Use of timolol in the preparation of a medicament for the prevention and / or treatment of liver fibrosis

Abstract

The application discloses application of tamibarotene in preparation of a medicine for preventing and / or treating liver fibrosis, and belongs to the technical field of biological medicine. The application finds that tamibarotene has a significant anti-liver fibrosis effect for the first time. Experimental results show that in two kinds of liver fibrosis animal models induced by CCl4 and TAA, tamibarotene can significantly down-regulate the expression of alpha-SMA and type I collagen, reduce the liver inflammatory response, and improve liver function. In particular, the anti-fibrosis effect of tamibarotene is significantly better than that of RARbeta selective agonist KCL-286 and RARalpha selective agonist Am580, and an unexpected technical effect is achieved. The application provides a new medicine selection for the treatment of liver fibrosis, and has a good clinical application prospect and development value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the use of tamibarbitine in the preparation of medicaments for the prevention and / or treatment of liver fibrosis. Background Technology

[0002] Liver fibrosis is an excessive repair response to various chronic liver injuries (such as chronic viral hepatitis, metabolic-related fatty liver disease, alcoholic liver disease, and autoimmune liver disease). Its core pathophysiological feature is the abnormal imbalance between the synthesis and degradation of the extracellular matrix in the liver, leading to the massive deposition of connective tissue rich in type I and type III collagen within the liver parenchyma. Without effective intervention, progressive liver fibrosis disrupts the normal lobular structure and microcirculation of the liver, eventually developing into cirrhosis. Current research indicates that liver fibrosis, as an important precancerous lesion of cirrhosis and liver cancer, is reversible in both clinical patients and experimental animal models. However, there are currently no approved specific treatments for liver fibrosis worldwide.

[0003] In the extremely complex liver microenvironment, abnormal activation of hepatic stellate cells (HSCs) is a central driver of liver fibrosis. Therefore, inhibiting HSC activation and restoring them to their resting state is widely recognized as the most effective anti-fibrotic strategy. However, existing interventions have significant limitations: anti-fibrotic drugs currently in clinical trials, such as pirfenidone, suffer from imprecise targeting, limited efficacy, and significant gastrointestinal side effects, making them unsuitable for long-term clinical use.

[0004] Retinoic acid (RA) is the main active metabolite of vitamin A (retinol) in the body. It forms heterodimers by binding to members of the nuclear receptor superfamily—retinoic acid receptors (RARs) and retinoid X receptors (RXRs)—thereby regulating the transcription of target genes and exerting various biological functions. RARs include three isoforms: RARα, RARβ, and RARγ, which differ significantly in tissue distribution, ligand affinity, and biological function. Basic research has shown that resting hepatic stellate cells store a large number of retinoic acid-rich lipid droplets, and the loss of these droplets is closely related to hepatic stellate cell activation. Therefore, research has begun to focus on the role of retinoids in liver fibrosis. Some studies have shown that retinoids can inhibit the proliferation and activation of hepatic fibrosis cells (HSCs): co-culturing HSCs that are about to be activated with retinol or all-trans retinoic acid (ATRA) can restore intracellular vitamin A reserves, reduce type I collagen synthesis, and inhibit the development of liver fibrosis. Furthermore, existing research has found that activation of RARα and RARβ alone may alleviate the progression of liver fibrosis, but the effects are limited.

[0005] However, traditional retinoic acid substances still face some obstacles in drug development: although supplementing with exogenous natural retinoic acid substances can inhibit the activation of hepatic stellate cells to a certain extent, these natural substances face drug development challenges such as poor water solubility, extremely large required doses, and significant systemic toxic side effects, making it difficult to transform them into clinical drugs.

[0006] In summary, there is still a technological gap in the current research and development of anti-liver fibrosis: there is a lack of an intervention strategy with known safety, well-defined pharmacokinetics, and the ability to safely and effectively reverse HSC activation. Summary of the Invention

[0007] This invention aims to provide a novel medical use for the known drug tamibarotene (trade name Amnolake, research code Am80), specifically, its use in the preparation of a medicament for the prevention and / or treatment of liver fibrosis. It provides a medicament for the treatment of liver fibrosis and related chronic liver diseases. Specifically, this invention addresses the following two key technical problems existing in the prior art: 1. To address the clinical problems of limited efficacy and significant side effects of existing anti-liver fibrosis drug candidates: Currently, there are no widely approved drugs specifically for treating liver fibrosis in clinical practice. Candidate drugs in clinical trials (such as pirfenidone PFD) suffer from drawbacks such as imprecise targeting, limited ability to reverse hepatic stellate cell activation, and poor patient tolerability (e.g., gastrointestinal adverse reactions). This invention addresses these issues by providing a novel intervention regimen with superior anti-fibrotic efficacy in in vivo animal models compared to existing clinical candidates. Furthermore, since Am80 is an already marketed drug, the safety risks of the finished product are significantly reduced. 2. Solving the technical challenges of high toxicity and difficulty in drug formulation of traditional retinoids: Existing research indicates that supplementing with exogenous natural retinoic acid can inhibit hepatic stellate cell activation to some extent. However, natural retinoic acid substances face technical obstacles such as poor water solubility, the need for extremely high doses, and severe systemic toxicity, making them difficult to translate into clinical drugs. This invention utilizes the artificially synthesized specific retinoic acid receptor (RAR) agonist Am80, circumventing the toxicity defects of natural ligands and solving the technical challenge of safely targeting hepatic stellate cells.

[0008] To achieve the above objectives, the present invention provides, in one aspect, the use of tamibarbitaline or its pharmaceutically acceptable salts, esters, solvates, or active metabolites in the preparation of medicaments for the treatment and / or prevention of liver diseases.

[0009] Furthermore, the liver diseases include chronic liver injury and the resulting liver fibrosis and cirrhosis.

[0010] Furthermore, the route of administration of the drug includes any one of oral administration, intravenous injection, intraperitoneal injection, subcutaneous injection, intramuscular injection, and local injection into the liver.

[0011] Furthermore, the dosage form for oral administration is selected from one or more of tablets, capsules, granules, and oral solutions.

[0012] Furthermore, the dosage of the tamilbarbitine is 2 mg / kg to 10 mg / kg daily.

[0013] Furthermore, the dosage of the tamilbarbitine is 4 mg / kg to 8 mg / kg daily, preferably 6 mg / kg.

[0014] Furthermore, the drug also comprises a pharmaceutically acceptable carrier and / or excipient.

[0015] Furthermore, the drug also contains one or more other active ingredients for treating liver fibrosis.

[0016] A second aspect of the present invention provides a pharmaceutical composition for treating liver fibrosis, comprising a therapeutically effective amount of tamibarbitine or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0017] Furthermore, the pharmaceutical composition comprises one or more other active ingredients for treating liver fibrosis.

[0018] A third aspect of the present invention provides the use of tamilbarbitine in the preparation of drugs that inhibit hepatic stellate cell activation and reduce extracellular matrix (ECM) deposition.

[0019] Furthermore, the extracellular matrix includes, but is not limited to, type I collagen, type III collagen, and α-smooth muscle actin (α-SMA).

[0020] The fourth aspect of this invention provides the use of tamibarbital in the preparation of drugs that upregulate cannabinoid receptor 2 (CNR2) expression.

[0021] Furthermore, the tamibarbitine described in this invention, as a retinoic acid receptor (RAR) agonist, enhances the sensitivity of hepatic stellate cells to antifibrinolytic signals by activating the RAR pathway and driving the transcriptional upregulation of the CNR2 receptor.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects: 1. Significant therapeutic effect, with anti-fibrotic efficacy superior to existing clinical candidate drugs. This invention confirms that tamibarbital (Am80) exhibits excellent anti-hepatic fibrosis activity both in vitro and in vivo. In two classic mouse models of liver fibrosis induced by CCl4 and TAA, Am80 (especially the 6 mg / kg dose group) effectively improved liver function, reduced liver tissue necrosis and inflammatory infiltration, and decreased the expression of type I and type III collagen and α-SMA in the liver. Its combined efficacy in inhibiting collagen deposition and reversing hepatic stellate cell activation is superior to pirfenidone, a currently popular anti-hepatic fibrosis drug in phase III clinical trials, providing a more effective alternative treatment option for clinical practice.

[0023] 2. The mechanism is novel, proposing and validating for the first time a precise targeting strategy of Am80 for CNR2. This invention reveals that Am80 specifically activates the retinoic acid receptor and potently drives the transcriptional upregulation of cannabinoid receptor 2 (CNR2). This mechanism weakens the activation capacity of hepatic stellate cells, achieving precise regulation of the hepatic stellate cell phenotype and providing a novel target pathway for the development of anti-hepatic fibrosis drugs.

[0024] 3. Short conversion cycle, extremely high drug-likeness and safety. This invention is a typical example of "repurposing an existing drug." Unlike the lengthy and risky development cycle of original new drugs, tamibarbitine (Am80), already approved for marketing in Japan for the treatment of acute promyelocytic leukemia, has had its pharmacokinetic characteristics, metabolic stability, and systemic safety in humans fully verified. This not only overcomes the shortcomings of traditional natural retinoic acid substances, such as poor water solubility and significant toxic side effects, but also lowers the threshold and time cost for advancing this invention to clinical trials for anti-liver fibrosis. Attached Figure Description

[0025] Figure 1 This is a phenotypic verification of Am80's ability to alleviate liver fibrosis in mice in one embodiment of the present invention. (A) Comparison of the ameliorative effects of Am80 and pirfenidone (PFD) on CCl4-induced liver fibrosis in mice. (B) Am80 inhibits the expression of liver fibrosis-related molecules at the protein level. (C) Am80 inhibits the expression of liver fibrosis-related molecules at the RNA level. Scalebars=50 µm (100×), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0026] Figure 2 This invention illustrates significant differences in gene expression in mouse liver tissue after Am80 (6 mg / kg) intervention in one embodiment. (A) Volcano map of differentially expressed genes in mice from the CCl4 model group and the Am80 (6 mg / kg) treatment group. (B) Global 3D PCA map of genes in mice from the NC group, CCl4 model group, and Am80 (6 mg / kg) treatment group. (C) Retinoic acid-related pathways were downregulated in the CCL4 model group but recovered and were enriched after Am80 (6 mg / kg) treatment. (D) Liver fibrosis-related pathways were upregulated in the CCL4 model group but decreased and were enriched after Am80 (6 mg / kg) treatment. (E) Heatmap of differentially expressed genes containing RARE elements that were significantly upregulated in the Am80 (6 mg / kg) treatment group.

[0027] Figure 3This invention describes the expression of downstream genes related to retinoic acid in the livers of mice in various groups, according to one embodiment. (A) Western blot (WB) showed increased protein levels of Klf4 in mice treated with Am80. (B) RT-qPCR showed increased transcription levels of downstream molecules of the retinoic acid pathway, Cyp26a1, Socs3, and Klf4, in mice treated with Am80. (C) Expression of Cnr2 in paraffin sections of mouse liver and quantitative analysis of positive area by immunohistochemistry. (D) Expression of Cnr2 and Klf4 in mice in various groups (WB). (E) RNA expression of Cnr2 in mice in various groups. Scalebars = 50 µm (100×), **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0028] Figure 4 This invention describes the effects of Am80 versus PFD on TGF-β-induced LX-2 cell activation, cytoskeleton remodeling, and related protein expression in one embodiment. (A) Comparison of Am80 and PFD in inhibiting fibrosis-related indicators at the protein level. (B) RT-qPCR results comparing the inhibitory effects of Am80 and PFD on activated LX-2 fibrosis-related indicators at the RNA level. (C) Phalloidin staining in the NC, TGF-β, and Am80 groups. (D) Protein expression of CNR2 and KLF4 in the NC, TGF-β-induced, Am80-treated, and BMS493-blocked groups, respectively. (E) RNA expression of CNR2, CYP26A1, SOCS3, and KLF4 in the NC, TGF-β-induced, Am80-treated, and BMS493-blocked groups, respectively. (F) Phalloidin staining in the NC, TGF-β-induced, Am80-treated, and BMS493-blocked groups. Scale bars=25 µm (200×), ***P<0.001, ****P<0.0001.

[0029] Figure 5 This invention describes the anti-fibrotic effect of in vitro CNR2 overexpression in one embodiment. (A) Protein expression of COLA2 and CNR2 after transfection with a CNR2-overexpressing plasmid using LX-2. (B) Verification of CNR2 overexpression mRNA level. (C) Inhibitory effect of CNR2 overexpression on liver fibrosis at the mRNA level. (D) Phalloidin staining to illustrate the important role of CNR2 in anti-fibrosis from a cellular phenotype perspective. Scale bars = 25 µm (200×), **P<0.01, ***P<0.001, ****P<0.0001.

[0030] Figure 6This invention describes the effect of CNR2 knockdown on the anti-fibrotic effect of Am80 in one embodiment. (A) Phalloidin staining of LX-2 cells activated by TGF-β after CNR2 knockdown following Am80 use and CNR2 overexpression. (B) CNR2 and fibrosis marker expression in LX-2 cells activated by TGF-β after CNR2 knockdown following Am80 use and CNR2 overexpression. Scale bars = 25 µm (200×), **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0031] Figure 7 This invention describes the effects of Am80, Am580, and KCL-286 on TGF-β-induced LX-2 cell activation, cytoskeleton remodeling, and related gene expression in one embodiment. (A) Comparison of the protein-level inhibition of fibrosis-related indicators by Am80, Am580, and KCL-286. (B) RT-qPCR results comparing the RNA-level inhibition of activated LX-2 fibrosis-related indicators by Am80, Am580, and KCL-286. (C) Phalloidin staining in the NC, TGF-β, Am80, Am580, and KCL-286 groups. (D) α-SMA immunofluorescence staining in the NC, TGF-β, Am80, Am580, and KCL-286 groups. Scale bars = 25 µm (200×), **P < 0.01, ***P < 0.001, ****P < 0.0001. Detailed Implementation

[0032] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0033] The tamibarbital (Am80) and its derivatives provided by this invention can be directly used to prepare clinical drugs for the prevention, relief, or reversal of liver fibrosis and cirrhosis caused by various acute / chronic liver injuries. Potential specific indications include, but are not limited to: Metabolic liver diseases: Metabolic-associated fatty liver disease (MASLD) and metabolic-associated steatohepatitis (MASH); Viral hepatitis: fibrosis complicated by chronic hepatitis B (HBV) and hepatitis C (HCV) infection; Chemical and toxic liver injury: alcoholic liver disease (ALD), drug-induced liver injury (DILI), and liver damage caused by environmental toxins; Autoimmune liver diseases: primary biliary cholangitis (PBC), autoimmune hepatitis (AIH), etc.; Parasitic liver diseases: such as liver fibrosis caused by schistosomiasis.

[0034] This invention clarifies the mechanism by which tamibarbital amplifies the CNR2 receptor on target cells, and can be applied to the development of novel combination drug regimens for treating liver diseases. Application method: Tamibarbital is prepared in combination with cannabinoid receptor 2 (CNR2) specific agonists (such as AM1241), antiviral drugs, lipid-lowering drugs, or hepatoprotective and anti-inflammatory drugs to create a compound preparation or combination drug kit for clinical application to achieve synergistic anti-fibrotic effects.

[0035] Based on the Am80 pathway disclosed in this invention, which upregulates CNR2 expression by activating retinoic acid receptors and thereby inhibits hepatic stellate cell activation, this pathway can be applied to in vitro drug screening platforms for pharmaceutical R&D companies. Application method: Using RAR activation and CNR2 expression levels as core pharmacodynamic biomarkers, it can be used to screen, evaluate, or confirm the pharmacological activity of other potential anti-hepatic fibrosis compounds.

[0036] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the raw materials used in the following embodiments are all disclosed in the prior art, such as those that can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0038] In the following experiments, the preparation of paraffin sections of mouse liver tissue, hematoxylin-eosin (HE) staining, Sirius Red staining, immunohistochemistry (IHC) staining, extraction of total protein and quantitative analysis of BCA from mouse liver tissue, extraction of cellular protein, Western blot (WB), tissue RNA extraction, and cellular RNA extraction were all performed in accordance with the reagent instructions or conventional methods in this field.

[0039] Experimental reagents: Carbon tetrachloride (CCl4) and olive oil are from Shanghai E-En Chemical Technology Co., Ltd. Thioacetamide (TAA), 30% gelling solution, 10% SDS, 1M Tris-HCl buffer (pH 6.8), 1.5M Tris-HCl buffer (pH 8.8), 20×TBST buffer, skim milk powder, bovine serum albumin, tris(hydroxymethyl)aminomethane, glycine, sodium lauryl sulfate, TEMED, RIPA, PMSF, blocked goat serum, and Triton X-100 were from Beijing Solarbio Science & Technology Co., Ltd. The 3% hydrogen peroxide was sourced from Jiangxi Jianbao Pharmaceutical Technology Co., Ltd. Xylene, anhydrous ethanol, and methanol were sourced from Shantou Xilong Scientific Co., Ltd. The universal enzyme-labeled goat anti-mouse / rabbit IgG polymer, immunohistochemical antibody diluent, and DAB colorimetric kit were from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. Citrate powder, PBS powder, ammonium persulfate, HRP Conjugated AffiniPure Goat Anti-mouse IgG (H+L) (BA1051), and HRP Conjugated AffiniPure Goat Anti-rabbit IgG (H+L) (BA1055) were sourced from Wuhan Boster Biological Engineering Co., Ltd. Hematoxylin staining solution, eosin staining solution, differentiation solution, and blueing solution were sourced from Nanchang Yulu Experimental Equipment Co., Ltd. Sirius Red Staining Kit is from Nanjing Senbega Biotechnology Co., Ltd.; The neutral resin is from Guangzhou Shuopu Biotechnology Co., Ltd. The nitrocellulose membrane was sourced from Stofavente, Inc., USA. 6×Protein Loading Buffer and GAPDH antibody (HC301-01) were obtained from Beijing TransGen Biotech Co., Ltd. The BCA protein quantification kit and BSA standard were sourced from Beijing Tiangen Biotech Co., Ltd. The phosphatase and protease inhibitors are from Wuhan Saiwei Biotechnology Co., Ltd. ProClean antibacterial and preservative agent is from Shanghai Beyotime Biotechnology Co., Ltd. FastKing cDNA First-Strand Synthesis Kit is from Beijing Tiangen Biotech Co., Ltd. 2×M5 HiPer SYBR Premix EsTaq is from Beijing Jumei Biotechnology Co., Ltd. The total RNA extraction kit for cells / tissues and the ECL chemiluminescence ultrasensitive colorimetric kit were provided by Yisheng Biotechnology (Shanghai) Co., Ltd. Collagen I antibody (ab270993) is from CST Biosciences, Inc., USA. The α-SMA antibody (ab5694) is from Abcam, a UK company. The CNR2 antibody (29371-1-AP) is from Wuhan Sanying Biotechnology Co., Ltd. The ECL chemiluminescence colorimetric reagent kit is from Thermo Fisher Scientific (China) Co., Ltd.

[0040] Establishment of an animal model of liver fibrosis: (1) Construction of a mouse liver fibrosis model based on carbon tetrachloride (CCl4) Forty-eight healthy male wild-type C57BL / 6J mice (approximately 6 weeks old, weighing 20–25 g) of SPF grade were selected. After one week of acclimatization in a standard laboratory animal facility, all animals were randomly assigned to three groups: a CCl4 modeling group (n=8), a solvent control group (Oil group, n=8), and treatment groups (treated with various concentrations of Am80 and PFD after modeling). During the modeling period, mice in both the modeling and treatment groups received intraperitoneal injections of 20% CCl4 solution at a dose of 1 μL / g, administered twice weekly for six weeks. Simultaneously, mice in the Oil group received the same volume and frequency of simple olive oil via intraperitoneal injection at the same time points to control for systematic errors caused by the solvent and the procedure.

[0041] (2) Construction of a mouse liver fibrosis model based on thioacetamide (TAA) Forty-eight healthy male wild-type C57BL / 6J mice, approximately 6 weeks old and weighing between 20-25g, were selected for this experiment. During a one-week acclimatization period, they were divided into a liver fibrosis model group (TAA group), a solvent control group (PBS group), and different Am80 or PFD administration groups, with eight mice in each group. During the model establishment process, the TAA group and all administration groups were administered 10% TAA solution (1μL / g) via intraperitoneal injection three times a week for eight weeks; while the control group was administered the same amount of PBS at the corresponding time points.

[0042] (3) Drug administration strategy for treatment group mice Two weeks after the induction of the model via intraperitoneal injection, each treatment group received drug treatment. The experiment consisted of four subgroups, with the specific protocols as follows: Am80 intervention group: Based on different dosages, they were divided into three dose gradient groups: low, medium, and high, receiving 4 mg / kg, 6 mg / kg, and 8 mg / kg of Am80 solution, respectively. The administration was performed once daily via gavage, and the dosing schedule continued throughout the entire modeling period.

[0043] PFD positive control group: This group of mice served as positive controls for the two model drug interventions. They were administered pirfenidone (PFD) by gavage daily at a dose of 150 mg / kg. The timing and duration of intervention were the same as in the Am80 group, until the experimental endpoint.

[0044] Mouse liver specimen processing: Experimental mice were first anesthetized with isoflurane using a mask method. After deep anesthesia, blood was collected from the eyeballs, followed by euthanasia via cervical dislocation. After euthanasia, the mice were rapidly transferred to a laminar flow hood. The surgical site was disinfected with 75% medical alcohol. A midline abdominal incision was made using sterile instruments to fully expose the abdominal contents. The liver parenchyma was carefully dissected using microsurgical methods, and the liver was divided into several regions or subunits according to the experimental objectives. During tissue sampling, the largest and most intact liver lobe was selected as the primary observation material, generally cut longitudinally into three parts. The central portion was prioritized for routine histological examination during paraffin embedding and sectioning. The remaining portion was cut into approximately 2mm × 2mm pieces, placed in two 2ml centrifuge tubes according to different biomarker requirements, flash-frozen in liquid nitrogen, and then stored at -80℃ for later use.

[0045] Example 1: The therapeutic effect of tamibarbitine (Am80) on liver fibrosis in vivo Experimental methods: CCl4- and TAA-induced mouse liver fibrosis models were established, respectively. Participants were randomly divided into a control group, a model group, a PFD-positive control group (150 mg / kg), and different Am80 dose groups (4, 6, and 8 mg / kg).

[0046] Experimental results: like Figure 1As shown in Figure A, the IHC results for Sirius red, α-SMA, and Collagen-III revealed that in the control group (NC group), weak α-SMA positive expression was only observed in the vascular smooth muscle layer of the liver, with almost no expression in the liver parenchyma. In the model group (CCl4 group), however, α-SMA positive areas (dark brown) showed extensive reticular and cord-like strong positive expression along the damaged central vein and newly formed fibrous septa, suggesting HSC activation in the liver. After Am80 treatment, the areas of strong positive expression of α-SMA and Collagen-III in liver tissue were significantly reduced. Compared with the control group, the immunohistochemical staining areas of α-SMA and Collagen-III in the liver tissue of the model group were significantly increased (P<0.0001); while the Am80 treatment group showed a significant downregulation trend compared with the model group (P<0.01).

[0047] Quantitative analysis of protein and transcription levels ( Figure 1 (B, C): Total RNA and total protein were extracted from liver tissues of each group and analyzed. The results showed that CCl4 induced overexpression of Acta2, Col1, and Col3a1 genes and their corresponding proteins (P<0.05). After intervention with Am80, the transcription and translation levels of these two core collagen proteins were effectively suppressed, showing a statistically significant decrease compared to the model group (P<0.05).

[0048] like Figure 1 As shown in Figure D, numerous deeply stained red collagen fibers were observed in the Sirius red stained sections of the livers of mice in the TAA model group. These collagen fibers extended and intersected, forming large, dense bridging fibrous septa that divided the liver lobules into nodules of varying sizes, indicating significant excessive collagen deposition. Am80 treatment significantly inhibited collagen fiber deposition. Microscopically, the red-stained collagen fibers were noticeably thinner and lighter, and the originally interwoven, large fibrous septa broke and disappeared. Collagen deposition was confined to the perivascular area and did not form a significant bridging network. Staining scores showed that the TAA model group had higher Sirius red staining scores and more stained collagen than the NC group. Intervention with Am80 at concentrations of 4, 6, and 8 mg / kg effectively reduced Sirius red staining scores.

[0049] In summary, this invention confirmed the efficacy of Am80 from three dimensions—morphology, protein expression, and gene transcription—by constructing two classic mouse models of liver fibrosis induced by CCl4 and TAA. Histopathological results showed that Am80 effectively reduced collagen deposition in the liver; immunohistochemical and molecular biological tests further confirmed that Am80 significantly inhibited the synthesis and expression of the activated hepatic stellate cell marker α-SMA and the core extracellular matrix (Collagen-I, Collagen-III). Compared with the clinical control drug pirfenidone, Am80 exhibited superior intervention potential, providing a phenotypic basis for subsequent mechanistic studies.

[0050] Example 2: Am80 reduces extracellular matrix deposition by inhibiting HSC activation 2.1 Experimental methods: Immunohistochemistry (IHC) and RT-qPCR were used to detect the levels of α-SMA and type III collagen in the liver tissue of mice in each group.

[0051] 2.2 RNA reverse transcription (1) Mix 0.2 μL of total RNA sample, 2 μL of 5×g DNA Buffer and 7.8 μL of nuclease-free water by shaking to prepare a genomic DNA removal system; (2) The reverse transcription reaction system consisted of 2 μL of FQ-RT Primer Mix, 2 μL of 10×King RT Buffer, 1 μL of FastKing RT Enzyme Mix, and nuclease-free water to a total volume of 10 μL. (3) The prepared genomic DNA removal system was set to 42℃ and reacted for 3 min in the PCR amplification instrument to remove DNA. The system prepared in step 2 was added to the system after DNA removal and thoroughly shaken and mixed. It was incubated at 42℃ for 15 min and at 95℃ for 3 min. After reverse transcription, it was stored at -80℃ for a long time.

[0052] 2.3 Quantitative Real-time PCR (qPCR) (1) Primer sequences were obtained from the PubMedNCBI database or PrimerBank and synthesized by Shandong Zhuoyue Biotechnology Co., Ltd. The primer sequences are shown in Table 1 below: Table 1 (2) Prepare the PCR reaction system: cDNA 2μL, SYBR 10μL, ROXDyeⅡ 0.4μL, RNase-Free-ddH2O 6.8μL, ForwardPrimer (10μM) 0.4μL, ReversePrimer (10μM) 0.4μL.

[0053] (3) Turn on the PCR instrument and set the program according to the instruction manual, as shown in Table 2 below: Table 2 (4) The CT value of the corresponding gene after the reaction is completed can be obtained by using the instrument. The subsequent analysis will use GAPDH as an internal reference to perform quantitative statistical analysis.

[0054] Experimental results: Compared with the model group, Am80 intervention significantly downregulated the protein abundance and mRNA level of α-SMA, demonstrating that it works by inhibiting the activation of hepatic stellate cells, and its ability to inhibit hepatic stellate cell activation is superior to that of pirfenidone (PFD).

[0055] Example 3: Target Screening and Discovery of CNR2 Based on Transcriptome Sequencing 3.1 Experimental Methods: In order to systematically analyze the molecular mechanism of tamibarbitine (Am80) in the fight against liver fibrosis at the whole genome level and to find potential key targets, total RNA was extracted from the liver tissues of mice in the control group (NC), model group (CCl4), and treatment group (CCL4+6mg / kg Am80), libraries were constructed, and high-throughput transcriptome sequencing (RNA-seq) was performed.

[0056] After completing the quality control and comparison of sequencing data, this invention used |log2(Fold Change)|>1 and the corrected P value (FDR)<0.05 as the threshold to screen differentially expressed genes among groups.

[0057] 3.2 Experimental Results: (1) Model-induced gene population changes: Volcano plot analysis results show that ( Figure 2 In the CCl4 model group (A), compared with the NC group, the expression of 242 genes in the liver tissue was significantly altered, including 94 downregulated genes and 148 upregulated genes. This indicates that CCl4 toxicity damage triggered a dramatic remodeling at the liver transcriptome level, with a large number of pro-inflammatory and pro-fibrotic genes being abnormally activated.

[0058] Am80 treatment-induced regression: After Am80 intervention, compared with the CCl4 model group, a total of 1553 differentially expressed genes were screened in the Am80 treatment group. Among them, 571 genes were significantly downregulated and 982 genes were significantly upregulated.

[0059] (2) To further and more intuitively evaluate the ability of Am80 to reverse the global expression pattern of the transcriptome, this invention also selected genes with significant differential expression among the groups (all DEGs) and performed hierarchical cluster analysis, and drew a global gene expression cluster heatmap. The color gradient and dendritic branch clustering results of the heatmap showed that each biological duplicate sample had extremely high similarity and clustering within the group, indicating that the sequencing data quality was reliable.

[0060] In intergroup comparisons ( Figure 2 In the gene expression profiles of the B group, the NC group, and the CCl4 model group, the boundaries between the color patches were clear; however, the overall gene expression pattern in the Am80 treatment group showed a significant regression. Furthermore, many genes that were suppressed in the model group and are essential for maintaining normal liver metabolism showed significant recovery after Am80 treatment.

[0061] (3) After clarifying that Am80 can cause a significant backsliding of the whole genome expression profile, in order to further reveal the key biological functions and signal transduction pathways involved by these differentially expressed genes (DEGs), this invention conducted in-depth joint analysis and KEGG pathway enrichment analysis on the sequencing data.

[0062] This invention introduces genes that are downregulated in the CCL4 model group but recover after Am80 treatment, and genes that are upregulated in the CCL4 model group but decrease after Am80 treatment, into an enrichment analysis platform for KEGG pathway annotation. The KEGG enrichment bar chart results show ( Figure 2 Genes downregulated in the CCL4 model group but restored after Am80 treatment were significantly enriched in multiple pathways related to endogenous metabolism and detoxification. Among them, the "retinol metabolism" pathway ranked high in enrichment. Genes upregulated in the CCL4 model group but decreased after Am80 treatment were significantly enriched in multiple pathways related to liver fibrosis progression, especially the extracellular matrix-receptor interaction (ECM-receptor interaction) and TGF-β signaling pathway, two pathways directly related to liver fibrosis, which ranked high in enrichment. Figure 2 The presence of D in the middle part of the sample indirectly proves the anti-liver fibrosis ability of Am80.

[0063] (4) After confirming that Am80 can effectively activate the liver retinoic acid signaling pathway, this invention further explored the differential gene set screened by transcriptome sequencing in order to find the core downstream executive molecules that mediate the anti-fibrotic effect of Am80.

[0064] Since this invention has demonstrated that Am80 activates the retinoic acid pathway and most likely regulates anti-liver fibrosis-related capabilities through this pathway, the intersection of the list of differentially expressed genes (DEGs) that were significantly upregulated after Am80 treatment with known genes containing RARE elements was obtained. This yielded the differentially expressed genes containing RARE elements that were significantly upregulated after Am80 treatment compared to the CCL4 model group. Figure 2 (E). Among them, cannabinoid receptor 2 (Cnr2) attracted the focus of this invention. CNR2 belongs to the G protein-coupled receptor family, and previous sporadic studies have suggested its potential protective role in immune regulation and inhibition of tissue fibrosis, but its upstream and downstream regulatory relationship with the retinoic acid receptor (RAR) signaling pathway remains unknown. Transcriptome sequencing data of this invention showed that, compared with the CCl4 model group, the abundance of Cnr2 transcripts in the liver tissue of mice treated with Am80 (6 mg / kg) was significantly increased (upregulated by approximately 3.6-fold). This finding makes CNR2 a candidate core target for the action of Am80.

[0065] (5) In the transcriptome sequencing results of this invention, no significant upregulation of the mRNA level of the retinoic acid receptor (RARα) itself was observed after Am80 treatment. From the perspective of nuclear receptor pharmacology, RARα, as a typical ligand-dependent transcription factor, is activated by Am80 through a core mechanism involving spatial conformational changes, dissociation of co-repressors, and recruitment of co-activators after ligand binding, rather than an increase in the abundance of receptor expression itself. Therefore, in order to conclusively demonstrate that Am80 successfully and specifically activates the retinoic acid signaling pathway in vivo, this invention selected downstream target genes with promoter regions rich in classical retinoic acid response elements (RAEs) for verification.

[0066] To confirm the activation of the retinoic acid pathway, this invention selected three downstream retinoic acid molecules—Cyp26A1, Klf4, and Socs3—for validation in mouse liver tissue. Cyp26A1, a member of the cytochrome P450 family, is a key retinoic acid metabolic enzyme whose transcription is directly positively regulated by the RAR and is a recognized biomarker for retinoic acid pathway activation. RT-qPCR results showed ( Figure 3Compared with the NC group and the CCl4 model group, the mRNA expression level of Cyp26A1 in the liver tissue of mice treated with Am80 was significantly upregulated (approximately 50-fold increase, P < 0.001). This change in transcriptional level provides direct molecular evidence that Am80 successfully binds to and activates RARα transcriptional function in target cells of damaged liver. Simultaneously, the upregulation of Socs3 and Klf4 also provides further evidence for the activation of the retinoic acid pathway, and Western blot experiments also showed a significant increase in Klf4 protein in mice after treatment with 6 mg / kg Am80. Figure 3 (A)

[0067] (6) To rule out false positives that may occur with high-throughput sequencing and to confirm the regulatory role of Am80 on CNR2, this invention first validated the effect at the tissue level using immunohistochemistry, protein imprinting, and RT-qPCR. Immunohistochemical results showed ( Figure 3 In liver tissue sections from groups C and D, NC, and CCl4 modeling group, only extremely weak basal expression of Cnr2 was observed. Positive signals (brownish granules) were mainly sparsely distributed in non-parenchymal cell areas, confirming that the protein abundance of endogenous Cnr2 in the liver was at a low level regardless of whether the liver was healthy or simply modeled. However, in paraffin sections of liver tissue from the Am80 treatment group, the positive staining area of ​​Cnr2 was significantly increased (p<0.0001).

[0068] Western blot results showed that the abundance of Cnr2 protein in the liver of mice treated with Am80 was increased, higher than that in the NC group and the CCl4 model group. RT-qPCR showed that the transcriptional level of Cnr2 in the liver of mice treated with Am80 was significantly increased, higher than that in the NC group (p<0.01) and the CCl4 model group (p<0.001), demonstrating that the addition of Am80 can upregulate the expression of Cnr2 in mice.

[0069] 3.3 Conclusion This invention utilizes high-throughput RNA-seq technology to systematically analyze the changes in the whole-genome expression profile before and after Am80 treatment. Pathway enrichment and molecular validation clearly indicate that Am80 reverts to the abnormal liver fibrosis gene network induced by CCl4, and that Am80 activates the expression of downstream molecules of the retinoic acid signaling pathway, namely Cyp26A1, Socs3, Klf4, and Cnr2, in vivo. Crucially, through cross-alignment of differentially expressed genes rich in RARE elements, this invention, for the first time, discovers and validates that cannabinoid receptor 2 (CNR2) is a core downstream target gene that is significantly upregulated in the Am80 regulatory network, thus identifying the molecular pathway of Am80's anti-liver fibrosis effect.

[0070] Example 4: In vitro validation of the regulatory mechanism of RAR-driven CNR2 upregulation 4.1 Experimental methods: TGF-β was used to induce activation of human LX-2 cells, followed by intervention with 20 μM Am80, and the RAR-specific antagonist BMS493 was introduced for blocking experiments.

[0071] 4.2 Cell culture, exogenous plasmid transfection (based on the jetPRIME® transfection system), siRNA dissolution and transfection (based on the GP-transfer-Mate system), and cytoskeleton fluorescence staining (phalloidin method) were all performed in accordance with the reagent instructions or conventional methods in the field.

[0072] 4.3 Drug administration and intervention strategies in in vitro cell models The cell culture and drug intervention process of this invention is carried out according to the following timeline: Day 1 (Plate preparation and transdyeing): LX-2 cells were seeded at the experimentally set density in multi-well culture plates and placed in a cell culture incubator to allow them to adhere naturally. For experimental groups that were designed to overexpress LX-2 cells with CNR2 and the AM1241 agonist using plasmids, transfection with the target plasmid was performed simultaneously after cell adhesion.

[0073] Day 2 (Hunger and Induced Activation): After the cells stabilized, the original culture medium was aspirated and discarded, and replaced with 2% low-serum culture medium for 6 hours of starvation treatment. After starvation, without changing the culture medium, TGF-β (10 ng / ml) was added directly to the original culture system to induce pathological activation of LX-2.

[0074] Day 3 (Pharmacological Intervention): After 24 h of continuous TGF-β induction, the following drug administration treatments were administered according to the specific experimental groups: In the conventional drug administration group, Am80 (40 μM) or PFD (50 μM) (as a positive control) was added directly to the drug. RNA was collected 24 hours after drug treatment and protein was collected 48 hours later. BMS493 pre-blocking group: To verify receptor specificity, 6 hours before the originally scheduled addition of Am80 (i.e., 18 hours after TGF-β induction), BMS493 at a final concentration of 0.5 μM was added for pre-blocking; after 6 hours of incubation, 40 μM of Am80 was added. AM1241 activation group: For cells that had completed plasmid transfection on Day 1, AM1241 was added to a final concentration of 1 μM for intervention at the time point 24 hours after TGF-β induction.

[0075] Each time the drug was added, the corresponding NC group or other groups that needed to be controlled were added with the corresponding solvent medium to balance all conditions. After the corresponding drug addition operation was completed, all groups continued to be cultured in a 37°C, 5% CO2 constant temperature incubator until the set observation endpoint was reached. Then the experiment was terminated and cell samples were collected for downstream detection.

[0076] 4.4 Statistical Analysis In this invention, the quantization and grayscale extraction of all original images were completed using ImageJ software, the statistical analysis relied on SPSS 26.0 and GraphPad Prism 8 software platforms, and the final chart layout and drawing were performed using GraphPad Prism 8 and Adobe Illustrator 2020 software.

[0077] For continuous data, normality and homogeneity of variance tests are routinely performed before comparing groups. For comparisons of two independent samples that are normally distributed and have homogeneous variances, the independent samples t-test is used; if the data are not normally distributed or have unequal variances, the Mann-Whitney U nonparametric rank-sum test is used. For comparisons of variables among three or more groups, one-way ANOVA is used if normality and homogeneity of variance are met; otherwise, the Kruskal-Wallis H test is used. The χ² test (chi-square test) is used to compare differences in distribution among groups for categorical (count) data. Pearson correlation analysis is used to assess linear relationships between bivariates; the Kaplan-Meier method is used for survival data analysis and survival curve plotting.

[0078] All hypothesis tests in this invention are two-tailed tests, with a significance level set at α = 0.05. A difference is considered statistically significant when P < 0.05.

[0079] 4.5 Experimental Results: (1) Based on the fact that Am80 significantly inhibits collagen deposition and fibrosis in animal models, this invention further detected the changes in the expression of HSCs activation and core markers of collagen deposition by RT-qPCR, Western Blot and phalloidin staining.

[0080] Changes in collagen component expression: COL1A1 and COL3A1 are the main components constituting pathological matrix deposition in the liver. Detection results show that ( Figure 4 In LX-2 cells, TGF-β significantly stimulated the transcription and translation of COL1 and COL3 (P<0.0001). However, after treatment with Am80, the mRNA and protein expression levels of these two core fibrotic collagens were significantly downregulated (P<0.001). Although PFD had an inhibitory effect on liver fibrosis, compared with Am80, the protein levels of α-SMA, COL1A1, and COL3A1 decreased more significantly in the Am80-treated group, and the mRNA levels of COL1A2 and ACTA2 also decreased more significantly (p<0.01).

[0081] Phalloidin-specific F-actin fluorescence staining results showed ( Figure 4 (C) NC group: The cells are mostly slender spindle-shaped or star-shaped, and the red fluorescent stress fibers inside are relatively thin and sparse; TGF-β model group: After TGF-β stimulation, LX-2 cells were significantly larger and spread out, and a large number of extremely thick, dense and parallel stress fiber bundles appeared in the cytoplasm, exhibiting the typical morphological characteristics of highly active myofibroblasts. Am80 treatment group: After Am80 intervention, TGF-β-induced abnormal cytoskeleton remodeling was inhibited; cell morphology re-shrinked, the large cell body became thinner, no coarse stress fiber network was observed, and the morphology was close to the resting state; PFD treatment group: After PFD intervention, TGF-β-induced activated LX-2 partially returned to resting state, cell morphology recovered and contracted, stress fiber network staining was reduced but still significant, and anti-LX-2 activation was weaker than Am80.

[0082] (2) Although Am80 is a recognized RAR-specific agonist, it is still unknown whether its effect of upregulating CNR2 and inhibiting extracellular matrix secretion in LX-2 cells in vitro depends on the classical RAR receptor pathway or is due to some non-specific off-target effect. In order to rigorously answer this key scientific question, this invention first verified the activation of the retinoic acid pathway in vitro, and then introduced the pan-retinoic acid receptor inverse agonist / antagonist BMS493 to verify the loss of function.

[0083] Am80 effectively activates the retinoic acid signaling pathway in hepatic stellate cells in vitro, consistent with the in vivo validation logic. First, this invention constructs TGF-β-induced LX-2 activated hepatic stellate cells to simulate liver fibrosis. Then, Am80 is used for treatment. Finally, total RNA and protein are extracted at the LX-2 cell level for molecular biological validation, and phalloidin staining is used to demonstrate the activation status of LX-2. This invention detects classic downstream target genes whose promoter regions contain retinoic acid response elements (RAREs). Western blot results show (…). Figure 4 In the study of retinoic acid (RT-D) proteins, the downstream genes CNR2 and KLF4 containing RARE elements, showed significant increases after the addition of Am80. RT-qPCR results showed that... Figure 4 After incubation with Am80, the mRNA level of CYP26A1, a core enzyme in retinoic acid metabolism, was significantly upregulated in LX-2 cells (P<0.0001). Simultaneously, the target genes KLF4 and SOCS3, which have anti-inflammatory and cellular quiescent functions, also showed a significant trend of transcriptional activation. This result conclusively demonstrates that Am80 can penetrate the LX-2 cell membrane and successfully enter the nucleus, exerting the targeted transcriptional activation function of RAR. Phalloidin staining results showed ( Figure 4 The addition of BMS493 caused Am80 to lose its function of maintaining cell morphology. The cells once again exhibited a large, spread-out activated morphology, and the robust F-actin stress fiber network that had previously subsided under Am80 treatment re-formed in large quantities, meaning that Am80's ability to combat liver fibrosis was blocked.

[0084] Furthermore, to demonstrate that the upregulation of CNR2 is a direct consequence of RAR pathway activation, this invention included a retinoic acid pathway blocking group. LX-2 cells were pretreated with the specific antagonist BMS493 (500 nM) before Am80 treatment to preemptively occupy and block intracellular RAR receptors. Western blotting and phalloidin staining showed that after BMS493 pretreatment to block the retinoic acid pathway, LX-2 cells that would normally be inhibited by Am80 were unaffected. The cells again exhibited a broad, extended activated morphology, and the stress fiber network that had previously subsided under Am80 treatment re-formed in large quantities. This indicates that Am80's anti-liver fibrosis ability was blocked, thus further reinforcing the evidence that Am80 works through the retinoic acid pathway.

[0085] 4.6 Conclusion This invention utilizes a TGF-β-induced human hepatic stellate cell (LX-2) activation model to demonstrate that Am80 significantly downregulates the expression of type I and type III collagen and promotes the disaggregation of large stress fibers within cells, restoring cells to a near-resting phenotype. Compared to the positive control drug pirfenidone, Am80 exhibits superior ability to inhibit profibrotic gene transcription at both protein and RNA levels. Mechanism reversal experiments further demonstrate that Am80 specifically activates the intracellular retinoic acid signaling pathway. Blocking the RAR receptor using BMS493 not only inhibits Am80's CNR2 transcription-promoting effect but also leads to a relapse of the activated cytoskeleton phenotype. This confirms that the in vitro antifibrotic efficacy of Am80 is highly dependent on the classical RAR receptor pathway.

[0086] Example 5: Forward and Reverse Rescue Experiment of CNR2 Targeted Intervention 5.1 Experimental methods: LX-2 cells were treated with siRNA knockdown of CNR2 expression or transfection with CNR2 overexpression plasmid and combined with receptor agonist AM1241.

[0087] 5.2 Experimental Results: (1) This invention constructed an overexpression plasmid (CNR2-OE) containing the human CNR2 sequence and established a blank control group. After transiently transfecting it into LX-2 cells, the transfection efficiency was confirmed by RT-qPCR. Figure 5 Compared with the blank group, the intracellular CNR2 mRNA level in the CNR2-OE group was increased by thousands of times (P<0.001), confirming the successful establishment of the in vitro overexpression cell model. Western blotting also showed that CNR2 protein expression in LX-2 cells increased after transfection with the OE-CNR2 plasmid.

[0088] At the molecular expression level, the detection results of Western Blot were consistent with morphological observations. Figure 5(A). Compared with the TGF-β model group or the Vector group, CNR2-OE alone only caused a slight decrease in COL1A1 expression, which was far from reaching the level of Am80 inhibiting LX-2 activation. Therefore, we hypothesized that since Am80-specifically activated RARα broadly regulates downstream molecules of the retinoic acid pathway, its effect may be far more than simply upregulating CNR2, but may also enhance the transcription of its ligands, achieving a dual anti-fibrotic effect of both making it more sensitive and activating more ligands. Therefore, we added the CNR2-specific activator AM1241 to the CNR2 overexpression to see if the combined effect could achieve the anti-fibrotic effect of Am80. The results showed that in the "OE + AM1241" combined intervention group, core collagen, whether at the protein level ( Figure 5 (B) or mRNA level ( Figure 5 Both C and N2 were strongly inhibited (P<0.01). This inhibition mimics the matrix intervention efficacy of Am80 monotherapy.

[0089] In a TGF-β-induced LX-2 activation model, this invention systematically evaluated the effects of different intervention strategies on cell morphology. Phalloidin cytoskeleton staining results showed ( Figure 5 D): The simple overexpression group (TGF-β + CNR2-OE): Although the cells contain extremely abundant CNR2 receptors, due to the lack of sufficient endogenous cannabinoid ligands in the culture system, the receptors are mostly in the resting conformation; at this time, the cells still maintain a large activated morphology, and the thick stress fibers do not show obvious regression. Combined intervention group (TGF-β + CNR2-OE + AM1241): When overexpressed CNR2 was combined with the specific receptor agonist Am1241, the cell phenotype was reversed. Cells shrank and thinned, and the coarse F-actin stress fiber network disappeared under the microscope. The degree of fibrosis recovery was even similar to that of the Am80 treatment group.

[0090] (2) In previous in vitro and in vivo experiments, this invention has confirmed that Am80 can significantly upregulate the expression of the target molecule CNR2 through the retinoic acid receptor (RAR) pathway. In order to clarify whether CNR2 plays a core role in the anti-fibrotic effect of Am80 or is merely an observer of the accompanying pathway activation, this invention introduced small interfering RNA (siRNA) technology in the in vitro LX-2 cell model to conduct a loss-of-function rescue experiment targeting CNR2.

[0091] Based on successful CNR2 knockdown, this invention administered TGF-β stimulation and Am80 treatment to each group of cells. Phalloidin cytoskeleton-specific staining results showed ( Figure 6 (A) si-NC rescue group (TGF-β + Am80): The cell morphology was consistent with that of the Am80-only treatment group, exhibiting a slender resting appearance, and the coarse F-actin stress fiber network was effectively inhibited; si-CNR2 rescue group (TGF-β + Am80 + Si-CNR2): When CNR2 expression was specifically blocked, the ability of Am80 to maintain cell morphology was significantly weakened. Microscopically, cells were observed to spread out and become broad and deformed again, and the previously diminished thick stress fiber bundles were reassembled and arranged in large quantities in the cytoplasm.

[0092] Meanwhile, this invention also attempted to overexpress CNR2 first, as shown in the figure ( Figure 6 The activation of LX2 in the B group was slightly inhibited, but its slightly enhanced anti-fibrotic effect was restored in the Si-CNR2 rescue group (TGF-β+OE-CNR2+Si-CNR2).

[0093] Transcription level shows ( Figure 6 (B) After using Si-CNR2 to knock down CNR2, both the strong anti-fibrotic effect of Am80 and the weak anti-fibrotic effect shown after CNR2 overexpression were reversed, suggesting that CNR2 plays a key role in the anti-liver fibrosis effect of Am80.

[0094] 5.3 Conclusion This invention explores the mediating role of CNR2 in the Am80 anti-fibrotic regulatory network by constructing cell rescue models with gain-of-function (plasmid overexpression) and loss-of-function (siRNA knockdown). Results show that receptor expansion (CNR2 overexpression) combined with its specific ligand activation (AM1241) perfectly reproduces the potent inhibition of matrix collagen secretion and reversal of cytoskeleton remodeling by Am80 monotherapy in LX-2 cells. Conversely, specific knockdown of CNR2 expression significantly weakens Am80's ability to maintain the resting morphology of HSCs, leading to the reassembly of heavily repressed robust stress fiber bundles. This series of forward and reverse rescue experiments rigorously confirms that CNR2 is a key downstream executive molecule in Am80's inhibition of HSC activation and remodeling.

[0095] Example 6: Parallel comparison based on receptor subtype specificity confirms the synergistic superiority of dual agonists 6.1 Reagents: Tamibarbitine (Am80): Chemical name 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl]benzoic acid, purity ≥98%, CAS number 94497-51-5, purchased from Selleck Chemicals (USA); Am580 (RARα selective agonist): purity ≥98%, CAS No. 10212-25-6, purchased from Med Chemicals Express (USA); KCL-286 (RARβ selective agonist): purity ≥98%, purchased from Selleck Chemicals (USA).

[0096] 6.2 Experimental Methods: To investigate the role of different RAR isoforms in antifibrosis, a TGF-β-induced LX-2 cell activation model was used. Cells were treated with appropriate concentrations of specific RARα agonists (Am580), specific RARβ agonists (KCL-286), and the RARα / β dual agonist tamibarbitine (Am80), respectively. The expression of activation markers in each group was then detected.

[0097] Grouping and drug intervention: Cells were divided into 5 groups: Control group: Only an equal volume of solvent was added; Model group (TGF-β1): Activated by stimulation with 10 ng / mL TGF-β1; Am580 group: Add 10 ng / mL TGF-β1, and add 1 μM Am580 after 24 hours; KCL-286 group: Add 10 ng / mL TGF-β1, and add 1 μM KCL-286 after 24 hours; Am80 group: Add 10 ng / mL TGF-β1, and add 40 μM Am80 after 24 hours; Intervention time: 24 hours after adding Am580, KCL-286 or Am80.

[0098] 6.3 Experimental Results: Cell morphology and cytoskeleton remodeling (phalloidin & α-SMA immunofluorescence): Fluorescence microscopy observation showed ( Figure 7In the TGF-β1-stimulated model group (CD), LX-2 cells showed a significant increase in volume and strong α-SMA expression (exhibiting bright red fluorescence). Phalloidin staining revealed a coarse and dense fiber network, indicating cell transformation into myofibroblasts. After intervention with 1 μM Am580 or 1 μM KCL-286, the fluorescence intensity of the stress fiber network and α-SMA decreased, but a large number of myofibroblast morphologies remained. In contrast, the α-SMA fluorescence signal in the 40 μM Am80 intervention group was significantly inhibited, and the cell morphology returned to a near-resting star-shaped pattern.

[0099] Inhibition of collagen secretion (WB and qPCR): RT-qPCR results showed that ( Figure 7 (B) TGF-β1 significantly upregulated the mRNA levels of COL1A1 and COL3A1. The single-target agonists Am580 and KCL-286 only moderately downregulated their expression. In contrast, the Am580 group showed superior inhibitory effects on COL1A1 and COL3A1 transcription, with a significant reduction in expression.

[0100] Western blot results showed that ( Figure 7 (A): The trend of COL1A1 protein expression level changes was consistent with the mRNA results. The gray value of the COL1A1 protein band in the Am80 group was lower than that in the groups that simply activated RARα or RARβ.

[0101] Data confirm that while single activation of RARα (Am580) or single activation of RARβ (KCL-286) can slightly downregulate α-SMA and collagen expression, their anti-activation efficacy is limited. In contrast, Am80, as a dual RARα / β agonist, exhibits significantly better inhibition rates against stress fiber depolymerization and matrix secretion than the aforementioned single-target agonists.

[0102] The above conclusions indicate that deep quiescence of HSCs requires the synergistic activation of RARα and RARβ. The Am80 selected in this invention overcomes the shortcomings of single-receptor subtype agonists in terms of efficacy, achieving superior anti-fibrotic effects due to its unique dual-target agonistic properties.

[0103] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. Use of tamibarbitine or its pharmaceutically acceptable salts, esters, solvates, or active metabolites in the preparation of medicines for the treatment and / or prevention of liver diseases.

2. The application according to claim 1, characterized in that, The liver diseases mentioned include chronic liver injury and the resulting liver fibrosis and cirrhosis.

3. The application according to claim 1, characterized in that, The drug can be administered via any one of the following routes: oral administration, intravenous injection, intraperitoneal injection, subcutaneous injection, intramuscular injection, or local injection into the liver.

4. The application according to claim 3, characterized in that, The dosage form for oral administration is selected from one or more of tablets, capsules, granules, and oral solutions.

5. The application according to claim 1, characterized in that, The dosage of tamilbarbitine is 4 mg / kg to 8 mg / kg daily.

6. The application according to claim 5, characterized in that, The recommended dosage of tamilbarbitine is 6 mg / kg daily.

7. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier and / or excipient.

8. A pharmaceutical composition for treating liver fibrosis, characterized in that, It contains a therapeutically effective amount of tamibarbitine or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

9. The use of tamibarbitine or its pharmaceutically acceptable salts, esters, solvates, or active metabolites in the preparation of drugs that inhibit hepatic stellate cell activation and reduce extracellular matrix deposition.

10. The use of tamibarbitaline or its pharmaceutically acceptable salts, esters, solvates, or active metabolites in the preparation of drugs that upregulate cannabinoid receptor 2 expression.

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