Application of plasmin inhibitor NKI10 in the preparation of drugs for preventing and treating excessive inflammatory responses

Abstract

This invention discloses the application of the plasmin inhibitor NKI10 in the preparation of drugs for preventing and treating excessive inflammatory responses, belonging to the field of biomedical technology. The amino acid sequence of the plasmin inhibitor NKI10 disclosed in this invention is shown in any one of SEQ ID NO. 1 to 10. The plasmin inhibitor NKI10 can significantly inhibit the production of IL-6, IL-1β, and TNF-α in animal and cellular inflammation models. Since the plasmin inhibitor NKI10 can inhibit excessive inflammatory responses, it can be prepared for use as a drug for preventing and treating excessive inflammatory responses.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the application of the plasmin inhibitor NKI10 in the preparation of drugs for preventing and treating excessive inflammatory responses. Background Technology

[0002] A cytokine storm, also known as cytokine release syndrome (CRS) or inflammatory storm, is an inflammatory response characterized by an excessive immune response. Multiple cytokines / chemokines (collectively referred to as cytokines) such as IL-6, IL-1, TNF-α, IL-12, IFN-α, IFN-γ, MCP-1, and IL-8 are rapidly and excessively produced in bodily fluids. It can be caused by pathogenic infections (such as viral or bacterial infections), chimeric antigen receptor T-cell therapy (CAR-T), cancer, and autoimmune diseases. Cytokine storms caused by excessive immune responses pose a serious threat to life, but currently, there are no specific treatments available. Inhibiting the excessive inflammatory response to reduce cytokine levels is one of the reliable strategies to block cytokine storms.

[0003] Acute lung injury (ALI) is an acute, diffuse, inflammatory lung injury caused by various etiologies, which can further develop into acute respiratory distress syndrome (ARDS), with a mortality rate as high as 40%. The etiology of ALI is complex, but excessive inflammation is a key pathogenic mechanism. Therefore, animal models of ALI are not only suitable for screening anti-ALI drugs but also ideal models for screening drugs that combat excessive inflammatory responses.

[0004] Plasmin is a key protease in the fibrinolytic system. Plasminogen is converted into plasmin by plasminogen activators, including tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). The main physiological function of plasmin is to degrade fibrin(ogen), and the degradation products are collectively called fibrin / fibrinogen degradation products (FDPs). Fibrinolytic therapy is currently considered an effective drug strategy for treating ALI. Liu Cong et al. searched PubMed, Embase, Web of Science, and CNKI databases to collect and analyze preclinical research data on the treatment of ALI with different fibrinolytic drugs (tPA, uPA, and plasmin). The results showed that fibrinolytic drug treatment can improve ALI symptoms and reduce lung damage, thus suggesting that fibrinolytic drug treatment may be a very promising method for treating ALI.

[0005] In the early stages of this invention, a highly effective and specific plasmin inhibitor, NKI10, was discovered, which can be used as an antifibrinolytic drug. In this invention, it was found that NKI10 significantly improved lung tissue damage in ALI mice induced by intratracheal infusion of lipopolysaccharide (LPS) (a TLR4 agonist) and Poly(I:C) (a TLR3 agonist), and reduced the level of inflammatory factors. That is, the antifibrinolytic drug NKI10 improved ALI, which is clearly different from the aforementioned fibrinolytic therapy considered an effective drug strategy for treating ALI. Further experimental studies using a macrophage inflammation model showed that NKI10 can inhibit the release of inflammatory factors from macrophages induced by LPS and Poly(I:C). Therefore, the results of this invention fully demonstrate that the plasmin inhibitor NKI10 can inhibit excessive inflammatory responses and can be prepared for use as a drug to prevent and treat diseases with excessive inflammatory responses.

[0006] Therefore, providing the application of the plasmin inhibitor NKI10 in the preparation of drugs to prevent and treat excessive inflammatory responses is a 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 the application of plasmin inhibitor NKI10 in the preparation of drugs for preventing and treating excessive inflammatory responses.

[0008] This invention found that the plasmin inhibitor NKI10 significantly improved lung tissue damage in ALI mice induced by intratracheal infusion of lipopolysaccharide (LPS) (TLR4 agonist) and Poly(I:C) (TLR3 agonist), and reduced serum levels of inflammatory factors IL-6, IL-1, and TNF-α.

[0009] This invention found that NKI10 can significantly inhibit the release of inflammatory factors IL-6, IL-1, and TNF-α from macrophages induced by LPS and Poly(I:C).

[0010] This invention found that NKI10 can inhibit the induced expression of TLR4 in macrophages.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] The application of plasmin inhibitor NKI10 in the preparation of drugs for preventing and treating excessive inflammatory responses, wherein the amino acid sequence of plasmin inhibitor NKI10 is shown in any one of SEQ ID NO.1 to 10.

[0013] SEQ ID NO.1 amino acid sequence (NKI10-1):

[0014] GHLCNGDLLPGPCRAKMERWGLDKESGKCKKFIYGGCGGNRNNFESEEKCKKRC KVD; SEQ ID NO.1.

[0015] The gene sequence encoding SEQ ID NO.1 (NKI10-1) is as follows:

[0016] SEQ ID NO.11.

[0017] SEQ ID NO.2 amino acid sequence (NKI10-2):

[0018] MGMKGSGHLCNGDLLPGPCRAKMERWGLDKESGKCKKFIYGGCGGNRNNFESEE KCKKRCKVD; SEQ ID NO.2.

[0019] The gene sequence encoding SEQ ID NO.2 (NKI10-2) is as follows:

[0020] atgggaatgaagggaagtggtcatctatgcaatggggatttactaccaggaccttgcagagctaaaatggaaagatggggattggat aaggaatcaggaaagtgcaaaaaattcatctacggtggttgcggtggaaacagaaacaattttgaaagtgaagagaaatgcaagaaacgtt gcaaagtggattga (contains stop codon tga); SEQ IDNO.12.

[0021] SEQ ID NO.3 amino acid sequence (NKI10-3):

[0022] CNGDLLPGPCRAKMERWGLDKESGKCKKFIYGGCGGNRNNFESEEKCKKRC; SEQ ID NO.3.

[0023] The gene sequence encoding SEQ ID NO.3 (NKI10-3) is as follows:

[0024] SEQ ID NO. 13.

[0025] SEQ ID NO.4 amino acid sequence (NKI10-4):

[0026] GHLCNGDLLPGPCKAKMERWGLDKESGKCKKFIYGGCGGNRNNFESEEKCKKRC KVD; SEQ ID NO.4.

[0027] The gene sequence encoding SEQ ID NO.4 (NKI10-4) is as follows:

[0028] SEQ ID NO.14.

[0029] SEQ ID NO.5 amino acid sequence (NKI10-5):

[0030] GHLCNGDLLPGPCKARMERWGLDKESGKCKKFIYGGCGGNRNNFESEEKCKKRC KVD; SEQ ID NO.5.

[0031] The gene sequence encoding SEQ ID NO.5 (NKI10-5) is as follows:

[0032] SEQ ID NO.15.

[0033] SEQ ID NO.6 amino acid sequence (NKI10-6):

[0034] GHLCNGDLLPGPCRARMERWGLDKESGKCKKFIYGGCGGNRNNFESEEKCKKRC KVD; SEQ ID NO.6.

[0035] The gene sequence encoding SEQ ID NO.6 (NKI10-6) is as follows:

[0036] ggtcatctatgcaatggggatttactaccaggaccttgcagagctagaatggaaagatggggattggataaggaatcaggaaagtg caaaaaattcatctacggtggttgcggtggaaacagaaacaattttgaaagtgaagagaaatgcaagaaacgttgcaaagtggattga(including the stop codon tga); SEQ ID NO.16.

[0037] Amino acid sequence of SEQ ID NO.7 (NKI10-7):

[0038] CNGDLLPGPCKAKMERWGLDKESGKCKKFIYGGCGGNRNNFESEEKCKKRC; SEQ ID NO.7.

[0039] Gene sequence encoding SEQ ID NO.7 (NKI10-7):

[0040] tgcaatggggatttactaccaggaccttgcaaagctaaaatggaaagatggggattggataaggaatcaggaaagtgcaaaaaattc atctacggtggttgcggtggaaacagaaacaattttgaaagtgaagagaaatgcaagaaacgttgc; SEQ ID NO.17.

[0041] Amino acid sequence of SEQ ID NO.8 (NKI10-8):

[0042] CNGDLLPGPCKARMERWGLDKESGKCKKFIYGGCGGNRNNFESEEKCKKRC; SEQ ID NO.8.

[0043] Gene sequence encoding SEQ ID NO.8 (NKI10-8):

[0044] tgcaatggggatttactaccaggaccttgcaaagctagaatggaaagatggggattggataaggaatcaggaaagtgcaaaaaatt catctacggtggttgcggtggaaacagaaacaattttgaaagtgaagagaaatgcaagaaacgttgc; SEQ ID NO.18.

[0045] SEQ ID NO.9 amino acid sequence (NKI10-9):

[0046] CNGDLLPGPCRARMERWGLDKESGKCKKFIYGGCGGNRNNFESEEKCKKRC; SEQ ID NO.9.

[0047] The gene sequence encoding SEQ ID NO.9 (NKI10-9) is as follows:

[0048] SEQ ID NO. 19.

[0049] SEQ ID NO.10 amino acid sequence (NKI10-10):

[0050] GHLCNGDLLPGPCRVKMERWGLDKESGKCKKFIYGGCGGNRNNFESEEKCKKRC KVD; SEQ ID NO.10.

[0051] The gene sequence encoding SEQ ID NO.10 (NKI10-10) is as follows:

[0052] SEQ ID NO.20.

[0053] Furthermore, a pharmaceutical formulation for preventing and treating excessive inflammatory responses comprises a plasmin inhibitor NKI10 as the active ingredient, either directly or with a pharmaceutically acceptable carrier; the amino acid sequence of the plasmin inhibitor NKI10 is shown in any one of SEQ ID NO. 1 to 10.

[0054] Furthermore, the application of plasmin inhibitor NKI10 in the preparation of drugs that inhibit the overexpression of inflammatory factors, wherein the amino acid sequence of plasmin inhibitor NKI10 is shown in any one of SEQ ID NO.1 to 10.

[0055] Furthermore, the inflammatory factors include, but are not limited to, IL-6, IL-1β, and TNF-α.

[0056] Furthermore, the application of NKI10 in the preparation of drugs for the prevention and treatment of acute lung injury, wherein the amino acid sequence of the plasmin inhibitor NKI10 is shown in any one of SEQ ID NO.1 to 10.

[0057] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of plasmin inhibitor NKI10 in the preparation of drugs for preventing and treating excessive inflammatory responses, which has the following beneficial effects:

[0058] The plasmin inhibitor NKI10 in this invention can significantly improve LPS and Poly(I:C)-induced lung tissue damage in ALI mice and reduce the levels of inflammatory factors IL-6, IL-1β, and TNF-α in the serum of ALI mice.

[0059] The plasmin inhibitor NKI10 in this invention can significantly reduce the levels of IL-6, IL-1, and TNF-α in the supernatant of LPS and Poly(I:C) induced macrophage culture cells.

[0060] The plasmin inhibitor NKI10 in this invention can inhibit the induced expression of TLR4 in cells, thereby suppressing the inflammatory response. Attached Figure Description

[0061] 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.

[0062] Figure 1 Protective effect of NKI10 against LPS-induced acute lung injury in mice

[0063] Wherein, A represents the wet-to-dry weight ratio of the right lower lobe of mice in each group; B represents the lung tissue damage score of mice in each group; CH represents the overall observation of lung tissue in mice in the sham-operated group, model group, TXA group, low-dose NKI10-1 group, medium-dose NKI10-1 group, and high-dose NKI10-1 group, respectively; IN represents the pathological changes of lung tissue in mice in the sham-operated group, model group, TXA group, low-dose NKI10-1 group, medium-dose NKI10-1 group, and high-dose NKI10-1 group, respectively; O represents the serum TNF-α level of mice in each group; P represents the serum IL-1β level of mice in each group; and Q represents the serum IL-6 level of mice in each group. *** P < 0.001, compared with the sham surgery group; ## P < 0.01, ### P < 0.001, compared with the model group; & P < 0.05 && P < 0.01, &&& P < 0.001, compared with the TXA group; ^^^ P < 0.001, compared with the low-dose NKI10 group; $$$ P < 0.001, compared with the medium-dose group of NKI10;

[0064] Figure 2 Experimental study on the effect of NKI10 on LPS-induced inflammation in RAW264.7 cells and THP-1-derived macrophages.

[0065]

[0066] Wherein, A represents the TNF-α level of RAW264.7 cells in each group; B represents the IL-1β level of RAW264.7 cells in each group; C represents the IL-6 level of RAW264.7 cells in each group; D represents the TNF-α level of THP-1-derived macrophages in each group; E represents the IL-1β level of THP-1-derived macrophages in each group; F represents the IL-6 level of THP-1-derived macrophages in each group; G represents the TNF-α level of RAW264.7 cells in each group; H represents the IL-1β level of RAW264.7 cells in each group; and I represents the IL-6 level of RAW264.7 cells in each group. ** P < 0.01, *** P < 0.001, compared with the blank control group; ## P < 0.01, ### P < 0.001, compared with the LPS group; &&& P < 0.001, compared with the TXA group; ^^^ P < 0.001, compared with the low-dose NKI10 group; $$$ P < 0.001, compared with the medium-dose group of NKI10;

[0067] Figure 3 The protective effect of NKI10 against Poly(I:C)-induced acute lung injury in mice.

[0068] Wherein, A represents the wet-to-dry weight ratio of the right lower lobe of mice in each group; B represents the lung tissue damage score of mice in each group; CE represents the overall observation of lung tissue in mice in the sham-operated group, Poly(I:C) group, and NKI10-1 group, respectively; FH represents the pathological changes of lung tissue in mice in the sham-operated group, Poly(I:C) group, and NKI10-1 group, respectively; I represents the serum TNF-α level of mice in each group; J represents the serum IL-1β level of mice in each group; and K represents the serum IL-6 level of mice in each group. ** P < 0.01, *** P < 0.001, compared with the sham surgery group; # P < 0.05 ## P < 0.01, ### P < 0.001, compared with the Poly(I:C) group;

[0069] Figure 4 The role of NKI10 in a Poly(I:C)-induced macrophage inflammation model

[0070] Wherein, A represents the TNF-α level of RAW264.7 cells in each group; B represents the IL-1β level of RAW264.7 cells in each group; C represents the IL-6 level of RAW264.7 cells in each group; D represents the TNF-α level of THP-1-derived macrophages in each group; E represents the IL-1β level of THP-1-derived macrophages in each group; and F represents the IL-6 level of THP-1-derived macrophages in each group. ** P < 0.01, compared with the blank control group; ## P < 0.01, compared with the Poly(I:C) group;

[0071] Figure 5 The effect of NKI10 on TLR4 expression levels in RAW264.7 cells (n = 3, 200 × 10⁻⁶).

[0072] In the image, AI represents the TLR4 expression in RAW264.7 cells of each group, with green fluorescence representing TLR4 expression; J represents the amount of green fluorescence in each group, which represents the TLR4 expression level. Specifically, A1-A3 are the Control group, B1-B3 are the Fgn group, C1-C3 are the Plm group, D1-D3 are the Fgn+Plm (FDPs) group, E1-E3 are the Fgn+Plm+NKI10-1 group, F1-F3 are the LPS group, G1-G3 are the LPS+NKI10-1 group, H1-H3 are the LPS+Fgn group, and I1-I3 are the LPS+Fgn+NKI10-1 group.*** P < 0.001, compared with the Control group; ### P < 0.001, compared with the Fgn+Plm group; && P < 0.01, &&& P < 0.001, compared with the LPS group; $$$ P < 0.001, compared with the LPS+Fgn group; Nucleus: blue fluorescence represents the nucleus; TLR4 expression level: green fluorescence represents the TLR4 expression level; The combination of both: the combination of the nucleus and the TLR4 expression level. Detailed implementation manners

[0073] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0074] Example 1 Protective effect of NKI10-1 on LPS-induced acute lung injury in mice

[0075] 1) Animals

[0076] 78 SPF-grade 2-month-old male C57BL / 6J mice, weighing 18-22 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd., license number: SCXK(Beijing)2020-0004. They were acclimated in the SPF environment of the Experimental Animal Center of Guangdong Medical University for 7 days before the experiment. All experiments were approved by the Experimental Animal Management and Ethics Committee of Guangdong Medical University, and the ethical review number: GDY2103018. The handling and experimental operation procedures of the mice were carried out in accordance with the ethical requirements of experimental animals.

[0077] 2) Instruments

[0078] The microplate reader (ELX 808, 800TS) is a product of Bio Tek Company, USA; the KD2268 microtome is from Cody Instruments Co., Ltd., Jinhua City, Zhejiang Province; the OLYMPUS CX23 microscope was purchased from Olympus Corporation, Japan.

[0079] 3) Drugs and reagents

[0080] Tranexamic acid (TXA) was purchased from Sigma-Aldrich, USA, lot number: LRAB8978; lipopolysaccharide (LPS) was purchased from Sigma-Aldrich, USA, lot number: 086M4159V; the mouse tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) ELISA kits were purchased from R&D Company, USA, lot numbers P340968, P138448, and P311746, respectively.

[0081] Preparation of recombinant NKI10-1 (rNKI10-1): The method for preparing the crude product refers to Chinese Patent Application No. 201810505110.1, followed by purification. ① Preparation of crude product: Taking the expression of the amino acid sequence shown in SEQ ID NO.1 (NKI10-1) as an example, in short: design the upstream primer NI10-1e (SEQ ID NO.21) and the downstream primer NI10-2e (SEQ ID NO.22) for recombinant expression encoding. Using the gene sequence encoding the amino acid sequence of SEQ ID NO.1 (SEQ ID NO.11) as a template, the gene sequence encoding the amino acid sequence shown in SEQ ID NO.1 is amplified. Amplification system: ddH2O 38μL, dNTP (2mmol / L) 5.0μL, 10×PCR buffer (containing Mg) 2+5.0 μL of primer, 0.6 μL each of forward and reverse primers (20 μmol / L), 0.5 μL of template, and 0.3 μL of PfuDNA polymerase (5 U / μL). Reaction conditions: 95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, for a total of 30 cycles. The obtained gene sequence was ligated into the prokaryotic expression vector pET32a-sumo, and the correct recombinant plasmid was constructed and transformed into Escherichia coli BL21(DE3). Expression was induced by IPTG. After isolation and sonication of the host cells, the expression product was purified by nickel affinity chromatography to obtain the fusion protein. The fusion partner was cleaved by SUMO protease, and the fusion partner was further removed by affinity chromatography to obtain the crude recombinant NKI10-1 product. ② Purification: The crude rNKI10-1 product was purified using an AKTA pure 150 (GE) protein purification system, followed by SP Bio-sep FF cation exchange chromatography and Superdex 75pg (Xi'an Baosai Hengcheng Biotechnology Co., Ltd.) gel filtration for further purification and endotoxin removal. The sterile double-distilled water and PB buffer used in reagent preparation were filtered through a 5Kd hollow fiber column (MicroKros mPES 5Kd, Spectrum product) to remove endotoxins. Glassware was first soaked in 0.5mol / L NaOH for 12 hours and then rinsed with sterile double-distilled water to remove endotoxins. Heat-resistant reagent bottles were baked at 200℃ for 3 hours and then cooled before use. Eppendorf tubes and pipette tips were soaked in 0.5mol / L NaOH and then rinsed with sterile double-distilled water to remove endotoxins. Reagents and containers were used within 48 hours. The purified rNKI10-1 was analyzed for protein concentration using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.), yielding a concentration of 3.18 mg / mL. Endotoxin levels were determined using a gel electrophoresis Limulus Amebocyte Lysate (LAL) assay kit (Zhanjiang Andus Biotechnology Co., Ltd.). The purified rNKI10-1 was aliquoted, lyophilized, and stored at -40°C for later use. Before use, the aliquots were prepared at the recommended dosage on ice.

[0082] 4) Preparation of LPS-induced acute lung injury model in mice

[0083] Mice were placed in a sealed anesthesia box and anesthetized with 5% isoflurane. After anesthesia, the mice were quickly removed and fixed supine on a 37°C constant-temperature operating table with their incisors and limbs exposed. Isoflurane gas was continuously administered through a face mask to maintain anesthesia. The hair around the neck was carefully shaved, and the neck skin was disinfected with 75% alcohol and povidone-iodine solution. The neck skin was longitudinally cut about 1 cm, and the trachea was carefully separated and exposed. A 1 mL insulin needle was used to puncture between the two tracheal cartilages towards the lungs, and LPS 5 mg / kg was infused, with a total infusion volume of 40 μL, to establish a mouse model of acute lung injury.

[0084] 5) Experimental grouping and drug administration

[0085] Seventy-eight 2-month-old male SPF-grade C57BL / 6J mice were randomly divided into six groups: sham-operated group (Control), model group (LPS: 5.0 mg / kg, Model), tranexamic acid (TXA: 100.0 mg / kg), low-dose NKI10-1 group (1.0 mg / kg, NKI10-1-L), medium-dose NKI10-1 group (2.0 mg / kg, NKI10-1-M), and high-dose NKI10-1 group (4.0 mg / kg, NKI10-1-H), with 13 mice in each group. Except for the sham-operated group, acute lung injury models were established in mice using the LPS intratracheal infusion method. The TXA group received the corresponding dose of the drug via tail vein injection 3 hours after LPS infusion, while the NKI10-1 group received the corresponding dose via tail vein injection 6 hours after LPS infusion. The sham-operated group and model group received an equal volume of saline via tail vein injection. Both grouping and drug administration were performed using a double-blind method. Mice were fed a normal diet during the experiment, and the condition of mice in each group was observed.

[0086] 6) Sample collection and processing

[0087] Twenty-four hours after intratracheal instillation, mice were placed in an anesthesia box and anesthetized with 5% isoflurane. After anesthesia, the mice were removed, and blood was collected by enucleation. The blood was incubated at 4°C for 2 hours, then centrifuged at 3500 rpm for 15 minutes to separate the serum, which was then stored at -80°C for later use. Nine mice were randomly selected from each group. The lower lobe of the right lung was isolated, washed with physiological saline, blotted dry with filter paper, and weighed as wet weight. The lower lobe of the right lung was baked in a 60°C oven for 72 hours until constant weight, and weighed as dry weight. The upper lobe of the right lung was taken, washed with physiological saline, blotted dry with filter paper, and then immersed in 10% neutral formaldehyde at room temperature for at least 24 hours. The lung tissue was then embedded in paraffin blocks for further pathological staining and photography. The remaining lung tissue was washed with physiological saline, blotted dry with filter paper, and stored at -80°C for later use.

[0088] 7) Lung tissue wet / dry ratio detection

[0089] Calculate the wet / dry weight ratio (W / D) of lung tissue, where W / D = wet weight / dry weight.

[0090] 8) Pathological observation of lung tissue

[0091] Mice were euthanized by cervical dislocation and immediately fixed in 10% neutral formaldehyde. After 24 hours of fixation, the trimmed lung tissue was dehydrated in ethanol of different concentrations and cleared in xylene, following these steps: 70% ethanol for 12 hours; 80% ethanol for 12 hours; 95% ethanol for 3 hours; anhydrous ethanol for 1 hour; and xylene for 2 minutes. The cleared tissue blocks were then placed in a paraffin bath (70°C) with pre-melted paraffin and kept warm for 3 hours. After the paraffin had completely penetrated the tissue and solidified, the embedded paraffin blocks were fixed on a paraffin microtome and cut into 5μm thick sections. The sections were spread in heated water (40°C), retrieved using a slide to prevent detachment, and finally dried in a 50°C oven. HE staining, microscopic observation, and photography were then performed. Lung tissue damage was assessed by a researcher unaware of our experiment. Specific scoring method: The severity of lung tissue damage is represented by a scale of 0 to 4: 0 indicates no damage, 1 indicates minor damage, 2 indicates moderate damage, 3 indicates severe damage, and 4 indicates very severe damage.

[0092] 9) Measurement of inflammatory factor levels in serum and BALF

[0093] Mouse serum was collected, and the levels of TNF-α, IL-1β, and IL-6 in the serum of each group of mice were detected according to the detection steps in the kit instructions.

[0094] 10) Statistical Analysis

[0095] All experimental data were expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 17.0 software. The t-test was used to test the significance of comparisons between two groups, and the LSD (Least Significant Range) method was used for comparisons among multiple groups in one-way ANOVA. A p-value < 0.05 was considered statistically significant.

[0096] 11) Results

[0097] like Figure 1 A. Compared with the sham-operated group, the wet-to-dry weight ratio of the right lower lobe of mice in the model group was significantly increased (P < 0.001). Compared with the model group, the wet-to-dry weight ratio of the right lower lobe of mice in the NKI10-1 group was significantly decreased (P < 0.01).

[0098] like Figure 1 In mice in groups C and I, the lung tissue of the sham-operated group was generally bright with no dark shadows, normal structure, thin alveolar walls, and clearly visible alveolar cavities, without hemorrhage or inflammatory cell infiltration. Compared with the sham-operated group, the lung tissue of mice in the model group showed obvious large dark spots, thickened alveolar walls and septa, and a large number of inflammatory cell infiltrations 24 hours after LPS intratracheal infusion, indicating significant damage. Figure 1D and J), lung tissue injury scores were significantly increased (P < 0.001) Figure 1 B). Compared with the model group, the NKI10-treated mice showed a significant reduction in lung tissue dysporinous patches, more normal morphology and structure, significantly reduced inflammatory cell infiltration, significantly decreased alveolar wall and septal thickness, and a significantly lower lung tissue damage score (P < 0.001). Figure 1 F~H and Figure 1 L~N).

[0099] like Figure 1 Compared with the sham-operated group, the serum levels of TNF-α, IL-1β, and IL-6 in the model group mice were significantly increased (P < 0.001). Compared with the model group, the serum levels of TNF-α, IL-1β, and IL-6 in the NKI10-1 group mice were significantly decreased (P < 0.001). Compared with the low-dose NKI10-1 group, the serum levels of TNF-α, IL-1β, and IL-6 in the medium-dose and high-dose NKI10-1 groups were significantly decreased (P < 0.001). Compared with the medium-dose NKI10-1 group, the serum levels of TNF-α, IL-1β, and IL-6 in the high-dose NKI10-1 group were significantly decreased (P < 0.001).

[0100] 12) Conclusion

[0101] NKI10-1 can significantly improve the symptoms of LPS-induced acute lung injury and reduce the level of inflammatory response. Therefore, NKI10 can be formulated for the prevention and treatment of acute lung injury and to inhibit excessive inflammatory response.

[0102] Example 2: Effect of NKI10-1 on LPS-induced macrophage inflammation model

[0103] 1) Cells

[0104] Human acute monocytic leukemia cells (THP-1 cells) were purchased from the American Type Culture Collection Center (ATCC); mouse mononuclear macrophages RAW264.7 were purchased from Wuhan Procell Biotechnology Co., Ltd.

[0105] 2) Instruments

[0106] The BB150 CO2 cell incubator is a product of Thermo Fisher Scientific, Inc., USA, and the microplate reader (ELX 808, 800TS) is a product of BioTek Inc., Inc., USA.

[0107] 3) Drugs and reagents

[0108] The human TNF-α ELISA kit, IL-1β ELISA kit, and IL-6 ELISA kit were products of R&D Company, USA, with batch numbers P284648, P274237, and P280369, respectively; the preparation of NKI10 was as follows; other drugs and reagents were the same as in Example 1.

[0109] Preparation of NKI10: The preparation of SEQ ID NO.1 (NKI10-1) is described in Example 1. The amino acid sequences shown in SEQ ID NO.2 (NKI10-2), SEQ ID NO.3 (NKI10-3), SEQ ID NO.4 (NKI10-4), SEQ ID NO.5 (NKI10-5), SEQ ID NO.6 (NKI10-6), SEQ ID NO.7 (NKI10-7), SEQ ID NO.8 (NKI10-8), SEQ ID NO.9 (NKI10-9), and SEQ ID NO.10 (NKI10-10) were prepared according to the method used to prepare SEQ ID NO.1 (NKI10-1). Using the corresponding coding sequences (as shown in SEQ ID NO.12-20) as templates, the primer sequences used to amplify each coding gene are shown in Table 1.

[0110] Table 1 Primers used to amplify the NKI10 coding sequence.

[0111]

[0112]

[0113] The amplified coding sequences of SEQ ID NO.2 (NKI10-2) were paired with NI10-3e and NI10-2e; the amplified coding sequences of SEQ ID NO.4 (NKI10-4), SEQ ID NO.5 (NKI10-5), SEQ ID NO.6 (NKI10-6), and SEQ ID NO.10 (NKI10-10) were paired with NI10-1e and NI10-2e; and the amplified coding sequences of SEQ ID NO.3 (NKI10-3), SEQ ID NO.7 (NKI10-7), SEQ ID NO.8 (NKI10-8), and SEQ ID NO.9 (NKI10-9) were paired with NI10-5e and NI10-6e. All prepared NKI10 concentrations were >2.0 mg / mL. After aliquoting, the NKI10 was stored at -40°C for later use. Before use, the NKI10 was prepared to a dosing concentration on ice.

[0114] 4) Grouping of the effects of NKI10-1 on LPS-induced inflammation in RAW264.7 cells and THP-1-derived macrophages

[0115] RAW264.7 cells were cultured in a DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator.

[0116] THP-1 cells were cultured in a 37°C, 5% CO2 incubator using 1640 high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. THP-1 cells were stimulated with 100 ng / mL PMA for 24 h to transform into macrophages.

[0117] The two cell types were divided into 6 groups (5 × 10⁻⁶ cells each). 4 Cells were seeded at 1 mL per well in 12-well plates, serving as a control group (100 ng / mL), an LPS group (100 ng / mL), a TXA group (25 μg / mL), a low-dose NKI10-1 group (5 μg / mL), a medium-dose NKI10-1 group (10 μg / mL), and a high-dose NKI10-H group (20 μg / mL). Each concentration group was replicated six times. Except for the control group, all other groups were co-stimulated with the drug and LPS for 3 h, followed by the detection of inflammatory markers in the cell supernatant.

[0118] 5) Grouping of NKI10 effects on LPS-induced inflammation in RAW264.7 cells

[0119] RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. The cells were divided into 12 groups (5 × 10⁶ cells per group). 4 Cells were seeded at 1 mL per well in 12-well plates, with the following groups: Control, LPS (100 ng / mL), LPS+NKI10⁻¹ (5 μg / mL), LPS+NKI10⁻² (5 μg / mL), LPS+NKI10⁻³ (5 μg / mL), LPS+NKI10⁻⁴ (5 μg / mL), LPS+NKI10⁻⁵ (5 μg / mL), LPS+NKI10⁻⁶ (5 μg / mL), LPS+NKI10⁻⁷ (5 μg / mL), LPS+NKI10⁻⁸ (5 μg / mL), LPS+NKI10⁻⁹ (5 μg / mL), and LPS+NKI10⁻¹⁰ (5 μg / mL). Each concentration group was replicated six times. Except for the control group, all other groups were co-stimulated with the drug and LPS for 3 h, followed by the detection of inflammatory markers in the cell supernatant.

[0120] 6) Measurement of inflammatory factor levels in cell supernatant

[0121] Two cell supernatants were collected, and the levels of TNF-α, IL-1β, and IL-6 in each group were detected according to the test procedure in the kit instructions.

[0122] 7) Statistical analysis: Same as Example 1.

[0123] 8) Results

[0124] like Figure 2 RAW264.7 cells (A-C): Compared with the blank control group, the levels of TNF-α, IL-1β, and IL-6 in the LPS group were significantly increased (P < 0.001). Compared with the LPS group, the levels of TNF-α, IL-1β, and IL-6 in the NKI10-1 group were significantly decreased (P < 0.001). Compared with the low-dose NKI10-1 group, the levels of TNF-α, IL-1β, and IL-6 in the medium-dose and high-dose NKI10-1 groups were significantly decreased (P < 0.001). Compared with the medium-dose NKI10 group, the levels of TNF-α, IL-1β, and IL-6 in the high-dose NKI10 group were significantly decreased (P < 0.001).

[0125] like Figure 2 D–F, THP-1-derived macrophages: Compared with the blank control group, the levels of TNF-α, IL-1β, and IL-6 in the LPS group were significantly increased (P < 0.001). Compared with the LPS group, the levels of TNF-α, IL-1β, and IL-6 in the NKI10-1 treatment group were significantly decreased (P < 0.001). Compared with the low-dose NKI10-1 group, the levels of TNF-α, IL-1β, and IL-6 in the medium-dose and high-dose NKI10-1 groups were significantly decreased (P < 0.001). Compared with the medium-dose NKI10-1 group, the levels of TNF-α, IL-1β, and IL-6 in the high-dose NKI10-1 group were significantly decreased (P < 0.001).

[0126] like Figure 2 G-I, RAW264.7 cells: Compared with the blank control group, the levels of TNF-α, IL-1β, and IL-6 in the LPS group were significantly increased (P < 0.01). Compared with the LPS group, the levels of TNF-α, IL-1β, and IL-6 in the NKI10-treated group were significantly decreased (P < 0.01).

[0127] 9) Conclusion

[0128] NKI10 significantly inhibited the levels of inflammatory factors in LPS-induced RAW264.7 cells and THP-1-derived macrophages, indicating that NKI10 can suppress excessive inflammatory responses.

[0129] Example 3: Protective effect of NKI10-1 against Poly(I:C)-induced lung injury in mice.

[0130] 1) Animals

[0131] Twenty-four 2-month-old male C57BL / 6J mice at the SPF level, weighing 18 - 22 g, were purchased from Beijing Speywood Biotechnology Co., Ltd., with the license number: SCXK(Beijing)2019-0010. Before the experiment, they were acclimated in the SPF environment of the Experimental Animal Center of Guangdong Medical University for 7 days. All experiments were approved by the Experimental Animal Management and Ethics Committee of Guangdong Medical University, with the ethical review number: GDY2203019. The handling and experimental operation procedures of the mice were carried out in accordance with the ethical requirements of experimental animals.

[0132] 2) Instruments: The same as in Example 1.

[0133] 3) Drugs and reagents

[0134] Poly(I:C) was purchased from Sigma Company, USA, batch number: 0000105459; others were the same as in Example 1.

[0135] 3) Preparation of a mouse model of lung injury induced by Poly(I:C)

[0136] The mice were placed in a closed anesthesia box, and anesthetized with 5% isoflurane. After anesthesia, the mice were quickly taken out, and their incisors and limbs were fixed face up on a surgical table maintained at 37°C. Isoflurane gas was continuously administered to the mice through a face mask to maintain anesthesia. The hair around the neck was carefully shaved, and the neck skin was disinfected with 75% alcohol and iodophor disinfectant. The neck skin was longitudinally incised about 1 cm, and the trachea was carefully separated and exposed. A 1 mL insulin syringe was used to puncture and drip 3 mg / kg of Poly(I:C) into the lungs from between the two tracheal cartilages, with a total drip volume of 40 μL to establish a mouse model of lung injury.

[0137] 4) Experimental grouping and drug administration

[0138] Twenty-four 2-month-old male C57BL / 6J mice at the SPF level were randomly divided into 3 groups, namely the sham operation group (Control), the model group (Poly(I:C): 3 mg / kg), and the NKI10-1 group (2 mg / kg), with 8 mice in each group. Except for the sham operation group, the mouse models of lung injury were established in the other groups by tracheal instillation of Poly(I:C). The NKI10 administration group was intravenously injected with the corresponding dose of the drug via the tail vein 6 h after the instillation of LPS. The sham operation group and the model group were intravenously injected with an equal volume of normal saline. The experimental grouping and drug administration were both carried out by double-blind methods. The mice were fed normally during the experiment, and the conditions of the mice in each group were observed.

[0139] 5) Sample collection and processing

[0140] Twenty-four hours after modeling, mice were anesthetized, and blood was collected by enucleation. The blood was allowed to stand at 4°C for 2 hours, then centrifuged at 3500 rpm for 15 minutes to separate the serum, which was then stored at -80°C for later use. The serum levels of TNF-α, IL-1β, and IL-6 in each group of mice were measured according to the kit instructions. Lung tissue was carefully removed, rinsed thoroughly with saline, and photographed. For the remaining mice, the lower lobe of the right lung was harvested, washed with saline, blotted dry with filter paper, and weighed as wet weight. The lower lobe was then baked in a 60°C oven for 72 hours until constant weight, and weighed as dry weight. The wet / dry weight ratio (W / D) of the lung tissue was calculated, W / D = wet weight / dry weight, to reflect the degree of lung edema. The upper lobe of the right lung was harvested, washed with saline, blotted dry with filter paper, and stored in 10% neutral formaldehyde. Histological sections were prepared according to the method in Example 1, stained with hematoxylin and eosin (HE), observed under a microscope, and photographed to assess lung tissue damage. The remaining lung tissue was washed with saline, blotted dry with filter paper, and stored at -80°C for later use.

[0141] 6) Statistical analysis: Same as Example 1.

[0142] 7) Results

[0143] like Figure 3 A. Compared with the sham-operated group, the wet-to-dry weight ratio of the right lower lobe of mice in the model group was significantly increased (P < 0.01). Compared with the model group, the wet-to-dry weight ratio of the right lower lobe of mice in the NKI10-1 group was significantly decreased (P < 0.05).

[0144] like Figure 3 In groups C and F, the lung tissue of sham-operated mice was generally bright with no dark shadows, normal structure, thin alveolar walls, and clearly visible alveolar cavities, without hemorrhage or inflammatory cell infiltration. Compared with the sham-operated group, the lung tissue of model group mice showed obvious large dark spots, thickened alveolar walls and septa, and a large number of inflammatory cell infiltrations 24 hours after Poly(I:C) intratracheal infusion, indicating significant damage. Figure 3 (D and G), lung tissue injury scores were significantly elevated (P < 0.001) Figure 3 B). Compared with the model group, the NKI10 group mice showed a significant reduction in lung tissue dysporinous patches, more normal morphology and structure, significantly reduced inflammatory cell infiltration, significantly reduced alveolar wall and septal thickness, and a significantly lower lung tissue damage score (P < 0.01). Figure 3 E and H).

[0145] like Figure 3 Compared with the sham-operated group, the serum levels of TNF-α, IL-1β, and IL-6 in the model group mice were significantly increased (P < 0.001). Compared with the model group, the serum levels of TNF-α, IL-1β, and IL-6 in the NKI10-1 group mice were significantly decreased (P < 0.01).

[0146] 8) Conclusion

[0147] NKI10-1 can significantly improve symptoms of Poly(I:C)-induced lung injury and inhibit Poly(I:C)-induced inflammatory response.

[0148] Example 4: Effect of NKI10 on Poly(I:C)-induced macrophage inflammation model

[0149] 1) Cells and instruments: Same as in Example 2.

[0150] 2) Drugs and reagents

[0151] Poly(I:C) was purchased from Sigma-Aldrich, USA, lot number: 0000105459; other components are the same as in Example 2.

[0152] 3) RAW264.7 cell culture and grouping

[0153] RAW264.7 cells were cultured as in Example 2, at a concentration of 5 × 10⁻⁶ cells / cells. 4 Cells were seeded at 1 mL per well in 12-well plates. Cells were divided into 12 groups: a control group, a Poly(I:C) group (20 μg / mL), and a Poly(I:C) + NKI10 group (NKI10-1 to 10 groups, 20 μg / mL). Each concentration group was replicated 6 times. Except for the control group, all other groups were co-stimulated with the drug and Poly(I:C) for 3 h, followed by the detection of inflammatory markers in the cell supernatant.

[0154] 4) THP-1 cell culture and grouping

[0155] THP-1-derived macrophages were cultured in the same manner as in Example 2, at 5 × 10⁻⁶ cells per cell line. 4 Cells were seeded at 1 mL per well in 12-well plates. Three groups were established: a control group, a Poly(I:C) group (20 μg / mL), and a Poly(I:C) + NKI10 group (NKI10-1 to 10, 20 μg / mL). Each concentration group was replicated six times. Except for the control group, all other groups were co-stimulated with the drug and Poly(I:C) for 3 h, followed by measurement of inflammatory markers in the cell supernatant.

[0156] 5) Measurement of inflammatory factor levels in cell supernatant

[0157] Two cell supernatants were collected, and the levels of TNF-α, IL-1β, and IL-6 in each group were detected according to the test procedure in the kit instructions.

[0158] 6) Statistical analysis: Same as Example 1.

[0159] 7) Results

[0160] like Figure 4 In both macrophage groups, compared with the blank control group, the levels of TNF-α, IL-1β, and IL-6 in the Poly(I:C) group were significantly increased (P < 0.001). Compared with the Poly(I:C) group, the levels of TNF-α, IL-1β, and IL-6 in the NKI10 group were significantly decreased (P < 0.001).

[0161] 8) Conclusion

[0162] NKI10 significantly inhibited the levels of inflammatory factors induced by Poly(I:C) in RAW264.7 cells and THP-1-derived macrophages.

[0163] Example 5: The intervention effect of NKI10 on TLR4 expression in RAW264.7 cells

[0164] 1) Cells

[0165] Mouse mononuclear macrophages RAW264.7 were purchased from Wuhan Procell Biotechnology Co., Ltd.

[0166] 2) Main medicines and reagents

[0167] Human fibrinogen (Fgn) was purchased from Shenzhen Weiguang Biological Products Co., Ltd., batch number: 20190805; human plasmin (Plm) was purchased from Prolytix (USA), batch number: NN0427; lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (USA), batch number: 086M4159V; TLR4 monoclonal antibody was purchased from Cell Signaling Technology (USA), batch number: 14358S; Alexa Fluor488 fluorescent secondary antibody was purchased from Beijing Solarbio Science & Technology Co., Ltd., batch number: 20210628; DAPI was purchased from Beijing Solarbio Science & Technology Co., Ltd., batch number: 20210824. NKI10-1 was prepared by the research group, see Example 1.

[0168] 3) Instruments and equipment

[0169] IXplore SpinSR rotating super-resolution laser confocal microscope (Olympus Corporation, Japan).

[0170] 4) Cell culture and grouping

[0171] RAW264.7 cells in the logarithmic growth phase were centrifuged, counted, and diluted to the appropriate cell density at 5 × 10⁻⁶ cells / cell. 4Cells were seeded at 1 mL per well in 12-well plates. Cells were divided into four groups: Control, Fibrinogen (Fgn), Plasmin (Plm), Fgn+Plm (FDPs), Fgn+Plm+NKI10-1, LPS, LPS+NKI10-1, LPS+Fgn, and LPS+Fgn+NKI10-1, with three replicates per group. For the Fgn+Plm and Fgn+Plm+NKI10-1 groups, the drugs were thoroughly mixed in vitro and incubated at 37°C for 30 min before being added to the corresponding cell groups. For the other groups, the drugs were added to the corresponding cell groups simultaneously. The cell culture plates were then incubated statically in a cell culture incubator for 12 h. The drug concentrations in each group were: Fgn 1 mg / mL; Plm 80 nmol / mL; NKI10-1 1 μg / mL; and LPS 1 μg / mL.

[0172] 5) Observation of TLR4 expression

[0173] After 12 hours of static culture in an incubator, the culture medium was discarded, and the cells were washed three times with PBS. The cells were then fixed with 4% neutral formaldehyde solution for 20 minutes, washed three times with PBS, treated with Triton X-100 for 20 minutes, and washed three times with PBS. After 30 minutes of blocking with 2% BSA, TLR4 antibody dilution (1:400) was added, and the cells were incubated overnight at 4°C. On the second day, the cells were washed three times with PBS, and Alexa Fluor 488 fluorescent secondary antibody dilution (1:300) was added, and the cells were incubated at room temperature in the dark for 1 hour. After discarding the secondary antibody, the nuclei were stained with DAPI staining solution (1:100) for 5 minutes, washed three times with PBS, and observed under a confocal microscope. The fluorescence intensity was analyzed using ImageJ 1.52v software.

[0174] 6) Statistical analysis: Same as Example 1.

[0175] 7) Results

[0176] like Figure 5Compared with the blank control group, the TLR4 expression level of cells in the Fgn+Plm(FDPs), LPS, and LPS+Fgn groups was significantly increased (P<0.001); compared with the Fgn+Plm(FDPs) group, the TLR4 expression level of cells in the Fgn+Plm+NKI10-1 group was significantly decreased (P<0.001), while the TLR4 expression level of cells in the LPS and LPS+Fgn groups was significantly increased (P<0.001); compared with the LPS group, the TLR4 expression level of cells in the LPS+NKI10-1 group was significantly decreased (P<0.001), while the TLR4 expression level of cells in the LPS+Fgn group was significantly increased (P<0.01); compared with the LPS+Fgn group, the TLR4 expression level of cells in the LPS+Fgn+NKI10-1 group was significantly decreased (P<0.001).

[0177] 8) Conclusion

[0178] Fibrinogen (Fgn) and plasmin (Plm) neither stimulated TLR4 expression in macrophages, but fibrinogen-derived fibrinogen-derived fibrinogen (FDPs) produced by plasmin degradation significantly stimulated TLR4 expression in macrophages. NKI10-1 inhibited the degradation of Fgn by Plm and significantly suppressed TLR4 expression in macrophages. NKI10-1 also significantly inhibited LPS-induced TLR4 expression in macrophages. These results indicate that NKI10 can inhibit the induction of TLR4 expression in cells, thereby suppressing the inflammatory response.

[0179] 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. Use of the plasmin inhibitor NKI 10 for the preparation of a medicament for the prevention and treatment of acute lung injury, characterized in that, The amino acid sequence of the plasmin inhibitor NKI10 is shown in SEQ ID NO.

1. The amino acid sequence of the plasmin inhibitor NKI10 is shown in SEQ ID NO. 1.

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