Use of protosappanin A in the preparation of a medicament for treating liver fibrosis

Abstract

The application discloses application of protosappanin A in preparation of a medicine for treating liver fibrosis, relates to the technical field of biological medicine, and is used for reducing liver fibrosis by targeting CHEK1-mediated proliferation of hepatic stellate cells; the protosappanin A is used for reducing inflammation infiltration, necrosis and collagen deposition of the liver in the liver fibrosis; the medicine comprises protosappanin A and pharmaceutically acceptable adjuvants; the medicine is used for inhibiting activation and / or proliferation of hepatic stellate cells; the medicine is used for reducing liver fibrosis by down-regulating expression levels of CHEK1; a medicine composition for treating liver fibrosis comprises an effective amount of protosappanin A and pharmaceutically acceptable adjuvants; the application discloses a new use of protosappanin A (PrA) in preparation of an anti-liver fibrosis medicine; in-vivo and in-vitro experiments prove that PrA can significantly reduce serum transaminase (ALT, AST) levels, and reduce inflammation infiltration, necrosis and collagen deposition of the liver.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of protohematoxylin A in the preparation of drugs for the treatment of liver fibrosis. Background Technology

[0002] Liver fibrosis, characterized by abnormal activation and proliferation of hepatic stellate cells (HSCs) and excessive extracellular matrix (ECM) deposition, is a common pathological basis of chronic liver diseases. Currently, effective anti-fibrotic drugs are lacking in clinical practice. Protosappanin A (PrA) is a biphenyloxacyclooctone active ingredient extracted from the traditional Chinese medicine Caesalpinia sappan L. In recent years, natural products have become a hot topic in anti-fibrotic drug development due to their multi-target and low toxicity characteristics. Cell cycle regulation plays a crucial role in the activation and proliferation of hepatic stellate cells (HSCs), but specific targets have not been fully explored. Checkpoint kinase 1 (CHEK1), as a core regulatory factor, has a largely unknown function in fibrosis. Therefore, elucidating whether PrA alleviates liver fibrosis by targeting CHEK1 to inhibit HSC proliferation has significant theoretical value and translational potential; an application of protosappanin A in the preparation of drugs for the treatment of liver fibrosis is needed. Summary of the Invention

[0003] In view of this, the present invention addresses the deficiencies of the existing technology, and its main objective is to provide an application of protohematoxylin A in the preparation of drugs for the treatment of liver fibrosis. It reveals a new use of protohematoxylin A (PrA) in the preparation of anti-liver fibrosis drugs. In vitro and in vivo experiments have confirmed that PrA can significantly reduce serum transaminase (ALT, AST) levels and alleviate liver inflammatory infiltration, necrosis and collagen deposition.

[0004] To achieve the above objectives, the present invention adopts the following technical solution: The application of protohematoxylin A in the preparation of drugs for the treatment of liver fibrosis.

[0005] As a preferred option: the protohematoxylin A is used to target CHEK1-mediated hepatic stellate cell proliferation to alleviate liver fibrosis.

[0006] As a preferred option, the proto-hematoxylin A is used to reduce inflammatory infiltration, necrosis and collagen deposition in the liver during liver fibrosis.

[0007] As a preferred embodiment, the drug comprises protohematoxylin A and pharmaceutically acceptable excipients; the drug is used to inhibit the activation and / or proliferation of hepatic stellate cells.

[0008] As a preferred option, the drug is used to downregulate the expression level of CHEK1 to alleviate liver fibrosis.

[0009] As a preferred embodiment, the drug is used to reduce the expression of liver fibrosis markers COL1A1 and / or α-SMA.

[0010] A pharmaceutical composition for treating liver fibrosis comprises an effective amount of protohematoxylin A or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0011] As a preferred embodiment, the dosage form of the pharmaceutical composition is selected from oral preparations or injections.

[0012] Compared with existing technologies, this invention has significant advantages and beneficial effects. Specifically, as shown in the above technical solution, this invention reveals a novel application of protohematoxylin A (PrA) in the preparation of anti-hepatic fibrosis drugs. In vitro and in vivo experiments have confirmed that PrA can significantly reduce serum transaminase (ALT, AST) levels, alleviate liver inflammation infiltration, necrosis, and collagen deposition, and effectively reverse CCl4-induced liver fibrosis in mice. This invention, through the integration of network pharmacology, quantitative proteomics, and molecular dynamics simulations, verifies that CHEK1 is a key direct target of PrA in anti-fibrosis, providing a structural basis for precise drug design. This invention elucidates that PrA downregulates CHEK1 expression and inhibits hepatic stellate cell proliferation, confirming that CHEK1 overexpression can reverse the anti-fibrotic effect of PrA, and establishing the PrA-CHEK1-proliferation axis as a novel regulatory node for fibrosis drug treatment. PrA is derived from traditional Chinese medicine, has good safety profile, mature preparation technology, and significant clinical translational potential.

[0013] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating how PrA reduces liver fibrosis levels in CCl4 mice according to the present invention. Figure 2 This is a schematic diagram illustrating how PrA of the present invention can inhibit the activation of hepatic stellate cells at in vitro and in vivo levels; Figure 3 This is a schematic diagram illustrating the integrated analysis and identification of four key targets of PrA using network pharmacology and proteomics in this invention. Figure 4 This is a schematic diagram illustrating the integration of molecular docking and molecular dynamics simulations in this invention to identify CHEK1 as the optimal target for PrA. Figure 5 This is a schematic diagram illustrating how CHEK1 of the present invention exacerbates liver fibrosis by promoting the proliferation of hepatic stellate cells; Figure 6 This is a schematic diagram illustrating how PrA of the present invention can inhibit the proliferation of hepatic stellate cells both in vivo and in vitro. Figure 7 This is a schematic diagram illustrating how PrA of the present invention inhibits the proliferation of hepatic stellate cells by downregulating CHEK1; Figure 8 This is a schematic diagram illustrating how the PrA treatment of the present invention reduces hepatic stellate cell proliferation, alleviates liver fibrosis, and reduces inflammation in the fibrotic liver by inhibiting CHEK1 expression. Figure 9 This is a schematic diagram showing the changes in mRNA expression of fibrosis and inflammation markers after in vitro and in vivo PrA treatment according to the present invention. Figure 10 This is a schematic diagram showing the changes in mRNA expression of proliferation-related markers in HSCs after knocking down CHEK1 according to the present invention. Figure 11 This is a schematic diagram showing the changes in mRNA expression of proliferation-related markers in mice after PrA treatment according to the present invention. Figure 12 This is a schematic diagram showing the changes in mRNA expression of proliferation-related markers in HSCs after PrA treatment according to the present invention; Figure 13 This is a schematic diagram showing the changes in mRNA expression of proliferation markers in HSCs after CHEK1 function recovery according to the present invention.

[0015] Explanation of reference numerals in the attached diagram: Figure 1 In the study: (A) H&E staining, Sirius red, and Masson staining (scale bar = 200 μM); (B) Quantitative analysis of Sirius red positive area; (C) Quantitative analysis of Masson tricolor positive area; (D) ALT level after PrA treatment (n=6); (E) AST level after PrA treatment (n=6). Data are expressed as mean ± SEM (n=6). # P<0.05 vs. Oil group * P<0.05 vs. CCl4 group; Figure 2 (A) Immunohistochemical staining of COL1A1 and α-SMA in liver tissue (scale bar = 200 μM) (B) Quantitative analysis of COL1A1 positive area (C) Quantitative analysis of α-SMA positive area (D) Western blot analysis of α-SMA and COL1A1 protein expression in liver tissue (E) Quantitative analysis of α-SMA protein level (F) Quantitative analysis of COL1A1 protein level (G) Western blot detection of α-SMA and COL1A1 protein expression in LX-2 cells (H) Quantitative analysis of COL1A1 protein level (I) Quantitative analysis of α-SMA protein level. Data are expressed as mean ± SEM. # P < 0.05 vs. Oil group vs. Control group * P < 0.05 vs. CCl4 group vs. TGF-β group; Figure 3 (A) Chemical structure of PrA (B) Intersection analysis of PrA targets and liver fibrosis-related genes (C) Venn diagram showing intersection targets (D) KEGG pathway enrichment analysis of intersection targets (E) GO enrichment analysis of intersection targets (F) Volcano plot showing differentially expressed proteins (DEPs) in the liver before and after PrA treatment (G) Heatmap showing the top 25 upregulated and downregulated DEPs (H) GO enrichment analysis of DEPs (I) KEGG pathway enrichment analysis of DEPs (J) Venn diagram showing the intersection of proteomics-derived DEPs and network pharmacology-derived targets (K) Heatmap showing the expression levels of CHEK1, CDK1, APP and PLA2G7; Figure 4 In the middle: Molecular docking affinity of PrA with (A)APP, (B)CHEK1, (C)CDK1 and (D)PLA2G7; (E) Root mean square deviation (RMSD) analysis results; (F) Root mean square fluctuation (RMSF) analysis results; (G) Hydrogen bond analysis results; (H) Radius of gyration (Rg) analysis results; Figure 5 (A) Immunohistochemical analysis of CHEK1 expression in liver tissue (scale bar = 20 μm). (B) Immunofluorescence colocalization of CHEK1 and α-SMA, a marker of hepatic stellate cell activation (scale bar = 100 μm). (C) Western blot analysis of proliferative and activation markers after CHEK1 knockdown. (D) Western blot analysis of proliferative and activation markers after CHEK1 overexpression. (E) Quantitative analysis of protein levels in (C). (F) Quantitative analysis of protein levels in (D). Data are expressed as mean ± standard error. #P<0.05 vs. Oil or Control group; *P<0.05 vs. CCl4 or TGF-β group; Figure 6 (A) Immunohistochemical staining of Ki67, Cyclin D1, and PCNA in liver tissue (scale bar = 200 μm). (B) Quantitative analysis of Ki67-positive cells. (C) Quantitative analysis of Cyclin D1 expression. (D) Quantitative analysis of PCNA expression. (E) Immunofluorescence colocalization of PCNA and α-SMA (scale bar = 150 μm). (F) EdU assay for hepatic stellate cell proliferation (scale bar = 200 μm). (G) Quantitative analysis of EdU-positive cells. Data are expressed as mean ± standard error. #P<0.05 vs. Oil or Control group; *P<0.05 vs. CCl4 or TGF-β group; Figure 7(A) Western blot analysis of CHEK1, fibrosis-related markers, and proliferation markers in liver tissue after PrA treatment. (B) Quantitative analysis of protein levels in (A). (C) Western blot analysis of CHEK1, fibrosis-related markers, and proliferation markers in LX-2 cells after PrA treatment. (D) Quantitative analysis of protein levels in (C). (E) Western blot analysis of proliferation and activation markers in PrA-treated cells after CHEK1 overexpression. (F) Quantitative analysis of protein levels in (E). Data are expressed as mean ± standard error. #P<0.05 vs. Oil or Control group; *P<0.05 vs. CCl4 or TGF-β group.

[0016] Figure 9 (A) Expression of Col1a1 mRNA in mouse liver. (B) Expression of Acta2 mRNA in mouse liver. (C) Screening of PrA concentration in vitro. (D) Expression of COL1A1 mRNA in LX-2 cells. (E) Expression of ACTA2 mRNA in LX-2 cells. (F) Expression of Tnf-α mRNA in mouse liver. (G) Expression of Il-6 mRNA in mouse liver. (H) Expression of Il-1β mRNA in mouse liver. Data are expressed as mean ± standard error (SEM). #P < 0.05 compared with Oil or Control. *P < 0.05 compared with CCl4 or TGF-β group; Figure 10 In the study: (A) Expression of CHEK1 mRNA in LX-2 cells. (B) Expression of COL1A1 mRNA in LX-2 cells. (C) Expression of ACTA2 mRNA in LX-2 cells. (D) Expression of CCND1 mRNA in LX-2 cells. (E) Expression of PCNA mRNA in LX-2 cells. Data are expressed as mean ± standard error (SEM). #P < 0.05 compared to Control. *P < 0.05 compared to TGF-β group; Figure 11 (A) Expression of Chek1 mRNA in mouse liver. (B) Expression of Ccnd1 mRNA in mouse liver. (C) Expression of Acta2 mRNA in mouse liver. (D) Expression of Col1a1 mRNA in mouse liver. (E) Expression of Pcna mRNA in mouse liver. Data are expressed as mean ± standard error (SEM). #P<0.05 compared with Oil; *P<0.05 compared with CCl4 group; Figure 12(A) Expression of COL1A1 mRNA in LX-2 cells. (B) Expression of ACTA2 mRNA in LX-2 cells. (C) Expression of PCNA mRNA in LX-2 cells. (D) Expression of CCND1 mRNA in LX-2 cells. (E) Expression of CHEK1 mRNA in LX-2 cells. Data are expressed as mean ± standard error (SEM). #P<0.05 compared with Control; *P<0.05 compared with TGF-β group; Figure 13 (A) Expression of COL1A1 mRNA in LX-2 cells. (B) Expression of ACTA2 mRNA in LX-2 cells. (C) Expression of CHEK1 mRNA in LX-2 cells. (D) Expression of CCND1 mRNA in LX-2 cells. (E) Expression of PCNA mRNA in LX-2 cells. Data are expressed as mean ± standard error (SEM). #P<0.05 compared with Control; *P<0.05 compared with TGF-β group. Detailed Implementation

[0017] The present invention is as follows Figure 1 As shown in Figure 13, a protohematoxylin A is used in the preparation of a drug for the treatment of liver fibrosis.

[0018] This proto-hematoxylin A is used to target CHEK1-mediated hepatic stellate cell proliferation and alleviate liver fibrosis.

[0019] Proto-hematoxylin A is used to reduce inflammatory infiltration, necrosis, and collagen deposition in the liver during liver fibrosis.

[0020] The drug comprises protohematoxylin A and pharmaceutically acceptable excipients; the drug is used to inhibit the activation and / or proliferation of hepatic stellate cells.

[0021] The structural formula of the original hematoxylin A is: ; This drug is used to downregulate CHEK1 expression levels to alleviate liver fibrosis.

[0022] This drug is used to reduce the expression of liver fibrosis markers COL1A1 and / or α-SMA.

[0023] A pharmaceutical composition for treating liver fibrosis comprises an effective amount of protohematoxylin A or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0024] The dosage form of the pharmaceutical composition is selected from oral preparations or injections.

[0025] Example: Application of protohematoxylin A in the preparation of a drug for treating liver fibrosis Materials and Methods: Drugs and reagents: Protosappanin A (PrA), product number: JOT-10793, batch number: 24050608, provided by Chengdu Pufeide Biotechnology Co., Ltd., with a purity of 99.28% as determined by HPLC. Silymarin (SiL), product number: HY-N7073, batch number: 65666-07-1, provided by MedChemExpress (MCE). Anti-CHEK1 antibody (2360s), anti-COL1A1 antibody (72026S), anti-PCNA antibody (13110), and anti-Ki67 antibody (9027) were all purchased from Cell Signaling Technology (USA). Anti-α-SMA antibody (ab124964), anti-GAPDH antibody (ab181602), anti-CyclinD1 (ab134175), and anti-COL1A1 (ab254113) were all purchased from Abcam (USA). High-performance tissue / cell RIPA lysis buffer, PMSF, Masson's three-color kit, and Sirius red kit were all purchased from Solarbio (China). Phosphatase inhibitor mixture and Tris-EDTA antigen retrieval solution were purchased from Proteintech (China). CCl4 (purity >99%) was purchased from Maclean's (China). BCA protein quantification kit and Protein Standards were purchased from Thermo Fisher (USA). Goat anti-rabbit IgG H&L / HRP and goat anti-mouse IgG H&L / HRP were purchased from Bioss (China). Hematoxylin, eosin, PBS buffer powder, environmentally friendly dewaxing solution, blocking goat serum, and DAB colorimetric kit were all purchased from Beijing Zhongshan Jinqiao Co., Ltd. (China).

[0026] Experimental animals and treatment methods: Thirty-six 8-week-old male C57BL / 6J mice, weighing 20-22g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Certificate No.: SCXK2021-0006). All animals were placed under standard conditions for 7 days of acclimatization before the experiment. All animals were handled according to the NIH Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 80-23, 1978 Revision). All animal procedures were approved according to the "Xinjiang Medical University Animal Experiment Ethics Form" (Approval No.: K202505-43). Animal grouping: Mice were randomly divided into six groups: Oil + solvent group, CCl4 group, CCl4 + silymarin group (100mg / kg), and CCl4 + PrA group (doses of 5, 10, and 15mg / kg, respectively), with six mice in each group. CCl4 model mice were intraperitoneally injected with 2.5 ml / kg of CCl4 diluted in 20% corn oil, three times a week for six consecutive weeks. Mice in the Oil group received an equal volume of corn oil. Four weeks after CCl4 injection, mice were injected with PrA and SiL dissolved in a solvent (physiological saline containing 5% DMSO, 5% Tween 80, and 40% PEG300). After treatment, serum samples were collected to measure liver function. Mice were then sacrificed, and liver samples were preserved at -80°C or in 4% paraformaldehyde for further analysis.

[0027] Cell culture: Human hepatic stellate cell line (LX-2) was purchased from the Shanghai Cell Bank (EP-CL-0560), China. Cells were cultured in LX-2-specific culture medium (Boster) at 37°C in incubators with 5% CO2 and 20% O2. Four groups were established: normal group, TGF-β group, and TGF-β+PrA (25 μM, 50 μM). The TGF-β stimulation group was stimulated with 10 ng / ml TGF-β1 for 48 h, while the TGF-β+PrA group was simultaneously induced with 10 ng / ml TGF-β1 and the corresponding concentration of PrA and cultured for 48 h.

[0028] Drug concentration screening: Using a long-term cell assay instrument (Incucyte CX3 Sartorius, China), 1×10⁶ cells were analyzed. 4 LX-2 cells were seeded in 96-well plates. When the cell confluence reached 60%–70%, 10–100 μM PrA and 10 ng / ml TGF-β1 were added respectively. Each group was repeated three times. Cells were observed for 48 h under a long-term cell monitoring instrument, and cell viability was calculated.

[0029] CHEK1 gene expression regulation: For in vitro knockdown experiments, small interfering RNA (siRNA) specifically targeting CHEK1 was purchased from GenePharma (Shanghai, China). LX-2 cells were cultured at 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 g / cm³ in 6-well plates and cultured until cell confluence reached 60%–70%. Transfection was performed according to the manufacturer's instructions using siRNA transfection reagent (siRNA-mate plus, G04026, GenePharma, China). The siRNA-lipid complex was added to the cells, and after incubation at 37°C and 5% CO₂ for 48 hours, cells were collected for qRT-PCR and Western blot analysis to assess knockdown efficiency.

[0030] For in vitro overexpression, lentiviral particles targeting CHEK1 were purchased from GenePharma (Shanghai, China). LX-2 cells were cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 g / well in 6-well plates and cultured until cell confluence reached 40%–50%. The medium was then replaced with complete medium containing 5 μg / mL polybrene and a lentiviral suspension with a multiplicity of infection (MOI) of 50. Twenty-four hours after infection, the medium was replaced with complete medium containing 2 μg / mL puromycin for 7 days of stability selection to establish a CHEK1 overexpressing cell line. Overexpression efficiency was confirmed by qRT-PCR and Western blot analysis.

[0031] Bioinformatics Analysis: The PrA structure file was obtained from the Pubchem database. Swisstargetprediction and Pharmmapper databases were used to predict and identify potential targets for PrA binding. Genes closely related to liver fibrosis were obtained from OMIM and Gene Cards for cross-analysis. The intersection of intervention targets and liver fibrosis disease targets was used to obtain core targets. A protein-protein interaction (PPI) network was constructed using the STRING database. GO functional enrichment analysis and KEGG pathway enrichment analysis were performed on the intersection targets using the Metascape database. P < 0.05 and FDR < 0.01 were used as screening criteria to obtain core biological processes and key signaling pathways. Key targets, major pathways, and intervention factors were integrated, and a visual regulatory network was constructed using Cytoscape. Hub genes were screened using degree values, intermediateness centrality, and proximity centrality.

[0032] Histopathological analysis: Liver tissue was rapidly isolated from euthanized mice, rinsed with pre-cooled PBS, and fixed in 4% paraformaldehyde solution for 48 h (Yang et al., 2026). After gradient dehydration and paraffin embedding, the tissue was serially sectioned at a thickness of 4 μm and baked at 60°C for 1 h before use. Routine histopathological evaluation was performed using hematoxylin-eosin (H&E) staining, while Sirius red and Masson's trichrome staining were used to assess the degree of fibrosis. For immunohistochemical staining, antigen retrieval was performed using EDTA or sodium citrate, followed by blocking with goat serum, incubation overnight with grade 4 primary antibody (COL1A1, Cyclin D1, PCNA, CHEK1, α-SMA, Ki67), followed by warming and incubation with secondary antibody for 2 h. DAB was used for development, and hematoxylin was used for counterstaining. The slides were then photographed using an automated slide scanner. Positive areas were analyzed using Image-Pro Plus 6.0 (Media Cybernetics) software.

[0033] Immunofluorescence analysis: After dewaxing and rehydration of paraffin sections, antigen retrieval was performed using EDTA, followed by staining according to the TSA three-label four-color kit instructions (G1226, Solarbio, China). Primary antibody was incubated overnight at 4°C, and secondary antibody was incubated at room temperature for 2 hours. Cell nuclei were stained using DAPI, and images were taken using a fluorescence inverted microscope (DMi8, Leica, Germany).

[0034] Western blot: Mouse liver tissue or LX-2 cells were lysed with RIPA lysis buffer and homogenized using a tissue homogenizer (KZ-06, Solarbio, China). Protein quantification was then performed using a BCA protein quantification kit (23227, Thermo Fisher Scientific, USA). 20 μg of protein was added to each sample, separated by 7.5%–10% SDS-PAGE gel, and transferred to a 0.45 μm PVDF membrane (Millipore, USA). The PVDF membrane was blocked with 5% skim milk powder and incubated overnight at 4°C with primary antibodies (COL1A1, CyclinD1, PCNA, CHEK1, α-SMA). The following day, after washing with TBST, the membrane was incubated with secondary antibodies at room temperature for 2 hours. Visualization analysis was performed using an ECL kit (Proteintech) and an e-BLOT contact chemiluminescence imaging system. Gray-scale quantification was performed using ImageJ software (1.54p) with GAPDH as an internal control.

[0035] Proteomics analysis: Liver tissue samples (n=5) from mouse Oil, CCl4, and CCl4+PrA groups (50 mg each) were ground into powder at low temperature, followed by lysis, precipitation, and trypsin digestion. Independent acquisition (DIA) mass spectrometry analysis was performed using the Orbitrap Astral platform (Novogene Biotech, Beijing, China). Raw data were processed by comparing with the UniProt database using DIA-NN software. P Differentially expressed proteins (DEPs) were identified using thresholds of < 0.05 and |log2FC| > 0.585. Functional annotation and pathway enrichment analyses were performed using GO and KEGG, and protein-protein interaction networks were predicted using the STRING database.

[0036] Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR): RNA was extracted using the Trizol method and reverse transcribed into cDNA using a reverse transcription kit (R323-01, Vazyme). Subsequently, 2 μL of cDNA was mixed with 18 μL of Master Mix (1129280, Vazyme, Nanjing, China) and qRT-PCR was performed on a real-time quantitative PCR instrument (4376592, StepOnePlus) using the SYBR Green I dye method (1129280, Vazyme). The housekeeping gene GAPDH was used as an internal control, and the relative gene expression level was calculated using the 2^(−ΔΔCt) method.

[0037] Statistical analysis: Graphpad 10.0 software was used to analyze the data. Statistical results for continuous data are expressed as mean ± standard deviation (Mean ± SEM). Before performing parametric analysis, the Shapiro-Wilk test was used to verify whether the data conformed to a normal distribution, and the Levene test was used to assess homogeneity of variance. Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. A p-value <0.05 was defined as statistically significant.

[0038] Discussion of Results: PrA can reduce CCl4-induced liver damage: To investigate the therapeutic effect of PrA on liver injury and inflammation in mice, a CCl4 mouse model of liver fibrosis was used. Results showed that PrA treatment significantly improved inflammatory infiltration and necrosis in the liver of mice, and significantly reduced collagen deposition in the liver. Figure 1 A- Figure 1 C), after mice were injected with CCl4, liver function ALT and AST levels increased significantly, and after PrA treatment, ALT and AST levels decreased significantly. Figure 1 D- Figure 1E). The above results indicate that PrA reduces CCl4-induced liver damage in mice.

[0039] Immunohistochemical analysis showed that CCl4 treatment increased the expression of Col1a1 and α-SMA in the liver of mice, while PrA treatment significantly reduced their expression. Figure 2 A- Figure 2 C). Consistent with these observations, CCl4 treatment significantly upregulated the protein and mRNA levels of Col1a1 and α-SMA in mouse liver tissue, and both were reduced by PrA in a dose-dependent manner. Figure 2 D- Figure 2 F and Figure 9 A, Figure 9 B).

[0040] Based on the cell viability assay results, drug concentrations (25 μM and 50 μM) that resulted in cell viability exceeding 50% were selected for in vitro experiments. Figure 9 C). In vitro experiments using human hepatic stellate cells LX-2 further demonstrated that PrA (25 μM and 50 μM) inhibited TGF-β1-induced upregulation of α-SMA and COL1A1 at both protein and mRNA levels in a dose-dependent manner (Figure 2G-Figure 2I). Figure 9 D、 Figure 9 E).

[0041] Furthermore, PrA treatment effectively inhibited the expression of inflammation-related genes TNF-α, IL-6, and IL-1β in mouse liver. Figure 9 F- Figure 9 In summary, these results indicate that PrA can alleviate CCl4-induced liver injury both in vitro and in vivo by inhibiting the activation of HSCs and suppressing the inflammatory response.

[0042] Integrated bioinformatics screening reveals CHEK1 as a key target in PrA anti-liver fibrosis therapy. To investigate the specific mechanism of PrA's anti-fibrotic effect, network pharmacology was used to screen the targets of PrA. Figure 3 A). First, 106 structural prediction targets of PrA were obtained using the Pharmmapper and Swisstargetprediction databases. Then, cross-analysis was performed between these prediction targets and liver fibrosis-related genes in the public disease database (genecards), resulting in 92 targets associated with liver fibrosis. Figure 3 B). Subsequently, protein-protein interaction (PPI) network analysis of these 92 targets identified core genes such as Egfr and Esr1, which may mediate signal regulation after PrA treatment. Figure 3C). Functional annotation of these 92 target genes was performed using GO and KEGG enrichment analyses. The results showed significant enrichment in pathways such as cell growth, serine / threonine kinase activity, and epithelial cell proliferation, indicating that PrA is involved in the regulation of cell proliferation during fibrosis. Figure 3 D、 Figure 3 E).

[0043] To further validate the bioinformatics predictions, proteomic analysis was performed on liver tissues of mice treated with CCl4 and with or without PrA treatment. Differential expression analysis (|log2 FC| ≥ 0.58, p < 0.05) showed that, compared with the control group, 77 proteins were upregulated and 550 proteins were downregulated in the PrA-treated group (Figure 3F, Figure 3G). GO and KEGG enrichment analyses of differentially expressed proteins (DEPs) indicated that the downregulated proteins were enriched in biological processes such as DNA replication initiation, phagocytosis, and chemotaxis, further supporting the hypothesis that PrA may inhibit fibrosis by suppressing cell proliferation and regulating the local immune microenvironment. Figure 3 H, Figure 3 I). By performing intersection analysis of DEPs with 92 PrA-related targets, four candidate targets were identified: CHEK1, CDK1, APP, and PLA2G7. Figure 3 J). All four targets were significantly upregulated in fibrotic liver and significantly downregulated after PrA treatment. Figure 3 K).

[0044] To assess the binding potential of PrA to the four targets mentioned above, virtual docking of proteins and molecules was performed one by one using AutoDock Vina. The results showed that the binding energy calculated by APP was -8.8 kcal / mol. Figure 4 A) The calculated binding energy of CHEK1 is -9.1 kcal / mol ( Figure 4 B) The calculated binding energy of CDK1 is -8.8 kcal / mol ( Figure 4 C) The calculated binding energy of PLA2G7 is -8.3 kcal / mol ( Figure 4 D), among which CHEK1 binds most stably to PrA. To further evaluate the target-drug binding stability, molecular dynamics simulations (total 100 ns) were performed. The results showed that CHEK1 was the optimal effective target among the four targets, with the root mean square deviation (RMSD) converging to 0.25 nm after 20 ns. Figure 4 E) indicates that the ligand can bind to the protein and maintain a relatively stable state; the radius of gyration (Rg) is stable at 1.98-2.02 nm, indicating the formation of a tight and stable complex. Figure 4H), Root mean square fluctuation (RMSF) showed that the flexibility of the binding pocket residues (80-140) was significantly lower than that of the flexible region, indicating that the structure of the complex formed by the drug and the target was more stable. Figure 4 F). The hydrogen bond diagram shows that the number of hydrogen bonds between proteins and small molecules is stable at 6, with a maximum of 8, forming a stable binding conformation (F). Figure 4 H).

[0045] In summary, among the four candidate targets, CHEK1 and PrA exhibit superior binding stability, which is reflected in the convergent RMSD, stable hydrogen bonds, compact Rg, and reduced flexibility within the binding pocket.

[0046] CHEK1 induces liver fibrosis by promoting the proliferation of hepatic stellate cells. CHEK1 is a serine / threonine kinase and a core regulator in the DNA damage response process. Its role is to regulate cell cycle checkpoints and maintain genomic stability, playing a crucial role in S phase, G2 / M transition, and M phase (Pang et al., 2025). To clarify the role of CHEK1 in fibrotic liver, immunohistochemistry and qRT-PCR were used to assess the changes in CHEK1 expression in fibrotic liver before and after PrA treatment. The results showed that CHEK1 was significantly upregulated in fibrotic liver, while its expression was extremely low in normal liver. Figure 5 A). The mRNA level of CHEK1 also showed an increasing trend in CCl4-induced fibrosis in mice. Figure 11 C). Furthermore, immunofluorescence colocalization analysis showed that CHEK1 colocalized with the hepatic stellate cell marker α-SMA, and the expression trend was consistent with that of α-SMA. Figure 5 B).

[0047] To further investigate the effect of CHEK1 on HSC proliferation, knockdown and overexpression experiments of CHEK1 were performed in LX-2 cells. Compared with the TGF-β treatment group, CHEK1 knockdown using siRNA significantly reduced the protein and mRNA levels of COL1A1 and α-SMA. CHEK1 knockdown also inhibited the expression of proliferation-related markers Cyclin D1 and PCNA. Figure 5 C Figure 5 E and Figure 10 Conversely, lentiviral-mediated CHEK1 overexpression increased the protein levels of COL1A1 and α-SMA, as well as Cyclin D1 and PCNA. Figure 5 D、 Figure 5 F). This indicates that CHEK1 promotes liver fibrosis by enhancing the proliferation of HSCs.

[0048] PrA alleviates liver fibrosis by inhibiting CHEK1-mediated hepatic stellate cell proliferation. To directly assess the effect of PrA on cell proliferation, the proliferation level in the livers of mice treated with PrA was examined. Immunohistochemical analysis showed that PrA treatment significantly reduced the expression of Cyclin D1, PCNA, and Ki67 in CCl4-induced fibrotic liver, indicating that PrA inhibits cell proliferation during liver fibrosis. Figure 6 A- Figure 6 D). Immunofluorescence co-localization analysis further confirmed that PCNA and α-SMA co-localized, indicating that PrA can alleviate CCl4-induced HSC proliferation (D). Figure 6 E). Notably, PCNA staining showed that PrA also inhibited the proliferation of hepatocytes in the liver of CCl4-treated mice, which may reflect a reduction in the severity of liver injury, thereby leading to a decrease in the liver's need for repair. To further verify the inhibitory effect of PrA on HSC proliferation, an in vitro EdU uptake experiment was performed, and the results also showed that PrA significantly inhibited TGF-β-induced HSC proliferation (E). Figure 6 F).

[0049] To determine whether CHEK1 mediates the inhibitory effect of PrA on HSC proliferation, the expression of CHEK1 after PrA treatment was first detected by Western blot and qRT-PCR. In a CCl4-induced mouse model, PrA administration significantly inhibited the expression of CHEK1 and related proliferation markers (Figures 7A, 7B, and 7C). Figure 11 Similarly, in LX-2 cells, PrA inhibited TGF-β-induced upregulation of CHEK1 and proliferation-related markers at both protein and mRNA levels in a dose-dependent manner. Figure 7 C Figure 7 D and Figure 12 The rescue experiment further confirmed that CHEK1 overexpression could reverse the inhibitory effect of PrA. Compared with the PrA-treated group, the protein and mRNA levels of CHEK1, COL1A1, α-SMA, CyclinD1, and PCNA were significantly increased in the PrA + CHEK1-OE group. Figure 7 E, Figure 7 F and Figure 13 In summary, this indicates that PrA alleviates liver fibrosis by reducing CHEK1 expression, thereby inhibiting HSC proliferation.

[0050] This application establishes a carbon tetrachloride (CCl4)-induced mouse liver fibrosis model to evaluate the in vivo therapeutic effect of PrA. The human hepatic stellate cell line LX-2 was stimulated with TGF-β1 to assess the in vitro effect of PrA on HSC activation. Network pharmacology and quantitative proteomics techniques were integrated to identify potential targets of PrA. Molecular docking and molecular dynamics simulations were used to verify the binding affinity and stability between PrA and candidate targets. CHEK1 expression in LX-2 cells was regulated using siRNA knockdown and lentiviral overexpression to explore its functional role in HSC proliferation. Histopathological staining (H&E, Sirius red, and Masson's trichrome staining), immunohistochemistry, immunofluorescence, Western blot, and qRT-PCR were used to evaluate liver fibrosis and the therapeutic effect of the drug.

[0051] PrA treatment significantly improved CCl4-induced liver inflammation, necrosis, and collagen deposition in mice, accompanied by a decrease in serum ALT and AST levels. PrA inhibited the expression of fibrosis markers (COL1A1 and α-SMA) and proliferation markers (CyclinD1, PCNA, and Ki67) in a dose-dependent manner both in vitro and in vivo. Comprehensive bioinformatics screening identified checkpoint kinase 1 (CHEK1) as a key target of PrA, exhibiting the strongest binding affinity (-9.1 kcal / mol) and best binding stability among four candidate targets. CHEK1 is aberrantly upregulated in fibrotic livers and co-localizes with α-SMA in activated hepatic stellate cells (HSCs). Knockdown of CHEK1 inhibited HSC proliferation and fibrosis, while overexpression of CHEK1 exacerbated these pathological processes. Mechanistically, PrA inhibits HSC proliferation by downregulating CHEK1 expression, thereby alleviating liver fibrosis. The rescue experiment confirmed that CHEK1 overexpression reverses the antifibrotic effect of PrA.

[0052] This application confirms that PrA is an antifibrotic drug that alleviates liver fibrosis by targeting and inhibiting CHEK1-mediated activation and proliferation of HSCs. It establishes the PrA-CHEK1-proliferation axis as a novel regulatory node in fibrosis formation and identifies CHEK1 as a promising therapeutic target for fibrotic diseases, highlighting the translational potential of PrA in antifibrotic drug development.

[0053] This application demonstrates that PrA, a natural polyphenolic compound isolated from *Sappanwood*, exhibits potent anti-fibrotic activity in a CCl4-induced mouse model of liver fibrosis. Through integrated network pharmacology, quantitative proteomics, and molecular dynamics simulations, CHEK1 was identified as the direct target of PrA, and it was elucidated that PrA alleviates liver fibrosis by inhibiting CHEK1-mediated proliferation of hepatic stellate cells (HSCs). Mechanistically, PrA stably binds to CHEK1, downregulating its expression, thereby inhibiting cell cycle progression in activated hepatic stellate cells and thus improving the fibrotic process. Figure 8 This study not only revealed the previously unknown role of CHEK1 in the pathogenesis of liver fibrosis, but also made PrA a promising lead compound in the development of antifibrotic drugs.

[0054] Activation and abnormal proliferation of hepatic stellate cells (HSCs) are core pathogenic drivers of liver fibrosis progression. Under the stimulation of inflammatory mediators and mechanical stress, resting HSCs transdifferentiate into myofibroblasts, characterized by excessive extracellular matrix (ECM) synthesis and dysregulation of proliferation, thereby continuously promoting the fibrotic process. TGF-β is the most potent endogenous stimulatory factor activating and promoting HSC proliferation, regulating ECM generation and deposition. In this application, we demonstrate that PrA can inhibit TGF-β1-induced HSC activation in a dose-dependent manner both in vitro and in vivo. Notably, carbon tetrachloride (CCl4)-induced fibrotic liver exhibits strong hepatic stellate cell activation and proliferation, as evidenced by significant upregulation of α-SMA, COL1A1, and proliferation markers including Cyclin D1, PCNA, and Ki67. PrA treatment effectively inhibits hepatic stellate cell activation and proliferation, establishing this dual blocking effect as the core mechanism of PrA's anti-fibrotic effect. This simultaneous inhibition of phenotypic transformation and proliferation distinguishes PrA from traditional antifibrotic drugs that primarily target a single pathological process.

[0055] This study demonstrated that PrA significantly ameliorated carbon tetrachloride-induced liver injury in a mouse fibrosis model, reduced inflammatory infiltration and collagen deposition, and inhibited hepatic stellate cell (HSC) activation in a dose-dependent manner. This integrative multi-omics study revealed that CHEK1 is a key mediator of PrA's anti-fibrotic activity. Rescue experiments confirmed that CHEK1 overexpression eliminated the inhibitory effect of PrA on HSC proliferation and fibrosis, thus establishing the PrA-CHEK1 axis as a specific regulatory node targeting HSC phenotypic transformation and dysregulated proliferation in the pro-fibrotic network.

[0056] This study is the first to demonstrate that CHEK1 is abnormally upregulated in fibrotic liver tissue and activated hepatic stellate cells, consistent with its established pro-tumor activity. The results of this study indicate that CHEK1 overexpression directly drives the proliferation of pathological HSCs and mediates the anti-fibrotic efficacy of PrA.

[0057] In summary, this application demonstrates that the natural polyphenol compound PrA is a potent anti-fibrotic drug that alleviates carbon tetrachloride (CCl4)-induced liver fibrosis by targeting and inhibiting CHEK1-mediated activation and proliferation of hepatic stellate cells (HSCs). Through integrated network pharmacology, quantitative proteomics, molecular dynamics simulations, and functional recovery experiments, the PrA-CHEK1-proliferation axis was established as a previously unrecognized regulatory node in the fibrosis process. This establishes CHEK1 as a novel therapeutic target for fibrosis and highlights the translational potential of PrA as a precision medicine candidate.

[0058] The key design focus of this invention is as follows: This invention reveals a novel application of protohematoxylin A (PrA) in the preparation of anti-hepatic fibrosis drugs. In vitro and in vivo experiments have confirmed that PrA can significantly reduce serum transaminase (ALT, AST) levels, alleviate liver inflammation, necrosis, and collagen deposition, and effectively reverse CCl4-induced liver fibrosis in mice. This invention integrates network pharmacology, quantitative proteomics, and molecular dynamics simulations to verify that CHEK1 is a key direct target of PrA in anti-fibrosis, providing a structural basis for precise drug design. This invention elucidates that PrA downregulates CHEK1 expression and inhibits hepatic stellate cell proliferation, confirming that CHEK1 overexpression can reverse the anti-fibrotic effect of PrA, and establishing the PrA-CHEK1-proliferation axis as a novel regulatory node for fibrosis drug treatment. PrA is derived from traditional Chinese medicine, has good safety profiles, mature preparation processes, and significant clinical translational potential.

[0059] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. The use of protohematoxylin A in the preparation of a drug for treating liver fibrosis.

2. The application according to claim 1, characterized in that: The proto-hematoxylin A is used to target CHEK1-mediated hepatic stellate cell proliferation and alleviate liver fibrosis.

3. The application according to claim 1, characterized in that: The proto-hematoxylin A is used to reduce inflammatory infiltration, necrosis, and collagen deposition in the liver during liver fibrosis.

4. The application according to claim 1, characterized in that: The drug comprises protohematoxylin A and pharmaceutically acceptable excipients; the drug is used to inhibit the activation and / or proliferation of hepatic stellate cells.

5. The application according to claim 1, characterized in that: The drug is used to downregulate CHEK1 expression levels to alleviate liver fibrosis.

6. The application according to claim 1, characterized in that: The drug is used to reduce the expression of liver fibrosis markers COL1A1 and / or α-SMA.

7. A pharmaceutical composition for treating liver fibrosis, characterized in that: The application comprises an effective amount of protohematoxylin A or a pharmaceutically acceptable salt thereof, as well as one or more pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterized in that: The dosage form of the pharmaceutical composition is selected from oral preparations or injections.

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