Use of grass uae in the preparation of liver fibrosis prevention and treatment drugs

Abstract

The application discloses application of delavaine in preparation of a medicine for preventing and treating liver fibrosis. The application first proposes and verifies that delavaine can promote cell ferroptosis, inhibit transcription and expression of fibrosis genes, effectively inhibit activity of cells abnormally activated in a liver fibrosis course, and avoid collagen deposition, so as to effectively relieve liver fibrosis progression while reducing liver damage, and has an excellent application prospect in clinical treatment of liver fibrosis.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry, and in particular to the application of aconitine in the preparation of drugs for the prevention and treatment of liver fibrosis. Background Technology

[0002] Liver fibrosis is a common pathological basis for the progression of various chronic liver diseases to cirrhosis and hepatocellular carcinoma, and has become a major global public health problem. The core pathological feature of liver fibrosis is the abnormal accumulation of extracellular matrix (ECM) in the liver parenchyma. Its progression is a complex process driven by multiple molecular mechanisms, among which the activation of hepatic stellate cells (HSCs) is considered a key step. When the liver suffers chronic injury, resting HSCs are activated into myofibroblasts, becoming the main source of ECM; simultaneously, hepatocytes and immune cells secrete large amounts of cytokines, further amplifying the inflammatory response and promoting HSC activation and ECM deposition. Although the academic community generally recognizes that liver fibrosis is potentially reversible, current anti-fibrotic interventions mainly focus on treating the underlying causes, and there is a lack of clinically widely accepted specific anti-fibrotic drugs.

[0003] Crassicauline A is a diterpenoid alkaloid extracted from plants of the Aconitum genus. It mainly exerts its analgesic and anti-inflammatory effects by inhibiting voltage-gated sodium channels and is commonly used to treat various chronic pain conditions. However, whether crassicauline A has anti-fibrotic effects in vivo is currently unclear. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide aconitine, which promotes ferroptosis in abnormally activated cells during the course of liver fibrosis and inhibits the transcription and expression of fibrosis genes, thereby effectively alleviating liver damage and slowing the progression of liver fibrosis. Based on this, its application in the preparation of drugs for the prevention and treatment of liver fibrosis is proposed.

[0005] Technical solution: The application of aconitine as described in this invention in the preparation of drugs for the prevention and treatment of liver fibrosis.

[0006] Preferably, the CAS number of the aconitine is 79592-91-9.

[0007] Preferably, the application is in the preparation of a drug for inhibiting collagen deposition in the liver during the course of liver fibrosis.

[0008] Preferably, the application is in the preparation of a drug that inhibits the expression of fibrosis-related proteins; more preferably, the fibrosis-related proteins include smooth muscle α-actin and / or type I collagen.

[0009] Preferably, the application is in the preparation of a drug that promotes ferroptosis in abnormally activated hepatic stellate cells.

[0010] Preferably, the drug contains aconitine or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients.

[0011] Preferably, the drug further contains pharmaceutically acceptable excipients; more preferably, the pharmaceutically acceptable excipients include excipients selected from one or more of diluents, lubricants, flow aids, wetting agents, emulsifiers, or pH buffers.

[0012] Preferably, the dosage form of the drug includes tablets, capsules, granules, oral liquids, syrups, powders, microcapsules, injections, powder injections, infusions, suspensions, ointments, creams, gels, sprays, eye drops, ear drops, nasal drops, patches, lotions, suppositories, dressings, nebulizing solutions, film-forming agents, implants, microemulsions, liposomes, nanoparticles, oral instant films, sponges, and capsules.

[0013] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention proposes and verifies for the first time that aconitine can effectively inhibit the activity of abnormally activated cells in the course of liver fibrosis by promoting cell ferroptosis and inhibiting the transcription and expression of fibrosis genes, and avoid collagen deposition. It can effectively alleviate the progression of liver fibrosis while reducing liver damage, and has excellent application prospects in the clinical treatment of liver fibrosis. Attached Figure Description

[0014] Figure 1 The image shows the cell viability assay results of human hepatic stellate cells LX2 after treatment with aconitine. Figure 2 The image shows the cell viability assay results of the LX2 liver fibrosis cell model after treatment with aconitine. Figure 3 Figure 1 shows the results of fibrosis-related mRNA level measurement in the LX2 liver fibrosis cell model after treatment with aconitine. Figure 4 Figure 1 shows the results of fibrosis-related protein level measurement in the LX2 liver fibrosis cell model after aconitine treatment. Figure 5 Representative images for measuring the proliferation capacity of LX2 liver fibrosis cell model after aconitine treatment; Figure 6 A quantitative analysis of the proliferation capacity of LX2 liver fibrosis cell model after treatment with aconitine. Figure 7 Figure 1 shows the flow cytometry results of the proportion of dead cells in the LX2 liver fibrosis cell model after treatment with aconitine. Figure 8 Figure 1 shows the ROS level flow cytometry analysis results of the LX2 liver fibrosis cell model after aconitine treatment. Figure 9 Figure 1 shows the flow cytometry results of lipid peroxidation levels in the LX2 liver fibrosis cell model after treatment with aconitine. Figure 10 The figure shows the results of ferrous ion level measurement in the LX2 liver fibrosis cell model after aconitine treatment. Figure 11 Figure 1 shows the CD71 protein level measurement results of the LX2 liver fibrosis cell model after aconitine treatment. Figure 12 The image shows the liver function test results of a mouse model of liver fibrosis after treatment with aconitine. Figure 13 Representative images of liver tissue stained with H&E in a mouse model of liver fibrosis after treatment with aconitine; Figure 14 Representative Masson staining images of liver tissue from a mouse model of liver fibrosis after treatment with aconitine; Figure 15 Representative images of Sirius red staining of liver tissue from a mouse model of liver fibrosis after treatment with aconitine; Figure 16 Representative images of 4-HNE immunohistochemical staining of liver tissue from mice with liver fibrosis model after aconitine treatment; Figure 17 Figure 1 shows the results of measuring the level of fibrosis-related mRNA in liver tissue of a mouse model of liver fibrosis after treatment with aconitine. Figure 18 Figure 1 shows the results of measuring the levels of fibrosis-related proteins in the liver tissue of a mouse model of liver fibrosis after treatment with aconitine. Figure 19 Representative images of immunofluorescence staining of lipid peroxidation levels in liver tissue of mice with liver fibrosis model after treatment with aconitine; Figure 20 Figure 1 shows the results of ferrous ion level measurement in liver tissue of a mouse model of liver fibrosis after treatment with aconitine. Figure 21 Figure 1 shows the results of CD71 protein level measurement in liver tissue of a mouse model of liver fibrosis after treatment with aconitine. Figure 22 This is a representative image of CD71 immunohistochemical staining in liver tissue of a mouse model of liver fibrosis after treatment with aconitine. Detailed Implementation

[0015] The technical solution of the present invention will be further described below.

[0016] Example 1: In vitro efficacy verification of crassicauline A (CRA) in the treatment of liver fibrosis 1. Assay of cell viability of human hepatic stellate cells LX2 after CRA treatment Human hepatic stellate cells LX2 (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number CL-0560) were cultured in DMEM medium supplemented with 10% fetal bovine serum in a dedicated incubator at 37°C and 5% carbon dioxide.

[0017] 1×10 LX2 cells in logarithmic growth phase 3 Cells were seeded at a density of 0.5% per well in 96-well plates. After cell adhesion, the original medium was replaced with DMEM complete medium containing 0, 0.25, 0.5, 1, 2.5, 5, 10, 25, or 50 µM CRA (purchased from MedChemExpress LLC., catalog number HY-N1924) (all containing 1‰ DMSO).

[0018] After 24 h of treatment, each well was replaced with 100 μL of DMEM basal medium containing 10% CCK8 reagent and incubated at 37 °C for 2 h. The absorbance was measured at 450 nm using a microplate reader, and the relative level of cell viability was calculated.

[0019] The results are as follows Figure 1 As shown, when the CRA treatment concentration does not exceed 5 µM, the cell viability can be maintained above 80%, while when it is greater than 5 µM, the cell viability is significantly reduced (below 70%). Therefore, the CRA treatment concentration selected in subsequent experiments was 5 µM.

[0020] 2. Construction of a liver fibrosis cell model and determination of cell viability of the liver fibrosis cell model after CRA treatment Human hepatic stellate cells (LX2) were cultured in DMEM medium supplemented with 10% fetal bovine serum in a dedicated incubator at 37°C and 5% carbon dioxide. When the cell density reached 70%, TGF-β1 (purchased from MedChemExpress LLC., catalog number HY-P70543) at a final concentration of 10 ng / mL was added to the medium for 24 h to construct a liver fibrosis cell model.

[0021] Normal LX2 cells or LX2 cells treated with TGF-β1 were used at a dose of 1×10⁻⁶. 3 Cells were seeded at a density per well in 96-well plates. After culture and adhesion, normal LX2 cells were treated by replacing the original medium with DMEM complete medium containing 1‰ DMSO (CTRL group). For TGF-β1-treated LX2 cells, the original medium was replaced with DMEM complete medium containing 1‰ DMSO (DMSO group), or CRA with a final concentration of 5µM (CRA group), or pirfenidone with a final concentration of 20µM (PFD group, pirfenidone purchased from MedChemExpress LLC., catalog number HY-B0673).

[0022] After 24 h of treatment, each well was replaced with 100 μL of DMEM basal medium containing 10% CCK8 reagent and incubated at 37 °C for 2 h. The absorbance was measured at 450 nm using a microplate reader, and the relative level of cell viability was calculated.

[0023] The results are as follows Figure 2 As shown, compared with the CTRL group cells, the activity of liver fibrotic cells (DMSO group cells) increased to 1.78, while the cell activity decreased to 0.72 after treatment with 5 µM CRA. The cell activity inhibition effect was similar to the therapeutic effect of the fibrosis-positive drug PFD, which preliminarily indicates that CRA can be used as a potential drug to inhibit the activity of fibrotic liver stellate cells.

[0024] 4. Determination of expression of key fibrosis markers in a liver fibrosis cell model after CRA treatment. Normal LX2 cells or LX2 cells treated with TGF-β1 were divided into groups of 5 × 10⁻⁶. 4 Cells were seeded at a density of 1‰ in 6-well plates. After culture and adhesion, normal LX2 cells were treated by replacing the original medium with DMEM complete medium containing 1‰ DMSO (CTRL group). For TGF-β1 treated LX2 cells, the original medium was replaced with DMEM complete medium containing 1‰ DMSO (DMSO group), or CRA with a final concentration of 5 µM (CRA group), or pirfenidone with a final concentration of 20 µM (PFD group).

[0025] 4.1 Determination of mRNA expression levels as a key indicator of fibrosis Cells were collected 24 h after treatment, and total RNA was extracted using the Novizan FreeZol Reagent Kit (catalog number R711-01). RNA was then extracted using Novizan HiScript IV RT SuperMix for qPCR (+gDNA). The wiper reagent (catalog number R423-01) was used for reverse transcription to cDNA, using the following primers: COL1A1 upstream primer: 5'-tggtttcgacttcagcttcc-3'; COL1A1 downstream primer: 5'-atgttctcgatctgctggct-3'; ACTA2 upstream primer: 5'-gaagctgataaacgtgggct-3'; ACTA2 downstream primer: 5'-tgctttatggctggaattggt-3'; GAPDH upstream primer: 5'-gtgaaggtcggtgtgaacg-3'; GAPDH downstream primer: 5'-tgccgtgagtggagtcatac-3'. qPCR detection was performed using the Novizan ChamQ Universal SYBR qPCR Master Mix reagent (catalog number Q711-02), with reaction conditions of 95℃ pre-denaturation for 30 s and 95℃ denaturation for 10 s. Annealing and extension at 60℃ for 30 s, 35 cycles. Fluorescence values ​​were collected during the extension phase of each cycle. Ct values ​​were calculated using GAPDH as an internal control, and analyzed by 2... -ΔΔCt The relative expression levels of COL1A1 and ACTA2 were calculated using this method.

[0026] The results are as follows Figure 3 As shown, compared with the CTRL group cells, the COL1A1 and ACTA2 gene transcription levels of the DMSO group cells were significantly increased, and CRA could effectively inhibit the transcription of the above genes, similar to the effect of the positive drug PFD.

[0027] 4.2 Determination of protein expression levels as key indicators of fibrosis Cells were collected 24 h after treatment and lysed using RIPA lysis buffer (Catalog No. WB3100) containing 0.1 volume of cyprotease inhibitor mixture (Catalog No. P001) and 0.1 volume of cyprotease inhibitor mixture (Catalog No. P003). The cells were lysed on ice with shaking for 30 min, centrifuged at 12000 rpm for 30 min at 4 °C, and the supernatant was collected. Quantification was performed using the cyprotease BCA protein quantification kit (Catalog No. WB6501). Subsequently, 5× Loading buffer (Catalog No. WB2001) was added proportionally, and the mixture was boiled in a metal bath for 10 minutes. After a period of time, SDS-PAGE electrophoresis was performed. After transfer and blocking, COL1A1 primary antibody (purchased from Proteintech, catalog number 67288-1-Ig) diluted 1:5000, ACTA2 primary antibody (purchased from Proteintech, catalog number 14395-1-AP) diluted 1:5000, or GAPDH primary antibody (purchased from Proteintech, catalog number 10494-1-AP) diluted 1:20000 were added and incubated overnight at 4°C. After rinsing, secondary antibody (purchased from Proteintech, catalog number SA00001-2) diluted 1:10000 was added and incubated at room temperature for 2 h. Finally, the chemiluminescence was developed and images were acquired using the New Semiconductor Ultrasensitive ECL Chemiluminescence Kit (catalog number P10100).

[0028] The results are as follows Figure 4 As shown, compared with the CTRL group cells, the DMSO group cells showed significantly increased levels of COL1A1 and ACTA2 protein expression. CRA effectively inhibited the expression of these proteins, similar to the effect of the positive control drug PFD. This result is consistent with the determination of gene transcription levels.

[0029] 5. Determination of cell proliferation capacity in a liver fibrosis cell model after CRA treatment Normal LX2 cells or LX2 cells treated with TGF-β1 were divided into groups of 5 × 10⁻⁶. 4 Cells were seeded at a density of 1‰ in 6-well plates. After culture and adhesion, normal LX2 cells were treated by replacing the original medium with DMEM complete medium containing 1‰ DMSO (CTRL group). For TGF-β1 treated LX2 cells, the original medium was replaced with DMEM complete medium containing 1‰ DMSO (DMSO group), or CRA with a final concentration of 5 µM (CRA group), or pirfenidone with a final concentration of 20 µM (PFD group).

[0030] Cells were collected 24 h after treatment, and 20 μL of EdU reagent (purchased from Novizan, catalog number A413-01) was added to each well. The cells were incubated at 37°C for 2 h, fixed with 4% paraformaldehyde solution at room temperature for 15 min, and then permeabilized with Beyotime immunostaining permeabilization solution (catalog number P0096) at room temperature for 20 min. Click reaction solution (purchased from Novizan, catalog number A413-01) was then added, and the cells were incubated in the dark for 30 min. The cells were then mounted with Beyotime anti-fluorescence quenching mounting solution (catalog number P0131). The cells were observed and images were acquired using a fluorescence microscope, and the percentage of EdU-positive cells was statistically analyzed.

[0031] The results are as follows Figure 5 , 6 As shown, compared with the CTRL group, the EdU fluorescence intensity and the number of positive cells in the DMSO group were significantly increased, indicating that the proliferative activity of hepatic stellate cells in liver fibrosis was significantly increased, aggravating the symptoms of liver fibrosis. However, after treatment with CRA or PFD, the EdU fluorescence intensity was significantly reduced, thereby inhibiting the cell activity of hepatic fibrotic stellate cells.

[0032] 6. Determination of cell death levels in a liver fibrosis cell model after CRA treatment Normal LX2 cells or LX2 cells treated with TGF-β1 were divided into groups of 5 × 10⁻⁶. 4 Cells were seeded at a density of 1‰ in 6-well plates. After culture and adhesion, normal LX2 cells were treated by replacing the original medium with DMEM complete medium containing 1‰ DMSO (CTRL group). For TGF-β1 treated LX2 cells, the original medium was replaced with DMEM complete medium containing 1‰ DMSO (DMSO group), or CRA with a final concentration of 5 µM (CRA group), or pirfenidone with a final concentration of 20 µM (PFD group).

[0033] Cells were collected 24 h after treatment and incubated for 30 min in the dark with a PI probe (purchased from MedChemExpress LLC., catalog number HY-D0815) at a final concentration of 2 μM. The intracellular PI positivity rate was analyzed by flow cytometry.

[0034] The results are as follows Figure 7 As shown, the PI positivity rate was low in both the CTRL and DMSO groups, but increased to 19.1% after CRA treatment, and 21.5% in the PFD treatment group. This indicates that CRA can significantly induce fibrotic cell death and ultimately alleviate the symptoms of liver fibrosis.

[0035] 7. Determination of reactive oxygen species (ROS) levels in a liver fibrosis cell model after CRA treatment. Normal LX2 cells or LX2 cells treated with TGF-β1 were divided into groups of 5 × 10⁻⁶. 4 Cells were seeded at a density of 1‰ in 6-well plates. After culture and adhesion, normal LX2 cells were treated by replacing the original medium with DMEM complete medium containing 1‰ DMSO (CTRL group). For TGF-β1 treated LX2 cells, the original medium was replaced with DMEM complete medium containing 1‰ DMSO (DMSO group), or CRA with a final concentration of 5 µM (CRA group), or pirfenidone with a final concentration of 20 µM (PFD group).

[0036] Cells were collected 24 h after treatment and stained with the DCFH-DA probe (purchased from MedChemExpress LLC., catalog number HY-D0940) at a final concentration of 5 μM for 30 min in the dark at 37°C. Cells were then detected and analyzed by flow cytometry.

[0037] The results are as follows Figure 8 As shown, compared with the DMSO group, the intracellular ROS level was significantly increased after CRA or PFD treatment, which can inhibit cell activity by inducing cells to produce higher levels of oxidative stress, thereby achieving the purpose of treating liver fibrosis.

[0038] 8. Determination of lipid peroxidation level in a liver fibrosis cell model after CRA treatment Normal LX2 cells or LX2 cells treated with TGF-β1 were divided into groups of 5 × 10⁻⁶. 4 Cells were seeded at a density of 1‰ in 6-well plates. After culture and adhesion, normal LX2 cells were treated by replacing the original medium with DMEM complete medium containing 1‰ DMSO (CTRL group). For TGF-β1 treated LX2 cells, the original medium was replaced with DMEM complete medium containing 1‰ DMSO (DMSO group), or CRA with a final concentration of 5 µM (CRA group), or pirfenidone with a final concentration of 20 µM (PFD group).

[0039] Cells were collected 24 h after treatment, washed with pre-cooled PBS, and stained with BODIPY 581 / 591 C11 probes (purchased from MedChemExpress LLC., catalog number HY-D1301) at 37°C in the dark for 30 min. Intracellular lipid peroxidation levels were detected by flow cytometry and analyzed.

[0040] The results are as follows Figure 9 As shown, CRA or PFD treatment can significantly induce higher levels of lipid peroxidation in cells, which can significantly induce ferroptosis.

[0041] 9. Determination of ferroptosis-related markers in a liver fibrosis cell model after CRA treatment Normal LX2 cells or LX2 cells treated with TGF-β1 were divided into groups of 5 × 10⁻⁶. 4 Cells were seeded at a density of 1‰ in 6-well plates. After culture and adhesion, normal LX2 cells were treated by replacing the original medium with DMEM complete medium containing 1‰ DMSO (CTRL group). For TGF-β1 treated LX2 cells, the original medium was replaced with DMEM complete medium containing 1‰ DMSO (DMSO group), or CRA with a final concentration of 5 µM (CRA group), or pirfenidone with a final concentration of 20 µM (PFD group).

[0042] After 24 h of treatment, cell culture supernatant and cells were collected separately. The collected cells were lysed with RIPA lysis buffer containing 0.1 volume of RIPA protease inhibitor mixture and 0.1 volume of RIPA phosphatase inhibitor mixture on ice for 30 min by shaking. After centrifugation at 12000 rpm for 30 min at 4 °C, the supernatant was collected to obtain cell protein supernatant, which was then quantified using the RIPA BCA protein quantification kit.

[0043] 9.1 Ferrous ion (Fe) 2+ ) Level measurement The obtained cell culture supernatant was analyzed using the Elite Total Iron Colorimetric Assay Kit (catalog number E-BC-K772-M) to detect Fe in the supernatant. 2+ The level.

[0044] The results are as follows Figure 10 As shown, the concentration of Fe in cells was 9.9 nmol / mgprot in the CTRL group, 11.9 nmol / mgprot in the DMSO group, 33.7 nmol / mgprot in the CRA group, and 34.7 nmol / mgprot in the PFD group, indicating that CRA can significantly increase Fe in the liver fibrosis model. 2+ It increases the content of iron, promotes ferrodeation of cells, and alleviates the symptoms of liver fibrosis.

[0045] 9.2 Determination of CD71 protein expression level The aforementioned cell protein supernatant was mixed with 5× Loading buffer from Neo-Syneema, heated to boiling in a metal bath for 10 min, and then subjected to SDS-PAGE electrophoresis. After transfer and blocking, CD71 primary antibody (purchased from Proteintech, catalog number 10084-2-AP) diluted 1:5000 or GAPDH primary antibody diluted 1:20000 was added and incubated overnight at 4°C. After rinsing, secondary antibody (purchased from Proteintech, catalog number SA00001-2) diluted 1:10000 was used and incubated at room temperature for 2 h. Finally, the cells were developed and images were acquired using the Neo-Syneema ultrasensitive ECL chemiluminescence kit.

[0046] The results are as follows Figure 11 As shown, compared to the CTRL group, the DMSO group had significantly lower CD71 protein levels, which inhibited Fe... 2+ It enters the cell from the extracellular space and inhibits ferroptosis. However, with treatment with CRA or PFD, the protein level of CD71 is significantly restored, promoting ferroptosis and thus inhibiting the activity of hepatic stellate cells.

[0047] Example 2: In vivo efficacy verification of aconitine in the treatment of liver fibrosis 1. Construction of animal models of liver fibrosis and drug treatment Male C57BL / 6 mice aged 6-8 weeks were purchased from the Experimental Animal Center of Nantong University. The mice were housed under standard laboratory conditions and had free access to food and water.

[0048] All mice were randomly divided into sham, saline, CRA, and PFD groups, with 12 mice in each group, and a liver fibrosis animal model was established. Mice in the saline, CRA, and PFD groups were injected intraperitoneally twice a week with 10% (v / v) CCl4 dissolved in corn oil (purchased from MedChemExpress LLC., catalog number HY-Y1888), at a dose of 5 μL / g, for 4 weeks. Mice in the sham group underwent the same procedure, but were injected with the same amount of corn oil.

[0049] After the successful establishment of the liver fibrosis animal model, the drug was administered via intraperitoneal injection once a day. Mice in the CRA group were given 20 mg / kg of aconitine, mice in the PFD group were given 20 mg / kg of pirfenidone, and mice in the saline group and sham-operated group were given the same volume of saline. The administration was continued for 4 weeks.

[0050] After the last administration, peripheral blood was collected from all mice after a 12-hour fast and serum was separated. The mice were then euthanized and liver tissue was collected for further analysis.

[0051] 2. Liver function tests The peripheral blood serum collected from the aforementioned mice was analyzed using Solarbio GPT / ALT Activity Assay Kit (catalog number BC1555), Solarbio GOT / AST Activity Assay Kit (catalog number BC1565), Solarbio Alkaline Phosphatase (AKP / ALP) Activity Assay Kit (catalog number BC2145), Solarbio Total Bile Acid (TBA) Content Assay Kit (catalog number BC6300), and Solarbio Total Bilirubin (TBIL) Content Assay Kit (catalog number BC5185) to detect the levels of ALT, AST, ALP, TBA, and TBIL in the serum.

[0052] The results are as follows Figure 12 As shown, CCl4 treatment induced severe liver damage, manifested by a significant increase in serum ALT, AST, ALP, TBA, and TBIL levels. CRA or PFD treatment significantly improved these abnormal increases, demonstrating a strong protective effect against liver damage.

[0053] 3. Liver tissue pathological analysis 3.1 H&E staining The collected mouse liver tissues were fixed in 4% paraformaldehyde solution for 48 h, dehydrated in a gradient manner, embedded in paraffin, and cut into 5 μm sections. After dewaxing and hydration, hematoxylin-eosin (H&E) staining was performed, and the sections were mounted with neutral resin and observed and images were acquired under a microscope.

[0054] The results are as follows Figure 13 As shown, the liver tissue of mice in the sham-operated group maintained typical lobular structure and normal cell morphology, while the liver tissue of mice in the saline group treated with CCl4 showed obvious pathological changes, including disordered lobular structure, extensive hepatocyte necrosis, and severe inflammatory cell infiltration in the lesion area. After treatment with CRA or PFD, these abnormal histological changes were moderately improved, indicating that CRA can significantly alleviate CCl4-induced liver fibrosis in mice.

[0055] 3.2 Masson staining The preliminary steps were the same as those for H&E staining. In the staining section, the collagen deposits in the liver tissue were stained using the Solarbio Masson Tricolor Staining Kit (catalog number G1340). After mounting with neutral resin, the tissue was observed and images were acquired under a microscope.

[0056] 3.3 Sirius Red Staining The preliminary steps were the same as those for H&E staining. In the staining section, the collagen fibers of the liver tissue were stained using the Solarb Sirius Red Staining Kit (catalog number G1472). After mounting with neutral resin, the tissue was observed and images were acquired under a microscope.

[0057] Masson staining results are as follows: Figure 14 As shown, the Sirius red staining results are as follows: Figure 15 As shown, compared with the liver tissue of the sham-operated group, the liver tissue of the saline group showed obvious collagen accumulation in the liver parenchyma. After CRA or PFD intervention, the degree of collagen deposition was significantly reduced.

[0058] 4. Immunohistochemical staining of 4-hydroxynonenoic acid (4-HNE) levels in liver tissue The preliminary steps were the same as for H&E staining. Liver tissue sections were antigen-retrieved for 20 min at 95°C using citrate buffer (pH 6.0). Endogenous peroxidase activity was inhibited for 10 min with 3% hydrogen peroxide solution, followed by blocking with 5% bovine serum albumin solution for 1 h. The sections were then incubated overnight at 4°C with a 1:500 dilution of 4-HNE primary antibody (Proteintech, catalog number 27309-1-AP). After washing, the sections were incubated with a 1:500 dilution of horseradish peroxidase (HRP) conjugated with secondary antibody (Proteintech, catalog number SA00004-2) at room temperature for 1 h. Signal detection was performed using the Beyotime DAB chromogenic reagent kit (catalog number P0202), and cell nuclei were counterstained with hematoxylin. Images were acquired using an optical microscope.

[0059] The results are as follows Figure 16 As shown, compared with the saline group, the CRA and PFD treatment groups showed significantly increased 4-HNE levels in liver tissue, indicating the presence of high levels of ferroptosis.

[0060] 5. Measurement of expression of key indicators of liver fibrosis 5.1 Determination of mRNA expression levels as a key indicator of fibrosis 100 mg of the previously collected mouse liver tissue was ground in liquid nitrogen using a mortar and pestle, and then 1 mL of Novizan FreeZolReagent kit was added to extract total RNA. The RNA was then reverse transcribed into cDNA using Novizan HiScript IV RT SuperMix for qPCR (+gDNAwiper) reagent, and the method described in Example 1 was followed. Gapdh The primers used for qPCR detection as an internal control are: Col1a1 Upstream primer: 5'-tcatacgcggaattcgagaaac-3'; Col1a1 Downstream primer: 5'-aattgtttgcagccgtccac-3'; Acta2 Upstream primer: 5'-gcgttcattaattcgctctatgc-3'; Acta2 :5'-gatctccttctgcatcctgt-3'; GapdhUpstream primer: 5'-aattcaacggcacagtcaagg-3'; Gapdh Downstream primer: 5'-accagtagactccacgacat-3'. Ct values ​​were calculated after qPCR detection using the ΔΔCt method. Col1a1 , Acta2 The relative expression level.

[0061] The results are as follows Figure 17 As shown, compared to the sham-operated group mice, the liver tissue of the saline group mice had significantly higher concentrations of... Col1a1 and Acta2 Transcription levels were significantly increased, while both CRA and PFD treatments effectively suppressed the transcription of the aforementioned genes.

[0062] 5.2 Determination of protein expression levels as key indicators of fibrosis 100 mg of the previously collected mouse liver tissue was ground in liquid nitrogen using a mortar and pestle. The liver tissue was then lysed in 1 mL of a mixture of 0.1-fold cytosine protease inhibitor and 0.1-fold cytosine phosphatase inhibitor in RIPA lysis buffer at 4°C for 30 min. After centrifugation at 12000 rpm at 4°C for 30 min, the total liver protein supernatant was collected. The expression levels of COL1A1 and ACTA2 were determined using the cytosine BCA protein quantification kit and according to the method described in Example 1.

[0063] The results are as follows Figure 18 As shown, compared with the sham-operated group mice, the levels of Col1a1 and Acta2 proteins in the liver tissue of the saline group mice were significantly increased, while CRA and PFD treatments could effectively reduce the expression levels of the above proteins, consistent with the trend of mRNA level changes.

[0064] 6. Measurement of lipid peroxidation level in liver tissue A portion of the collected mouse liver tissue was embedded in OCT embedding medium and rapidly frozen in liquid nitrogen. Subsequently, 4 μm thick frozen sections were prepared, fixed with 4% paraformaldehyde for 30 min, washed with PBS, and incubated with 0.1% (v / v) Triton X-100 PBS at room temperature for 10 min. Then, the sections were stained with 5 µM BODIPY 581 / 591 C11 probe (MCE, HY-D1301) staining solution in the dark for 30 min. Finally, the sections were mounted with Beyotime anti-fluorescence quenching mounting solution (containing DAPI) (P0131) and observed and images were acquired by fluorescence microscopy.

[0065] The results are as follows Figure 19As shown, the intensity of green fluorescence, representing lipid peroxidation, was significantly increased in mice with liver fibrosis after treatment with CRA or PFD, indicating that CRA and PFD can induce ferroptosis and ultimately achieve the goal of treating liver fibrosis.

[0066] 7. Measurement of hepatic tissue ferroptosis-related indicators 7.1 Determination of Ferrous Ion Levels 100 mg of mouse liver tissue was collected, and 1 mL of pre-cooled physiological saline was added to prepare a tissue homogenate. The homogenate was centrifuged at 12000 rpm for 15 min, and the supernatant was collected. The Fe concentration in the supernatant was detected using the Elite Total Iron Colorimetric Assay Kit. 2+ The level.

[0067] The results are as follows Figure 20 As shown, in the liver fibrosis model, CRA can significantly increase Fe 2+ The concentration of [specific substance] further promotes ferroptosis in cells, consistent with in vitro experiments.

[0068] 7.2 Determination of CD71 protein expression level The expression level of CD71 was determined by taking the total protein supernatant from the aforementioned mouse liver tissue and measuring it according to the method described in Example 1.

[0069] The results are as follows Figure 21 As shown, CD71 protein levels are significantly reduced in CCl4-induced liver fibrosis, while treatment with CRA or PFD can restore CD71 protein levels, thereby successfully inducing ferroptosis.

[0070] 7.3. CD71 Immunohistochemical Staining The initial steps were consistent with 4-HNE level immunohistochemical staining. Antibody incubation was performed using a 1:500 dilution of CD71 primary antibody (Proteintech, catalog number 10084-2-AP) incubated overnight at 4°C. After washing, the sections were incubated with a 1:500 dilution of horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. Signal detection was performed using the Beyotime DAB chromogenic reagent kit, and cell nuclei were counterstained with hematoxylin. Images were acquired using an optical microscope.

[0071] The results are as follows Figure 22 As shown, consistent with the results of Western blot, CRA or PFD can significantly increase the expression level of CD71, promote ferroptosis, and ultimately achieve the goal of treating liver fibrosis.

Claims

1. The application of aconitine in the preparation of drugs for the prevention and treatment of liver fibrosis.

2. The application according to claim 1, characterized in that, The CAS number for the aconitine is 79592-91-9.

3. The application according to claim 1, characterized in that, The application is in the preparation of drugs that inhibit collagen deposition in the liver during the course of liver fibrosis.

4. The application according to claim 3, characterized in that, The application is in the preparation of drugs that inhibit the expression of fibrosis-related proteins.

5. The application according to claim 4, characterized in that, The fibrosis-related proteins include smooth muscle α-actin and / or type I collagen.

6. The application according to claim 1, characterized in that, The application is in the preparation of drugs that promote ferroptosis in abnormally activated hepatic stellate cells.

7. The application according to claim 1, characterized in that, The drug contains aconitine or its pharmaceutically acceptable salts, solvates, or hydrates as active ingredients.

8. The application according to claim 7, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipients include excipients selected from one or more of diluents, lubricants, flow aids, wetting agents, emulsifiers, or pH buffers.

10. The application according to claim 1, characterized in that, The dosage forms of the drugs include tablets, capsules, granules, oral liquids, syrups, powders, microcapsules, injections, powder injections, infusions, suspensions, ointments, creams, gels, sprays, eye drops, ear drops, nasal drops, patches, lotions, suppositories, dressings, nebulizing solutions, film-forming agents, implants, microemulsions, liposomes, nanoparticles, oral instant films, sponges, and capsules.

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