Application of agrimonolide in preparation of medicine for treating cholestatic liver disease

Abstract

The invention discloses an application of agrimonolide in preparation of a medicine for treating cholestatic liver diseases. It is found for the first time that the agrimonolide promotes bile acid efflux by up-regulating expression and / or functions of bile acid efflux transporter, so that small bile duct hyperplasia is inhibited, hepatic fibrosis is reduced, macrophage infiltration is relieved, a unique liver protection effect is generated, and a new medication strategy is brought to treatment of cholestatic liver diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of agrimony lactone in the preparation of drugs for treating cholestatic liver disease. Background Technology

[0002] Cholestatic liver disease encompasses a diverse range of hepatobiliary disorders characterized by impaired hepatocyte transport and bile acid secretion. This damage leads to the accumulation of bile acids, bilirubin, and cholesterol. Currently, the preferred and most established medical treatment is the natural bile acid ursodeoxycholic acid (UDCA), which primarily improves bile production in hepatocytes and bile duct cells through a complex post-transcriptional mechanism. However, the limited efficacy of UDCA in various cholestatic liver diseases, such as primary sclerosing cholangitis, underscores the need to develop novel treatments. Recent research has focused on novel transcriptional regulators of bile production, including nuclear hormone receptors such as FXR and their agonist obeticholic acid (OCA). In a recent study, patients with primary biliary cholangitis (PBC) who did not respond well to UDCA treatment were treated with doses of 10, 25, or 50 mg of OCA. While continuing UDCA treatment, OCA reduced alkaline phosphatase levels by 20% in 70% of patients. However, 50% of PBC patients in this study experienced itching, a significant side effect of OCA that has been mentioned in several trials.

[0003] Agrimonia lactone (AM) is an isocoumarin found in the herb Agrimonia pilosa. According to existing literature, Agrimonia lactone (AM) possesses a wide range of pharmacological activities, including hepatoprotective, anti-inflammatory, antioxidant, and cholesterol metabolism-regulating effects. The herb Agrimonia pilosa has been used as a traditional medicine since ancient times to treat syndromes such as hepatobiliary diseases and inflammatory disorders. However, there are currently no reports on the use of Agrimonia lactone for the treatment of cholestatic liver disease. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of the prior art by providing the application of agrimony lactone in the preparation of a medicament for treating cholestatic liver disease.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The first aspect is the use of agrimonolactone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of cholestatic liver disease, which promotes bile acid efflux by upregulating the expression and / or function of bile acid efflux transporters, thereby inhibiting small bile duct proliferation, reducing liver fibrosis and alleviating macrophage infiltration.

[0007] Furthermore, when treated with the bile acid efflux and transport inhibitor BMS-986020, the therapeutic effect of the agrimonolactone on cholestatic liver disease was significantly weakened or reversed.

[0008] Furthermore, the dosage of the agrimony lactone is 10 mg / kg to 90 mg / kg.

[0009] Furthermore, the cholestatic liver disease was induced by the DDC diet.

[0010] The second aspect is to provide a pharmaceutical composition for treating cholestatic liver disease, comprising a therapeutically effective amount of agrimonolactone or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0011] Furthermore, the pharmaceutical composition promotes bile acid efflux by upregulating the expression and / or function of bile acid efflux transport proteins, thereby inhibiting small bile duct proliferation, reducing liver fibrosis, and alleviating macrophage infiltration.

[0012] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0013] This invention is the first to discover that agrimony lactone promotes bile acid efflux by upregulating the expression and / or function of bile acid efflux transport proteins, thereby inhibiting small bile duct proliferation, reducing liver fibrosis, and alleviating macrophage infiltration, producing a unique hepatoprotective effect and bringing a new medication strategy to the treatment of cholestatic liver disease. Attached Figure Description

[0014] Figure 1 The flowchart shows the mouse cholestatic liver disease model and the treatment with agrimony lactone (AM).

[0015] Figure 2 The study showed the changes in body weight on day 28 after treatment with three doses of agrimony lactone (AM) in a cholestatic liver injury model. Statistical tests were performed using one-way ANOVA and Tukey's multiple comparison test. *P<0.032, **P<0.0021, ***P<0.0002, ****P<0.0001 (the same below).

[0016] Figure 3 Histograms showing the percentage of liver weight to body weight on day 28 in a cholestatic liver injury model treated with three doses of agrimony lactone (AM).

[0017] Figure 4 Histograms of serum biochemical parameters, including ALT, AST, and ALP levels, on day 28 of a cholestatic liver disease model treated with three doses of agrimony lactone (AM).

[0018] Figure 5 Histograms of serum total bilirubin and direct bilirubin levels on day 28 of a cholestatic liver disease model treated with three doses of agrimony lactone (AM).

[0019] Figure 6 The liver histopathology results, including HE and Masson staining, show the characteristics of cholestatic liver injury, including ductal proliferation, inflammatory cell infiltration, intraductal embolism, pericholangitis, onion-skin-like periductal fibrosis, and biliary fibrosis.

[0020] Figure 7 The volcano plot shows the transcriptomic changes induced by agrimonolactone (AM) treatment on day 28 in a cholestatic liver disease model, including 197 upregulated genes and 254 downregulated genes.

[0021] Figure 8 GO analysis revealed the biological processes (BP) / cellular components (CC) / molecular functions (MF) involved in the significant alterations in genes induced by agrimonolactone (AM) treatment on day 28 in a cholestatic liver disease model.

[0022] Figure 9 GSEA analysis revealed significant alterations in extracellular matrix tissue, collagen-containing extracellular matrix, and extracellular matrix degradation caused by agrimonolactone (AM) treatment on day 28 in a cholestatic liver disease model.

[0023] Figure 10 The liver hydroxyproline levels on day 28 of a cholestatic liver disease model treated with three doses of agrimony lactone (AM) are shown.

[0024] Figure 11 The expression levels of liver fibrosis markers α-Sma, Col1a1, Timp1, Epc, and Mmp2 were shown on day 28 of a cholestatic liver disease model treated with three doses of agrimony lactone (AM).

[0025] Figure 12 Typical liver histopathological results of α-SMA expression on day 28 in a cholestatic liver disease model treated with three doses of agrimony lactone (AM).

[0026] Figure 13 GSEA analysis revealed significant alterations in genes involved in day 28 of agrimonol (AM) treatment in a cholestatic liver disease model, including reprogramming of cytokine pathways, myeloid cell development, and monocyte pathways.

[0027] Figure 14 Histograms showing the liver gene expression levels of cytokines Tnf-α, Il-6, and Il-1β on day 28 of a cholestatic liver disease model treated with three doses of agrimony lactone (AM).

[0028] Figure 15Histograms of liver Tgfb1 gene expression levels on day 28 in a cholestatic liver disease model treated with three doses of agrimony lactone (AM).

[0029] Figure 16 The results of liver immunohistochemistry of CD45 and CD11b in a cholestatic liver disease model treated with agrimony lactone (AM) are shown.

[0030] Figure 17 The results of liver immunohistochemistry of CK19 in a cholestatic liver disease model treated with agrimony lactone (AM) are shown.

[0031] Figure 18 Serum cholesterol levels, serum BA levels, and liver BA levels were shown on day 28 of treatment with three doses of agrimony lactone (AM) in a cholestatic liver disease model.

[0032] Figure 19 The expression of genes involved in the classical BA synthesis pathway, including Cyp7a1 and Cyp8b1, in the liver on day 28 after induction of cholestatic liver injury and treatment with three doses of agrimony lactone (AM) was shown.

[0033] Figure 20 The expression of genes involved in the classical BA synthesis pathway, including Cyp27a1 and Cyp7b1, in the liver on day 28 after induction of cholestatic liver injury and treatment with three doses of agrimony lactone (AM) was shown.

[0034] Figure 21 The levels of 15 bile acids in mouse serum and liver samples were shown on day 28 after treatment with three doses of agrimony lactone (AM).

[0035] Figure 22 The liver gene expression of BA to bile efflux, including Bsep and Mrp2, was shown on day 28 in a cholestatic liver injury model treated with three doses of agrimony lactone (AM).

[0036] Figure 23 The liver gene expression of BA entering the bloodstream in a cholestatic liver injury model on day 28 after treatment with three doses of agrimony lactone (AM) was shown, including Oatp2, Mrp3 and Mrp4; *P<0.032.

[0037] Figure 24 The study showed the expression of genes related to BA uptake in hepatocytes, including Ntcp and Oatp1, on day 28 of a cholestatic liver injury model and treatment with three doses of agrimony lactone (AM).

[0038] Figure 25The expression of genes involved in the first stage of BA metabolism (including Cyp3a11 and Cyp2b10) in the liver was shown in a cholestatic liver injury model and on day 28 after treatment with three doses of agrimony lactone (AM).

[0039] Figure 26 The liver gene expression of genes involved in the second phase of BA metabolism (including Ugt1a1 and Sult2a1) in a cholestatic liver injury model and on day 28 of treatment with three doses of agrimony lactone (AM) was shown.

[0040] Figure 27 The percentage of liver weight to body weight on day 28 after treatment with BMS-986020 and agrimony lactone (AM) in a cholestatic liver injury model is shown.

[0041] Figure 28 Serum biochemical results of ALT, AST and ALP levels on day 28 after treatment with BMS-986020 and agrimony lactone (AM) in a cholestatic liver injury model are shown.

[0042] Figure 29 Serum and liver BA levels were shown on day 28 after treatment with BMS-986020 and agrimony lactone (AM) in a cholestatic liver injury model.

[0043] Figure 30 Serum and liver BA levels were shown on day 28 after treatment with BMS-986020 and agrimony lactone (AM) in a cholestatic liver injury model.

[0044] Figure 31 This study showed liver gene expression associated with bile effusion from BA, including Bsep and Mrp2, on day 28 after treatment with BMS-986020 and agrimony lactone (AM) in a cholestatic liver injury model.

[0045] Figure 32 The study showed liver gene expression, including Oatp2, Mrp3, and Mrp4, on day 28 after treatment with BMS-986020 and agrimony lactone (AM) in a cholestatic liver injury model.

[0046] Figure 33 The expression of liver genes, including Ntcp and Oatp1, associated with hepatocyte uptake of BA was shown on day 28 after treatment with BMS-986020 and agrimony lactone (AM) in a cholestatic liver injury model.

[0047] Figure 34 HE and Masson staining results of liver tissue pathology on day 28 after treatment with BMS-986020 and agrimony lactone (AM) in a cholestatic liver injury model.

[0048] Figure 35 The liver hydroxyproline level on day 28 after treatment with BMS-986020 and agrimony lactone (AM) in a cholestatic liver injury model is shown.

[0049] Figure 36 The gene expression levels of α-Sma, Col1a1, Timp1, and Epc agrimonolactone (AM) were shown on day 28 after treatment with BMS-986020 and agrimonolactone (AM) in a cholestatic liver injury model.

[0050] Figure 37 This shows the Tgfb1 gene expression level on day 28 after treatment with BMS-986020 and agrimony lactone (AM) in a cholestatic liver injury model.

[0051] Figure 38 Immunohistochemical results of CK19 and CD45 in the liver on day 28 after treatment with BMS-986020 and agrimony lactone (AM) in a cholestatic liver injury model.

[0052] Figure 39 The percentages of myeloid cells and lymphocytes in immune cells of a cholestatic liver injury model after treatment with BMS-986020 and agrimony lactone (AM) are shown.

[0053] Figure 40 The percentages of CD4+ T cells and CD8+ T cells in lymphocytes in the BMS-986020 and Agrimonia pilosa lactone (AM) treatment groups were shown in a cholestatic liver injury model.

[0054] Figure 41 Typical pseudocolor images of Kupffer cells (CD11b Middle, F4 / 80hgih) and monocyte-derived macrophages (MDMs, CD11b High, F4 / 80middle) from immune cells.

[0055] Figure 42 The percentages of Kupffer cells and MDM cells in bone marrow cells of mice treated with BMS-986020 and agrimony lactone (AM) in a cholestatic liver injury model are shown. Detailed Implementation

[0056] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0057] This invention provides the use of agrimonolactone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cholestatic liver disease. The medicament promotes bile acid efflux by upregulating the expression and / or function of bile acid efflux transport proteins, thereby inhibiting small bile duct proliferation, reducing liver fibrosis, and alleviating macrophage infiltration. When treated with the bile acid efflux and transport inhibitor BMS-986020, the therapeutic effect of the agrimonolactone on cholestatic liver disease is significantly weakened or reversed. This was verified through the following specific examples.

[0058] The materials used in the following embodiments are as follows:

[0059] (1) Chemical reagents and kits

[0060] Agrimonia lactone (AM) was purchased from Selleck (Shanghai, China). Primary antibodies used for immunohistochemistry included CD11b (ab133357, Abc Agrimonia lactone (AM)), CD45 (ab10558, Abc Agrimonia lactone (AM)), F4 / 80 (ab6640, Abc Agrimonia lactone (AM)), α-SMA (GB111364), CD11b (GB11058), and CK19 (GB11197) (Servicebio, Wuhan, Hubei, China). Immunofluorescent antibodies used for flow cytometry analysis were obtained from BioLegend (San Jose, California, USA) and included PerCP / Cyanine5.5 anti-mouse CD45 (30-F11, Cat#:103132), PE / Cyanine7 anti-mouse / human CD11b (M1 / 70, Cat#:101216), APC anti-mouse F4 / 80 (BM8, Cat#:123116), PE anti-mouse CD3 (17A2, Cat#:100206), Brilliant Violet 510 anti-mouse CD8b (53-6.7, Cat#:100751), and PerCP / Cyanine5.5 anti-mouse CD45 recombinant (QA17A26, Cat#:157612) antibodies. DDC(137030) reagents and BA standards were purchased from Sigma-Aldrich (Supplementary Table S1). All other chemical reagents were from commercial suppliers.

[0061] (2) Animals

[0062] Specific pathogen-free (SPF) male C57BL / 6J mice were purchased from Shanghai Xipu Bekalin Laboratory Animal Co., Ltd. (Shanghai, China). All animals were housed at the Animal Center of Shanghai Sixth People's Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, under a standard 12-hour light-dark cycle. Animals had free access to food and water and were housed until 8 weeks of age (20–25 g). Mice were randomly assigned to groups of 5 per cage and allowed a one-week acclimatization period. A cholestatic liver injury model was established by feeding a diet containing 0.08% DDC for four weeks. The DDC-containing diet (cereal-based rodent diet, 4.5% fat, 0.08% DDC) and the control diet (cereal-based rodent diet, 4.5% fat) were provided by Shanghai Sixth People's Hospital. All experiments were conducted in a blinded manner to minimize bias in treatment and sample collection. The experimental protocol was approved by Shanghai Sixth People's Hospital in accordance with the Ethical Guidelines for the Care and Use of Laboratory Animals (Approval No.: DWSY2020-179).

[0063] The specific methods used in the embodiments of the present invention are as follows:

[0064] 1) Agrimonia lactone (AM) treatment and sample collection

[0065] To evaluate the dose-dependent hepatoprotective effect of agrimony lactone (AM), animals in each group were administered three doses (10, 30, and 90 mg / kg) of agrimony lactone (AM) daily via intraperitoneal injection from day 1 to day 28 after the start of the DDC diet. Mice were monitored daily and their weight was recorded. After four weeks of DDC diet feeding and corresponding agrimony lactone (AM) treatment, animals were anesthetized with sodium pentobarbital (50 mg / kg, intraperitoneal injection), and blood samples were collected for serum and peripheral blood mononuclear cell analysis. The liver was removed, rinsed with phosphate-buffered saline (PBS), blotted dry, and weighed. The left lobe was separated from the remaining liver tissue before freezing, while sections of the right and middle lobes were fixed with 4% paraformaldehyde for histological examination.

[0066] To investigate the pharmacological mechanism of agrimonylactone (AM), BMS-986020 was selected as a bile acid transport inhibitor due to its potential to inhibit BSEP and MRP4. The dosage of BMS-986020 was 30 mg / kg. In addition to the DDC-induced cholestatic liver injury group and the AM treatment group, mice with AM-induced cholestatic liver injury were also administered BMS-986020 via daily intraperitoneal injection. At the experimental endpoint, after anesthesia with sodium pentobarbital (50 mg / kg, intraperitoneal injection), blood samples were collected for serum and peripheral blood mononuclear cell analysis. The liver was removed, rinsed with PBS, blotted dry, and weighed again. The left lobe was separated from the rest of the liver before freezing, and sections of the right and middle lobes were fixed with 4% paraformaldehyde for histological examination.

[0067] 2) Serum liver function index measurement

[0068] The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and bilirubin in the serum of mice in designated groups were determined by enzymatic methods using an automated biochemical analyzer (Nanjing Jiancheng Bioengineering Institute, China) and in accordance with the manufacturer's instructions.

[0069] 3) Histological staining

[0070] Mouse liver tissue was fixed with 4% paraformaldehyde and cut into 4 μm thick sections. Masson's trichrome staining and hematoxylin-eosin (HE) staining were used to assess histological damage, inflammatory infiltration, and collagen deposition. For immunohistochemical (IHC) analysis of tissue samples, sections were incubated with antibodies against α-SMA (GB111364, Servicebio), CD11b (GB11058, Servicebio), CD11b (ab133357, Abc agrimony lactone (AM)), CD45 (ab10558, Abc agrimony lactone (AM)), F4 / 80 (ab6640, Abc agrimony lactone (AM)), and CK19 (GB11197, ServiceBio). Immunohistochemical staining was performed according to the standard methods described previously. The main experimental steps included fixation, paraffin embedding, dewaxing, dehydration, antigen retrieval, endogenous peroxidase activity blocking, blocking, primary antibody incubation, washing, secondary antibody incubation, colorimetric detection, nuclear reverse staining, dehydration, mounting, and observation. Representative images were acquired using panoramic scanning (3DHISTECH, Budapest, Hungary) of stained sections via blind scanning. The images were then analyzed and quantified using ImageJ software (version 2.9.0; Bethesda, USA).

[0071] 4) Enzyme-linked immunosorbent assay (ELISA)

[0072] Serum levels of TNF-α, IL-1β, and IL-6 in mice were measured by ELISA in the control group, cholestatic liver injury group, and corresponding agrimonylactone (AM) treatment groups. Analysis was performed according to the instructions of the commercial kits provided by eBioscience (San Diego, CA).

[0073] 5) RNA extraction and quantitative real-time PCR

[0074] Total RNA was extracted using TRIzol reagent (Invitrogen, USA) and subsequently transcribed into cDNA using SuperScript II reverse transcriptase (Invitrogen, USA) according to the manufacturer's instructions. Real-time PCR was performed using SYBR Green Premix ExTaq reagent (TaKaRaBio, Japan). Gene expression levels were normalized to Gapdh or 18S rRNA in the same sample and expressed as fold changes relative to matched control values.

[0075] 6) RNA sequencing

[0076] Total RNA from mouse liver was extracted using TRIzol reagent according to standard protocols. After quality assessment using an Agilent 2100 Bioanalyzer (catalog number G2939AA, California, USA), 1 μg of total RNA was purified twice using Oligo(dT) (Vazyme, catalog number N401-01; Nanjing, China). 1000 ng of total RNA was fragmented and used for first-strand cDNA synthesis after the addition of first-strand reaction buffer and a random primer mixture, followed by second-strand cDNA synthesis. The purified double-stranded cDNA was then ligated with aptamers. Samples were amplified using barcoded primers for multiplex amplification. PCR conditions consisted of one cycle of 98°C for 30 seconds, followed by 11 cycles of 98°C for 10 seconds and 65°C for 75 seconds, with a final incubation at 65°C for 5 minutes. The adapter-ligated libraries were then purified and sent for quality assessment. The libraries were sequenced using a NovaSeq system (Illumina Inc., San Diego, CA, USA). The raw sequencing files are available from SRA and include mRNA-seq metadata. Each sample is analyzed in five biological replicates.

[0077] 7) Data collection and analysis

[0078] We obtained a batch RNA sequencing dataset of mouse livers (GSEX) from QianTang Biotechnology Co., Ltd. (Suzhou, China) by analyzing the original FASTQ files. Transcriptome reference sequences were obtained via Ensembl. To estimate transcript abundance, HISAT was used to align reads and summarize gene-level transcript abundance. StringTie was used for transcript prediction, while Bowtie2 was used to align sequencing reads with long reference sequences. For read matrix analysis, clustering, and differential gene expression analysis, we used the R package "Seurat". A unique counting threshold was applied to filter out cell dimers with counts exceeding 5,000 or below 500. Normalization for each cell was performed using the "LogNormalize" method based on total expression levels. Upregulated and downregulated genes were identified using the DESeq2, EBseq, NOIseq, and PoissonDis packages. KEGG and GO analyses were performed using ClusterProfiler. Gene set enrichment analysis (GSEA) was performed using a Java application from the Broad Institute. The complete gene set obtained from the differential gene expression analysis was sorted by "beta" value and used as input for GSEA pre-sorting analysis, referencing the C5 Gene Ontology-Bioprocesses (GO-BP) database. Visualization analyses, including volcano plots, bar charts, and Venn diagrams, were performed using the standard R package ggplot2. Enriched and depleted genes in the differential gene expression analysis were defined using an adjusted p-value threshold of 0.05 and a fold change threshold of 1.5.

[0079] 8) Determination of liver hydroxyproline content

[0080] A fresh right hepatic lobe tissue sample (50 mg) was homogenized with 6N hydrochloric acid solution and hydrolyzed overnight in a 110°C water bath. After cooling, 10 μL of indicator was added to each tube to adjust the pH to 7.0. The hydrolysate was diluted with 10 mL of double-distilled water. Subsequently, 4 mL of the diluted hydrolysate was filtered, neutralized with 1N sodium hydroxide solution, and then oxidized with chloramine-T. It was then reacted with perchloric acid and p-dimethylaminobenzaldehyde to generate a chromophore for quantitative analysis. The supernatant (200 μL) of each sample was transferred to a 96-well plate, centrifuged, and measured spectrophotometrically at 565 nm.

[0081] 9) Extraction and determination of bile acids in serum and liver

[0082] Accurately weigh 100 mg of liver sample and add 1000 μL of extraction solvent (methanol:water = 4:1). The sample was homogenized for 6 minutes at -20°C and 60 Hz using a cryo-tissue homogenizer, followed by sonication at 4°C for 30 minutes. After standing at -20°C for 30 minutes, the mixture was centrifuged at 4°C and 12,000 rpm for 15 minutes. 100 μL of the supernatant was then collected for analysis. For serum samples, 50 μL was mixed with 150 μL of methanol. The mixture was then sonicated at 4°C for 30 minutes. After an additional 30 minutes of incubation at -20°C, the mixture was centrifuged at 4°C and 12,000 rpm for 15 minutes. Finally, 150 μL of the supernatant was collected for analysis. The total bile acid content in liver and serum samples was determined using a total bile acid kit (MAK309).

[0083] (Sigma–Aldrich, St. Louis, Missouri) was measured according to the manufacturer’s agreement.

[0084] 10) Target determination was performed using LC-MS.

[0085] The type and concentration of bile acids (BA) in liver and serum samples were analyzed using liquid chromatography-mass spectrometry (LC-MS). Supernatants from all samples were injected into a UHPLC-Q / Exactive instrument (Thermo Scientific, USA) equipped with a Hypersil GoldDim column (100 × 2.1 mm, 1.9 μm). Mobile phase A was acetonitrile, and mobile phase B was an aqueous solution containing 5 mM ammonium carbamate and 0.1% formic acid. The flow rate was set to 0.3 mL / min, with the following gradient program: 0–2 min, 5% A; 2–5 min, 30% A; 5–16 min, 55% A; 16–18 min, 95% A; 18–20 min, 5% A. The mass spectrometer was equipped with an electrospray ionization source, and all alkaloids were detected in negative ion mode. For sample preparation, 180 μL of ice-cold alkaline acetonitrile (containing 5% NH4OH) was added to 20 μL of biological sample (bile samples required a 100-fold dilution). After vortexing, the mixture was centrifuged at 15,000 rpm for 30 minutes at 4°C. The supernatant was collected, evaporated under vacuum, and then redissolved in 40 μL of 50% acetonitrile. Subsequently, 10 μL of the supernatant was injected into an LC-MS / MS system for quantitative analysis. The mixed standard solution was prepared by weighing the standard, adding methanol to a constant volume, and mixing thoroughly. The working solution was prepared by diluting the mixed standard solution with 50% methanol. We detected 53 bile acids using consistent chromatographic-mass spectrometric parameters through relative quantitative comparisons.

[0086] 11) Flow cytometry

[0087] The antibodies used in this study included: PerCP / Cyanine5.5 anti-mouse CD45 (30-F11, Cat#:103132), PE / Cyanine7 anti-mouse / human CD11b (M1 / 70, Cat#:101216), APC anti-mouse F4 / 80 (BM8, Cat#:123116), PE anti-mouse CD3 (17A2, Cat#:100206), Brilliant Violet510 anti-mouse CD8b (53-6.7, Cat#:100751), PerCP / Cyanine5.5 anti-mouse CD45 recombinant (QA17A26, Cat#:157612), and anti-mouse CD16 / CD32 PerCP-Cyanine5.5 (45-0161-80, eBioscience). To assess the specificity of flow cytometry (FCM) results, a fluorescence minus-one control was used. Cells were blocked at 4°C for 15 minutes before the addition of other antibodies. Analysis was performed using a Beckman Coulter CytoFLEX flow cytometer (USA). Cell dimers were excluded by electronic gating based on the forward scatter to pulse width ratio, and data were analyzed using FlowJo 10.8.1 software.

[0088] 12) Statistical Analysis

[0089] Data were analyzed using GraphPad Prism software (version 8.0; Northampton, Massachusetts, USA) and are expressed as mean ± standard deviation (SD). Differences between groups were assessed using one-way ANOVA (Tukey's test), two-way ANOVA (Tukey's test), or Student's t-test. Statistical significance was determined using the following thresholds: *P < 0.032, **P < 0.0021, ***P < 0.0002, and ****P < 0.0001. Significant differences were indicated in the results and marked in the figures.

[0090] The experimental results obtained from the embodiments of the present invention are as follows:

[0091] 1. Agrimonia lactone (AM) improves cholestatic liver injury in a dose-dependent manner.

[0092] To evaluate the dose-dependent hepatoprotective effect of agrimonolactone (AM), we first established a chronic cholestatic liver injury model in mice by feeding them DDC daily for four weeks. From day 1 to day 28 after model induction, mice were administered three doses of agrimonolactone (AM) (10, 30, and 90 mg / kg) daily via intraperitoneal injection to designated groups. Figure 1 The DDC diet resulted in significant weight loss; however, administration of agrimony lactone (AM) alleviated these changes in a dose-dependent manner. Figure 2 Furthermore, the liver-to-body weight ratio increased from 4.5% to 11.2%. These results are consistent with previous studies that the DDC diet can induce weight loss and hepatomegaly. Agrimonia pilosa lactone (AM) treatment alleviated the abnormal increase in liver weight relative to body weight in a dose-dependent manner. Figure 3 Serum biochemical tests showed that the DDC diet led to abnormally elevated levels of ALT, AST, and ALP, while treatment with agrimony lactone (AM) resulted in a dose-dependent decrease in these serum markers. Figure 4 Furthermore, DDC diet-induced levels of total bilirubin and direct bilirubin were significantly elevated, showing dose-dependent remission after treatment with agrimony lactone (AM). Figure 5 Histopathological analysis showed that the DDC diet induced small bile duct hyperplasia, macrophage infiltration, intraductal embolism, pericholangitis, onion-skin-like pericholangial fibrosis, and bile duct fibrosis. Agrimonia pilosa lactone (AM) treatment alleviated small bile duct hyperplasia, macrophage infiltration, and bile duct fibrosis. Figure 6 In summary, these results indicate that agrimonolactone (AM) improves cholestatic liver injury in a dose-dependent manner.

[0093] 2. DDC-induced liver fibrosis and bile duct hyperplasia were alleviated by treatment with agrimony lactone (AM).

[0094] Masson staining of liver tissue in the DDC group showed that all mice exhibited typical fibrotic features. Figure 6 Agrimonia pilosa lactone (AM) treatment alleviated liver fibrosis and bile duct hyperplasia in a dose-dependent manner. Whole-genome RNA sequencing identified 254 downregulated genes and 197 upregulated genes, with a fold change of 1.5 and a significance level of p < 0.001. Figure 7 Gene ontology (GO) analysis revealed that biological processes associated with DNA repair, DNA replication, and DNA recombination were significantly affected. Figure 8Cellular component analysis revealed that the altered genes primarily acted on chromosomal regions, replication forks, and chromosomes. Notably, the molecular functions of differentially expressed genes (DEGs) were mainly related to catalytic activity, particularly DNA, ATPase activity, and helicase activity. Combined with the results of major GO analysis, we found that the DDC diet induced DNA damage repair, RNA transcription, and translation. Gene set enrichment analysis (GSEA) showed significant enrichment in extracellular matrix tissues, collagen-containing extracellular matrix, and extracellular matrix degradation-related pathways. Figure 9 These findings highlight the typical perichondral and bile ductal fibrosis features in DDC diet-induced chronic cholestatic liver injury.

[0095] We used biochemical methods to determine the level of hydroxyproline, a marker of liver fibrosis. DDC diet feeding led to a fourfold increase in hydroxyproline levels in liver tissue from 26.4 μg / mg to 86.8 μg / mg. Figure 10 Agrimonia pilosa lactone (AM) treatment showed a dose-dependent reduction in hepatic hydroxyproline accumulation. Furthermore, we assessed the expression levels of liver fibrosis markers α-Sma, Col1a1, Timp1, Epc agrimony pilosa lactone (AM), and Mmp2 using real-time quantitative PCR analysis. Our results showed that the expression of these markers was significantly increased in the DDC group compared to the normal control group. Figure 11 However, after 4 weeks of high-dose agrimonylactone (AM) treatment, the expression of α-SMA, Col1a1, Timp1, Epc, and Mmp2 significantly decreased. Immunohistochemical analysis showed that DDC-induced chronic cholestasis led to a significant increase in the protein expression of the liver fibrosis marker α-SMA, while this expression was significantly reduced after 4 weeks of agrimonylactone (AM) treatment. Figure 12 Overall, 4 weeks of agrimony lactone (AM) treatment significantly reduced the degree of fibrosis.

[0096] 3. Agrimonia lactone (AM) alleviated liver inflammation in DDC-induced chronic cholestatic liver injury.

[0097] Compared with the normal control group, DDC treatment led to reprogramming of cytokine pathways, myeloid cell development, and non-cellular pathways, which was confirmed by gene set enrichment analysis (GSEA). Figure 13 Subsequently, we measured the mRNA expression levels of inflammatory factors (including tumor necrosis factor-α (Tnf-α), interleukin-6 (Il-6), and interleukin-1β (Il-1β)) in the livers of mice with chronic cholestasis. In DDC-induced chronic cholestasis mice, the levels of Tnf-α, Il-6, and Il-1β were significantly elevated. Figure 14Conversely, agrimonolactone (AM) treatment reduced the hepatic upregulation of these inflammatory cytokines. Transforming growth factor-β1 (Tgfb1) gene expression was significantly upregulated in the DDC diet group, while agrimonolactone (AM) treatment effectively reduced its expression. Figure 15 Immunopathological results showed that the DDC-induced chronic cholestatic liver injury group exhibited excessive bile duct proliferation and excessive infiltration of leukocytes and myeloid cells. Figure 16 Notably, four weeks of agrimony lactone (AM) treatment inhibited bile duct hyperplasia, macrophage infiltration, and intraductal embolism, while also reducing liver fibrosis.

[0098] 4. Agrimonia pilosa lactone (AM) alleviates sterol metabolism disorders in DDC-induced chronic cholestasis mice. CK19 staining immunopathological results confirmed excessive bile duct proliferation in the DDC diet group. Agrimonia pilosa lactone (AM) treatment alleviated excessive proliferation in DDC-induced chronic cholestasis mice in all dosage groups. Figure 17 Agrimonia lactone (AM) treatment resulted in a dose-dependent remission of bile duct hyperplasia. Serum biochemical tests showed that DDC-fed mice with chronic cholestasis had significantly elevated serum cholesterol and bile acid levels, reflecting impaired bile duct proliferative function. Figure 18 Notably, after four weeks of high-dose agrimonolactone (AM) treatment, bile acid accumulation was significantly reduced. Hepatic bile acid levels were significantly elevated in the DDC diet group. Agrimonia pilosa (AM) treatment resulted in a dose-dependent decrease in hepatic bile acid levels. We identified primary and secondary bile acids (…). Figure 18 Quantitative PCR was used to determine the key enzymes Cyp7a1, Cyp8b1, Cyp27a1, and Cyp7b1 involved in bile acid synthesis in both classical and alternative cholesterol pathways. Enzymes Cyp7a1 and Cyp8b1 are responsible for the first step in the classical bile acid synthesis pathway and are rate-limiting enzymes, while enzymes Cyp27a1 and Cyp7b1 are responsible for the alternative bile acid synthesis pathway. The results showed that these enzymes were downregulated in all DDC diet-fed groups. Figure 19 Agrimonia pilosa lactone (AM) treatment increased the expression of Cyp7a1, Cyp8b1, and Cyp7b1 in a dose-dependent manner. High-dose agrimony pilosa lactone (AM) (90 mg / kg) reversed DDC-induced downregulation of Cyp27a1, while the effects of 10 and 30 mg / kg agrimony pilosa lactone (AM) were limited. Figure 20 ).

[0099] 5. Agrimonia lactone (AM) reprograms bile acid metabolism and increases the expression of bile acid transporters in the liver of DDC-induced chronic cholestasis mice.

[0100] The concentrations of primary and secondary hepatic bile acids (BAs) were determined by LC-MS / MS. Specifically, the levels of 15 bile acids in mouse serum and liver samples were measured. Figure 21 Compared with the normal control group, the levels of unconjugated bile acids (CA), α-MCA, and β-MCA were significantly increased in the DDC treatment group. Agrimonia pilosa lactone (AM) treatment inhibited the increase of TCA, T-α-MCA, β-MCA, and T-β-MCA. Notably, the DDC diet induced the accumulation of T-CDCA, T-DCA, and T-UDCA, an effect that could only be reversed by high-dose agrimony pilosa lactone (AM) (90 mg / kg).

[0101] We further evaluated transport proteins involved in bile acid efflux and found that the expression of Bsep and Mrp2 genes was downregulated in the DDC diet group. Figure 22 Agrimonia pilosa lactone (AM) treatment reversed the downregulation of Bsep at all three concentrations, with 30 mg / kg and 90 mg / kg AM increasing Bsep expression. Among the transport proteins responsible for bile acid efflux from the circulatory system, the DDC diet led to upregulation of Oatp2, Mrp3, and Mrp4 gene expression. Figure 23 Agrimonia lactone (AM) treatment consistently reduced the expression of Oatp2, Mrp3, and Mrp4 in a dose-dependent manner. Furthermore, the DDC diet led to a decrease in the expression of Ntcp and Oatp1, transport proteins involved in bile acid uptake in hepatocytes. Figure 24 Treatment with agrimony lactone (AM) at doses of 30 and 90 mg / kg increased the expression of Ntcp and Oatp1. Furthermore, after DDC diet feeding, the gene expression of phase I bile acid metabolizing enzymes Cyp3a11 and Cyp2b10 was upregulated. Figure 25 Agrimonia lactone (AM) treatment alleviated the upregulation of these phase I enzymes in a dose-dependent manner. Conversely, gene expression of phase II bile acid metabolism enzymes Ugt1a1 and Sult2a1 was suppressed after DDC diet feeding, and Agrimonia lactone (AM) treatment reversed the suppression of Ugt1a1 and Sult2a1 gene expression. Figure 26 ).

[0102] 6. BMS-986020 attenuated the hepatoprotective effect of agrimonolactone (AM) mediated on cholestatic liver injury.

[0103] To investigate the pharmacological mechanism of agrimonolactone (AM), we used BMS-986020 as a bile acid transport inhibitor due to its potential as a BSEP and MRP4 inhibitor. The dose of BMS-986020 was 30 mg / kg, while the dose of agrimonolactone (AM) was 90 mg / kg. In addition to the previously described DDC-induced cholestatic liver injury group and the AM-treated cholestatic liver injury group, mice with AM-treated cholestatic liver injury received daily intraperitoneal injections of BMS-986020. Consistent with previous results, DDC diet feeding led to an abnormally high liver-to-body weight ratio of 10.96%, which partially recovered to 5.31% after AM treatment. Although BMS-986020 treatment did not significantly affect the liver-to-body weight ratio in the DDC diet group, it reversed the AM-mediated relief of hepatomegaly. Figure 27 Furthermore, serum biochemical tests showed that agrimony lactone (AM) treatment normalized ALT, AST, and ALP levels. Conversely, BMS-986020 treatment did not significantly alter serum ALT, AST, and ALP levels, nor did it counteract the allergic reaction to agrimony lactone (AM) treatment. Figure 28 Agrimonia pilosa lactone (AM) treatment also increased the accumulation of serum and hepatic bile acids, while BMS-986020 significantly counteracted these AM-mediated increases in levels. Figure 29 Furthermore, compared with the agrimony lactone (AM) treatment group, the serum cholesterol level in the BMS-986020 group was significantly higher. Figure 30 Treatment with agrimony lactone (AM) increased the expression of Bsep and Mrp2, indicating improved bile acid excretion into bile. However, BMS-986020 decreased the expression of these excretion genes. Figure 31 Furthermore, the Oatp2, Mrp3, and Mrp4 genes, responsible for the efflux of bile acids into the circulatory system, were downregulated after treatment with agrimonylactone (AM). Figure 32 BMS-986020 significantly inhibited its expression. Notably, compared with agrimony lactone (AM) alone, the combination of BMS-986020 and agrimony lactone (AM) resulted in increased expression of Oatp2 and Mrp4. Furthermore, the genes Ntcp and Oatp1, responsible for bile acid uptake in hepatocytes, were upregulated after agrimony lactone (AM) treatment, while BMS-986020 significantly counteracted the AM-mediated upregulation of these genes. Figure 33 In summary, these results indicate that agrimonolactone (AM) has a hepatoprotective effect on cholestatic liver injury in mice, while BMS-986020 weakens this pharmacological effect by inhibiting the efflux of bile acids into the bile and circulatory system.

[0104] 7. BMS-986020 exacerbated liver fibrosis and inflammation, thereby counteracting the hepatoprotective effect mediated by agrimonolactone (AM) in cholestatic mice.

[0105] Based on the fibrotic changes observed in the liver tissue of mice in the DDC group and the hepatoprotective effect of agrimony lactone (AM), BMS-986020 alone did not cause significant changes in pathological features. Figure 34 However, the combined use of BMS-986020 and agrimony lactone (AM) significantly exacerbated liver fibrosis and inflammation, as confirmed by HE and Masson staining. We measured hepatic hydroxyproline levels and confirmed the anti-fibrotic effect of agrimony lactone (AM), which reduced hepatic hydroxyproline levels from 81.3 μg / mg to 32.3 μg / mg. Figure 35 Conversely, BMS-986020 combined with agrimonylactone (AM) significantly increased hepatic hydroxyproline levels to 45.2 μg / mg, indicating that the anti-fibrotic effect mediated by agrimonylactone (AM) was reversed. In the DDC diet-fed group, the expression of fibrosis marker genes (including α-Sma, Col1a1, Timp1, and Epc agrimonylactone (AM)) was significantly upregulated. However, agrimonylactone (AM) treatment inhibited the fibrotic process by reducing the expression of these genes. Figure 36 BMS-986020 treatment counteracted the antifibrotic effects mediated by agrimony lactone (AM), leading to a significant increase in the expression of α-Sma, Col1a1, Timp1, and Epc agrimony lactone (AM).

[0106] Tgf-b1 is known to regulate hepatocyte growth and stimulate extracellular matrix synthesis in various liver diseases. Agrimonia pilosa lactone (AM) inhibits Tgfb1 gene expression, while BMS-986020 attenuates the AM-mediated inhibition of hepatocyte growth and extracellular matrix synthesis. Figure 37 Immunohistochemical staining of CK19 and CD45 confirmed agrimonolactone (AM)-mediated ductal proliferation, intraductal embolism, inflammatory cell infiltration, and remission of pericholangitis. Figure 38 Compared with treatment with agrimony lactone (AM) alone, the combined use of BMS-986020 and agrimony lactone (AM) resulted in increased tubular hyperplasia, intraductal embolism, inflammatory cell infiltration, and pericholangitis.

[0107] We developed a flow cytometry strategy for the simultaneous detection of lymphocytes and myeloid cells. Previous studies have shown that the main characteristic of DDC diet-induced chronic cholestatic liver injury is increased myeloid cells and aberrant macrophage infiltration. CD11b expression showed that agrimonolactone (AM) significantly reduced the proportion of myeloid cells and increased the proportion of lymphocytes, indicating that the inflammatory process was alleviated. Figure 39 BMS-986020 reversed the agrimony lactone (AM)-mediated decrease in the proportion of abnormal lymphocytes. The proportions of CD4+ and CD8+ lymphocytes were also assessed, and the results showed that agrimony lactone (AM) inhibited the infiltration of these lymphocytes. Figure 40 However, BMS-986020 increased the proportion of infiltrating CD4+ and CD8+ lymphocytes. Immunohistochemistry, however, showed weak CD3+ staining, indicating that lymphocytes were less distinct and fewer in number among immune cells, with macrophages being the dominant inflammatory immune cells. For the major macrophage population, we identified two types of Kupffer cells (moderately expressed CD11b and highly expressed F4 / 80) and monocyte-derived macrophages (MDMs, highly expressed CD11b and moderately expressed F4 / 80) based on pseudocolor results. Figure 41 Agrimonia pilosa lactone (AM) treatment significantly reduced the number of Kupffer cells and MDMs, while the combination of BMS-986020 and agrimony pilosa lactone (AM) reversed the proportion of these macrophage populations. Figure 42 In summary, agrimonolactone (AM) exhibits antifibrotic and anti-inflammatory pharmacological effects in DDC-induced chronic cholestatic liver injury. The bile acid efflux inhibitor BMS-986020 attenuated the hepatoprotective effect of AM, and combined treatment resulted in the loss of both antifibrotic and anti-inflammatory effects.

[0108] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of agrimonolactone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cholestatic liver disease, characterized in that, The drug promotes bile acid efflux by upregulating the expression and / or function of bile acid efflux transport proteins, thereby inhibiting small bile duct proliferation, reducing liver fibrosis, and alleviating macrophage infiltration.

2. The application according to claim 1, characterized in that, When treated with the bile acid efflux and transport inhibitor BMS-986020, the therapeutic effect of Agrimonia pilosa lactone on cholestatic liver disease was significantly weakened or reversed.

3. The application according to claim 1, characterized in that, The dosage of the agrimony lactone is 10 mg / kg to 90 mg / kg.

4. The application according to claim 1, characterized in that, The cholestatic liver disease was induced by a DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) diet.

5. A pharmaceutical composition for treating cholestatic liver disease, comprising a therapeutically effective amount of agrimonolactone or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition promotes bile acid efflux by upregulating the expression and / or function of bile acid efflux transport proteins, thereby inhibiting small bile duct proliferation, reducing liver fibrosis, and alleviating macrophage infiltration.

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