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

Abstract

The application provides an application of pentagalloyl glucose in preparation of a medicine for preventing and / or treating liver fibrosis, and belongs to the technical field of biological medicine. Through in-vitro cell experiments, organoid models and animal models, it is found that PGG can significantly inhibit the activation, migration, contraction and extracellular matrix (ECM) deposition of hepatic stellate cells (HSC), and the mechanism of action involves targeting the FAK-Paxillin adhesion plaque signal axis and intervening the core pathological link of liver fibrosis. As a natural polyphenol compound, PGG has the advantages of high safety and low side effects, and provides a new candidate drug for liver fibrosis treatment. The application also clearly defines the effective dose range and the administration scheme of PGG, and has a significant clinical conversion value.

Description

Technical Field

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

[0002] Hepatic fibrosis (HF) is an essential pathological process in the progression of chronic liver disease to cirrhosis and hepatocellular carcinoma. Essentially, it stems from an imbalance in the repair response triggered by chronic liver injury, leading to pathological tissue remodeling. Currently, there are no specific anti-fibrotic drugs available. Existing treatments can only slow disease progression in some patients and generally suffer from limited efficacy, significant side effects, and narrow indications. Epidemiological studies show that liver fibrosis is a crucial underlying cause of hepatocellular carcinoma, resulting in approximately one million deaths worldwide each year. Therefore, the development of safe and effective anti-fibrotic drugs is an urgent clinical need.

[0003] Pentagalloylglucose (PGG) is a hydrolyzable tannin compound widely found in various medicinal plants. Publicly available studies have confirmed its diverse pharmacological activities, including anti-inflammatory, antioxidant, antitumor, and hepatoprotective effects. Some studies have also reported its ability to regulate inflammation-related signaling pathways and alleviate renal fibrosis. However, the application value of PGG in the treatment of liver fibrosis has not yet been systematically elucidated. Existing research has not clarified its effective dosage, administration regimen, core targets, and molecular regulatory mechanisms in combating liver fibrosis, nor has it demonstrated its ability to reverse existing liver fibrosis. Therefore, it is insufficient to support the drug development and clinical translation of PGG in the field of anti-liver fibrosis. Summary of the Invention

[0004] The purpose of this invention is to provide an application of pentagalloglucopyranoside in the preparation of drugs for the prevention and / or treatment of liver fibrosis and cirrhosis, to clarify its core target, molecular mechanism, safe and effective dosage and regimen for anti-liver fibrosis, to fill the gap in the application of PGG in the treatment of liver fibrosis in the prior art, and to provide a novel, safe and efficient candidate drug for the clinical treatment of liver fibrosis.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of pentagalloglucopyranoyl glucose in the preparation of medicaments for the prevention and / or treatment of liver fibrosis.

[0006] Preferably, the liver fibrosis includes liver fibrosis caused by chronic liver injury, non-alcoholic steatohepatitis, alcoholic liver disease, or viral hepatitis.

[0007] Preferably, the drug uses pentagalloglucopyranoside as the sole active ingredient.

[0008] Preferably, the drug further contains one or more pharmaceutically acceptable carriers or excipients.

[0009] Preferably, the pharmaceutically acceptable carrier or excipient is selected from one or more of diluents, disintegrants, binders, lubricants, pH adjusters, osmotic pressure adjusters, solubilizers, antioxidants, antibacterial agents, and buffers.

[0010] Preferably, the dosage form of the drug is an oral preparation or an injection.

[0011] Beneficial effects

[0012] This invention systematically reveals for the first time the application value of pentagalloglucopyranoyl glucose (PGG) in anti-liver fibrosis, clarifying its target, molecular mechanism, safe and effective dosage and administration regimen. Compared with the prior art, it has the following significant beneficial effects: (1) This invention confirms that PGG can dose-dependently inhibit the pathological process of liver fibrosis and reduce the fibrotic burden of liver tissue at the in vitro cellular level, in human liver fibrosis organoid models, and in vivo animal models. It can not only delay the progression of liver fibrosis, but also has a clear potential to reverse existing liver fibrosis. PGG can selectively act on activated hepatic stellate cells (HSCs) and has extremely low toxicity to normal hepatocytes. Within a dose window with no obvious cytotoxicity, it can stably inhibit the proliferation, contraction, chemotactic migration ability of activated HSCs, downregulate the expression and secretion of pro-fibrotic markers such as α-SMA, type I collagen, and type III collagen, and block the abnormal deposition and remodeling of extracellular matrix (ECM).

[0013] (2) This invention is the first to clearly identify the core target of PGG in anti-liver fibrosis as focal adhesion kinase (FAK). By binding to the key pocket of the FAK kinase domain, it inhibits autophosphorylation at the FAK Tyr397 site, thereby weakening the phosphorylation of downstream Paxillin and the maturation of focal adhesion, blocking the mechanical signal transduction mediated by the "FAK-Paxillin" signaling axis, and ultimately inhibiting HSC activation and ECM deposition. This mechanism breaks through the indirect intervention mode of traditional drugs that only target upstream liver damage, directly targeting the core pathological link of fibrosis. At the same time, it can block the positive feedback loop of fibrosis maintenance of "ECM sclerosis-enhanced mechanical stimulation-maintainment of fibrotic phenotype-continued ECM accumulation", providing a new action strategy and target for the treatment of intermediate and advanced liver fibrosis.

[0014] (3) PGG is a natural polyphenol compound that is widely found in a variety of medicinal plants and has good biocompatibility. Compared with conventional antifibrotic drugs (such as single-target kinase inhibitors and chemically synthesized hepatoprotective drugs), PGG has no obvious liver and kidney toxicity, no off-target effects, and is less likely to cause drug resistance with long-term use. It can significantly broaden the safe use window and improve patients' compliance and tolerance to long-term medication.

[0015] (4) This invention clarifies the safe and effective working concentrations and dosing regimens of PGG at the cellular level (10 μM), in organoid models (30 μM), and in vivo in animals (10~50 mg / kg / day), providing sufficient experimental evidence for subsequent formulation development and clinical translation. PGG can be developed into various clinically applicable dosage forms, which can be used as a monotherapy for the entire course of liver fibrosis caused by different etiologies, or in combination with existing etiology-controlling drugs to further improve the effectiveness and safety of clinical treatment of liver fibrosis, and have extremely high clinical translational value and market application prospects. Attached Figure Description

[0016] Figure 1 This is a graph showing the inhibitory effect of PGG on normal hepatocytes and hepatic stellate cells in Example 1 and its activation state dependence. Figure 2 This is a graph showing the effect of PGG on the contractile and chemotactic migration phenotypes of activated LX-2 cells within a low-toxicity dose window in Example 1. Figure 3 The image shows the results of establishing the human liver fibrosis organoid model and identifying the expression of fibrosis-related biomarkers in Example 1. Figure 4 The graph shows the results of the inhibitory effect of PGG on the activity of human liver fibrosis organoids in Example 1. Figure 5 This is a diagram showing the results of PGG inhibiting TGF-β1-induced human liver organoid fibrosis and reducing YAP nuclear translocation in Example 1; Figure 6 This is a diagram showing the construction and histological evaluation results of the rat liver fibrosis model in Example 2; Figure 7 This is a diagram showing the results of screening gene modules that showed the strongest positive correlation with the liver fibrosis / cirrhosis phenotype in Example 2; Figure 8 This is a diagram showing the results of the core gene set for liver fibrosis in Example 2; Figure 9 and Figure 10 This is a diagram showing the validation results of the core gene set in Example 2; Figure 11 The graph shows the regulatory effect of PGG on the core transcriptional program of liver fibrosis in Example 2. Figure 12This is a graph showing the results of functional enrichment analysis of the core gene set in Example 2; Figure 13 This is a figure showing the effect of PGG on the expression of p-FAK(Y397) and Paxillin in activated LX-2 cells within a low-toxicity dose window in Example 3; Figure 14 This is a graph showing the effect of PGG on the expression of profibrotic molecules in activated LX-2 cells within a low-toxicity dose window in Example 3; Figure 15 The figure shows the molecular docking results of PGG and FAK in Example 3 and the comparison results with PF-573228. Detailed Implementation

[0017] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0018] Example 1

[0019] 1. Detection of PGG's sensitivity differences in cell type and activation state.

[0020] ① Cell culture and activation model construction: The normal human liver epithelial cell line THLE-2 and the human liver stellate cell line LX-2 were routinely cultured, and LX-2 cells were treated with 5 ng / mL TGF-β1 to establish an HSC activation model.

[0021] ② Drug intervention and cell viability assay: THLE-2, resting LX-2, and TGF-β1-activated LX-2 cells were treated with gradient concentrations of PGG for 48 h. Cell viability was assessed using the CCK-8 assay, dose-response curves were fitted, and the half-maximal inhibitory concentration (IC50) was calculated. 50 ).

[0022] ③ Proliferation capacity detection: The CCK8 assay was used to detect the effect of different concentrations of PGG (5μM, 10μM, 20μM) on the proliferation activity of activated LX-2 cells, and to clarify the concentration dependence of its proliferation inhibition.

[0023] The results showed that the IC50 values ​​of THLE-2, LX-2, and LX-2 activated by TGF-β1 were significantly higher than those of THLE-2 and LX-2. 50 The concentrations were 48.07 μmol / L (μM), 22.48 μM, and 13.47 μM, respectively. Figure 1 A) suggests that PGG is more sensitive to LX-2 cells than normal hepatocytes, and this sensitivity is further enhanced in the activated state. Furthermore, in activated LX-2 cells, the inhibition of proliferation activity by PGG shows a concentration-dependent increase, meaning that the higher the concentration, the stronger the inhibitory effect. Figure 1 B).

[0024] 2. Detection of the inhibitory effect of low-toxicity dose intra-window PGG on key phenotypes of activated HSC fibrosis

[0025] ① Cell model and experimental grouping: Human LX-2 cells were routinely cultured, and two HSC activation models were set up: TGF-β1 continuous stimulation co-treatment model: 5 ng / mL TGF-β1 and drug intervention were added simultaneously after cell adhesion for 48 h; ②TGF-β1 pre-activation therapy model: Cells were pretreated with 5 ng / mL TGF-β1 for 24 h to complete activation, followed by drug intervention for 48 h. The experiment included a blank control group, a TGF-β1 model group, a 10 μM PGG intervention group, and a positive control group for the FAK-specific inhibitor PF-573228, with three biological replicates for each group.

[0026] ③ Cell contraction ability detection: Collagen gel contraction experiment was used. After 24 h of cell intervention, the contraction area and contraction rate of collagen gel were quantitatively detected to evaluate the effect of PGG on the contraction ability of activated HSCs.

[0027] ④ Cell migration ability detection: Transwell chemotaxis assay was used. LX-2 cells were seeded in the upper chamber, and TGF-β1 chemotactic conditions were set in the lower chamber. After simultaneous drug intervention for 24 h in each group, the number of migrating cells was detected to evaluate the effect of PGG on the chemotactic migration ability of activated HSCs. The results are as follows: Figure 2 As shown (where A represents the results of the collagen gel shrinkage experiment, and B represents the results of the Transwell chemotactic migration experiment), P<0.05, P<0.01, P<0.001, P<0.0001).

[0028] Depend on Figure 2 It can be seen that, regardless of whether it is in the continuous stimulation mode or the pre-activation mode, TGF-β1 can significantly enhance the contraction and migration ability of LX-2, while PGG can significantly inhibit the above phenotype at low toxicity effective doses, and its inhibitory effect is comparable to that of the FAK-specific inhibitor PF-573228. Figure 2 The results suggest that PGG has a stable inhibitory effect on the pro-fibrotic effect of activated HSCs.

[0029] 3. Initial screening of PGG intervention conditions in human liver fibrosis organoid models

[0030] ① Construction of human liver fibrosis organoid model: After culturing human liver organoids and inducing maturation, they were treated with 5 ng / mL TGF-β1 for 72 h to establish a human liver fibrosis organoid model.

[0031] ② Model identification: The growth status of liver organoids in the control group and the fibrotic group was observed under a bright-field microscope. HE staining was used to detect the morphology of the liver fibrotic organoids. Masson staining was used to detect the fibrosis level of the human liver fibrotic organoids. Immunofluorescence staining was used to detect the expression of liver fibrosis markers such as α-SMA, Fibronectin, and COL1A1 in the human liver fibrotic organoids. Results are as follows: Figure 3 As shown (where A is a growth diagram of liver organoids, B is HE staining and Masson staining, and C~E are immunofluorescence single staining detection of the expression of α-SMA (C), Fibronectin (D) and COL1A1 (E) in human liver fibrosis organoids).

[0032] The results showed that the liver organoids in the control group were regular spherical or nearly spherical with clear and smooth edges; the liver organoids in the fibrosis group were shrunken, grape-like, and had decreased translucency. Figure 3 A). HE staining revealed cell nuclei (purple-blue) and cytoplasm (pink). Masson staining showed that in the control group, blue collagen fibers were sparsely distributed, with only a small amount of fine, light blue-green staining visible at the edges or in the background of some organoid structures; in the fibrosis-induced group, blue collagen fibers were significantly increased and thickened, with a large amount of blue collagen matrix filling the area, indicating significant fibrosis. Figure 3 B). Immunofluorescence results showed that the control group weakly expressed α-SMA, Fibronectin, and COL1A1, while the fibrosis-induced group showed significantly enhanced expression of α-SMA, Fibronectin, and COL1A1, exhibiting strong positivity. Figure 3 CE).

[0033] ③ Drug intervention and morphological observation: Liver fibrotic organoids were treated with gradient concentrations of PGG (0, 7.5, 15, 30, 60, 120 μM) for 48 h. The morphological changes of organoids before and after treatment with different concentrations of PGG were observed and recorded under a bright field microscope.

[0034] ④ Organoid activity detection and working concentration determination: The activity levels of organoids treated with different concentrations of PGG were detected using CellCounting-Lite® 3D luminescence immunoassay. Dose-response curves were plotted and IC50 was calculated. 50 Based on the detection results, the optimal working concentration for subsequent experiments was determined. Liver fibrosis organoids were treated with 0, 7.5, 15, 30, 60, and 120 μM PGG for 48 h. Morphological observation showed that with increasing concentration, the organoids exhibited structural fragmentation and increased debris. Figure 4 A). Organoid activity was assessed using CellCounting-Lite® 3D luminescence immunoassay. The results showed that organoid activity decreased in a dose-dependent manner with increasing PGG concentration. Based on this, the IC50 was calculated. 50 Approximately 100.2 μM ( Figure 4 B). Based on this initial screening result, subsequent validation of the mechanism and fibrosis indicators will use 30 μM as the working concentration (lower than IC50). 50 And it is within the operable effective concentration range.

[0035] ⑤ Effect of drug intervention on the fibrosis level of human liver organoids: A control group (normally cultured human liver organoids), a fibrosis induction group (human liver organoids treated with 5 ng / ml TGF-β for 72 h, followed by continued culture), and a fibrosis induction + PGG intervention group (human liver organoids treated with 5 ng / ml TGF-β for 72 h, followed by treatment with 30 μM PGG for 48 h) were set up. HE staining was used to detect the morphology of liver fibrosis organoids, Masson staining was used to detect the fibrosis level of human liver fibrosis organoids, immunofluorescence single staining was used to detect the expression of α-SMA, Fibronectin and COL1A1 in human liver fibrosis organoids, and immunofluorescence was used to detect the YAP nuclear localization of organoids.

[0036] Results showed that HE staining revealed cell nuclei (blue-purple) and cytoplasm (pink). Masson staining showed that, compared with the control group, the fibrosis group showed deeper blue staining, while PGG treatment resulted in lighter blue staining. Figure 5 A). Immunofluorescence results showed that the control group expressed low levels of α-SMA, Fibronectin, and COL1A1, while the fibrosis group showed increased expression of α-SMA, Fibronectin, and COL1A1. PGG treatment resulted in decreased expression of α-SMA, Fibronectin, and COL1A1. Figure 5 Immunofluorescence results showed that YAP was mainly expressed in the cytoplasm in the control group, while its expression was enhanced in the nucleus in the fibrosis group. PGG treatment reduced the nuclear localization of YAP. Figure 5 E).

[0037] Example 2

[0038] 1. Establishment, histological verification, and acquisition of differentially expressed genes from liver tissue transcriptome in rats with liver fibrosis / cirrhosis.

[0039] ① Animal modeling: 6-8 week old SPF-grade male SD rats, weighing 180-220g, were randomly divided into a control group and a modeling group. The modeling group was given a subcutaneous injection of 40% CCl4 / olive oil suspension in the back (twice a week for 8 consecutive weeks), while the control group was injected with an equal volume of olive oil. CCl4 stimulation was stopped at the end of the 8th week after histological confirmation of fibrosis. Samples were collected at the end of the experiment at the 14th week.

[0040] ② Histological verification: Rat liver tissue was subjected to HE staining and Masson staining to assess the degree of collagen deposition and fibrosis in the liver tissue, verifying the successful construction of the model.

[0041] ③ Transcriptome sequencing: Transcriptome sequencing was performed on liver fibrosis tissue from successfully modeled rats and normal liver tissue from the control group to screen for differentially expressed genes related to liver fibrosis.

[0042] The results are as follows Figure 6 As shown (where A and B are the gross morphology and HE staining images of liver tissue from normal control group rats and liver fibrosis tissue from model group rats, respectively).

[0043] 2. Open cohort data mining and screening of cross-species liver fibrosis core gene sets

[0044] ① Cohort data selection: The GSE14323 dataset with cirrhosis (late-stage fibrosis) as the main phenotype was selected from the GEO database, and transcriptome data from 19 normal liver samples and 41 cirrhosis samples were included.

[0045] ②WGCNA analysis: Weighted gene co-expression network analysis was performed on the dataset to construct gene co-expression modules and screen for gene modules that showed the strongest positive correlation with the liver fibrosis / cirrhosis phenotype. For example... Figure 7 As shown in Figures A and B, the Turquoise module was identified as having the strongest positive correlation with the liver fibrosis / cirrhosis phenotype (r=0.94, P=5×10). -28 This module contains 1684 genes.

[0046] ③ Construction of cross-species core gene set: The intersection of the above module genes and the upregulated differentially expressed genes in the transcriptome of the rat liver fibrosis model was obtained to obtain a core gene set of liver fibrosis with consistent expression trends in humans and rats. A total of 637 core genes with consistent changes in humans and rats were obtained. Figure 8 ).

[0047] 3. Multi-system reliability verification of the core gene set

[0048] ① Clinical sample validation: Seven human liver cirrhosis (advanced fibrosis) tissues and four normal liver tissues were collected. Total RNA was extracted and bulk RNA-seq sequencing was performed to verify the expression characteristics of the core gene set in human liver cirrhosis tissues.

[0049] ② Cell model validation: Human LX-2 cells were stimulated with 5 ng / mL TGF-β1 to establish an HSC activation model. Total RNA was extracted and transcriptome sequencing was performed to verify the expression characteristics of the core gene set in activated HSCs.

[0050] ③ Bioinformatics analysis verification: The ssGSEA and GSVA algorithms were used to calculate the liver fibrosis core gene set score of the samples. Combined with GSEA analysis, the enrichment trend of the core gene set in cirrhotic tissue and activated HSCs was verified to be related to the disease.

[0051] Based on the above human tissue bulk RNA-seq data, and combined with transcriptome data of LX-2 cells before and after TGF-β1 activation for validation, the results showed that this core gene set was stably elevated in cirrhotic tissue and under HSC activation state. Figure 9 and Figure 10 This supports its good disease relevance and cross-system consistency.

[0052] 4. Analysis of the regulatory effect of PGG on the core transcriptional program in liver fibrosis

[0053] Transcriptome data of TGF-β1-activated LX-2 cells after PGG intervention were obtained. The core gene set scores of liver fibrosis in the PGG-treated group and the control group were calculated using ssGSEA and GSVA algorithms. The differences between the groups were statistically analyzed using t-tests. GSEA was used to analyze the enrichment characteristics of the core gene set in the PGG-treated group, and the normalized enrichment score (NES) and false discovery rate (FDR) were calculated to clarify the regulatory effect of PGG on the core transcriptional program of liver fibrosis.

[0054] Based on the 637 core genes, programmed liver fibrosis / cirrhosis scores were calculated for different samples. In transcriptomic data from TGF-β1-activated LX-2 cells treated with PGG, both ssGSEA and GSVA algorithms showed that the liver fibrosis / cirrhosis scores in the PGG-treated group were significantly lower than those in the control group. Figure 11 A~B). GSEA analysis further showed that this core gene set was negatively enriched in the PGG treatment group (NES=-1.314, FDR < 0.05). Figure 11 (C) suggests that PGG can suppress core molecular features associated with liver fibrosis / cirrhosis at the transcriptional level.

[0055] 5. Functional enrichment analysis of the core gene set

[0056] The core gene set for liver fibrosis identified through screening was subjected to Gene Ontology (GO) functional enrichment analysis (covering biological processes (BP), cellular components (CC), and molecular functions (MF)) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to clarify the pro-fibrotic biological processes and key signaling pathways associated with the core gene set.

[0057] Functional enrichment analysis of the aforementioned core genes showed that this gene set is mainly associated with profibrotic biological processes such as ECM deposition and remodeling, and cell migration, and is significantly enriched in cell-matrix interface-related pathways such as adhesion plaques, ECM-receptor interactions, and cytoskeleton regulation. Figure 12 ).

[0058] In summary, this core gene set can stably characterize transcriptional programs related to late-stage fibrosis / cirrhosis, and PGG can significantly suppress these programs and show a negative enrichment trend in HSC cell models, suggesting that PGG has an inhibitory effect on molecular programs related to fibrosis progression (including late-stage fibrosis / cirrhosis). Its mechanism of action may be related to the regulation of cell-matrix interface (ECM-focal adhesion / skeleton) related pathways, providing a basis for subsequent mechanism verification focusing on key nodes.

[0059] Example 3

[0060] 1. Experimental Methods

[0061] (1) Western blot (WB) detection of FAK-Paxillin signaling axis protein expression

[0062] Cell Models and Grouping: Human LX-2 cells were routinely cultured, and two HSC activation models were established: ① TGF-β1 continuous stimulation model: After cell adhesion, 5 ng / mL TGF-β1 and the drug were added simultaneously for 48 h; ② TGF-β1 pre-activation model: Cells were first pretreated with 5 ng / mL TGF-β1 for 24 h to complete activation, and then the culture medium was changed and the drug was added for 48 h. The experiment included a blank control group, a TGF-β1 model group, a 10 μM PGG intervention group, and a 1 μM FAK-specific inhibitor PF-573228 positive control group, with three biological replicates in each group.

[0063] Western blot (WB) detection procedure: Total protein was extracted from cells in each group. After protein quantification using the BCA method, the cells were subjected to SDS-PAGE electrophoresis, wet transfer, and blocking with 5% skim milk. The cells were then incubated sequentially with p-FAK (Y397), total FAK, p-Paxillin (Tyr118), total Paxillin, and the internal control GAPDH primary antibody, as well as the corresponding HRP-labeled secondary antibody. The cells were then subjected to ECL chemiluminescence imaging to quantitatively analyze the relative expression level of the target protein.

[0064] The results showed that Western blotting indicated that PGG reduced p-FAK (Y397), accompanied by a decrease in total Paxillin and phosphorylation: in both TGF-β1 continuous stimulation and pre-activation models, 10 μM PGG treatment for 48 h showed a decreasing trend in p-FAK (Y397), while total FAK did not change significantly; simultaneously, p-Paxillin decreased, accompanied by a decrease in total Paxillin (…). Figure 13 The co-influence of the FAK kinase inhibitor PF-573228 suggests that PGG may inhibit FAK activation, reduce Paxillin-related plaque signal readout, and potentially affect Paxillin protein homeostasis.

[0065] 2. Real-time quantitative PCR (RT-qPCR) detection of mRNA expression of profibrosis-related genes

[0066] Cell treatment: The cell model, grouping and intervention conditions were the same as in 1 above. 10 μM PGG was used for intervention for 48 h. Each group was set up with 3 biological replicates and the experiment was independently repeated 3 times.

[0067] RT-qPCR detection procedure: Total RNA was extracted from cells in each group and reverse transcribed to synthesize cDNA; target gene-specific primers were designed and synthesized, and the detection targets included ECM component-related genes, myofibroblast-like activation markers, matrix crosslinking / remodeling-related factors, etc., with GAPDH as an internal reference gene; real-time quantitative PCR amplification was performed using the SYBR Green method. ^-ΔΔCt The relative mRNA expression levels of each gene were calculated using the method, and the differences between groups were statistically analyzed. A p-value < 0.05 was considered statistically significant.

[0068] The results showed that RT-qPCR indicated a stable downregulation of pro-fibrotic molecules, exhibiting activation-dependent selectivity. RT-qPCR results showed that under both continuous TGF-β1 stimulation and pre-activation modes, PGG (10 μM) treatment for 48 h consistently downregulated the output of typical pro-fibrotic molecules in activated HSCs, including ECM components and related genes (COL1A1 / COL1A2, COL3A1, COL6A3, FN1, VCAN), myofibroblast-like markers (ACTA2), and matrix crosslinking / remodeling-related factors (LOXL1, THBS1, SPARC, PDGFRB, ADAMTS2). Further analysis suggested that PGG exhibited activation-dependent selective regulation of some molecules: inhibition of MMP2, TIMP1, and CCN2 was not significant, while downregulation of MMP9, COL4A1, COL4A2, SNAI1, and FAP only occurred under TGF-β pre-activation conditions. Figure 14 Pre-TGF-β1: LX-2 cells were pre-activated with TGF-β1 for 24 h; TGF-β1: LX-2 cells were continuously stimulated with TGF-β1; PGG: pentagalloglucopyranoside; PF-573228: positive control, a specific inhibitor of FAK. P<0.05, P<0.01, P<0.001, (P<0.0001), indicating that PGG selectively suppresses the maintenance / remodeling process after activation, rather than simply downregulating it broadly.

[0069] 3. Molecular docking analysis of the binding characteristics of PGG to the FAK kinase domain.

[0070] Structural pretreatment: High-resolution crystal structures of human FAK kinase domains were obtained from the RCSB PDB database, and chemical structures of PGG and PF-573228 were obtained from the PubChem database.

[0071] Docking and Analysis: A full-protein multi-pocket search was performed on the pretreated FAK protein. A docking grid covering all candidate pockets was set, and the molecular docking was completed using the Lamarck genetic algorithm. The conformation with the lowest binding free energy was taken as the optimal docking result. The correlation between the binding pockets and binding orientations of PGG and PF-573228 was compared and analyzed. The docking results were visualized in three dimensions using PyMOL software.

[0072] The results showed that molecular docking suggested that PGG might act on candidate pockets of the FAK kinase domain, providing structural evidence for competition and occupancy verification, and structural clues for subsequent interaction and site association verification. After searching multiple pockets of the FAK protein, we scored the docking conformations of PGG and the FAK inhibitor PF-573228 in each candidate pocket. The results showed that the optimal docking results of both ligands pointed to the same candidate pocket, and the binding orientation within that pocket had a certain similarity. Figure 15 (where A represents the molecular docking result between PGG and FAK, and B represents the molecular docking result between PF-573228 and FAK), suggesting that PGG may have overlap, proximity, or allosteric association with the target region of PF-573228. Given that PF-573228 is an ATP-competitive FAK kinase inhibitor, this result structurally supports the possibility that PGG may act on the FAK kinase domain and affect its catalytic function, providing verifiable structural evidence for subsequent pharmacological competition experiments and functional verification of key residues.

[0073] 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. The use of pentagalloglucopyranoside in the preparation of medicaments for the prevention and / or treatment of liver fibrosis.

2. The application according to claim 1, characterized in that, The liver fibrosis includes liver fibrosis and cirrhosis caused by chronic liver injury, non-alcoholic steatohepatitis, alcoholic liver disease, or viral hepatitis.

3. The application according to claim 2, characterized in that, The drug uses pengaloyl glucose as its sole active ingredient.

4. The application according to claim 3, characterized in that, The drug also contains one or more pharmaceutically acceptable carriers or excipients.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable carrier or excipient is selected from one or more of the following: diluents, disintegrants, binders, lubricants, pH adjusters, osmotic pressure adjusters, solubilizers, antioxidants, antibacterial agents, and buffers.

6. The application according to claim 5, characterized in that, The drug is available in oral or injectable form.

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