Application of PAI-1 inhibitors in the preparation of drugs for the treatment of liver ischemia-reperfusion injury

Abstract

The present invention discloses an application of a PAI-1 inhibitor in the preparation of a drug for treating liver ischemia-reperfusion injury, and relates to the field of biomedicine technology. The present invention selects PAI-1 gene knockout mice and C57BL / 6 wild-type mice as experimental subjects, constructs mouse liver ischemia-reperfusion injury models respectively, and studies the function of PAI-1 in liver ischemia-reperfusion injury. The study found that after PAI-1 knockout, it can reduce liver ischemia-reperfusion injury by affecting the formation and recruitment of liver neutrophil induction network, inhibiting the inflammatory response of the liver. PAI-1 is a key gene that affects neutrophil activation and is an important target for regulating inflammatory response during the reperfusion period. Animal experiments have verified that the PAI-1 specific inhibitor PAI-039 can reduce the degree of injury of mouse liver ischemia-reperfusion, providing new ideas for the development of new drugs for preventing, alleviating and / or treating tissue ischemia-reperfusion injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of a PAI-1 inhibitor in the preparation of a drug for treating liver ischemia-reperfusion injury. Background Art

[0002] Hepatic ischemia-reperfusion injury (I / R) is an unavoidable complication of liver transplantation, hepatectomy, and hepatic artery ligation or embolization. It is also a major cause of early transplant failure, tissue damage, organ rejection, and even liver failure. Statistical analysis has found that 10% of early liver transplant failures are due to I / R. I / R is primarily manifested by sinusoidal congestion, hepatocyte swelling, and inflammatory cell infiltration in the portal areas. In severe cases, apoptosis and necrosis may occur, resulting in irreversible liver damage and severely hindering the application and therapeutic efficacy of liver transplantation. Currently, I / R remains an unsolved clinical challenge. Due to the complex and diverse mechanisms of action of I / R and its influence by various factors, the prevention and treatment of I / R are diverse. In addition to traditional preventive measures such as pre-ischemic conditioning, a number of small molecule inhibitors, such as prolyl hydroxylase inhibitors and adenosine receptor agonists, have been used. However, the effectiveness of these treatments has been mixed, and to date, no effective intervention has been established. Therefore, exploring the pathogenesis of liver I / R and finding early intervention methods for liver I / R are not only the basis for developing new therapies for liver I / R, but also one of the hot topics in liver I / R research.

[0003] In clinical practice, neutrophils are used as one of the biomarkers to evaluate the degree of transplant liver injury. Therefore, we believe that the strong innate immune response caused by neutrophils during the reperfusion period is considered to be the core factor leading to severe liver function damage. We will focus on the behavioral changes of neutrophils at this stage. However, the process of neutrophil activation inducing liver injury is very complex, and the specific mechanism of neutrophils in the inflammatory response is still unclear. Therefore, in-depth exploration of the regulatory mechanism of neutrophils in the liver inflammatory response induced by liver I / R is crucial for us to understand the immune inflammatory response during liver I / R reperfusion in essence.

[0004] Plasminogen activator inhibition 1 (PAI-1) is a multi-domain, single-chain glycoprotein encoded by the Serpine1 gene. It is a physiological inhibitor of plasminogen activators (PAs), particularly urokinase-prothrombin activator (uPA) and tissue-type prothrombin activator (tPA). PAI-1 is primarily synthesized and secreted by hepatocytes, endothelial cells, and monocytes, and accumulates in circulating platelets. In addition to inhibiting fibrinolysis, PAI-1 also regulates numerous important physiological and pathological processes, including fibrinolysis, coagulation, cell-matrix remodeling, cell adhesion, and migration. Recent studies have revealed that PAI-1 plays a crucial regulatory role in various inflammatory diseases, including myocardial infarction, sepsis, and acute lung injury. As a stress gene, PAI-1 is not only a key factor that is highly expressed in the early stages of inflammation, but also a key gene that influences inflammatory cell chemotaxis and participates in the formation of inflammatory factor networks. Current research suggests that during acute liver injury, the inflammatory cascade is primarily initiated by immune cells in the liver microenvironment. Neutrophils, Kupffer cells, natural killer cells, and CD4 + T cells are the primary cellular players in ischemia-reperfusion injury, but PAI-1's regulation of immune cells in the liver microenvironment remains poorly understood. Therefore, further investigation is needed to determine which components of the liver's immune microenvironment PAI-1 regulates during liver I / R to drive the proinflammatory response. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of PAI-1 in liver ischemia-reperfusion injury, determine the relationship between PAI-1 gene expression and liver ischemia-reperfusion injury, reveal the new function of PAI-1 in liver ischemia-reperfusion injury, and provide the application of PAI-1 gene inhibitors in the preparation of drugs for the treatment of liver ischemia-reperfusion injury.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides the use of a PAI-1 inhibitor in preparing a drug for treating liver ischemia-reperfusion injury.

[0008] Preferably, the inhibitor is selected from a PAI-1 gene antagonist, a PAI-1 enzyme activity inhibitor, a shRNA that inhibits PAI-1 gene expression, or a gene editing vector that inhibits PAI-1 gene expression.

[0009] More preferably, the nucleotide sequence of the shRNA is as shown in SEQ ID NO.1.

[0010] More preferably, the gene editing vector is CRISPR-CAS9 gene editing.

[0011] The present invention also provides the use of PAI-1 gene or PAI-1 protein as a target in the preparation of a drug for treating liver ischemia-reperfusion injury.

[0012] Compared with the prior art, the present invention has the following technical effects:

[0013] The present invention selects PAI-1 gene knockout mice (C57BL / 6JGpt-Serpine1em1Cd4033 / Gpt) and C57BL / 6J wild-type mice (WT); selects to construct adeno-associated virus-mediated PAI-1 liver-specific knockdown mice (pAAV8-TBG-sfEFG-3xFLAG-mir30shRNA (Serpine1)-WPRE) and empty control (NC) mice; selects PAI-1 specific inhibitor PAI-039-treated and solvent-controlled C56BL / 6J mice to respectively construct mouse liver ischemia-reperfusion injury models to study the function of PAI-1 in liver ischemia-reperfusion injury.

[0014] Through liver function test, HE staining, immunofluorescence, ELISA, q-PCR, statistical analysis and other tests and analyses, the results showed:

[0015] (1) Compared with the sham group, the levels of PAI-1 mRNA and protein in the liver tissue of the ischemia-reperfusion injury group and the secretion of serum PAI-1 were significantly increased; the expression of PAI-1 in the liver of clinical liver transplant patients was significantly increased compared with that in normal and liver transplant donor samples;

[0016] (2) Compared with the WT group, the liver necrosis area in the PAI-1-KO group was significantly reduced. PAI-1 gene knockout reduced the infiltration of neutrophils in the liver, inhibited the liver inflammatory response, and alleviated the degree of liver ischemia-reperfusion injury.

[0017] (3) Compared with the NC group, the liver necrosis area in the AAV8-shPAI-1 group was significantly reduced.

[0018] (4) Compared with the solvent control group, the liver necrosis area, liver neutrophil infiltration, and liver inflammatory response in the PAI-039-treated group were significantly reduced.

[0019] The present invention discovered a new function of the PAI-1 gene in liver ischemia-reperfusion injury. PAI-1 knockout or activity inhibition can alleviate liver ischemia-reperfusion injury by inhibiting neutrophil and NETosis-induced inflammatory responses. Therefore, PAI-1 can be used to screen or prepare drugs for preventing / treating liver ischemia-reperfusion injury, providing a new drug target for liver ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Serpine1 expression in RNA-seq data of liver I / R mice (A) and microarray data of liver transplant patients (B);

[0021] Figure 2 The expression of serum PAI-1 protein level (A) and liver Serpine1 mRNA (B) in WT mice after 1 h of ischemia and reperfusion at different time points were detected by ELISA and q-PCR.

[0022] Figure 3 Immunofluorescence staining was used to detect the expression of Serpine1 in the liver of WT mice after 1h of ischemia and 12h of reperfusion.

[0023] Figure 4 The protein (A) and mRNA (B) expression of PAI-1 in the liver of PAI-1-cKO and control mice were detected by WB and qPCR;

[0024] Figure 5 The levels of serum ALT (A) and AST (B) in PAI-1-cKO and control mice 12 h after IRI were detected by ELISA;

[0025] Figure 6 The liver histopathological structure of PAI-1-cKO and control mice 12 hours after IRI was detected by H&E method (A), and the liver necrosis area of ​​mice in each group was statistically analyzed using Image pro plus software (B);

[0026] Figure 7 The mRNA expression of inflammatory factors in the liver of PAI-1-cKO and control mice 12 hours after IRI was measured by qPCR;

[0027] Figure 8 Figure 2 shows the anatomical structure of the liver of mice in the PAI-1-039 treatment group and the solvent control group 12 hours after IRI (A) and the pathological results of H&E staining of liver tissue (B);

[0028] Figure 9 This is the 12-hour survival curve of IRI in mice treated with PAI-1-039 and the solvent control group;

[0029] Figure 10 The ELISA test shows the serum ALT and AST levels in the PAI-1-039 treated group and the solvent control group 12 hours after IRI.

[0030] Figure 11 The mRNA expression of inflammatory factors in the liver of mice treated with PAI-1-039 and the solvent control group was measured by qPCR 12 hours after IRI.

[0031] Figure 12 Diagram of the clustering strategy established for flow cytometry analysis of immune cells in the liver of PAI-1-shPAI-1 and control mice 12 hours after IRI;

[0032] Figure 13 Myeloid cells and lymphoid T cells occupy CD45 in the liver + Ratio diagram of immune cells;

[0033] Figure 14 Myeloid cells including macrophages, neutrophils and inflammatory monocytes occupy CD11b + Cell scale diagram;

[0034] Figure 15 T lymphocytes including CD4 + and CD8 + CD3 + Cell ratio and CD4 + / CD8 + Wetting ratio diagram;

[0035] Figure 16 Immunofluorescence staining was used to detect the expression of Ly6G in the liver of PAI-1-shPAI-1 and control mice 12 hours after IRI.

[0036] Figure 17 Immunofluorescence staining was used to detect the expression of Ly6G in the liver of mice in the PAI-1-039-treated group and the solvent control group 12 hours after IRI.

[0037] Figure 18 Multiple immunofluorescence staining was used to detect Ly6G in the liver of PAI-1-shPAI-1 and control mice 12 hours after IRI. + H3Cit expression in cells. DETAILED DESCRIPTION

[0038] The present invention provides the use of a PAI-1 gene inhibitor in the preparation of a drug for treating liver ischemia-reperfusion injury.

[0039] In the present invention, the inhibitor is selected from a PAI-1 gene antagonist, a PAI-1 enzyme activity inhibitor, a shRNA that inhibits PAI-1 gene expression, or a gene editing vector that inhibits PAI-1 gene expression. In the present invention, the PAI-1 enzyme activity inhibitor can be selected from Tiplaxtinin and PAI-039.

[0040] In the present invention, the nucleotide sequence of the shRNA is shown in SEQ ID NO.1. Preferably, the present invention uses Sigma and Invitrogen Block-iT to design the shRNA sequence, and clones the Serpine gene sequence into the adeno-associated virus vector pAAV-TBG-sfEGFP-3×FLAG-WPRE to obtain pAAV-TBG-sfEGFP-3×FLAG-mir30shRNA(Serpine1)-WPRE. The above plasmid is transfected into AAV-293 cells using liposomes, and the supernatant and intracellular viruses are collected 72 hours after transfection. The virus is purified and concentrated according to standard procedures and used to construct liver-specific Serpine1 knockout mice in vivo.

[0041] In the present invention, the gene editing vector is CRISPR-Cas9 gene editing. Currently, CRISPR-Cas9 can be used to edit DNA in vivo in adult animals. However, the lack of suitable vectors to deliver CRISPR-Cas9 into animals has limited its application. In the present invention, as a feasible approach, Cas9 and gRNA are integrated into an adenoviral vector, and the CRISPR-Cas9 adenovirus is packaged for in vivo editing in adult animals to construct PAI-1 knockout mice.

[0042] In a specific embodiment of the present invention, the PAI-1 gene knockout or inhibition of PAI-1 gene expression by the inhibitor can reduce the area of ​​plaque necrosis in the liver of hepatic I / R mice and alleviate the degree of edema and bleeding.

[0043] In a specific embodiment of the present invention, the PAI-1 gene knockout or inhibitor inhibiting PAI-1 gene expression can reduce the expression of serum ALT and AST in liver I / R mice and alleviate the degree of liver function damage.

[0044] In a specific embodiment of the present invention, the PAI-1 gene knockout or inhibitor inhibits PAI-1 gene expression, which can inhibit liver inflammatory response and the release of proinflammatory factors; the proinflammatory factors include IL-6, TNF-α, IL-β, IFN-γ, Mcp1, IL-1β, and CXCL2.

[0045] In a specific embodiment of the present invention, the PAI-1 gene knockout or inhibitor inhibits PAI-1 gene expression and can reduce CD45 + Immune cells and myeloid CD11b + Cell infiltration.

[0046] In a specific embodiment of the present invention, the PAI-1 gene knockout or inhibitor inhibiting PAI-1 gene expression can reduce the infiltration of neutrophils in damaged liver sites.

[0047] The present invention provides an application of a PAI-1 gene or a PAI-1 protein as a target in the preparation of a drug for treating liver ischemia-reperfusion injury.

[0048] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] Example 1

[0050] Serpine1 knockout mice (C57BL / 6JGpt-Serpine1) were purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd. em1Cd4033 C57BL / 6J wild-type mice (WT mice) (male, 8-12 weeks, weight: 25-27 g) were housed in the SPF-grade experimental animal room of the Experimental Animal Center of Wenzhou Medical University. Mice were housed in a temperature-controlled (22-24°C), humidity-controlled (40% to 70%), and light-controlled room and were allowed free access to a standard diet. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0051] Mouse liver I / R injury model

[0052] Surgery: A mouse model of hepatic I / R injury was established using a classic method. Mice were fasted for 12 hours before surgery and had free access to water. Mice were anesthetized with 3% sodium pentobarbital and their limbs were secured to a mouse operating table. The surgical area was disinfected with 75% ethanol. A ventral midline incision was made into the abdomen (layer by layer), exposing the hepatic pedicles of the left and middle lobes of the liver. After laparotomy, vascular clamps were used to clamp the trifurcations of the hepatic artery, portal vein, and bile duct in the left and middle lobes of the liver, resulting in approximately 70% hepatic ischemia. If the blocked lobe turns white compared to the intact right lobe, blood flow obstruction is successful and the procedure was performed correctly. Ischemia was then initiated and maintained for 1 hour. Sham group mice did not have hepatic blood flow obstruction. After ischemia, the vascular clamps were removed, and hepatic blood flow was restored. Following the procedure, the medial and lateral skin were sutured continuously with polypropylene sutures. Sham-operated mice underwent the same surgical procedure, except for the vascular clamps. After the abdominal cavity was closed, the mice were placed in a clean cage at 37°C for observation.

[0053] Sampling: Mice were removed from the sham group (Sham group) and the I / R group at designated postoperative times and anesthetized with 3% sodium pentobarbital. One ml of blood was collected from the orbital venous plexus, and serum was separated. The middle lobe of the liver was collected and frozen in liquid nitrogen, and the left lobe was fixed in 4% paraformaldehyde for 48 h, dehydrated, embedded, and prepared into paraffin sections.

[0054] Plasmid construction

[0055] The Serpine gene sequence was cloned into the adeno-associated virus vector pAAV-TBG-sfEGFP-3×FLAG-WPRE to obtain pAAV-TBG-sfEGFP-3×FLAG-mir30shRNA(Serpine1)-WPRE. The primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.3. The above plasmid was transfected into AAV-293 cells using liposomes. The supernatant and intracellular viruses were collected 72 hours after transfection, and the viruses were purified and concentrated according to standard procedures. The virus titer was determined by TCID50 method, and the virus titer should not be less than 1.0*10 13 (vg / ml), and empty particles without Serpine1 gene sequence were used as control adenovirus (pAAV-TBG-sfEGFP-3×FLAG-mir30shRNA(NC)-WPRE).

[0056] Construction of adeno-associated virus-mediated Serpine1 liver-specific knockdown mice

[0057] pAAV8-sh(Serpine1) virus particles (1*10 12 vg per mouse) was injected into WT mice via the tail vein to knock down Serpine1 in the mouse liver tissue. A control group was injected with the same dose of pAAV8-shRNA(NC) particles. Twenty-one days after injection, two mouse livers were selected to verify the liver knockdown efficiency and then a mouse liver I / R model was established using the method described in 2 above.

[0058] Generation of gene knockout mice using CRISPR / Cas9 technology

[0059] CRISPR / Cas9 uses a small RNA to recognize and cut DNA to degrade foreign nucleic acid molecules. In the CRISPR-Cas9 system, sgRNA is formed by fusing crRNA and tracrRNA sequences to recognize the PAM (5'-NGG-3') site, a three-nucleotide region at the end of the target sequence of the genome, to guide the Cas9 endonuclease to generate a double-stranded DNA break in the target fragment. The RNA-DNA recognition mechanism of the CRISPR-Cas9 system provides a simple and powerful tool for genome engineering research. The Serpine1 gene has 3 transcripts (as shown in the table below). According to the structure of the Serpine1 gene, the exon 3-exon 5 (ENSMUST0000041388.10) transcript of Serpine1-2010 is recommended as the knockout region, which contains a 628bp coding sequence. Knocking out this region will lead to the destruction of protein function. The present invention uses CRISPR / Cas9 technology to modify the Serpine1 gene. Through CRISPR-Cas9-mediated homologous recombination (HR) reaction, the target gene, selection marker or other genetic factors can be integrated into the ROSA26 safe harbor site on mouse chromosome 6, ensuring long-term and stable expression of the transferred gene.

[0060] Table 1 Three transcripts of Serpine1 gene

[0061]

[0062] Analysis of liver function and serum biochemical indicators

[0063] Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using Nanjing Jiancheng kits. PAI-1 expression and enzyme activity in mouse serum were determined using ELISA kits from R&D Co., Ltd. Specific procedures were performed according to the kit instructions.

[0064] HE staining

[0065] Liver sections (4 μm) were stained with H&E, and necrotic areas were analyzed using Image Pro Plus software. The percentage of necrotic area relative to the total area of ​​the tissue section was blindly quantified in at least five sections per mouse. A pathologist who was unaware of the experimental protocol provided morphological assessments.

[0066] Immunofluorescence

[0067] Fresh animal tissue was fixed with 4% paraformaldehyde, embedded, and serially sectioned in paraffin. Immunofluorescence staining with antibodies against PAI-1, Ly6G, and H3Cit was used to detect neutrophil infiltration into liver sections and further examine the expression of neutrophil-attracting networks (NETs). Sections were blocked with PBST containing 10% goat serum before antibody application. The sections were incubated with the primary antibody to be tested overnight at 4°C, followed by incubation with the secondary antibody at room temperature for 1 hour in the dark. Cell nuclei were labeled with DAPI. Immunofluorescence images were captured and analyzed using Image Pro Plus (version 6.0).

[0068] Western blotting

[0069] Identical protein samples were added to loading buffer and separated by 10% SDS-PAGE electrophoresis. After electrophoresis, the proteins were transferred to a PVDF membrane. The membrane was blocked with 5% nonfat dry milk for approximately 1 hour at room temperature, followed by the addition of a primary antibody and incubation at 4°C overnight. A secondary antibody raised against the corresponding species was added, incubated for 1 hour at room temperature, and the blot was prepared using an enhanced chemiluminescence system and recorded on light-sensitive imaging film. Signals were acquired using a gel imaging system (Chemidoc-XRS+). Protein expression levels were quantified using ImageJ software.

[0070] qRT-PCR

[0071] Fresh tissues were lysed with Trizol, and RNA was extracted. Primers were designed and 2 μg of mRNA was taken. The cDNA was synthesized using the Prime Script™ RT reagent Kit with gDNA Eraser. qRT-PCR (Real-Time-polymerase chain reaction, qRT-PCR) was performed using the cDNA as a template. β-actin was used as an internal reference to detect gene expression levels. Each gene was analyzed. The reaction conditions were set as follows: incubation at 95°C for 10 minutes, followed by 40 cycles of incubation at 95°C for 10 seconds and at 60°C for 1 minute. The data obtained were calculated using Formula 2. -ΔΔ CT calculation expression.

[0072] Flow cytometry

[0073] Flow cytometry was used to assess liver immune cell infiltration. 0.5 g of tumor tissue was weighed from each mouse liver, and the remaining tissue was aliquoted. Tumor digestion solution (0.1 mg / mL collagenase, 0.25 mg / mL DNase I, Hanks formulation, prepared freshly as the enzymes are easily degraded) was collected, 4 mL per mouse, and the tumor was minced and incubated at 37°C at 100 rpm for 30 minutes. The sample was then centrifuged at 1200 rpm for 5 minutes, the supernatant discarded, and the sample was washed once with PBS. The sample was blocked with 5% BSA on ice for 20 minutes. The sample was then centrifuged at 2000 rpm for 5 minutes, the BSA discarded, and the sample was washed three times with PBS. The supernatant was then centrifuged to harvest the cells. Antibodies against CD45, CD11b, CD3, CD4, CD8, Ly6G, and Ly6C were prepared in a 1:1000 ratio in either PBS or 5% BSA. The sample was incubated at 4°C in the dark for 30 minutes. The sample was then centrifuged at 2000 rpm for 5 minutes, the supernatant discarded, and the sample was washed once with PBS. Note that when staining, separate the tubes into single positive and minus one control tubes. Discard the supernatant, add 200 μL PBS to resuspend, filter, and then load onto the microscope.

[0074] Statistical analysis

[0075] All data are expressed as mean ± standard error. SPSS 19.0 software was used for all statistical analyses. One-way ANOVA was used to compare statistical differences between two or more groups, followed by Bonferroni analysis (for data with homogeneity of variance) or Tamhane's T2 analysis (for data showing heteroscedasticity). The statistical differences between the two groups were compared using a two-tailed Student's T test. P < 0.05 was statistically significant.

[0076] Result Analysis

[0077] PAI-1 expression is upregulated during hepatic I / R, and downregulation of PAI-1 expression aggravates liver injury in the mouse I / R model. To analyze whether PAI-1 is involved in I / R-induced liver dysfunction, the present invention first determined the expression of PAI-1 in the liver of I / R mice. In the mouse liver I / R model, RNA-seq results showed that PAI-1 mRNA expression was upregulated 12 hours after reperfusion ( Figure 1 A). At the same time, in clinical liver transplant samples, gene chip data showed that PAI-1 mRNA expression was upregulated 24 hours after liver transplantation ( Figure 1 B). Consistent with this, the protein and gene levels of PAI-1 in serum and liver of mice increased in a time-dependent manner after 1h ischemia and reperfusion (2, 6, and 12h) ( Figure 2 AB). 12 hours after I / R, immunofluorescence staining results showed that PAI-1 was significantly overexpressed mainly in hepatic sinusoidal cells ( Figure 3The significant changes in PAI-1 expression strongly suggest that PAI-1 is a key regulatory factor in the pathological process of liver injury after hepatic I / R.

[0078] To investigate the role of PAI-1 in hepatic I / R injury, the present invention constructed Serpine1 knockout mice. Western blot and q-PCR analysis confirmed that PAI-1 gene knockout expression ( Figure 4 Serpine1 knockout mice (C57BL / 6JGpt-Serpine1em1Cd4033 / Gpt) and wild-type (WT) mice were used to establish a liver I / R injury model. Compared with WT ischemia-reperfusion mice, serum AST and ALT levels in PAI-1 knockout mice were significantly decreased ( Figure 5 AB), the pathological findings of the mouse liver, such as plaque necrosis area, edema and hemorrhage, were significantly restored. And the relatively normal liver structure was maintained ( Figure 6 AB). Therefore, it can be considered that PAI-1 gene knockout plays a key role in the progression of I / R injury. In addition, the present invention also detected the expression of inflammatory factors in the liver of gene knockout mice. The results showed that after PAI-1 knockout, the release of pro-inflammatory factors (IL-6, TNF-α, IL-β, etc.) in the liver of liver I / R mice can be significantly inhibited, and the release of anti-inflammatory factor IL-10 can be inhibited ( Figure 7 ), these results suggest that PAI-1 gene knockout improves liver I / R injury.

[0079] To further confirm the important role of PAI-1 in liver I / R injury, the present invention administered a PAI-1 specific inhibitor (PAI-039) and a corresponding solvent control to WT ischemia-reperfusion mice to observe their therapeutic effects. According to the HE staining results, after 12 hours, the degree of liver damage and necrosis area in the PAI-039 group were significantly reduced ( Figure 8 AB), 100% ischemic overall survival was significantly improved ( Figure 9 ), and ALT and AST levels were significantly decreased ( Figure 10 The present invention detected the inflammatory response of the liver after ischemia-reperfusion in PAI-039 and solvent control mice. In the PAI-039 mouse liver I / R injury model, the mRNA expression levels of inflammatory factors such as TNF-α, IL-6, IL-1β, CCL2 and IFN-γ in liver tissue were significantly reduced 12 hours after injury ( Figure 11 These results suggest that hepatic PAI-1 expression plays an important role in regulating liver dysfunction. The protective effect of PAI-1 deficiency against hepatic I / R may be related to its ability to suppress hepatic inflammatory responses.

[0080] It is well known that in the acute stage of liver injury, the inflammatory cascade reaction is mainly caused by the immune cells in the liver microenvironment. + T cells and natural killer cells are the main cellular participants in liver I / R. Therefore, the present invention further analyzed the infiltration of inflammatory immune cells in the liver microenvironment after liver I / R in mice with adeno-associated virus AAV8 liver-specific knockout of Serpine1 [pAAV-TBG-sfEGFP-3×FLAG-mir30shRNA (Serpine1)-WPR] and control mice by flow cytometry. The clustering strategy is as follows Figure 12 As shown. Flow cytometry results showed that all CD45 + In cells, CD11b + The proportion of total myeloid cells decreased significantly, and CD3 + There was no significant difference in the proportion of T cells ( Figure 13 ). Based on the above research results, the present invention further analyzed the changes in myeloid cells and T cell subsets. The results showed that in CD11b + Among the total myeloid cell subsets, macrophages (CD11b + f / 4 / 80 + ) and inflammatory monocytes (CD11b + Ly6C + ) had no significant changes, while neutrophils (CD11b + Ly6G + ) ratio decreased significantly ( Figure 14 ). In CD3 + Among T cell subsets, CD4 + T and CD8 + There was no significant change in the proportion of T cells ( Figure 15 In conclusion, overexpression of PAI-1 in hepatocytes can significantly inhibit neutrophil infiltration in damaged livers.

[0081] Next, the present invention further explored the effect of PAI-1 on the function of neutrophils recruited to the liver after liver I / R. According to current literature reports, NETs are a new form of activation of neutrophils that induce immune inflammatory responses in many diseases, and are currently receiving widespread attention. Therefore, we further analyzed the protein expression of liver Ly6G (neutrophil marker) and H3Cit (neutrophil-induced NET marker) in adeno-associated virus AAV8 liver-specific Serpine1 knockout mice [AAV8-TBG-shPAI-1] and control mice after liver I / R by multiple immunofluorescence staining. The results showed that compared with the control group, the protein expression of Ly6G in the liver of mice in the PAI-1 knockdown group was significantly reduced ( Figure 16 ), and the same conclusion was obtained in the PAI-039 treatment group ( Figure 17), suggesting that PAI-1 can significantly inhibit the number of neutrophils in the liver. So, whether PAI-1 can regulate the recruitment of NETs to promote liver I / R injury, this study co-stained Ly6G and H3Ci in AAV8-TBG-shPAI-1 and control mice liver I / R mouse model. The results showed that PAI-1 knockdown can significantly inhibit the co-expression of Ly6G and H3Ci ( Figure 18 ), the results of this study suggest that PAI-1 knockout can improve the progression of liver I / R and may be related to the formation and recruitment of NETs.

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of PAI-1 inhibitors in the preparation of drugs for the treatment of liver ischemia-reperfusion injury; The inhibitor is a PAI-1 enzyme activity inhibitor or a shRNA that inhibits PAI-1 gene expression. The PAI-1 enzyme activity inhibitor is PAI-039, and the nucleotide sequence of the shRNA is shown in SEQ ID NO.1.

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