Fusion protein and application thereof in preparation of medicine for intervening hepatic fibrosis process

Abstract

The invention discloses a fusion protein and application thereof in preparation of a medicine for intervening in a hepatic fibrosis process, and relates to the field of preparation of biological products. The invention designs and constructs a novel fusion protein which has targeted binding capacity and efficient thrombolysis capacity. Besides, according to the invention, a gene for coding the fusion protein and a polypeptide gene capable of specifically neutralizing plasminogen activator inhibitor 1 (PAI-1) are creatively connected through a P2A self-cleavage peptide and constructed in a single expression vector, so that co-expression of two functional proteins is realized. The co-expression system can directly and efficiently activate plasminogen through fusion protein in a hepatic fibrosis pathological microenvironment on one hand, and remove endogenous inhibition through PAI-1 polypeptide binding inhibitor on the other hand, so that degradation of fibrous protein and extracellular matrix is doubly and efficiently promoted; and a new active molecule and gene technology strategy is provided for the intervention of the hepatic fibrosis related pathological process.

Description

Technical Field

[0001] This invention relates to the field of biological product preparation, and more specifically to a fusion protein and its application in the preparation of drugs that intervene in the process of liver fibrosis. Background Technology

[0002] Liver fibrosis is a complex wound repair response initiated by the liver after persistent damage (such as viral infection, alcohol abuse, metabolic abnormalities, or autoimmune attack). Its core pathological feature is an imbalance between the synthesis and degradation of the extracellular matrix (ECM) in the liver, leading to excessive deposition of ECM, particularly collagen (such as type I and type III collagen). Under the stimulation of chronic injury, hepatic stellate cells (HSCs) are activated and transdifferentiate into myofibroblasts, which are the main source of ECM. Myofibroblasts not only synthesize and secrete large amounts of ECM components such as collagen and fibronectin, but also express α-smooth muscle actin (α-SMA), acquiring contractile ability and further exacerbating the distortion of tissue structure. As fibrosis progresses, the normal liver lobule structure is destroyed, forming fibrous septa and pseudolobules, which may eventually develop into cirrhosis, leading to portal hypertension, liver failure, and even liver cancer, seriously threatening human health.

[0003] The fibrinolytic system is an important proteolytic system in the body, with its core functions being the dissolution of blood clots and the maintenance of vascular patency. This system mainly consists of plasminogen (PLG), plasminogen activators (PAs), and plasminogen activator inhibitors (PAIs). Insufficient activity of the fibrinolytic system inhibits the degradation of the extracellular matrix, leading to excessive deposition and promoting fibrosis progression. Conversely, enhancing the activity of the fibrinolytic system can potentially break the vicious cycle of extracellular matrix deposition and promote the reversal of fibrosis.

[0004] Mounting evidence suggests that the fibrinolytic system plays a dual and complex role in the development and progression of liver fibrosis. On the one hand, plasminogen activator (PAI) expression may be upregulated in the early stages of liver injury, creating conditions for hepatocyte regeneration and tissue repair. However, as injury persists and fibrosis progresses, PAI-1 expression is significantly upregulated, far exceeding the increase in plasminogen activator expression, leading to suppression of the overall activity of the fibrinolytic system. This suppression reduces the degradation capacity of the extracellular matrix, thereby exacerbating net extracellular matrix deposition. Conversely, PAI-1 gene knockout mice can delay the progression of liver fibrosis.

[0005] PAI-1 is a member of the serine protease inhibitor superfamily and is the most important physiological inhibitor of plasminogen activator. It is a single-chain glycoprotein with a molecular weight of approximately 50 kDa, composed of 379 amino acids. The active site of PAI-1 is located at the Arg346-Met347 peptide bond, which can form a stable 1:1 covalent complex with the active site of tPA / uPA, thereby irreversibly inhibiting its activity. PAI-1 activity is regulated by various factors, including its own conformational changes (active state, latent state, and substrate state), binding to vitronectin, and interaction with cell surface receptors. Under physiological conditions, PAI-1 is mainly synthesized and secreted by the liver, vascular endothelial cells, and platelets, playing a crucial role in maintaining fibrinolytic homeostasis. Under pathological conditions, such as inflammation, tumors, and fibrosis, various cytokines (such as TGF-β and IL-1β) can induce PAI-1 expression, leading to inhibition of fibrinolytic activity. PAI-1 inhibits the activity of plasminogen activator, reduces plasmin production, thereby inhibiting the degradation of extracellular matrix (ECM) and promoting net ECM accumulation. This is its main mechanism for promoting fibrosis.

[0006] Therefore, how to efficiently and specifically enhance plasmin activity to address the insufficient degradation of extracellular matrix during the pathological process of liver fibrosis is a core technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a fusion protein and its application in the preparation of drugs that intervene in the process of liver fibrosis.

[0008] To solve the above-mentioned technical problems, this application adopts the following technical solution: The primary objective of this invention is to provide a novel, molecularly designed and optimized fusion protein designed to efficiently and specifically enhance plasmin activity, addressing the core issue of insufficient extracellular matrix degradation during liver fibrosis. This fusion protein can more effectively locate and degrade deposited fibrin and extracellular matrix within the liver fibrosis microenvironment, thus providing a potent molecular tool for reversing liver fibrosis.

[0009] A fusion protein, named HepaFibrinAct, has the amino acid sequence shown in SEQ ID No. 1.

[0010] The second core objective of this invention is to provide a co-expression system capable of achieving synergistic intervention. Considering the complexity of the pathological mechanisms of liver fibrosis and the limitations of single targets, this invention not only designed the aforementioned fusion protein, but also further designed a co-expression vector capable of simultaneously expressing the fusion protein and a PAI-1 peptide binding inhibitor.

[0011] A co-expression system for co-expressing the above-mentioned fusion protein and a PAI-1 peptide binding inhibitor.

[0012] Furthermore, the amino acid sequence of the PAI-1 peptide binding inhibitor is shown in SEQ ID No. 2.

[0013] This invention utilizes P2A self-cleaving peptide technology to separate the fusion protein and the PAI-1 peptide-binding inhibitor into two independent functional proteins after translation. This design combines two functional modules: targeted localization and fibrinolytic cascade triggering. In the model of this invention's embodiments, a synergistic improvement on liver fibrosis-related indicators was observed: on the one hand, the fusion protein directly enhances plasmin production; on the other hand, the PAI-1 peptide-binding inhibitor neutralizes endogenous PAI-1, relieving the inhibitory effect on the fibrinolytic system, thereby creating a more favorable microenvironment for the fusion protein's activity. This invention aims to achieve dual and efficient regulation of the fibrinolytic system through this synergistic effect, resulting in a superior anti-fibrotic effect compared to single-therapy approaches.

[0014] Furthermore, the gene expression cassette structure of the co-expression system is as follows: [EF1α short promoter]-[exotropic HepaFibrinAct open reading frame]-[P2A]-[exotropic anti-PAI-1 active binding polypeptide open reading frame]-[P2A]-[puromycin N-acetyltransferase]-[mini SV40 polyA signal].

[0015] Furthermore, the nucleotide sequence of the expression cassette is shown in SEQ ID No. 3.

[0016] The above-mentioned fusion protein and co-expression system are used in the preparation of drugs with active molecular molecules that intervene in liver fibrosis.

[0017] Compared with the prior art, the present invention has the following advantages: This invention successfully designed and constructed a novel fusion protein (HepaFibrinAct) and its co-expression system with a PAI-1 peptide-binding inhibitor through innovative protein engineering and gene intervention strategies. This system integrates two functions: enhancing plasminogen activation and reversing its key inhibition, achieving dual and synergistic regulation of the fibrinolytic system in the pathological microenvironment of liver fibrosis. Experimental data show that this co-expression system exhibits highly efficient plasminogen activating activity and PAI-1 inhibitory activity in vitro; at the cellular level, it significantly enhances the degradation capacity of the extracellular matrix (ECM); and in two classic animal models of liver fibrosis, it effectively improves liver function and significantly reduces ECM deposition. Its anti-fibrotic effect is superior to single-component intervention and is superior to existing positive controls under the specified model, dosage, and statistical test conditions. These results fully demonstrate the great potential of this invention in intervening in clinical processes related to liver fibrosis. This strategy not only overcomes the limitations of existing single-target interventions but also provides a new approach and strong experimental evidence for developing safer, more effective, and more precise anti-fibrotic biological agents, with broad clinical application prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 The SPR binding curve in Embodiment 1 of the present invention; Figure 2 This is the ELISA binding curve in Example 1 of the present invention; Figure 3 This is a 3D structural image of the PAI-1 peptide binding inhibitor in Example 2 of the present invention; Figure 4 This is an image showing the interaction between the PAI-1 peptide binding inhibitor and PAI-1 in Example 2 of the present invention, where the pink part represents the inhibitor and the blue part represents PAI-1. Figure 5 This refers to the binding affinity of the PAI-1 peptide binding inhibitor in Example 2 of the present invention; Figure 6 This is the PAI-1 peptidase inhibition curve in Example 2 of the present invention; Figure 7 This is the complete structure of the gene expression cassette in Example 3 of the present invention; Figure 8 The result shows the area of ​​the transparent halo in Embodiment 5 of the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1 Construction of HepaFibrinAct fusion protein One of the core technical solutions of this invention is the design of a novel fusion protein. This fusion protein, named "HepaFibrinAct," meaning "liver fibrinolysis activator," has undergone protein reconstruction and genetic engineering optimization to enhance its efficacy in intervening in liver fibrosis. The fusion protein consists of two key functional domains: one is the "FibrinAff Domain," which binds with high affinity to fibrin and ECM, guiding the entire fusion protein to specifically accumulate in liver fibrosis lesions; the other is the "PlasCata Domain," possessing highly efficient catalytic activity. This domain is the core engine for activating plasminogen and initiating the fibrinolytic cascade reaction. This "targeting + catalysis" design allows the HepaFibrinAct fusion protein to achieve lesion localization and locally enhance plasminogen activation and ECM degradation efficiency, thereby achieving precise and efficient ECM degradation.

[0022] (1) Design of HepaFibrinAct fusion protein The amino acid sequence of the HepaFibrinAct fusion protein is as follows: GVCIKKPTMSCDDHPCHNGTCSDGIGSFQCICMKGWEGRLCDHESYPCKENGCHPFCYPDEDTQQRYCSCAPGYQESDNPTECKPVVNFPCGKPKVKYPTPNFEELRIRISGGKPANKGDSPWQVILLDSRSKLKCGGVLIHSSWVLTAAHCVE HPKYLRVRLGEYDIRRYENSEMDFNIQKAIVHPNYTKSTSDNDIALLYLDKSVVFSKYILPICLPNLGLAHRELMKEGTETVVTGWGRQFEESRNRTYTLRFIKIPLASYTKCSQTMQNSMSENMLCAGMLGDTRDACEGDSGGPMVTELR, SEQ ID No.1.

[0023] Among them, SEQ ID No. 1, 1~176bp is the FibrinAff Domain structure, and SEQ ID No. 1, 177~305bp is the PlasCata Domain structure.

[0024] (2) Expression and purification of HepaFibrinAct fusion protein The designed HepaFibrinAct fusion protein gene sequence (exocrine HepaFibrinAct ORF) was cloned into an EF1α Short promoter-driven mammalian expression vector (containing a multiple cloning site and a polyA signal) to ensure efficient transcription and translation of the target gene. The constructed EF1α Short promoter-driven expression vector was transfected into mouse bone marrow MSCs (Mbm-MSCs) via liposomes for transient expression and secretion identification; stable expression was achieved using the EF1α Short-lentivirus co-expression system described later. Cells transfected with the constructed EF1α Short promoter-driven expression vector were cultured in serum-free mouse bone marrow MSC expression medium for 48–72 h, and the cell culture supernatant was collected. The fusion protein in the culture supernatant was purified by affinity chromatography. The purified protein was identified by SDS-PAGE and Western blotting to confirm its molecular weight and purity. Finally, the protein was replaced in PBS buffer (10 mM phosphate, 150 mM NaCl, pH 7.4) by ultrafiltration, and its concentration was determined for subsequent experiments.

[0025] (3) Assessment of the targeting ability of the HepaFibrinAct fusion protein To evaluate the targeting ability of the HepaFibrinAct fusion protein to fibrin, we used surface plasmon resonance (SPR) technology to determine its binding affinity to immobilized fibrin. The results showed that the binding affinity (KD) of the HepaFibrinAct fusion protein to fibrin was 2.5 ± 0.3 nM ( Figure 1 ).

[0026] Considering that collagen is a major component of the extracellular matrix (ECM) in liver fibrosis, we further examined the binding ability of the HepaFibrinAct fusion protein to type I collagen. ELISA experiments showed that the HepaFibrinAct fusion protein can bind to type I collagen, and its ECM... 50 The value is 150 ± 20 nM ( Figure 2Although the affinity is lower than that for fibrin, it still indicates that the HepaFibrinAct fusion protein has some targeting ability for the major components of the ECM, which helps it to function in fibrotic regions.

[0027] Example 2 Screening and optimization of PAI-1 peptide binding inhibitors Another key technical solution of this invention is the design and screening of a polypeptide that can specifically bind to and neutralize PAI-1 activity, which we call the "PAI-1 polypeptide binding inhibitor". This polypeptide, designed with computer aids and experimentally verified, can bind to the active site of PAI-1 with high affinity, thereby preventing PAI-1 from binding to plasminogen activator and effectively relieving the inhibitory effect of PAI-1 on the fibrinolytic system. The design of the PAI-1 polypeptide binding inhibitor aims to overcome the shortcomings of natural PAI-1 inhibitors in terms of stability and specificity, providing a new tool for precise regulation of the fibrinolytic system. The PAI-1 polypeptide binding inhibitor works synergistically with the HepaFibrinAct fusion protein, enhancing plasmin production on the one hand and relieving its inhibition on the other, thereby maximizing the overall activity of the fibrinolytic system.

[0028] (1) Screening method for PAI-1 peptide binding inhibitors The PAI-1 peptide binding inhibitors of this invention were obtained through a combination of computer-aided design and high-throughput screening. First, using molecular docking and virtual screening techniques, peptide sequences that might bind to the active site of PAI-1 were screened from a large peptide library to competitively inhibit PAI-1 activity. Subsequently, these candidate peptides were synthesized using solid-phase synthesis (SPPS). To verify their activity, in vitro enzyme inhibition assays were performed. Specifically, different concentrations of candidate peptides were pre-incubated with a fixed concentration of PAI-1, followed by the addition of plasminogen activator. The residual activity of the plasminogen activator was assessed by detecting the cleavage rate of the substrate (S-2288). Peptides that significantly restored plasminogen activator activity were considered effective PAI-1 inhibitors and selected as candidate molecules for further optimization.

[0029] (2) Optimization of PAI-1 peptide binding inhibitors The selected candidate peptides may exhibit poor stability and low affinity, thus requiring further optimization. Optimization strategies include: 1) Amino acid substitution: Identifying crucial amino acid residues in the peptide sequence for binding through alanine scanning or site-directed mutagenesis, and attempting to replace them with other amino acids to improve affinity or stability. 2) Structural modification: Introducing non-natural amino acids, D-type amino acids, or cyclizing the peptide at its N- or C-terminus to enhance its resistance to proteases and prolong its in vivo half-life.

[0030] The optimized amino acid sequence of the PAI-1 peptide binding inhibitor is as follows: AETYIIWVIMIRIDIVCNSSGMPEKYLNRVLPKCNIAPAPAPAPCAVILVVSKCHPNKIYGRAAMGSSIKDHDGRTNFMV, SEQ ID No. 2.

[0031] The optimized 3D structure image of the PAI-1 peptide binding inhibitor is shown below. Figure 3 As shown.

[0032] The optimized PAI-1 peptide binding inhibitor interacts with PAI-1 as shown in the image below. Figure 4 As shown.

[0033] (3) Validation of PAI-1 peptide binding inhibitors The optimized peptide needs to be validated again through in vitro enzyme inhibition experiments to confirm its enhanced activity and provide a basis for subsequent in vivo experiments. After obtaining the final optimized peptide sequence, the binding affinity between the PAI-1 peptide binding inhibitor and immobilized PAI-1 was determined using SPR technology. The results showed that the binding affinity (KD) between the PAI-1 peptide binding inhibitor and PAI-1 was 5.8 ± 0.7 nM, indicating a high-affinity interaction between the two. Figure 5 ).

[0034] We evaluated the inhibitory effect of PAI-1 peptide-binding inhibitors on PAI-1 activity using in vitro enzyme inhibition assays. The results showed that PAI-1 peptide-binding inhibitors effectively inhibited the inhibitory effect of PAI-1 on tPA, with an IC50 value of [missing information]. 50 The value is 12.5 ± 1.5 nM ( Figure 6 ).

[0035] Example 3 Design of co-expression vectors We designed a co-expression vector based on a P2A self-cleaving peptide for the HepaFibrinAct fusion protein and the PAI-1 peptide binding inhibitor: an EF1α Short promoter to drive transcription of the entire expression cassette; a gene encoding the HepaFibrinAct fusion protein; a coding sequence for a P2A self-cleaving peptide; a gene encoding the PAI-1 peptide binding inhibitor; a coding sequence for a second P2A self-cleaving peptide; a selection marker gene (puromycin N-acetyltransferase gene, PuroR) for selecting stably transfected cells; and a polyA signaling sequence to ensure mRNA stability and termination of translation.

[0036] To achieve the synergistic effect of the HepaFibrinAct fusion protein and the PAI-1 peptide binding inhibitor, this invention designs a co-expression system based on a single vector. The gene expression cassette structure of this system is as follows: [EF1α short promoter]-[exocrine HepaFibrinAct fusion protein open reading frame]-[P2A]-[exocrine PAI-1 peptide binding inhibitor open reading frame]-[P2A]-[puromycin N-acetyltransferase (with stop codon)]-[mini SV40 polyA signal (polyA tail)].

[0037] The complete structure of the gene expression cassette is as follows Figure 7 As shown.

[0038] In this system, the EF1α short promoter (Elongation Factor 1 Alpha Short) drives the transcription of the entire expression cassette, producing a long mRNA. During translation, a "ribosome skipping" phenomenon occurs when the ribosome encounters the coding sequence of the P2A self-cleaving peptide, causing peptide chain synthesis to be interrupted at this point and translation to restart downstream. Ultimately, three independent proteins are translated from a single mRNA molecule: the HepaFibrinAct fusion protein, the PAI-1 peptide binding inhibitor, and puromycin N-acetyltransferase (PuroR). The entire expression cassette is cloned into a high-copy-number plasmid vector to facilitate subsequent viral packaging and cell infection.

[0039]

[0040] Among them, SEQ ID No. 3, 1-212bp is the EF1α short promoter, SEQ ID No. 3, 213-1187bp is the exotropic HepaFibrinAct ORF, SEQ ID No. 3, 1188-1253bp is P2A, SEQ ID No. 3, 1254-1499bp is the exotropic PAI-1 peptide binding inhibitor ORF, SEQ ID No. 3, 1500-1565bp is P2A, SEQ ID No. 3, 1566-2216bp is the puromycin N-acetyltransferase pac ORF, and SEQ ID No. 3, 2217-2265bp is mini SV40polyA.

[0041] Example 4 Identification of co-expression vectors and engineered mesenchymal stem cells (1) Culture and selection of engineered mesenchymal stem cells (mouse bone marrow MSC cell line: D1).

[0042] (2) Construction of co-expression vector and introduction of expression cassette: The co-expression vector was constructed using standard molecular cloning techniques. First, the HepaFibrinAct fusion protein gene, the PAI-1 peptide binding inhibitor gene, and the P2A sequence were obtained by PCR amplification. Then, using restriction endonucleases and DNA ligases, these fragments were sequentially cloned into the vector backbone in the designed order. The constructed vector was verified by colony PCR, enzyme digestion identification, and DNA sequencing to ensure that all sequences were correct.

[0043] To verify the function of the co-expression vector, it was transfected into mesenchymal stem cells (mouse bone marrow MSCs, which were expanded and cultured in conventional culture medium, and passaged when the cell confluence reached 70%~90%) via lentivirus transfection.

[0044] (3) Secretion verification: 48 h post-transfection, specific bands consistent with those of the HepaFibrinAct fusion protein (≈45 kDa) and the PAI-1 peptide binding inhibitor (≈10 kDa) were detected in the culture supernatant, indicating that the co-expression system could simultaneously produce two target proteins. The proteins produced by the co-expression system were purified by multi-step chromatography. The purity of the final HepaFibrinAct fusion protein and the PAI-1 peptide binding inhibitor were both greater than 95%, meeting the requirements for subsequent in vitro and in vivo experiments.

[0045] The expression cassette containing the EF1 promoter was then introduced into mouse bone marrow MSCs via lentiviral transduction. After introduction, the cells were screened and expanded according to selection markers to obtain a stable expression cell population.

[0046] (4) Cell quality control: Before the experiment, the viability of engineered mouse bone marrow MSCs was tested (trypan blue exclusion method), and the cell morphology and proliferation status were recorded to ensure that the cells were in good condition.

[0047] Example 5 Detection of fibrin degradation capacity at the cellular level To directly assess the cells' ability to degrade fibrin, we employed a fibrin gel cover assay. Stable co-expressing cells were mixed with plasminogen and then plated onto a plate containing fibrinogen and thrombin, forming a fibrin gel. After incubation at 37°C for 60 min, if the cells were able to degrade fibrin, a transparent "halo" would form around the cells. By measuring the area of ​​the "halo," the cells' fibrin degradation capacity could be quantitatively assessed.

[0048] This experiment was performed in three independent biological replicates, each containing eight independent samples (n=8). Measurements from each sample were recorded independently and used for statistical analysis. Quantitative results are expressed as mean ± standard deviation (mean ± SD). Two-tailed t-tests were used for comparisons between the two groups, and p < 0.001 is indicated by *** in the figure.

[0049] Statistical analysis results: Control group: 0.484 ± 0.052 mm², co-expression group: 2.530 ± 0.114 mm².

[0050] The two-group comparison (Welch's t test, two-sided) yielded P≈8.87×10⁻⁶. -13 It is less than 0.001.

[0051] Compared with the control group, the co-expression group showed a larger area of ​​transparent region around the cells.

[0052] Cellular-level fibrin degradation capacity data: In the fibrin gel coverage assay, the area of ​​the transparent "halo" formed around the co-expression group cells was 2.530 ± 0.114 mm. 2 The size of the cells was significantly larger than that of the control group (0.484 ± 0.052 mm). 2 ( Figure 8 This directly demonstrates that the co-expression system can significantly enhance the cell's ability to degrade fibrin.

[0053] Example 6 Evaluation of the biological mechanisms of action of animal models To systematically evaluate the properties of the fusion protein and its co-expression system described in this invention in intervening in liver fibrosis, we employed two classic rodent liver fibrosis models with complementary pathological mechanisms: a chemically induced carbon tetrachloride (CCl4) model and a surgically induced bile duct ligation (BDL) model. These two models respectively simulate the pathological process of liver fibrosis in humans caused by toxic damage and cholestasis, enabling a comprehensive evaluation of the anti-fibrotic effects of the candidate protein under different pathological microenvironments. All animal experiments strictly adhered to animal welfare and ethical guidelines and were approved by the institution's animal ethics committee.

[0054] (1) Mouse model of liver fibrosis induced by carbon tetrachloride (CCl4) The CCl4 model is one of the most commonly used and mature animal models for studying liver fibrosis. It repeatedly induces hepatocyte necrosis and inflammatory responses, activating hepatic stellate cells (HSCs), ultimately leading to excessive deposition of extracellular matrix (ECM) and the formation of fibrous septa. Its pathological process is highly similar to the progression of human chronic hepatitis to liver fibrosis. In this case study, we selected 6-8 week old male C57BL / 6J mice weighing 19-21 g as experimental animals. This strain of mice showed stable and highly reproducible responses to CCl4-induced liver injury and fibrosis. The specific procedures for establishing the model were as follows: CCl4 and olive oil were mixed at a volume ratio of 1:9 to prepare a 10% CCl4 solution. Mice in the model group were administered 5 mL / kg body weight of the CCl4 solution twice a week (Mondays and Thursdays) via intraperitoneal injection (ip) for 8 consecutive weeks. Control group mice were given an equal volume of pure olive oil.

[0055] (2) Cholestatic liver fibrosis rat model induced by bile duct ligation (BDL) The BDL model is the gold standard model for simulating liver fibrosis caused by cholestatic liver diseases in humans (such as primary biliary cholangitis, biliary atresia, etc.). This model involves surgically ligating the common bile duct, obstructing bile excretion, leading to the accumulation of toxic substances such as bile acids in the liver, thereby triggering bile duct epithelial cell proliferation, portal vein inflammation, and significant fibrotic reactions. In this case study, we selected male Sprague-Dawley (SD) rats weighing 250-300 g. This strain of rats has a moderate size, the surgical procedure is relatively simple, and they tolerate BDL surgery well. The specific procedure for establishing the model was as follows: Under isoflurane anesthesia, the common bile duct was exposed through a midline abdominal incision. The common bile duct was ligated using 4-0 silk sutures near the porta hepatis and near the duodenum, and then transected between the two ligation points to ensure complete obstruction of bile flow. Rats in the sham group (Sham group) only underwent laparotomy and common bile duct exposure, without ligation or transecting. Postoperatively, all animals were given standard feed and water, and their weight, activity level, and jaundice were closely monitored. This model can develop significant liver fibrosis centered on the portal vein area within 2-4 weeks postoperatively, accompanied by marked cholestasis and liver function impairment, making it an ideal model for evaluating the efficacy of antifibrotic proteins in cholestatic liver fibrosis.

[0056] (3) Experimental grouping and experimental design In the CCl4 model, at week 4 of modeling (when early fibrosis had already formed), the model mice were randomly divided into 7 groups of 10 mice each based on their body weight and serum ALT / AST levels, and interventions were initiated for 4 weeks until the end of the experiment. The specific grouping and experimental protocol are as follows: ① Normal control group: 5 mL / kg body weight of olive oil was injected intraperitoneally, and physiological saline was given daily.

[0057] ② Model control group: 5 mL / kg body weight of 10% CCl4 solution was injected intraperitoneally, and physiological saline was given daily.

[0058] ③ Positive control group: 5 mL / kg body weight of 10% CCl4 solution was injected intraperitoneally, and tPA+uPA fusion protein (5 mg / kg, intraperitoneal injection) was given daily as a reference.

[0059] ④ Mouse-derived bone marrow MSCs-empty vector control group: Intraperitoneal injection of 5 mL / kg body weight, 10% CCl4 solution, and daily administration of wild-type MSCs (5 × 10⁻⁶). 6 1 cell / dose, intraperitoneal injection).

[0060] ⑤ Mouse-derived bone marrow MSCs-HepaFibrinAct group: Intraperitoneal injection of 5 mL / kg body weight, 10% CCl4 solution, and daily administration of genetically modified MSCs capable of stably secreting HepaFibrinAct fusion protein (5 × 10⁻⁶ cells). 6 1 cell / dose, intraperitoneal injection).

[0061] ⑥ Mouse-derived bone marrow MSCs - inhibitory peptide group: Intraperitoneal injection of 5 mL / kg, 10% body weight CCl4 solution, and daily administration of genetically modified MSCs capable of stably secreting PAI-1 peptide binding inhibitors (5 × 10⁻⁶). 6 1 cell / dose, intraperitoneal injection).

[0062] ⑦ Mouse-derived bone marrow MSCs - co-expression group: Intraperitoneal injection of 5 mL / kg of 10% CCl4 solution, and daily administration of MSCs capable of simultaneously secreting HepaFibrinAct fusion protein and PAI-1 peptide binding inhibitor (5 × 10⁻⁶ cells). 6 1 cell / dose, intraperitoneal injection).

[0063] In the BDL model, at week 4 of modeling, surviving rats were randomly divided into 7 groups of 12 rats each, and intervention began and lasted for 3 weeks. Specific grouping and protocols are as follows: ①Sham surgery group: A sham surgery was performed, and normal saline (5 mg / kg, intraperitoneal injection) was administered daily.

[0064] ② Model control group: underwent BDL surgery and were given normal saline (5 mg / kg, intraperitoneal injection) daily.

[0065] ③ Positive control group: BDL surgery was performed, and tPA+uPA fusion protein (5 mg / kg, intraperitoneal injection) was given daily.

[0066] ④ Mouse-derived bone marrow MSCs-empty vector control group: underwent BDL surgery and were given wild-type MSCs (5×10⁻⁶ cells) daily. 6 1 cell / dose, intraperitoneal injection).

[0067] ⑤ Mouse bone marrow MSCs-HepaFibrinAct group: Patients underwent BDL surgery and were given 5 × 10⁻⁶ genetically modified MSCs (5 × 10⁻⁶ cells) daily that could stably secrete the HepaFibrinAct fusion protein. 6 1 cell / dose, intraperitoneal injection).

[0068] ⑥ Mouse bone marrow MSCs-inhibitory peptide group: After BDL surgery, MSCs that are genetically modified to stably secrete PAI-1 peptide binding inhibitors (5×10⁻⁶ cells) are administered daily. 6 1 cell / dose, intraperitoneal injection).

[0069] ⑦ Mouse bone marrow MSCs - co-expression group: Patients underwent BDL surgery and were given daily MSCs (5 × 10⁻⁶ cells) capable of simultaneously secreting HepaFibrinAct fusion protein and PAI-1 peptide binding inhibitor. 6 1 cell / dose, intraperitoneal injection).

[0070] All proteins and cells were prepared with sterile saline and administered at fixed times daily. At the end of the experiment (week 8 for the CCl4 model and week 3 post-surgery for the BDL model), all animals were euthanized by intraperitoneal injection of an excessive amount of sodium pentobarbital after a 12-hour fast with free access to water. Blood and liver tissue samples were collected for subsequent testing.

[0071] (4) Evaluation indicators of biological mechanism of action At the end of the experiment, blood and liver tissue samples were collected from mice to evaluate the following biological mechanisms of action: ① Liver function tests: Detect the levels of ALT, AST, ALP and TBil in serum to assess the degree of liver damage.

[0072] ② Histopathological analysis: Liver tissue was stained with HE, Masson's trichrome stain and Sirius red stain to observe the pathological structure of the liver, inflammatory cell infiltration and collagen fiber deposition, and a semi-quantitative fibrosis score was performed.

[0073] ③ Hydroxyproline (HYP) content determination: The HYP content in liver tissue was determined to quantitatively assess the total amount of collagen.

[0074] ④Western Blot: Detect the expression levels of α-SMA and Collagen I in liver tissue to assess the degree of HSC activation and ECM deposition.

[0075] (5) Results ① Changes in liver function indicators Serum biochemical indicators of liver function are the most direct and commonly used indicators for evaluating the degree of liver damage and hepatoprotective effects. In this case study, we measured the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBil). ALT and AST are mainly found in the cytoplasm and mitochondria of hepatocytes. When hepatocytes are damaged, these two enzymes are released into the blood in large quantities, leading to a sharp increase in serum levels, making them sensitive indicators of the degree of hepatocyte necrosis. ALP and TBil are important indicators of cholestasis and bile duct injury, and their levels are significantly elevated in the BDL model.

[0076] Table 1. Changes in liver function indicators in mice of different groups in the CCl4 model.

[0077] Note: Data are expressed as mean ± standard deviation (Mean ± SD), n=10. Compared with the normal control group, *P<0.05, **P<0.01, **P<0.001. Compared with the model control group, #P<0.05, ##P<0.01, ###P<0.001. One-way ANOVA was used to detect overall inter-group differences (two-tailed), and Tukey's HSD post-hoc test was used.

[0078] Table 2. Changes in liver function indicators in rats of the BDL model.

[0079] Note: Data are expressed as mean ± standard deviation (Mean ± SD), n=12. Compared with the sham surgery group, *P<0.05, **P<0.01, **P<0.001. Compared with the model control group, #P<0.05, ##P<0.01, ###P<0.001. One-way ANOVA was used to detect overall inter-group differences (two-tailed), and Tukey's HSD post-hoc test was used.

[0080] In both the CCl4 and BDL models, the levels of ALT, AST, ALP, and TBil in the model control group were significantly higher than those in the normal control / sham-operated group, indicating the successful establishment of the liver injury and cholestasis model. Compared with the model control group, all liver function indicators in the HepaFibrinAct intervention group and the co-expression system group showed significant improvement. Among them, the improvement effect of the murine bone marrow MSC cell-co-expression group was the most significant, with ALT and AST levels decreasing by 72.7% and 68.2% respectively in the CCl4 model, and by 70.0% and 65.5% respectively in the BDL model. Its effect was not only superior to the murine bone marrow MSC cell-HepaFibrinAct group, but also superior to the positive control group. This fully demonstrates that by co-expressing the PAI-1 peptide binding inhibitor, the highly expressed PAI-1 in the pathological microenvironment was neutralized, the inhibition of the fibrinolytic system was relieved, thereby enhancing the hepatoprotective effect of the HepaFibrinAct fusion protein, demonstrating the advantage of synergistic effect.

[0081] ② Histopathological analysis We used hematoxylin-eosin (HE) staining, Masson's trichrome staining, and Sirius Red staining for pathological evaluation of liver tissue sections. HE staining was used to observe the overall liver structure, hepatocellular damage, and inflammatory cell infiltration. Masson's trichrome staining and Sirius Red staining can specifically visualize collagen fibers. Sirius Red staining, in particular, can distinguish between different types of collagen fibers (type I and type III) under polarized light, making it a classic method for quantitatively analyzing the degree of liver fibrosis. We used the Ishak scoring system to semi-quantitatively score the degree of liver fibrosis and used image analysis software such as ImageJ to quantitatively analyze the collagen-positive area.

[0082] Table 3. Percentage of collagen area and fibrosis score in liver tissue of mice in each group of the CCl4 model.

[0083] Note: Data are expressed as mean ± standard deviation (Mean ± SD), n=10. Compared with the normal control group, *P<0.05, **P<0.01, **P<0.001. Compared with the model control group, #P<0.05, ##P<0.01, ###P<0.001. One-way ANOVA was used to detect overall inter-group differences (two-tailed), and Tukey's HSD post-hoc test was used.

[0084] The results showed that the collagen area in the CCl4 model was highest in the control group (15.8%), and significantly reduced in the mouse bone marrow MSC cell co-expression group (5.5%). Regarding the changes in Ishak fibrosis score in the CCl4 model, the mouse bone marrow MSC cell co-expression group (1.8) was closest to normal.

[0085] Table 4. Percentage of collagen area and fibrosis score in liver tissue of mice in each group of the BDL model.

[0086] Note: Data are expressed as mean ± standard deviation (Mean ± SD), n=10. Compared with the sham surgery group, *P<0.05, **P<0.01, **P<0.001. Compared with the model control group, #P<0.05, ##P<0.01, ###P<0.001. One-way ANOVA was used to detect overall inter-group differences (two-tailed), and Tukey's HSD post-hoc test was used.

[0087] The results showed that the collagen area in the BDL model was highest in the control group (19.5%), while it was significantly reduced in the mouse bone marrow MSC cell co-expression group (4.8%).

[0088] ③ Hydroxyproline (HYP) content determination Hydroxyproline (HYP) is an amino acid unique to collagen, and its content is directly proportional to the total amount of collagen in tissues. Therefore, measuring the HYP content in liver tissue is a reliable biochemical indicator for quantitatively assessing the degree of liver fibrosis. We used the Sigma-Aldrich (product number: JX01066689) hydroxyproline assay kit, following the instructions, and determined the HYP concentration by alkaline hydrolysis and chloramine-T oxidation, finally using a colorimetric method.

[0089] Table 5. Hydroxyproline (HYP) content in the liver of mice in each group of the CCl4 model (μg / g liver tissue)

[0090] Note: *Data are expressed as mean ± standard deviation (Mean ± SD), n=10. Compared with the normal control group, *P<0.05, **P<0.01, ***P<0.001. Compared with the model control group, #P<0.05, ##P<0.01, ###P<0.001. One-way ANOVA was used to detect overall inter-group differences (two-tailed), and Tukey's HSD post-hoc test was used.

[0091] Table 6. Hydroxyproline (HYP) content in the liver of mice in each group of the BDL model (μg / g liver tissue)

[0092] Note: *Data are expressed as mean ± standard deviation (Mean ± SD), n=12. Compared with the sham surgery group, *P<0.05, **P<0.01, ***P<0.001. Compared with the model control group, #P<0.05, ##P<0.01, ###P<0.001. One-way ANOVA was used to detect overall inter-group differences (two-tailed), and Tukey's HSD post-hoc test was used.

[0093] The results showed that in both models, the hydroxyproline content was significantly reduced in the mouse bone marrow MSC cell co-expression group.

[0094] ④Western Blot Analysis α-Smooth muscle actin (α-SMA) is a specific marker of activated hepatic stellate cells (HSCs), and its expression level directly reflects the degree of HSC activation. Collagen I is a major component of the hepatic stromal cell (ECM), and its expression level is closely related to the degree of fibrosis. We observed the protein localization and expression intensity of α-SMA and Collagen I in liver tissue using immunohistochemical staining, and quantitatively detected their protein expression levels using Western blotting.

[0095] Table 7. Relative expression levels of α-SMA and Collagen I proteins in the liver of mice in each group of the CCl4 model (Western Blot)

[0096] *Note: Data are expressed as mean ± standard deviation (Mean ± SD), n=10. Compared with the normal control group, *P<0.05, **P<0.01, **P<0.001. Compared with the model control group, #P<0.05, ##P<0.01, ###P<0.001. One-way ANOVA was used to detect overall inter-group differences (two-tailed), and Tukey's HSD post-hoc test was used.

[0097] Table 8. Relative expression levels of α-SMA and Collagen I proteins in the liver of rats in each group of the BDL model (Western Blot)

[0098] *Note: Data are expressed as mean ± standard deviation (Mean ± SD), n=12. Compared with the sham surgery group, *P<0.05, **P<0.01, ***P<0.001. Compared with the model control group, #P<0.05, ##P<0.01, ###P<0.001. One-way ANOVA was used to detect overall inter-group differences (two-tailed), and Tukey's HSD post-hoc test was used.

[0099] In the CCl4 model, the α-SMA expression level was highest in the model control group (5.50), while it was significantly lower in the mouse bone marrow MSC cell co-expression group (1.52). Regarding Collagen I expression changes in the CCl4 model, the mouse bone marrow MSC cell co-expression group (1.83) was closest to normal. In the BDL model, the α-SMA expression level was increased in the model control group (7.26), while it decreased most significantly in the mouse bone marrow MSC cell co-expression group (2.20). Regarding Collagen I expression in the BDL model, the mouse bone marrow MSC cell co-expression group (2.53) had the lowest expression level.

[0100] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0101] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A fusion protein, characterized in that, The fusion protein is named HepaFibrinAct, and its amino acid sequence is shown in SEQ ID No.

1.

2. A co-expression system, characterized in that, The fusion protein of claim 1 and the PAI-1 peptide binding inhibitor are co-expressed.

3. The co-expression system as described in claim 2, characterized in that, The amino acid sequence of the PAI-1 peptide binding inhibitor is shown in SEQ ID No.

2.

4. The co-expression system as described in claim 2, characterized in that, The gene expression cassette structure of the co-expression system is as follows: [EF1α short promoter] - [exotropic HepaFibrinAct open reading frame] - [P2A] - [exotropic anti-PAI-1 active binding polypeptide open reading frame] - [P2A] - [puromycin N-acetyltransferase] - [mini SV40 polyA signal].

5. The co-expression system as described in claim 4, characterized in that, The nucleotide sequence of the expression cassette is shown in SEQ ID No.

3.

6. The use of the fusion protein of claim 1 and the co-expression system of any one of claims 2 to 5 in the preparation of drugs with active molecular molecules that intervene in liver fibrosis.

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