Application of ancylostoma duodenum polypeptide AdKI1 in preparation of medicine for resisting excessive inflammatory reaction

Abstract

The invention discloses application of ancylostoma duodenum polypeptide AdKI1 in preparation of drugs for resisting excessive inflammatory reaction, and belongs to the technical field of biological medicines. The invention discloses an application of ancylostoma duodenum polypeptide AdKI1 in preparation of a medicine for resisting excessive inflammatory reaction. The amino acid sequence of the AdKI1 is shown as SEQ ID NO.1 or SEQ ID NO.2. The invention further discloses a preparation method of the ancylostoma duodenum polypeptide AdKI1. The ancylostoma duodenum polypeptide AdKI1 can efficiently inhibit the activity of neutrophile granulocyte elastase, cathepsin G and protease 3, and can be applied to preparation of drugs for resisting excessive inflammatory reaction. AdKI1 can significantly improve lung tissue injury of LPS-induced acute lung injury mice and reduce inflammatory response; adKI1 can be used for remarkably inhibiting LPS (Lipopolysaccharide)-induced macrophages from secreting TNF (Tumor Necrosis Factor)-alpha, IL-6 and IL-1beta and promoting the secretion of IL-10.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more particularly to the use of a hookworm polypeptide AdKI1 in the preparation of a drug for resisting excessive inflammatory response. Background Art

[0002] Neutrophils are the most numerous white blood cells in the blood and are involved in the occurrence and development of a variety of infectious and non-infectious inflammatory diseases. Their abnormal activation or imbalanced regulation can promote tissue damage and inflammatory cascade reactions, playing an important role in the occurrence and development of inflammatory diseases.

[0003] There is a vast market demand for drugs targeting neutrophils. The world's first neutrophil-targeted drug, brensocatib, is expected to receive FDA approval on August 12, 2025. This blockbuster therapy, targeting the neutrophil pathway, not only promises to end the plight of bronchiectasis, which remains untreatable, but also potentially usher in a new era of targeted treatment for dozens of inflammatory diseases, including COPD, asthma, autoimmune diseases (including rheumatoid arthritis and systemic lupus erythematosus), inflammatory bowel disease, acute lung injury (ALI) / acute respiratory distress syndrome (ARDS), and more. Industry media outlet Evaluate listed it as one of the "Top 10 Potential Blockbuster Therapies of 2025," predicting sales of $2.8 billion by 2030. Some market forecasts predict that peak sales for the non-cystic fibrosis bronchiectasis (NCFBE) indication alone will exceed $5 billion. brensocatib will be the first marketed dipeptidyl peptidase 1 (DPP1) inhibitor, an indirect inhibitor of neutrophil serine proteases (NSPs). Its mechanism of action is to inhibit DPP1, thereby reducing the activation of three key NSPs: neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (Cat G), thereby inhibiting NSP-mediated tissue damage and excessive inflammation. However, it is worth noting that in the human body, DPP1 functions beyond activating NSPs and is involved in other physiological functions. Patients with congenital DPP1 deficiency (known as Papillon-Lefèvre syndrome) experience thickening of the skin on the palms and soles, as well as severe gum disease, leading to tooth loss. While brensocatib achieved its primary endpoint in both treatment arms of the clinical trial, treatment resulted in a high incidence of hyperkeratosis, thickening of the outer layer of the skin. This is consistent with the thickening of the skin on the palms and soles seen in patients with congenital DPP1 deficiency and may be a side effect of drugs targeting DPP1. This suggests that indirectly inhibiting NSPs through DPP1 inhibition may lead to complications (side effects), making drugs that directly inhibit NSPs a more effective option. Furthermore, direct NSP inhibition offers the advantage of rapid onset of action for some acute conditions, such as acute lung injury (ALI).

[0004] Acute lung injury (ALI) is a serious lung disease characterized by an uncontrolled host immune response to injury—an excessive inflammatory reaction. Among NSPs, nephrase (NE) was the first to be recognized for its ability to directly damage lung tissue and amplify the inflammatory cascade. Sivelestat sodium, a selective inhibitor of nephrase (NE), is currently the only drug approved globally for the treatment of ALI / ARDS. It was launched in my country in 2020 under the fast-track approval pathway. However, clinical studies have shown significant limitations in its effectiveness. Understandably, while sivelestat sodium selectively inhibits nephrase activity, it is unable to effectively inhibit the tissue-damaging and inflammatory effects of two other key NSPs—PR3 and Cat G. Therefore, there is an urgent need for drugs that can simultaneously inhibit nephrase, PR3, and Cat G.

[0005] Therefore, providing the application of the hookworm polypeptide AdKI1 in the preparation of anti-excessive inflammatory response drugs is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides the use of the Ancylostoma duodenale polypeptide AdKI1 in the preparation of a drug for resisting excessive inflammatory response.

[0007] The present invention team studied the blood-sucking parasite hookworm as a drug resource, and discovered that the duodenal hookworm peptide AdKI1 can simultaneously and efficiently inhibit NE, PR3 and Cat G. At very low dose levels, it can significantly improve lung tissue damage in mice with LPS-induced acute lung injury and reduce inflammatory responses. It can significantly reduce the level of LPS-induced macrophage inflammatory factors and promote the expression of the anti-inflammatory factor IL-10.

[0008] The duodenal hookworm polypeptide AdKI1 of the present invention can significantly inhibit the release of inflammatory factors TNF-α, IL-6, and IL-1β in mice and macrophages with acute lung injury induced by lipopolysaccharide (LPS), and promote the release of IL-10, indicating that the duodenal hookworm polypeptide AdKI1 can be used as a drug for preparing anti-excessive inflammatory response.

[0009] The duodenal hookworm polypeptide AdKI1 of the present invention can significantly improve lung tissue damage in mice with acute lung injury induced by LPS, indicating that the duodenal hookworm polypeptide AdKI1 can be used as a drug for preventing and treating acute lung injury.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions: The invention relates to the use of the hookworm polypeptide AdKI1 in the preparation of a drug for resisting excessive inflammatory response, wherein the amino acid sequence of the hookworm polypeptide AdKI1 is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0011] Furthermore, a pharmaceutical preparation for resisting excessive inflammatory response is prepared by using the duodenal hookworm polypeptide AdKI1 as an active ingredient directly or in combination with a pharmaceutically acceptable carrier; the amino acid sequence of the duodenal hookworm polypeptide AdKI1 is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0012] Furthermore, the use of the duodenal hookworm polypeptide AdKI1 in the preparation of a drug for inhibiting overexpression of inflammatory factors, wherein the amino acid sequence of the duodenal hookworm polypeptide AdKI1 is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0013] Furthermore, the inflammatory factors include but are not limited to TNF-α, IL-6, and IL-1β.

[0014] Furthermore, the use of the duodenal hookworm polypeptide AdKI1 in the preparation of a drug for preventing and treating acute lung injury, wherein the amino acid sequence of the duodenal hookworm polypeptide AdKI1 is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0015] Furthermore, a pharmaceutical preparation for preventing and treating acute lung injury is provided, wherein the duodenal hookworm polypeptide AdKI1 is used as an active ingredient and is prepared directly or with a pharmaceutically acceptable carrier; the amino acid sequence of the duodenal hookworm polypeptide AdKI1 is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0016] The amino acid sequence of SEQ ID NO.1 is: EKKLSDEERCNAPTHLEGPQCMAFFKRYTYNKEKCECEEFVYGGCHPSPNNFETLEECKKTCVKK; SEQ ID NO.1.

[0017] The gene sequence encoding SEQ ID NO.1 is: gagaagaagctatcagacgaag agagatgtaatgctccgactcacctagaaggaccacaatgcatggcgttcttcaagaggtacacctacaataaggagaagaaagaatgcgaagaatttgtttatggaggatgccacccatctccaaacaactttgagactttggaggagtg caagaagacttgcgttaagaag ; SEQ ID NO.3.

[0018] The amino acid sequence of SEQ ID NO.2 is: CNAPTHLEGPQCMAFFKRYTYNKEKCECEEFVYGGCHPSPNNFETLEECKKTC; SEQ ID NO.2.

[0019] The gene sequence encoding SEQ ID NO.2 is: tgtaatgctccgactcacc tagaaggaccacaatgcatggcgttcttcaagaggtacacctacaataaggagaagaaagaatgcgaagaatttgtttatggaggatgccacccatctccaaacaactttgagactttgg aggag tgcaagaagacttgc ; SEQ ID NO.4.

[0020] Among them, SEQ ID NO.3 and SEQ ID NO.4 are gene sequences encoding SEQ ID NO.1 and SEQ ID NO.2 amino acid sequences in duodenal hookworm, respectively. Technicians can synthesize the encoding gene sequences based on SEQ ID NO.1 and SEQ ID NO.2 amino acid sequences.

[0021] In the present invention, we found that the H. duodenalis peptide AdKI1 can effectively inhibit the enzyme activities of NE, PR3 and Cat G, with IC50 values ​​of (19.34±1.05) nmol / L and (34.93±1.07) nmol / L for NE and CG, respectively. At an equimolar concentration of 200 nmol / L, it can inhibit 58.8% of PR3 activity.

[0022] In the present invention, we found that the Ancylostoma duodenale polypeptide AdKI1 can significantly improve the symptoms of LPS-induced acute lung injury in mice in a dose-dependent manner. Therefore, the Ancylostoma duodenale polypeptide AdKI1 of the present invention can be prepared as a drug for preventing and treating acute lung injury.

[0023] In this study, we found that the duodenal hookworm polypeptide AdKI1 significantly inhibited LPS-induced inflammatory responses in mice, reducing the levels of inflammatory factors TNF-α, IL-6, and IL-1β in serum and bronchoalveolar lavage fluid (BALF), while increasing IL-10 levels in a dose-dependent manner. Furthermore, the duodenal hookworm polypeptide AdKI1 significantly reduced the levels of inflammatory factors TNF-α, IL-6, and IL-1β in LPS-induced macrophage culture supernatants, while promoting an increase in IL-10 levels. Therefore, the duodenal hookworm polypeptide AdKI1 of this invention can be used as a drug for treating excessive inflammatory responses.

[0024] As can be seen from the above technical solutions, compared to the prior art, the present invention discloses the use of the hookworm peptide AdKI1 in the preparation of a drug for treating excessive inflammatory responses. The hookworm peptide AdKI1 significantly reduces the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum and bronchoalveolar lavage fluid (BALF) of mice with LPS-induced ALI. Compared with a 30 mg / kg dose of sivelestat sodium, a 40 μg / kg dose of AdKI1 significantly reduced serum TNF-α levels and increased IL-10 levels. Compared with a 100.0 μg / mL dose of sivelestat sodium, a 0.5 μg / mL dose of the hookworm peptide AdKI1 significantly reduced TNF-α and IL-6 levels in macrophage culture supernatant. Therefore, the hookworm peptide AdKI1 significantly inhibits the inflammatory response of mice and macrophages in LPS-induced ALI and significantly improves lung tissue damage in mice with LPS-induced ALI, demonstrating superior efficacy to sivelestat sodium, a currently used clinical drug for the treatment of ALI. Therefore, the duodenal hookworm polypeptide AdKI1 can be prepared and used as a drug for anti-excessive inflammatory response or prevention and treatment of ALI.

[0025] The present invention discloses the use of the Ancylostoma duodenale polypeptide AdKI1 in the preparation of a drug for the prevention and treatment of acute lung injury. The Ancylostoma duodenale polypeptide AdKI1 significantly improves the symptoms of LPS-induced acute lung injury in mice at very low doses, with a far superior effect compared to sivelestat sodium. The half-maximal inhibition rate (IC50) of AdKI1 in inhibiting NE activity was approximately (19.34 ± 1.05) nmol / L (equivalent to 148.0 ± 8.0 μg / L, SEQ ID NO. 1), while the IC50 of sivelestat sodium in inhibiting NE activity was approximately 25.6 ± 3.1 nmol / L (equivalent to 1.17 ± 0.14 μg / L). In terms of mass activity, the inhibitory effect of AdKI1 on NE activity is much weaker than that of sivelestat sodium, but the effect of the 20μg / kg dose of AdKI1 group on improving lung tissue damage in LPS-induced ALI mice is significantly stronger than that of the 30mg / kg dose of sivelestat sodium, that is, AdKI1 has a very significant anti-acute lung injury effect at a very low dose, which is far superior to sivelestat sodium. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0027] Figure 1Effect of AdKI1 on the wet-to-dry weight ratio of the right lower lung of mice with LPS-induced ALI ( ±s, n=6); the groups were: sham operation group (Control), model group (LPS: 5.0 mg / kg), sivelestat sodium group (SVLS, 30 mg / kg), low-dose AdKI1 group (10 μg / kg), medium-dose AdKI1 group (20 μg / kg), and high-dose AdKI1 group (40 μg / kg); ***P<0.001, compared with the Control group; ## P<0.01, compared with the Model group.

[0028] Figure 2 Effects of AdKI1 on overall observation, pathological changes and lung injury scores of LPS-induced ALI mice ( ±s, n=9); A and G are Control groups; B and H are Model groups; C and I are sivelestat sodium groups (SVLS, 30 mg / kg); D and J are AdKI1 low-dose groups (10 μg / kg); E and K are AdKI1 medium-dose groups (20 μg / kg); F and L are AdKI1 high-dose groups (40 μg / kg); M is the lung tissue injury score of mice in each group; ***P < 0.001, compared with the Control group; ### P < 0.001, compared with Model; && P < 0.01, &&& P < 0.001, compared with the low-dose AdKI1 group; ^^^ P < 0.001, compared with the medium-dose AdKI1 group; @@@ P<0.001, compared with the AdKI1 high-dose group.

[0029] Figure 3 Effects of AdKI1 on serum TNF-α, IL-1β, IL-6, and IL-10 levels in LPS-induced ALI mice ( ±s, n=10); where A is the serum TNF-α level of mice in each group; B is the serum IL-1β level of mice in each group; C is the serum IL-6 level of mice in each group; D is the serum IL-10 level of mice in each group; *** P < 0.001, compared with the Control group; Sivelestat sodium group (SVLS, 30 mg / kg); ### P < 0.001, compared with the Model group; && P < 0.01, &&& P < 0.001, compared with the AdKI1 low-dose (10 μg / kg) group; ^ P<0.05,^^ P < 0.01, ^^^ P < 0.001, compared with the medium-dose AdKI1 (20 μg / kg) group; @ P<0.05, @@@ P<0.001, compared with the high-dose AdKI1 (40 μg / kg) group.

[0030] Figure 4 Effects of AdKI1 on cytokine levels in BALF of LPS-induced ALI mice ( ±s, n=4); where A is the TNF-α level of mice in each group; B is the IL-1β level of mice in each group; C is the IL-6 level of mice in each group; D is the IL-10 level of mice in each group; *** P < 0.001, compared with the Control group; Sivelestat sodium group (SVLS, 30 mg / kg); ### P < 0.001, compared with the Model group; & P<0.05, && P < 0.01, &&& P < 0.001, compared with the low-dose AdKI1 (10 μg / kg) group; ^ P<0.05, ^^ P < 0.01, ^^^ P < 0.001, compared with the medium-dose AdKI1 (20 μg / kg) group; @@@ P<0.001, compared with the high-dose AdKI1 (40 μg / kg) group.

[0031] Figure 5 Effects of AdKI1 on the levels of TNF-α, IL-1β, IL-6, and IL-10 in the culture supernatant of RAW264.7 cells induced by LPS ( ±s, n=6); where A is the level of TNF-α in cells of each group; B is the level of IL-1β in cells of each group; C is the level of IL-6 in cells of each group; D is the level of IL-10 in cells of each group; SVLS group (SVLS, 30 mg / kg); *** P < 0.001, compared with the Control group; ### P < 0.01, ### P < 0.001, compared with Model; & P<0.05, && P < 0.01, &&& P < 0.001, compared with the low-dose AdKI1 (10 μg / kg) group; ^ P<0.05, ^^ P < 0.01, ^^^P < 0.001, compared with the medium-dose AdKI1 (20 μg / kg) group; @@@ P<0.001, compared with the high-dose AdKI1 (40 μg / kg) group. DETAILED DESCRIPTION

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. 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.

[0033] Example 1 Protective effect of AdKI1 on LPS-induced acute lung injury in mice 1) Animals Seventy-eight SPF-grade, two-month-old male C57BL / 6J mice, weighing 18-22 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd., license number: SCXK (Liao) 2020-0001. Mice were acclimated to the SPF environment at the Guangdong Medical University Laboratory Animal Center for 7 days before the experiment. Mice were housed in cages of five at a temperature of 22 ± 2°C, humidity of 50% ± 15%, a 12-hour circadian cycle, and free access to food. All experiments were approved by the Laboratory Animal Care Ethics Committee of Guangdong Medical University, ethics review number: GDMU-2023-000001. Mouse handling and experimental procedures were performed in accordance with ethical standards for laboratory animals.

[0034] 2) Instruments The microplate reader (ELX 808, 800TS) was purchased from Bio-Tek, USA; the KD2268 microtome was purchased from Kedi Instrument Co., Ltd., Jinhua City, Zhejiang Province; and the OLYMPUS CX23 microscope was purchased from Olympus Corporation, Japan.

[0035] 3) Drugs and reagents Sivelestat sodium (SVLS) was purchased from Shanghai Huilun Jiangsu Pharmaceutical Co., Ltd., batch number: Z00095; lipopolysaccharide (LPS) was purchased from Sigma, USA, batch number: 086M4159V; mouse tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and interleukin 10 (IL-10) ELISA detection kits were purchased from R&D Company, USA, batch numbers: P340968, P138448, P311746, and P322031, respectively; human neutrophil protease (NE) was purchased from Applichem, Germany, batch number: S91676; PR3 was purchased from Sigma, USA, batch number: 039K1237V; cathepsin G (Cat) was purchased from R&D Company, USA, batch number: G) was purchased from Lee Biosolutions, USA, batch number: P53334; the substrate M4765 (batch number: 098K5054V) for detecting NE and PR3 activity and the substrate S7388 (batch number: 031M5003V) for detecting CatG activity were both purchased from Sigma, USA.

[0036] Recombinant preparation of AdKI1: The crude product preparation method is described in the reference: [Reference: Shao Zheng, et al. Isolation, expression and activity study of cysteine ​​protease inhibitor of hookworm. Chinese Journal of Pathogenic Biology, 2022, 17(11):1278-1282], and then purified. ① Preparation of crude product: Design upstream primer dI1e: 5'-CA to amplify the gene encoding the amino acid sequence shown in SEQ ID NO.1 GGATCC GAGAAGAAGCTATCAGACGAAG-3'; SEQ ID NO.5 (underlined for the BamHI restriction site), dI2e: 5'-GC AAGCTT ACTTCTTAACGCAAGTCTTCTTG-3'; SEQ ID NO.6 (the underline indicates the Hind III restriction site); upstream primer dI3e: 5'-CA designed to amplify the gene encoding the amino acid sequence shown in SEQ ID NO.2 GGATCC TGTAATGCTCCGACTCACC-3'; SEQ ID NO.7 (underlined for the BamHI restriction site), dI4e: 5'-TG AAGCTTAGCAAGTCTTCTTGCACTCCT-3'; SEQ ID NO. 8 (the HindIII restriction site is underlined). Using Ancylostoma duodenale cDNA as a template, the gene sequences encoding the amino acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 were amplified, respectively. The amplified coding genes were ligated into the prokaryotic expression plasmid pET32a-SUMO to construct recombinant plasmids. The recombinant plasmids were extracted and transformed into the expression host Escherichia coli (E. coli) to obtain genetically engineered strains expressing recombinant AdKI1. The constructed E. coli strain expressing recombinant AdKI1 was inoculated into LB medium containing ampicillin (100 μg / mL) and cultured at 37°C and 150 rpm to an optical density (OD) of approximately 0.6. Subsequently, isopropyl β-D-1-thiogalactopyranoside (IPTG, 100 μg / mL) was added, and expression was induced at 35°C for 6 h. After isolation and ultrasonic disruption of the host bacteria, the expressed product was purified by nickel affinity chromatography to obtain the fusion protein. The fusion partner was cleaved on a SUMO protease column (4°C, 3 h), and crude recombinant AdKI1 was eluted. ② Purification: The crude recombinant AdKI1 product was purified using an AKTApure 150 protein purification system (GE). It was further purified and endotoxin-free by SP Bio-sep FF cation exchange chromatography (Xi'an Baosai Hengcheng Bioengineering Co., Ltd.). The purified recombinant AdKI1 was aliquoted, lyophilized, and stored at -40°C until use. The concentration for administration was prepared on ice before use. During the purification process, sterile double-distilled water and buffers used in reagent preparation were filtered through a 5Kd hollow fiber column (MicroKros mPES 5Kd, Spectrum, USA) to remove endotoxins. Required glassware was soaked in 0.5 mol / L NaOH for 12 hours and then rinsed with endotoxin-free sterile double-distilled water. Heat-resistant reagent bottles were baked at 200°C for 3 hours before cooling. Eppendorf tubes and pipette tips were soaked in 0.5 mol / L NaOH and then rinsed with endotoxin-free sterile double-distilled water. Reagents and containers were used within 48 hours. The protein concentration was determined to be 3.2 mg / mL using a BCA protein assay kit (Beijing Solebao Technology Co., Ltd.). Endotoxin levels were determined to be <5 EU / mL using a gel-based Limulus amebocyte lysate assay (Zhanjiang Andus Biological Co., Ltd.). The inhibitory activity of AdKI1 against human neutrophil protease (NE), cathepsin G (Cat G), and proteinase 3 (PR3) was determined using a chromogenic substrate assay.

[0037] Method for determining the inhibition of NE, Cat G and PR3 enzyme activities by AdKI1: The chromogenic substrate method was used to detect the NE, Cat G and PR3 activities of AdKI1. The method referred to the literature published by the research team, "Identification and characterization of NaKuI3, a broad-spectrum serine protease inhibitor of Necator americanus. Zhong Fangfang et al., Chinese Journal of Pathogenic Biology, Volume 12, Issue 6, June 2017)". Briefly, the reaction system was 100 μL. The blank group consisted of 10 μL of PBS, 50 μL of enzyme solution (final concentration of Cat G was 50.0 nmol / L; final concentration of NE was 20.0 nmol / L; final concentration of PR3 was 200.0 nmol / L), and 40 μL of substrate (final concentration of M4765 or S7388 solution was 200 μmol / L). The experimental group consisted of 10 μL of AdKI1, 50 μL of enzyme solution (final concentration of Cat G was 50.0 nmol / L; final concentration of NE was 20.0 nmol / L; final concentration of PR3 was 200.0 nmol / L), and 40 μL of substrate (final concentration of M4765 or S7388 solution was 200 μmol / L). Add 10 μL of a specific concentration of AdKI1 and 50 μL of enzyme to a 96-well microplate. After 15 minutes, add 40 μL of the corresponding substrate (M4765 or S7388). Measure the A405 value continuously for 5 minutes using an ELX 808 IU microplate reader. Record the enzyme-substrate reaction rate (V) using BioTek Gen 5 software (V0 represents the reaction rate of the blank control group, and V represents the reaction rate of the experimental group). Perform the assay three times in parallel, and calculate the average value. Inhibition rate = (V0 - V) / V0. Calculate the IC50 value using GraphPad Prism 8.0 software. AdKI1 (SEQ ID NO. 1) showed IC50 values ​​of (19.34±1.05) nmol / L and (34.93±1.07) nmol / L for inhibition of NE and CG, respectively. At an equimolar ratio of AdKI1 to PR3 (200 nmol / L), PR3 activity was inhibited by 58.8%, and at a 100-fold molar ratio, PR3 activity was completely inhibited. There was no significant difference in the inhibitory activity of SEQ ID NO. 2 and SEQ ID NO. 1 against NE, CG, and PR3 depending on the molar concentration. The IC50 value of sivelestat sodium for inhibition of NE was approximately (25.6±3.1) nmol / L.

[0038] 4) Preparation of LPS-induced acute lung injury model in mice Mice were placed in a sealed anesthesia box and anesthetized with 5% isoflurane. After anesthesia, the mice were quickly removed and fixed supinely on a 37°C operating table with their incisors and limbs fixed. Anesthesia was maintained by continuous isoflurane infusion via a face mask. The hair around the neck was carefully shaved, and the skin was disinfected with 75% alcohol and iodine solution, respectively. The skin was longitudinally incised approximately 1 cm, and the trachea was carefully separated and exposed. Using a 1 mL insulin needle, a puncture was made between the two tracheal cartilages toward the lungs, and LPS (5 mg / kg) was instilled in a total volume of 40 µL to establish a mouse model of acute lung injury.

[0039] 5) Experimental grouping and drug administration Sixty two-month-old SPF male C57BL / 6 mice were randomly divided into six groups: sham-operated (Control), model (LPS: 5.0 mg / kg), sivelestat sodium (SVLS, 30 mg / kg), low-dose AdKI1 (10 μg / kg), medium-dose AdKI1 (20 μg / kg), and high-dose AdKI1 (40 μg / kg), with 10 mice in each group. Except for the sham-operated group, all other groups were treated with LPS via tracheal instillation to establish an acute lung injury model. Two hours after LPS instillation, mice in the sivelestat sodium and AdKI1 groups were injected with the corresponding dose of drug via the tail vein. Mice in the sham-operated and model groups were injected with an equal volume of saline via the tail vein. The experimental grouping and drug administration were double-blind. Mice were fed a normal diet during the experiment, and their condition was observed.

[0040] 6) Sample collection and processing 24 hours after tracheal instillation, mice were placed in an anesthesia box and anesthetized with 5% isoflurane. After anesthesia, the mice were removed, their eyes were removed, and blood was collected. After 2 hours of incubation at 4°C, the blood was centrifuged at 3500 rpm for 15 minutes, and the serum was separated and stored at -80°C until further use. Four mice were randomly selected from each group for independent bronchoalveolar lavage fluid (BALF) extraction (bronchial alveolar lavage was performed three times with 0.8 mL of sterile saline pre-cooled at 4°C). The BALF was recovered, centrifuged at 1000 rpm for 15 minutes, and the supernatant was separated and stored at -80°C until further analysis. The right lower lobe of the lung was isolated from the remaining six mice in each group, washed with saline, blotted dry with filter paper, and weighed as the wet weight. The right lower lobe was then oven-baked at 60°C for 72 hours to a constant weight, and weighed as the dry weight. The wet / dry weight ratio of lung tissue was calculated to reflect the degree of lung edema. The right upper lobe of the lung was removed, washed with saline, blotted dry with filter paper, and then immersed in 10% neutral formaldehyde at room temperature. After fixation for at least 24 hours, the lung tissue was embedded in paraffin blocks for further pathological staining and photography. The remaining lung tissue was washed with saline, blotted dry with filter paper, and stored at -80°C until further use.

[0041] 7) Measurement of lung tissue wet / dry ratio The right lower lobe of the lung was removed from all mice in each group. Blood was rinsed from the lung tissue surface with saline, and the surface moisture was blotted with filter paper and weighed (wet weight). The right lower lobe was then oven-baked at 60°C for 72 hours. After drying to a constant weight, the lung tissue was weighed again (dry weight). The wet / dry ratio (W / D) of the lung tissue was calculated (W / D = wet weight / dry weight).

[0042] 8) Lung tissue pathological observation Mice were sacrificed by cervical dislocation and immediately fixed in pre-prepared 10% neutral formaldehyde in the right upper lung lobe. After 24 hours of fixation, the trimmed lung tissue was dehydrated in various concentrations of ethanol and cleared in xylene as follows: 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 placed in a paraffin melting chamber (70°C) for 3 hours. After the paraffin completely permeated the tissue and solidified into a block, the embedded blocks were mounted on a paraffin microtome and cut into 5 μm thick sections. The slices were spread in heated water (40°C), then removed using a slide with a non-slipping glass slide. Finally, the labeled slides were dried in a 50°C incubator. Hematoxylin and eosin staining, microscopic observation, and photography were performed. Lung tissue damage was assessed by a researcher blinded to the experimental data. Specific scoring method: 0 to 4 represents the severity of lung tissue damage: 0 means no damage, 1 means mild damage, 2 means moderate damage, 3 means severe damage, and 4 means very severe damage.

[0043] 9) Determination of cytokine levels in serum and BALF Mouse serum and BALF were collected, and the levels of TNF-α, IL-1β, IL-6, and IL-10 in the serum and BALF of mice in each group were detected according to the detection steps in the kit instructions.

[0044] 10) Statistical analysis All experimental data were expressed as mean ± standard deviation and analyzed statistically using SPSS software. The t-test was used to test significance between two groups, and the least significant difference (LSD) method was used to test differences between multiple groups. A P < 0.05 was considered statistically significant.

[0045] 11) Results like Figure 1Compared with the Control group, the wet-to-dry weight ratio of the right lower lung of the 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 lung of the mice in the AdKI1 administration group and the SVLS group was significantly decreased (P < 0.05).

[0046] like Figure 2 The lung tissues of mice in the control group were bright overall, without shadows, with normal structure, thin alveolar walls, clear alveolar cavities, and no bleeding or inflammatory cell infiltration ( Figure 2 A and Figure 2 G). Compared with the Control group, the lung tissue of the Model group mice showed obvious large dark spots 24 hours after LPS tracheal instillation, thickening of the alveolar wall and septum, infiltration of a large number of inflammatory cells, and obvious damage ( Figure 2 B, 2H and 2M), lung tissue injury scores were significantly increased (P < 0.001) ( Figure 2 M); Compared with the Model group, the dark spots in the lung tissue of the mice in the AdKI1-treated group and the SVLS group were significantly reduced, the morphological structure was relatively normal, the degree of inflammatory cell infiltration was significantly reduced, the thickness of the alveolar wall and septum was significantly reduced, and the lung tissue injury score was significantly reduced (P < 0.001) ( Figure 2 C-2F, 2I-2L and 2M); Compared with the low-dose AdKI1 group, the lung tissue damage of mice in the medium-dose AdKI1 group and the high-dose AdKI1 group was significantly improved, and the lung tissue damage score was significantly reduced (P < 0.01) ( Figure 2 D-2F, 2J-2L and 2M); Compared with the medium-dose AdKI1 group, the lung tissue damage of the mice in the SVLS group was significantly aggravated, and the lung tissue damage score was significantly increased (P < 0.01), while the lung tissue damage of the mice in the high-dose AdKI1 group was significantly improved, and the lung tissue damage score was significantly decreased (P < 0.001) ( Figure 2 C, 2E, 2F, 2I, 2K, 2L and 2M); Compared with the high-dose AdKI1 group, the lung tissue damage of mice in the SVLS group was significantly aggravated, and the lung tissue injury score was significantly increased (P < 0.001) ( Figure 2 C, 2F, 2I, 2L and 2M).

[0047] like Figure 3Compared with the Control group, the serum TNF-α, IL-1β, IL-6 and IL-10 levels of the mice in the Model group were significantly increased (P < 0.001); compared with the Model group, the serum TNF-α, IL-1β and IL-6 levels of the mice in the SVLS group and the AdKI1 administration group were significantly decreased, while the IL-10 level was significantly increased (P < 0.001); compared with the AdKI1 low-dose group, the serum TNF-α and IL-1β levels of the mice in the SVLS group, the AdKI1 medium-dose group and the AdKI1 high-dose group were significantly decreased, while the IL-10 level was significantly increased (P < 0.01); compared with the AdKI1 medium-dose group, the serum TNF-α and IL-6 levels of the mice in the AdKI1 high-dose group were significantly decreased, while the IL-10 level was significantly increased (P < 0.05); compared with the AdKI1 high-dose group, the serum TNF-α level of the mice in the SVLS group was significantly increased, while the IL-10 level was significantly decreased (P < 0.05).

[0048] like Figure 4 Compared with the Control group, the levels of TNF-α, IL-1β, IL-6 and IL-10 in the BALF of the mice in the Model group were significantly increased (P < 0.001). Compared with the Model group, the levels of TNF-α, IL-1β and IL-6 in the BALF of the mice in the SVLS group and the AdKI1 administration group were significantly decreased, while the level of IL-10 was significantly increased (P < 0.001). Compared with the low-dose AdKI1 group, the levels of TNF-α, IL-1β and IL-6 in the BALF of the mice in the SVLS group and the high-dose AdKI1 group were significantly decreased, while the level of IL-10 was significantly increased (P < 0.05). Compared with the medium-dose AdKI1 group, the level of IL-6 in the BALF of the mice in the high-dose AdKI1 group was significantly decreased, while the level of IL-10 was significantly increased (P < 0.05). Compared with the high-dose AdKI1 group, the level of IL-10 in the BALF of the mice in the SVLS group was significantly decreased (P < 0.001).

[0049] 12) Conclusion AdKI1 can significantly improve the symptoms of LPS-induced acute lung injury and reduce the inflammatory response in a dose-dependent manner. In this example, the effect of AdKI1 at doses of 20 μg / kg and 40 μg / kg on improving ALI lung tissue damage was significantly better than that of sivelestat sodium at a dose of 30 mg / kg. Therefore, AdKI1 can be prepared as a drug for the prevention and treatment of acute lung injury, with better efficacy than the current clinical drug - sivelestat sodium.

[0050] Example 2 Effect of AdKI1 on LPS-induced RAW264.7 cell inflammation model 1) Cells Mouse mononuclear macrophage leukemia cells (RAW264.7 cells) were purchased from Wuhan Procell Life Science Technology Co., Ltd. (Procell).

[0051] 2) Instruments The BB150 CO2 cell incubator was a product of Thermo Fisher Scientific, USA; the microplate reader (ELX 808, 800TS) was a product of Bio-Tek, USA.

[0052] 3) Drugs and reagents Sivelestat sodium (SVLS) was purchased from Shanghai Huilun Jiangsu Pharmaceutical Co., Ltd. with batch number Z00095. Lipopolysaccharide (LPS) was purchased from Sigma, USA with batch number 086M4159V. Mouse tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and interleukin 10 (IL-10) ELISA kits were purchased from R&D Company, USA with batch numbers P340968, P138448, P311746, and P322031, respectively.

[0053] 4) RAW264.7 cell culture and grouping RAW264.7 cells were cultured in a 37°C, 5% CO2 incubator using DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were divided into 6 groups: blank control group (Control), LPS group (1 μg / mL), SVLS group (100 μg / mL), AdKI1 low-dose group (0.5 μg / mL), AdKI1 medium-dose group (1 μg / mL), and AdKI1 high-dose group (2 μg / mL). 2×10 4 Cells were co-stimulated for 3 h, and inflammatory markers in the cell supernatants were measured.

[0054] 5) Determination of cytokine levels in cell supernatant The cell supernatant was collected and the levels of TNF-α, IL-1β, IL-6 and IL-10 in each group were detected according to the detection steps in the kit instructions.

[0055] 6) Statistical analysis All experimental data were expressed as mean ± standard deviation and analyzed statistically using SPSS software. The t-test was used to test significance between two groups, and the least significant difference (LSD) method was used to test differences between multiple groups. A P < 0.05 was considered statistically significant.

[0056] 7) Results like Figure 5 Compared with the control group, the levels of TNF-α, IL-1β, IL-6, and IL-10 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 SVLS and AdKI1 groups were significantly decreased, while the level of IL-10 was significantly increased (P < 0.01). Compared with the low-dose AdKI1 group, the levels of TNF-α and IL-6 in the SVLS group were significantly increased, while the level of IL-1β was significantly decreased (P < 0.05). The level of TNF-α in the medium-dose AdKI1 group was significantly decreased, while the level of IL-10 was significantly increased (P < 0.01). The level of TNF-α, IL-1β, and IL-6 in the high-dose AdKI1 group was significantly decreased, while the level of IL-10 was significantly increased (P < 0.01). Compared with the medium-dose AdKI1 group, the level of TNF-α and IL-6 in the SVLS group was significantly increased, while the level of IL-1β was significantly decreased (P < 0.05). The levels of TNF-α, IL-1β, and IL-6 in the high-dose AdKI1 group were significantly decreased, while the level of IL-10 was significantly increased (P < 0.05). Compared with the high-dose AdKI1 group, the levels of TNF-α and IL-6 in the SVLS group were significantly increased, while the level of IL-10 was significantly decreased (P < 0.001).

[0057] 8) Conclusion AdKI1 significantly reduced LPS-induced inflammatory cytokines TNF-α, IL-6, and IL-1β in the supernatant of RAW264.7 cells and increased the anti-inflammatory cytokine IL-10 in a dose-dependent manner. AdKI1 at doses of 0.5 μg / mL, 1.0 μg / mL, and 2.0 μg / mL was significantly more effective in inhibiting TNF-α and IL-6 than 100 μg / mL sivelestat sodium. Therefore, AdKI1 has significant anti-inflammatory effects and could be used as a drug to combat excessive inflammatory responses.

[0058] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one 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 present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of the duodenal hookworm polypeptide AdKI1 in the preparation of an anti-excessive inflammatory response drug, characterized in that: The amino acid sequence of the Ancylostoma duodenale polypeptide AdKI1 is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. A pharmaceutical preparation for resisting excessive inflammatory response, characterized in that: The duodenal hookworm polypeptide AdKI1 is used as an active ingredient to prepare a preparation directly or with a pharmaceutically acceptable carrier; the amino acid sequence of the duodenal hookworm polypeptide AdKI1 is shown in SEQ ID NO.1 or SEQ ID NO.

2.

3. Use of the duodenal hookworm polypeptide AdKI1 in the preparation of a drug for inhibiting overexpression of inflammatory factors, characterized in that: The amino acid sequence of the Ancylostoma duodenale polypeptide AdKI1 is shown in SEQ ID NO.1 or SEQ ID NO.

2.

4. The use according to claim 3, characterized in that The inflammatory factors are TNF-α, IL-6 and IL-1β.

5. Use of the duodenal hookworm polypeptide AdKI1 in the preparation of a drug for preventing and treating acute lung injury, characterized in that: The amino acid sequence of the Ancylostoma duodenale polypeptide AdKI1 is shown in SEQ ID NO.1 or SEQ ID NO.

2.

6. A pharmaceutical preparation for preventing and treating acute lung injury, characterized in that: The duodenal hookworm polypeptide AdKI1 is used as an active ingredient to prepare a preparation directly or with a pharmaceutically acceptable carrier; the amino acid sequence of the duodenal hookworm polypeptide AdKI1 is shown in SEQ ID NO.1 or SEQ ID NO.2.

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