technology, and more specifically, to the application and method of Aloxe3 in promoting hepatocyte transplantation and liver regeneration. Background Technology

[0002] Hepatocyte transplantation, as a potential treatment strategy for end-stage liver disease, still faces the critical bottleneck of low reproliferation efficiency, fundamentally due to an incomplete understanding of the molecular regulatory network of liver regeneration. Transplanted hepatocytes need to colonize and proliferate in the host liver to form functional cell clusters—a process known as "reproliferation"—which relies on a deep understanding of hepatocyte proliferation regulation and death signals. Research shows that hepatocyte proliferation is the foundation of liver regeneration. During liver regeneration, metabolic remodeling of both non-parenchymal and parenchymal cells is a necessary condition for cell division and liver regeneration. The process of hepatocytes progressing from the quiescent state of the G0 phase to massive proliferation and precise restoration of the original liver mass is a complex and intricate molecular system woven by extracellular signaling networks, intracellular signal transduction pathways, and cell cycle regulatory mechanisms. Liver regeneration requires enormous energy; therefore, hepatocytes utilize fat mobilization, absorbing large amounts of fat and oxidizing stored fat to meet their energy needs. In the early stages of liver regeneration, hepatocytes accumulate lipids such as triglycerides and cholesterol in lipid droplets, resulting in transient regeneration-related steatosis. Studies show that changes in lipid accumulation perfectly match the time window of cell division; when lipid accumulation is affected, liver regeneration capacity is weakened. Early liver regeneration involves changes in lipid metabolism signaling pathways; however, the relationship between the two is currently unclear. The key factors and signaling pathways affecting liver regeneration are still unknown; therefore, it is urgent to start with lipid metabolism and use appropriate methods to identify the key factors and signaling pathways influencing liver regeneration.

[0003] In recent years, the rapid development of CRISPR gene editing technology has provided a powerful tool for functional gene screening. Compared with traditional gene editing technologies, CRISPR / Cas9 has significant advantages such as simple design, low cost, high editing efficiency, and the ability to target multiple genes simultaneously. Depending on the screening strategy, it can be divided into positive screening and negative screening. CRISPR screening technology, especially in vivo CRISPR screening, has demonstrated unique advantages and broad application prospects in liver research, providing unprecedented research tools for elucidating liver regeneration, metabolic regulation, and disease mechanisms.

[0004] Ferroprelation is a non-apoptotic form of programmed cell death discovered in recent years, primarily caused by lipid peroxidation triggered by cystine depletion and excessive production of lipid reactive oxygen species. Ferroprelation is finely regulated by multiple signaling pathways and metabolic networks, which collectively shape the ferroptotic phenotype of cells by influencing iron metabolism, lipid metabolism, or the function of the antioxidant system. Glutathione peroxidase 4 (GPX4) plays a crucial role in feroprelation, directly reducing lipid peroxides and thus inhibiting its occurrence. ACSL4 is a key enzyme in the ferroptosis execution pathway, catalyzing the production of coenzyme A derivatives from long-chain polyunsaturated fatty acids, promoting the incorporation of these fatty acids into membrane phospholipids, and providing substrates for lipid peroxidation. ACSL4 expression levels are positively correlated with cellular sensitivity to feroprelation. Numerous studies have shown that feroprelation is closely related to liver damage; excessive iron accumulation is a major characteristic of most liver diseases. Due to the liver's susceptibility to oxidative damage, feroprelation has rapidly gained significant attention in the field of liver disease. In the context of hepatocyte transplantation, transplanted hepatocytes undergo multiple stresses in the recipient's liver, including ischemia-reperfusion, oxidative stress, and immune attack. These stresses can induce ferroptosis. Therefore, the ferroptosis sensitivity of transplanted hepatocytes directly affects their colonization and reproliferation efficiency.

[0005] Arachidonic acid lipoxygenase 3 (Aloxe3) is an important member of the lipoxygenase family. The enzyme encoded by this gene belongs to the arachidonic acid lipoxygenase family and does not directly oxidize free fatty acids. Instead, it acts as a hydroperoxide isomerase, participating in the synthesis of key lipids such as ceramides, and is indispensable for the formation and homeostasis of the skin barrier. Mutations in this gene are one of the main causes of autosomal recessive congenital ichthyosis. Previous research on Aloxe3 has primarily focused on its role in skin barrier function, with little reported on its function in the liver. As a member of the lipoxygenase family, Aloxe3 catalyzes the oxidation of polyunsaturated fatty acids and is closely related to ferroptosis. The association between Aloxe3 and ferroptosis, and whether Aloxe3 can affect hepatocyte regeneration efficiency by regulating ferroptosis, has not been reported in current technology.

[0006] In summary, there is currently a lack of technical solutions to enhance the ferroptosis resistance of transplanted hepatocytes by regulating Aloxe3, thereby improving the reproliferation efficiency of hepatocytes after transplantation. Summary of the Invention

[0007] In view of this, the present invention proposes the application and method of Aloxe3 in promoting hepatocyte transplantation and liver regeneration, so as to solve the problems existing in the prior art.

[0008] To achieve the above objectives, the present invention proposes the following technical solution:

[0009] In a first aspect, the present invention provides the use of an Aloxe3 inhibitor in the preparation of a medicament for promoting hepatocyte transplantation and regeneration.

[0010] Optionally, the aforementioned Aloxe3 inhibitors promote hepatocyte regeneration by enhancing the hepatocytes' resistance to ferroptosis.

[0011] Optionally, the Aloxe3 inhibitors mentioned above are selected from any of the following: shRNA targeting the Aloxe3 gene, siRNA targeting the Aloxe3 gene, antisense oligonucleotides targeting the Aloxe3 gene, CRISPR-Cas9 gene editing system targeting the Aloxe3 gene, or small molecule inhibitors of the Aloxe3 protein.

[0012] Optionally, the aforementioned Aloxe3 inhibitor is a lentivirus or adeno-associated virus vector carrying shRNA targeting the Aloxe3 gene.

[0013] In a second aspect, the present invention provides a pharmaceutical composition for promoting repopulation of hepatocytes after transplantation, comprising an Aloxe3 inhibitor and a pharmaceutically acceptable carrier.

[0014] Thirdly, the present invention provides a cell preparation for hepatocyte transplantation, comprising hepatocytes with inhibited Aloxe3 gene expression and a pharmaceutically acceptable carrier.

[0015] Fourthly, the present invention provides a method for preparing a cell preparation for hepatocyte transplantation, comprising the step of contacting donor hepatocytes with an Aloxe3 inhibitor under in vitro conditions to obtain hepatocytes with inhibited Aloxe3 expression.

[0016] Optionally, the donor hepatocytes are primary hepatocytes.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] This invention is the first to discover, based on in vivo CRISPR screening, that Aloxe3 is a negative regulator of liver regeneration and reveals the molecular mechanism by which it affects hepatocyte reproliferation by regulating ferroptosis. Based on this, a new strategy is proposed to promote hepatocyte reproliferation after transplantation by using reagents that inhibit Aloxe3 gene expression or activity.

[0019] This invention, through in vivo CRISPR screening, for the first time discovered that Aloxe3 is a negative regulator of liver regeneration, providing a novel target for improving the reproliferation efficiency of hepatocyte transplantation. In vivo transplantation experiments confirmed that Aloxe3 knockdown can increase the reproliferation efficiency from approximately 20% to approximately 35%, an increase of approximately 1.75 times, effectively breaking through the core bottleneck of low hepatocyte transplantation reproliferation efficiency. This invention also elucidates for the first time the molecular mechanism by which Aloxe3 affects hepatocyte reproliferation by regulating ferroptosis; knockdown of Aloxe3 can upregulate GPX4 and downregulate ACSL4, enhancing the ferroptosis resistance of hepatocytes. Based on the above findings, this invention provides three industrially viable intervention strategies: in vitro gene modification, small molecule inhibitors, and combination therapy, with promising prospects for clinical translation. Attached Figure Description

[0020] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. In the drawings:

[0021] Figure 1 The images show the immunohistochemical staining results of FAH+ and ALOXE3+ hepatocytes at different time points during transplantation in this embodiment of the invention.

[0022] Figure 2 This is a cell state diagram of AML-12 cells after RSL3-induced ferroptosis in an embodiment of the present invention;

[0023] Figure 3 This is a diagram showing the expression of GPX4 and ALOXE3 in AML-12 cells before and after induction, as detected by Western blotting in an embodiment of the present invention.

[0024] Figure 4 This is a diagram showing the changes in density and viability of AML-12 cells before and after induction using CCK8 in an embodiment of the present invention.

[0025] Figure 5 These are bright-field and fluorescence images of three types of transfected cells after puromycin screening in this embodiment of the invention.

[0026] Figure 6 This is a diagram showing the expression of ALOXE3 in AML-12 cells before and after transfection, as detected by Western blot in an embodiment of the present invention.

[0027] Figure 7 This is a diagram showing the changes in density and viability of AML-12 cells before and after transfection, as detected by CCK8 in an embodiment of the present invention.

[0028] Figure 8 This is a diagram showing the expression of GPX4 and ACSL4 in AML-12 cells before and after Aloxe3 knockdown, as detected by Western blotting in an embodiment of the present invention.

[0029] Figure 9 This is a diagram illustrating the co-localization of FAH and ALOXE3 in regenerated hepatocytes as detected by multiplex immunofluorescence staining in an embodiment of the present invention.

[0030] Figure 10 This is an image showing the immunohistochemical staining results of FAH+ hepatocytes 4 weeks post-transplantation in an embodiment of the present invention.

[0031] Figure 11 This is a diagram showing the colocalization of FAH, ALOXE3, and ACSL4 in regenerated hepatocytes as detected by multiplex immunofluorescence staining in an embodiment of the present invention.

[0032] Figure 12 This is a graph showing the changes in the expression of ALOXE3 and ferroptosis-related indicators 4 weeks after transplantation, as detected by Western blotting in an embodiment of the present invention. Detailed Implementation

[0033] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the disclosure to those skilled in the art. It should be noted that, unless otherwise specified, the embodiments and features described herein can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0034] In vivo CRISPR screening revealed that Aloxe3 is a negative regulator of liver regeneration.

[0035] This invention first constructs a Fah-based - / - An in vivo CRISPR screening platform was developed in a mouse model of induced liver injury, and its genome-wide functional screening of lipid metabolism-related genes was performed. Through large-scale parallel screening and deep sequencing analysis, a number of candidate genes significantly enriched or deleted during liver regeneration were identified. Among them, the arachidonic acid lipoxygenase encoding gene Aloxe3 was predicted as a potential negative regulator of liver regeneration.

[0036] The mice used in the CRISPR experiment were male wild-type C57BL / 6J mice (8-10 weeks old) purchased from Shanghai Kelings Biotechnology Co., Ltd. - / -Mice were maintained in a normal physiological state and used in experiments at 8-10 weeks of age. All experimental procedures involving animals were reviewed and approved by the Ethics Committee of the Tongji University Animal Center to ensure that the experiments complied with animal welfare and ethical standards. The reagents used included FAH antibody, PTEN antibody, Ki67 antibody, ALOXE3 antibody, and a universal secondary antibody for rabbits and mice; unless otherwise specified, all reagents and instruments used in this invention are commercially available products.

[0037] In CRISPR experiments, CRISPR knockout libraries targeting lipid metabolism-related genes were screened in vivo. A total of 450 genes related to lipid metabolism pathways were extracted from the KEGG database. Combined with a commercial metabolic gene library (covering 2529 genes), 337 high-confidence lipid metabolism-related target genes were obtained through intersection analysis. The selected genes cover key lipid metabolism pathways such as fatty acid synthesis, degradation, transport, lipid droplet formation, cholesterol metabolism, and phospholipid metabolism.

[0038] Eight sgRNAs were designed for each target gene. 336 non-target control sgRNAs were designed, including sgRNAs targeting non-mammalian genes (such as fluorescent protein genes) and genomically safe sites (such as AAVS1). Control sgRNAs were used to evaluate the background editing efficiency and system noise of the selection system. All sgRNA sequences were synthesized via high-throughput and cloned into plasmid vectors. The constructed sgRNA expression vectors simultaneously expressed the Cas9 and Fah genes, with the Fah gene serving as a selection marker for in vivo selection. After NTBC removal, only hepatocytes successfully transformed with and expressing the Fah gene could survive and proliferate, thus achieving competitive selection of hepatocytes carrying different sgRNAs.

[0039] Select male Fah puppies aged 8-10 weeks, weighing 20-25 grams. - / - (Fumaroyl acetoacetate hydrolase gene knockout) mice were used as an in vivo screening model. Fah deficiency leads to the accumulation of toxic intermediates in tyrosine metabolism, causing persistent liver damage. All mice were supplemented with NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione) in their daily drinking water to maintain normal survival before the experiment. Withdrawal of NTBC induced synchronous and controllable liver damage and regeneration stress. In the experiment, fluid dynamics tail vein injection was used for delivery. A large volume of solution (equivalent to 8-10% of the mouse's body weight) was injected into the tail vein in a short time (5-8 seconds) to generate instantaneous hydrostatic pressure, allowing the substances in the solution to enter the hepatocytes efficiently and specifically. The core components of the delivery included a plasmid vector and Sleeping Beauty transposase SB100.

[0040] A mixture of 5 µg plasmid vector and 1 µg SB100 was dissolved in sterile 0.9% NaCl solution, with the final volume adjusted to 10% of the mouse's body weight. Using a syringe, the mixture was injected into the mouse's tail vein at a uniform rate over 5–8 seconds. Immediately after injection, the NTBC was removed from the drinking water, initiating the liver injury stress and screening process. Four weeks post-injection was set as the screening endpoint and sample collection time point. After NTBC removal, hepatocytes without Fah gene correction gradually died, creating strong selective stress. Under this stress, the differences in survival and proliferation capacity of hepatocytes carrying different sgRNAs (i.e., knocked-out different genes) were amplified, thus reflecting at the population level through sgRNA enrichment or depletion. Four weeks post-injection, mouse livers were isolated, and a portion of each hepatic lobule was immediately fixed in 4% paraformaldehyde for subsequent histological analysis. The remaining liver tissue was immediately flash-frozen in liquid nitrogen and then transferred to -80°C for long-term storage for genomic DNA extraction.

[0041] In the mixed CRISPR knockout selection experiment, the above plasmids and transposase SB100 were mixed at a ratio of 5 µg:1 µg to ensure that each hepatocyte could only be transfected with one type of plasmid. Ten 8-10 week old Fah cells were selected. - / - Mice underwent hydrodynamic tail vein injection (HDT), with the NTBC removed immediately afterward. Four weeks later, mouse liver tissue was collected. Immunohistochemical staining revealed FAH-positive hepatocytes in all liver tissue samples, confirming the successful entry of the mixed knockout selection system carrying the Fah gene marker into mouse hepatocytes. This system drove clonal expansion of hepatocytes under the selective pressure of NTBC removal, which can be used for further sequencing analysis. Furthermore, inter-individual differences in the size of FAH-positive clones, and the heterogeneity of different clone sizes within the same liver, preliminarily reflect the differences in fitness of hepatocytes carrying different sgRNAs under regenerative pressure. These differences will be quantified as sgRNA enrichment or deletion signals in subsequent high-throughput sequencing.

[0042] sgRNA enrichment analysis and candidate gene screening: Fah - / -After NTBC removal in mice, hepatocytes that successfully integrated and expressed the exogenous Fah gene carried different sgRNAs, which competed for these genes during liver regeneration. Hepatocytes with knocked-out genes negatively regulating liver regeneration exhibited greater clonal proliferation during liver regeneration, while those with knocked-out genes positively regulating liver regeneration showed less clonal proliferation. Following in vivo CRISPR knockout screening, mouse liver tissue samples were collected for genome extraction and PCR amplification, followed by NGS sequencing. The screening of genes affecting liver regeneration was based on sgRNA enrichment and deletion analysis. Targeted knockout of the target gene with sgRNA resulted in sgRNA enrichment in the sequencing results, indicating that cells with targeted knockout of this gene were in a proliferative state during liver regeneration. This gene knockout actually promoted hepatocyte proliferation, suggesting that the gene itself inhibits hepatocyte proliferation. Conversely, sgRNA deletion after targeted gene knockout indicated that the gene knockout promoted hepatocyte death, suggesting that the gene itself may promote hepatocyte proliferation. The sequencing results were analyzed using the MAGeCK tool. Robust Rank Aggregation (RRA) scores were calculated to assess the enrichment or scarcity of each gene during screening. A lower RRA score indicates a higher degree of gene enrichment and greater importance. Genes were ranked according to their RRA scores. Combining the results of gene dominance screening using the sgrankview function, the enrichment of sgRNAs after screening, volcano plot results, and RRA score ranking results, candidate genes negatively regulating liver regeneration were identified in this screening. The results show that in vivo CRISPR screening successfully identified a batch of candidate genes related to liver regeneration, with Aloxe3 ranking highly in multiple rankings and identified as a core candidate gene.

[0043] Functional annotation and pathway enrichment analysis of candidate genes: After identifying a list of candidate genes significantly related to liver regeneration through MAGeCK bioinformatics analysis, functional annotation and pathway enrichment analysis were further performed on these genes to analyze their biological functions and enriched signaling pathways, and to verify the reliability of the screening results from the perspective of systems biology. The top 100 candidate genes (ranked based on RRA scores) were imported into bioinformatics analysis platforms such as DAVID, Metascape and clusterProfiler for the following three types of enrichment analysis: (1) KEGG pathway enrichment analysis: to analyze the metabolic and signal transduction pathways significantly enriched by candidate genes; (2) Reactome pathway enrichment analysis: to further analyze gene functions from the perspective of chemical reactions and pathways; (3) GO functional enrichment analysis: to classify candidate genes from three dimensions: biological processes, cellular components and molecular functions. KEGG pathway enrichment analysis revealed that candidate genes were significantly enriched in multiple pathways closely related to lipid metabolism, primarily including: fatty acid metabolism-related pathways (fatty acid degradation and elongation), lipid metabolism regulatory pathways (PPAR signaling pathway and cholesterol metabolism), and cell death-related pathways (ferroptosis). Reactome pathway enrichment analysis results corroborated the KEGG analysis, further strengthening the association between candidate genes and lipid metabolism. Mitochondrial fatty acid β-oxidation was one of the most enriched pathways, involving the transport of fatty acids into mitochondria and the catalytic enzymes involved in each step of β-oxidation. Arachidonic acid is an important representative of polyunsaturated fatty acids; its metabolites are both precursors to inflammatory mediators and substrates for ferroptosis and lipid peroxidation. PPARα activation of gene transcription corresponds to the PPAR signaling pathway in the KEGG analysis. GO enrichment analysis comprehensively analyzed the functional characteristics of candidate genes from three dimensions: (1) biological processes: the biological processes significantly enriched by candidate genes include fatty acid β-oxidation, lipid homeostasis, oxidative stress response, and cell proliferation regulation; (2) cellular components: the proteins encoded by candidate genes are mainly located in the mitochondrial matrix and inner mitochondrial membrane (the main site of fatty acid oxidation), peroxisomes (the site of very long chain fatty acid oxidation), endoplasmic reticulum membrane (the main site of lipid synthesis), lipid droplets (the organelles for lipid storage), and cell membrane (the localization region of membrane phospholipids); (3) molecular functions: the molecular functions significantly enriched by candidate genes include lipoxygenase activity (Aloxe3, Alox15, etc. have this function), oxidoreductase activity, fatty acid binding, long chain fatty acid coenzyme A ligase activity (Acsl4 has this function), and phospholipid binding. The above results indicate that the screened candidate genes are highly correlated with lipid metabolism and ferroptosis pathway, verifying the biological reliability of the screening results. These analytical results also reveal the potential importance of the ferroptosis pathway in liver regeneration.Both KEGG and Reactome enrichment analyses showed significant enrichment of ferroptosis-related pathways, and GO analysis also showed significant enrichment of the "lipid peroxidation regulation" process. This finding suggests that ferroptosis may play an important role in liver regeneration. Ferroptosis is a form of cell death driven by lipid peroxidation, and early liver regeneration is accompanied by significant lipid accumulation and metabolic remodeling. The spatiotemporal correlation between these two factors suggests that ferroptosis may affect regeneration efficiency by regulating the survival threshold of hepatocytes under oxidative stress. Among the candidate genes mentioned above, Aloxe3 ranked highly in all evaluation dimensions. This gene encodes arachidonic acid lipoxygenase, a key enzyme in the arachidonic acid metabolic pathway, which catalyzes the formation of peroxides from polyunsaturated fatty acids and their metabolites, and is closely related to ferroptosis. Therefore, Aloxe3 is an important target for ferroptosis research. Thus, it is hypothesized that in vivo CRISPR knockout of the Aloxe3 gene helps damaged hepatocytes develop ferroptosis resistance, thereby promoting their survival and ultimately promoting liver regeneration.

[0044] Aloxe3, a member of the lipoxygenase family, is located at the intersection of lipid metabolism and ferroptosis pathways, in Fah - / - In the context of iron overload in mice, the expression level of Aloxe3 has a significant impact on hepatocyte fate. This invention, through in vivo CRISPR screening, reveals for the first time the negative regulatory role of Aloxe3 in liver regeneration, and selects Aloxe3 as a target for further research. As a member of the lipoxygenase family, Aloxe3 is closely related to arachidonic acid metabolism and the ferroptosis pathway, and its function warrants further in-depth investigation.

[0045] Function and mechanism of Aloxe3 gene in liver regeneration:

[0046] Based on CRISPR screening data, this invention proposes the following scientific hypothesis: Under liver injury and regenerative stress, Aloxe3 expression in hepatocytes may negatively regulate hepatocyte survival and proliferation by promoting the generation of specific lipid oxidation products, exacerbating lipid peroxidation, and inducing ferroptosis; inhibiting Aloxe3 function may enhance hepatocyte ferroptosis resistance, thereby promoting their reproliferative efficiency. To verify this hypothesis and elucidate its molecular mechanism, this chapter will conduct systematic in vitro and in vivo functional studies. First, Aloxe3 will be specifically knocked down in the mouse hepatocyte line (AML-12), and the effect of Aloxe3 deficiency on hepatocyte ferroptosis sensitivity and proliferation capacity will be clarified in vitro using ferroptosis inducers. Second, liver-specific Aloxe3 knockdown will be achieved in mice using an adeno-associated virus vector, and the results will be analyzed in Fah... - / -In a mouse hepatocyte transplantation model, the promoting effect of Aloxe3 function inhibition on the reproliferation efficiency of transplanted hepatocytes was directly evaluated, and changes in hepatocyte proliferation and ferroptosis markers were detected by histological and molecular biological methods.

[0047] Materials and reagents included the following: The cell line used was the normal mouse hepatocyte line AML-12, purchased from Wuhan Shangen Biotechnology Co., Ltd. Ferrocyte inducer RSL3 was used. Cell proliferation was detected using the CCK-8 kit, and reactive oxygen species (ROS) levels were detected using the DCFH-DA fluorescent probe kit. Primary antibodies used included ALOXE3 antibody, GPX4 antibody, ACSL4 antibody, and GAPDH antibody. Protein extraction was performed using RIPA lysis buffer containing 1% protease inhibitors, and PVDF membranes were used for transfer. Reagents used for in situ liver perfusion included Liver Perfusion Medium, EBSS buffer, and HEPES.

[0048] Experimental methods:

[0049] (1) Western blot detection of proteins

[0050] Cell or liver tissue samples were collected, and RIPA lysis buffer containing 1% protease inhibitor was added. The samples were lysed on ice for 30 minutes, centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. 5× protein loading buffer was added, and the samples were heated in a metal bath at 100°C for 10 minutes. After separation by SDS-PAGE gel electrophoresis, the proteins were transferred to a PVDF membrane using the wet transfer method. The membrane was blocked with rapid blocking buffer at room temperature for 30 minutes. ALOXE3 antibody, GPX4 antibody, ACSL4 antibody, or GAPDH antibody were added as primary antibodies, and the membrane was incubated overnight at 4°C. After washing with TBST, the corresponding secondary antibody was added and the membrane was incubated at room temperature for 1 hour. After washing, chemiluminescent substrate was added for development, and grayscale analysis was performed using GAPDH as an internal control.

[0051] (2) Immunohistochemical staining

[0052] Liver tissue was fixed in 4% paraformaldehyde for 72 hours, dehydrated with graded ethanol, cleared with xylene, and embedded in paraffin to prepare 3 μm thick paraffin sections. After dewaxing with xylene and rehydration with graded ethanol, the sections were placed in EDTA antigen retrieval solution and microwaved for antigen retrieval. After cooling, the sections were washed with PBS, incubated with 3% hydrogen peroxide at room temperature in the dark for 15 minutes to eliminate endogenous peroxidase activity, blocked with 3% bovine serum albumin, and then FAH antibody or ALOXE3 antibody was added as the primary antibody. The sections were incubated overnight at 4°C, warmed to room temperature the next day, washed with PBS, and incubated at 37°C for 30 minutes with the corresponding secondary antibody. After washing with PBS, DAB chromogenic solution was added for staining, counterstained with hematoxylin, dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, and images were acquired using a slide scanner.

[0053] (3) Multiple fluorescent staining

[0054] Pretreatment steps for paraffin sections, including dewaxing, rehydration, and antigen retrieval, are the same as for immunohistochemical staining. After blocking with 3% bovine serum albumin, the first round of primary antibody (FAH antibody, ALOXE3 antibody, or ACSL4 antibody) is added and incubated overnight at 4°C. The next day, after warming to room temperature, the sections are washed with PBS, and the secondary antibody provided in the kit is added and incubated at 37°C for 50 minutes. After washing with PBS, fluorescent dye (520 / 570 / 690) is added and stained at room temperature in the dark for 8 minutes. After washing with PBS, antigen retrieval is performed again, and the above steps are repeated for the second and third rounds of labeling. After three rounds of labeling, DAPI staining solution is added to counterstain the cell nuclei, and the images are observed and acquired under a fluorescence microscope.

[0055] (4) Cell proliferation detection (CCK-8 assay)

[0056] AML-12 cells were spaced at 2 × 10⁶ cells per well. 4 Cells were seeded in 96-well plates and induced for 48 hours with 20 μM RSL3 after cell attachment. Cells with the same volume of DMSO served as the control group, and unseeded wells served as the blank control group. After induction, the old culture medium was discarded, and 100 μL of fresh culture medium containing 10% CCK-8 solution was added to each well. The plates were incubated at 37°C in the dark for 1 hour. The absorbance at 450 nm and 650 nm was measured using a microplate reader, and the relative viability of each group was calculated.

[0057] (5) Detection of reactive oxygen species (ROS) in cells

[0058] AML-12 cells were seeded in 6-well plates. After cell attachment, 20 μM RSL3 was added for induction for 24 hours. The culture medium was discarded, and 1 mL of DCFH-DA fluorescent probe diluted with PBS was added to each well. The cells were incubated at 37°C for 20 minutes. The cells were washed three times with PBS to remove probes that did not enter the cells. 1 mL of Hoechst staining solution was added to each well to counterstain the cell nuclei. The cells were observed and images were acquired under a fluorescence microscope.

[0059] (6) Screening for puromycin

[0060] Lentivirally infected AML-12 cells were seeded into 6-well plates and cultured for 72 hours. Initial selection was performed by adding 10 μg / mL puromycin, followed by replacement with fresh medium after 3 hours. After cell confluence, cells were passaged. When the cell density reached approximately 50%, the medium was changed daily with 6 μg / mL puromycin for 4 consecutive days. After selection, GFP fluorescence intensity was detected under a fluorescence microscope to obtain stable transfected cell lines.

[0061] (7) In situ perfusion of liver and isolation of primary hepatocytes

[0062] Prepare perfusion solution I (25 mL Liver Perfusion Medium), perfusion solution II (50 mL EBSS + 0.5 mL HEPES), and digestion solution III (0.04 g collagenase + 50 mL EBSS + 0.5 mL HEPES), and preheat them in a 37°C water bath. After anesthetizing the mice, make a "V"-shaped incision in the abdominal cavity to expose the inferior vena cava and portal vein. Insert a cannula into the inferior vena cava, cut open the portal vein, and perfuse with perfusion solution I, II, and digestion solution III sequentially at a flow rate of 10 mL / min. Stop perfusion when 20 mL of digestion solution remains. Cut off the liver and immerse it in a culture dish containing digestion solution. Disperse the hepatocytes with forceps, filter through a 70 μm cell sieve, centrifuge at 50 g for 2 minutes, discard the supernatant, and resuspend the cells in DMEM medium. After washing once, resuspend the cells in DMEM medium containing 10% fetal bovine serum and 5% penicillin-dextrose antibodies, count the cells, and set aside for later use.

[0063] (8) Spleen injection for hepatocyte transplantation

[0064] receptor Fah - / - Mice were anesthetized and fixed in a right lateral decubitus position. A longitudinal incision of approximately 1 cm was made at the level of the lower edge of the last rib on the left abdomen to expose the spleen. 100 μL of cell suspension (containing 1 × 10⁶ cells) was aspirated using an insulin syringe. 6 (One cell), insert the needle into the spleen parenchyma at a very shallow angle parallel to the spleen surface, about 2-3 mm from the upper third of the spleen. Inject the cell suspension very slowly, leave the needle in place for 10-15 seconds, and then slowly withdraw it. Gently press the injection site with a dry cotton swab to stop bleeding. Gently return the spleen to its original position in the abdominal cavity, and suture the muscle layer and skin sequentially using 4-0 absorbable sutures. After suturing, place the mouse on a 37°C heating pad for resuscitation and house it individually.

[0065] (9) Statistical analysis

[0066] All experiments were independently repeated three times, and results are expressed as mean ± standard error. The t-test was used for comparisons between two groups, and one-way or multi-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant. GraphPad Prism 10.1.2 was used as the statistical software.

[0067] The results of the above experiments are shown below:

[0068] Preliminary verification of Aloxe3:

[0069] The previously constructed in vivo CRISPR screening system carries the Fah gene marker. This screening system is similar to an animal model in which Fah gene-deficient mice are transplanted with hepatocytes that normally express the Fah gene. Therefore, this embodiment first examines Aloxe3 expression in this model. Immunohistochemical staining results are as follows: Figure 1As shown, FAH-positive regions in liver tissue samples taken 1, 2, and 3 weeks after transplantation corresponded to ALOXE3-negative regions, while samples taken 4 weeks after transplantation showed FAH-positive regions corresponding to ALOXE3-positive regions. This indicates that ALOXE3 expression is suppressed in the early stages of hepatocyte proliferation after liver injury, while ALOXE3 expression gradually returns to normal in the later stages of proliferation. This is consistent with previous findings through in vivo CRISPR knockout screening that this gene is a negative regulator of liver regeneration after injury.

[0070] Figure 1 Fah at 1 week, 2 weeks, 3 weeks, and 4 weeks post-transplant - / - Representative images of multiplex immunofluorescence staining of liver tissue from recipient mice, scale bar = 100 μm. FAH-labeled reproduced hepatocytes derived from transplantation, ALOXE3-labeled target protein, and DAPI (blue)-labeled cell nuclei. One week after transplantation, scattered single or small clusters of FAH-positive hepatocytes were observed, but ALOXE3 positivity was not observed. Two weeks after transplantation, the number of FAH-positive cells increased, forming small clonal clusters, but ALOXE3 positivity was still not observed. Three weeks after transplantation, the area of ​​FAH-positive clones further expanded, the cells were densely packed, and ALOXE3 positivity began to appear. Four weeks after transplantation, FAH-positive clones occupied most of the liver lobules, forming large fused reproducible areas, with strong ALOXE3 signal and a significant increase in the co-localization area with FAH, showing a high level of co-expression.

[0071] Inducing and validating ferroptosis in the AML-12 cell line:

[0072] Ferroplasty was induced in the AML-12 cell line using the ferroptosis inducer RSL3. GPX4 is a key enzyme in lipid metabolism in mammalian cells. GPX4 converts glutathione to oxidized glutathione and reduces cytotoxic lipid peroxides to their corresponding alcohols. Therefore, inhibition of GPX4 activity leads to the accumulation of lipid peroxides. RSL3 can directly act on GPX4 and inhibit its activity, thereby reducing the cell's antioxidant capacity and accumulating ROS, ultimately leading to ferroptosis. Three control groups and three experimental groups were set up. The experimental groups were supplemented with 20 μM RSL3, and the control groups were supplemented with the same volume of DMSO. After 48 h of induction, subsequent experiments were conducted for verification. Figure 2 As shown, after 48 hours of induction, the cell density in the experimental group was significantly lower than that in the control group, and the cells in the experimental group exhibited phenomena such as membrane rupture and blistering.

[0073] Figure 2Phase-contrast microscopy images of AML-12 mouse hepatocytes from the control group (CTL-48h) after 48 hours of culture (scale bar = 100 μm). Cells exhibit typical epithelial-like morphology, good adhesion, plump cell bodies, clear cell boundaries, visible nucleoli, and tight intercellular junctions; no obvious floating or dead cells were observed. Phase-contrast microscopy images of AML-12 cells from the RSL3 treatment group (20 μM, 48 hours). Compared to the control group, RSL3-treated cells exhibited typical ferroptosis morphology: cells became shrunken and rounded, decreased adhesion, and some cells floated; vacuolar structures appeared in the cytoplasm, cell membrane integrity was impaired; cell density was significantly reduced, and the proportion of dead cells was significantly increased.

[0074] To demonstrate that the induction conditions could successfully induce ferroptosis in AML-12 cells, this example validated the results through a series of experiments. The first was a Western blotting experiment. After 48 hours of induction, cellular proteins were extracted, and changes in the ferroptosis-related marker GPX4 were detected using Western blotting. Figure 3 As shown, GPX4 expression was significantly downregulated after induction, preliminarily indicating successful ferroptosis in AML-12 cells. Furthermore, ALOXE3 expression was significantly upregulated, further demonstrating the close relationship between ALOXE3 and ferroptosis.

[0075] Figure 3 In the table, A: Western blot analysis of ALOXE3 and GPX4 protein expression levels in AML-12 cells from the control group (CTL) and the RSL3-treated group (20 μM, 48 hours). GAPDH was used as an internal control. B: Bar chart of ALOXE3 protein band grayscale quantitative analysis. The results were normalized to the control group (set to 1), showing that ALOXE3 expression level increased after RSL3 treatment. C: Bar chart of GPX4 protein band grayscale quantitative analysis, showing that GPX4 expression level decreased after RSL3 treatment.

[0076] Next, we performed a cell proliferation assay (Cell Counting Kit-8, CCK8). Experimental results... Figure 4 As shown, compared with the control group, the OD450nm value decreased significantly after 48 hours of induction with 20μM. Combined with the previous WB validation results, this indicates that ferroptosis was successfully induced in AML-12 cells after 48 hours of induction with 20μM RSL3.

[0077] Figure 4In the table, A: Representative phase-contrast microscopy images of AML-12 cells in the control group (20 μM DMSO treatment for 48 hours) and the RSL3 treatment group (20 μM RSL3 treatment for 48 hours), scale bar = 100 μm. Control group cells adhered well, exhibiting typical epithelial-like morphology and high density; RSL3 treatment group cells were significantly shrunken and rounded, with increased floating cells and a significantly decreased cell density, showing typical morphological changes of ferroptosis. B: Quantitative results of cell viability detection in both groups using the CCK8 assay. The ordinate represents relative cell viability. The results showed that compared with the control group, after 48 hours of treatment with 20 μM RSL3, cell viability was significantly reduced to approximately 0.5 times that of the control group. These results indicate that RSL3 successfully induced ferroptosis in AML-12 cells, significantly inhibiting cell viability, providing a reliable cell model for subsequent research on the function of Aloxe3 in ferroptosis.

[0078] Finally, ROS detection was performed. The ROS fluorescence intensity in the experimental group was significantly higher than that in the control group, indicating that lipid peroxidation occurred in AML-12 cells after induction with 20 μM RSL3 for 24 hours, consistent with the characteristics of ferroptosis. The above experimental results fully demonstrate that induction with 20 μM RSL3 for 48 hours can successfully induce ferroptosis in cells.

[0079] Constructing an Aloxe3 knockdown model in the AML-12 cell line using lentivirus:

[0080] To further investigate the relationship between Aloxe3 and ferroptosis and hepatocyte proliferation, this study used lentiviruses to construct a knockdown model targeting the Aloxe3 gene. To ensure the effectiveness of the knockdown, three knockdown targets were designed, corresponding to lentivirus numbers 166, 519, and 1755. AML-12 cells were infected with each of the three lentiviruses, and selection was performed using puromycin to construct stably transfected Aloxe3 knockdown cells. Figure 5 The results after puromycin screening show that after infecting AML-12 cells with the three lentiviruses, the transfection rate was over 80% after puromycin screening.

[0081] Figure 5In the image, representative images of AML-12 cells infected with three lentiviruses after puromycin selection are shown (scale bar = 100 μm). The left image is a bright-field image showing cell morphology and density; the middle image is a fluorescence field image showing lentivirus-infected positive cells carrying the GFP reporter gene; the right image is a superimposed image of bright-field and fluorescence fields used to assess infection efficiency. The three sets of images show that after puromycin selection, the proportion of GFP-positive cells in AML-12 cells infected with Aloxe3-shRNA-166, 519, and 1755 lentiviruses all reached over 80%, indicating high lentivirus infection efficiency and successful puromycin selection yielding a stable transfected cell population.

[0082] To determine which of the three knockdown targets was most effective, Western blotting was used to detect the ALOXE3 protein expression levels in three stably transfected cell lines. Results Figure 6 As shown, the expression level of ALOXE3 protein was significantly downregulated in all three stable cell lines, with the most significant downregulation observed in cell line 1755, suggesting that the lentivirus corresponding to cell line 1755 exhibits the best knockdown effect.

[0083] Figure 6 In the figure, A: Western blot analysis of ALOXE3 protein expression levels in control group (CTL) and AML-12 cells stably transfected with Aloxe3-shRNA-166, 519, and 1755, with GAPDH as an internal control; B: Bar chart of quantitative analysis of ALOXE3 protein band grayscale values. The vertical axis represents the relative protein expression level (normalized to 1 with CTL group as the baseline). The results show that compared with the CTL group, the grayscale value of ALOXE3 protein bands was significantly reduced after transfection with the three shRNA target sites, indicating that effective gene knockdown was achieved at each target site.

[0084] In addition, CCK8 assays were used to detect changes in cell viability of three stably transfected cell lines compared to the control group. Results Figure 7 As shown, the OD450nm values ​​of the three transfection groups were significantly higher than those of the control group. It is speculated that after knocking down the Aloxe3 gene, the ferroptosis resistance of stable transfected cells increased. Therefore, after adding the ferroptosis inducer RSL3, the viability of stable transfected cells was higher than that of the control group, further indicating that Aloxe3 is closely related to ferroptosis.

[0085] Figure 7In the figures, A: Representative phase-contrast microscopy images of AML-12 cells after treatment with 20 μM RSL3 for 48 hours, scale bar = 100 μm. Control group cells, after RSL3 treatment, exhibited typical morphological changes associated with ferroptosis: cells were significantly shrunken and rounded, with decreased adhesion, increased floating cells, and significantly reduced cell density. The three Aloxe3 knockdown groups showed relatively better cell morphology, with more adherent cells and fewer floating cells. The shRNA-519 group showed the best cell condition, approaching normal cell morphology. B: Bar chart of OD450nm values ​​of cells after treatment with 20 μM RSL3 for 48 hours, detected by CCK8 assay. The ordinate represents the OD450nm absorbance value, reflecting the number of viable cells and metabolic activity. The results showed that the OD450nm values ​​of all three transfected groups were higher than those of the control group, indicating that knocking down the Aloxe3 gene enhanced the resistance of AML-12 cells to ferroptosis. Therefore, after adding the ferroptosis inducer RSL3, the survival rate of stable transfected cells was higher and their viability was stronger.

[0086] Validating the function of Aloxe3 in the AML-12 cell line:

[0087] Having confirmed the effectiveness of all three knockdown targets, the effect of Aloxe3 knockdown on ferroptosis in AML-12 cells was first examined using Western blotting. Experimental Results Figure 8 As shown, compared with the control group, the expression levels of GPX4, a ferroptosis-related marker, were significantly increased in all three groups of Aloxe3 knockdown cells, while the expression levels of ACSL4 were significantly decreased. These results collectively indicate that Aloxe3 knockdown enhances the ferroptosis resistance of AML-12 cells.

[0088] Figure 8 In the figures, A: Western blot analysis of GPX4, ACSL4, and ALOXE3 protein expression levels in the control group (CTL) and three stably transfected AML-12 cells, with GAPDH used as an internal control; B: Bar chart of GPX4 protein band grayscale value quantitative analysis. The vertical axis represents the relative protein expression level (normalized to 1 with the CTL group as the baseline). The results showed that the GPX4 expression level in the three knockdown groups was significantly higher than that in the control group; C: Bar chart of ACSL4 protein band grayscale value quantitative analysis. The results showed that the ACSL4 expression level in the three knockdown groups was significantly lower than that in the control group.

[0089] In addition, the effect of Aloxe3 knockdown on ferroptosis in AML-12 cells was detected by ROS analysis. The ROS fluorescence intensity of the three Aloxe3 knockdown groups was slightly higher than that of the control group, further indicating that Aloxe3 knockdown enhanced the ferroptosis resistance of AML-12 cells.

[0090] Representative ROS fluorescence staining images of three stable cell lines after treatment with 20 μM RSL3 for 24 hours in the control group (untreated) and the control group (untreated). Scale bar = 100 μm. Each row, from left to right, shows: ROS fluorescent probe (green) detecting intracellular reactive oxygen species levels, Hoechst nuclear staining (blue), and a merged image of the two. In Aloxe3 knockdown AML-12 cells, after induction with 20 μM RSL3 for 24 hours, the ROS fluorescence intensity in all three experimental groups was higher than that in their respective untreated control groups, indicating that RSL3 successfully induced oxidative stress.

[0091] A knockdown model of Aloxe3 was established in mice using adeno-associated virus (AAV8):

[0092] To verify the function of the Aloxe3 gene in liver regeneration in vivo, this embodiment uses adeno-associated virus serotype 8 (AAV8) mediated RNA interference technology to specifically knock down Aloxe3 expression in mouse liver.

[0093] To visually validate the AAV8-sh-Aloxe3-mediated gene knockdown effect at the single-cell level and simultaneously observe the reproliferation of transplanted hepatocytes, this example used Fah cells 4 weeks post-transplantation. - / - Multiplex immunofluorescence staining was performed on mouse liver tissue. The co-localization of FAH and ALOXE3 in the liver tissues of the control group (transplanted with AAV8-sh-CTL pretreated hepatocytes) and the knockdown group (transplanted with AAV8-sh-Aloxe3 pretreated hepatocytes) was analyzed. Results Figure 9 As shown, distinct FAH-positive hepatocyte clones were observed in the liver tissue of the control group. These clones were clustered and clearly defined. Strong ALOXE3 fluorescence signals were detected in FAH-positive reproduced hepatocytes, indicating a significant co-localization relationship between ALOXE3 and FAH. However, in the knockdown group, the ALOXE3 fluorescence signal was significantly weakened in FAH-positive reproduced hepatocytes, with almost no ALOXE3 expression detected in the vast majority of FAH-positive cells. The co-localization of FAH and ALOXE3 essentially disappeared in the knockdown group. These results demonstrate that AAV8-sh-Aloxe3 achieves efficient and specific Aloxe3 knockdown in transplanted hepatocytes, and the knockdown effect remains stable for 4 weeks post-transplantation.

[0094] Figure 9 In the study, the control group (CTL-TR4W, transplanted with AAV8-shCTL pretreated hepatocytes) and the knockdown group (Aloxe3-KD-TR4W, transplanted with AAV8-shAloxe3 pretreated hepatocytes) showed significant differences in liver function at 4 weeks post-transplantation. - / -Representative images of multiplex immunofluorescence staining of mouse liver tissue, scale bar = 100 μm. From left to right: DAPI nuclear staining (blue), FAH (red) labeling of transplanted regenerated hepatocytes, ALOXE3 (green) labeling of the target protein, three-color overlay image (Merge), and magnification image. These results indicate that AAV8-shAloxe3 achieved efficient and specific Aloxe3 knockdown in transplanted hepatocytes, and the knockdown effect remained stable for 4 weeks post-transplantation.

[0095] In Fah - / - Validating the function of Aloxe3 in a mouse transplantation model:

[0096] Due to Fah - / - Mouse hepatocytes lacking the Fah gene cannot survive after NTBC removal; only successfully transplanted and proliferating donor-derived hepatocytes can form a clonal proliferating cell population in the liver. Therefore, the area of ​​the FAH-positive region can directly reflect the reproliferation efficiency of transplanted cells.

[0097] FAH immunohistochemical staining was performed on paraffin sections of the livers of mice in the control group and Aloxe3 knockdown group 4 weeks after transplantation. Results Figure 10 As shown, scattered FAH-positive hepatocyte clones were observed in the liver of the control group (transplanted AAV8-sh-CTL hepatocytes), with the FAH-positive area accounting for approximately 20% of the total tissue section area. In the Aloxe3 knockdown group (transplanted AAV8-sh-Aloxe3 hepatocytes), the area of ​​FAH-positive hepatocyte clones was significantly increased, with the FAH-positive area accounting for approximately 35% of the total tissue section area. These results indicate that Aloxe3 knockdown significantly enhances the reproliferative capacity of transplanted hepatocytes in damaged livers. Previous in vivo CRISPR screening showed significant enrichment of Aloxe3-targeting sgRNAs, meaning that Aloxe3 knockout confers a growth advantage to hepatocytes. This transplantation experiment reproduced this phenotype at the single-gene level, validating the reliability of the screening results. Furthermore, multiplex immunofluorescence staining... Figure 11 It has been confirmed that ALOXE3 expression almost completely disappeared in the transplanted cells of the knockdown group. The FAH staining results in this section further show that it is these cells with low ALOXE3 expression that have gained stronger reproliferative capacity, establishing a complete chain of evidence from gene knockdown to protein expression reduction and then to enhanced functional phenotype.

[0098] Figure 10 In the middle, A: Fah, 4 weeks post-transplantation, between the control group (CTL-TR4W) and the Aloxe3 knockdown group (Aloxe3-KD-TR4W). - / -Representative images of FAH immunohistochemical staining in mouse liver tissue, scale bar = 100 μm. FAH positive signals appear brownish-yellow, and hematoxylin counterstaining cell nuclei appears blue. Scattered FAH-positive hepatocyte clones can be observed in the control group liver tissue, with the positive area occupying a small proportion of the total liver parenchyma. In the Aloxe3 knockdown group, the area of ​​FAH-positive hepatocyte clones is significantly increased, with some clones fusing into sheets, occupying most of the liver lobules, and the positive area is significantly enlarged; B: Quantitative analysis bar chart of the percentage of FAH-positive area to the total liver parenchyma area. The vertical axis represents the percentage of FAH-positive area (%). The results show that Aloxe3 knockdown significantly enhances the reproliferative capacity of transplanted hepatocytes in damaged livers, increasing the reproliferative efficiency by approximately 1.75 times.

[0099] Preliminary investigation into the molecular mechanism by which Aloxe3 regulates liver regeneration:

[0100] The aforementioned functional experiments have confirmed that Aloxe3 knockdown significantly promotes the regeneration of transplanted hepatocytes, suggesting that Aloxe3 is a negative regulator of liver regeneration. To preliminarily explore its molecular mechanism, this embodiment focuses on the ferroptosis pathway—Aloxe3, as a member of the lipoxygenase family, participates in the peroxidation of polyunsaturated fatty acids, and ferroptosis is a form of programmed cell death driven by the accumulation of lipid peroxidation. This embodiment hypothesizes that Aloxe3 may affect the survival and proliferation of hepatocytes under regenerative stress by regulating ferroptosis-related molecules. Therefore, this embodiment performed multiplex immunofluorescence staining and Western blot analysis on liver tissue (without Aloxe3 knockdown, i.e., samples transplanted with control hepatocytes) 4 weeks post-transplantation to analyze the expression and localization relationship between Aloxe3 and key ferroptosis proteins.

[0101] To investigate the expression and spatial distribution of ALOXE3 and ferroptosis executive protein ACSL4 in regenerated hepatocytes at the single-cell level, this example studies the expression and spatial distribution of Fah cells 4 weeks post-transplantation. - / - Multiplex immunofluorescence staining was performed on liver tissues from mice (control group, transplanted with AAV8-sh-CTL pretreated hepatocytes). Results Figure 11 The results showed that strong ALOXE3 fluorescence signal was detected in the FAH-positive recolonized hepatocyte region, and ALOXE3 and FAH exhibited a significant co-localization relationship. Similarly, ACSL4 fluorescence signal was significantly enriched in the FAH-positive recolonized hepatocyte clone, showing high co-localization with FAH. These results indicate that both ALOXE3 and the ferroptosis executive protein ACSL4 are highly expressed in successfully recolonized hepatocytes, providing important morphological clues for the involvement of ALOXE3 in the regulation of ferroptosis.

[0102] Figure 11 In the control group, 4 weeks post-transplantation, Fah - / -Representative images of multiplex immunofluorescence staining of mouse liver tissue, scale bar = 100 μm. From left to right: DAPI nuclear staining (blue), FAH (red) labeling of transplanted repopulated hepatocytes, ALOXE3 (green) labeling of the target protein, ACSL4 (green) labeling of ferroptosis executive protein, three-color overlay image (Merge), and magnification image. Strong ALOXE3 and ACSL4 fluorescence signals were detected in the FAH-positive repopulated hepatocyte clonal regions, and ALOXE3 and ACSL4 showed significant co-localization with FAH.

[0103] To further verify the status of the ferroptosis pathway in regenerated hepatocytes at the protein level, liver tissue was extracted from normal C57BL / 6J wild-type mice (C57WT) and liver tissue from Fah cells 4 weeks after transplantation. - / - The expression levels of ALOXE3, ferroptosis inhibitor GPX4, and ferroptosis executive factor ACSL4 were detected by Western blotting in liver tissue (representing regenerated liver tissue mainly composed of regenerated hepatocytes) from mice (control group). Results Figure 12 The results showed that, compared with the C57WT group, the expression of both ALOXE3 and ACSL4 proteins was significantly downregulated, while the expression of GPX4 protein was significantly upregulated in the liver tissue of mice transplanted 4 weeks prior. These results indicate that ferroptosis resistance increased in the liver 4 weeks after transplantation, suggesting that at this stage, cells increased ferroptosis resistance by reducing ALOXE3 expression to help cells achieve faster proliferation.

[0104] Figure 12 In the middle, A: Western blot analysis of liver tissue from normal C57BL / 6J wild-type mice (C57WT) and liver tissue 4 weeks post-transplantation. - / - Protein expression levels of ALOXE3, GPX4, and ACSL4 in liver tissue of mice (tdTR4W). GAPDH was used as an internal control. The left lane represented the C57WT group, and the right lane represented the tdTR4W group. B: Bar chart of quantitative analysis of ALOXE3 protein band grayscale values. The vertical axis represents the relative protein expression level (normalized to 1 with C57WT group as the baseline). The results showed that the ALOXE3 expression level in the tdTR4W group was significantly lower than that in the C57WT group. C: Bar chart of quantitative analysis of GPX4 protein band grayscale values. The results showed that the GPX4 expression level in the tdTR4W group was significantly higher than that in the C57WT group. D: Bar chart of quantitative analysis of ACSL4 protein band grayscale values. The results showed that the ACSL4 expression level in the tdTR4W group was significantly lower than that in the C57WT group.

[0105] The most significant finding of this invention is that Aloxe3 knockdown can significantly promote the growth of transplanted hepatocytes in Fah. - / -The reproliferative efficiency in mouse liver, a functional phenotype, was validated at multiple levels. In vivo CRISPR screening showed significant enrichment of sgRNAs targeting Aloxe3, suggesting that its knockout confers a growth advantage to hepatocytes; AAV8-shRNA-mediated Aloxe3 knockdown was stably maintained both in vivo and in vitro; immunohistochemical staining confirmed that the clonal area of ​​FAH-positive hepatocytes in the knockdown group was significantly larger than that in the control group, with a reproliferative efficiency increase of approximately 1.75 times. These results collectively establish Aloxe3 as a negative regulator of liver regeneration.

[0106] Previous research on Aloxe3 has mainly focused on its role in skin barrier function. This study is the first to link Aloxe3 with hepatocyte reproliferation and reveal its key role in regulating transplanted hepatocyte reproliferation, expanding our understanding of this gene's function. The ferroptosis resistance conferred by Aloxe3 knockdown allows transplanted cells to regenerate in the Fah... - / - The mice gained a significant competitive reproliferative advantage in the microenvironment of iron overload and oxidative stress. This discovery not only provides a new perspective for understanding the molecular regulation of hepatocyte transplantation reproliferation, but also provides a potential intervention strategy for optimizing transplantation efficacy.

[0107] In Fah - / - In mouse transplantation models, transplanted cells exhibit a significant competitive reproliferative advantage over host cells. This advantage stems primarily from several factors. The selective pressure of the Fah model provides a unique competitive environment for transplanted cells. - / - In mice, after NTBC removal, toxic metabolites accumulate in hepatocytes, leading to progressive hepatocyte death. Transplanted hepatocytes, carrying the normal Fah gene, can metabolize these toxic products, thus gaining a survival advantage during the mass death of host hepatocytes and exhibiting clonal expansion. Therefore, the Fah model essentially constructs a competitive environment of "selective expansion," where any gene modification that enhances cell survival or proliferation is significantly amplified during reproliferation. Iron metabolism disorders exacerbate the vulnerability of host hepatocytes; previous studies have shown that Fah... - / - Mice exhibited severe iron overload after NTBC withdrawal, with liver iron content increasing approximately fivefold and transferrin saturation rising from 63% to 77%. Iron overload catalyzes the generation of hydroxyl radicals via the Fenton reaction, initiating a lipid peroxidation chain reaction and placing host hepatocytes in a vulnerable state of oxidative stress. Against this backdrop, transplanted cells with stronger antioxidant or anti-ferroptosis capabilities will have a significant competitive advantage. This competitive reproliferative advantage is quantified in reproliferative efficiency, as demonstrated in Fah... - / -In mouse transplantation models, reproliferation efficiency is essentially the result of competition between transplanted cells and host cells under selective pressure. Although the control group's transplanted cells also carry the normal Fah gene, their resistance to ferroptosis is limited due to high Aloxe3 expression. In contrast, the Aloxe3 knockdown group's cells, while retaining Fah function, exhibit stronger resistance to ferroptosis, resulting in a significantly improved reproliferation efficiency. The difference in reproliferation efficiency between the two groups, competing in the same host microenvironment, directly reflects the competitive advantage brought about by Aloxe3 knockdown. This result also indirectly confirms the role of Fah... - / - The mouse model's high sensitivity to differences in transplant cell fitness validates its unique value in screening liver regeneration regulators.

[0108] To investigate the association between Aloxe3 and ferroptosis, this invention examined the expression status of ferroptosis-related proteins in successfully regenerated hepatocytes. Western blot results showed that, compared with normal C57WT liver tissue, in liver tissue mainly composed of regenerated hepatocytes 4 weeks after transplantation, the expression of ALOXE3 and the ferroptosis executive protein ACSL4 was synchronously downregulated, while the expression of the ferroptosis inhibitor GPX4 was significantly upregulated. This change in expression profile has important functional implications. GPX4 is currently recognized as a core negative regulator of ferroptosis, which blocks ferroptosis by reducing phospholipid peroxides to corresponding alcohols to clear lipid peroxidation products. ACSL4 is a key enzyme in the ferroptosis executive pathway, which promotes the incorporation of long-chain polyunsaturated fatty acids into membrane phospholipids by catalyzing the production of coenzyme A derivatives, thus providing substrates for lipid peroxidation. The expression level of ACSL4 is positively correlated with the sensitivity of cells to ferroptosis, and knockout or inhibition of ACSL4 can significantly protect cells from ferroptosis damage. Therefore, the downregulation of ACSL4 and the upregulation of GPX4 together constitute a dual protection for cells to resist ferroptosis, while ALOXE3 itself is also downregulated, suggesting that regenerating hepatocytes may protect themselves from ferroptosis damage by actively downregulating the activity of each lipid peroxidation pathway.

[0109] To further clarify the relationship between ALOXE3 and the ferroptosis pathway, this study performed multiplex immunofluorescence staining on liver tissue 4 weeks post-transplantation. The results showed that the fluorescence signals of ALOXE3 and ACSL4 highly overlapped within the clonal regions of FAH-positive regenerated hepatocytes. This spatial co-localization has important functional implications: ACSL4 is responsible for "transferring" polyunsaturated fatty acids to membrane phospholipids, while ALOXE3 directly oxidizes polyunsaturated fatty acids in membrane phospholipids to generate lipid peroxides. The coexistence of the two in regenerated cells suggests that they may synergistically act in the same metabolic pathway, jointly regulating intracellular lipid peroxidation levels. This finding provides direct cellular evidence for the involvement of ALOXE3 in the regulation of ferroptosis. To further verify the causal relationship between Aloxe3 and ferroptosis, this study constructed a stable Aloxe3 knockdown model in AML-12 cells in vitro and induced ferroptosis using RSL3. The results showed that the survival rate of cells in the Aloxe3 knockdown group was significantly higher than that in the control group after RSL3 treatment, and ferroptosis-related indicators changed significantly: GPX4 expression was upregulated and ACSL4 expression was downregulated. These results indicate that Aloxe3 knockdown does indeed enhance the cells' resistance to ferroptosis.

[0110] Based on the above evidence, this study proposes a molecular mechanism model for Aloxe3-regulated liver regeneration: In regenerated hepatocytes from the control group, ALOXE3 and ACSL4 co-localize and synergistically act. ACSL4 catalyzes the coenzyme A conversion of polyunsaturated fatty acids and their incorporation into membrane phospholipids, while ALOXE3 directly oxidizes PUFAs in membrane phospholipids to generate lipid peroxides. Under the iron overload environment of the Fah model, iron ions catalyze the generation of highly reactive lipid free radicals from lipid peroxides via the Fenton reaction, initiating a lipid peroxidation chain reaction that ultimately leads to ferroptosis, limiting hepatocyte survival and clonal expansion. In regenerated hepatocytes from the Aloxe3 knockdown group, Aloxe3 knockdown leads to downregulation of ACSL4 expression, reducing PUFA incorporation into membrane phospholipids and decreasing the supply of lipid peroxidation substrates. Simultaneously, GPX4 expression is upregulated, enhancing the ability to scavenge lipid peroxides. The synergistic effect of these two mechanisms endows cells with ferroptosis resistance. In the same iron overload microenvironment as the Fah model, these cells can effectively resist oxidative damage, survive, and proliferate efficiently, ultimately demonstrating a significant improvement in regeneration efficiency.

[0111] The Aloxe3-ferroptosis regulatory axis discovered in this study forms a good connection with previous research. Existing studies have revealed the mechanism of iron overload in the Fah gene defect model and its role in liver injury, providing important background for understanding the microenvironmental stress faced by transplanted cells in this study. Recent studies have shown that ALOXE3, as a target gene of YAP-TEAD, can increase the sensitivity of hepatocellular carcinoma cells to ferroptosis through overexpression, which is highly consistent with the conclusion in this study that high expression of ALOXE3 promotes ferroptosis. This study is the first to extend this mechanism to the field of physiological liver regeneration, revealing a novel mechanism by which lipid metabolism enzymes affect hepatocyte fate by regulating ferroptosis.

[0112] In terms of clinical translation, as a member of the lipoxygenase family, the active site of Aloxe3 and its catalytic products have clear small molecule targeting potential. Compared with gene editing strategies, small molecule inhibitors have advantages such as convenient administration, reversible effects, and controllable dosage, making them more feasible for clinical translation.

[0113] In summary, this study identified Aloxe3 as a negative regulator of liver regeneration through in vivo CRISPR screening and systematically verified its function in influencing hepatocyte regeneration by regulating ferroptosis. This discovery expands the function of lipid metabolism enzymes from the traditional areas of energy metabolism and structural synthesis to the regulatory level of cell fate determination, revealing a direct link between metabolism and death signaling. Simultaneously, this study provides a novel intervention approach for hepatocyte transplantation, enhancing transplant efficacy by regulating the ferroptosis sensitivity of donor cells in vitro. This strategy holds promise for combined application with other regeneration-promoting methods to jointly advance the clinical translation of hepatocyte transplantation technology.

[0114] in conclusion

[0115] This embodiment focuses on the function and mechanism of Aloxe3 in liver regeneration, and draws the following main conclusions.

[0116] Aloxe3 is a negative regulator of liver regeneration. In vivo CRISPR screening identified Aloxe3 as a candidate gene, and functional validation showed that Aloxe3 knockdown significantly promoted the regeneration of transplanted hepatocytes in Fah. - / - The reproliferative efficiency in mouse liver was significantly increased by the area of ​​FAH-positive regions.

[0117] Successfully reproduced hepatocytes exhibited molecular characteristics of ferroptosis resistance. Western blot results showed that, compared with the control group, the expression of ALOXE3 and ferroptosis executive protein ACSL4 was downregulated in reproduced hepatocytes, while the expression of ferroptosis inhibitor GPX4 was upregulated, suggesting that the reproduced cells established a state of ferroptosis resistance.

[0118] FAH was co-localized with ALOXE3 and ACSL4 in recolonized hepatocytes. Multiplex immunofluorescence staining showed that the fluorescence signals of FAH, ALOXE3, and ACSL4 highly overlapped within the recolonized hepatocyte clones, providing spatial evidence for their synergistic regulation of ferroptosis.

[0119] This study preliminarily elucidates the molecular mechanism by which Aloxe3 regulates liver regeneration. Based on the above evidence, a working model is proposed: Aloxe3, through synergistic action with ACSL4, regulates lipid peroxidation levels, affects the ferroptosis sensitivity of hepatocytes, and thus determines their survival and proliferative fate under regenerative stress.

[0120] In summary, this chapter, through in vitro and in vivo functional verification and mechanistic investigation, clarifies the negative regulatory role of Aloxe3 in liver regeneration and preliminarily reveals the molecular mechanism by which it affects hepatocyte regeneration by regulating ferroptosis. These findings not only deepen our understanding of the metabolic regulatory network of liver regeneration but also provide new intervention targets and theoretical basis for improving the efficacy of hepatocyte transplantation.

[0121] This invention demonstrates clear innovation in its technical methods, research approach, and scientific findings, primarily in the following aspects: First, technological innovation: establishing an in vivo CRISPR screening platform suitable for liver regeneration research. Traditional CRISPR screening is mostly conducted in in vitro cell lines, which struggles to simulate the complex microenvironment during liver regeneration. This study utilizes Fah... - / - By combining an induced liver injury model with fluid dynamics delivery technology, an in vivo platform capable of high-throughput functional gene screening under physiological regenerative stress was constructed, providing a new technical means for analyzing gene function in a real pathophysiological context. Secondly, the approach is innovative: it intersects and integrates three major research areas: lipid metabolism, ferroptosis, and liver regeneration. Previous studies on liver regeneration have largely focused on growth factor signaling pathways, with relatively insufficient attention paid to metabolic regulation. This study uses lipid metabolism as a starting point, discovering Aloxe3 through in vivo CRISPR screening, and then revealing a new mechanism by which it affects liver regeneration by regulating ferroptosis. This is the first time that lipid metabolic remodeling, ferroptosis sensitivity regulation, and hepatocyte reproliferation efficiency have been linked, providing a new perspective for understanding the metabolic regulatory network of liver regeneration. Thirdly, the discovery is innovative: it is the first time that Aloxe3 has been identified as a negative regulator of liver regeneration and its mechanism has been elucidated. Previously, Aloxe3 was mainly studied for its role in skin barrier function, and its function in the liver has been rarely reported. This study is the first to discover that Aloxe3 plays a negative regulatory role in liver regeneration and reveals the molecular mechanism by which it affects hepatocyte survival and proliferation by regulating ferroptosis sensitivity. This expands our understanding of the function of this gene and provides a new intervention target for optimizing the efficacy of hepatocyte transplantation.

[0122] The aforementioned functional validation experiments have confirmed that targeting Aloxe3 to enhance the ferroptosis resistance of transplanted hepatocytes can significantly improve their reproliferation efficiency. Based on this, this embodiment further provides an application scheme for the Aloxe3 inhibition strategy in clinical hepatocyte transplantation.

[0123] First, the in vitro gene modification strategy. In clinical hepatocyte transplantation, donor hepatocytes can be subjected to in vitro Aloxe3 knockdown treatment before transplantation (e.g., using shRNA, siRNA, or CRISPR technology). Specifically, isolated primary donor hepatocytes are co-incubated in vitro with a viral vector (such as AAV8 or lentivirus) carrying shRNA targeting Aloxe3 or chemically synthesized siRNA to achieve transient or stable Aloxe3 knockdown. After washing to remove unbound vectors or siRNA, a cell preparation suitable for hepatocyte transplantation is obtained. This in vitro treatment strategy has better safety than direct in vivo injection of viral vectors, avoiding potential immune responses or off-target effects caused by the widespread distribution of viral vectors in the recipient body.

[0124] Second, the small molecule inhibitor strategy. As a member of the lipoxygenase family, Aloxe3's active site and its catalytic products have clear small molecule targeting potential. Compared to gene editing strategies, small molecule inhibitors offer advantages such as convenient administration, reversible action, and controllable dosage, making them more feasible for clinical translation. During the perioperative period of hepatocyte transplantation (pre-transplantation conditioning or short-term post-transplantation administration), administering Aloxe3-specific small molecule inhibitors can inhibit the enzyme activity of Aloxe3 in donor hepatocytes or recipient livers, reduce lipid peroxidation levels, and enhance the ferroptosis resistance of hepatocytes, thereby improving the reproliferation efficiency of transplanted cells. Small molecule inhibitors can be used alone or in combination with in vitro gene knockdown strategies.

[0125] Third, combination therapy strategies. Aloxe3 targeted inhibition can be combined with existing strategies to promote regeneration. Existing strategies to promote liver regeneration include growth factor supplementation (such as HGF and EGF), anti-inflammatory therapy, and MKK4 inhibitors. Aloxe3 inhibitors work by enhancing the ferroptosis resistance of transplanted hepatocytes, and their mechanism of action differs from the above strategies, showing the potential to synergistically improve the efficacy of transplantation. By combining Aloxe3 inhibition with the above-mentioned regeneration-promoting methods, it is expected to synergistically promote hepatocyte reproliferation from two levels: enhancing the survival ability of transplanted cells and improving the recipient microenvironment, thereby further improving the overall efficacy of hepatocyte transplantation.

[0126] In summary, this embodiment demonstrates through in vitro and in vivo experiments that Aloxe3 knockdown can increase the reproliferation efficiency of transplanted hepatocytes from approximately 20% to approximately 35%, representing a reproliferation efficiency increase of approximately 1.75 times. This discovery not only reveals a new link between lipid metabolism and ferroptosis in liver regeneration but also provides a potential intervention target and novel strategy for improving the efficacy of hepatocyte transplantation. This strategy can be combined with existing regenerative methods such as growth factor supplementation and anti-inflammatory therapy to synergistically enhance therapeutic effects, showing promising clinical translational potential.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. The use of an Aloxe3 inhibitor in the preparation of a drug for promoting hepatocyte transplantation and regeneration.

2. The application according to claim 1, characterized in that, The Aloxe3 inhibitor promotes hepatocyte regeneration by enhancing the hepatocytes' resistance to ferroptosis.

3. The application according to claim 1, characterized in that, The Aloxe3 inhibitor is selected from any of the following: shRNA targeting the Aloxe3 gene, siRNA targeting the Aloxe3 gene, antisense oligonucleotide targeting the Aloxe3 gene, CRISPR-Cas9 gene editing system targeting the Aloxe3 gene, or small molecule inhibitor of Aloxe3 protein.

4. The application according to claim 1, characterized in that, The Aloxe3 inhibitor is a lentivirus or adeno-associated virus vector carrying shRNA targeting the Aloxe3 gene.

5. A pharmaceutical composition for promoting regeneration of hepatocytes after transplantation, characterized in that, It contains an Aloxe3 inhibitor and a pharmaceutically acceptable carrier.

6. A cell preparation for hepatocyte transplantation, characterized in that, Hepatocytes with suppressed Aloxe3 gene expression and pharmaceutically acceptable vectors.

7. A method for preparing a cell preparation for hepatocyte transplantation, characterized in that, This includes the step of contacting donor hepatocytes with an Aloxe3 inhibitor under in vitro conditions to obtain hepatocytes with inhibited Aloxe3 expression.

8. The method according to claim 7, characterized in that, The donor hepatocytes were primary hepatocytes.

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