Application of interference NLK gene target spot in preparation of drugs for improving inflammatory activation of macrophages

Abstract

The invention belongs to the field of biological medicine, provides application of an interference NLK gene target spot in preparation of a medicine for improving macrophage inflammatory activation, and discloses application of an NLK gene in macrophage differential polarization regulation of sepsis inflammatory response period. A mouse in-vivo experiment proves that in a sepsis state, NLK gene up-regulation mediates inflammatory activation of macrophages; interference on NLK expression can promote the inflammatory activation of macrophages to be converted into an anti-inflammatory activation state, and the inflammatory response and the survival rate of sepsis mice are effectively improved. Research results show that the NLK gene is expected to become a drug target for treating sepsis.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and biomedicine, and relates to the application of interfering with NLK gene target in preparing a drug for improving inflammatory activation of macrophages, in particular, the application of NLK gene as a target in preparing a drug for improving inflammatory activation of mammalian sepsis macrophages. Background Art

[0002] Sepsis often follows severe clinical emergencies such as severe burns, trauma, infection, and shock, and can develop into severe sepsis, septic shock, and multiple organ dysfunction. In the past few decades, despite the global understanding of the pathophysiological mechanisms of sepsis and the progress in the treatment methods and technologies for sepsis patients, the incidence and mortality of sepsis remain high. Immune response dysfunction is the core mechanism of the occurrence and development of sepsis. It includes two stages and states: excessive inflammatory response and immunosuppression, which occur sequentially or simultaneously in the course of sepsis.

[0003] At present, the clinical treatment of sepsis usually relies on early antibiotic use, fluid resuscitation, vasoactive drugs and other basic treatment measures. Targeted regulatory treatment for different stages of sepsis to maintain immune balance at each stage has become a hot topic in sepsis research. In the inflammatory response period 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 from pathways such as the nuclear transcription 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 the damage in the inflammatory phase of sepsis. In the immunosuppressive phase of sepsis, targeted increase in M1 polarization and reduction of M2-like macrophages can effectively eliminate host immune paralysis and reduce the occurrence of opportunistic infections.

[0004] Nemo-like kinase (NLK) is a member of the MAPKs family. Recent studies have shown that NLK is involved in immune cells and their immune regulation. For example, NLK knockout of Tregs leads to reduced Treg cell-mediated immunosuppression in vivo, and NLK-deficient Treg cell animals will develop more severe experimental autoimmune encephalomyelitis. In T cell lineage NLK conditional knockout mice, single positive CD8 + The significant decrease in T cells indicated that NLK +NLK plays an important role in the survival of T cells (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 has a certain regulatory role in the adaptive immune response. However, how NLK regulates immunity in sepsis has not been reported.

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

[0006] The present invention aims to interfere with, inhibit, knock out or change the regulatory effect of NLK gene in improving inflammatory activation of sepsis macrophages, and further provide the use of NLK gene as a target in the preparation of a drug for improving inflammatory activation of sepsis macrophages.

[0007] The present invention is achieved through the following technical solutions:

[0008] Application of interfering with NLK gene target in preparing drugs for improving macrophage inflammatory activation.

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

[0010] The invention relates to the use of NLK gene as a target in the preparation of a drug for improving inflammatory activation of macrophages in sepsis in mammals, wherein the mammals are humans, monkeys, dogs, cats, rats or mice.

[0011] By interfering with the NLK gene, macrophages in sepsis are prompted to change from M1 inflammatory activation to M2 anti-inflammatory activation; the interference method is one of 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] A drug obtained based on any of the above applications.

[0013] A method for improving inflammatory activation of mammalian sepsis macrophages, comprising the following steps:

[0014] Query the location of the NLK gene; interfere with the NLK gene, promote the transformation of macrophages from M1 inflammatory activation to M2 anti-inflammatory activation during sepsis, and alleviate the inflammatory infiltration and pathological damage of the liver and kidney of mice. The interference method is one of RNA interference, gene knockout, CRISPR / Cas9 technology, zinc finger nuclease, gene editing of transcription activator-like effector, antisense oligonucleotide, transcription factor inhibition and drug interference. The drug obtained based on the above application and the method for improving the inflammatory activation of mammalian sepsis macrophages.

[0015] The medicine contains an effective amount of NLK gene inhibitor and a pharmaceutically acceptable carrier, and the carrier can be a buffer or a culture solution or a preservative.

[0016] The drug is a substance that reduces NLK gene expression, including reagents for interfering with NLK, drug intermediates, and NLK gene inhibitors; the drug includes a combination of one or more of small molecule drugs, antibodies, polypeptides, proteins (such as cytokines, hormones, soluble receptors and non-specific proteins), oligonucleotides (such as DNA and RNA encoding polypeptides, double-stranded RNA and antisense RNA), and peptidomimetics.

[0017] The phrase "pharmaceutically acceptable" as used in this application refers to those agents, compounds, materials, compositions and / or dosage forms that are consistent with sound medical judgment, suitable for use in contact with human or animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, and have a reasonable benefit / risk ratio.

[0018] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or excipient, such as a liquid or solid filler, diluent, vehicle or solvent encapsulating material, which participates in the delivery or transport of an agent from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious 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 tragacanth; (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) glycols, such as Such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffer agents, 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:

[0020] The present invention provides an application of the NLK gene in the preparation of a drug for improving inflammatory activation of macrophages in mammalian sepsis. The applicant and the inventor have found through research that sepsis can induce inflammatory activation of macrophages and their infiltration in the liver and kidneys of mice through upregulation of NLK, resulting in aggravated pathological damage to the liver and kidneys of mice, and seriously impairing the 7-day survival rate of mice; interfering with the NLK gene can effectively promote the conversion of macrophages from M1 inflammatory activation to M2 anti-inflammatory activation during sepsis, effectively alleviate the inflammatory infiltration and pathological damage of the liver and kidneys of mice, and significantly improve the survival rate of mice; in addition, interfering with the NLK gene is easy to operate, providing a new method for the clinical treatment of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 CD11b is the CD11b of spleen-derived macrophages (SDMs) from macrophage NLK conditional knockout mice (NKO) and their littermate wild-type mice (WT) after sham surgery and CLP sepsis model treatment. + F4 / 80 +Flow cytometry identification results and Western blot detection results of NLK protein expression levels. (A) SDMs flow cytometry identification of CD11b + F4 / 80 + The ratio of double positive cells. (B) Cell morphology of macrophages under the field of view of fluorescence microscope. (CD) Western Blot detection of NLK expression in SDMs of WT and NLK-deficient mice with or without CLP treatment and statistical analysis of the results.

[0022] Figure 2 The effect diagram of the macrophage polarization state of the macrophage NLK conditional knockout mice and their littermate control wild-type mice in the sham operation and CLP sepsis model was shown in Figure 2. (A) Flow cytometry was used to detect the expression of F4 / 80 and CD86 in spleen-derived macrophages of each group of mice. (B) Pro-inflammatory macrophages (F4 / 80 + CD86 + (C) Flow cytometry was used to detect the expression of F4 / 80 and CD206 in spleen-derived macrophages of each group of mice. (D) The percentage of anti-inflammatory macrophages (F4 / 80 + CD206 + The results of statistical analysis of the percentage of double positive).

[0023] Figure 3 To illustrate the results of the detection of serum inflammatory factors and the 7-day survival rate of macrophage NLK conditional knockout mice and their littermate control wild-type mice under sham surgery and CLP sepsis model treatment in the present invention, to indicate the inflammatory response and survival of each group of mice. (AD) Elisa detected 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 each group of mice after surgery.

[0024] Figure 4 To illustrate the HE staining and macrophage infiltration of the liver and kidney of the macrophage NLK conditional knockout mice and their littermate control wild-type mice under sham surgery and CLP sepsis model treatment. (A) HE staining shows the morphological structural changes and pathological damage of the liver and kidney organs of each group of mice; multi-channel fluorescence staining shows the distribution and infiltration of inflammatory (red) and anti-inflammatory (green) macrophages in various organs. (BC) Mouse liver injury score and macrophage fluorescence statistics. (DE) Mouse kidney injury score and macrophage fluorescence statistics. DETAILED DESCRIPTION

[0025] The present application is further described below in conjunction with specific embodiments:

[0026] Example

[0027] The experimental plan is as follows:

[0028] (1) Acquisition and identification of macrophage NLK conditional knockout mice (NKO):

[0029] Macrophage NLK conditional knockout mice (NKO) were customized from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. (License No.: SCXK(Su)2023-0009).

[0030] The parent NKO mice were paired and caged at a ratio of 2 / 1 between male and female, and the genotypes were identified when the offspring were bred to 7-10 days of age. Mouse gene identification operation: Before the operation, the gloves of both hands were disinfected with alcohol spray, and the skin on the back of the mouse's neck was grasped with the left hand, so that the mouse's abdomen was facing up and the limbs were fixed and opened, and the mouse was ear-tagged with ear-tagged forceps. Use alcohol cotton balls to disinfect the toes to be cut off, and use sterile ophthalmic scissors in the right hand to cut the toes into a 1.5ml sterile EP tube. After marking the EP tube and recording the mouse ear tag, the EP tube was inserted into the ice box, and the mouse was returned to the cage for continued feeding. After customizing the primers according to the primer information recorded in Table 1, the primers were prepared with sterile RNase-Free water and then aliquoted and stored at -20℃. Use the mouse genotype rapid identification kit recorded in Table 2 (Beijing Sunshine Yingrui Biotechnology Co., Ltd.), use Adv.Buffer and Sup.Protease to prepare tissue digestion solution to fully digest the mouse toe tissue in the EP tube, and place the EP tube in a 75℃ metal bath for digestion for 15min. After digestion, the supernatant was taken as a PCR template at 12000rpm×2min. According to the PCR reaction system described in Table 3 and the reaction conditions described in Table 4, the samples were added and PCR reaction was performed, and then agarose gel electrophoresis was performed; samples of each model mouse were taken and PCR reaction was performed using Flox primer pair (0.5μL Flox-F+0.5μL Flox-R), iCre1 primer pair (0.5μL iCre-F1+0.5μL iCre-R1), and iCre2 primer pair (0.5μLiCre-F2+0.5μL iCre-R2). 1.2g agarose powder was measured in a conical flask, 60μL 1×TAE buffer was added, and 2% agarose buffer was obtained after heating in a microwave oven several times. After the agarose buffer was slightly cooled, 5μL Safe Green nucleic acid dye was added, mixed, poured into the gel tank, and a comb was inserted. It was left to stand at room temperature for 30min until the agarose gel was formed. Take out the gel and put it into the horizontal electrophoresis tank, with the sample well close to the negative electrode. Add 1×TAE buffer, spot the PCR sample, load 10μL per well, load 5μL DNAMarker, electrophoresis at 150V, and observe in the BioRad gel imaging system after 25 minutes. Determine the mouse genotype based on the identification results. The presence of both Flox (375bp) and iCre (350bp, 2195bp) bands is NKO mouse; the presence of only Flox without iCre is the wild-type control mouse of the same litter.

[0031] Table 1 Primer information for NKO mice

[0032]

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

[0034]

[0035] Table 3 PCR reaction system

[0036]

[0037] Table 4 PCR reaction conditions

[0038]

[0039] (2) Establishment of the mouse cecal ligation and puncture (CLP) sepsis model:

[0040] 6-8 weeks old, 20-22 g macrophage NLK conditional knockout mice (NKO) and control mice (WT) were fasted for 12 h. After intraperitoneal injection of 50 mg / kg pentobarbital to anesthetize the mice, the mice were fixed in a supine position on the experimental table and the abdomen was disinfected with iodine tincture; CLP model operation: a midline incision was made along the abdominal wall to expose the contents of the abdominal cavity, the end of the cecum was gently pulled out with toothless forceps, and the surface blood vessels of the mesentery were separated. After that, a sterile No. 3 silk thread was used to ligate the ileocecal valve and the midpoint of the cecum. A 21G sterile needle was used to penetrate and puncture the cecal wall twice at the ligation site and the midpoint of the cecal apex. The end of the cecum was gently squeezed, and the cecum was returned to the abdominal cavity after squeezing out a small amount of intestinal contents. The abdominal cavity was closed and the surgical incision was sutured layer by layer. In the sham group (Sham group), only the end of the cecum was exposed and squeezed, and then the cecum was returned and the abdomen was closed. No cecal ligation and puncture were performed. The remaining operations were the same as those in the surgical group (CLP group). The incision was disinfected with iodine again, and the whole operation was strictly sterile. Postoperative operation and observation indicators: 50mL / kg normal saline was injected subcutaneously in the neck for fluid resuscitation, 0.05mg / kg buprenorphine was used for postoperative analgesia, and the mice were kept warm. The respiration and body temperature of the mice were closely monitored after the operation. After the mice woke up, they were put back into the cage for continued feeding. Routine observation of mice in each group after surgery showed that compared with the mice in the Sham group, the mice in the CLP group gradually developed a series of clinical symptoms of mouse sepsis, such as erected dorsal hair, mental depression, slow movement, weakened resistance to arrest, 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) in mice with sepsis

[0043] Place the mouse spleen in a 10cm cell culture dish filled with 4℃ PBS buffer, use pointed forceps to remove the connective tissue and visceral capsule around the spleen; transfer the spleen to a 40μm cell sieve, grind the spleen with a grinder and repeatedly rinse the cell sieve with 4℃ PBS buffer, and filter the grinding liquid into a 15mL centrifuge tube. Centrifuge at 1500rpm for 5min and discard the supernatant; resuspend the cell pellet with 2mL PBS buffer. Use a 10mL sterile siliconized centrifuge tube in the lymphocyte separation fluid kit and add 4mL of separation fluid; draw the cell suspension along the tube wall and slowly add it to the sterile siliconized centrifuge tube, keep the liquid surface separation layer, and centrifuge at 20℃, 400g for 30min. After centrifugation, the liquid in the siliconized tube is divided into four layers from top to bottom: diluent layer, lymphocyte layer (ring-shaped milky white), separation liquid layer, and red blood cell layer; carefully aspirate the lymphocyte layer into another 15mL centrifuge tube, add 5mL of washing solution, and mix well by blowing; centrifuge at 400g for 10min, and discard the supernatant; take 5mL of washing solution to resuspend the cells, centrifuge at 250g for 10min, discard the supernatant, wash again, count the cells, centrifuge and discard the supernatant to obtain spleen lymphocytes.

[0044] Every 10 7 Resuspend lymphocytes in 90 μL PBS buffer. 7 Add 10 μL F4 / 80 + Magnetic beads, mix well, place in a 4℃ refrigerator, incubate for 15min away from light; after the incubation, 7 The cells were washed with 1-2 mL PBS, centrifuged at 300 g for 10 min, and the supernatant was discarded; 8 Resuspend each cell in 500 μL PBS buffer; install the filter column on the magnetic stand, wet the filter column once with 2-3 mL PBS, add the cell suspension, and use a 10 cm cell culture dish to collect the F4 / 80 filtered out of the filter column. - cells; add 500 μL or 3 mL PBS to wash the filter column, repeat 3 times; F4 / 80 - After the cells are filtered, remove the filter column and install it in a 15 mL centrifuge tube. Add 1 mL of PBS buffer to the MS tube and 5 mL of PBS buffer to the LS tube. Push the push rod to transfer the cells adsorbed on the wall of the filter column to the 15 mL centrifuge tube to obtain F4 / 80. + Spleen-derived macrophages (SDMs).

[0045] 3.2 Identification of macrophages in mouse sepsis

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

[0047] Collect the SDMs and resuspend the cells in 100 μL PBS.6 0.25 μg TruStain FcX TM PLUS antibody, mix well, incubate on ice for 5-10 minutes to block Fc receptors to reduce nonspecific binding of flow cytometry antibodies and cell surface receptors; set up double negative tubes, APC-CD11b single staining tubes, and FITC-F4 / 80 single staining tubes; the remaining groups were added with 1μCD11b and 1μL F4 / 80 antibody, incubated at room temperature in the dark for 30 minutes, then added with 1mL PBS buffer to wash the cells, centrifuged, discarded the supernatant, added 300μL 1% paraformaldehyde to fix at 4℃, and waited for detection on the machine.

[0048] 3.2.2SDMs polarization identification

[0049] Collect the SDMs and resuspend the cells in 100 μL PBS. 6 0.25 μg TruStain FcX TM PLUS antibody, mix well, incubate on ice for 5-10 minutes to block Fc receptors; 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, and it needs to be fixed before flow antibody staining and then stained after membrane permeabilization; use Cyto-Fast TM Fix / Perm Buffer Set kit was used to fix and permeabilize SDMs; 10×Cyto-Fast was diluted with deionized water. TM Wash buffer 10 times, take 100μL to resuspend SDMs; add 150μL Fix / Perm Buffer, let stand at room temperature for 20min; add 1mL 1×Wash buffer after membrane permeabilization, centrifuge at 1200rpm for 5min, discard the supernatant, and repeat washing once; add 100μL 1×Wash buffer to resuspend cells; add 1μL PE-CD86 and 1μL APC-CD206 antibodies to each tube of the control group and the experimental group, respectively, and incubate on ice in the dark for 15-20min; wash the cells with 2mL PBS buffer, centrifuge at 1200rpm for 5min, and repeat washing once; fix the cells with 300μL 1% paraformaldehyde, seal the flow tube and store it at 4℃ in the dark until it is detected by flow cytometer.

[0050] 3.3 Histopathology and immunofluorescence analysis of sepsis in mice

[0051] 3.3.1. Collection of liver and kidney samples from mice with sepsis

[0052] The mice were killed by cervical dislocation; soaked in 75% alcohol for 30 seconds, placed on the operating table, and the abdominal, chest 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 the free liver was immersed in 4% paraformaldehyde after cutting the portal blood vessels; the adipose tissue around the kidney was separated, the kidney was cut and immersed in 4% paraformaldehyde for cleaning, and then replaced with fresh 4% paraformaldehyde for full immersion.

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

[0054] Mouse liver and kidney were cut into 3-4 mm tissue blocks in one direction, placed in 4% paraformaldehyde, and fixed at 4°C for 48h. The tissue blocks were rinsed with running water 3 times, 5 min each time; the tissue blocks were placed in 30% ethanol, 50% ethanol, 70% ethanol, 95% ethanol (first time), 95% ethanol (second time), and anhydrous ethanol for dehydration; the dehydrated tissue blocks were placed in 50% ethanol + 50% xylene, xylene (first time), xylene (second time), and xylene (third time) solutions for transparent treatment for 5 min-30 min; the tissue blocks were immersed in paraffin embedding agent 3 times, the first time was about 15 min, and the latter two times could be extended to 30 min-1 h; the tissue blocks were embedded using an embedding machine and placed in a 4°C refrigerator overnight. Use a slicer to slice the liver and kidneys in paraffin; spread and bake the slices, and then place the slices in xylene solution for 10 minutes, xylene solution for 10 minutes, anhydrous ethanol for 5 minutes, anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, 70% ethanol for 5 minutes, and pure ethanol for 5 minutes. Soak the slices in hematoxylin solution for 4 minutes; rinse the slices with running water for 10 minutes; differentiate the slices in hydrochloric acid ethanol for 3 seconds, and then rinse the slices with running water for 10 minutes to return to blue; soak the slices in eosin solution for 25 seconds-1 minute; soak the slices in anhydrous ethanol for 5 minutes to dehydrate, and repeat the dehydration 3 times; after dehydration, soak the slices in xylene for 10 minutes to permeabilize, and repeat 3 times; use neutral resin to directly seal the slices, and after they are naturally dried, they can be placed under a microscope for observation.

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

[0056] CD68, CD86, CD206 antibodies and a four-color multiplex fluorescent staining kit were used to indicate the infiltration and distribution of total macrophages, M1 pro-inflammatory macrophages, and M2 anti-inflammatory macrophages in each organ tissue slice. Put the dewaxing solution No. 1 cylinder and the slices into a 55°C constant temperature incubator and let them stand for 30 minutes for baking; take them out and let them stand for 5 minutes at room temperature, then immerse them in the room temperature dewaxing solution No. 2 cylinder for 5 minutes; continue to put the slices and slice racks into the room temperature dewaxing solution No. 2 cylinder, room temperature dewaxing solution No. 3 cylinder, anhydrous ethanol No. 1 cylinder, anhydrous ethanol No. 2 cylinder, and anhydrous ethanol No. 3 cylinder in turn, and soak them in each cylinder for 5 minutes each time; wash the slices with running water for 5 minutes after the end. Immerse the slices in a repair box containing repair solution, and heat the whole in a microwave oven at medium-high heat for 3 minutes; place the repair box in the microwave oven for 5 minutes, heat it again at high heat for 3 minutes, and then let it stand for 5 minutes; then heat it at medium-low heat for 1 minute and let it stand for 5 minutes; finally, take out the slices and the antigen repair box and slowly cool them to room temperature. Use 5% blank goat serum to block the slices, place them in a wet box, and incubate them in a 37°C constant temperature and humidity incubator for 30 minutes; continue to incubate CD86, CD68 and CD206 primary antibodies in sequence, and incubate them at 4°C overnight. After all primary antibody incubation and washing are completed, add the fluorescent secondary antibody working solution corresponding to the primary antibody species to the sample, protect from light, and incubate at 37°C for 1 hour; repeatedly use TBS buffer to wash the slices 3 times, 5 minutes each time; add DAPI working solution to the sample; protect from light, incubate at room temperature for 10 minutes, then wash with TBST buffer for 5 minutes; wash with TBS buffer 3 times, 5 minutes each time; finally, add anti-fluorescence attenuation mounting agent, cover with coverslip, observe and collect images under a fluorescence microscope.

[0057] (4) Calculation method:

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

[0059] After CLP and Sham treatment, mice in each group were returned to their cages and continued to be raised for one week. The mental and behavioral states of the mice were observed daily, and the number of mice surviving and dying was counted daily. The final results were analyzed and the 7-day survival rate curve of mice was plotted using GraphPad Prism8.0 software.

[0060] 4.2 Statistical methods

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

[0062] (5) Experimental results

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

[0064] Flow cytometry was used to identify the CD11b of SDMs extracted from this experimental protocol. + F4 / 80 + The double positive rate was as high as 97.8%, indicating that the obtained macrophages were of high purity. Protein immunoblotting showed that sepsis significantly induced the increased expression of NLK in SDMs. Figure 1 Shown in A: SDMs flow cytometry identification of CD11b + 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 25μm) and 2× magnified field of view (scale bar 10μm); CD, Western Blot detection of NLK expression in SDMs of WT and NLK-deficient mice with or without CLP treatment and statistical analysis of the results, n=3, compared with the control group WT-Sham and NKO-Sham groups, *P<0.05, **P<0.01, ns means P>0.05.

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

[0066] Flow cytometry revealed that F4 / 80 + 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; when NLK gene was knocked out, F4 / 80 + CD86 + The proportion of double-positive macrophages decreased significantly, while F4 / 80 + CD206 + On the contrary, the proportion of double-positive macrophages increased significantly. Figure 2As shown, NLK knockout can promote the transformation of sepsis-induced macrophage inflammatory activation to an anti-inflammatory activation state. The figure shows the expression of surface molecular markers CD86 and CD206 of SDMs in each group detected by flow cytometry and their statistical analysis n=3, comparison of differences among groups, *P<0.05, **P<0.01.

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

[0068] 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. The results showed that sepsis significantly induced an increase in the expression of serum TNF-α, IL-6, IL-1β and IL-18 in mice, and the 7-day survival rate of mice after surgery was only 41.67%; while the expression levels of serum TNF-α, IL-6, IL-1β and IL-18 in NLK gene-deficient mice were significantly decreased, and the 7-day survival rate of mice after surgery was 75%. As shown in the attached Figure 3 As shown, AD: Elisa detected the production of serum inflammatory factors TNF-α, IL-6, IL-1β, and IL-18 in each group of mice, n=6; E: The survival rate of mice in each group 7 days after surgery, n=8; Comparison between the groups, *P<0.05, **P<0.01.

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

[0070] HE staining showed obvious pathological damage to the liver and kidney of wild-type septic mice, including hemorrhage, hepatocyte edema, structural abnormalities, and changes in inflammatory cell infiltration; immunofluorescence staining showed a large number of M1 inflammatory macrophages recruited and infiltrated in the liver and kidney (indicated by red fluorescence). The degree of liver and kidney damage in NLK-deficient mice during sepsis was significantly milder than that of their wild-type littermates, and the liver and kidney were mainly infiltrated by anti-inflammatory macrophages (indicated by green fluorescence). Figure 4 As shown, A: HE staining showed the morphological and structural changes and pathological damage of the liver and kidney of mice in each group; multi-channel fluorescence staining showed the polarization and distribution infiltration of macrophages in various organs, CD68 (yellow) marked total macrophages, CD86 (red) marked M1 macrophages, and CD206 (green) marked M2 macrophages (scale bar 100μm); BC: mouse liver injury score and macrophage fluorescence statistics; DE: mouse kidney injury score and macrophage fluorescence statistics (n=3), comparison of differences among the groups, *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 and significant infiltration in the liver and kidney of mice, ultimately impairing the survival rate of mice. NLK gene knockout can significantly increase the proportion of anti-inflammatory polarization of macrophage M2; NLK gene-deficient sepsis mice have significantly reduced macrophage inflammatory infiltration and tissue damage in the liver and kidneys, and significantly improved the survival rate of mice.

[0073] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. Application of interfering with NLK gene target in the preparation of drugs for improving macrophage inflammatory activation.

2. Application of NLK gene as a target in the preparation of drugs to improve inflammatory activation of macrophages in sepsis.

3. The use of NLK gene as a target in the preparation of a drug for improving inflammatory activation of mammalian sepsis macrophages, characterized in that: The mammal is a human, a monkey, a dog, a cat, a rat or a mouse.

4. The use according to claim 2 or 3, characterized in that: By interfering with the NLK gene, macrophages in sepsis are prompted to change from M1 inflammatory activation to M2 anti-inflammatory activation; the interference method is any one of 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.

5. A medicine obtained by the use according to any one of claims 1 to 3.

6. The drug according to claim 5, characterized in that The drug is an agent or drug intermediate for interfering with NLK or an NLK gene inhibitor; the drug is a combination of one or more of small molecules, antibodies, polypeptides, cytokines, hormones, soluble receptors, non-specific proteins, DNA and RNA encoding polypeptides, double-stranded RNA and antisense RNA, and peptidomimetics.

7. The drug according to claim 5, characterized in that The drug comprises a pharmaceutically acceptable carrier, which comprises a buffer or a culture solution or a preservative.

8. A method for improving inflammatory activation of mammalian sepsis macrophages, characterized in that: The steps include: 1) Query the location of the NLK gene; 2) Interfering with the NLK gene, prompting macrophages to switch from M1 inflammatory activation to M2 anti-inflammatory activation during sepsis, alleviating inflammatory infiltration and pathological damage in the liver and kidneys of mice, wherein the interference method is any one of 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.

Images