Use of polypeptide NAPTin in preparation of anti-sepsis drugs

Abstract

The application discloses application of a polypeptide NAPTin in preparation of an anti-sepsis drug and belongs to the technical field of biological medicines.The application discloses the application of the polypeptide NAPTin in preparation of the anti-sepsis drug, and the amino acid sequence of the polypeptide NAPTin is shown as SEQ ID NO.1.The polypeptide NAPTin can significantly improve organ injury of a mouse sepsis caused by a cecal ligation puncture operation, reduce mortality, inhibit an inflammatory reaction, and can significantly inhibit an inflammatory reaction of an in-vitro cultured macrophage.The polypeptide NAPTin can be prepared into a drug and applied to the anti-sepsis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the application of the polypeptide NAPTI in the preparation of antiseptic drugs. Background Technology

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection. In severe cases, it can lead to multiple organ dysfunction syndrome (MODS), a serious and complex clinical syndrome that can be life-threatening. Sepsis is a global public health problem; it is estimated that more than 30 million people worldwide contracted sepsis annually from 1979 to 2015, and 5.3 million people may die from it each year. The pathogenesis of sepsis is extremely complex, involving multiple factors, including excessive inflammatory response, immunosuppression, microcirculatory disturbances and ischemia-hypoxia, cellular metabolic disorders, and intestinal barrier dysfunction. The treatment principle for sepsis is early identification and timely intervention, employing comprehensive treatment measures, including controlling infection, supporting organ function, and modulating the immune response. Currently, there are no specific immunomodulatory drugs. Glucocorticoids can be used depending on the condition for septic shock that has not responded well to fluid resuscitation and vasoactive drug therapy. Other drugs, such as biologics and immune enhancers, are still under investigation.

[0003] Sepsis is a systemic inflammatory response caused by infection, which can lead to coagulation disorders, and these disorders can reinforce each other. Therefore, anticoagulation therapy is considered one of the important intervention approaches. Clinically, sepsis patients have a significantly increased risk of venous thromboembolism (VTE) due to factors such as bed rest and vascular endothelial damage. Low molecular weight heparin is commonly used for anticoagulation to prevent thrombosis. In severe sepsis or early septic shock, if a hypercoagulable state is present (e.g., elevated D-dimer, depletion of coagulation factors), some guidelines recommend short-term use of unfractionated heparin or low molecular weight heparin to inhibit microthrombus formation and improve organ perfusion. However, whether anticoagulants can significantly improve prognosis (e.g., reduce mortality, reduce organ dysfunction) remains controversial. Some studies have shown that anticoagulants may reduce D-dimer levels, but have no significant impact on survival. Therefore, anticoagulation therapy alone may not be effective in treating sepsis. We speculate that if a drug could both inhibit the development of excessive inflammation in sepsis by reducing inflammation and inhibit the formation of microthrombi in sepsis by anticoagulation, thus having both anti-inflammatory and anticoagulant effects, it might produce unexpected antiseptic effects. However, there are currently no relevant drug studies.

[0004] Therefore, providing the application of the peptide NAPtin in the preparation of antiseptic drugs is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides the application of the polypeptide NAPTI in the preparation of antiseptic drugs.

[0006] The team discovered the anticoagulant peptide AcaNAP10 from the blood-sucking parasite hookworm, which exhibits strong inhibitory activity against both fVIIa / TF and fXIa. After mutation, NAPTI was obtained, showing no significant inhibitory activity against fVIIa / TF but retaining its inhibitory effect on fXIa. This invention has subsequently been granted European and American patents (European Patent: EP 2698378; US Patent: US 9243044). In sepsis-induced MODS, the lungs are typically the most significantly affected and earliest affected organ. This invention established a mouse sepsis model using the cecal ligation and puncture (CLP) method. The effects of NAPTI on sepsis were observed in terms of lung injury, pathological changes in lung tissue, changes in in vivo inflammatory factor levels, and differences in mouse mortality. The results showed that NAPTI has a significant protective effect against sepsis and can significantly improve the survival rate of septic mice. Further experiments showed that NAPTI can significantly inhibit the LPS-induced inflammatory response in cultured macrophages.

[0007] This invention has found that the peptide NAPTI has a significant protective effect against sepsis in mice undergoing cecal ligation and puncture (CLP) and can inhibit LPS-induced inflammatory responses in macrophages in vitro, indicating that the peptide NAPTI can be used as an anti-septic drug.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The application of the polypeptide NAPTI in the preparation of antiseptic drugs, wherein the amino acid sequence of the polypeptide NAPTI is shown in SEQ ID NO.1.

[0010] Furthermore, an antiseptic pharmaceutical formulation comprises a polypeptide NAPTI as the active ingredient, either directly or with a pharmaceutically acceptable carrier; the amino acid sequence of said polypeptide NAPTI is shown in SEQ ID NO.1.

[0011] The amino acid sequence of the polypeptide NAPTI is as follows:

[0012] SCGENERHDECSRKECDPKCKYDGTEEKDDEKPVVCLTRVCYGDCICRDGFLRNKNGACVKAEDCELD; SEQ ID NO.1.

[0013] The encoded gene sequence is as follows:

[0014] agctgtggtgagaatgaaaggcatgatgagtgcagtagaaaggagtgcgatcccaagtgtaaatatgacggaactgaggagaaagacgacgagaaacctgtggtatgcctaacacgtgtgtgttatggcgattgcatatgcagagatggattcctcagaaacaaaaatggcgcctgtgtgaaagc agaagactgcgaacttgactaa (Including the stop codon taa); SEQ ID NO.2.

[0015] As can be seen from the above technical solution, compared with the prior art, this invention discloses the application of the peptide NAPTI in the preparation of antiseptic drugs. NAPTI can significantly improve lung tissue damage in CLP mice, increase the survival rate of CLP mice, reduce the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in plasma, and increase the level of the anti-inflammatory factor IL-10 in a dose-dependent manner, and the required effective dose level is very low. NAPTI can significantly inhibit the secretion of TNF-α, IL-1β, and IL-6 by LPS-induced in vitro cultured alveolar macrophages (MH-S cells), and promote the secretion of IL-10 in a dose-dependent manner. NAPTI can directly inhibit the inflammatory response of in vitro cultured cells, indicating that NAPTI has an anti-inflammatory pathway independent of inhibiting fXIa. Combined with the anticoagulant effect of NAPTI that can inhibit fXIa, NAPTI can exert its antiseptic effect through both anti-inflammatory and anticoagulant pathways, and may therefore produce a significant antiseptic effect. In summary, the polypeptide NAPTI has a significant protective effect against septicemia in mice, and therefore, it can be prepared and used as an anti-septic drug. Attached Figure Description

[0016] 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.

[0017] Figure 1 The effect of NAPTIn on the wet-to-dry weight ratio of the right lower lobe in CLP septic mice ( ±s, n=6); where the groups are: sham surgery group (Control), model group (Model), low-dose NAPTI group (NAPTin-L, 25 μg / kg), medium-dose NAPTI group (NAPTin-M, 50 μg / kg), and high-dose NAPTI group (NAPTin-H, 100 μg / kg). *** P < 0.001, compared with the control group; # P < 0.05 ### P < 0.001, compared with the Model group.

[0018] Figure 2 The effects of NAPTIn on the overall lung tissue, pathological changes, and lung injury scores in CLP septic mice ( (±s, n=6), where A and F are Control groups; B and G are Model groups; C and H are NAPtin-L groups (0.25 μg / kg); D and I are medium-dose NAPtin groups (NAPTin-M, 50 μg / kg); E and J are high-dose NAPtin groups (NAPTin-H, 100 μg / kg); arrows indicate thrombosis status; K represents the lung tissue damage score of mice in each group; *** P < 0.001, compared with the control group; # P < 0.05 ## P < 0.01, ### P < 0.001, compared with the Model; & P < 0.05, compared with the NAPtin-L group.

[0019] Figure 3 The effect of NAPTIn on plasma TNF-α, IL-1β, IL-6 and IL-10 levels in CLP-induced sepsis mice ( (±s, n=10), where A represents the plasma TNF-α level in each group of mice; B represents the plasma IL-1β level in each group of mice; C represents the plasma IL-6 level in each group of mice; and D represents the plasma IL-10 level in each group of mice. The groups are: sham-operated group (Control), model group, low-dose NAPtin group (NAPTin-L, 25 μg / kg), medium-dose NAPtin group (NAPTin-M, 50 μg / kg), and high-dose NAPtin group (NAPTin-H, 100 μg / kg). * P < 0.05 *** P < 0.001, compared with the control group; # P < 0.05 ### P < 0.001, compared with the Model group; &&P < 0.01, &&& P < 0.001, compared with the NAPtin-L group; ^^ P < 0.01, ^^^ P < 0.001, compared with the NAPtin-M group.

[0020] Figure 4 The effect of NAPTIn on the survival rate of CLP-induced sepsis mice ( ±s, n=10); where the groups are: sham surgery group (Control), model group (Model), low-dose NAPTI group (NAPTin-L, 25 μg / kg), medium-dose NAPTI group (NAPTin-M, 50 μg / kg), and high-dose NAPTI group (NAPTin-H, 100 μg / kg). * P < 0.05, compared with the control group; # P < 0.05, compared with the Model group.

[0021] Figure 5 The effect of NAPTI on the levels of TNF-α, IL-1β, IL-6 and IL-10 in the culture supernatant of LPS-induced MHS cells ( (±s, n=3), where A is the TNF-α level; B is the IL-1β level; C is the IL-6 level; D is the IL-10 level; the groups are: blank control group (Control), LPS group (1 μg / mL), Resaprolite group (RSTV, 1 μmol / L), low-dose NAPTI group (NAPTin-L, 100 ng / mL), medium-dose NAPTI group (NAPTin-M, 200 ng / mL), and high-dose NAPTI group (NAPTin, 400 ng / mL). *** P < 0.001, compared with the control group; ### P < 0.001, compared with the LPS group; & P < 0.05 && P < 0.01, &&& P < 0.001, compared with the NAPtin-L group; ^ P < 0.05 ^^^ P < 0.001, compared with the NAPtin-M group. $$$ P < 0.001, compared with the NAPtin-H group. Detailed Implementation

[0022] 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.

[0023] Example 1: Protective effect of peptide NAPTIn on acute lung injury in CLP-infected mice.

[0024] 1) Animals

[0025] One hundred and eighteen SPF-grade 2-month-old male C57BL / 6 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-2024-000088. Mouse handling and experimental procedures were conducted in accordance with ethical requirements for laboratory animals.

[0026] 2) Instruments

[0027] 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.

[0028] 3) Drugs and reagents

[0029] Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich, USA, lot number: 086M4159V; the 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, lot numbers P340968, P138448, P311746, and P322031, respectively.

[0030] Recombinant preparation of NAPTI: The preparation method of crude product can refer to the relevant published literature: "Prokaryotic expression, purification and activity study of Kunitz-type serine protease inhibitor IsKuI-1" (Cui Hongdi, Shao Zheng, Deng Li, et al., China Biotechnology Journal, 2014, 34(12):30-35), and then purification is performed. ① Preparation of crude product: Using primer NT-1e: 5'-CA GGATCC AGCTGTGGTGAGAATGAAAG-3'; SEQ ID NO.3 (underlined is the BamH I restriction site) and NT-2e: 5'-CG AAGCTT The sequence SEQ ID NO. 2 was paired with SEQ ID NO. 4 (underlined Hind III restriction site), amplified by PCR, and ligated into the prokaryotic expression plasmid pET32a-sumo. The constructed recombinant plasmid was then transformed into the expression host *Escherichia coli* (E. coli). *E. coli* strains expressing recombinant NAPTI were inoculated into LB medium containing ampicillin (100 μg / mL) and cultured at 37°C and 150 rpm until the optical density (OD) reached 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 sonication of the host cells, the expression product was purified by nickel affinity chromatography to obtain the fusion protein. The fusion chaperone was cleaved on a SUMO protease column (4°C, 3 h), and the crude recombinant NAPTI product was obtained by elution. ② Purification: The obtained crude recombinant NAPTI was purified using an AKTA pure 150 (GE) protein purification system, followed by further purification and endotoxin removal via DEAE ion exchange chromatography (Xi'an Baosai Hengcheng Biotechnology Co., Ltd.). All sterile double-distilled water and buffer solutions used in 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 1.1 mg / mL using a BCA protein concentration assay kit (Beijing Solarbio Science & Technology Co., Ltd.), and the endotoxin concentration was <10.0 EU / mL using a gel electrophoresis Limulus amebocyte lysate assay kit (Zhanjiang Andus Biotechnology Co., Ltd.). The purified NAPTI was aliquoted, lyophilized, and stored at -40°C for later use. Before use, it was prepared to the recommended dosage on ice.

[0031] 3) Preparation of a mouse sepsis model induced by cecal ligation and puncture (CLP)

[0032] Mice were anesthetized with 5% isoflurane and placed supine on a 37°C constant-temperature experimental table. The fur on the mouse's abdomen was shaved with electric clippers. After disinfecting the abdominal skin with povidone-iodine, a 1 cm incision was made along the midline of the abdomen to open the abdominal cavity and locate the cecum. The cecum was ligated with No. 4 silk suture 1 cm from the cecum end. A 10 mL syringe needle was used to pierce the cecum once at the middle of the ligation site (i.e., 0.5 cm from the cecum end). The cecum was gently squeezed to ensure that feces were expelled and that the puncture site was patent. The cecum was then placed back into the abdominal cavity and the abdominal muscle layer and skin were sutured. The wound was disinfected again with povidone-iodine, and the mouse was marked with a marker. The mice were then returned to their cages, kept supine, kept warm, and their condition was closely observed.

[0033] 4) Experimental grouping and drug administration

[0034] Pharmacodynamic experiments: Fifty-eight SPF-grade 2-month-old male C57BL / 6 mice were randomly divided into 5 groups: sham-operated group (Control), model group (Model), low-dose NAPTI group (NAPTin-L, 25 μg / kg), medium-dose NAPTI group (NAPTin-M, 50 μg / kg), and high-dose NAPTI group (NAPTin-H, 100 μg / kg). Except for the sham-operated group (n=10), each of the other groups had 12 mice. Except for the sham-operated group, mouse sepsis models were established in all other groups using the CLP method. Mice in the NAPTI-treated groups received the corresponding dose of NAPTI via tail vein injection 2 hours after model establishment, while mice in the sham-operated and model groups received an equal volume of saline via tail vein injection. The experimental grouping and administration were double-blind. Mice were fed normally during the experiment, and the condition of each group was observed. Samples were collected 24 hours after model establishment.

[0035] Survival rate experiment: Mouse grouping, modeling, and drug administration were the same as in the pharmacodynamic experiment, with 10 mice in each group, for a total of 60 mice. The condition of the mice was observed continuously for 72 hours, and the mortality rate was recorded.

[0036] 5) Sample collection and processing

[0037] Twenty-four hours after modeling (pharmacodynamic experiment), mice were anesthetized with isoflurane, and blood was collected by enucleation. The collected blood samples were placed in 1.5 mL anticoagulant EP tubes and incubated at 4°C for 2 hours. After the supernatant plasma separated, the 1.5 mL anticoagulant EP tubes were centrifuged at 3500 rpm for 15 minutes at 4°C. Lung tissue was carefully separated, washed with physiological saline, and weighed. Based on the mortality of the mice, six mice from each group were randomly selected, and the upper lobe of the right lung was harvested. The lung tissue was washed with physiological saline, blotted dry with filter paper, and then soaked in 10% formaldehyde solution for 24 hours. After fixation, the lung tissue was embedded in paraffin for further pathological staining and photography. The lower lobe of the right lung was separated, 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 then weighed as dry weight. The wet weight / dry weight ratio of the lung tissue was calculated to reflect the degree of edema in the lung tissue. The remaining lung tissue was washed with physiological saline and dried with filter paper, then stored at -80℃ for later use.

[0038] 6) Measurement of wet / dry ratio of lung tissue

[0039] 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.

[0040] 7) Pathological observation of lung tissue

[0041] 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.

[0042] 8) Measurement of cytokine levels in plasma

[0043] Mouse plasma was collected, and the levels of TNF-α, IL-1β, IL-6, and IL-10 in each group of mice were measured according to the test steps in the kit instructions.

[0044] 9) Changes in mouse survival

[0045] Mice in each group were observed for 72 hours (survival rate experiment), and the mortality rate of mice was recorded.

[0046] 10) Statistical analysis

[0047] 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. Survival analysis was used to statistically analyze and plot survival curves. A p-value < 0.05 was considered statistically significant.

[0048] 11) Results

[0049] like Figure 1 The wet-to-dry weight ratios of mice in each group were as follows: 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 NAPTI-treated groups was significantly decreased (P < 0.05).

[0050] like Figure 2 Pathological changes in lung tissue of mice in each group: The lung tissue of mice in the control group was generally bright, without shadows, with normal structure, thin alveolar walls, and clearly visible alveolar cavities. No hemorrhage, thrombosis, or inflammatory cell infiltration was observed. Figure 2 A and Figure 2 F). Compared with the control group, the model group mice showed obvious large dark spots in their lung tissue, thickening of alveolar walls and septa, extensive infiltration of inflammatory cells, and obvious thrombosis in the vascular lumen, indicating significant damage and a significantly higher lung tissue damage score (P < 0.001). Figure 2 A, 2B, 2F, 2G and Figure 2 K); Compared with the Model group, the NAPTI-treated group of mice showed a significant reduction in lung dysfocal bands, more normal morphology and structure, significantly reduced inflammatory cell infiltration, and significantly reduced alveolar wall, septal thickness, and thrombus formation (indicated by arrows), and a significantly lower lung tissue damage score (P < 0.05). Figure 2 (B-2E, 2G-2J, and 2K), among which the NAPtin-H group showed the most significant improvement in the above symptoms; compared with the NAPtin-L group, the NAPtin-H group mice showed significantly improved lung tissue damage and significantly lower lung tissue damage scores (P < 0.05). Figure 2 C, 2E, 2H, 2J, and 2K).

[0051] like Figure 3 Plasma cytokine levels in mice of different groups: Compared with the Control group, the plasma TNF-α, IL-1β, and IL-6 levels in the Model group were significantly increased (P < 0.001); compared with the Model group, the plasma TNF-α, IL-1β, and IL-6 levels in the NAPTI-treated group were significantly decreased (P < 0.001), while IL-10 was significantly increased (P < 0.05); compared with the NAPTI-L group, the plasma TNF-α, IL-1β, and IL-6 levels in the NAPTI-M and NAPTI-H groups were significantly decreased (P < 0.01), while IL-10 was significantly increased (P < 0.05); compared with the NAPTI-M group, the plasma TNF-α, IL-1β, and IL-6 levels in the NAPTI-H group were significantly decreased (P < 0.01), while IL-10 was significantly increased (P < 0.001).

[0052] Survival rate experiment: Statistical analysis of the survival rates of the five groups of mice was performed. Compared with the control group, the survival rate of the model group was significantly lower (P < 0.05); compared with the model group, the survival rate of the NAPTI-treated group was significantly higher (P < 0.05). Figure 4 Approximately 4 hours after modeling, the mice began to show decreased activity, exhibiting symptoms such as chills, reduced food and water intake, disheveled fur, and lethargy, which gradually worsened over time. Around 24 hours later, mice began to die, with the highest mortality rate between 24 and 48 hours after modeling. Data analysis after 3 days revealed that all 10 mice in the Model group ultimately died, with a survival rate of 0%; the survival rate on the first day was 50%, on the second day 20%, and on the third day 0%. In the NAPtin-L group, 5 out of 10 mice survived, a survival rate of 50%; the survival rates on the first day, second day, and third day were 70%, 50%, and 50%, respectively. In the NAPtin-M group, 5 out of 10 mice survived, a survival rate of 50%; the survival rates on the first day, second day, and third day were 70%, 60%, and 50%, respectively. In the NAPtin-H group, 6 out of 10 mice survived, a survival rate of 60%; the survival rates on the first day, second day, and third day were all 60%.

[0053] 12) Conclusion

[0054] NAPTin significantly improved the symptoms of acute lung injury in septic mice, reduced the inflammatory response, and significantly improved the survival rate of septic mice. Therefore, NAPTin can be formulated as an antiseptic drug for application.

[0055] Example 2: Effect of NAPTIn on LPS-induced MH-S cell inflammation model

[0056] 1) Cells

[0057] Mouse alveolar macrophages (MH-S cells) were purchased from Wuhan Procell Biotechnology Co., Ltd.

[0058] 2) Instruments

[0059] 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.

[0060] 3) Drugs and reagents

[0061] Resatorvaline (RSTV) was purchased from MedChemExpress, USA, lot number: 66229; Lipopolysaccharide (LPS) was purchased from Sigma, USA, lot number: 086M4159V; ELISA kits for mouse tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and interleukin 10 (IL-10) were purchased from R&D Company, USA, lot numbers: P340968, P138448, P311746, and P322031, respectively.

[0062] 4) MH-S cell culture and grouping

[0063] MH-S 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), a Resapoxetine group (RSTV, 1 μmol / L), a low-dose NAPTI group (NAPTin-L, 100 ng / mL), a medium-dose NAPTI group (NAPTin-M, 200 ng / mL), and a high-dose NAPTI group (NAPTin, 400 ng / mL). The cell number was 5 × 10⁶ cells / mL. 5 / well, with 3 replicates for each concentration group. The blank control group (Control) was added with an equal volume of physiological saline; the LPS group was added with an equal volume of physiological saline and LPS (1 μg / mL); the Resapotevi group, the low-dose NAPTI group, the medium-dose NAPTI group, and the high-dose NAPTI group were added with LPS (1 μg / mL) and the corresponding concentration of the drug; co-stimulation culture was performed for 24 h, and then inflammatory markers in the cell supernatant were detected.

[0064] 5) Measurement of cytokine levels in cell supernatant

[0065] 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.

[0066] 6) Statistical analysis

[0067] 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.

[0068] 7) Results

[0069] 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 RSTV group and the NAPtin-treated group were significantly decreased (P < 0.001), while the level of IL-10 was significantly increased (P < 0.001); compared with the NAPtin-L group, the levels of TNF-α, IL-1β, and IL-6 in the RSTV group, NAPtin-M group, and NAPtin-H group were significantly decreased (P < 0.05), while the level of IL-10 in the NAPtin-H group was significantly increased (P < 0.001); compared with the NAPtin-M group, the level of IL-1β in the NAPtin-H group was significantly decreased (P < 0.001), while the level of IL-10 was significantly increased (P < 0.001); compared with the NAPtin-H group, the level of IL-10 in the RSTV group was significantly decreased (P < 0.001).

[0070] 8) Conclusion

[0071] NAPTI significantly reduced the levels of inflammatory cytokines TNF-α, IL-1β, and IL-6 in the supernatant of LPS-induced cultured MH-S cells in a dose-dependent manner, while increasing the level of the anti-inflammatory cytokine IL-10. This result indicates that NAPTI can directly inhibit the inflammatory response. Combined with NAPTI's anticoagulant effect against fXIa, it is clear that NAPTI exerts its effects in sepsis through both anti-inflammatory and anticoagulant pathways, thus producing a significant antiseptic effect.

[0072] 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 the polypeptide NAPTI in the preparation of antiseptic drugs, characterized in that, The amino acid sequence of the polypeptide NAPTI is shown in SEQ ID NO.1.

Images