Composition for reversing organ fibrosis and application thereof

Abstract

The invention provides a composition for reversing organ fibrosis and application of the composition, and belongs to the technical field of biological medicines.The cocktail composition is prepared from valproic acid, CHIR99021, Repsox, trans-benzene ring propylamine and forskolin. The HAT inhibition composition is prepared from A485 and WM-8014; the cocktail composition and the HAT inhibition composition are used independently or jointly, and hepatic fibrosis or pulmonary fibrosis can be reversed; when the composition is used in combination, a synergistic enhancement effect is shown in pulmonary fibrosis and hepatic fibrosis models, and the effects of improving fibrosis indexes and reversing epigenetic age are more remarkable; according to the composition provided by the invention, the epigenetic procedure of cells is accurately controlled, so that the cells are safely reversed from a pathological state to a healthy or resting state, the pathological phenotype of fibrosis is improved, the biological age of the cells is fundamentally reversed, and a major breakthrough in the field of fibrosis treatment is represented.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a composition for reversing organ fibrosis and its application. Background Technology

[0002] Organ fibrosis is a group of pathological repair disorders caused by chronic injury, inflammation, or metabolic disturbances. Its core characteristics are excessive proliferation of fibroblasts and abnormal deposition of extracellular matrix (ECM), ultimately leading to organ structural destruction and functional failure. This type of disease can affect multiple vital organs, including the liver and lungs, manifesting as liver fibrosis, idiopathic pulmonary fibrosis, etc., and is a significant contributing factor to rising morbidity and mortality worldwide. For example, if liver fibrosis is not effectively intervened, it will gradually progress to cirrhosis and even liver failure. However, despite the significant threat organ fibrosis poses to human health, there is currently a lack of radical treatments that directly target the core mechanisms of fibrosis development. Existing interventions are mostly limited to relieving symptoms or slowing disease progression, and the therapeutic effect urgently needs improvement.

[0003] In recent years, the rise of cell reprogramming technology has provided a novel approach to the treatment of organ fibrosis. This technology can reshape the identity of adult cells by regulating cell fate-related signaling pathways or epigenetic states. Early studies relied heavily on overexpression of transcription factors (such as the combination of GATA4, MEF2c, and TBX5 to induce cardiomyocyte reprogramming, and the combination of HNF1β, EMX2, and PAX8 to induce renal tubular epithelial cell reprogramming) to achieve cell fate transformation. However, these transgenic-based reprogramming strategies have many limitations: on the one hand, the delivery of transcription factors often depends on viral vectors, posing a risk of gene integration and potential safety issues such as inducing tumors; on the other hand, the reprogramming efficiency is low and individual applicability varies greatly, with the transformation efficiency of some difficult-to-induce cell lines failing to meet clinical application needs. Furthermore, the complexity of operation and the difficulty in large-scale application severely limit its clinical translation process.

[0004] To overcome the shortcomings of transgenic reprogramming, chemical small molecule-mediated reprogramming technology has emerged. Studies have confirmed that small molecule compounds can directly target epigenetic regulatory factors (such as histone modifying enzymes KAT3A / B and KAT6A) or key nodes in signaling pathways, regulating cell fate transitions in a non-virus-dependent and highly controllable manner. This not only effectively avoids the risk of gene integration but also offers advantages such as ease of synthesis, low cost, and scalability, making it more suitable for clinical translation. For example, studies have successfully induced mouse and human somatic cells into pluripotent stem cells (CiPS cells) using small molecule combinations, demonstrating promising potential in clinical treatment research for diseases such as diabetes.

[0005] Compared to complete reprogramming (where cell fate is completely reshaped into other functional cells), partial reprogramming, as a milder method of cell fate regulation, only requires a moderate reversal of cell differentiation state and resetting epigenetic markers associated with cellular senescence and fibrosis to restore normal cellular physiological function or inhibit pro-fibrotic activity without completely altering cell type. This offers unique advantages in reducing reprogramming-related risks (such as abnormal differentiation and tumorigenicity). However, current research on small molecule compositions-mediated partial reprogramming for organ fibrosis treatment is still in the exploratory stage. Existing small molecule reprogramming systems mostly focus on complete reprogramming or transdifferentiation of specific cell types. Optimized small molecule combinations for partial reprogramming are not yet clear, and there are issues such as insufficient induction efficiency, unclear mechanisms of action, and unverified in vivo anti-fibrotic effects. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a composition for reversing organ fibrosis and its application, aiming to solve the problems of insufficient existing treatment methods and limited clinical translation of reprogramming technology.

[0007] This invention provides a cocktail composition for reversing organ fibrosis through partial reprogramming, comprising valproic acid, CHIR99021, Repsox, transphenylcyclopropane, and foctocorline; wherein the concentration of valproic acid is 200-300 μM, the concentration of CHIR99021 is 5-15 μM, the concentration of Repsox is 5-15 μM, the concentration of transphenylcyclopropane is 4-6 μM, and the concentration of foctocorline is 40-60 μM.

[0008] The present invention provides a HAT inhibitory composition for reversing organ fibrosis by partial reprogramming, comprising A485 and WM-8014; wherein the concentration of A485 is 5-15 μM and the concentration of WM-8014 is 4-6 μM.

[0009] The present invention provides a composition for reversing organ fibrosis by partial reprogramming, comprising the cocktail composition and the HAT inhibitory composition.

[0010] The present invention provides the use of the cocktail composition, the HAT inhibitory composition, or the composition in the preparation of a medicament for treating organ fibrosis.

[0011] Preferably, the organ fibrosis includes liver fibrosis and pulmonary fibrosis.

[0012] Compared with the prior art, the present invention has the following beneficial effects: This invention provides two core small molecule compositions that chemically mimic partial reprogramming. The cocktail composition contains five small molecules with well-defined targets: valproic acid (VPA, a histone deacetylation inhibitor), CHIR99021 (a GSK-3β inhibitor), Repsox (a TGF-β signaling pathway inhibitor), tranylcypromine (an LSD1 inhibitor), and Forskolin (an adenylate cyclase activator). These five molecules act as a sophisticated regulatory team, working synergistically across multiple pathways; VPA loosens chromatin structure by inhibiting histone deacetylation, facilitating reprogramming factors' access to DNA; CHIR99021 activates the Wnt / β-catenin pathway by inhibiting GSK-3β, promoting cell fate transition; and Repsox directly counteracts the core driving signals of fibrosis by inhibiting the TGF-β pathway (e.g., reducing p-Smad2 / 3 levels). Transphenylcyclopropane regulates histone methylation levels by inhibiting LSD1, further altering the epigenetic state; foscoline provides necessary second messenger support for reprogramming by activating the cAMP pathway. This invention, by regulating the concentration and dosage of these molecules, enables them to work synergistically to create a suitable chemical environment within cells for "partial reprogramming," ultimately achieving effects similar to gene therapy in reversing fibrosis and epigenetic age.

[0013] The HAT inhibition combination provided by this invention comprises A485 (KAT3A / B inhibitor) and WM-8014 (KAT6A inhibitor); its mechanism of action is more direct, namely, by inhibiting histone acetyltransferase (HAT), it specifically reduces the enrichment of activating histone markers (such as H3K27ac, H3K9ac) in the promoter regions of fibrosis genes (such as COL1A1, ACTA2) and reduces chromatin accessibility (confirmed by ATAC-seq), thereby “turning off” the expression of these harmful genes at the epigenetic level.

[0014] Furthermore, this invention combines the aforementioned cocktail composition with the HAT-inhibiting composition, demonstrating a synergistic enhancement effect in both lung and liver fibrosis models. The effects on improving fibrosis markers and reversing epigenetic age are significantly superior to either combination alone. This proves that multi-target intervention through different mechanisms (such as activating necessary pathways and inhibiting harmful epigenetic markers) can achieve therapeutic goals more efficiently and thoroughly. In summary, the technical solution of this invention precisely manipulates the epigenetic program of cells through chemical means, safely reversing them from a pathological state to a healthy or resting state. This not only improves the pathological phenotype of fibrosis but also fundamentally reverses its biological age, representing a major breakthrough in the field of fibrosis treatment.

[0015] Existing technologies typically disclose cocktail compositions for complete in vitro reprogramming, which reverses somatic cells into pluripotent stem cells (iPSCs), resetting their epigenetic age to "0" and fundamentally altering their cellular identity. This invention, however, achieves partial reprogramming in the in vivo environment. Its aim is to reverse fibrosis-related pathological states and epigenetic age without changing the original cellular identity (e.g., hepatocytes remain hepatocytes and are not converted into iPSCs). Therefore, this invention represents a precise and safe in vivo epigenetic remodeling strategy, fundamentally different from the goals, scenarios, and biological outcomes of existing technologies. Detailed Implementation

[0016] This invention provides a cocktail composition for reversing organ fibrosis through partial reprogramming, comprising valproic acid, CHIR99021, Repsox, transphenylcyclopropane, and foctocorline; wherein the concentration of valproic acid is 200-300 μM, the concentration of CHIR99021 is 5-15 μM, the concentration of Repsox is 5-15 μM, the concentration of transphenylcyclopropane is 4-6 μM, and the concentration of foctocorline is 40-60 μM.

[0017] In this invention, the concentration of valproic acid used is 200-300 μM, preferably 220-280 μM, more preferably 250 μM; the concentration of CHIR99021 used is 5-15 μM, preferably 8-13 μM, more preferably 10 μM; the concentration of Repsox used is 5-15 μM, preferably 8-12 μM, more preferably 10 μM; the concentration of transphenylcyclopropylamine used is 4-6 μM, preferably 4.5-5.5 μM, more preferably 5 μM; and the concentration of fosco-coal used is 40-60 μM, preferably 45-55 μM, more preferably 50 μM.

[0018] The present invention provides a HAT inhibitory composition for reversing organ fibrosis by partial reprogramming, comprising A485 and WM-8014; wherein the concentration of A485 is 5-15 μM, preferably 8-12 μM, more preferably 10 μM, and the concentration of WM-8014 is 4-6 μM, preferably 4.5-5.5 μM, more preferably 5 μM.

[0019] The present invention also provides a composition for reversing organ fibrosis through partial reprogramming, comprising the cocktail composition and the HAT inhibitory composition. The present invention combines the cocktail composition and the HAT inhibitory composition, reducing the dosage and achieving or exceeding the full-dose efficacy of either combination while exhibiting better animal tolerability.

[0020] In this invention, the composition is preferably a liquid formulation, and more preferably an injectable formulation. This invention does not limit the solvent of the composition; any pharmaceutically acceptable solvent may be used.

[0021] The present invention provides the use of the cocktail composition, the HAT inhibitory composition, or the composition in the preparation of a medicament for treating organ fibrosis.

[0022] In this invention, the organ fibrosis includes liver fibrosis and pulmonary fibrosis.

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1

[0025] A cocktail composition that reverses organ fibrosis through partial reprogramming.

[0026] Components, working concentration and molar ratio: Valproic acid: working concentration 250 µM, molecular weight 144.21 g / mol.

[0027] CHIR99021: Working concentration 10 µM, molecular weight 465.33 g / mol.

[0028] Repsox: working concentration 10 µM, molecular weight 295.33 g / mol.

[0029] Refractory cyclopropane: working concentration 5 µM, molecular weight 133.19 g / mol.

[0030] Foscoline: working concentration 50 µM, molecular weight 410.50 g / mol.

[0031] Molar ratio: VPA : CHIR99021 : Repsox : Tranylcypromine : Forskolin = 50 : 2 : 2 : 1 : 10.

[0032] Preparation method and dosage: Stock solution: Dissolve each component in DMSO to prepare a high-concentration stock solution (VPA 500 mM, CHIR99021 10 mM, Repsox 10 mM, transphenylcyclopropane 5 mM, foscolin 50 mM), and store at -20°C protected from light.

[0033] Working solution (in vivo administration): Before use, mix each stock solution in physiological saline containing 10% Kolliphor HS-15 (or equivalent solvent) in proportion to ensure that the final DMSO concentration is <5%. Filter sterilize through a 0.22 µm filter membrane.

[0034] In vivo dosage (intraperitoneal injection): Calculated based on animal body weight, the daily injection volume is 5-10 mL / kg. The corresponding dosages for each component in this volume are approximately: VPA 18-36 mg / kg, CHIR99021 2.3-4.7 mg / kg, Repsox 1.5-3.0 mg / kg, transphenylcyclopropionamide 0.33-0.66 mg / kg, and foctocorine 10.3-20.6 mg / kg.

[0035] The specific dosages for individual use are: VPA 25mg / kg, CHIR99021 3.5mg / kg, Repsox 2.2mg / kg, tranylcypromine 0.5mg / kg, and foctocorine 15mg / kg; the injection volume is calculated based on the dosage used.

[0036] Treatment course: Pulmonary fibrosis once daily for 14-21 days; Liver fibrosis once daily for 28 days.

[0037] Example 2

[0038] HAT inhibitory composition that reverses organ fibrosis through partial reprogramming

[0039] Components, working concentration and molar ratio: A485: Working concentration 10 µM, molecular weight 452.50 g / mol.

[0040] WM-8014: Working concentration 5 µM, molecular weight 478.93 g / mol.

[0041] Molar ratio: A485 : WM-8014 = 2 : 1.

[0042] Preparation method and dosage: Stock solution: Dissolve each component in DMSO to prepare a high-concentration stock solution (A485 10 mM, WM-8014 5 mM), and store at -20°C protected from light.

[0043] Working solution (in vivo administration): Before use, mix the stock solution with physiological saline containing 10% Kolliphor HS-15 to ensure the final DMSO concentration is <5%. Sterilize by filtration through a 0.22 µm filter membrane.

[0044] The specific dosages for use alone are: A485 20 mg / kg, WM-8014 10 mg / kg, with the injection volume calculated based on the dosage used.

[0045] Treatment course: once daily or every other day for 3-4 weeks.

[0046] Example 3

[0047] Combination therapy regimen (cocktail combination + HAT inhibitor combination combination)

[0048] Combination therapy principle: To achieve a synergistic effect, two compositions are used in combination. When using combination therapy, the dosage of each individual drug can be maintained or reduced to control potential toxicity while achieving synergistic effects.

[0049] Specific combined dosage regimen: Option A (Full-dose combination): Cocktail composition: Prepared according to the above single-use dosages (VPA 25mg / kg, CHIR99021 3.5mg / kg, Repsox 2.2mg / kg, tranylcypromine 0.5mg / kg, foctocorine 15mg / kg).

[0050] HAT inhibitory composition: Prepared according to the above single-use dosage (A485 20mg / kg, WM-8014 10mg / kg).

[0051] Administration method: Inject the two working solutions separately (at least 1 hour apart), or mix them under strict aseptic conditions and then inject them at once (ensure that the components are stable and do not precipitate after mixing, and that the total injection volume and solvent concentration are within the safe range).

[0052] Option B (Reduction and Collaborative Integration - Optimal): Cocktail composition: Use 70% of the single drug dose (e.g., VPA 17.5 mg / kg, CHIR99021 2.45 mg / kg, Repsox 1.54 mg / kg, tranylcypromine 0.35 mg / kg, foctocorine 10.5 mg / kg).

[0053] HAT inhibitory composition: Use 70% of the single drug dose (A485 14 mg / kg, WM-8014 7 mg / kg).

[0054] Administration method: A single injection after mixing is recommended to simplify the procedure. This regimen has been validated in preliminary experiments, demonstrating efficacy comparable to or better than that of a single drug at full dose, while exhibiting superior tolerability in animals.

[0055] Combined drug treatment course: in sync with the monotherapy regimen, pulmonary fibrosis once daily for 14-21 consecutive days; liver fibrosis once daily for 28 consecutive days.

[0056] Experimental Example 1

[0057] Animal model establishment

[0058] (I) Bleomycin (BLM)-induced mouse pulmonary fibrosis model

[0059] This model is the most widely recognized and classic model in the world, and it can well simulate the key pathological features of human idiopathic pulmonary fibrosis (IPF), including alveolar epithelial damage, inflammatory cell infiltration, fibroblast proliferation and excessive collagen deposition.

[0060] Animal strain: C57BL / 6J mice. This strain has moderate sensitivity to bleomycin, can form a stable fibrosis model, and has good reproducibility.

[0061] Age and weight: 8-10 weeks old, weight 20-25 grams.

[0062] Husbandry environment: Husbands were housed in a standard SPF-grade environment with free access to food and water. The experimental protocol was reviewed and approved by the institution's animal ethics committee.

[0063] Main reagents and instruments

[0064] Modeling reagent: Bleomycin, which should be prepared to the required concentration with sterile physiological saline before use.

[0065] Anesthetic: Sodium pentobarbital (1%) or isoflurane inhalation anesthesia system.

[0066] Main instruments: small animal operating table, laryngoscope, miniature pipette, animal heating pad, surgical sutures, etc.

[0067] Modeling steps

[0068] 1. Grouping and Anesthesia: Mice were randomly divided into a control group (Sham group) and a model group (BLM group).

[0069] Mice were anesthetized by intraperitoneal injection of sodium pentobarbital or inhalation of isoflurane. After complete anesthesia (as indicated by no response to toe pinching), the mice were fixed to the operating board in a supine position.

[0070] 2. Tracheal exposure and infusion: Gently pull the mouse's incisors with tape to straighten its neck. Disinfect the skin on the front of the neck with alcohol.

[0071] Make a longitudinal incision of about 0.5-1 cm along the midline of the neck, bluntly dissect the subcutaneous tissue and glands, and expose the trachea.

[0072] Using an insulin needle or microinjector, insert the needle into the tracheal lumen at a 30-45° angle to the body, between the tracheal cartilage rings. Control group mice were infused with 50 μL of sterile saline, while model group mice were infused with 50 μL of saline solution containing bleomycin.

[0073] Key modeling parameters: Bleomycin dosage: 3.0 mg / kg body weight. This dosage range has been determined in preliminary experiments to ensure animal survival while inducing significant fibrosis.

[0074] Infusion volume: 50 μL / unit.

[0075] Quickly remove the needle and immediately rotate the mouse upright to ensure the medication is evenly distributed in both lungs.

[0076] 3. Suturing and resuscitation: The incision was closed layer by layer using surgical sutures.

[0077] The mice were placed on a 37°C constant temperature mat in a lateral position until they were fully awakened from anesthesia and then returned to their cages.

[0078] Model Validation and Evaluation Metrics

[0079] The peak period of fibrosis formation was from day 21 to 28 after modeling. At this time, the animals were sacrificed for the following analysis to verify whether the model was successfully established.

[0080] 1. General observation: The model group mice exhibited typical symptoms such as weight loss, disheveled fur, reduced activity, and rapid breathing.

[0081] 2. Gross observation of lung tissue: After euthanasia, the lungs were harvested. The lung tissue in the model group was found to be reduced in volume, lacked elasticity, and had a rough and uneven surface, with diffuse pale white nodules visible.

[0082] 3. Histopathological analysis (core indicators): Hematoxylin-eosin (H&E) staining: to observe the overall structural damage, inflammatory cell infiltration and fibrosis of the lungs.

[0083] Masson trichrome staining or Sirius Red staining: specifically displays blue collagen fibers (Masson) or red collagen fibers (Sirius Red), visually reflecting the degree of collagen deposition.

[0084] Ashcroft score: A double-blind, semi-quantitative scoring method (0-8 points) is used to assess the severity of fibrosis in Sirius red-stained lung tissue sections. The Ashcroft score in the model group should be significantly higher than that in the control group (usually >5 points).

[0085] 4. Hydroxyproline content determination (biochemical quantification of collagen deposition): Hydroxyproline is a characteristic amino acid of collagen, and its content can accurately reflect the total collagen content in the lungs.

[0086] Hydroxyproline was extracted from lung tissue using alkaline hydrolysis and quantified using a kit.

[0087] The hydroxyproline content in the lung tissue of the model group should be significantly higher than that of the control group (usually 1.5 to 2 times higher).

[0088] 5. Molecular biological indicators: α-Smooth muscle actin (α-SMA): Localized and semi-quantitatively analyzed by immunohistochemical staining, it is a marker of activated myofibroblasts. The number of α-SMA-positive cells should be significantly increased in the model group.

[0089] Fibrosis-related gene expression: The mRNA expression levels of fibrosis-related genes (such as Col1a1, Acta2, Tgfb1) were detected by real-time quantitative PCR. The expression levels in the model group were significantly upregulated.

[0090] Model success criteria

[0091] A pulmonary fibrosis model is considered successfully established if all of the following conditions are met: Ashcroft score: The model group score was significantly higher than that of the control group (p < 0.01), and the mean was > 5.

[0092] Hydroxyproline content: The content in the model group was significantly higher than that in the control group (p < 0.01).

[0093] Histological evidence: H&E and Masson / Sirius red staining showed severe damage to alveolar structure and extensive collagen deposition in the model group.

[0094] The results are shown in Table 1.

[0095] Table 1. Validation results of the bleomycin-induced mouse pulmonary fibrosis model (n=8)

[0096] (I) Establishment of an animal model of liver fibrosis

[0097] In this embodiment of the invention, a carbon tetrachloride (CCl4)-induced mouse liver fibrosis model is mainly used. This model is a classic and reliable model for studying the pathogenesis of liver fibrosis and evaluating the efficacy of drugs. It can well simulate the core pathological processes of human liver fibrosis, including hepatocyte damage, inflammatory response, hepatic stellate cell activation, and excessive deposition of extracellular matrix.

[0098] laboratory animals

[0099] Animal strain: C57BL / 6J mouse. C57BL / 6J is a standard inbred strain with a well-defined genetic background and is sensitive to fibrosis induction.

[0100] Age and weight: Mice: 6-8 weeks old, weight 18-22 grams.

[0101] Housing environment: Animals are housed in a standard SPF-grade environment with free access to food and water. All animal experimental procedures have been reviewed and approved by the institution's animal ethics committee.

[0102] Main reagents and instruments

[0103] Modeling reagents: carbon tetrachloride (CCl4), olive oil.

[0104] Syringe (1 mL for mice), iodine solution, alcohol swabs.

[0105] Instruments: balance, tissue homogenizer, paraffin microtome, microscope, etc.

[0106] Modeling steps (intraperitoneal injection method)

[0107] This method is simple to operate, has good repeatability, and can produce diffuse liver fibrosis.

[0108] 1. Solution preparation: The CCl4 stock solution was mixed with olive oil as a dispersant at a certain volume ratio and then thoroughly vortexed.

[0109] Key modeling parameters: CCl4 concentration: Mice were given a 10%-20% (v / v) CCl4 olive oil solution; the specific concentration needs to be determined through preliminary experiments, so as to produce significant fibrosis while ensuring the survival rate of the animals.

[0110] Injection dose: 5 μL / g body weight (calculated based on CCl4 volume).

[0111] Injection frequency: twice a week (Monday and Thursday).

[0112] Modeling cycle: 6-8 weeks of continuous injections. The degree of fibrosis worsens with prolonged injection cycles.

[0113] 2. Grouping and Injection: Animals were randomly divided into a control group (Control) and a model group (CCl4).

[0114] Control group: Intraperitoneal injection of an equal volume of pure olive oil.

[0115] Model group: Intraperitoneal injection of CCl4 olive oil solution of corresponding concentration.

[0116] The animal must be properly secured during injection to avoid damaging its internal organs.

[0117] Model Validation and Evaluation Metrics

[0118] Animals were sacrificed 48–72 hours after the last injection, and the following analyses were performed to verify the successful establishment of the model.

[0119] 1. General condition and gross observation of the liver: The model group animals exhibited slow or decreased weight gain, sparse and dull fur, and lethargy.

[0120] After euthanasia, the liver was removed. The livers of the model group were found to be enlarged or shrunken, with a rough and uneven surface, yellowish-brown or dark red color, hardened texture, and diffuse granular or nodular changes.

[0121] 2. Serum biochemical indicators: Blood is collected and serum is separated.

[0122] The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured. The ALT and AST activities in the model group should be significantly higher than those in the control group, indicating significant hepatocellular damage.

[0123] 3. Histopathological analysis (core indicators): Hematoxylin-eosin (H&E) staining: to observe the destruction of liver lobule structure, fatty degeneration of hepatocytes, ballooning degeneration, necrotic foci and inflammatory cell infiltration.

[0124] Sirius Red staining: Under a polarized light microscope, type I collagen appears as a bright yellow or red birefringence, while type III collagen appears as a green birefringence. This staining is the "gold standard" for evaluating collagen fiber deposition and distribution.

[0125] Semi-quantitative fibrosis scoring: The Ishak scoring system (0-6 points) is more refined and can better distinguish between moderate and severe fibrosis.

[0126] Metavir scoring system (F0-F4): Commonly used in clinical practice.

[0127] The scoring was performed in a double-blind manner by at least two pathologists who were unaware of the experimental group. A successful model group should have an Ishak score of ≥4 (significant fibrosis) or a Metavir score of F3 (severe fibrosis) or higher.

[0128] 4. Determination of hydroxyproline content in liver tissue (biochemical quantification of collagen deposition): Hydroxyproline is a characteristic amino acid of collagen.

[0129] A portion of liver tissue was taken, and hydroxyproline was extracted using acid hydrolysis. The quantification was performed using a kit.

[0130] The hydroxyproline content in the liver tissue of the model group should be significantly higher than that of the control group (usually 2-3 times higher).

[0131] 5. Molecular biological indicators: α-Smooth muscle actin (α-SMA): As shown by immunohistochemistry or immunofluorescence staining, it is a marker of hepatic stellate cells activating into myofibroblasts. The number of α-SMA-positive cells in the portal area and fibrous septa should be significantly increased in the model group.

[0132] Fibrosis-related gene expression: The mRNA expression levels of Col1a1 (type I collagen), Acta2 (α-SMA), Timp1 (tissue inhibitor of matrix metalloproteinase 1), and Tgfb1 (transforming growth factor β1) were detected by real-time quantitative PCR. The expression levels were significantly upregulated in the model group.

[0133] Model success criteria

[0134] A liver fibrosis model is considered successfully established if all of the following conditions are met: Histological score: The Ishak score of the model group was significantly higher than that of the control group (p < 0.01), and the mean score was ≥ 4.

[0135] Hydroxyproline content: The content in the model group was significantly higher than that in the control group (p < 0.01).

[0136] Serological markers: ALT / AST levels were significantly elevated in the model group (p < 0.01).

[0137] Molecular markers: α-SMA positive area and / or fibrosis gene expression were significantly upregulated in the model group.

[0138] The results are shown in Table 2.

[0139] Table 2. Validation results of the carbon tetrachloride-induced liver fibrosis model in mice (n=8)

[0140] After the model was successfully constructed, treatment was administered, with control group, model group, cocktail group, HAT inhibition group, and combination therapy group included.

[0141] Cocktail Set: 1. Preparation of small molecule stock solutions and working solutions Valproic acid (VPA): Dissolve in sterile water or DMSO to prepare a 500 mM stock solution.

[0142] CHIR99021: Dissolve in DMSO to prepare a 10 mM stock solution.

[0143] Repsox: Dissolve in DMSO to prepare a 10 mM stock solution.

[0144] Refractory cyclopropane: Dissolve in sterile water or DMSO to prepare a 5 mM stock solution.

[0145] Foscoline: Dissolve in DMSO to prepare a 50 mM stock solution.

[0146] Preparation of working solution: Before use, mix the above stock solution in sterile saline or a carrier solution containing a small amount (e.g., <5%) of solvent (e.g., Cremophor EL / ethanol) in proportion, vortex to mix, and prepare a cocktail composition containing all five components. The final concentrations are: VPA 250 μM, CHIR99021 10 μM, Repsox 10 μM, transphenylcyclopropane 5 μM, and foscolin 50 μM.

[0147] Animal Experiment Design and Grouping

[0148] Animal models: Bleomycin-induced pulmonary fibrosis model or carbon tetrachloride-induced liver fibrosis model established using the methods described above.

[0149] Experimental groups: at least 8 animals per group.

[0150] Control Group

[0151] Without fibrosis modeling, patients received the same volume of solvent (such as saline or olive oil) as the model group, and an equal volume of carrier solution (containing no drug components) during treatment.

[0152] Objective: To serve as a normal baseline for assessing the physiological status of animals in the absence of fibrosis and treatment intervention.

[0153] Dosage regimen: Control group of pulmonary fibrosis model: intraperitoneal injection of normal saline once a day for 14 consecutive days.

[0154] Control group of the liver fibrosis model: intraperitoneal injection of olive oil once a day for 28 consecutive days.

[0155] Model Group

[0156] Handling method: After successful fibrosis modeling (such as bleomycin-induced pulmonary fibrosis or CCl4-induced liver fibrosis), only an equal volume of drug carrier solution (containing no active ingredients) is administered during treatment.

[0157] Purpose: As a disease control group, it was used to assess the natural progression of fibrosis and the effect of the carrier solution on the model.

[0158] Dosage regimen: Pulmonary fibrosis model group: Carrier solution was injected intraperitoneally once a day for 14 consecutive days.

[0159] Liver fibrosis model group: The carrier solution was injected intraperitoneally once a day for 28 consecutive days.

[0160] Cocktail Treatment Group

[0161] Handling method: After successful fibrosis modeling, administer the working solution of the cocktail composition (containing VPA, CHIR99021, Repsox, transphenylcyclopropane, and foctocorline) daily.

[0162] Drug preparation: Prepared according to the method in Example 1, the final concentrations were: VPA 250 µM, CHIR99021 10 µM, Repsox 10 µM, transphenylcyclopropane 5 µM, and foctocorline 50 µM.

[0163] Dosage regimen: Pulmonary fibrosis cocktail group: intraperitoneal injection, once daily for 14 consecutive days.

[0164] Liver fibrosis cocktail group: intraperitoneal injection, once daily for 28 consecutive days.

[0165] The injection dosage is described in Example 1.

[0166] 4. HAT Inhibition Group

[0167] Handling method: After successful fibrosis modeling, the patient was given a working solution of the HAT inhibitory composition (containing A485 and WM-8014) daily.

[0168] Drug preparation: Prepared according to the method in Example 2, the final concentrations were: A485 10 µM, WM-8014 5 µM.

[0169] Dosage regimen: HAT inhibition group for pulmonary fibrosis: intraperitoneal injection, once daily for 14 consecutive days.

[0170] Liver fibrosis HAT inhibition group: tail vein injection (liver-targeted), once daily for 3–4 weeks.

[0171] The injection dosage is described in Example 2.

[0172] Combination Treatment Group

[0173] Handling method: After successful fibrosis modeling, a combined working solution of the cocktail composition and the HAT inhibitory composition is administered daily.

[0174] Drug preparation: Option B (Reduction and Collaborative Approach): Cocktail composition: 70% of the single-drug dose (VPA 17.5 mg / kg).

[0175] HAT inhibitory composition: 70% of the single-drug dose (A485 14 mg / kg, WM-8014 7 mg / kg).

[0176] The working solution is mixed and injected in a single injection.

[0177] Dosage regimen: Combined treatment group for pulmonary fibrosis: intraperitoneal injection, once daily for 14 consecutive days.

[0178] Combined treatment group for liver fibrosis: intraperitoneal injection, once daily for 28 consecutive days.

[0179] The injection dosage is described in Example 3, Scheme B.

[0180] 2. Treatment cycle: Administer medication after successful model establishment.

[0181] Pulmonary fibrosis: Usually begins on the 7th day after tracheal infusion of bleomycin, administered once daily for 14 consecutive days.

[0182] Liver fibrosis: Start 4 weeks after CCl4 injection (when obvious fibrosis has formed), administer once daily for 28 consecutive days.

[0183] 3. Sample collection and testing methods

[0184] 4. Animals were sacrificed 24 hours after the last administration, and samples were collected for the following analysis: Detection metrics: Ashcroft / Ishak score Measurement method: 1. Tissue processing: Take the left lobe of the lung / liver, fix it with 4% paraformaldehyde, embed it in paraffin, and section it (4-5 μm).

[0185] 2. Staining: Perform H&E staining and Sirius red staining.

[0186] 3. Double-blind scoring: Two pathologists, whose groups are unknown, independently score 5-10 randomly selected fields of view of each slide under an optical microscope according to the Ashcroft (lung, 0-8 points) or Ishak (liver, 0-6 points) scoring criteria, and the average value is taken.

[0187] 4. Data processing and grouping: Compare the scores of the model group and the treatment group, calculate the percentage reduction, and perform statistical tests (such as t-tests).

[0188] Test indicator: Hydroxyproline content

[0189] Measurement method: 1. Tissue hydrolysis: Take about 50 mg of lung / liver tissue, add 6N HCl, and hydrolyze at 110℃ for 24 hours.

[0190] 2. Neutralization and derivatization: The hydrolysate was neutralized with NaOH, and the oxidation with chloramine T and the colorimetric method with p-dimethylaminobenzaldehyde were used.

[0191] 3. Measurement: The absorbance was measured at a wavelength of 560 nm using a spectrophotometer. The hydroxyproline content was calculated based on the standard curve. The results were expressed in μg / gram wet weight tissue or per leaf organ.

[0192] Data processing and grouping: Comparing the levels in the model group and the treatment group demonstrated a reduction in collagen deposition.

[0193] Detection indicator: Number of α-SMA positive cells

[0194] Measurement method (detailed steps): 1. Immunohistochemistry / immunofluorescence: Antigen retrieval is performed on paraffin sections, followed by incubation with anti-α-SMA primary antibody, and then detection with the corresponding HRP or fluorescent secondary antibody.

[0195] 2. Image Analysis and Quantification: Images were acquired using a microscope. A uniform threshold was set using ImageJ or equivalent software to calculate the percentage of positively stained area relative to the total tissue area. At least three random, non-overlapping fields of view were analyzed for each slide.

[0196] Data processing and grouping: Compare the percentage of positive area between the model group and the treatment group to assess the reversal of myofibroblast activation.

[0197] Detection indicators: expression of fibrosis gene mRNA (TGF-β1, COL1A1, TIMP1)

[0198] Measurement method: 1. RNA extraction: Total RNA was extracted from approximately 30 mg of tissue using the TRIzol method, and its concentration and purity were determined.

[0199] 2. Reverse transcription: cDNA was synthesized using a reverse transcription kit.

[0200] 3. Real-time quantitative PCR (qPCR): Amplification was performed on a quantitative PCR instrument using SYBR Green or TaqMan probes. Gapdh or β-actin was used as an internal reference gene, and 2^ (-ΔΔCt) The relative expression level of the target gene is calculated using this method.

[0201] Data processing and grouping: The expression levels of the treatment group were normalized to those of the model group (set to 1.0), and the downregulation fold was displayed.

[0202] Detection indicators: Levels of proteins involved in the mechanism of action (H3K9ac, β-catenin, p-Smad2 / 3)

[0203] Measurement method: 1. Protein extraction: The tissue was homogenized in RIPA lysis buffer, centrifuged, and the supernatant was collected to determine the protein concentration.

[0204] 2. Western Blot: Load equal amounts of protein, perform SDS-PAGE electrophoresis, transfer to a membrane, and incubate with specific primary antibodies (anti-H3K9ac, anti-β-catenin, anti-p-Smad2 / 3) and corresponding secondary antibodies.

[0205] 3. Development and Quantification: Chemiluminescent substrate was used for development, and the gray values ​​of the bands were quantified using ImageJ software. Total protein (e.g., GAPDH) or total Smad2 / 3 was used as an internal control for homogenization.

[0206] Data processing and grouping: The protein levels in the treatment group were compared with those in the model group (a multiple of the control group) to demonstrate the pathway regulation effect.

[0207] Specific epigenetic detection methods

[0208] Detection indicators: Histone modification enrichment (H3K27ac, H3K9ac)

[0209] Measurement method: 1. Chromatin immunoprecipitation (ChIP): Fresh or frozen tissue is taken, cross-linked, and then the chromatin is broken down by sonication to 200-500 bp fragments.

[0210] 2. Immunoprecipitation: Chromatin fragments were co-incubated with anti-H3K27ac or anti-H3K9ac antibodies and Protein A / G magnetic beads were used to capture the complex.

[0211] 3. Washing, decrosslinking and purification: Obtain DNA fragments that bind to specific histone modifications.

[0212] 4. Quantitative PCR (ChIP-qPCR): qPCR analysis was performed using primers specific to the promoter regions of fibrosis genes (COL1A1, ACTA2). The enrichment fold was calculated using the input DNA as a reference.

[0213] Data processing and grouping: The enrichment of the treatment group at specific gene loci was compared with that of the model group (a multiple of the control group), demonstrating a reduction in activation markers.

[0214] Detection indicator: Chromatin accessibility (ATAC-seq analysis)

[0215] Measurement method: 1. Nuclear extraction and transposition reaction: Cell nuclei are extracted from fresh tissue, and sequencing adapters are inserted into open chromatin regions using Tn5 transposase.

[0216] 2. Library construction and sequencing: Purify the transposon products, construct sequencing libraries by PCR amplification, and perform high-throughput sequencing on the Illumina platform.

[0217] 3. Bioinformatics analysis: sequence alignment, identification of open regions (peaks), and comparison of peak signal intensity at fibrosis gene sites (such as COL1A1) among different groups.

[0218] Data processing and grouping: The treatment group showed significantly reduced chromatin accessibility at key fibrosis gene loci compared to the model group, confirming epigenetic silencing at a global level.

[0219] Through the specific and reproducible experimental methods described above, this invention demonstrates that both small molecule compositions can safely and effectively reverse organ fibrosis and achieve significant reversal of epigenetic age by regulating epigenetic state.

[0220] The experimental results are shown in Tables 3 and 4.

[0221] Table 3. Efficacy data of combined therapy for pulmonary fibrosis (n=8)

[0222] Table 4. Efficacy data of combined treatment for liver fibrosis (n=8)

[0223] As can be seen from the above embodiments, the technical solution of the present invention precisely manipulates the epigenetic program of cells through the chemical means of small molecule compositions, safely reversing them from a pathological state to a healthy or resting state. This not only improves the pathological phenotypes of pulmonary fibrosis and liver fibrosis, but also reverses their biological age from the root cause.

[0224] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A cocktail composition for reversing organ fibrosis through partial reprogramming, characterized in that, The active ingredients include valproic acid, CHIR99021, Repsox, transphenylcyclopropylamine, and foctocorline; the concentrations used for valproic acid are 200-300 μM, CHIR99021 is 5-15 μM, Repsox is 5-15 μM, transphenylcyclopropylamine is 4-6 μM, and foctocorline is 40-60 μM.

2. A HAT inhibitory composition for reversing organ fibrosis through partial reprogramming, characterized in that, It includes A485 and WM-8014; the concentration of A485 used is 5~15μM, and the concentration of WM-8014 used is 4~6μM.

3. A composition for reversing organ fibrosis through partial reprogramming, characterized in that, Includes the cocktail composition of claim 1 and the HAT-inhibiting composition of claim 2.

4. Use of the cocktail composition of claim 1, the HAT-inhibiting composition of claim 2, or the composition of claim 3 in the preparation of a medicament for treating organ fibrosis.

5. The application according to claim 4, characterized in that, The organ fibrosis includes liver fibrosis and pulmonary fibrosis.

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