Function regulator based on sheep neutrophil release extracellular trap net and application thereof

Abstract

The invention belongs to the technical field of animal immune regulation, and particularly relates to a function regulator based on a sheep neutrophil release extracellular trapping net and application of the function regulator. The functional regulator is prepared from the following components: a Jacquard trophozoite, DPI (Diisopropylamine), U0126, SB202190, C29 and TLR-IN-C34 (Toll Like Receptor-IN-C34). Through multi-channel cooperative regulation and control, an agonist giardia trophozoite and channel inhibitors such as NADPH oxidase, MAPK and TLR are integrated, and accurate dynamic regulation of NETs release is achieved; livestock medicine pertinence: the sheep neutrophile granulocytes are specially designed, and the research blank of NETs regulation and control of economic animals is filled up; the application scene is expanded, and the method has the bidirectional treatment potential of infectious diseases and autoimmune diseases and is superior to a traditional single-function method. According to the method, the release of the neutrophil NETs of the sheep is accurately regulated and controlled in a mode of combining pretreatment of the inhibitor and stimulation of the giardia trophozoite, and a new strategy is provided for prevention and treatment of diseases of the sheep.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal immune regulation, and particularly relates to a functional regulator based on the extracellular trap released by sheep neutrophils and its application. Background Art

[0002] Neutrophil extracellular traps (NETs) are a reticular structure composed of chromatin fibers, histones, and granule proteins released by neutrophils in response to pathogen infections. NETs can capture and kill pathogens such as bacteria, fungi, and parasites, and are an important part of the innate immune system. However, the excessive release or dysfunction of NETs can also lead to tissue damage, exacerbated inflammatory responses, and even participate in the occurrence and development of various diseases, such as infectious diseases, autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis), and thrombotic diseases, etc.

[0003] In the health management of sheep, the regulation of NETs is of great significance. As an important economic animal, the immune status of sheep directly affects production performance and disease resistance. Research shows that the release of NETs by sheep neutrophils is regulated by multiple signaling pathways, including the NADPH oxidase, MAPK (such as ERK and p38 MAPK), and Toll-like receptor (TLR) signaling pathways, etc. These pathways play key roles in the formation of NETs. Therefore, inhibitors or agonists targeting these pathways can be important tools for regulating the release of NETs.

[0004] In recent years, a variety of inhibitors have been developed to study the regulatory mechanisms of NETs and their roles in diseases. For example: DPI, as an inhibitor of NADPH oxidase, blocks the formation of NETs by inhibiting the production of reactive oxygen species (ROS). U0126, as an inhibitor of the MEK / ERK signaling pathway, blocks downstream signal transduction by inhibiting the phosphorylation of ERK, thereby inhibiting the release of NETs. The ERK signaling pathway plays important roles in various cellular processes, including cell proliferation, differentiation, and immune responses. SB202190, as a p38 MAPK inhibitor, regulates inflammatory responses and the formation of NETs by inhibiting the activity of p38 MAPK. p38 MAPK plays a key role in stress and inflammatory responses. TLR4-IN-C34, as a specific inhibitor of TLR4, inhibits the release of NETs by blocking the TLR4 signaling pathway. TLR4 is an important receptor for recognizing pathogen-associated molecular patterns (PAMPs) and plays a core role in innate immunity.

[0005] In addition, agonists such as C29 (a TLR2 agonist) and Giardia have also been used to study the promoting effect of NETs. C29 promotes the release of NETs by activating the TLR2 signaling pathway; while Giardia, as a parasite, can directly stimulate neutrophils to release NETs, providing a model for studying the role of NETs in parasitic infections.

[0006] Although the regulatory mechanism of NETs has been extensively studied in humans and mice, the research in economically important animals such as sheep is still relatively limited. Developing functional regulators for NETs release in sheep not only helps to deeply understand the role of NETs in the immune response of sheep, but also provides new strategies for the prevention and treatment of sheep diseases. For example, in infectious diseases, enhancing the pathogen clearance ability by promoting NETs release; in autoimmune diseases, reducing inflammatory damage by inhibiting NETs release. Therefore, studying the functional regulators of sheep neutrophil NETs has important theoretical and application values. Summary of the Invention

[0007] Aiming at the above problems, the purpose of the present invention is to provide a functional regulator based on the release of extracellular traps by sheep neutrophils and its application.

[0008] The technical content of the present invention is as follows: The present invention provides a functional regulator based on the release of extracellular traps by sheep neutrophils, and the composition of the functional regulator includes Giardia trophozoites, DPI, U0126, SB202190, C29 and TLR-IN-C34; The usage amount of the Giardia trophozoites is a mass ratio of 1:1 with sheep neutrophils; The usage concentrations of each component in the functional regulator are: DPI 10 μM, U0126 50 μM, SB202190 10 μM, C29 100 μM and TLR-IN-C34 10 μM.

[0009] The present invention also provides an application of the above functional regulator in the preparation of a product for regulating extracellular traps of sheep neutrophils; The application method includes separating sheep peripheral blood neutrophils, co-incubating the sheep neutrophils and the above functional regulator for a period of time, and then detecting the release amount of neutrophil extracellular traps (NETs).

[0010] The beneficial effects of the present invention are as follows: The functional regulator based on extracellular trap release by sheep neutrophils of the present invention is a regulator targeting sheep neutrophils, and its composition includes Giardia trophozoites, DPI, U0126, SB202190, C29 and TLR-IN-C34, and has the following advantages: ① Multi-pathway synergistic regulation: Integrating the agonist Giardia trophozoites and inhibitors of pathways such as NADPH oxidase, MAPK, and TLR to achieve precise dynamic regulation of NETs release; ② Specificity for veterinary medicine: Specifically designed for sheep neutrophils, filling the research gap in the regulation of NETs in economic animals; ③ Expansion of application scenarios: Having the potential for two-way treatment of both infectious diseases (promoting NETs) and autoimmune diseases (inhibiting NETs), which is superior to traditional single-function methods. The present invention precisely regulates the release of NETs by sheep neutrophils through a combination of inhibitor pretreatment and Giardia trophozoite stimulation, providing a new strategy for the prevention and treatment of sheep diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is for the component CitH3 of extracellular trap released by Giardia trophozoite-stimulated sheep neutrophils; Figure 2 It is for the component NE of extracellular trap released by Giardia trophozoite-stimulated sheep neutrophils; Figure 3 It is for the release of sheep neutrophils induced by different ratios of Giardia trophozoites; Figure 4 It is for the generation of ROS induced by different ratios of Giardia trophozoites; Figure 5 It is for DPI, U0126, SB202190 to inhibit the extracellular trap and ROS generation of Giardia trophozoite-stimulated sheep neutrophils; Figure 6 It is for C29, TLR-IN-34 to inhibit the extracellular trap and ROS generation of Giardia trophozoite-stimulated sheep neutrophils. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be further described in detail below through specific implementation cases and the description of the drawings. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention. After reading the present invention, various equivalent modifications made by those skilled in the art fall within the scope defined by the appended claims of this application.

[0013] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents on the conventional market.

[0014] In the present invention: ① Isolation of sheep neutrophils Sheep neutrophils were isolated using a sheep neutrophil isolation kit (Solarbio, China). After isolation, the neutrophils were resuspended in RPMI 1640 (Solarbio, China), counted, and seeded in a 96-well plate at a certain density. The cells were allowed to adhere for at least 30 minutes for subsequent experiments.

[0015] ② Culture of Giardia trophozoites Giardia trophozoites (WB strain, assemblage A, ATCC #30957) were cultured in TYI-S-33 medium at 37°C. Trophozoites were enriched by centrifugation at 1000 g for 10 minutes and resuspended in RPMI 1640 for subsequent experiments.

[0016] ③ Immunofluorescence analysis Giardia trophozoites and sheep neutrophils (sheep neutrophils: Giardia trophozoites = 1:1) were co-incubated on polylysine-coated coverslips (14 mm in diameter) at 27°C for 2 hours. The coverslip samples were treated with 4% paraformaldehyde and 0.1% Triton X-100, and then blocked with BSA (bovine serum albumin, Biosharp, China) at room temperature for 2 hours. After blocking, the samples were incubated with rabbit anti-histone antibody (1:200, Abcam, UK) and rabbit anti-elastase antibody (1:200, Abcam, UK) overnight at 4°C. After incubation with the primary antibodies, the samples were co-incubated with anti-rabbit IgG-FITC antibody (1:200, Proteintech, USA) at room temperature for 2 hours, and then stained with Sytox Orange dye (5 μm, Invitrogen, USA) for DNA. Finally, the samples were observed under a confocal microscope. Yeast sugar (1 mg / mL) was used as a positive control in the experiment.

[0017] ④ Detection of NETs release Giardia trophozoites and sheep neutrophils (sheep neutrophils: Giardia trophozoites = 1:0.5, 1:1, 1:2) were co-incubated at 37°C for 2 hours. The samples were treated with the Pico Green® dye kit (Invitrogen, USA) and detected at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a multimode microplate reader for statistical analysis.

[0018] ⑤ Detection of ROS level Giardia trophozoites and sheep neutrophils (sheep neutrophils: Giardia trophozoites = 1:0.5, 1:1, 1:2) were co-incubated at 37 °C for 2 hours. The samples were treated with 2,7 dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich, USA), and detected at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a multi-functional microplate reader for statistical analysis.

[0019] ⑥ Inhibitor experiment After the sheep neutrophils adhered, they were pretreated with the NADPH oxidase inhibitor Diphenyleneiodonium chloride (DPI, 10 μM, Sigma-Aldrich, USA), the ERK1 / 2 inhibitor U0126 (50 μM, Sigma-Aldrich, USA), the p38 MAPK inhibitor SB202190 (10 μM, Sigma-Aldrich, USA), the TLR2 inhibitor C29 (100 μM, MedChemExpress, USA), and the TLR4 inhibitor TLR3-IN-C34 (10 μM, MedChemExpress, USA) for 30 minutes, and then Giardia trophozoites were added (sheep neutrophils: Giardia trophozoites = 1:1) and co-incubated for 2 hours. The samples were treated with the Pico Green® dye kit or DCFH-DA probe, and the NETs release and ROS production were detected at an excitation wavelength of 485 nm and an emission wavelength of 535 nm using a multi-functional microplate reader for statistical analysis.

[0020] ⑦ Statistical analysis Data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for data analysis, and Tukey's multiple comparison test was performed using GraphPad 9.0 software. The differences between treatments were statistically significant (P < 0.05).

[0021] Example 1 Demonstration that Giardia trophozoites can induce the release of extracellular traps and ROS production in sheep neutrophils Using Giardia trophozoites at different ratios as stimulants, they were co-incubated with sheep neutrophils. The experimental methods and results are as follows: Isolate sheep peripheral blood neutrophils, establish a Giardia trophozoite infection model (sheep neutrophils: Giardia trophozoites = 1:1), co-incubate at 37 °C for 2 hours, incubate overnight at 4 °C with rabbit-derived CitH3 and NE antibodies respectively, then incubate at room temperature with anti-rabbit for 2 hours, stain the DNA and mount the slides, and observe under a confocal microscope. It can be seen that Giardia trophozoites stimulate sheep neutrophils to generate a reticular structure, and the components include DNA, CitH3, and NE (Figure 1 , Figure 2 ).

[0022] Establish a model of Giardia trophozoite-infected sheep neutrophils (sheep neutrophils: Giardia trophozoite = 1:0.5, 1:1, 1:2). After 2 hours, directly detect the content of extracellular DNA and the level of ROS. The results show that Giardia trophozoites significantly increase the release of extracellular traps from sheep neutrophils ( Figure 3 ), and also increase the production of ROS ( Figure 4 ). The generation of NETs has been proven to be related to the production of ROS, and ROS depends on activated neutrophils to increase the production of NADPH oxidase.

[0023] Example 2 Prove that Giardia trophozoites induce the generation of extracellular traps in sheep neutrophils and are related to the NADPH oxidase, ERK1 / 2, p38 MAPK signaling pathways, TLR2 and TLR4 receptors Treat sheep neutrophils stimulated with Giardia trophozoites with different inhibitors. The experimental methods and results are as follows: Isolate sheep peripheral blood neutrophils, pretreat them with DPI (10 μM), U0126 (50 μM), SB202190 (10 μM), C29 (100 μM) and TLR-IN-C34 (10 μM) for 30 minutes, and then add Giardia trophozoites for stimulation (Giardia trophozoite: sheep neutrophils = 1:1) for 2 hours. Detect the content of extracellular DNA and the level of ROS in the samples. The results show that the above inhibitors significantly inhibit the generation of NETs and ROS induced by Giardia trophozoites ( Figure 5 , Figure 6 ), indicating that the generation of Giardia trophozoite-stimulated sheep neutrophils is related to the NADPH oxidase, ERK1 / 2, p38 MAPK signaling pathways, TLR2 and TLR4 receptors. These pathways or receptors can be targeted to regulate the generation of extracellular traps in sheep neutrophils.

[0024] All of the above experiments used yeast sugar (1 mg / mL) as a positive control. The data are expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used for data analysis, and Tukey multiple comparison tests were performed using GraphPad 9.0 software. The differences between treatments were statistically significant (P < 0.05).

[0025] Based on the above results, Giardia trophozoites have the ability to induce the release of NETs from sheep neutrophils. This process is positively correlated with the accumulation of ROS and the activation of TLR2 / TLR4, NADPH oxidase, ERK1 / 2, and p38 MAPK signaling pathways, suggesting that these functional inducers can regulate the generation of extracellular traps by sheep neutrophils, providing insights into the regulatory mechanism of the release of extracellular traps by sheep neutrophils.

Claims

1. A functional regulator for the release of extracellular traps by sheep neutrophils, characterized in that, The composition of the functional regulator includes Giardia trophozoites.

2. The functional regulator according to claim 1, wherein The composition of the functional regulator also includes DPI, U0126, SB202190, C29, and TLR-IN-C34.

3. Use of the functional regulator according to claim 1 or 2 in the preparation of a product for regulating neutrophil extracellular traps in sheep.

4. Use of the functional regulator according to claim 3 in the preparation of a product for regulating neutrophil extracellular traps in sheep, characterized in that, The application method includes separating neutrophils from sheep peripheral blood, co-incubating the sheep neutrophils with the above functional regulator for a period of time, and then detecting the release amount of neutrophil extracellular traps (NETs).

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