Application of Akebia quinata phenylethanol glycoside B in the preparation of drugs for the prevention and treatment of liver fibrosis

Abstract

This invention belongs to the field of biomedicine, specifically relating to the use of Akebia quinata phenylethanoidin B in drugs for treating liver fibrosis. Using a TGF-β1-induced human hepatic stellate cell line LX-2 activation model, this invention demonstrates that Akebia quinata phenylethanoidin B inhibits hepatic stellate cell activation. Furthermore, Akebia quinata phenylethanoidin B can bind to and regulate the function of G3BP1. Using a CCl4-induced mouse model of liver fibrosis, in vivo, it was demonstrated that Akebia quinata phenylethanoidin B significantly improves liver function indicators, reduces collagen deposition in liver tissue, and inhibits the expression of hepatic stellate cell activation markers. Therefore, both in vitro and in vivo, Akebia quinata phenylethanoidin B can effectively inhibit hepatic stellate cell activation, thereby inhibiting the progression of liver fibrosis. It has promising applications in the preparation of drugs for treating liver fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the use of Akebia quinata phenylethanoid B in drugs for the treatment of liver fibrosis. Background Technology

[0002] Liver fibrosis is a key pathological stage in the progression of chronic liver disease to cirrhosis. Its core characteristic is the excessive deposition of extracellular matrix (ECM), leading to liver structural damage and functional impairment. The sustained activation of hepatic stellate cells (HSCs) is a central event in the development of liver fibrosis. In a normal liver, HSCs are quiescent; when the liver is damaged by factors such as viruses, alcohol, and metabolism, HSCs are activated, transforming into myofibroblasts. These cells highly express α-smooth muscle actin (α-SMA) and secrete large amounts of ECM components such as type I collagen (COL1A1), ultimately forming fibrotic scars. Transforming growth factor β1 (TGF-β1) is the most potent known pro-fibrotic factor and plays a crucial role in HSC activation. Therefore, finding compounds that can effectively inhibit TGF-β1-induced HSC activation is an important strategy for developing anti-liver fibrosis drugs. Currently, there are no specifically approved anti-liver fibrosis drugs in clinical practice; existing treatments mainly focus on etiological control and symptomatic supportive care. Therefore, developing novel drugs that can directly target HSC activation, reverse or delay the progression of liver fibrosis is of great clinical significance.

[0003] Calceolarioside B is a phenylethanol glycoside compound derived from traditional Chinese medicinal materials such as Akebia quinata, Angelica dahurica, and Codonopsis pilosula. Its bioactive components and potential molecular mechanisms for anti-liver fibrosis have not yet been explored. Summary of the Invention

[0004] One of the objectives of the invention is to enable G3BP1 to be used as a drug target in the preparation of drugs for the prevention and treatment of liver fibrosis.

[0005] Second objective of the invention: This invention, through liver fibrosis cell models and mouse liver fibrosis models, discovers a novel use of Akebia trifoliata phenylethanol glycoside B in anti-liver fibrosis by directly binding to G3BP1 protein.

[0006] Technical solution: Application of G3BP1 as a drug target in the preparation of drugs for the prevention and treatment of liver fibrosis.

[0007] The use of akebia phenethyl glycoside B or a pharmaceutically acceptable salt or ester thereof in the preparation of medicaments for the prevention or treatment of liver fibrosis, wherein the structural formula of akebia phenethyl glycoside B is shown below: .

[0008] The use of a pharmaceutical composition in the preparation of a medicament for the prevention or treatment of liver fibrosis, characterized in that the pharmaceutical composition comprises akebia phenylethanol glycoside B or a pharmaceutically acceptable salt or ester thereof, and pharmaceutically acceptable excipients.

[0009] The application is characterized in that the phenylethanoid glycoside B of Akebia trifoliata combines with G3BP1 to achieve anti-liver fibrosis.

[0010] Furthermore, akebia phenylethanoid glycoside B binds to G3BP1 and inhibits the activation of hepatic stellate cells.

[0011] The application is characterized in that the dosage form of Akebia quinata phenylethanoid B is an oral dosage form and a non-oral dosage form.

[0012] The application is characterized in that the oral dosage form is a tablet, capsule, powder, granule, suspension or oral liquid; and the non-oral dosage form is an injection, lyophilized powder for injection, ointment or gel.

[0013] The application is characterized in that Akebia quinata phenylethanoid glycoside B inhibits the activation of hepatic stellate cells by directly binding to G3BP1 protein, thereby achieving an anti-hepatic fibrosis effect.

[0014] The application is characterized in that akebia phenylethanoid glycoside B binds to the NTF2-like domain of G3BP1.

[0015] The application is characterized in that the pharmaceutical composition further comprises one or more other anti-hepatic fibrosis drugs, wherein the other anti-hepatic fibrosis drugs are selected from one or more of silymarin, colchicine, and polyene phosphatidylcholine.

[0016] The application is characterized in that the liver fibrosis disease includes one or more of the following: viral hepatitis-associated liver fibrosis, alcoholic liver fibrosis, non-alcoholic fatty liver fibrosis, and autoimmune liver disease-associated liver fibrosis.

[0017] The dosage of Akebia quinata phenylethanol glycoside B described in this invention can be adjusted by comprehensively considering factors such as the administration method, the severity of the patient's condition, the patient's age, and whether there is a history of the disease.

[0018] When this invention is used to prepare a medicine for the prevention and treatment of liver fibrosis, the excipients and preparation methods can be of any pharmaceutically acceptable form.

[0019] Research Approach: This invention uses TGFβ1-induced activation of LX2 cells to mimic hepatic stellate cell activation, thereby evaluating the in vitro anti-hepatic fibrosis effect of Akebia quinata phenylethanoidin B. The cytotoxicity of Akebia quinata phenylethanoidin B on LX2 cells was analyzed using the CCK-8 assay. The effects of Akebia quinata phenylethanoidin B on the expression levels of liver fibrosis-related genes and proteins were analyzed using RT-qPCR and Western blot.

[0020] A carbon tetrachloride (CCl4)-induced liver fibrosis animal model was constructed to further evaluate the in vivo anti-liver fibrosis effect of Akebia quinata phenylethanoidin B. In the CCl4 animal model, 6-8 week old C57BL / 6J mice were intraperitoneally injected with olive oil or 10% CCl4 three times a week. Drug administration began after 4 weeks. Mice were divided into a control group, a model group, and a group receiving Akebia quinata phenylethanoidin B. Blood samples were collected 4 weeks after administration for biochemical analysis. Liver tissue was collected for H&E staining, Masson staining, and Sirius red staining. The hydroxyproline content in the liver tissue was detected.

[0021] To further explore the mechanism of Akebia quinata phenylethanoid glycoside B in resisting liver fibrosis, the binding of Akebia quinata phenylethanoid glycoside B to G3BP1 was discovered and confirmed using methods such as Docking, DARTS, CETSA, and BLI.

[0022] Beneficial Effects: G3BP1 is an evolutionarily conserved RNA-binding protein and a core component of stress granules (SG), highly expressed in various tumor tissues and cells. Existing research indicates that G3BP1 can bind to viral RNA and participate in regulating viral replication, the RIG-I signaling pathway, and type I interferon-mediated host innate immune responses. Furthermore, G3BP1 can promote tumor proliferation and metastasis through mechanisms such as regulating aging-associated secretory phenotype (SASP), long non-coding RNA (lncRNA) function, and protein stability. Currently, research on G3BP1 mainly focuses on oncology, with reports showing that the expression levels of G3BP1 protein and its mRNA in hepatocellular carcinoma (HCC) tissues are higher than in normal liver tissues, and that high expression is closely associated with poor prognosis in HCC patients. However, the biological functions of G3BP1 in hepatic stellate cell activation and the progression of liver fibrosis have not yet been clearly reported. Current research also fails to reveal the potential of G3BP1 as a therapeutic target for liver fibrosis. Therefore, clarifying the function of G3BP1 in liver fibrosis and developing corresponding targeted drugs is of great significance for the treatment of liver fibrosis.

[0023] This invention marks the first discovery of the application of G3BP1 as a drug target in the preparation of drugs for the prevention and treatment of liver fibrosis. Further, it reveals the application of Akebia quinata phenylethanoid glycoside B in the preparation of anti-liver fibrosis drugs, which exert their anti-liver fibrosis effect by binding to G3BP1. Akebia quinata phenylethanoid glycoside B binds to the NTF2-like domain of G3BP1, interfering with protein-protein interactions or stress granule formation, thereby inhibiting hepatic stellate cell activation and achieving an anti-liver fibrosis effect. This has not been previously reported in the literature.

[0024] Specifically, this invention demonstrates, using a TGF-β1-induced human hepatic stellate cell line LX-2 activation model, that Akebia quinata phenylethanoidin B can effectively inhibit hepatic stellate cell activation. Using a CCl4-induced mouse model of liver fibrosis, this invention further verifies that Akebia quinata phenylethanoidin B can significantly improve liver function indicators, reduce collagen deposition in liver tissue, and downregulate the expression of hepatic stellate cell activation markers. Furthermore, this invention clarifies the key mechanism by which Akebia quinata phenylethanoidin B exerts its anti-liver fibrosis effect by binding to G3BP1. These results indicate that Akebia quinata phenylethanoidin B can effectively inhibit hepatic stellate cell activation both in vitro and in vivo, thereby inhibiting the progression of liver fibrosis, demonstrating promising drug development prospects and clinical application value. Attached Figure Description

[0025] Figure 1 Figure 1 shows the experimental results of akebia phenethyl glycoside B inhibiting the activation of the hepatic stellate cell line LX-2; where a represents the relative mRNA expression level of ACTA2, b represents the relative mRNA expression level of COL1A1, and c represents the relative mRNA expression level of TIMP1; d shows the results of Western blot detection and quantitative analysis of α-SMA and COL1A1 protein expression levels; n=3, * P<0.05 , **P< 0.01 , ***P<0.001 , ****P<0.0001 ; Figure 2 Figure 1 shows the experimental results of Akebia trifoliata phenylethanoid glycoside B improving liver pathological morphology, collagen deposition, and liver function damage in mice with CCl4-induced liver fibrosis. Figure 2 shows H&E staining, Masson staining, and SiriusRed staining images of the livers of mice in each group; Figure 3 shows the statistical analysis of the positive areas of Masson and Sirius Red staining; Figure 4 shows the detection results of hydroxyproline content in liver tissue; Figure 5 shows the detection results of serum ALT content; Figure 6 shows the detection results of serum AST content; n=8. **P<0.01 , ****P< 0.0001 ; Figure 3 Figure 1 shows the experimental results of akebia phenylethanoid glycoside B inhibiting the expression of CCl4-induced liver fibrosis-related proteins and genes in mice. In figure 1, a shows the expression levels and quantitative analysis of α-SMA and COL1A1 proteins in liver tissue detected by Western blot; b shows the relative expression level of Acta2 mRNA in liver tissue detected by RT-qPCR; c shows the relative expression level of Col1a1 mRNA; d shows the relative expression level of Timp1 mRNA; n=8. **P<0.01 , ***P<0.001 , ****P<0.0001 ; Figure 4Figure 1 shows the experimental results of Akebia quinata phenylethanoid glycoside B inhibiting hepatic stellate cell activation via G3BP1. Specifically, a) Western blot analysis shows that Akebia quinata phenylethanoid glycoside B (CB) inhibits hepatic stellate cell activation in a G3BP1-dependent manner; b) molecular docking predicts the binding site of Akebia quinata phenylethanoid glycoside B (CB) to G3BP1; c) Cell thermal transfer assay (CETSA) detects the potential binding effect of Akebia quinata phenylethanoid glycoside B (CB) to G3BP1; d) Drug affinity response target stability assay (DARTS) detects the potential binding effect of Akebia quinata phenylethanoid glycoside B (CB) to G3BP1; and e) Surface plasmon resonance (SPR) assay detects the affinity of Akebia quinata phenylethanoid glycoside B (CB) to G3BP1. Detailed Implementation

[0026] This invention provides the application of Akebia trifoliata phenylethanoid B in the preparation of a drug for treating liver fibrosis. The technical solution provided by this invention is described in detail below with reference to embodiments, but should not be construed as limiting the scope of protection of this invention.

[0027] Calceolarioside B, a commercially available product, has a purity of ≥98%.

[0028] Preparation of Akebia quinata phenylethanoid B solution for cell culture: Dissolve 1 mg of Akebia quinata phenylethanoid B powder in an appropriate amount of DMSO to prepare a 50 mM stock solution. Dilute the stock solution to the required concentration using PBS buffer, filter to sterilize, aliquot, and store at -20°C for later use. When using, dilute to the working concentration with DMEM medium.

[0029] Preparation of Akebia quinata phenylethanoid glycoside B injection for animals: Dissolve Akebia quinata phenylethanoid glycoside B powder in an appropriate amount of DMSO to prepare stock solutions of different concentrations. Prepare and use immediately.

[0030] Example 1: Akebia trifoliata phenylethanoid glycoside B inhibits the activation of human hepatic stellate cell line LX-2: I. Materials and methods required for the experiment in Example 1 1. Experimental Materials The human hepatic stellate cell line LX-2 was used as the cell model in this experiment. Akebia quinata phenylethanoid glycoside B (purity ≥98%, CAS: 105471-98-5) was purchased from Shanghai Yuanye Biotechnology. TGF-β1 was purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd. DMEM culture medium and fetal bovine serum were purchased from Gibco, USA. The CCK8 kit was purchased from Shanghai Haoyuan Biomedical Technology Co., Ltd. The COL1A1 promoter luciferase reporter gene plasmid was constructed in our laboratory. The dual-luciferase reporter gene assay kit was purchased from Novizan Biosciences Co., Ltd. RT-qPCR reagents were purchased from Novizan Biosciences Co., Ltd. Antibodies used in Western blot, including anti-α-SMA and anti-COL1A1, were purchased from Abcam, USA.

[0031] 2. Cell Culture Human hepatic stellate cell line LX-2 was cultured in DMEM medium containing 10% fetal bovine serum and incubated at 37°C in a 5% CO2 incubator. Cells were passaged when they reached 80%–90% confluence.

[0032] 3. CCK8 method Take LX-2 cells in the logarithmic growth phase and distribute them at a density of 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 100 μL per well in 96-well plates and cultured for 24 h. The culture medium was discarded, and serum-free medium containing different concentrations of akebia phenylethanoid B (0, 2.5, 5, 10, 20, 40, 80, 160 μM) was added, with three replicates per group. After another 24 h of culture, 10 μL of CCK8 solution was added to each well, and the cells were incubated for 2 h. The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability was calculated as follows: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%.

[0033] 4. COL1A1 promoter luciferase reporter gene assay LX-2 cells in the logarithmic growth phase were cultured in 6-well plates until they reached approximately 90%–95% confluence, at which point transfection was performed. 24 h after transfection, cells were seeded at a concentration of 1 × 10⁶ cells per well. 4Cells were seeded at a density of 100 μL per well in 96-well plates and cultured for 24 h. The culture medium was discarded, and serum-free medium containing different concentrations of akebia phenylethanoid B (0, 3, 10, 30, 100 μM) and 2 ng / mTGF-β1 was added to each well (100 μL), and the cells were cultured for another 24 h. The culture medium was then discarded, and the activities of firefly luciferase and Renilla luciferase were measured sequentially using a microplate reader according to the instructions of the dual-luciferase reporter gene assay kit. The ratio of these two activities was used as the relative luciferase activity, reflecting the activity of the COL1A1 promoter.

[0034] 5. Cell treatment and grouping Take LX-2 cells in the logarithmic growth phase, and distribute them at a ratio of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 mg / mL in 6-well plates and cultured for 4 h. The culture medium was discarded, and the cells were washed twice with PBS buffer. 2 mL of serum-free DMEM medium was added to each well, and the cells were starved for 24 h. Cells were then divided into a control group, a TGF-β1 model group, and a treatment group containing 10.0 μM, 20.0 μM, and 40.0 μM of akebia phenethyl glycoside B (10.0 μM, 20.0 μM, and 40.0 μM). Except for the control group, all other groups were treated with serum-free medium containing 2 ng / mL TGF-β1 and the corresponding concentration of akebia phenethyl glycoside B for 24 h.

[0035] 6. RT-qPCR detection Cells from each group were collected, total RNA was extracted, and after reverse transcription into cDNA, the relative mRNA expression levels of hepatic stellate cell activation markers ACTA2, COL1A1, and TIMP1 were detected using real-time quantitative PCR (RT-qPCR). GAPDH was used as an internal reference gene, and the relative expression levels were calculated using the 2-ΔΔCt method.

[0036] Table 1 Primer sequences involved in RT-qPCR .

[0037] 7. Western blot detection Cells from each group were collected, total protein was extracted, separated by SDS-PAGE electrophoresis, and transferred to PVDF membranes. After blocking with 5% skim milk powder, the membranes were incubated overnight at 4°C with α-SMA, COL1A1, and β-actin primary antibodies, respectively. The next day, after washing with PBST, the corresponding secondary antibodies were added, and the membranes were incubated at room temperature for 1 h. Chemiluminescence was used for imaging, and images were acquired using a gel imaging system. The relative expression levels of the target proteins were analyzed using β-actin as an internal control.

[0038] II. Experimental Results: 1. Phenylehnic acid glycoside B of Akebia trifoliata has no cytotoxic effect on LX-2 cells. The cytotoxicity of Akebia trifoliata phenylethanoidin B to LX-2 cells was detected using the CCK8 assay. The results showed that Akebia trifoliata phenylethanoidin B had no significant cytotoxicity to LX-2 cells within the concentration range of 3.0–100.0 μM, and the cell viability remained above 90%. Figure 1 a). Meanwhile, within the concentration range of 10.0–50.0 μM, Akebia quinata phenylethanoid glycoside B exhibited good COL1A1 promoter inhibitory activity ( Figure 1 b). Accordingly, subsequent experiments selected 10.0, 20.0, and 40.0 μM as low, medium, and high dosing concentrations of Akebia quinata phenylethanoid B, respectively.

[0039] 2. Akebia trifoliata phenylethanoid B inhibits the mRNA expression of hepatic stellate cell activation markers. The mRNA expression levels of activation markers in cells of each group were detected by RT-qPCR. The results showed that, compared with the control group, the relative expression levels of ACTA2, COL1A1, and TIMP1 mRNA in the TGF-β1 model group were significantly upregulated (P<0.001), indicating the successful construction of the hepatic stellate cell activation model. Compared with the TGF-β1 model group, each dose group of Akebia trifoliata phenylethanoid B (10.0 μM, 20.0 μM, 40.0 μM) significantly inhibited the upregulation of the mRNA expression of the above three markers (P<0.05 or P<0.01), and showed a certain dose-dependent effect. Figure 1 c~e).

[0040] 3. Akebia trifoliata phenylethanoid B inhibits the protein expression of hepatic stellate cell activation markers. Western blot was used to detect the expression levels of α-SMA and COL1A1 proteins in each group of cells. The results showed that TGF-β1 treatment significantly induced the upregulation of α-SMA and COL1A1 protein expression in LX-2 cells; while treatment with akebia phenylethanoid glycoside B resulted in a dose-dependent decrease in the expression levels of both α-SMA and COL1A1 proteins. Figure 1 f). The above results indicate that Akebia trifoliata phenylethanoid B can inhibit hepatic stellate cell activation at the protein level.

[0041] The above results indicate that Akebia trifoliata phenylethanoid B can significantly downregulate the expression of hepatic stellate cell activation-related markers, thereby effectively inhibiting the activation of LX-2 cells.

[0042] Example 2: Akebia quinata phenylethanoid glycoside B improves liver pathology and liver function in mice with CCl4-induced liver fibrosis: I. Materials and methods required for the experiment 1. Laboratory animals Forty 6-week-old male C57BL / 6J mice, weighing approximately 20–24 g, were housed in an SPF-grade enclosure at the animal testing center. The housing conditions were: temperature 25°C, humidity 70%, and automatic lighting with a controlled light cycle (8:00–20:00). All animal experiments were approved by the institution's laboratory animal ethics committee.

[0043] 2. Animal grouping and model establishment Mice were randomly divided into 5 groups: control group, CCl4 model group, low-dose group of akebia phenylethanoid B (10 mg / kg), medium-dose group of akebia phenylethanoid B (20 mg / kg), and high-dose group of akebia phenylethanoid B (40 mg / kg), with 8 mice in each group. Except for the control group, the mice in the other groups were intraperitoneally injected with 10% CCl4 (dissolved in olive oil, injection dose of 0.5 mL / 100 g body weight) three times a week for 4 consecutive weeks to establish a liver fibrosis model. The control group mice were injected with an equal volume of olive oil.

[0044] 3. Dosing regimen Starting from week 4, the treatment group received an intraperitoneal injection of the corresponding dose of Akebia quinata phenylethanol glycoside B daily, while the model group received an intraperitoneal injection of an equal volume of solvent daily, for a total of 4 weeks.

[0045] 4. Sample Collection After the experiment, all mice were euthanized, and serum and liver tissue were collected. Serum was used for liver function tests; a portion of the liver tissue was fixed with 4% paraformaldehyde for histopathological staining, while the remainder was stored at -80°C for hydroxyproline and protein content analysis.

[0046] 5. Histopathological staining After fixation, dehydration, and embedding, liver tissue was prepared into paraffin sections. Hematoxylin-eosin (H&E), Masson staining, and Sirius Red staining were performed to observe the pathological morphology and collagen deposition of the liver tissue.

[0047] 6. Hydroxyproline content detection An appropriate amount of liver tissue was taken, and the hydroxyproline content was determined using a hydroxyproline assay kit to assess the total collagen content of the liver tissue.

[0048] 7. Serum biochemical marker detection Serum was collected from each group of mice, and serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected using a kit.

[0049] II. Experimental Results 1. Akebia quinata phenylethanoid glycoside B improves CCl4-induced liver tissue pathological damage. H&E staining results showed that the liver tissue of the control group mice had normal structure, regular hepatocyte morphology, and no obvious lesions. The liver tissue of the CCl4 model group mice showed obvious hepatocyte degeneration and necrosis, inflammatory cell infiltration, and fibrous septum formation. Compared with the model group, the liver pathological damage in each treatment group of Akebia quinata phenylethanoid B was significantly reduced, and the degree of hepatocyte necrosis and inflammatory infiltration was significantly improved. Figure 2 a).

[0050] 2. Akebia quinata phenylethanoid glycoside B reduces collagen deposition in liver tissue. Masson staining and Sirius red staining results showed that a large number of blue or red collagen fibers were deposited in the liver tissue of mice in the CCl4 model group, and the collagen deposition area was significantly increased. The collagen deposition area was significantly reduced in all treatment groups of Akebia quinata phenylethanoid B in a dose-dependent manner. Figure 2 b).

[0051] 3. Akebia trifoliata phenylethanoid B reduces the content of hydroxyproline in the liver. The results of hydroxyproline content detection showed that the hydroxyproline content in the liver tissue of mice in the CCl4 model group was significantly higher than that in the control group (P<0.0001), indicating a significant increase in liver collagen content. The hydroxyproline content in all treatment groups of Akebia trifoliata phenylethanoid B was significantly lower than that in the model group, and the difference was statistically significant (P<0.0001). Figure 2 c).

[0052] 4. Akebia quinata phenylethanoid B improves liver function indicators. Serum ALT and AST levels were significantly higher in the CCl4 model group than in the control group (P<0.0001), indicating significant liver damage. Serum ALT and AST levels were significantly lower in all treatment groups of Akebia trifoliata phenylethanoid B compared to the model group (P<0.0001). Figure 2 d~e).

[0053] The above results indicate that Akebia quinata phenylethanoid B can significantly improve the pathological morphology of CCl4-induced mouse liver, reduce collagen deposition in liver tissue, and effectively reduce serum transaminase levels, suggesting that it has good anti-liver fibrosis and liver protection effects.

[0054] Example 3: Akebia trifoliata phenylethanoid glycoside B inhibits CCl4-induced activation of mouse hepatic stellate cells: I. Required Materials and Methods 1. Experimental Materials Liver tissues from each group of mice in Example 2 were used to detect the expression of hepatic stellate cell activation markers. The antibodies and RT-qPCR reagents used for Western blot were the same as those used in Example 1.

[0055] 2. Western blot detection Liver tissues from mice in each group were collected, and total protein was extracted using RIPA lysis buffer. Protein concentration was determined using the BCA method. Subsequent experimental procedures were the same as in Example 1.

[0056] 3. RT-qPCR detection Liver tissues were collected from mice in each group, and total RNA was extracted using an RNA kit. cDNA was then synthesized via reverse transcription. Subsequent experimental procedures were the same as in Example 1.

[0057] Table 2 Primers involved in RT-qPCR .

[0058] II. Experimental Results 1. Akebia trifoliata phenylethanoid B inhibits CCl4-induced expression of α-SMA and COL1A1 proteins in mouse liver tissue. Western blot was used to detect the expression levels of α-SMA and COL1A1 proteins in the liver tissues of mice in each group. The results showed that the expression levels of α-SMA and COL1A1 proteins were low in the liver tissues of the control group; the expression levels of these two proteins were significantly increased in the CCl4 model group, indicating activation of hepatic stellate cells. Compared with the model group, the expression levels of α-SMA and COL1A1 proteins in the liver tissues of mice in each treatment group of Akebia trifoliata phenylethanoid B (10 mg / kg, 20 mg / kg, 40 mg / kg) were significantly reduced, and showed a certain dose-dependent effect. Figure 3 a). The above results indicate that Akebia trifoliata phenylethanoid B inhibits hepatic stellate cell activation at the protein level.

[0059] 2. Akebia trifoliata phenylethanoid B downregulates the mRNA expression of activation markers in CCl4-induced mouse liver tissue. The relative mRNA expression levels of Acta2, Col1a1, and Timp1 in the liver tissues of mice in each group were detected by RT-qPCR. The results showed that, compared with the control group, the mRNA expression levels of Acta2, Col1a1, and Timp1 in the liver tissues of mice in the CCl4 model group were significantly upregulated (P<0.0001), confirming the successful establishment of the liver fibrosis model. Compared with the model group, the mRNA expression levels of the above three genes in the liver tissues of mice in each treatment group of Akebia quinata phenylethanoid B (10 mg / kg, 20 mg / kg, 40 mg / kg) were significantly downregulated (P<0.0001). Figure 3 b~d).

[0060] The above results demonstrate that Akebia trifoliata phenylethanoid B can significantly inhibit CCl4-induced activation of hepatic stellate cells in vivo, thereby alleviating liver fibrosis.

[0061] Example 4: Akebia trifoliata phenylethanoid glycoside B inhibits hepatic stellate cell activation via G3BP1: I. Materials and methods required for the experiment in Example 4 1. Cell Culture and Processing Human hepatic stellate cell line LX-2 was cultured in DMEM medium containing 10% fetal bovine serum and incubated at 37°C in a 5% CO2 incubator. Cells were divided into a control group, a TGF-β1 model group, a benzoyl peroxide phenylethanol glycoside B (CB) treatment group, an siRNA knockdown group, and an overexpression group, with specific treatment methods as follows.

[0062] 2. siRNA knockdown and overexpression experiments G3BP1 expression was knocked down using small interfering RNA (siRNA) technology, followed by transfection with either G3BP1-specific siRNA or negative control siRNA. Overexpression experiments were performed using G3BP1 overexpression plasmids or empty vector plasmids. After transfection, cells were treated with TGF-β1 and / or CB, and the expression levels of COL1A1 and α-SMA proteins were detected by Western blot.

[0063] 3. Molecular docking Molecular docking was performed using Autodock Vina software to predict potential binding sites between CB and G3BP1. The crystal structure of G3BP1 was obtained from the Protein Database (PDB). After removing water molecules and adding hydrogen atoms using software, a docking box was set to cover the NTF2-like domain of G3BP1. Docking results were analyzed using binding free energy (kcal / mol) and hydrogen bond interactions.

[0064] 4. Cell heat transfer assay (CETSA) LX-2 cells were collected and treated with either CB or a control solvent. After lysis, the cell lysate was aliquoted into PCR tubes and heated at different temperatures (46℃ to 53℃). Soluble proteins were then separated by centrifugation. The expression level of G3BP1 protein was detected by Western blot to analyze the effect of the drug on the thermostability of the target protein.

[0065] 5. Drug Affinity Response Target Stability (DARTS) Experiment LX-2 cells were collected, lysed, and the protein lysate was incubated with either CB or a control solvent. Different concentrations of streptomycin were added for limited enzymatic digestion. The degree of G3BP1 protein degradation was detected by Western blot to analyze the protective effect of the drug against the target protein's digestion.

[0066] 6. Surface Plasmon Resonance (SPR) Analysis The binding affinity between CB and G3BP1 was detected using surface plasmon resonance (SPR) technology. Full-length G3BP1 protein, the NTF2-like domain (NTF2L), and its truncated mutant were coupled to a sensor chip. Different concentrations of CB were injected as the mobile phase, binding and dissociation curves were recorded, and the kinetic dissociation constant (KD) was calculated.

[0067] II. Experimental Results 1. G3BP1 is a key target for the inhibition of hepatic stellate cell activation by akebia phenethyl glycoside B. To clarify the target of akebia phenylethanoid glycoside B (CB), G3BP1 expression was knocked down using siRNA. Western blot results showed that G3BP1 knockdown significantly inhibited TGF-β1-induced COL1A1 and α-SMA protein expression and completely eliminated the inhibitory effect of CB on hepatic stellate cell activation. Conversely, overexpression of G3BP1 exacerbated TGF-β1-induced hepatic stellate cell activation and significantly weakened the inhibitory effect of CB on COL1A1 and α-SMA. Figure 4 a). The above results indicate that G3BP1 is a key target for CB to inhibit hepatic stellate cell activation.

[0068] 2. Molecular docking prediction of the binding site between CB and G3BP1 Molecular docking was performed using Autodock Vina software to predict potential binding sites between CB and G3BP1. The results showed that the binding free energy between CB and G3BP1 was -7.0 kcal / mol, indicating a strong binding affinity. Conformational analysis revealed hydrogen bonds between CB and amino acid residues L36, G58, L59, and A32 of G3BP1, suggesting that CB may inhibit hepatic stellate cell activation by binding to the NTF2-like domain of G3BP1, interfering with protein-protein interactions or stress granule formation. Figure 4 b).

[0069] 3. CETSA experimental verification of the binding of CB and G3BP1 The binding effect of CB to G3BP1 was verified using a cell heat transfer assay (CETSA). The results showed that the expression level of G3BP1 gradually decreased with increasing temperature; compared with the control group, the expression level of G3BP1 in the CB-treated group was significantly increased at temperatures exceeding 49℃, indicating that CB can increase the thermostability of G3BP1. Figure 4 c). The above results support the existence of a direct interaction between CB and G3BP1.

[0070] 4. DARTS experiments verify the binding of CB to G3BP1. The binding interaction between CB and G3BP1 was further verified using Drug Affinity Response Target Stability (DARTS) technology. Results showed that under streptomycin treatment, the G3BP1 protein band in the control group gradually weakened with increasing protease concentration; while in the CB-treated group, G3BP1 protein degradation was significantly inhibited, and the protein band was significantly stronger than that in the control group. Figure 4 d). The above results indicate that CB can bind directly to the G3BP1 protein.

[0071] 5. SPR assay to detect the binding affinity of CB to G3BP1 The binding affinity between CB and G3BP1 was determined using surface plasmon resonance (SPR) technology. The results showed that the kinetic dissociation constant (KD) for the binding of CB to the full-length G3BP1 protein was [missing value]. KD The value is 23.25 × 10 −6 M, KD combined with the NTF2-like structural domain (NTF2L) of G3BP1 KD The value is 15.40 × 10 −6 M, while no significant binding was detected in the truncated mutant of this domain. KD KD The smaller the value, the higher the binding stability. Figure 4 e). The above results indicate that CB and the NTF2L domain of G3BP1 have a strong binding relationship.

[0072] Based on the above experimental results, and using multi-level experimental evidence such as thermal stability of protein-protein interactions (CETSA), drug affinity response target stability (DARTS), and surface plasmon resonance (SPR), G3BP1 is demonstrated to be a potential target for the inhibition of hepatic stellate cell activation by akebia phenylethanoid glycoside B.

[0073] Conclusion: As shown in the above examples, Akebia quinata phenylethanoid glycoside B can significantly inhibit the expression of hepatic stellate cell activation markers, exhibiting good inhibitory effects on hepatic stellate cell activation both in vitro and in vivo, and can significantly improve CCl4-induced liver fibrosis in mice. Therefore, Akebia quinata phenylethanoid glycoside B can be used in the preparation of drugs for treating liver fibrosis and has good application prospects. 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. Application of G3BP1 as a drug target in the preparation of drugs for the prevention and treatment of liver fibrosis.

2. The use of akebia phenylethanoid glycoside B or its pharmaceutically acceptable salt or ester in the preparation of drugs for the prevention or treatment of liver fibrosis, characterized in that, The structural formula of the akebia phenylethanol glycoside B is shown below: 。 3. The use of a pharmaceutical composition in the preparation of a drug for the prevention or treatment of liver fibrosis, characterized in that, The pharmaceutical composition comprises akebia phenylethanol glycoside B or a pharmaceutically acceptable salt or ester thereof, and pharmaceutically acceptable excipients.

4. The application according to claim 2 or 3, characterized in that, The mass percentage of the akebia phenylethanol glycoside B or its pharmaceutically acceptable salt or ester in the drug or pharmaceutical composition is 0.1% to 99.9%.

5. The application according to claim 2 or 3, characterized in that, The dosage forms of Akebia quinata phenylethanol glycoside B are oral and non-oral.

6. The application according to claim 5, characterized in that, The oral dosage forms are tablets, capsules, powders, granules, suspensions, or oral liquids; the non-oral dosage forms are injections, lyophilized powder injections, ointments, or gels.

7. The application according to claim 2 or 3, characterized in that, Akebia quinata phenylethanoid glycoside B exerts its anti-hepatic fibrosis effect by directly binding to the G3BP1 protein, thereby inhibiting the activation of hepatic stellate cells.

8. The application according to claim 7, characterized in that, Akebia quinata phenylethanoid B binds to the NTF2-like domain of G3BP1.

9. The application according to claim 3, characterized in that, The pharmaceutical composition also includes one or more other anti-hepatic fibrosis drugs selected from one or more of silymarin, colchicine, and polyene phosphatidylcholine.

10. The application according to claim 2 or 3, characterized in that, The liver fibrosis diseases include one or more of the following: viral hepatitis-associated liver fibrosis, alcoholic liver fibrosis, non-alcoholic fatty liver fibrosis, and autoimmune liver disease-associated liver fibrosis.

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