Polypeptide with anti-hepatic fibrosis effect and application thereof

Abstract

The application discloses a polypeptide with an anti-hepatic fibrosis effect and application thereof. The polypeptide has a significant anti-hepatic fibrosis effect in various mouse models, can inhibit abnormal activation of hepatic stellate cells, down-regulate expression of hepatic fibrosis related markers, inhibit collagen synthesis and excessive deposition of extracellular matrix. The polypeptide has low toxic and side effects, good safety, can be used for preparing medicine for preventing and / or treating hepatic fibrosis or chronic liver disease with hepatic fibrosis, can be used for preparing health care products for delaying progression of hepatic fibrosis, and can be used as a functional component combined with a diagnosis, drug delivery or intervention device to prepare medical devices or medical device compositions for hepatic fibrosis related treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a polypeptide with anti-liver fibrosis effect and its application. Background Technology

[0002] Liver fibrosis is a common pathological process in the progression of various chronic liver diseases, including viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease / metabolic fatty liver disease, drug-induced liver injury, and autoimmune liver disease. Its main characteristic is abnormal deposition and remodeling of the extracellular matrix, especially the excessive accumulation of collagen components, represented by type I and type III collagen, in liver tissue (Bai R, et al. In Situ Assembly of Fluorogenic RNA for Screening Natural Anti-Liver Fibrosis Compounds. Adv Sci, 2025, 12, e02850). Progressive liver fibrosis can further develop into cirrhosis and even increase the risk of hepatocellular carcinoma, making it a significant factor contributing to poor clinical outcomes and increased disease burden in patients with chronic liver disease.

[0003] Currently, clinical intervention for liver fibrosis primarily focuses on etiological treatment and risk factor management, such as antiviral therapy, alcohol abstinence, weight control, and intervention for metabolic risk factors (Zhang CY, et al. Treatment of Liver Fibrosis: Past, Current, and Future. World J Hepatol, 2023, 15, 755-774). Although the FDA has approved resmetirom and semaglutide for certain adult patients with moderate to advanced liver fibrosis in non-cirrhotic MASH patients (Keam SJ. Resmetirom: First Approval. Drugs, 2024, 84, 729-735), the applicable etiologies, disease stages, and patient populations for these approved drugs remain relatively limited, and their long-term clinical benefits require further validation. Therefore, developing novel, safe, and effective anti-liver fibrosis drugs suitable for liver fibrosis caused by different etiologies has significant clinical and commercialization value. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a polypeptide with anti-liver fibrosis effect.

[0005] Another object of the present invention is to provide the application of the above-mentioned polypeptide with anti-liver fibrosis effect.

[0006] This invention is achieved through the following technical solution:

[0007] A polypeptide with anti-liver fibrosis activity has the structure shown in Formula I:

[0008] ;

[0009] In Equation I: R1 is Asn, Lys, Thr-Leu, His-Lys, Pro-Asn, Gly-Thr-Leu, Gly-Tyr-Lys, Pro-Tyr-Lys, Pro-Gly-Thr-Leu, Ala-Pro-Gly-Thr-Leu, Ser-Lys-Thr-Asp-Val, Pro-Ala-Pro-Gly-Thr-Leu, Pro-Asn-Asn-Pro-Tyr-Gly, Ala-Pro-Ala-Pro-Gly-Thr-Leu, Leu-Ala-Pro-Ala-Pro-Gly-Thr-Leu, or does not exist;

[0010] R2 is Gly-Pro, Glu-Leu, Asp-Phe, Val-Ala, Gly-Val-Ala-Gly, Ser-Ser-Gly-Gly-Glu-Leu, Leu-Ile-Glu-Glu-Gly-Lys, Leu-Pro-Asn-Gly-Arg-Ala-Ala or Ser-Arg-Asp-Gly-Met-Asn-Ala-Ala;

[0011] R3 is Ala, Arg, Leu, Lys, Ser, Arg-Gly, Arg-Thr, Lys-Ala, Lys-Glu or Pro-Asp-Gly-Glu-Glu-Leu-Lys;

[0012] When the two Cys side chain thiol groups form an intramolecular disulfide bond, the polypeptide is a disulfide bond cyclic peptide.

[0013] Furthermore, in a preferred embodiment of the present invention, the polypeptide includes, but is not limited to, AHF1 to AHF20 as shown in the following structures:

[0014] .

[0015] The polypeptide can be prepared using polypeptide preparation methods known in the art, including but not limited to chemical synthesis methods using solid-phase polypeptide synthesis technology. Alternatively, the corresponding coding nucleotide sequence of the polypeptide can be deduced based on its amino acid sequence, a recombinant expression vector can be constructed, and biosynthesis can be carried out in prokaryotic or eukaryotic host cells.

[0016] The above-mentioned peptides are used in the preparation of products for the prevention and / or treatment of liver fibrosis or chronic liver disease with liver fibrosis.

[0017] The liver fibrosis mentioned includes, but is not limited to, liver fibrosis caused by factors such as chronic viral hepatitis, alcoholic liver disease, metabolic dysfunction-related fatty liver disease or hepatitis, drug or toxic liver injury, autoimmune liver disease, cholestasis or biliary tract disease, hereditary or metabolic liver disease, vascular or congestive liver disease, etc.

[0018] The products mentioned are pharmaceuticals, health products, or medical devices.

[0019] The medicine is used for the prevention and / or treatment of liver fibrosis or chronic liver disease with liver fibrosis.

[0020] The aforementioned health products are suitable for liver function regulation and early intervention in high-risk groups for liver fibrosis or patients with chronic liver disease.

[0021] The medical device may be a diagnostic and therapeutic device, a drug delivery device, an implantable device or an interventional device containing the polypeptide as a functional component, or an in vitro diagnostic device or test kit that uses the polypeptide as a recognition molecule or functional ligand.

[0022] The aforementioned pharmaceuticals and health products contain one or more of the above-mentioned polypeptides, or pharmaceutically acceptable salts, stereoisomers, prodrugs, or metabolites thereof, with the remainder being excipients and / or other compatible drugs.

[0023] The excipients are at least one of solvents, slow-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, adsorbent carriers, surfactants, preservatives, and lubricants.

[0024] The other compatible drugs mentioned can be at least one of natural drugs, chemical drugs, and biological drugs with antiviral, antioxidant, anti-inflammatory, or hepatoprotective effects, in order to achieve combined drug use or synergistic therapeutic effects.

[0025] The aforementioned drugs or health products can be prepared into conventional pharmaceutical dosage forms such as tablets, capsules (including sustained-release capsules), oral solutions, granules, injections, lyophilized powder injections, controlled-release preparations, or liver-targeted drug delivery preparations to meet different clinical application scenarios.

[0026] The medical device comprises one or more of the above-mentioned polypeptides, or their pharmaceutically acceptable salts, stereoisomers, prodrugs, or metabolites as functional components, for interventional and / or local treatment of liver fibrosis.

[0027] A pharmaceutical composition comprising the above-mentioned polypeptide and a pharmaceutically acceptable carrier.

[0028] Compared with the prior art, the present invention has the following advantages and effects:

[0029] (1) The polypeptide provided by the present invention can inhibit TGF-β1-induced activation of hepatic stellate cells and significantly downregulate the expression of liver fibrosis-related markers such as α-smooth muscle actin (α-SMA) and type I collagen α1 chain (COL1A1), thereby reducing excessive deposition of collagen and extracellular matrix.

[0030] (2) In various animal models of liver fibrosis, the polypeptide provided by the present invention showed good in vivo anti-liver fibrosis effect.

[0031] (3) The polypeptide provided by the present invention has no obvious acute toxicity to experimental animals at higher doses, and no obvious adverse reactions have been observed, indicating good safety.

[0032] (4) The polypeptides provided by the present invention can be stably prepared by mature chemical synthesis or bioengineering expression methods, which are suitable for large-scale production and have good industrialization and application prospects. Attached Figure Description

[0033] Figure 1 This is a high-resolution mass spectrum of the polypeptide AHF1; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0034] Figure 2 This is a high-resolution mass spectrum of polypeptide AHF2; where A is the primary mass spectrum and B to C are the secondary mass spectra.

[0035] Figure 3 This is a high-resolution mass spectrum of polypeptide AHF3; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0036] Figure 4 This is a high-resolution mass spectrum of polypeptide AHF4; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0037] Figure 5 This is a high-resolution mass spectrum of peptide AHF5; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0038] Figure 6 This is a high-resolution mass spectrum of polypeptide AHF6; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0039] Figure 7 This is a high-resolution mass spectrum of polypeptide AHF7; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0040] Figure 8This is a high-resolution mass spectrum of polypeptide AHF8; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0041] Figure 9 This is a high-resolution mass spectrum of polypeptide AHF9; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0042] Figure 10 This is a high-resolution mass spectrum of polypeptide AHF10; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0043] Figure 11 This is a high-resolution mass spectrum of polypeptide AHF11; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0044] Figure 12 This is a high-resolution mass spectrum of the polypeptide AHF12; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0045] Figure 13 This is a high-resolution mass spectrum of polypeptide AHF13; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0046] Figure 14 This is a high-resolution mass spectrum of polypeptide AHF14; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0047] Figure 15 This is a high-resolution mass spectrum of polypeptide AHF15; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0048] Figure 16 This is a high-resolution mass spectrum of polypeptide AHF16; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0049] Figure 17 This is a high-resolution mass spectrum of polypeptide AHF17; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0050] Figure 18 This is a high-resolution mass spectrum of the polypeptide AHF18; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0051] Figure 19 This is a high-resolution mass spectrum of the polypeptide AHF19; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0052] Figure 20 This is a high-resolution mass spectrum of polypeptide AHF20; where A is the primary mass spectrum and B to D are the secondary mass spectra.

[0053] Figure 21 This figure shows the effect of peptides AHF1–AHF20 on TGF-β1-induced ACTA2 mRNA expression levels in LX-2 cells; data are expressed as mean ± standard deviation (Mean ± SD, n = 3); compared with the normal control group, ### ** indicates P < 0.001; compared with the model group, ** indicates P < 0.01, and *** indicates P < 0.001.

[0054] Figure 22 This figure shows the effect of peptides AHF1–AHF20 on TGF-β1-induced COL1A1 mRNA expression levels in LX-2 cells; data are expressed as mean ± standard deviation (Mean ± SD, n = 3); compared with the normal control group, ### *** indicates P < 0.001; compared with the model group, *** indicates P < 0.001. Detailed Implementation

[0055] The substantive content of the present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the content of the present invention is not limited thereto.

[0056] In the following examples, unless otherwise specified, the experimental methods were performed under standard experimental conditions or according to the manufacturer's recommendations. All materials and reagents used are commercially available products unless otherwise stated.

[0057] Example 1: Synthesis of polypeptides AHF1-AHF20

[0058] (1) Solid-phase synthesis of polypeptide AHF1

[0059] 2.0 g of 2-chlorotriphenylmethyl resin (0.40 mmol / g) with a degree of substitution of 0.40 mmol / g was weighed and placed in a reactor. 20 mL of dichloromethane (DCM) was added, and the resin was allowed to swell at room temperature for 20 min. The solvent was then removed by filtration. 0.88 mmol of Fmoc-Arg(Pbf)-OH and 15 mL of N,N-dimethylformamide (DMF) were added, followed by the dropwise addition of 2 mL of N,N-diisopropylethylamine. The mixture was stirred at room temperature for 1.5 h. After the reaction was complete, the resin was washed three times with DMF. 4 mL of methanol and 15 mL of DCM were added, and the mixture was reacted at room temperature for 35 min to block the unreacted active sites of the resin. The reaction solution was removed by filtration, and the resin was washed three times with DMF. Subsequently, 20 mL of a 20% (v / v) piperidine / DMF solution was added to the resin, and the mixture was stirred for 10 min to remove Fmoc. After filtering to remove the reaction solution, another 20 mL of the same piperidine / DMF solution was added, and the mixture was stirred for another 5 min. After filtering to remove the reaction solution, the resin was washed 5 times with DMF. Fmoc-Thr(tBu)-OH and 1-hydroxybenzotriazole were weighed at 3 molar equivalents of the resin's degree of substitution, dissolved in an appropriate amount of DMF, and activated with N,N′-diisopropylcarbodiimide (DIC) for about 10 min. Then, the mixture was added to the resin for condensation, and stirred at room temperature for 1 h. After the reaction was completed, the reaction solution was filtered to remove the reaction solution, and the mixture was washed 5 times with DMF. Following the cycle of "Fmoc removal → amino acid condensation", each amino acid in the AHF1 sequence was condensed sequentially until the N-terminal asparagine residue was condensed and Fmoc removal was completed. The mixture was then washed sequentially with DMF and methanol and dried to obtain 3.20 g of resin-loaded peptide. Prepare 15 mL of lysis buffer for every 1 g of resin-loaded peptide. Mix deionized water, triisopropylsilane (TIS), 1,2-ethylenedithiol (EDT), and trifluoroacetic acid (TFA) at a volume ratio of TFA:TIS:EDT:H₂O = 95:2:2:1 (v / v / v / v) to prepare a TFA / TIS / EDT / water lysis buffer. Pre-cool the lysis buffer to 0–10 °C, then add the resin-loaded peptide while stirring. After the system temperature stabilizes, continue stirring at 20–25 °C for 2 h to complete lysis. Filter to remove the resin and collect the filtrate. Slowly add 10 volumes of ice-cold anhydrous diethyl ether to precipitate the peptide. Centrifuge to collect the precipitate and wash four times with cold anhydrous diethyl ether. Dry under reduced pressure at room temperature to obtain 700 mg of crude peptide. The crude polypeptide was dissolved in a 20% (v / v) acetonitrile / water solution, the pH was adjusted to 7-8 with dilute ammonia, and the mixture was stirred and left to stand overnight at room temperature to complete the oxidation of disulfide bonds. Dilute acetic acid was then added to adjust the pH to approximately 6.0.The crude peptide was purified by preparative reversed-phase C18 column (10 μm, 50 mm × 250 mm) high-performance liquid chromatography (HPLC). The solution was equilibrated with 10% acetonitrile for 10 min in a 0.1% (v / v) TFA aqueous solution / 0.1% (v / v) TFA-acetonitrile system. The oxidized peptide solution was then injected and purified by linear gradient elution with 10%–40% acetonitrile. The target peak fraction was collected. The collected target fraction was analyzed by analytical HPLC. After removing the organic solvent under reduced pressure, the fraction was lyophilized to obtain 92 mg of white AHF1 powder. Mass spectrometry analysis confirmed that the molecular weight was consistent with the theoretical value, and the purity was 99.25%.

[0060] (2) Solid-phase synthesis of polypeptides AHF2 to AHF20

[0061] The synthesis methods for peptides AHF2 to AHF20 are basically the same as the solid-phase synthesis process for AHF1. Using the aforementioned Fmoc solid-phase synthesis strategy, the peptides are synthesized by sequentially performing Fmoc removal, amino acid activation condensation, and DMF washing from the C-terminus to the N-terminus, linking each amino acid residue in the peptide sequence one by one to 2-chlorotriphenylmethyl resin to obtain the corresponding resin-loaded peptides. Subsequently, the resin-loaded peptides are cleaved using the same TFA / TIS / EDT / water cleavage system as for AHF1, removing the resin carrier and side-chain protecting groups. After oxidation, they are purified by preparative reversed-phase C18 column high-performance liquid chromatography, finally obtaining pure peptides AHF2 to AHF20.

[0062] Example 2: Mass spectrometry identification of peptides AHF1 to AHF20

[0063] Appropriate amounts of peptides AHF1 to AHF20 were weighed and dissolved in deionized water to prepare sample solutions with a mass concentration of 0.10 mg / mL. After removing particulate impurities by filtering each sample solution through a 0.22 μm aqueous microporous membrane, they were injected into a Q Exactive Plus high-resolution orbital trap mass spectrometer equipped with a heated electrospray ionization source for analysis.

[0064] Mass spectrometry analysis was performed in HESI positive ion mode, with the following key parameters set: spray voltage +3.5 kV, capillary temperature 320 °C, spray probe auxiliary heating temperature 350 °C, sheath gas flow rate 35 arb, auxiliary gas flow rate 12 arb, and S-Lens RF level set to 55.0. Mass spectrometry acquisition employed full scan (Full MS) combined with high-energy collisional dissociation (HCD) tandem mass spectrometry (MS / MS) mode. First, the multi-charge precursor ion peak of the peptide was acquired in Full MS mode. Then, the target precursor ion was selected for HCD fragmentation, with the normalized collision energy (NCE) set to 27%. The b-ion and y-ion series in the acquired MS / MS spectra were compared with the theoretical amino acid residue sequences to verify the amino acid sequences and molecular weights of peptides AHF1–AHF20. The corresponding mass spectra are shown below. Figures 1-20 The amino acid sequences of the polypeptides AHF1 to AHF20, as shown in the technical solution described above, were confirmed.

[0065] Example 3: Effects of peptides AHF1-AHF20 on TGF-β1-induced collagen synthesis and fibrosis-related gene expression in hepatic stellate cells

[0066] Logarithmic growth phase human hepatic stellate cell line LX-2 (purchased from Shanghai Fuheng Biotechnology Co., Ltd.) was collected by routine trypsin digestion and centrifugation, and resuspended in complete culture medium (10% fetal bovine serum FBS + 1% glutamine Gln + high glucose DMEM). The cells were then cultured at 4.5 × 10⁻⁶ cells / day. 5 Cells were seeded at a density of / well in 6-well plates and cultured at 37 ℃ in a 5% CO2 incubator until adherence. When cell confluence reached approximately 50%–60%, the old culture medium was discarded, and the cells were washed 2–3 times with PBS. Subsequently, normal control, model, and AHF1–AHF20 treatment groups were established. The normal control group was given fresh complete culture medium without TGF-β1 and peptides; the model group was given fresh complete culture medium containing TGF-β1 (final concentration 5 ng / mL), and incubated for 24 h to establish a hepatic stellate cell activation model. Each treatment group was incubated with 1 μM peptide AHF1–AHF20 after TGF-β1 (5 ng / mL) induction, and incubated for another 24 h.

[0067] After incubation, the culture medium was discarded, cells were washed with PBS, TRIzol was added to lyse the cells, and total RNA was extracted using the standard chloroform / isopropanol precipitation method. After washing with 75% ethanol, the RNA was dissolved in RNase-free water, and RNA concentration and A were measured using a NanoDrop 2000. 260 / A280 Samples with a ratio of approximately 1.7 to 2.0 were selected for subsequent experiments.

[0068] Following the reverse transcription kit instructions, a 20 μL reverse transcription reaction system was prepared using 1000 ng of total RNA. This system consisted of 1000 ng of total RNA, 11 μL of RevertAid reverse transcription premix, and RNase-free ddH2O, with the RNase-free ddH2O brought to a final volume of 20 μL. The reverse transcription conditions were: 42 °C for 30 min, followed by 95 °C for 5 min to terminate the reaction, and then storage at 4 °C to obtain first-strand cDNA. The obtained cDNA was diluted to 5 ng / μL with RNase-free ddH2O and used as a template for real-time quantitative PCR (RT-qPCR) using the SYBR Green system. Each PCR reaction system consisted of 20 μL of SYBR premix, including 10 μL of SYBR premix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 2 μL of cDNA template, and 7 μL of RNase-free ddH2O. PCR amplification conditions were: 95 ℃ pre-denaturation for 2 min; 95 ℃ denaturation for 15 s, 60 ℃ annealing and extension for 1 min, for a total of 40 cycles. Melting curve analysis was performed after amplification, with reaction conditions of 95 ℃ for 15 s, 60 ℃ for 1 min, and 95 ℃ for 15 s. Using GAPDH as an internal reference gene, the mRNA expression levels of the myofibroblast marker gene ACTA2 and the type I collagen α1 chain encoding gene COL1A1 were detected. The primer sequences used are as follows: ACTA2-F (5′-ACTGAGCGTGGCTATTCCTCCGTT-3′), ACTA2-R (5′-GCAGTGGCCATCTCATTTTCA-3′), COL1A1-F (5′-GTGCGATGACGTGATCTGTGA-3′), COL1A1-R (5′-CGGTGGTTTCTTGGTCGGT-3′), GAPDH-F (5′-AAGAAGGTGGTGAAGCAGGC-3′), GAPDH-R (5′-TCCACCACCCTGTTGCTGTA-3′). After amplification, the Ct values ​​of each sample were obtained, and a 2-T value was used. -△△Ct The relative expression levels of each treatment group relative to the normal control group were calculated, and the differences between the model group and each treatment group were statistically analyzed.

[0069] Experimental results showed that, under TGF-β1 induction conditions, treatment with 1 μM peptides AHF1–AHF20 significantly downregulated the mRNA expression levels of ACTA2 and COL1A1 in LX-2 cells compared to the model group (see [link to data]). Figure 21 and Figure 22 Among them, ACTA2 encodes α-smooth muscle actin (α-SMA), and COL1A1 encodes type I collagen α1 chain, both of which are key molecular markers in the activation of hepatic stellate cells and the process of liver fibrosis. These results indicate that peptides AHF1–AHF20 can significantly inhibit the abnormal increase in TGF-β1-induced hepatic stellate cell activation and collagen synthesis-related genes, thereby inhibiting the transformation of hepatic stellate cells into myofibroblast phenotypes and demonstrating clear anti-liver fibrosis activity.

[0070] Example 4: Therapeutic effect of peptides AHF1-AHF5 on carbon tetrachloride (CCl4)-induced liver fibrosis model mice

[0071] (1) Establishment and grouping of the CCl4 liver fibrosis model: SPF-grade 6-week-old male C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After the mice were acclimatized, they were randomly divided into a normal control group, a model group, a low-dose group (5 mg / kg) of AHF1 to AHF5 and a high-dose group (25 mg / kg), with 10 mice in each group. Mice in the normal control group were injected intraperitoneally with 200 μL of olive oil, while the other groups were injected intraperitoneally with 200 μL of 12.5% ​​CCl4 (dissolved in olive oil), once every other day to establish the model. Two weeks after the model was established (starting from the first injection of CCl4), the AHF1 to AHF5 groups were administered 5 mg / kg or 25 mg / kg of the corresponding polypeptide solution by gavage, while the normal control group and the model group were administered an equal volume of physiological saline by gavage. The administration volume was 0.1 mL / mouse, and the administration continued until the model was established for 6 weeks. The body weight of the mice was recorded weekly during the experiment. Mice were euthanized after modeling, whole blood was collected and serum was separated. At the same time, liver tissue was quickly separated. A portion of the liver tissue was used for the determination of hydroxyproline content, and the remaining liver tissue was fixed in 4% paraformaldehyde solution for pathological analysis.

[0072] (2) Determination of liver function and hydroxyproline (Hyp) content in liver tissue: The alanine aminotransferase (ALT) level in serum samples was detected using a fully automated biochemical analyzer to reflect the degree of hepatocyte damage. Approximately 50 mg of liver tissue from each group of mice was weighed and processed according to the hydroxyproline detection kit instructions. After acid hydrolysis (6 mol / L HCl, 110 ℃, 16 h), the samples were cooled and centrifuged. The supernatant was collected and neutralized to neutral with sodium hydroxide solution. Subsequently, the oxidation and colorimetric reactions were completed according to the kit instructions, and the absorbance was measured at 550 nm. A standard curve was plotted using hydroxyproline standards to calculate the Hyp content of the samples.

[0073] (3) Sirius Red staining and pathological observation of liver tissue: Liver tissue fixed with 4% paraformaldehyde was routinely dehydrated, paraffin-embedded, and sectioned. After dewaxing and hydration, the sections were stained with collagen fibers according to the instructions of the Sirius Red staining kit. The structural changes of liver tissue, including inflammatory cell infiltration, fibrous septum formation, and destruction of liver lobule structure, were observed under an optical microscope. The area of ​​Sirius Red positive collagen fibers was analyzed by image analysis, and the percentage of collagen-positive area to the total field of view (PSR⁺ area, %) was calculated as a morphological evaluation indicator of the degree of liver fibrosis.

[0074] The experimental results showed that, compared with the normal control group, the serum ALT level of mice in the model group was significantly increased after CCl4 modeling, and the Hyp content in the liver tissue was significantly increased, indicating that the mice had significant hepatocyte damage and collagen deposition, and the liver fibrosis model was successfully established. Compared with the model group, the serum ALT level and liver tissue Hyp content of mice were significantly decreased after administration of AHF1 to AHF5 at each dose, with the high-dose group showing a more significant improvement, indicating that AHF1 to AHF5 can effectively alleviate CCl4-induced liver damage and inhibit excessive collagen deposition (see Table 1).

[0075] Sirius Red staining results showed that almost no collagen fiber deposition was observed in the liver tissue of the normal control group. In the model group, after 6 weeks of modeling, a large number of red-stained collagen fibers appeared in the portal and ductal areas, the fibrous septa were significantly thickened, and the PSR⁺ area (%) was significantly increased. After treatment with AHF1 to AHF5, collagen fiber deposition in the liver tissue was significantly reduced, the red staining intensity was weakened, the fibrous septa were thinned, and the PSR⁺ area (%) was significantly lower than that in the model group, suggesting that all five peptides can significantly improve the degree of CCl4-induced liver fibrosis (see Table 1).

[0076] Table 1. Effects of peptides AHF1-AHF5 on CCl4-induced liver fibrosis model mice.

[0077]

[0078] Note: Data are expressed as mean ± standard deviation (Mean ± SD); compared with the normal control group, ### This indicates that P < 0.001; compared to the model group, ** This indicates that P < 0.01. *** This indicates that P < 0.001.

[0079] Example 5: Therapeutic effects of peptides AHF1-AHF5 on a mouse model of liver fibrosis induced by bile duct ligation (BDL).

[0080] Six-week-old male SPF-grade C57BL / 6J mice were randomly divided into four groups: sham-operated group, BDL model group, low-dose BDL+AHF1-AHF5 group (5 mg / kg), and high-dose BDL+AHF1-AHF5 group (25 mg / kg). Mice were fasted for 12 hours preoperatively, and their body weight was recorded. Anesthesia was administered via intraperitoneal injection of 8% tribromoethanol. After adequate anesthesia, routine skin preparation and disinfection were performed. An abdominal incision was made along the midline, and the liver lobe and part of the intestine were gently separated to expose the common bile duct parallel to the portal vein. A short segment of the common bile duct was bluntly dissected using ophthalmic forceps, and double-ligated at its proximal end with 6-0 surgical sutures. The bile duct was then cut between the ligations, completing the bile duct ligation model. The organs were then repositioned, and the abdominal wall was sutured layer by layer. In the sham-operated group, the common bile duct was exposed only after anesthesia and abdominal incision; no ligation or cutting was performed. After repositioning, the abdomen was closed following the same steps. Postoperatively, the mice were placed in a dry, warm environment for recovery and allowed free access to food and water. Starting from day 2 post-surgery, the peptide treatment group received the corresponding peptide solution via gavage at doses of 5 mg / kg or 25 mg / kg, while the sham-operated group and BDL model group received an equal volume of physiological saline via gavage. The administration volume was 0.1 mL per mouse, once daily for 14 consecutive days. The general condition of the mice was observed daily during the administration period, and body weight was recorded weekly. After the last administration, the mice were anesthetized with isoflurane, blood was collected via the orbital puncture, and the mice were euthanized. The liver was rapidly separated, and approximately 30 mg of liver tissue was collected. The mRNA expression levels of ACTA2 and COL1A1 in the liver tissue were detected using the aforementioned RT-qPCR method.

[0081] The experimental results showed that, compared with the sham-operated group, the mRNA expression levels of ACTA2 and COL1A1 in the liver tissue of mice in the BDL model group were significantly upregulated, suggesting that bile duct ligation can induce significant activation of hepatic stellate cells and promote type I collagen synthesis. Compared with the BDL model group, the relative expression levels of ACTA2 and COL1A1 in the liver tissue of mice after administration of AHF1 to AHF5 were significantly reduced, and the inhibitory effect was more obvious in the high-dose group (see Table 2), indicating that the five peptides also have a clear in vivo anti-hepatic fibrosis effect in the cholestatic liver fibrosis model.

[0082] Table 2. Effects of peptides AHF1-AHF5 on liver tissue in mice with BDL-induced liver fibrosis model.

[0083]

[0084] Note: Data are expressed as mean ± standard deviation (Mean ± SD); compared with the sham surgery group, ### This indicates that P < 0.001; compared with the BDL model group, *** This indicates that P < 0.001.

[0085] Example 6: Acute toxicity test of peptides AHF1-AHF5

[0086] Six-week-old male SPF-grade C57BL / 6J mice were randomly divided into four groups (AHF1, AHF2, AHF3, AHF4, and AHF5) after a one-week acclimatization period, with ten mice in each group. Each group was administered a single oral gavage dose of peptides AHF1–AHF5 at a dose of 100 mg / kg, with a gavage volume of 0.20 mL per mouse. Mice were observed for 14 days post-administration, and their general condition (mental state, coat color, diet, water intake, and behavior), excretion, and any signs of poisoning such as respiratory abnormalities or convulsions were recorded. The time of death and the number of mice that died were also recorded to assess acute toxicity.

[0087] The experimental results showed that during the 14-day observation period, mice in the AHF1-AHF5 groups were in good condition, with glossy fur, normal diet and water intake, normal behavior, and normal excretion without diarrhea. No obvious toxic reactions or deaths were observed (see Table 3). The above experimental results suggest that the peptides AHF1-AHF5 have low acute toxicity and good safety.

[0088] Table 3. Results of acute toxicity tests on peptides AHF1–AHF5

[0089]

[0090] Example 7: Preparation of tablets

[0091] Weigh 0.1 g of peptide AHF2 and mix it evenly with 39.9 g of lactose and 59.8 g of pregelatinized starch. The mixture is then sieved through an 80-mesh sieve to remove agglomerated particles. The resulting mixture is dried in a 50 °C forced-air drying oven for 2 h until the moisture content is below 3.0%. After drying, 0.2 g of magnesium stearate is added to the mixture as a lubricant, and the mixture is mixed thoroughly in a V-type mixer at 20 rpm for 5 min. The mixed material is then placed in a single-punch tablet press and compressed into tablets at a pressure of 8 kN and a speed of 30 rpm, yielding 100 tablets, each containing 1 mg of peptide AHF2.

[0092] Example 8: Preparation of Injectable Form

[0093] Weigh 0.1 g of peptide AHF2 and add it to 50 g of propylene glycol. Place the mixture on a magnetic stirrer and stir at 300 rpm for 10 min until fully dissolved. Then add 100 mL of water for injection and continue stirring until the system is homogeneous. Add 9 g of sodium chloride to the resulting solution and stir at room temperature until completely dissolved. Then add water for injection to bring the volume to 1000 mL. Adjust the pH of the solution to 6.0–6.5 using 0.1 mol / L sodium hydroxide solution. After adjustment, the solution is sterilely filtered through a 0.22 μm sterile microporous membrane and aseptically dispensed into sterilized injection vials, sealed, to obtain 500 injection vials, each containing 0.2 mg of peptide AHF2.

[0094] Example 9: Preparation of Controlled-Release Capsules

[0095] Weigh 0.99 g of starch, add an appropriate amount of purified water and stir to disperse. Heat to 80–90 °C and stir continuously to fully gelatinize the starch. After cooling to room temperature, add purified water to make up to a total mass of 9.9 g, obtaining a starch slurry with a mass fraction of 10%, for later use. Weigh 0.1 g of peptide AHF2 and 40 g of lactose, mix evenly and add to a rotary granulator. Spray the above 9.9 g starch slurry as a binder. Prepare peptide-loaded particles under spray pressure of 0.2 MPa and inlet air temperature of 45 °C. Mix isopropanol and purified water at a mass ratio of 90:10 as a solvent, add ethyl cellulose to prepare a coating solution with a mass fraction of 15% ethyl cellulose. Add 5% poloxamer 188 (by mass of ethyl cellulose) as a wetting agent and dispersing stabilizer, and stir evenly. Spray the coating solution onto the surface of the drug-loaded particles at a rate of 2.0 mL / min to obtain AHF2 sustained-release particles with an average particle size of approximately 450 μm. After drying the obtained sustained-release granules to a constant weight, they were uniformly packed into hard capsule shells to obtain controlled-release capsules containing 1 mg of polypeptide AHF2 per capsule.

[0096] Example 10: Medical device composition containing peptide AHF2 (hepatic artery interventional perfusion catheter)

[0097] Hepatic artery perfusion catheters were fabricated using medical-grade polyurethane (PU) material. After being cut to standard dimensions of 80 cm in length and 0.8 mm in inner diameter, the catheter lumen was modified using plasma surface activation treatment to form a hydrophilic active layer on its surface. A polypeptide AHF2 was weighed and dissolved in 0.01 mol / L phosphate-buffered saline (PBS) at pH 7.4 to prepare a polypeptide solution with a mass concentration of 1 mg / mL, which was then filtered through a 0.22 μm sterile microporous membrane. Under aseptic conditions, the surface-activated and sterilized catheter lumen was completely filled with the AHF2 solution and incubated at 25 °C for 12 h to allow AHF2 adsorption and loading onto the catheter lumen surface. After incubation, the catheter lumen was slowly flushed with sterile phosphate-buffered saline to remove unadsorbed free AHF2. The AHF2 loading on the catheter lumen surface was calculated by measuring the change in polypeptide content in the AHF2 solution before and after loading. AHF2-loaded catheters are dried and sealed under aseptic conditions to obtain hepatic artery perfusion catheters containing the novel polypeptide AHF2. In use, the catheter is inserted into the target area via the hepatic artery interventional route. During fluid perfusion, the AHF2 adsorbed on the inner surface of the catheter is gradually desorbed and released, thereby achieving local delivery of AHF2.

[0098] The above description represents a preferred embodiment of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A polypeptide with anti-liver fibrosis activity, characterized in that: The structure of the polypeptide is shown in Formula I: ； In Equation I: R1 is Asn, Lys, Thr-Leu, His-Lys, Pro-Asn, Gly-Thr-Leu, Gly-Tyr-Lys, Pro-Tyr-Lys, Pro-Gly-Thr-Leu, Ala-Pro-Gly-Thr-Leu, Ser-Lys-Thr-Asp-Val, Pro-Ala-Pro-Gly-Thr-Leu, Pro-Asn-Asn-Pro-Tyr-Gly, Ala-Pro-Ala-Pro-Gly-Thr-Leu, Leu-Ala-Pro-Ala-Pro-Gly-Thr-Leu, or does not exist; R2 is Gly-Pro, Glu-Leu, Asp-Phe, Val-Ala, Gly-Val-Ala-Gly, Ser-Ser-Gly-Gly-Glu-Leu, Leu-Ile-Glu-Glu-Gly-Lys, Leu-Pro-Asn-Gly-Arg-Ala-Ala or Ser-Arg-Asp-Gly-Met-Asn-Ala-Ala; R3 is Ala, Arg, Leu, Lys, Ser, Arg-Gly, Arg-Thr, Lys-Ala, Lys-Glu or Pro-Asp-Gly-Glu-Glu-Leu-Lys; When the two Cys side chain thiol groups form an intramolecular disulfide bond, the polypeptide is a disulfide bond cyclic peptide.

2. The polypeptide with anti-liver fibrosis effect according to claim 1, characterized in that: The structure of the polypeptide is shown in any one of AHF1 to AHF20: 。 3. The use of the polypeptide of claim 1 or 2 in the preparation of products for the prevention and / or treatment of liver fibrosis or chronic liver disease with liver fibrosis.

4. The use of the polypeptide according to claim 3 in the preparation of products for the prevention and / or treatment of liver fibrosis or chronic liver disease with liver fibrosis, characterized in that: The products mentioned are pharmaceuticals, health products, or medical devices.

5. The use of the polypeptide according to claim 4 in the preparation of products for the prevention and / or treatment of liver fibrosis or chronic liver disease with liver fibrosis, characterized in that: The liver fibrosis mentioned is caused by at least one of the following factors: chronic viral hepatitis, alcoholic liver disease, metabolic dysfunction-related fatty liver disease or hepatitis, drug or toxic liver injury, autoimmune liver disease, cholestasis or biliary tract disease, hereditary or metabolic liver disease, vascular or congestive liver disease. The medicine is used for the prevention and / or treatment of liver fibrosis or chronic liver disease with liver fibrosis; The aforementioned health products are used for liver function regulation and early intervention in high-risk groups for liver fibrosis or patients with chronic liver disease; The medical device is a diagnostic and therapeutic device, drug delivery device, implantable device or interventional device containing the polypeptide as a functional component, or an in vitro diagnostic device or test kit that uses the polypeptide as a recognition molecule or functional ligand.

6. The use of the polypeptide according to claim 4 in the preparation of products for the prevention and / or treatment of liver fibrosis or chronic liver disease with liver fibrosis, characterized in that: The pharmaceuticals and health products described herein contain one or more of the polypeptides described in claim 1 or 2, or pharmaceutically acceptable salts, stereoisomers, prodrugs, or metabolites thereof, with the remainder being excipients and / or other compatible drugs.

7. The use of the polypeptide according to claim 6 in the preparation of products for the prevention and / or treatment of liver fibrosis or chronic liver disease with liver fibrosis, characterized in that: The excipients are at least one of the following: solvent, sustained-release agent, excipient, filler, binder, wetting agent, disintegrant, absorption promoter, adsorbent carrier, surfactant, preservative and lubricant; The other compatible drugs may be at least one of natural drugs, chemical drugs, and biological drugs with antiviral, antioxidant, anti-inflammatory, or hepatoprotective effects.

8. The use of the polypeptide according to claim 6 in the preparation of products for the prevention and / or treatment of liver fibrosis or chronic liver disease with liver fibrosis, characterized in that: The dosage forms of the aforementioned drugs and health products are tablets, capsules, oral solutions, granules, injections, lyophilized powder injections, controlled-release preparations, or liver-targeted drug delivery preparations.

9. The use of the polypeptide according to claim 4 in the preparation of products for the prevention and / or treatment of liver fibrosis or chronic liver disease with liver fibrosis, characterized in that: The medical device comprises one or more of the polypeptides described in claim 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, prodrug, or metabolite thereof as a functional component.

10. A pharmaceutical composition, characterized in that: It contains the polypeptide as described in claim 1 or 2 and a pharmaceutically acceptable carrier.

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