Use of duodenal hookworm polypeptide AdKI1 in preparation of anti-excessive inflammatory reaction drugs

Abstract

The application discloses application of a duodenum hookworm polypeptide AdKI1 in preparation of an anti-excessive inflammation reaction medicine and belongs to the technical field of biological medicines. The application discloses the application of the duodenum hookworm polypeptide AdKI1 in preparation of the anti-excessive inflammation reaction medicine, and the amino acid sequence of AdKI1 is shown as SEQ ID NO. 1 or SEQ ID NO. 2. The duodenum hookworm polypeptide AdKI1 can efficiently inhibit activities of neutrophil elastase, cathepsin G and proteinase 3, and can be applied in preparation of the anti-excessive inflammation reaction medicine. AdKI1 can significantly improve lung tissue damage of a mouse with LPS-induced acute lung injury and reduce an inflammation reaction. AdKI1 can significantly inhibit LPS-induced secretion of TNF-alpha, IL-6 and IL-1beta by macrophages and promote secretion of IL-10.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the application of hookworm polypeptide AdKI1 in the preparation of drugs against excessive inflammatory response. Background Technology

[0002] Neutrophils are the most numerous white blood cells in the blood and participate in the occurrence and development of various infectious and non-infectious inflammatory diseases. Abnormal activation or regulatory imbalance of neutrophils can promote tissue damage and inflammatory cascades, 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, brenscotib, is expected to receive FDA approval on August 12, 2025. This blockbuster therapy targeting the neutrophil pathway not only has the potential to end the "untreatable" predicament of bronchiectasis, but also could usher in a new era of targeted therapy for dozens of inflammatory diseases, including COPD, asthma, autoimmune diseases (including rheumatoid arthritis and systemic lupus erythematosus), inflammatory bowel disease, and acute lung injury (ALI) / acute respiratory distress syndrome (ARDS). Industry media 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 suggest that peak sales for the single noncystic fibrotic bronchiectasis (NCFBE) indication will exceed $5 billion. Brenscotib will be the first marketed dipeptidyl peptidase-1 (DPP1) inhibitor, an indirect inhibitor of neutrophil serine proteases (NSPs). Its mechanism of action involves reducing the activation of three key NSPs—neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (Cat G)—by inhibiting DPP1, thereby suppressing NSP-mediated tissue damage and excessive inflammatory responses. However, it is important to note that in the human body, DPP1 functions in addition to activating NSPs and is involved in other physiological functions. Patients with congenital DPP1 deficiency (known as Papillon-Lefèvre syndrome) experience thickened skin on the palms and soles, as well as severe gingival disease leading to tooth loss. Brensocatib met its primary endpoint in both treatment groups of the clinical trial, but the treatment resulted in a higher incidence of hyperkeratosis and thickening of the outer layer of skin. This is consistent with the thickening of the skin on the palms and soles of patients with congenital DPP1 deficiency, and may be a side effect of DPP1-targeting drugs. This also suggests that indirectly inhibiting NSP activity by inhibiting DPP1 may lead to complications (side effects), and drugs that directly inhibit NSPs are a better choice. Moreover, direct inhibition of NSPs has the advantage of rapid onset of action in some acute conditions such as acute lung injury (ALI).

[0004] Acute lung injury (ALI) is a severe lung disease essentially caused by an uncontrolled immune response to injury—an excessive inflammatory reaction. Among non-inflammatory inflammatory factors (NSPs), norepinephrine (NE) was first recognized as directly damaging lung tissue and amplifying the inflammatory cascade. Svillestat sodium, a selective inhibitor of NE, is currently the only drug approved globally for the treatment of ALI / ARDS. It was approved for marketing in my country through the fast-track approval process in 2020. However, clinical studies have shown significant limitations in the efficacy of svillestat sodium. Understandably, while svillestat sodium selectively inhibits NE activity, it cannot effectively inhibit the tissue-damaging and inflammatory amplification effects of the other two key NSPs—performer PR3 and Cat G. Therefore, there is an urgent need for drugs that can simultaneously inhibit NE, PR3, and Cat G.

[0005] Therefore, providing the application of the duodenal hookworm polypeptide AdKI1 in the preparation of drugs against excessive inflammatory responses is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides the application of the duodenal hookworm polypeptide AdKI1 in the preparation of drugs against excessive inflammatory response.

[0007] The team studying this invention investigated hookworm, a blood-sucking parasite, as a drug resource. They discovered that the duodenal hookworm polypeptide 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 response. It can also 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 this invention can significantly inhibit the release of inflammatory factors TNF-α, IL-6, and IL-1β in mice and macrophages induced by lipopolysaccharide (LPS) acute lung injury, and promote the release of IL-10, indicating that the duodenal hookworm polypeptide AdKI1 can be used as a drug to combat excessive inflammatory response.

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

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] The application of hookworm polypeptide AdKI1 in the preparation of drugs against excessive inflammatory response, wherein the amino acid sequence of hookworm polypeptide AdKI1 is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0012] Furthermore, a pharmaceutical formulation for combating excessive inflammatory response comprises a duodenal hookworm polypeptide AdKI1 as the active ingredient, either 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.

[0013] Furthermore, the application of the duodenal hookworm polypeptide AdKI1 in the preparation of drugs that inhibit the 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.

[0014] Furthermore, the inflammatory factors include, but are not limited to, TNF-α, IL-6, and IL-1β.

[0015] Furthermore, the application of the duodenal hookworm polypeptide AdKI1 in the preparation of drugs for the prevention and treatment of 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.

[0016] Furthermore, a pharmaceutical formulation for the prevention and treatment of acute lung injury comprises a duodenal hookworm polypeptide AdKI1 as the active ingredient, either 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.

[0017] The amino acid sequence of SEQ ID NO.1 is as follows:

[0018] EKKLSDEERCNAPTHLEGPQCMAFFKRYTYNKEKCECEEFVYGGCHPSPNNFETLEECKKTCVKK; SEQ ID NO.1.

[0019] The gene sequence encoding SEQ ID NO.1 is as follows:

[0020] gagaagaagctatcagacgaagagagatgtaatgctccgactcacctagaaggaccacaatgcatggcgttcttcaagaggtacacctacaataaggagaagaaagaatgcgaagaatttgtttatggaggatgccacccatctccaaacaactttgagactttggaggagtg caagaagacttgcgttaagaag ; SEQ ID NO.3.

[0021] The amino acid sequence of SEQ ID NO.2 is as follows:

[0022] CNAPTHLEGPQCMAFFKRYTYNKEKCECEEFVYGGCHPSPNNFETLEECKKTC; SEQ ID NO.2.

[0023] The gene sequence encoding SEQ ID NO.2 is as follows:

[0024] tgtaatgctccgactcacc tagaaggaccacaatgcatggcgttcttcaagaggtacacctacaataaggagaagaaagaatgcgaagaatttgtttatggaggatgccacccatctccaaacaactttgagactttgg aggag tgcaagaagacttgc ;SEQ ID NO.4.

[0025] Among them, SEQ ID NO.3 and SEQ ID NO.4 are gene sequences from hookworm of the duodenum that encode the amino acid sequences of SEQ ID NO.1 and SEQ ID NO.2, respectively. Technicians can synthesize the encoding gene sequences based on the amino acid sequences of SEQ ID NO.1 and SEQ ID NO.2.

[0026] In this invention, we found that the duodenal hookworm polypeptide AdKI1 can effectively inhibit the enzyme activities of NE, PR3 and Cat G. Its IC50 for inhibiting NE and CG are (19.34±1.05) nmol / L and (34.93±1.07) nmol / L, respectively. At an equimolar concentration (200 nmol / L), it can inhibit 58.8% of PR3 activity.

[0027] In this invention, we discovered that the duodenal hookworm polypeptide AdKI1 significantly improved LPS-induced acute lung injury symptoms in mice in a dose-dependent manner. Therefore, the duodenal hookworm polypeptide AdKI1 of this invention can be prepared as a drug for the prevention and treatment of acute lung injury.

[0028] In this invention, we found that the duodenal hookworm polypeptide AdKI1 significantly inhibited LPS-induced inflammatory responses in mice, reduced the levels of inflammatory cytokines TNF-α, IL-6, and IL-1β in serum and bronchoalveolar lavage fluid (BALF), and increased IL-10 levels in a dose-dependent manner. Furthermore, AdKI1 significantly reduced the levels of inflammatory cytokines TNF-α, IL-6, and IL-1β in LPS-induced macrophage culture supernatant and promoted an increase in IL-10 levels. Therefore, the duodenal hookworm polypeptide AdKI1 of this invention can be prepared for use as an anti-excessive inflammatory drug.

[0029] As can be seen from the above technical solution, compared with the prior art, this invention discloses the application of hookworm polypeptide AdKI1 in the preparation of drugs against excessive inflammatory response. Hookworm polypeptide AdKI1 can significantly reduce the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the serum and bronchoalveolar lavage fluid (BALF) of LPS-induced ALI mice. Compared with 30 mg / kg of cefelestat sodium, 40 μg / kg of AdKI1 can significantly reduce serum TNF-α levels and increase IL-10 levels. Compared with 100.0 μg / mL of cefelestat sodium, 0.5 μg / mL of hookworm polypeptide AdKI1 can significantly reduce TNF-α and IL-6 levels in macrophage culture supernatant. Therefore, hookworm polypeptide AdKI1 can significantly inhibit the inflammatory response of LPS-induced ALI mice and macrophages; it can significantly improve lung tissue damage in LPS-induced ALI mice, and its effect is superior to that of cefelestat sodium, currently used clinically for the treatment of ALI. Therefore, the duodenal hookworm polypeptide AdKI1 can be prepared for use as a drug to combat excessive inflammatory response or prevent ALI.

[0030] This invention discloses the application of the duodenal hookworm polypeptide AdKI1 in the preparation of drugs for the prevention and treatment of acute lung injury. AdKI1 significantly improves the symptoms of LPS-induced acute lung injury in mice at very low doses, with effects far superior to cefelestat sodium. The half-maximal inhibitory concentration (IC50) of AdKI1 against norepinephrine (NE) activity is approximately (19.34 ± 1.05) nmol / L (equivalent to 148.0 ± 8.0 μg / L, SEQ ID NO.1 sequence), while the IC50 of cefelestat sodium against NE activity is approximately 25.6 ± 3.1 nmol / L (equivalent to 1.17 ± 0.14 μg / L). In terms of mass activity, AdKI1's inhibitory effect on NE activity is much weaker than that of cevelexat sodium. However, in terms of improving LPS-induced lung tissue damage in ALI mice, the 20 μg / kg dose of AdKI1 was significantly stronger than that of 30 mg / kg dose of cevelexat sodium. This means that AdKI1 has a very significant anti-acute lung injury effect at very low doses, which is far superior to cevelexat sodium. Attached Figure Description

[0031] 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.

[0032] Figure 1 The effect of AdKI1 on the wet-to-dry weight ratio of the right lower lobe in LPS-induced ALI mice ( ±s, n=6); where the groups were: sham surgery group (Control), model group (LPS: 5.0 mg / kg), severexostat 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.

[0033] Figure 2 The effects of AdKI1 on the overall lung tissue, pathological changes, and lung injury scores in LPS-induced ALI mice ( (±s, n=9), where A and G are Control groups; B and H are Model groups; C and I are sevimestat sodium groups (SVLS, 30 mg / kg); D and J are low-dose AdKI1 groups (10 μg / kg); E and K are medium-dose AdKI1 groups (20 μg / kg); F and L are high-dose AdKI1 groups (40 μg / kg); M is the lung tissue damage score of mice in each group; ***P<0.001, compared with the Control group; ### P < 0.001, compared with the 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 high-dose AdKI1 group.

[0034] Figure 3 The effect 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 each group of mice; B is the serum IL-1β level of each group of mice; C is the serum IL-6 level of each group of mice; and D is the serum IL-10 level of each group of mice. *** P < 0.001, compared with the Control group; Cevelexa sodium group (SVLS, 30 mg / kg). ### P < 0.001, compared with the Model group; && 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 (20 μg / kg) group of AdKI1; @ P < 0.05 @@@ P < 0.001, compared with the high-dose AdKI1 (40 μg / kg) group.

[0035] Figure 4 The effect of AdKI1 on cytokine levels in BALF of LPS-induced ALI mice ( (±s, n=4), where A represents the TNF-α level in each group of mice; B represents the IL-1β level in each group of mice; C represents the IL-6 level in each group of mice; and D represents the IL-10 level in each group of mice. ***P < 0.001, compared with the Control group; Cevelexa 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 (20 μg / kg) group of AdKI1; @@@ P < 0.001, compared with the high-dose AdKI1 (40 μg / kg) group.

[0036] Figure 5 The effect of AdKI1 on the levels of TNF-α, IL-1β, IL-6 and IL-10 in the culture supernatant of LPS-induced RAW264.7 cells ( (±s, n=6), where A is the TNF-α level of cells in each group; B is the IL-1β level of cells in each group; C is the IL-6 level of cells in each group; D is the IL-10 level of cells in each group; Cevelexa sodium group (SVLS, 30 mg / kg). *** P < 0.001, compared with the control group; ### P < 0.01, ### P < 0.001, compared with the 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 (20 μg / kg) group of AdKI1; @@@ P < 0.001, compared with the high-dose AdKI1 (40 μg / kg) group. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1: Protective effect of AdKI1 against LPS-induced acute lung injury in mice

[0039] 1) Animals

[0040] Seventy-eight SPF-grade 2-month-old male C57BL / 6J mice, weighing 18-22g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd., license number: SCXK (Liaoning) 2020-0001. Mice were acclimatized to the SPF environment for 7 days prior to the experiment at the Experimental Animal Center of Guangdong Medical University. Hull conditions: 5 mice per cage, temperature 22±2℃, humidity 50%±15%, diurnal cycle: 12 h / 12 ​​h, free access to food. All experiments were approved by the Experimental Animal Management Ethics Committee of Guangdong Medical University, ethics review number: GDMU-2023-000001. Mouse handling and experimental procedures were performed in accordance with ethical requirements for laboratory animals.

[0041] 2) Instruments

[0042] The microplate reader (ELX 808, 800TS) was a product of Bio Tek Corporation, 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.

[0043] 3) Drugs and reagents

[0044] Severecitabine sodium (SVLS) was purchased from Shanghai Huilun Jiangsu Pharmaceutical Co., Ltd., batch number: Z00095; lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (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 kits were purchased from R&D Systems (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-Aldrich (USA), batch number: 039K1237V; cathepsin G (Cat G) was purchased from Lee Biosolutions, USA, lot number: P53334; substrate M4765 (lot number: 098K5054V) for detecting NE and PR3 activity and substrate S7388 (lot number: 031M5003V) for detecting CatG activity were both purchased from Sigma, USA.

[0045] Recombinant preparation of AdKI1: The method for preparing the crude product is described in the reference: [Reference: Shao Zheng, et al. Isolation, expression and activity study of cysteine ​​protease inhibitors of hookworm. Chinese Journal of Pathogenic Biology, 2022, 17(11):1278-1282], and then purification is performed. ① Preparation of crude product: Design upstream primer dI1e: 5'-CA for amplifying the amino acid sequence shown in SEQ ID NO.1. GGATCC GAGAAGAAGCTATCAGACGAAG-3'; SEQ ID NO.5 (underlined is the BamHI restriction site), dI2e: 5'-GC AAGCTT ACTTCTTAACGCAAGTCTTCTTG-3'; SEQ ID NO. 6 (underlined is the Hind III restriction site); Design upstream primer dI3e: 5'-CA for amplifying the gene encoding the amino acid sequence shown in SEQ ID NO. 2. GGATCC TGTAATGCTCCGACTCACC-3'; SEQ ID NO.7 (underlined is the BamHI restriction site), dI4e: 5'-TG AAGCTTAGCAAGTCTTCTTGCACTCCT-3'; SEQ ID NO.8 (underlined is the HindIII restriction site). Using hookworm cDNA as a template, gene sequences encoding the amino acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2 were amplified. The amplified coding genes were ligated into the prokaryotic expression plasmid pET32a-sumo to construct a recombinant plasmid. The recombinant plasmid was extracted and transformed into the expression host *Escherichia coli* (E. coli) to obtain a genetically engineered bacterium 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℃ and 150 rpm until the optical density (OD) was approximately 0.6. Subsequently, isopropyl β-D-1-thiogalactopyranoside (IPTG, 100 μg / mL) was added, and expression was induced at 35℃ for 6 h. After separation and sonication of the host cells, the expression product was purified by nickel affinity chromatography to obtain the fusion protein. The fusion chaperone was then cleaved on a SUMO protease column (4℃, 3h), and the crude recombinant AdKI1 product was obtained by elution. ② Purification: The obtained crude recombinant AdKI1 product was purified using an AKTApure 150 (GE) protein purification system, and further purified and detoxified using SP Bio-sep FF cation exchange chromatography (Xi'an Baosai Hengcheng Biotechnology Co., Ltd.). The purified recombinant AdKI1 was aliquoted, lyophilized, and stored at -40℃ for later use. Before use, it was prepared to a dosage concentration on ice. During purification, the sterile double-distilled water and buffer solutions used for reagent preparation were filtered through a 5Kd hollow fiber column (MicroKros mPES 5 Kd, Spectrum, USA) to remove endotoxins. Glassware was first soaked in 0.5 mol / L NaOH for 12 h and then rinsed with sterile double-distilled water to remove endotoxins. Heat-resistant reagent bottles were baked at 200℃ for 3 h and then cooled before use. Eppendorf tubes and pipette tips were soaked in 0.5 mol / L NaOH and then rinsed with sterile double-distilled water to remove endotoxins. Reagents and containers were used within 48 h. The protein concentration was determined to be 3.2 mg / mL using the BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.). Endotoxin levels were <5 EU / mL using the gel permeation assay kit (Zhanjiang Andus Biotechnology Co., Ltd.). The inhibitory activity of AdKI1 against human neutrophil protease (NE), cathepsin G (Cat G), and protease 3 (PR3) was determined using the chromogenic substrate method.

[0046] Methods for determining the inhibitory activities of AdKI1 on NE, Cat G and PR3 enzymes: The chromogenic substrate method was used to detect the NE, Cat G and PR3 activities of AdKI1, referring to the literature published by the research team, "Identification and characterization of NaKuI3, a broad-spectrum serine protease inhibitor of Hookworm (Zhong Fangfang et al., Chinese Journal of Pathogenic Biology, June 2017, Vol. 12, No. 6). In summary, the reaction system consisted of 100 μL of the following solutions: Blank group: 10 μL PBS, 50 μL enzyme solution (Cat G final concentration 50.0 nmol / L; NE final concentration 20.0 nmol / L; PR3 final concentration 200.0 nmol / L), and 40 μL substrate (M4765 or S7388 solution final concentration 200 μmol / L); Experimental group: 10 μL AdKI1, 50 μL enzyme solution (Cat G final concentration 50.0 nmol / L; NE final concentration 20.0 nmol / L; PR3 final concentration 200.0 nmol / L), and 40 μL substrate (M4765 or S7388 solution final concentration 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). Continuously measure the A405 value for 5 minutes using an ELX 808 IU microplate reader. Record the enzyme-substrate reaction rate V using BioTek Gen 5 software (V0 is the reaction rate measured in the blank control group, and V is the reaction rate measured in the experimental group). Perform the assay in triplicate and take the mean. Inhibition rate = (V0 - V) / V0. Calculate the IC50 value using GraphPad Prism 8.0 software. The IC50 values ​​of AdKI1 (SEQ ID NO.1) for inhibiting NE and CG were (19.34±1.05) nmol / L and (34.93±1.07) nmol / L, respectively. At an equimolar concentration of AdKI1 and PR3 (200 nmol / L), it inhibited 58.8% of PR3 activity; at a 100-fold molar ratio of AdKI1 to PR3, it completely inhibited PR3 activity. There was no significant difference in the molar concentration inhibitory activity of SEQ ID NO.2 and SEQ ID NO.1 sequences for NE, CG, and PR3. The IC50 of cetylrestat sodium for inhibiting NE was approximately (25.6±3.1) nmol / L.

[0047] 4) Preparation of LPS-induced acute lung injury model in mice

[0048] 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 in a supine position. 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.

[0049] 5) Experimental grouping and drug administration

[0050] Sixty SPF-grade, 2-month-old male C57BL / 6 mice were randomly divided into 6 groups: sham-operated group (Control), model group (LPS: 5.0 mg / kg), sevilegata 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), with 10 mice in each group. Except for the sham-operated group, all other groups established a mouse model of acute lung injury via LPS intratracheal infusion. Two hours after LPS infusion, mice in the sevilegata sodium group and AdKI1 administration group received the corresponding dose of drug via tail vein injection, while mice in the sham-operated group and model group received an equal volume of physiological saline via tail vein injection. The experimental grouping and drug administration were double-blind. Mice were fed a normal diet during the experiment, and the condition of each group was observed.

[0051] 6) Sample collection and processing

[0052] Twenty-four hours after tracheal 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 then incubated at 4°C for 2 hours, centrifuged at 3500 rpm for 15 minutes, and the serum was separated and stored at -80°C for later use. Four mice from each group were randomly selected for bronchoalveolar lavage fluid (BALF) extraction (bronchial and bronchoalveolar lavage was performed using 0.8 mL of sterile saline pre-cooled at 4°C, repeated 3 times). The BALF was recovered, centrifuged at 1000 rpm for 15 minutes, and the supernatant was separated and stored at -80°C for analysis. The remaining six mice from each group had their right lower lobe isolated, washed with saline, blotted dry with filter paper, and weighed as wet weight. The right lower lobe was then baked in a 60°C oven for 72 hours until constant weight, weighed, and the wet / dry weight ratio of the lung tissue was calculated to reflect the degree of lung edema. The upper lobe of the right lung was harvested, 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.

[0053] 7) Measurement of wet / dry ratio of lung tissue

[0054] After harvesting the lower lobe of the right lung from each group of mice, the lung tissue surface was rinsed with physiological saline to remove bloodstains. The surface moisture was then blotted dry with filter paper and weighed, recorded as wet weight. The lower lobe was then placed in a 60℃ oven for 72 hours until it reached constant weight, and the lung tissue was weighed again, recorded as dry weight. The wet / dry weight ratio (W / D) of the lung tissue was calculated: W / D = wet weight / dry weight.

[0055] 8) Pathological observation of lung tissue

[0056] 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 5 μm 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.

[0057] 9) Measurement of cytokine levels in serum and BALF

[0058] 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 each group of mice were detected according to the detection steps in the kit instructions.

[0059] 10) Statistical analysis

[0060] All experimental data were expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 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.

[0061] 11) Results

[0062] like Figure 1 Compared with the Control 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 AdKI1 administration group and the SVLS group was significantly decreased (P < 0.05).

[0063] like Figure 2 In the control group, the lung tissue of mice was generally bright, without shadows, with normal structure, thin alveolar walls, and clearly visible alveolar cavities. No hemorrhage or inflammatory cell infiltration was observed. Figure 2 A and Figure 2 G). Compared with the control group, the model group mice showed obvious large dark spots in their lung tissue 24 h after LPS intratracheal instillation, thickening of alveolar walls and septa, and extensive infiltration of inflammatory cells, indicating significant damage. Figure 2 B, 2H, and 2M) significantly increased lung tissue injury scores (P < 0.001). Figure 2 M); Compared with the Model group, the AdKI1-treated group and the SVLS group 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 significantly lower lung tissue damage scores (P < 0.001). Figure 2 C-2F, 2I-2L, and 2M); compared with the low-dose AdKI1 group, the medium-dose and high-dose AdKI1 groups showed significantly improved lung tissue damage and significantly lower lung tissue damage scores (P < 0.01). Figure 2 D-2F, 2J-2L, and 2M); Compared with the medium-dose AdKI1 group, the SVLS group showed significantly more severe lung tissue damage and a significantly higher lung tissue damage score (P < 0.01), while the high-dose AdKI1 group showed significantly improved lung tissue damage and a significantly lower lung tissue damage score (P < 0.001). Figure 2 C, 2E, 2F, 2I, 2K, 2L, and 2M; compared with the high-dose AdKI1 group, the SVLS group showed significantly more severe lung tissue damage and a significantly higher lung tissue damage score (P < 0.001). Figure 2 C, 2F, 2I, 2L, and 2M).

[0064] like Figure 3Compared with the Control group, the serum levels of TNF-α, IL-1β, IL-6, and IL-10 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 SVLS group and the AdKI1 administration group mice were significantly decreased, while the IL-10 level was significantly increased (P < 0.001); compared with the low-dose AdKI1 group, the serum levels of TNF-α and IL-1β in the SVLS group, the medium-dose AdKI1 group, and the high-dose AdKI1 group mice were significantly decreased, while the IL-10 level was significantly increased (P < 0.01); compared with the medium-dose AdKI1 group, the serum levels of TNF-α and IL-6 in the high-dose AdKI1 group mice were significantly decreased, while the IL-10 level was significantly increased (P < 0.05); compared with the high-dose AdKI1 group, the serum levels of TNF-α in the SVLS group mice were significantly increased, while the IL-10 level was significantly decreased (P < 0.05).

[0065] like Figure 4 Compared with the control group, the levels of TNF-α, IL-1β, IL-6, and IL-10 in the BALF of 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 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 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 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 mice in the SVLS group was significantly decreased (P < 0.001).

[0066] 12) Conclusion

[0067] AdKI1 significantly improved symptoms of LPS-induced acute lung injury and reduced inflammatory response in a dose-dependent manner. In this embodiment, AdKI1 at doses of 20 μg / kg and 40 μg / kg showed significantly better efficacy in improving lung tissue damage in ALI than cevelexat sodium at a dose of 30 mg / kg. Therefore, AdKI1 can be formulated as a drug for the prevention and treatment of acute lung injury, exhibiting better efficacy than the currently used clinical drug, cevelexat sodium.

[0068] Example 2: Effect of AdKI1 on LPS-induced inflammation model in RAW264.7 cells

[0069] 1) Cells

[0070] Mouse mononuclear macrophage leukemia cells (RAW264.7 cells) were purchased from Wuhan Procell Biotechnology Co., Ltd.

[0071] 2) Instruments

[0072] The BB150 CO2 cell incubator is a product of Thermo Fisher Scientific, Inc., USA; the microplate reader (ELX 808, 800TS) is a product of BioTek Inc., Inc., USA.

[0073] 3) Drugs and reagents

[0074] Severelistat sodium (SVLS) was purchased from Shanghai Huilun Jiangsu Pharmaceutical Co., Ltd., batch number: Z00095; lipopolysaccharide (LPS) was purchased from Sigma-Aldrich, 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 kits were purchased from R&D Company, USA, batch numbers P340968, P138448, P311746, and P322031, respectively.

[0075] 4) RAW264.7 cell culture and grouping

[0076] RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells were divided into six groups: a blank control group (Control), an LPS group (1 μg / mL), an SVLS group (100 μg / mL), a low-dose AdKI1 group (0.5 μg / mL), a medium-dose AdKI1 group (1 μg / mL), and a high-dose AdKI1 group (2 μg / mL). Cells were seeded in 24-well plates at 2 × 10⁻⁶ cells / well. 4 Cells / wells were prepared in 6 replicates for each concentration group. The control group was treated with an equal volume of physiological saline; the LPS group was treated with an equal volume of physiological saline and LPS (1 μg / mL); the SVLS group, low-dose AdKI1 group, medium-dose AdKI1 group, and high-dose AdKI1 group were treated with LPS (1 μg / mL) and the corresponding concentration of the drug. Cells were co-stimulated and cultured for 3 h, and then inflammatory markers in the cell supernatant were measured.

[0077] 5) Measurement of cytokine levels in cell supernatant

[0078] Collect cell supernatant and, according to the kit instructions, detect the levels of TNF-α, IL-1β, IL-6, and IL-10 in each group.

[0079] 6) Statistical analysis

[0080] All experimental data were expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 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.

[0081] 7) Results

[0082] 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 group and the AdKI1 administration group 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 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 < 0.01). Compared with the medium-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). In the high-dose AdKI1 group, the levels of TNF-α, IL-1β and IL-6 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).

[0083] 8) Conclusion

[0084] AdKI1 significantly reduced the levels of LPS-induced inflammatory cytokines TNF-α, IL-6, and IL-1β in the supernatant of RAW264.7 cells, while increasing the level of the anti-inflammatory cytokine IL-10 in a dose-dependent manner. The inhibitory effects of AdKI1 at concentrations of 0.5 μg / mL, 1.0 μg / mL, and 2.0 μg / mL were significantly better than those at 100 μg / mL cefelestat sodium in inhibiting TNF-α and IL-6. Therefore, AdKI1 possesses significant anti-inflammatory activity and can be formulated as a drug to combat excessive inflammatory responses.

[0085] 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. The application of hookworm polypeptide AdKI1 in the preparation of drugs for the prevention and treatment of acute lung injury, characterized in that, The amino acid sequence of the hookworm polypeptide AdKI1 is shown in SEQ ID NO.1 or SEQ ID NO.

2.

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

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