Application of interfering with NLK gene targets in the preparation of drugs that improve macrophage inflammatory activation.

Abstract

This invention belongs to the field of biomedicine and provides the application of interfering with the NLK gene target in the preparation of drugs that improve macrophage inflammatory activation. It discloses the application of the NLK gene in regulating the differential polarization of macrophages during the inflammatory response phase of sepsis. In vivo experiments in mice demonstrate that under sepsis, NLK gene upregulation mediates macrophage inflammatory activation; interfering with NLK expression can induce macrophages to shift from an inflammatory activated state to an anti-inflammatory activated state, effectively improving the inflammatory response and survival rate in septic mice. The research results of this invention indicate that the NLK gene holds promise as a drug target for the treatment of sepsis.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and biomedicine, and relates to the application of interfering with the NLK gene target in the preparation of drugs that improve the inflammatory activation of macrophages, especially the application of the NLK gene as a target in the preparation of drugs that improve the inflammatory activation of macrophages in mammalian sepsis. Background Technology

[0002] Sepsis often develops secondary to severe burns, infections, shock, and other acute and critical clinical conditions, and can progress to severe sepsis, septic shock, and multiple organ dysfunction. Despite significant advancements in global understanding of the pathophysiological mechanisms of sepsis and improvements in treatment methods and techniques over the past few decades, the incidence and mortality rates of sepsis remain persistently high. Impaired immune response is the core mechanism in the occurrence and development of sepsis. It includes two phases and states: excessive inflammatory response and immunosuppression, which occur sequentially or simultaneously during the course of sepsis.

[0003] Currently, clinical treatment of sepsis typically relies on early antibiotic use, fluid resuscitation, vasoactive drugs, and other basic treatment measures. However, targeted regulatory therapy for different stages of sepsis to maintain immune balance at each stage has become a hot topic in sepsis research. During the inflammatory phase of sepsis, targeted inhibition of M1 inflammatory macrophage polarization and promotion of M2 anti-inflammatory macrophage polarization can significantly reduce the release of inflammatory factors, thereby reducing tissue damage, patient severity, and mortality. Recent studies have shown that inhibiting macrophage inflammatory activation through pathways such as the nuclear factor κB (NF-κB) pathway (Tong Y et al., 2021), the mitogen-activated protein kinase (MAPK) signaling pathway (Chen XS et al., 2022), and the Notch signaling pathway (Bai X et al., 2020) can significantly improve sepsis inflammatory phase damage. During the immunosuppressive phase of sepsis, targeted increase of M1 polarization and reduction of M2-like macrophages can effectively eliminate host immune paralysis and reduce opportunistic infections.

[0004] Nemo-like kinase (NLK) is a member of the MAPK family. Recent studies have shown that NLK is involved in immune cells and their immune regulation. For example, NLK knockout of Treg cells leads to reduced Treg cell-mediated immunosuppression in vivo, and NLK-deficient Treg cell animals develop more severe experimental autoimmune encephalomyelitis. In T-cell lineage NLK conditional knockout mice, reduced phosphorylation of lymphocyte enhancement-binding factor 1 (LEF1) and histone deacetylase 1 (HDAC1) resulted in single-positive CD8+. + A significant decrease in T cells indicates that NLK is involved in CD8. +NLK plays a crucial role in T cell survival (Renée Daams et al., 2020). NLK strongly inhibits the production of type I interferon by phosphorylating mitochondrial antiviral signaling protein (MAVS), leading to its degradation and subsequent inactivation of downstream signaling pathways, suggesting that NLK is essential for the cellular homeostasis control of innate immunity (Li SZ et al., 2019). These findings indicate that NLK plays a regulatory role in adaptive immune responses. However, the role of NLK in the immune regulation of sepsis remains unreported.

[0005] This application proposes for the first time that NLK can regulate the inflammatory response phase of sepsis by mediating differential polarization of sepsis macrophages, which is expected to provide a new theoretical target for the treatment of sepsis. Summary of the Invention

[0006] The purpose of this invention is to provide the application of the NLK gene as a target in the preparation of drugs that improve the inflammatory activation of septic macrophages by interfering with, inhibiting, knocking out or altering the regulatory role of the NLK gene in improving the inflammatory activation of septic macrophages.

[0007] This invention is achieved through the following technical solution:

[0008] Application of interfering with NLK gene targets in the preparation of drugs that improve macrophage inflammatory activation.

[0009] Application of the NLK gene as a target in the preparation of drugs that improve inflammatory activation of sepsis macrophages.

[0010] The application of the NLK gene as a target in the preparation of drugs that improve inflammatory activation of macrophages in mammals with sepsis, wherein the mammals are humans, monkeys, dogs, cats, rats, or mice.

[0011] Interfering with the NLK gene induces a shift in macrophage activation from M1 inflammatory activation to M2 anti-inflammatory activation during sepsis; the interference method is one of the following: RNA interference, gene knockout, CRISPR / Cas9 technology, zinc finger nuclease, gene editing of transcription activator-like effector factors, antisense oligonucleotides, transcription factor inhibition, and drug interference.

[0012] Drugs obtained based on any of the above applications.

[0013] A method to improve inflammatory activation of macrophages in mammalian sepsis includes the following steps:

[0014] The location of the NLK gene was determined; interference with the NLK gene induced a shift from M1 inflammatory activation to M2 anti-inflammatory activation in macrophages during sepsis, alleviating inflammatory infiltration and pathological damage in the liver and kidneys of mice. The interference method included one of the following: RNA interference, gene knockout, CRISPR / Cas9 technology, zinc finger nucleases, gene editing of transcription activator-like effector factors, antisense oligonucleotides, transcription factor inhibition, and drug interference. A drug based on the aforementioned applications and its method for improving inflammatory activation of macrophages in mammalian sepsis were also developed.

[0015] The drug contains an effective amount of an NLK gene inhibitor and a pharmaceutically acceptable carrier, which may be a buffer solution, culture medium, or preservative.

[0016] The drug is a substance that reduces NLK gene expression, including reagents for interfering with NLK, pharmaceutical intermediates, and NLK gene inhibitors; the drug includes one or more combinations of small molecule drugs, antibodies, peptides, proteins (e.g., cytokines, hormones, soluble receptors and nonspecific proteins), oligonucleotides (e.g., DNA and RNA encoding peptides, double-stranded RNA and antisense RNA), and peptides.

[0017] The phrase “pharmaceutically acceptable” as used in this application means reagents, compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human or animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, and have a reasonable benefit / risk ratio, in accordance with sound medical judgment.

[0018] The phrase “pharmaceutically acceptable carrier” as used in this application refers to pharmaceutically acceptable materials, compositions, or excipients, such as liquid or solid fillers, diluents, excipients, or solvent encapsulation materials, which participate in the delivery or transport of reagents from one organ or part of the body to another organ or part of the body. Each carrier must be “acceptable,” meaning it is compatible with other components of the formulation and will not be harmful to the patient. Examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered astragalus gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, castor oil, sesame oil, olive oil, corn oil, and soybean oil; (10) diols, such as… Examples include propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffer solution; (21) polyesters, polysaccharide esters and / or polyanhydrides; and (22) other non-toxic biocompatible substances used in pharmaceutical preparations.

[0019] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0020] This invention relates to the application of the NLK gene in the preparation of drugs that improve the inflammatory activation of macrophages in mammalian sepsis. The applicant and inventors discovered through research that sepsis can induce inflammatory activation of macrophages and their infiltration in the liver and kidneys of mice through NLK upregulation, leading to aggravated pathological damage in the liver and kidneys and severely impairing the 7-day survival rate of mice. Interfering with the NLK gene can effectively induce macrophages in sepsis to shift from M1 inflammatory activation to M2 anti-inflammatory activation, effectively alleviating inflammatory infiltration and pathological damage in the liver and kidneys of mice, and significantly improving the survival rate of mice. Furthermore, interfering with the NLK gene is easy to perform, providing a new method for the clinical treatment of sepsis. Attached Figure Description

[0021] Figure 1 This invention refers to CD11b from spleen-derived macrophages (SDMs) derived from NLK conditional knockout mice (NKO) and their littermate wild-type control mice (WT) after sham surgery and CLP sepsis model treatment, respectively. + F4 / 80 +Flow cytometry identification results and Western blot analysis of NLK protein expression levels. (A) Flow cytometry identification of CD11b in SDMs. + F4 / 80 + (B) Proportion of double-positive cells. (C) Cell morphology of macrophages under a fluorescence microscope. (D) Western blot analysis of NLK expression in SDMs of WT and NLK-deficient mice with and without CLP treatment and statistical results.

[0022] Figure 2 This image shows the effects of NLK conditional knockout mice and their littermate wild-type control mice on macrophage polarization under sham surgery and CLP sepsis models, respectively. (A) Flow cytometry analysis of the expression of F4 / 80 and CD86 in spleen-derived macrophages in each group of mice. (B) Pro-inflammatory macrophages (F4 / 80) in each group of mice. + CD86 + Statistical analysis results of the percentage of double positives. (C) Flow cytometry detection of the expression of F4 / 80 and CD206 in spleen-derived macrophages of mice in each group. (D) Anti-inflammatory macrophages (F4 / 80) in each group of mice. + CD206 + Statistical analysis results of the percentage of double positives.

[0023] Figure 3 To illustrate the detection of serum inflammatory factors and the 7-day survival rate of macrophage NLK conditional knockout mice and their littermate wild-type control mice under sham surgery and CLP sepsis models in this invention, this study aims to indicate the inflammatory response and survival status of mice in each group. (AD) ELISA was used to detect the expression levels of serum inflammatory factors TNF-α, IL-6, IL-1β, and IL-18 in each group of mice. (E) Statistical results of the 7-day survival rate of mice in each group after surgery.

[0024] Figure 4 To illustrate the HE staining and macrophage infiltration of the liver and kidney of NLK conditional knockout mice and their littermate wild-type control mice under sham surgery and CLP sepsis models in this invention. (A) HE staining shows the morphological and structural changes and pathological damage of the liver and kidneys in each group of mice; multichannel fluorescence staining shows the distribution and infiltration of inflammatory (red) and anti-inflammatory (green) macrophages in various organs. (BC) Liver damage score and macrophage fluorescence statistics in mice. (DE) Kidney damage score and macrophage fluorescence statistics in mice. Detailed Implementation

[0025] The present application will be further described below with reference to specific embodiments:

[0026] Example

[0027] The experimental procedure is as follows:

[0028] (1) Obtaining and identifying macrophage NLK conditional knockout mice (NKO):

[0029] Macrophage NLK conditional gene knockout mice (NKO) were custom-made by Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (License No.: SCXK(Su)2023-0009).

[0030] NKO mice were paired at a 2:1 female-to-male ratio and housed together. Genotyping of offspring was performed at 7-10 days of age. Mouse genotyping procedure: Before the procedure, gloves were disinfected with alcohol spray. The mouse was held by the skin on the back of its neck with the left hand, its abdomen facing upwards and limbs spread out. Ear tags were then used to mark the mouse's ears. The toes to be removed were disinfected with an alcohol swab. The toes were then removed with sterile ophthalmic scissors using the right hand and inserted into a 1.5ml sterile EP tube. The EP tube and mouse ear tag were marked, and the EP tube was placed in an ice box. The mouse was then returned to its cage for continued housing. Primers were customized according to the primer information in Table 1 and prepared using sterile RNase-free water, then aliquoted and stored at -20℃. Using the mouse genotyping rapid identification kit (Beijing Sunshine Yingrui Biotechnology Co., Ltd.) listed in Table 2, tissue digestion solution was prepared using Adv. Buffer and Sup. Protease to fully digest the mouse toe tissue in the EP tube. The EP tube was then placed in a 75℃ metal bath for 15 minutes for digestion. After digestion, the sample was processed at 12000 rpm for 2 min, and the supernatant was used as a PCR template. Samples were added according to the PCR reaction system described in Table 3 and the reaction conditions described in Table 4, and PCR was performed followed by agarose gel electrophoresis. Samples from each model mouse were used for PCR reactions with Flox primer pairs (0.5 μL Flox-F + 0.5 μL Flox-R), iCre1 primer pairs (0.5 μL iCre-F1 + 0.5 μL iCre-R1), and iCre2 primer pairs (0.5 μL iCre-F2 + 0.5 μL iCre-R2). 1.2 g of agarose powder was placed in an Erlenmeyer flask, and 60 μL of 1×TAE buffer was added. After heating repeatedly in a microwave oven, a 2% agarose buffer was obtained. After the agarose buffer cooled slightly, 5 μL of Safe Green nucleic acid dye was added, mixed well, poured into a gel casting tank, a comb was inserted, and the mixture was allowed to stand at room temperature for 30 min until the agarose gel solidified. Transfer the gel to a horizontal electrophoresis tank, with the sample wells near the negative electrode. Add 1×TAE buffer, load 10 μL of PCR samples into each well, and 5 μL of DNA marker into each well. Perform electrophoresis at 150 V and observe the gel using a BioRad gel imaging system after 25 min. Determine the mouse genotype based on the identification results. Mice with both Flox (375 bp) and iCre (350 bp, 2195 bp) bands are NKO mice; mice with only Flox and no iCre are wild-type control mice from the same littermate.

[0031] Table 1 Primer information for NKO mice

[0032]

[0033] Table 2. Components of the Mouse Genotype Rapid Identification Kit

[0034]

[0035] Table 3 PCR reaction system

[0036]

[0037] Table 4 PCR reaction conditions

[0038]

[0039] (2) Establishment of a mouse cecal ligation-perforation (CLP) sepsis model:

[0040] 6-8 week old NLK conditional knockout mice (NKO) and control mice (WT) with 20-22g macrophages were fasted for 12 hours. Mice were anesthetized with an intraperitoneal injection of 50mg / kg pentobarbital, then fixed in a supine position on the experimental table. The abdomen was disinfected with povidone-iodine. CLP model procedure: A midline incision was made along the abdominal wall to expose the abdominal contents. The distal cecum was gently pulled out with toothless forceps. After separating the mesenteric vessels, a sterile No. 3 silk suture was used to ligate the ileocecal valve at the midpoint between the ileocecal valve and the cecum. A 21G sterile needle was used to puncture the cecal wall twice at the midpoint between the ligation site and the cecal apex. The distal cecum was gently squeezed to expel a small amount of intestinal contents before being returned to the abdominal cavity. The abdominal cavity was closed, and the surgical incision was sutured layer by layer. In the sham surgery group (Sham group), only the distal cecum was exposed, squeezed, returned to the cecum, and the abdomen was closed. Cecal ligation and perforation treatment were not performed. The remaining procedures were the same as in the surgical group (CLP group). The incision site was disinfected again with povidone-iodine, and strict aseptic technique was maintained throughout the surgery. Postoperative procedures and observation indicators: Fluid resuscitation was performed by subcutaneous injection of 50 mL / kg physiological saline in the neck. Postoperative analgesia was administered with 0.05 mg / kg buprenorphine. The mice were kept warm, and their respiration and body temperature were closely monitored after the operation. After the mice regained consciousness, they were returned to their cages for continued rearing. Routine observation of mice in each group was performed postoperatively. Compared with the Sham group, the CLP group mice gradually developed a series of clinical symptoms of mouse sepsis, including ruffled fur, lethargy, bradykinesia, weakened arrest resistance, inability to fully open eyes, increased secretions, and labored breathing, indicating that the CLP model was successfully established.

[0041] (3) Effect evaluation

[0042] 3.1 Extraction and purification of spleen-derived macrophages (SDMs) from mice with sepsis

[0043] Mouse spleens were placed in 10cm cell culture dishes containing 4°C PBS buffer. The surrounding connective tissue and visceral capsule were removed using pointed forceps. The spleen was then transferred to a 40μm cell strainer, and the spleen was homogenized using a homogenizer. The strainer was repeatedly rinsed with 4°C PBS buffer, and the homogenate was filtered into a 15mL centrifuge tube. After centrifugation at 1500rpm for 5 min, the supernatant was discarded. The cell pellet was resuspended in 2mL PBS buffer. Using a 10mL sterile silanized centrifuge tube from the lymphocyte separation kit, 4mL of separation buffer was added. The cell suspension was slowly added along the tube wall to the sterile silanized centrifuge tube, maintaining a separation layer on the surface. The tube was centrifuged at 20°C, 400g for 30 min. After centrifugation, the liquid in the silicated tube separated into four layers from top to bottom: dilution layer, lymphocyte layer (ring-shaped milky white), separation layer, and erythrocyte layer. Carefully aspirate the lymphocyte layer into another 15mL centrifuge tube, add 5mL of washing buffer, and mix by pipetting. Centrifuge at 400g for 10min and discard the supernatant. Resuspend the cells in 5mL of washing buffer, centrifuge at 250g for 10min, discard the supernatant, wash again, count the cells, centrifuge again, discard the supernatant, and obtain spleen lymphocytes.

[0044] Every 10 7 Each lymphocyte was resuspended in 90 μL of PBS buffer, and every 10 7 Add 10 μL LF4 / 80 to each cell + Mix the magnetic beads thoroughly and incubate at 4°C in the dark for 15 minutes; after incubation, repeat every 10 minutes. 7 Cells were washed with 1-2 mL of PBS, centrifuged at 300 g for 10 min, and the supernatant was discarded; every 10 8 Each cell was resuspended in 500 μL of PBS buffer; the filter column was mounted on a magnetic rack, moistened once with 2-3 mL of PBS, and the cell suspension was added. The filtered F4 / 80 cells were collected in 10 cm cell culture dishes. - Cells; wash the filter column with 500 μL or 3 mL PBS, repeat 3 times; F4 / 80 - After cell filtration, remove the filter column and install it into a 15mL centrifuge tube. Add 1mL of PBS buffer to the MS tube and 5mL to the LS tube. Push the plunger to transfer the cells adsorbed on the filter column wall into the 15mL centrifuge tube to obtain F4 / 80. + Spleen-derived macrophages (SDMs).

[0045] 3.2 Identification of Sepsis-Induced Macrophages in Mice

[0046] 3.2.1 CD11b of SDMs + F4 / 80 + Identification

[0047] Collect SDMs, resuspend cells in 100 μL PBS, and repeat every 10...6 Add 0.25 μg TruStain FcX to each cell. TM PLUS antibody, mix well, incubate on ice for 5-10 min to block Fc receptors to reduce non-specific binding of flow cytometry antibody to cell surface receptors; set up double negative tubes, APC-CD11b single staining tubes, and FITC-F4 / 80 single staining tubes; add 1 μL LCD11b and 1 μL F4 / 80 antibody to the remaining groups, incubate at room temperature in the dark for 30 min, wash cells with 1 mL PBS buffer, centrifuge, discard supernatant, add 300 μL 1% paraformaldehyde and fix at 4℃, ready for flow cytometry analysis.

[0048] 3.2.2 Polarization Identification of SDMs

[0049] Collect SDMs, resuspend cells in 100 μL PBS, and repeat every 10... 6 Add 0.25 μg TruStain FcX to each cell. TM PLUS antibody, mix well, incubate on ice for 5-10 min for Fc receptor blocking; set up double-negative tubes, APC-CD206 single-staining tubes, and PE-CD86 single-staining tubes for control and compensation adjustment; CD206 is a transmembrane protein receptor, so it needs to be fixed before flow cytometry antibody staining and stained after membrane perforation; use Cyto-Fast... TM Fix / Perm Buffer Set kit for SDMs fixation and membrane perforation; dilute 10×Cyto-Fast with deionized water. TM Wash buffer 10 times, resuspend SDMs in 100 μL; add 150 μL Fix / Perm Buffer, incubate at room temperature for 20 min; after membrane rupture, add 1 mL 1×Wash buffer, centrifuge at 1200 rpm for 5 min, discard the supernatant, and repeat the washing once; resuspend cells in 100 μL 1×Wash buffer; add 1 μL PE-CD86 and 1 μL L APC-CD206 antibody to each tube of the control and experimental groups, respectively, and incubate on ice in the dark for 15-20 min; wash cells with 2 mL PBS buffer, centrifuge at 1200 rpm for 5 min, and repeat the washing once; fix cells with 300 μL 1% paraformaldehyde, seal the flow cytometry tubes, and store at 4℃ in the dark for later flow cytometry analysis.

[0050] 3.3 Histopathological and immunofluorescence analysis of mouse sepsis tissue

[0051] 3.3.1. Liver and kidney sampling in mice with sepsis

[0052] Mice were euthanized by cervical dislocation; after soaking in 75% alcohol for 30 seconds, they were placed on a worktable, and the abdominal and thoracic skin and peritoneum were cut along the midline of the abdomen using ophthalmic scissors to open the abdominal cavity; the liver was separated using toothless forceps, and after cutting the hepatic hilar vessels, the free liver was soaked in 4% paraformaldehyde; the adipose tissue around the kidneys was separated, and the kidneys were cut off and soaked in 4% paraformaldehyde for cleaning, and then replaced with fresh 4% paraformaldehyde for thorough soaking.

[0053] 3.3.2 HE staining of liver and kidney in mice with sepsis

[0054] Mouse livers and kidneys were unidirectionally cut into tissue blocks approximately 3-4 mm in size and fixed in 4% paraformaldehyde at 4°C for 48 h. The tissue blocks were rinsed three times with running water for 5 min each time. The tissue blocks were then dehydrated sequentially by soaking them in 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol (first time), 95% ethanol (second time), and anhydrous ethanol. After dehydration, the tissue blocks were then cleared sequentially by soaking them in a solution of 50% ethanol + 50% xylene, xylene (first time), xylene (second time), and xylene (third time) for 5 min-30 min. The tissue blocks were then immersed in paraffin embedding medium three times, the first time for about 15 min, and the subsequent two times for 30 min-1 h. The tissue blocks were then embedded using an embedding machine and stored overnight at 4°C. Paraffin sections of the liver and kidneys were prepared using a microtome. After mounting and baking the continuously cut tissue sections, the sections were sequentially immersed in xylene solution for 10 min, xylene solution for 10 min, anhydrous ethanol for 5 min, anhydrous ethanol for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, 70% ethanol for 5 min, and pure ethanol for 5 min. The sections were then immersed in hematoxylin solution for 4 min; rinsed with running water for 10 min; immersed in hydrochloric acid ethanol for 3 s to differentiate, then rinsed with running water for 10 min to achieve blue reversion; immersed in eosin solution for 25 s–1 min; immersed in anhydrous ethanol for 5 min to dehydrate, repeating dehydration 3 times; after dehydration, immersed in xylene for 10 min for permeation, repeating 3 times; mounted directly with neutral resin and allowed to air dry before being photographed and observed under a microscope.

[0055] 3.3.3 Fluorescence detection of macrophage polarization in the liver and kidneys of mice with sepsis

[0056] Using CD68, CD86, and CD206 antibodies and a four-color multiplex fluorescent staining kit, the infiltration and distribution of total macrophages, M1 pro-inflammatory macrophages, and M2 anti-inflammatory macrophages in tissue sections of various organs were indicated, respectively. The dewaxing solution (tank #1) and sections were placed together in a 55°C incubator and allowed to stand for 30 minutes for baking. After being removed and allowed to stand at room temperature for 5 minutes, the sections were immersed in room temperature dewaxing solution (tank #2) for 5 minutes. The sections and slide holders were then sequentially immersed in room temperature dewaxing solution (tank #2), room temperature dewaxing solution (tank #3), anhydrous ethanol (tank #1), anhydrous ethanol (tank #2), and anhydrous ethanol (tank #3), for 5 minutes in each tank. Finally, the sections were rinsed with running water for 5 minutes. Immerse the slides in a retrieval chamber containing retrieval solution, and microwave on high for 3 minutes. Let the retrieval chamber stand in the microwave for 5 minutes, then microwave on high for another 3 minutes, followed by another 5 minutes of resting. Next, microwave on medium-low for 1 minute, then let stand for 5 minutes. Finally, remove the slides and antigen retrieval chamber and allow them to cool slowly to room temperature. Block the slides with 5% blank goat serum and incubate them in a humidified chamber at 37°C for 30 minutes. Continue incubation with CD86, CD68, and CD206 primary antibodies sequentially at 4°C overnight. After all primary antibody incubation and washing are completed, add the working solution of the fluorescent secondary antibody corresponding to the species of the primary antibody to the sample, and incubate at 37°C for 1 hour in the dark; wash the slide three times with TBS buffer for 5 minutes each time; add DAPI working solution to the sample; incubate at room temperature in the dark for 10 minutes, and then wash with TBST buffer for 5 minutes; wash three times with TBS buffer for 5 minutes each time; finally, add anti-fluorescence attenuation mounting medium, cover with a coverslip, and observe and acquire images under a fluorescence microscope.

[0057] (4) Calculation method:

[0058] 4.1 Survival rate of mice 7 days after surgery

[0059] After each group of mice was treated with CLP and Sham, they were returned to their cages and kept for another week. The mice's mental and behavioral status was observed daily, and the number of mice that survived and died each day was counted. The final results were analyzed using GraphPad Prism 8.0 software to plot the 7-day survival rate curve of the mice.

[0060] 4.2 Statistical Methods

[0061] Unless otherwise specified, all data are expressed as mean ± standard error. The data were presented in [data missing]. Statistical analysis was performed using IBM SPSS Statistics 20 and GraphPad Prism 8.0 software. All data were tested for normality and homogeneity of variance. One-way ANOVA and Bonferroni multiple comparisons were used to verify differences between groups. All data were standardized relative to the control group. P < 0.05 was considered statistically significant.

[0062] (5) Experimental Results

[0063] 5.1 Sepsis induces increased production of NLK protein in macrophages.

[0064] CD11b in SDMs extracted using this experimental protocol was identified by flow cytometry. + F4 / 80 + The double-positive rate was as high as 97.8%, indicating high purity of the obtained macrophages. Immunoblot analysis showed that sepsis significantly induced increased NLK expression in SDMs. (See attached image) Figure 1 Figure A: Flow cytometry identification of CD11b using SDMs + F4 / 80 + The proportion of double-positive cells was 97.8% ± 0.16 (n = 3); B: Cell morphology of macrophages under a fluorescence microscope at 630× field of view (scale bar 25 μm) and 2× magnified field of view (scale bar 10 μm); CD: Western blot analysis of NLK expression in SDMs of WT and NLK-deficient mice with and without CLP treatment and statistical results, n = 3. Compared with the control groups WT-Sham and NKO-Sham, *P<0.05, **P<0.01, ns indicates P>0.05.

[0065] 5.2. NLK gene deficiency can cause macrophages in sepsis to polarize from an M1 inflammatory state to an M2 anti-inflammatory state.

[0066] Flow cytometry analysis revealed that F4 / 80 was present in a sepsis state. + CD86 + The proportion of double-positive macrophages increased, while F4 / 80 + CD206 + The proportion of double-positive macrophages was significantly reduced, indicating that sepsis induced inflammatory activation of macrophages; after NLK gene knockout, the F4 / 80 ratio was reduced. + CD86 + The proportion of double-positive macrophages decreased significantly, while F4 / 80 + CD206 + The proportion of double-positive macrophages increased significantly. (See attached image) Figure 2As shown, NLK knockout can induce a shift from sepsis-induced inflammatory activation of macrophages to an anti-inflammatory activation state. The figure illustrates the expression of the surface molecular markers CD86 and CD206 of SDMs in each group as detected by flow cytometry and their statistical analysis. n=3, intergroup differences, *P<0.05, **P<0.01.

[0067] 5.3. NLK gene deficiency can reduce the expression levels of inflammatory factors in septic mice and effectively improve the survival rate of mice.

[0068] The expression levels of serum inflammatory factors TNF-α, IL-6, IL-1β, and IL-18 in mice of each group were detected by ELISA. The results showed that sepsis significantly induced increased expression of serum TNF-α, IL-6, IL-1β, and IL-18 in mice, with a 7-day survival rate of only 41.67% after surgery. In contrast, the expression levels of serum TNF-α, IL-6, IL-1β, and IL-18 in NLK gene-deficient mice were significantly decreased, with a 7-day survival rate of 75%. (See attached image) Figure 3 As shown, AD: ELISA was used to detect the production of inflammatory factors TNF-α, IL-6, IL-1β, and IL-18 in the serum of mice in each group, n=6; E: Survival rate of mice in each group 7 days after surgery, n=8; Intergroup comparison, *P<0.05, **P<0.01.

[0069] 5.4. NLK deficiency can reduce the infiltration of inflammatory macrophages and pathological damage in the liver and kidneys of septic mice.

[0070] HE staining revealed significant pathological damage to the liver and kidneys of wild-type septic mice, including hemorrhage, hepatocyte edema, structural abnormalities, and inflammatory cell infiltration. Immunofluorescence staining showed abundant recruitment and infiltration of M1 inflammatory macrophages in the liver and kidneys (indicated by red fluorescence). In contrast, NLK-deficient mice exhibited significantly less liver and kidney damage than their littermate wild-type counterparts, with predominantly anti-inflammatory macrophage infiltration in the liver and kidneys (indicated by green fluorescence). (See attached image) Figure 4 As shown, A: HE staining shows the morphological and structural changes and pathological damage of the liver and kidneys of mice in each group; multichannel fluorescence staining shows the polarization and distribution infiltration of macrophages in various organs, with CD68 (yellow) labeling total macrophages, CD86 (red) labeling M1 macrophages, and CD206 (green) labeling M2 macrophages (scale bar 100μm); BC: Liver damage score and macrophage fluorescence statistics of mice; DE: Kidney damage score and macrophage fluorescence statistics of mice (n=3). Intergroup differences were compared, *P<0.05, **P<0.01.

[0071] Summarize:

[0072] Sepsis can induce a significant upregulation of NLK expression, leading to inflammatory activation of macrophage M1 cells and significant infiltration into the liver and kidneys of mice, ultimately impairing mouse survival. NLK gene knockout, on the other hand, significantly increases the proportion of macrophage M2 cells undergoing anti-inflammatory polarization; NLK-deficient septic mice show significantly reduced macrophage inflammatory infiltration and tissue damage in the liver and kidneys, and a significantly improved survival rate.

[0073] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. Use of a knockout reagent for NLK gene in the manufacture of a medicament for ameliorating sepsis, characterized in that, Knocking out NLK gene promotes the transformation of macrophages from M1 inflammatory activation to M2 anti-inflammatory activation during sepsis.

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