DDX49-based novel lipid-induced assembled particle as well as preparation method and application of DDX49-based novel lipid-induced assembled particle

Abstract

The invention belongs to the field of biotechnology and medicine, and particularly relates to a DDX49-based novel lipid-induced assembled particle as well as a preparation method and application of the DDX49-based novel lipid-induced assembled particle. The invention aims at liquid-liquid phase separation potential research of a DDX family member DDX49 for assembling a membrane-free organelle in an MASLD process, and finds that the DDX family member DDX49 is positioned in cytoplasm of liver cells, and liquid-liquid phase separation occurs and is assembled into particles, namely lipid-induced particles (LIG) when lipid is accumulated in the fatty degeneration liver cells during the MASLD process. The LIG assembly in fatty degenerative liver cells inhibits the activation of hepatic stellate cells and the progress of hepatic fibrosis by inhibiting the generation of a fibrosis-promoting hepatic cell factor TIMP2. The discovery is verified in both the human liver and in a mouse model. Therefore, a new strategy and new application for treating hepatic fibrosis by regulating and controlling lipid-induced particle (LIG) assembly are provided.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, specifically relating to a novel lipid-induced assembly particle based on DDX49, its preparation method, and its application. Background Technology

[0002] Eukaryotic cells contain a variety of membraneless organelles, including nucleoli, promyelocytic leukemia (PML) bodies, Cajal bodies, nuclear spots, and nuclear stress bodies in the nucleus, and stress granules (SGs), processing bodies (P-bodies), myogranules, and neuronal transport granules in the cytoplasm. These membraneless organelles are executors of spatiotemporal coordination of various intracellular biological processes. In the nucleus, they participate in chromatin organization, maintenance of genome stability, DNA damage response and repair, and transcriptional regulation; in the cytoplasm, they participate in signal transduction, intracellular transport, metabolic programming, mRNA modification and translation, and protein modification and functional regulation. Recent studies have confirmed that these intracellular membraneless organelles play a crucial role in health and disease. For example, stress granules can rapidly assemble under various environmental stresses such as chemical stimulation, ultraviolet radiation, heat shock, and oxygen free radicals, temporarily sealing untranslated mRNA and its related proteins, thereby preventing abnormal translation and promoting post-stress recovery. Processing bodies mainly participate in mRNA modification and degradation, persist under normal physiological conditions, and further increase when stress or other factors inhibit mRNA translation. Dysregulation or dysfunction of stress granules and processing bodies is associated with a variety of diseases, including neurodegenerative diseases, cancer, and infections. Nevertheless, the discovery of novel membraneless organelles and the elucidation of their biological functions remain active and highly anticipated research areas.

[0003] Membraneless organelles are rich in various proteins and RNAs and are reversibly and dynamically assembled through liquid-liquid phase separation (LLPS). Proteins with LLPS potential can serve as scaffolds for assembling membraneless organelles; for example, GTPase activator protein SH3 domain-binding protein 1 (G3BP1) can assemble stress granules. LLPS is a spontaneous phase separation phenomenon driven by multivalent interactions between biomolecules, typically involving non-covalent and low-affinity binding. This process is often regulated by modifications of intrinsically disordered regions (IDRs) within proteins. LLPS depends on the concentration of macromolecules in aqueous solution and is also influenced by intrinsic macromolecular properties (such as length, hydrophobicity, and charge distribution) and external biophysical conditions (such as temperature, pH, and ionic strength). Further in-depth research is needed to identify proteins with LLPS potential, especially those serving as scaffolds for membraneless organelle assembly.

[0004] The global prevalence of metabolic-associated fatty liver disease (MASLD) is increasing annually, with an estimated adult incidence of 30%. Metabolic-associated steatohepatitis (MASH) is a severe form of MASLD, characterized not only by steatosis but also by hepatocellular damage and inflammation. Driven by repeated hepatocellular injury and chronic inflammation, MASH progresses to liver fibrosis, marked by excessive deposition of extracellular matrix (ECM) produced by activated hepatic stellate cells (HSCs). Cirrhosis, the end stage of fibrosis, leads to liver failure and malignant tumors, causing more than one million deaths globally each year. However, the existence of novel membraneless organelles assembled from scaffold proteins with liquid-liquid phase separation potential remains a key question in the progression of MASLD to MASH and liver fibrosis.

[0005] Members of the DEAD-box RNA helicase (DDX) family have been shown to form RNA-containing phase-separating organelles in both prokaryotes and eukaryotes. These members typically contain inherently disordered regions, giving them liquid-liquid phase-separation potential to regulate a variety of intracellular biological processes. However, in the context of liver disease, little is known about the membraneless organelles formed by DDX family members through liquid-liquid phase separation.

[0006] In conclusion, given the current critical question of whether novel membraneless organelles assembled from scaffold proteins with liquid-liquid phase separation potential exist in the progression of MASLD and its progression to MASH and liver fibrosis, there is an urgent need to explore this issue and provide potential treatment options for patients with liver fibrosis. Summary of the Invention

[0007] To address the aforementioned problems, this invention investigates the liquid-liquid phase separation potential of DDX49, a member of the DDX family, during MASLD (Massage-Induced Lactation). It was found that DDX49 is located in the cytoplasm of hepatocytes and undergoes liquid-liquid phase separation and assembly into granules, namely lipid-induced granules (LIGs), during lipid accumulation in steatotic hepatocytes in MASLD. LIG assembly in steatotic hepatocytes inhibits the production of the pro-fibrotic hepatocyte cytokine TIMP2, thereby suppressing hepatic stellate cell activation and the progression of liver fibrosis.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the use of lipid-inducible particles (LIG) in the preparation of medicaments for the prevention and / or treatment of liver fibrosis.

[0009] Furthermore, the lipid-inducible particles (LIG) are obtained by lysing steatotic hepatocytes with lysis buffer.

[0010] Furthermore, the drug comprises a single active ingredient, lipid-inducible particles (LIG), and a pharmaceutically acceptable carrier.

[0011] Furthermore, the drug also includes excipients, which include one or more of fillers, disintegrants, binders, sweeteners, and flavorings.

[0012] Secondly, the present invention also provides a method for preparing the lipid-induced particles (LIG) described in the first aspect, the method comprising the following steps: Step 1: Treat hepatocytes with oleic acid and palmitic acid for 22-26 hours, or treat hepatocytes with MCD medium for 22-26 hours to obtain hepatocytes with liposome accumulation; preferably, treat for 24 hours. Step 2: Lyse the hepatocytes accumulated by liposomes obtained in Step 1 with lysis buffer for 28-22 minutes, centrifuge at 2000g for 5 minutes, and collect the supernatant. Step 3, Step 2: Lyse the hepatocytes accumulated by liposomes obtained in Step 1 with lysis buffer for 28-22 minutes, centrifuge at 1800-2200g for 3-8 minutes, and collect the supernatant; Step 3: Centrifuge the supernatant obtained in Step 2 at 18000-22000g for 18-22 minutes to obtain an insoluble precipitate. Resuspend it in lysis buffer and centrifuge at 18000-22000g for 3-8 minutes to purify it. Then resuspend it in lysis buffer and centrifuge at 800-900g for 3-8 minutes. The supernatant is the LIG component. Further, the supernatant obtained in step 2 was centrifuged at 20000g for 20 minutes to obtain an insoluble precipitate. After resuspending in lysis buffer, it was centrifuged at 20000g for 5 minutes for purification. Then, it was resuspended in lysis buffer and centrifuged at 850g for 5 minutes. The supernatant was the LIG component.

[0013] Further, the concentration of oleic acid is 0.8-1.5 mM, and the concentration of palmitic acid is 0.3-0.8 mM.

[0014] Further, the concentration of oleic acid is 0.9, 1.0, 1.1, 1.2, 1.3, or 1.4 mM, preferably 1 mM; the concentration of palmitic acid is 0.4, 0.5, 0.6, 0.7, or 0.8 mM, preferably 0.5 mM.

[0015] Further, the lysis buffer comprises: 50 mM Tris pH 7.6, 50 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 1 mM β-mercaptoethanol, 1% protease inhibitor, and 0.4 U / mL RNase inhibitor.

[0016] Thirdly, the present invention also provides a pharmaceutical composition for the prevention and / or treatment of liver fibrosis, the pharmaceutical composition comprising a single active ingredient, lipid-inducible particles (LIG), and a pharmaceutically acceptable carrier.

[0017] Furthermore, the lipid-induced particles (LIG) are obtained by the preparation method according to any one of claims 5-7.

[0018] Furthermore, the pharmaceutical composition further includes excipients, which include one or more of fillers, disintegrants, binders, sweeteners, and flavorings.

[0019] Furthermore, the dosage form of the pharmaceutical composition is any pharmaceutically acceptable dosage form.

[0020] This invention, compared to existing technologies, focuses on the liquid-liquid phase separation potential of DDX49, a member of the DDX family, during MASLD (Massage-Induced Lactation) assembly of membrane-free organelles. It reveals that DDX49 is located in the cytoplasm of hepatocytes and undergoes liquid-liquid phase separation and assembly into granules, namely lipid-inducible granules (LIGs), during lipid accumulation in steatotic hepatocytes under MASLD. Inhibition of DDX49 liquid-liquid phase separation exacerbates MASH-induced hepatic stellate cell activation and liver fibrosis, demonstrating that LIG assembly by DDX49 in steatotic hepatocytes feedback-inhibits MASH-induced liver fibrosis. LIG assembly in steatotic hepatocytes inhibits hepatic stellate cell activation and the progression of liver fibrosis by suppressing the production of the pro-fibrotic hepatocyte cytokine TIMP2. This finding has been validated in both human livers and mouse models. Therefore, this provides a novel strategy and application for treating liver fibrosis by regulating lipid-inducible granule (LIG) assembly. Attached Figure Description

[0021] Figure 1This data represents the IDR-dependent liquid-liquid phase separation results of DDX49 in steatotic hepatocytes during MASLD. A: Immunofluorescence staining of DDX49 and hepatocytes in mouse liver tissue sections; B: Immunofluorescence staining of Flag-tagged DDX49 after stimulation of transfected HHL5 cells and primary hepatocytes with OA / PA (oleic acid / palmitic acid) or MCD medium for 24 hours, followed by treatment with 5% 1,6-hexanediol (1,6-HD) for 1 minute; C: Immunofluorescence staining of endogenous DDX49 after stimulation of HHL5 cells and primary hepatocytes with OA / PA or MCD medium for 24 hours, followed by treatment with 5% 1,6-HD for 1 minute; D: Fluorescence recovery-after-photobleaching (FRAP) analysis of GFP-DDX49 particles in live HHL5 cells stably expressing GFP-DDX49; EF: GFP-DDX49 particle splitting (E) or fusion (F) in single live HHL5 cells stably expressing GFP-DDX49. G: Time-lapse microscopy images of GFP-DDX49 (50 μM) after treatment with 5% 1,6-HD in vitro (50 mM NaCl, room temperature conditions); H: Immunofluorescence staining of Flag-tagged DDX49 and its truncated variants in HHL5 cells after treatment with OA / PA for 24 hours; I: Immunofluorescence staining of endogenous DDX49 and G3BP1 in HHL5 cells after treatment with sodium arsenite (NaAsO2, 60 μM for 1 hour), hydrogen peroxide (H2O2, 2 mM for 1 hour), OA / PA, or MCD medium for 24 hours; J: Immunofluorescence staining of endogenous DDX49 in liver tissue sections from mice fed with normal diet (ND), high-fat diet (HFD), choline-deficient high-fat diet (CD-HFD), Western diet (WD), or MCD diet.

[0022] Figure 2 LIG assembly in hepatocytes inhibits MASH-induced liver fibrosis through a feedback mechanism. (AF): DDX49 f / f Mouse and hepatocyte-specific DDX49 knockout (DDX49 hep- / - After mice were fed an MCD diet, Sirius red staining (A), Masson staining (B), and α-smooth muscle actin (α-SMA) staining (C) were performed on liver tissue sections (top image shows staining, bottom image shows quantitative analysis, n=6 mice / group); mRNA levels of fibrosis-related genes in liver tissue (D, n=6 mice / group); protein levels of α-SMA, type I collagen α1 chain (COL1A1), and type III collagen α1 chain (COL3A1) in liver tissue (E, n=6 mice / group); hydroxyproline content in liver tissue (F, n=6 mice / group). Scale bar: 40 μm. GL: DDX49ΔIDRf / f After being fed an MCD diet, mice and hepatocyte-specific DDX49ΔIDR knockout (DDX49ΔIDRhep) mice underwent Sirius red (G), Masson (H), and α-SMA (I) staining of liver tissue sections (top image shows staining, bottom image shows quantitative analysis, n=6 mice / group); mRNA levels of fibrosis-related genes in liver tissue (J, n=6 mice / group); protein levels of α-SMA, COL1A1, and COL3A1 in liver tissue (K, n=6 mice / group); and hydroxyproline content in liver tissue (L, n=6 mice / group). Scale bar: 40 μm. Data are expressed as mean ± SD. **P < 0.01, ns indicates no statistical significance, and Student's test was used for statistical analysis (AD, FJ, L).

[0023] Figure 3 To investigate the integration of pro-fibrotic TIMP2 mRNA into lipid-inducible granules (LIG) and the inhibition of its translation. A: Schematic diagram of the cell co-culture system; B: Protein levels of fibrosis-related genes in primary hepatocyte stellate cells (HSCs) cultured in supernatant from primary hepatocytes treated with OA / PA (oleic acid / palmitic acid) for different time periods (n=2 / group); C: Schematic diagram of the cell co-culture system; D: Primary DDX49 cells treated with OA / PA or MCD medium for 24 hours. f / f Hepatocytes and DDX49 hep- / -Protein levels of α-smooth muscle actin (α-SMA), type I collagen α1 chain (COL1A1), and type III collagen α1 chain (COL3A1) in primary HSCs after culturing hepatocyte supernatant (n=2 / group); E: Left: RIP-seq heatmap of DDX49 particle-enriched mRNA in HHL5 cells overexpressing Flag-DDX49 and treated with OA / PA; Right: Differential proteomics analysis of secreted proteins between primary DDX49f / f hepatocytes and DDX49hep- / - hepatocytes treated with OA / PA (n=3); Bottom: Venn diagram showing DDX49 particle-enriched mRNA and DDX49hep- / - hepatocytes. Overlap of upregulated proteins in supernatant; F: Fold change in expression of the four overlapping proteins in Figure E in the supernatant of primary DDX49hep- / - hepatocytes and DDX49f / f hepatocytes treated with OA / PA; GH: Protein level of TIMP2 in the supernatant of HHL5 cells (G) and primary hepatocytes (H) transfected with Flag-DDX49 and treated with OA / PA or MCD medium (n=3 / group); I: Protein level of TIMP2 in the supernatant of primary DDX49f / f hepatocytes and DDX49hep- / - hepatocytes treated with OA / PA or MCD medium (n=3 / group); J: DDX49 cells fed with a choline-deficient high-fat diet (CD-HFD) or MCD diet. f / f Mice and DDX49 hep- / - Serum TIMP2 levels in mice (n=3 mice / group); KL: After OA / PA treatment, the binding of DDX49 to TIMP2 mRNA in HHL5 cells or primary hepatocytes transfected with Flag-DDX49 was detected by RIP-qRT-PCR (n=3 / group); M: In OA / PA-treated HHL5 cells, the binding of DDX49 to TIMP2 mRNA was detected by RNA pull-down assay; N: Primary DDX49f / f hepatocytes and DDX49... hep- / - In hepatocytes, the relative distribution of TIMP2 mRNA in each ribosomal component was analyzed by qRT-PCR (n=3 / group); O: TIMP2 protein level in the supernatant of primary DDX49ΔIDRf / f hepatocytes and DDX49ΔIDRhep hepatocytes treated with OA / PA or MCD medium (n=3 / group); P: DDX49 ΔIDR hepatocytes fed with CD-HFD or MCD diet. f / f Serum TIMP2 levels in mice and DDX49 ΔIDRhep mice (n=3 mice / group); PQ: Primary DDX49 ΔIDR treated with OA / PA. f / fThe relative distribution of TIMP2 mRNA in each ribosomal component was analyzed by qRT-PCR in hepatocytes and DDX49ΔIDRhep hepatocytes (n=3 / group). Data are expressed as mean ± SD. *P<0.05, **P<0.01, statistical analysis was performed using Student's test (GJ, NQ) or one-way ANOVA combined with Dunnett's multiple comparison test (K, L).

[0024] Figure 4 Lipid-inducible granules (LIGs) are assembled in human steatotic hepatocytes and are negatively correlated with fibrosis progression. In Figures AC: Liver tissue sections from healthy controls (A, n=6), patients with hepatic steatosis (B, n=12), and MASH patients (C, n=12), LIG ​​assembly in hepatocytes was detected using immunofluorescence staining with endogenous DDX49; Figure DF: Liver tissue sections from patients with hepatic steatosis (D) and MASH patients (E), showing immunofluorescence staining with DDX49 (red), YBX1 (green), and RNA-scope imaging of TIMP2 mRNA (white) (n=12); Figure F shows representative cases of hepatic steatosis patient #1 and MASH patient #1, showing DDX49 separately. Channel images of X49, YBX1, and TIMP2 mRNA; G: Quantitative analysis of LIG assembly in liver tissues of healthy controls, patients with hepatic steatosis, and MASH patients (n=21); H: Pearson correlation coefficient analysis of LIG quantity in liver tissue sections of MASH patients with NAFLD fibrosis score (NFS) (n=21); I: Spearman correlation coefficient analysis of LIG quantity in liver tissue sections of MASH patients with fibrosis score 4 (FIB-4) (n=21); J: Pearson correlation coefficient analysis of LIG quantity in liver tissue sections of MASH patients with serum TIMP2 level (n=21). Data are expressed as mean ± SD. R² and P values ​​are directly labeled in the figures. Statistical analysis was performed using Welch ANOVA combined with Games-Howell test (G), Pearson correlation coefficient analysis (H, J), or Spearman correlation coefficient analysis (I).

[0025] Figure 5 This diagram illustrates the mechanism by which the lipid-inducible granule (LIG) assembly of this invention inhibits hepatic stellate cell activation and the progression of liver fibrosis by suppressing the production of the pro-fibrotic hepatocyte cytokine TIMP2. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the embodiments of the present invention. The following embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0027] In the following embodiments, the animal models with the target gene knocked out were created by the company based on the inventors' conformation.

[0028] 1. Laboratory mice 4-6 week old C57BL / 6J mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd. (Ddx49) f / f Mice, Ddx49ΔIDR f / f Mice were constructed by Cyagen Biosciences (Suzhou), and Alb-Cre mice were constructed by Shanghai Southern Model Biotechnology Co., Ltd. All animal experiments were conducted in accordance with the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals.

[0029] Genotyping was performed using PCR analysis of mouse tail tip genomic DNA. Primer sequences are shown in Table 1.

[0030] Table 1 Primer sequences used for genotyping

[0031] 2. Mouse model construction MASLD and MASH models: 4-week-old male mice were fed a 4-month HFD (60kcal% fat), a 2-month CD-HFD (60kcal% fat), a 4-week MCD (60kcal% fat), or a 6-month WD (40kcal% fat) diet, respectively.

[0032] All mice were housed in SPF-grade animal facilities at a temperature of 23±2℃ and a relative humidity of 40-70%, with a 12-hour light-dark cycle.

[0033] 3. Cell Culture and Transfection Human hepatocyte cell line HHL5 was cultured in DMEM medium containing 10% fetal bovine serum (FBS); human immortalized hepatic stellate cell line LX-2 was cultured in DMEM medium containing 2% FBS.

[0034] The DDX49-GFP sequence was cloned into the pLent-EF1a-FH-CMV-Puro vector, and cells were infected with TransDux™MAX lentiviral transduction reagent. GFP-positive cells were screened by single-cell sorting (Sony SH800s) 48 hours after transfection.

[0035] HHL5 cells or primary hepatocytes were treated with 1 mM oleic acid (OA) + 0.5 mM palmitic acid (PA) for 24 hours, or cultured in MCD medium for 24 hours.

[0036] 4. Liver histology, immunohistochemistry and immunofluorescence Immunofluorescence: Sections or cells were stained with antibodies such as DDX49, HNF4α, and G3BP1, and the nuclei were stained with DAPI. The images were taken using a Leica MDI8 confocal microscope, and the number of DDX49 aggregates was quantified using ImageJ.

[0037] 5. Fluorescence Recovery After Photobleaching (FRAP) HHL5 cells stably expressing GFP-DDX49 were seeded in glass-bottomed culture dishes and, after treatment with OA / PA or MCD, were examined using a Leica DMI8 confocal microscope. Purified protein was diluted in a buffer solution containing 20 mM Tris-HCl, 10% PEG8000, and 50 mM NaCl, and loaded into the imaging chamber. The target region was bleached with a 488 nm laser (100% power) for 2 seconds, and continuous imaging was performed over 180 seconds. Data were analyzed using a single-exponential model fitted with GraphPad Prism8.

[0038] 6. In vitro liquid-liquid phase separation (LLPS) experiment The purified protein was diluted to different concentrations (containing 20 mM Tris-HCl, a specified concentration of PEG8000 and NaCl), loaded into the imaging chamber, and the phase separation was observed using a Leica DMI8 confocal microscope.

[0039] Experimental results are as follows Figure 1 As shown, the expression of DDX49 in the liver was detected, and immunofluorescence confirmed that DDX49 was mainly localized in parenchymal hepatocytes ( ). Figure 1 (A). In the HHL5 hepatocyte cell line and primary hepatocytes, forced expression of the tagged DDX49 demonstrated its ability to form cytoplasmic aggregates upon exposure to OA / PA or methionine-choline-deficient (MCD) media, and that these aggregates could be disrupted by 5% 1,6-hexanediol (1,6-HD). Figure 1 (See section B), indicating that DDX49 undergoes liquid-liquid phase separation in steatotic hepatocytes. Further validation of endogenous DDX49 liquid-liquid phase separation upon exposure to OA / PA or MCD media was performed in HHL5 cells and primary hepatocytes. Figure 1 (C). To determine whether the aggregates formed by DDX49 possess liquid-like properties, HHL5 cells stably overexpressing the GFP tag DDX49 were constructed. It was found that their fluorescence recovery after photobleaching (FRAP) was rapid (C). Figure 1 (D), and DDX49 condensates can effectively split and fuse ( Figure 1 Adding 5% 1,6-hexanediol to (E, F) will disrupt droplet formation ( Figure 1 (G).

[0040] These results indicate that DDX49 undergoes liquid-liquid phase separation in the cytoplasm of steatotic hepatocytes, and that proteins containing inherently disordered regions possess the potential for liquid-liquid phase separation. Constructing a truncated form of DDX49 confirmed that liquid-liquid phase separation of DDX49 depends on its inherently disordered regions, and that these regions can independently undergo liquid-liquid phase separation. Figure 1 (H).

[0041] To compare the localization, size, and number of DDX49 aggregates with known intracellular non-membrane organelles, it was found that OA / PA or MCD-induced DDX49 aggregates did not colocalize with stress granules. Figure 1 In addition, in liver tissues induced by high-fat diet (HFD)-induced MASLD and MASH-induced CD-HFD, Western diet (WD), or MCD, particles formed by endogenous DDX49 assembly were observed in vivo. Figure 1 (J).

[0042] In summary, during MASLD and MASH, a novel particle is assembled in steatotic hepatocytes via liquid-liquid phase separation of DDX49, a process that depends on their inherent disordered regions.

[0043] Example 2: LIG assembly in hepatocytes inhibits MASH-induced liver fibrosis through a feedback mechanism. 1. The experimental mice were the same as in Example 1. 2. The mouse model was constructed in the same manner as in Example 1.

[0044] 3. Cell Culture and Transfection Human hepatocyte cell line HHL5 was cultured in DMEM medium containing 10% fetal bovine serum (FBS); human immortalized hepatic stellate cell line LX-2 was cultured in DMEM medium containing 2% FBS.

[0045] Primary mouse hepatocytes and HHL5 cells were transfected with plasmids using Jet-PRIME transfection reagent (24 hours); HHL5 cells were transfected with DDX49 siRNA using LipofectamineRNAiMAX (48 hours), with a final siRNA concentration of 40 nM. The siRNA sequences were: sense strand 5'-GAGAGUGUGAGAUCAAACUTT-3' (SEQ ID NO. 7), antisense strand 5'-AGUUUGAUCUCACACUCUCTT-3' (SEQ ID NO. 8).

[0046] The DDX49-GFP sequence was cloned into the pLent-EF1a-FH-CMV-Puro vector, and cells were infected with TransDux™MAX lentiviral transduction reagent. GFP-positive cells were screened by single-cell sorting (Sony SH800s) 48 hours after transfection.

[0047] HHL5 cells or primary hepatocytes were treated with 1 mM oleic acid (OA) + 0.5 mM palmitic acid (PA) for 24 hours, or cultured in MCD medium for 24 hours.

[0048] 4. Liver histology and immunohistochemistry Paraffin-embedded liver tissue sections were stained with H&E, Sirius red, and Masson staining. The area of ​​liver fibrosis (positive area / total section area) was quantified using ImageJ software.

[0049] Immunohistochemistry: Sections were stained with α-SMA antibody, and the area of ​​α-SMA positivity was quantified using ImageJ.

[0050] 5. RNA extraction and quantitative PCR Total RNA was extracted from tissues / cells using TRIzol reagent, and qRT-PCR was performed using the SYBRRT-PCR kit (Takara) and LightCycler (Roche). ΔΔCt The relative expression level was calculated using the method (internal reference gene normalization), and the primer sequences are shown in Table 2.

[0051] Table 2 qRT-PCR primer sequences

[0053] 6. Western Blot (WB) After quantification by BCA, tissue / cell lysates were separated by SDS-PAGE, transferred to a membrane, and incubated with antibodies (α-SMA, COL1A1, COL3A1, etc.) before imaging.

[0054] 7. Quantitative and Statistical Analysis Data are expressed as mean ± SD or as shown in the figure. The unpaired t-test was used for comparison between two groups, and the one-way ANOVA + Tukey HSD / Dunnett test was used for comparison of multiple groups. P < 0.05 was considered significant. The number of animals / samples in each group is shown in the legend.

[0055] The above experiments prove that DDX49 hep- / - MASH-induced liver fibrosis was significantly aggravated in mice, manifested by increased collagen deposition and enhanced activation of hepatic stellate cells. Figure 2 The results (AF) indicate that DDX49 deficiency in hepatocytes may exacerbate MASH-induced hepatic stellate cell activation and liver fibrosis by disrupting LIG assembly.

[0056] To further explore DDX49 hep- / -To investigate whether the exacerbation of LIG-mediated liver fibrosis is attributable to the disruption of LIG assembly, we constructed mice with hepatocyte-specific deletions of the intrinsic disordered region of DDX49 (responsible for its liquid-liquid phase separation activity and LIG assembly) (DDX49ΔIDR). hep In these mice, MASH-induced liver fibrosis was significantly aggravated. Figure 2 (in GL), and in DDX49 hep- / - The findings were consistent across mouse species. Therefore, loss of DDX49 liquid-liquid phase separation activity exacerbates MASH-induced hepatic stellate cell activation and liver fibrosis, suggesting that LIG feedback assembly of DDX49 in steatotic hepatocytes inhibits MASH-induced liver fibrosis.

[0057] Example 3: Lipid-inducible granules (LIG) integrate pro-fibrotic TIMP2 mRNA and inhibit its translation. 1. The experimental mice were the same as in Example 2.

[0058] 2. The mouse model was constructed in the same manner as in Example 2.

[0059] 3. Cell culture and transfection are the same as in Example 2.

[0060] 4. Protein Expression and Purification The gene sequence was cloned into the pET28a vector, and a His tag was added to the C-terminus of DDX49, or His6-EGFP / His6-TurboRFP tags were added respectively. Recombinant protein expression was induced for 18 hours at 22°C with 0.8 mM IPTG. After bacterial homogenization and lysis, the supernatant was purified by nickel affinity chromatography, eluted with a linear imidazole gradient (200-500 mM), and the purity was verified by SDS-PAGE. The purified protein was then dialyzed into pre-cooled storage buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol, 1 mM MSF) and stored at -80°C.

[0061] 5. RNA extraction and quantitative PCR Total RNA was extracted from tissues / cells using TRIzol reagent, and qRT-PCR was performed using the SYBRRT-PCR kit (Takara) and LightCycler (Roche). ΔΔCt The relative expression level was calculated using the method (internal reference gene normalization). The newly added primer sequence was Timp2, forward: 5'-GCAACCCCATCAAGAGGATTC-3', reverse: 5'-GGGGCCGTGTAGATAAACTCG-3'.

[0062] 6. RNA pull-down Biotin-labeled TIMP2 mRNA probes (Geneseed) were incubated with HHL5 cell lysate, and streptavidin magnetic beads captured RNA-protein complexes. Western blot was used to detect the binding proteins.

[0063] 7. Western Blot (WB) After quantification by BCA, tissue / cell lysates were separated by SDS-PAGE, transferred to a membrane, and incubated with antibodies (α-SMA, COL1A1, COL3A1, DDX49, etc.) before imaging.

[0064] 8. RNA immunoprecipitation (RIP)-seq and RIP-qRT-PCR Cells were lysed after UV cross-linking and RIP was performed using the MagnaRIP® kit. The co-precipitated RNA was extracted with TRIzol and then sequenced (RIP-seq) or qRT-PCR (RIP-qRT-PCR, IgG was used as a negative control).

[0065] 9. LIGs protein sample preparation Hepatocytes were washed with PBS and lysed for 20 minutes in 1.5 mL of lysis buffer (50 mM Tris, pH 7.6, 50 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 1 mM β-mercaptoethanol, 1% protease inhibitor, 0.4 U / mL RNase inhibitor). One-tenth of the lysis buffer was used as the total cell fraction, and the remainder was centrifuged at 2000g for 5 minutes to separate the cytoplasm (supernatant) and nucleus (precipitate). One mL of the cytoplasmic supernatant was centrifuged at 20000g for 20 minutes to obtain an insoluble precipitate. This precipitate was resuspended, centrifuged at 20000g for 5 minutes for purification, and then resuspended in 300 μL of lysis buffer. The precipitate was centrifuged at 850g for 5 minutes, and the supernatant was the LIGs fraction. LIGs samples were subjected to 10X proteomics analysis (PTMBIO), and the data were uploaded to ProteomeXchange (PXD064747). Simultaneously, LIGs samples were frozen and thawed, and metabolites were detected by ELISA.

[0066] 10. Quantitative Proteomics Analysis of Cell Supernatant (10X) Ddx49 f / f and Ddx49 hep- / - Primary hepatocytes were treated with OA / PA for 24 hours, and the supernatant was collected and centrifuged at 12000g for 10 minutes for 10X proteomics analysis (PTMBIO). LC-MS / MS data were uploaded to ProteomeXchange (PXD064823).

[0067] 11. Polyribosome profiling analysis Cells were lysed after treatment with 100 μg / mL CHX for 7 minutes, centrifuged at 15000g to obtain supernatant, spread on a 10-50% sucrose gradient, centrifuged at 39000 rpm for 90 minutes using an SW41Ti rotor, separated into 12 fractions using GradientStation, and the absorbance was measured at 260 nm using NanoDrop. RNA was extracted with TRIzol and analyzed by qRT-PCR.

[0068] 12. Ribosome profiling analysis (Ribo-seq) In primary hepatocytes treated with OA / PA, polyribosomes were digested with RNase I to obtain ribosome protective fragments (RPFs), which were then purified and used to construct libraries for sequencing (OMbiotech). The data were trimmed with Cutadapt, aligned with STAR, and the translation efficiency (RPF abundance / RNA abundance) was analyzed using RiboParser.

[0069] 13. ELISA Commercial kits can be used to detect TIMP2, PKM, LDHB, and NPNT in mouse / human serum, as well as metabolites such as arachidonic acid, 6-keto-PGF1α, and PGF2α in cells / liver tissues.

[0070] 14. Quantitative and Statistical Analysis Data are expressed as mean ± SD or as shown in the figure. The unpaired t-test was used for comparison between two groups, and the one-way ANOVA + Tukey HSD / Dunnett test was used for comparison of multiple groups. P < 0.05 was considered significant. The number of animals / samples in each group is shown in the legend.

[0071] This study investigated how the loss of hepatocyte ligation (LIG) in steatotic hepatocytes enhances hepatic stellate cell activation, thereby exacerbating liver fibrosis. Notably, hepatocytes produce and secrete a series of cytokines (called hepatocyte cytokines) that promote the progression of liver fibrosis. To verify whether the anti-fibrotic effect of LIG in hepatocytes is mediated by regulating hepatocyte cytokine production and subsequently interacting with hepatic stellate cells, a co-culture system of hepatocytes and hepatic stellate cells was established. Figure 3 (A). Furthermore, treatment of HHL5 cell supernatant with OA / PA overexpressing DDX49 inhibited LX-2 cell activation ( Figure 3 (B), while OA / PA processes DDX49. hep- / - The supernatant of primary hepatocytes promotes the activation of primary hepatic stellate cells. Figure 3 (C, D) indicates that hepatic LIG assembly inhibits hepatic stellate cell activation by regulating the production of hepatocyte cytokines in hepatocytes.

[0072] Subsequently, the hepatocyte-cytokines regulated by LIG in steatotic hepatocytes were investigated. Given that RNA is typically integrated into intracellularly assembled particles, RNA immunoprecipitation sequencing (RIP-seq) was performed using immunoprecipitation of DDX49 in steatotic hepatocytes, identifying 289 mRNAs. Figure 3 (E). DDX49 for OA / PA processing. f / f and DDX49 hep- / - Proteomic analysis of primary hepatocyte supernatant revealed differential levels of 54 proteins. The intersection of the two datasets yielded four candidate hepatocyte cytokines, including pyruvate kinase M1 / 2 (PKM), lactate dehydrogenase B (LDHB), renin (NPNT), and tissue inhibitor of metalloproteinases 2 (TIMP2), with TIMP2 showing the most significant elevation in DDX49 deficiency. Figure 3 EF). DDX49 overexpression inhibits the production and secretion of TIMP2 in hepatocytes, while its absence promotes this process. Figure 3 GI). In the MASLD and MASH mouse models, DDX49 hep- / - It also significantly increased serum TIMP2 levels ( Figure 3 (J). Therefore, LIG ​​assembly in steatotic hepatocytes inhibits hepatic stellate cell activation and the progression of liver fibrosis by suppressing the production of the pro-fibrotic hepatocyte cytokine TIMP2.

[0073] Next, we investigated how LIG assembled in steatotic hepatocytes inhibits TIMP2 expression. After OA / PA treatment, RIP-qRT-PCR and RNA pull-down experiments confirmed that TIMP2 mRNA was integrated into LIG assembled by DDX49. Figure 3 (Middle KM). The translation of TIMP2 mRNA after integration into LIG was evaluated. Polynucleotide profiling analysis of TIMP2 mRNA revealed that DDX49... hep- / - Enhance its translation efficiency ( Figure 3 (N). Furthermore, the LIG assembly inducers—arachidonic acid metabolites 6-keto-prostaglandin F1α and prostaglandin F2α—inhibit the translation of TIMP2 mRNA. Similar results were observed in hepatocytes lacking the intrinsic disordered region of DDX49 in vitro and in DDX49 ΔIDRhep mice in vivo. Figure 3 (OQ). In summary, LIG ​​assembled in steatotic hepatocytes integrates TIMP2 mRNA and inhibits its translation, thereby reducing TIMP2 production and feedback-inhibiting hepatic stellate cell activation during MASLD and MASH.

[0074] Example 4: LIG was assembled in human steatotic hepatocytes and was negatively correlated with the progression of fibrosis.

[0075] 1. Tissue sample Normal human liver tissue was obtained from patients who underwent liver surgery for benign liver diseases such as hepatic hemangioma, hepatic cysts, or gallstones at Naval Medical University (Shanghai, China). Human MASLD (metabolic steatohepatitis) and MASH (metabolic steatohepatitis) tissue and serum samples were also obtained from patients who underwent surgery for the aforementioned benign liver diseases at the same hospital. All human liver tissue samples underwent histological examination to confirm the pathological diagnosis and were fabricated into paraffin-embedded tissue microarrays for subsequent analysis. Written informed consent was obtained from all patients for this study, and the experiment was approved by the Institutional Research Ethics Committee of Naval Medical University (No.: EHBHKY2023G-H001-P001).

[0076] In MASH patients, the formulas for calculating liver fibrosis markers (NFS and FIB-4) are as follows: NFS = -1.675 + [0.37 × age (years)] + [0.094 × BMI (kg / m²)] 2 )]+[1.13×impaired fasting glucose (or diabetes, yes=1, no=0)]+(0.99×AST / ALT)-[0.013×platelet count(×10 9 [0.66 × albumin (g / dL)] - [0.66 × albumin (g / dL)]; FIB-4 = [Age (years) × AST (U / L)] / [Platelet count (1000 cells / μL) × ALT (U / L)] 1 / 2 ].

[0077] 2. Liver histology, immunohistochemistry and immunofluorescence Immunofluorescence: Sections or cells were stained with DDX49, YBX1, and TIMP2 antibodies, and the nuclei were stained with DAPI. The images were taken using a Leica MDI8 confocal microscope, and the number of DDX49 aggregates was quantified using ImageJ.

[0078] 3. LIGs protein sample preparation After washing hepatocytes with PBS, they were lysed for 20 minutes in 1.5 mL of lysis buffer (50 mM Tris, pH 7.6, 50 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 1 mM β-mercaptoethanol, 1% protease inhibitor, 0.4 U / mL RNase inhibitor), and vortexed to mix. One-tenth of the lysis buffer was used as the total cell fraction, and the remainder was centrifuged at 2000g for 5 minutes to separate the cytoplasm (supernatant) and nucleus (precipitate). One mL of the cytoplasmic supernatant was centrifuged at 20000g for 20 minutes to obtain an insoluble precipitate, which was resuspended and centrifuged at 20000g for 5 minutes for purification. It was then resuspended in 300 μL of lysis buffer and centrifuged at 850g for 5 minutes. The supernatant was the LIGs fraction. The LIGs samples were subjected to 10X proteomics analysis (PTMBIO), and the data were uploaded to ProteomeXchange (PXD064747). At the same time, the LIGs samples were frozen and thawed, and metabolites were detected by ELISA.

[0079] 4. ELISA Commercial kits for detecting TIMP2 in mouse / human serum.

[0080] To investigate whether LIG is assembled in hepatocytes of patients with MASLD and MASH, DDX49 was stained in tissue sections of normal human liver, steatotic liver, and MASH liver. The results showed that LIG was assembled in hepatocytes of steatotic and MASH livers, but was almost undetectable in normal livers. Figure 4 The study confirmed the presence of LIG assembly in human MASLD and MASH liver tissues. Furthermore, in human steatosis and MASH liver tissues, Y-box binding protein 1 (YBX1) and TIMP2 mRNA were observed to be integrated into the LIG assembled by DDX49. Figure 4 (Df) demonstrated that LIG assembled by DDX49 integrates YBX1 and TIMP2 mRNA in steatotic hepatocytes.

[0081] Subsequently, to analyze whether LIG assembly in hepatocytes of MASH patients is related to the progression of liver fibrosis, LIG ​​in liver slices was quantified. Figure 4 The study investigated the correlation between LIG (liver fibrosis index) and liver fibrosis scores (including non-alcoholic fatty liver disease fibrosis score (NFS) and fibrosis-4 score (FIB-4)) in MASH patients. Correlation analysis showed that the number of LIGs in liver slices was negatively correlated with severe NFS and FIB-4 scores. Figure 4 The presence of LIG assembly in hepatocytes (HI) indicates a negative correlation between LIG assembly and fibrosis progression in MASH patients. Furthermore, the number of LIGs observed in liver sections from MASH patients was negatively correlated with serum TIMP2 levels. Figure 4 (J), further confirming the conclusion that LIG assembly inhibits TIMP2 production in hepatocytes.

[0082] Therefore, LIG ​​assembly in human MASH livers predicts a lower risk of liver fibrosis and cirrhosis, consistent with the feedback inhibition of MASH-induced fibrosis observed in mouse models.

[0083] In summary, this invention investigates the liquid-liquid phase separation potential of DDX49, a member of the DDX family, in assembling membraneless organelles during MASLD. It reveals that DDX49 is located in the cytoplasm of hepatocytes and undergoes liquid-liquid phase separation and assembly into granules, namely lipid-induced granules (LIGs), during lipid accumulation in steatotic hepatocytes in MASLD. LIG assembly in steatotic hepatocytes inhibits the production of the pro-fibrotic hepatocyte cytokine TIMP2, thereby suppressing hepatic stellate cell activation and the progression of liver fibrosis (e.g., ...). Figure 5 (As shown).

[0084] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. Application of lipid-inducible particles (LIG) in the preparation of drugs for the prevention and / or treatment of liver fibrosis.

2. The application according to claim 1, characterized in that, The lipid-inducible particles (LIGs) were obtained by lysing hepatocytes containing liposomes using a lysis buffer.

3. The application according to claim 2, characterized in that, The drug comprises a single active ingredient, lipid-inducible particles (LIG), and a pharmaceutically acceptable carrier.

4. The application according to claim 1, characterized in that, The drug also includes excipients, which include one or more of fillers, disintegrants, binders, sweeteners, and flavorings.

5. The method for preparing lipid-induced particles (LIG) according to claim 1, characterized in that, The preparation method includes the following steps: Step 1: Treat hepatocytes with oleic acid and palmitic acid for 22-26 hours, or treat hepatocytes with MCD medium for 22-26 hours to obtain hepatocytes with liposome accumulation; Step 2: Lyse the hepatocytes accumulated by liposomes obtained in Step 1 with lysis buffer for 28-22 minutes, centrifuge at 1800-2200g for 3-8 minutes, and collect the supernatant. Step 3: Centrifuge the supernatant obtained in Step 2 at 18000-22000g for 18-22 minutes to obtain an insoluble precipitate. Resuspend it in lysis buffer and centrifuge at 18000-22000g for 3-8 minutes to purify it. Then resuspend it in lysis buffer and centrifuge at 800-900g for 3-8 minutes. The supernatant is the LIG component.

6. The preparation method according to claim 5, characterized in that, The concentration of oleic acid is 0.8-1.5 mM, and the concentration of palmitic acid is 0.3-0.8 mM.

7. The preparation method according to claim 5, characterized in that, The lysis buffer comprises: 50 mM TrispH 7.6, 50 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 1 mM β-mercaptoethanol, 1% protease inhibitor, and 0.4 U / mL RNase inhibitor.

8. A pharmaceutical composition for the prevention and / or treatment of liver fibrosis, characterized in that, The pharmaceutical composition comprises a single active ingredient, lipid-inducible particles (LIG), and a pharmaceutically acceptable carrier, wherein the lipid-inducible particles (LIG) are obtained by the preparation method according to any one of claims 5-7.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition further includes excipients, which include one or more of fillers, disintegrants, binders, sweeteners, and flavorings.

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