Application of inhibiting Caspase-8 expression in preparation of medicine for improving macrophage pan-apoptosis

Abstract

The invention belongs to the technical field of biomedicine, and provides application of inhibition of Caspase-8 expression in preparation of a medicine for improving macrophage pan-apoptosis. The invention also discloses an application of the NLK gene in preparation of a medicine for improving Caspase-8 dependent macrophage pan apoptosis. In-vitro experiments prove that under the state of sepsis, up-regulated NLK can regulate and control generation of Caspase-8-dependent macrophage pan apoptosis through protein interaction; through NLK knockout, the occurrence of Caspase-8 dependent pan apoptosis can be inhibited, and the death of macrophages is effectively reduced. Therefore, the research results show that the target regulation of Caspase-8-dependent pan apoptosis by intervening the NLK gene is expected to provide benefits for the treatment of sepsis; and a new method is provided for clinical treatment of sepsis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to the application of inhibiting Caspase-8 expression in preparing a drug for improving macrophage pan-apoptosis, and in particular to the application of inhibiting Caspase-8 protein expression through NLK gene interference, thereby alleviating Caspase-8-dependent septic macrophage pan-apoptosis. Background Art

[0002] A global epidemiological study on sepsis in 2020 showed that 189 patients per 100,000 person-years were hospitalized for sepsis treatment worldwide, with a mortality rate of approximately 26.7%; the incidence of sepsis treated in the ICU was approximately 58 cases per 100,000 person-years, of which 41.9% died before discharge. The high incidence and mortality of sepsis have become a major global health threat and have placed a heavy burden on global medical expenditures. Actively seeking effective means of preventing and treating sepsis and / or molecular targets, and developing related therapeutic drugs, are of great practical significance.

[0003] Pan-apoptosis is a new type of inflammatory cell death program, characterized by the simultaneous characterization of pyroptosis, apoptosis, and necroptosis in cells. Although the mechanism of pan-apoptosis in sepsis has not been fully clarified, new studies have shown that pan-apoptosis plays a vital role in the treatment of sepsis. For example, studies have shown that targeting non-inflammasome-dependent RIPK1 kinase activation and pan-apoptosis can provide therapeutic benefits for sepsis and TAK1-deficient myeloid cell proliferation (Malireddi RKS, 2020). Pan-apoptosis can be inhibited through the ursodeoxycholic acid interferon gene stimulator (STING) pathway to reduce sepsis-induced lung injury (He YQ, 2023), etc. Actively exploring the related mechanisms of pan-apoptosis in sepsis will hopefully provide effective potential molecular targets for the treatment of sepsis.

[0004] As a key molecule in the apoptosis and necroptosis pathways, cysteine-aspartic acid protease 8 (Caspase-8) is one of the earliest discovered bridges between different forms of cell death. Recent studies have shown that Caspase-8 is a key conversion molecule between pyroptosis, apoptosis, and necroptosis (Newton K, 2019; Fritsch M, 2019). Simply put, Caspase-8 can mediate the occurrence of pyroptosis by cleaving GSDMD and NLRP3 through active Caspase-8 (Sarhan J, 2018). When Caspase-8 is inactivated, the pyroptosis induced in the active state is replaced by necroptosis (Newton K, 2019). This Caspase-8-mediated conversion between different programmed cell deaths adds a crucial step to the immune plasticity induced by cell death. Caspase-8 has good application prospects as a molecular therapeutic target for sepsis. Summary of the invention

[0005] The first object of the present invention is to provide an application of inhibiting Caspase-8 expression in the preparation of a drug for improving macrophage pan-apoptosis; since sepsis can induce the occurrence of macrophage pan-apoptosis, Caspase-8 can serve as a key regulatory molecule to promote the generation of pyroptosis, apoptosis and necroptosis in pan-apoptosis during sepsis.

[0006] The ways in which caspase-8 expression is inhibited or interfered with include: RNA interference through small interfering RNA (siRNA) or short hairpin RNA (shRNA), CRISPR / Cas9 technology of gene knockout or CRISPR interference (CRISPRi), gene editing of zinc finger nucleases (ZFN) or transcription activator-like effector elements (TALEN), antisense oligonucleotides, transcription factor inhibition and drug interference.

[0007] The second purpose of the present invention is to provide an application of inhibiting Caspase-8 protein expression by NLK gene interference, thereby alleviating Caspase-8-dependent macrophage pan-apoptosis through the key regulatory role of NLK and Caspase-8 protein interaction in promoting sepsis and pan-apoptosis in sepsis discovered by the present invention.

[0008] Regulating Caspase-8 by knocking out NLK can effectively inhibit the occurrence of macrophage pan-apoptosis during sepsis and significantly rescue the death of sepsis-induced macrophages.

[0009] The technical solution of the present invention is as follows:

[0010] Application of inhibiting Caspase-8 expression in preparing drugs for improving macrophage pan-apoptosis.

[0011] Application of inhibiting Caspase-8 expression in preparing drugs for improving pan-apoptosis of sepsis macrophages.

[0012] Caspase-8 protein expression was inhibited by NLK gene interference, thereby alleviating Caspase-8-dependent macrophage pan-apoptosis.

[0013] Caspase-8 protein expression was inhibited by NLK gene interference, thereby alleviating Caspase-8-dependent pan-apoptosis of septic macrophages.

[0014] Inhibiting Caspase-8 protein expression by NLK gene interference can alleviate Caspase-8-dependent pan-apoptosis of mammalian septic macrophages.

[0015] And the medicine obtained by the above application.

[0016] The method for improving Caspase-8-dependent macrophage pan-apoptosis in mammalian sepsis comprises the following steps: interfering with NLK gene expression by RNA interference of small interfering RNA (siRNA) and short hairpin RNA (shRNA), gene knockout and CRISPR interference (CRISPRi) CRISPR / Cas9 technology, gene editing of zinc finger nuclease (ZFN) or transcription activator-like effector (TALEN), antisense oligonucleotides, transcription factor inhibition and drug interference, thereby reducing Caspase-8-dependent macrophage pan-apoptosis during sepsis, significantly reducing macrophage cell death rate, and alleviating sepsis symptoms.

[0017] The drug is a reagent for interfering with NLK, a drug intermediate and / or an NLK antibody.

[0018] The drug comprises a pharmaceutically acceptable carrier, which comprises a buffer or a culture solution or a preservative.

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

[0020] The present invention provides an application of inhibiting Caspase-8 expression in the preparation of a drug for improving macrophage pan-apoptosis, and an application of inhibiting Caspase-8 protein expression through NLK gene interference, thereby alleviating Caspase-8-dependent macrophage pan-apoptosis; experiments have shown and confirmed that upregulated NLK can regulate the occurrence of Caspase-8-dependent macrophage pan-apoptosis through protein interactions; NLK knockout can inhibit the occurrence of Caspase-8-dependent pan-apoptosis, effectively reducing macrophage death. Targeting Caspase-8 can effectively inhibit the occurrence of macrophage pan-apoptosis, thereby alleviating sepsis-induced cell death; therefore, targeted regulation of Caspase-8-dependent pan-apoptosis by, for example, intervening in the NLK gene is expected to provide benefits for sepsis treatment; and a new method is provided for the clinical treatment of sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The fluorescence protein co-localization imaging results of NLK-Caspase-8 in macrophages with or without LPS stimulation are shown in the present invention;

[0022] Figure 2 The results of the exogenous immunoprecipitation experiment of NLK and Caspase-8 in the present invention;

[0023] Figure 3 The Western blot test results of the expression levels of NLK, Caspase-8 and pan-apoptotic proteins in macrophages of the present invention, including the protein expression results of NLK, Caspase-8, NLRP3, Caspase-1, GSDMD, Caspase-3, Caspase-7, MLKL and p-MLKL in macrophages with or without LPS stimulation;

[0024] Figure 4 The statistical analysis results of the expression levels of NLK, Caspase-8 and pan-apoptotic proteins in macrophages of the present invention are shown in Figure 2. Figure 4 AH) Statistical results of NLK, Caspase-8, NLRP3, Caspase-1, GSDMD, Caspase-3, Caspase-7 and p-MLKL / MLKL protein expressions, n = 3, comparison among groups, ns means P > 0.05, *P < 0.05, **P < 0.01;

[0025] Figure 5 The changes in the morphology of pan-apoptotic macrophages in the present invention with or without LPS stimulation (scale bar 0.1 μm);

[0026] Figure 6 The present invention uses Calcein / PI to detect the macrophage mortality rate in each group. DETAILED DESCRIPTION

[0027] The present application is further described below in conjunction with specific embodiments:

[0028] Example

[0029] The experimental plan and specific details are as follows:

[0030] 1. Myeloid NLK conditional knockout mice (NLK lyz-fl / fl-iCre ) and NLK lyz-fl / fl Acquisition and identification of control mice:

[0031] NLK lyz-fl / fl-iCre Mice were customized from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (License No.: SCXK(Su)2023-0009).

[0032] The parent NLK lyz-fl / fl-iCreMice were paired and caged at a ratio of 2 / 1 between male and female, and genotype identification was performed 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 open, 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, the primers were prepared with sterile RNase-Free water and stored at -20℃. Use the mouse genotype rapid identification kit (Beijing Sunshine Yingrui Biotechnology Co., Ltd., C190801), 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. Samples were added and PCR reactions were performed according to the following PCR reaction system and reaction conditions; samples from each model mouse were taken and PCR reactions were performed using Flox primer pair (0.5μL Flox-F+0.5μL Flox-R), iCre1 primer pair (0.5μL iCre-F1+0.5μLiCre-R1), and iCre2 primer pair (0.5μL iCre-F2+0.5μL iCre-R2). Agarose gel electrophoresis: 1.2g agarose powder was weighed into 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 SafeGreen 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. Remove the gel to a 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 DNA Marker, perform electrophoresis at 150V, and observe in the BioRad gel imaging system after 25 minutes. Determine the mouse genotype based on the identification results. If both Flox (484bp) and iCre-Mut (700bp) positive bands appear, it is NLK. fl / fl-iCre Homozygous mice; mice with both Flox (484 bp) and iCre-WT (346 bp) positive bands are heterozygous mice; mice with only Flox but no iCre are wild-type control mice.

[0033] Table 1 NLKlyz-fl / fl-iCre mouse primer information

[0034]

[0035] Table 2 Components of Mouse Genotype Rapid Identification Kit

[0036]

[0037] Table 3 PCR reaction system

[0038]

[0039] Table 4 PCR reaction conditions

[0040]

[0041] 2. Extraction and culture of bone marrow-derived macrophages (BMDMs):

[0042] Take 6-8w NLK lyz-fl / fl-iCre Mouse and its NLK lyz-fl / fl Primary macrophages were extracted from the lower limb bones of control mice; after being killed by cervical dislocation, the mice were placed in 75% alcohol for disinfection for 1 min. Wearing sterile gloves, using sterile pointed forceps and ophthalmic scissors, cut the abdominal skin along the lower abdomen of the mouse horizontally, tear off the lower abdominal skin, expose the lower limbs, abduct and externally rotate the femur to dislocate it, separate it from the hip joint, and cut off the attached muscle tissue, free the lower limbs, fold the knee joint, and separate the femur and tibia and fibula; during the separation process, pay attention to keep the lower limb bones intact and no bone marrow exposed. After separating the attached muscle tissue of the lower limb bones with wet gauze, soak the femur and tibia and fibula in a 15mL centrifuge tube filled with pre-cooled PBS buffer for 1-3min, and then transfer them to a 15mL centrifuge tube filled with pre-cooled 75% alcohol and soak for 3min; transfer the lower limb bones to the pre-cooled PBS buffer in the clean bench and wash them twice. Use sterile pointed forceps to clamp the femur and tibia in turn, cut off the epiphysis at both ends with ophthalmic scissors, and use a 5mL syringe with a 1mL syringe needle to absorb pre-cooled RPMI 1640 culture medium (containing HEPES, glutamine, penicillin-streptomycin) to repeatedly flush the bone marrow cavity until the bone marrow turns white and transparent. Collect the bone marrow flushing fluid and filter it into a 50mL centrifuge tube using a 70μm cell sieve, mark it, and centrifuge it at 12000rpm for 5min; discard the supernatant, resuspend the cell pellet with 3-5mL red blood cell lysis buffer for each 1-2 mice, let it stand at room temperature for 5min, add double volume of PBS buffer to stop lysis, centrifuge it at 12000rpm for 5min, and discard the supernatant. Resuspend the cell pellet with 1mL complete culture medium (RPMI 1640 culture medium + 10% FBS + 50ng / mL M-CSF) and count the cells. Inoculate 1×10 per 6cm cell culture dish. 7 The cells were plated, labeled, and incubated in a 37°C, 5% CO2 incubator for one week. On the third day of cell incubation, half of the medium was replaced, and on the fifth day of incubation, the medium was fully replaced. On the seventh day, the cells were differentiated and mature BMDMs.

[0043] 3. Construction of in vitro bacterial lipopolysaccharide (LPS) sepsis model:

[0044] Use sterile deionized water to prepare LPS powder to a storage concentration of 1 μg / mL and store it in a refrigerator at -20°C. After discarding the BMDMs culture medium and washing the cells with PBS buffer, add 3 mL of complete culture medium and add an appropriate amount of LPS according to specific experimental conditions to construct a sepsis model.

[0045] 4. Calcein / PI cell activity detection:

[0046] The Calcein / PI cell viability and cytotoxicity detection kit (including detection buffer, 1000×CalceinAM and 1000×PI) was used to detect cell viability and cell death. Calcein AM itself has no fluorescence. After entering living cells, it can be decomposed into Calcein by endogenous esterases in the cells and retained in the cells to emit green fluorescence. Propidium iodide (PI) cannot penetrate the membrane of living cells and can only stain dead cells red. Image J software was used to count cell death rates.

[0047] 5. Protein extraction and BCA method to measure protein concentration:

[0048] 1) Cell lysis: Collect SDMs, add appropriate amount of cell lysis buffer (RIPA strong lysis buffer + 1× protease inhibitor + 1× phosphatase inhibitor), shake and mix, and put into ice box; use ultrasonic cell disruptor to ultrasonically disrupt cell membrane, wash the ultrasonic probe with deionized water, insert the probe under the sample liquid surface for ultrasonic disruption for 2-4s; wash the probe and replace the sample; centrifuge at 4℃ and 12000rpm for 30min, collect the supernatant of cell lysate into a new 1.5mL EP tube, mark it, and put it into ice box; 2) Preparation of BCA protein standard: Use bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology) (containing solution A, solution B, protein standard, protein standard diluent) to measure the protein concentration; take an appropriate amount of 25 mg / mL protein standard, dilute it with diluent to a final concentration of 0.5 mg / mL for standby use; take a 96-well plate, add 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, 20 μL BSA protein standard respectively, add the remaining diluent to make a total of 20 μL per well, shake gently to mix, then the concentration of protein standard in each well is 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.5 mg / mL; 3) Protein sample preparation and Concentration determination; take 2 μL of the protein sample to be tested into a 96-well plate, add 18 μL of diluent to each well to make up to a total of 20 μL, and shake gently to mix; 200 μL BCA working solution is required for each protein sample, and A solution and B solution are mixed in a ratio of 50:1 to prepare a light green BCA working solution and add it to each well. After mixing, incubate in a 37°C biochemical incubator for 30 minutes; after the incubation, place the 96-well plate in an ELISA reader, measure the absorbance of each well solution at 560 nm, and obtain the corresponding OD value; according to the OD value of the protein standard, use an EXCEL table to calculate the standard curve and the protein content of the sample to be tested in each well, calculate and add the 5× loading buffer required for each protein sample, and shake to mix; mark the protein sample name and extraction date; place it in a 100°C metal bath for 10 minutes to denature the protein, insert it into an ice box for 5 minutes, and then store it at -20°C.

[0049] 6. Transmission electron microscopy (TEM) observation of cell ultrastructure:

[0050] 1) Cell fixation: After the experimental treatment, BMDMs were collected, washed twice with 4°C PBS buffer, discarded, and an appropriate amount of Gluta fixative (special for electron microscopy, 2.5%) was added to fix the cells at 4°C for 1-2h; 2) Osmium acid fixation: The cells were centrifuged at 12000rpm and 4°C for 2min, the supernatant was discarded, and the cell pellet was collected; the cell pellet was rinsed with 0.1M phosphate buffer (pH7.4) 3 times, 15min each time; the cell clumps were fixed with 1% osmium acid precooled at 4°C at room temperature for 2-3h; the cell clumps were rinsed again with 0.1M phosphate buffer (pH7.4) 3 times, 15min each time; 3) Dehydration: Dehydrated with gradient alcohol (30%, 50%, 70%, 80%, 85%, 90%, 95%) , 100% twice) to dehydrate the cell pellet, each time for 15-20 min; 4) Infiltration: acetone: epoxy resin (2:1), acetone: epoxy resin (1:1), and epoxy resin were used to infiltrate the samples in a 37°C incubator in sequence, each time for 8-12 h; 5) Embedding and sectioning: the infiltrated samples were placed in an embedding plate, and the embedding agent epoxy resin was added. After polymerization in a 60°C incubator for 48 h, ultrathin sections were made using an ultrathin microtome (Leica, EMUC7, Germany), and the section thickness was generally 80-100 nm; 6) Staining: the sections were double-stained with 2% uranyl acetate saturated aqueous solution and lead citrate at room temperature for 15 min, and dried at room temperature overnight; 7) Image acquisition: a transmission electron microscope (Hitachi TEM system, HT7800, Japan) was used to observe the cell morphology and structure.

[0051] 7. Western Blot:

[0052] Western blotting experiments were performed using BIO-RAD (Bio-Rad, USA) mini vertical electrophoresis tank (Mini-PROTEANTetra), mini transfer tank (MiniTransBlotCell), universal electrophoresis instrument (PowerPacUniversal), and 1.5 mm glass plates (Mini-PROTEANGlassplates Catalog#1653312, Catalog#1653308).

[0053] 1) Preparation of separation gel and concentrated gel: Use CFAS any KD PAGE protein electrophoresis gel preparation kit (PE008, Zhonghui Hecai) to prepare separation gel and concentrated gel. The product components of the kit are as shown in Table 5:

[0054] Table 5

[0055]

[0056] Take 5mL of deionized water and add it to the ammonium persulfate (APS) test tube, shake and mix, and store it at 4℃ for a long time; install two pairs of 1.5mm glass plates on the matching fixing frame and prepare two matching 15-hole combs; take two 50mL centrifuge tubes, mark them as separation liquid and concentrate respectively, take 8mL of separation liquid A and 8mL of separation liquid B to the first centrifuge tube, add 160μL APS (the volume ratio of separation liquid and APS is 100:1), shake and mix; take 3mL of concentrated gel A and 3mL of concentrated gel B to another centrifuge tube, add 60μL APS (the volume ratio of concentrate and APS is 100:1), shake and mix; use a 1mL pipette to slowly add the separation solution into the glass plate along the plate wall to avoid bubbles, then draw the concentrate and slowly add it to the separation solution, flatten the separation solution surface, and insert the comb; after standing at room temperature for 25 minutes, the separation gel and concentrated gel are formed, removed from the fixing device, loaded on the electrophoresis rack, placed in the electrophoresis tank, and prepared for electrophoresis; 2) Preparation of electrophoresis fluid: use deionized water to dilute 10× electrophoresis fluid (HX1896, huaxingbio) into 1× electrophoresis fluid, add it to the electrophoresis tank, and slowly pull out the comb horizontally upward.Use a 1mL pipette to draw the electrophoresis solution to rinse the residual colloid and bubbles in each well; 3) Protein sample preparation: Take the protein sample out of the -20℃ refrigerator in advance and put it in an ice box to thaw; Place the protein sample in a 100℃ metal bath and heat it for 10 minutes to denature the protein; Immediately after denaturation, insert the protein sample into the ice box for 3-5 minutes, and centrifuge it at 4℃10000rpm for 1 minute; 4) Sample spotting: Load 5μL protein marker (26616, Invitrogen or HX6001, huaxingbio) in the first and last wells of the gel block, and load 30μg protein sample in the remaining wells from left to right; 5) Electrophoresis: Install the electrophoresis tank cover, turn on the power, and perform 100V stabilized voltage electrophoresis for 95 minutes to end the electrophoresis; 6) Transfer solution preparation: During the electrophoresis, use 700mL deionized water + 100mL 10× transfer solution (HX1895, huaxingbio) + 200mL methanol to prepare 1L in advance. 1× transfer solution, put in a 4℃ refrigerator for later use; 7) Transfer: After the electrophoresis is completed, use a scraper to remove the short glass plate and cut off the excess glue blocks; cut the filter paper and 0.22μm PVDF membrane (Iseq00010, Millipore) of the same size as the glue blocks to be transferred; put the PVDF membrane in a small box and add an appropriate amount of methanol to activate it for 3 minutes; pour the refrigerated 1× transfer solution into the plate, and place the following in the plate in order: negative transfer plate, sponge, filter paper, glue blocks, PVDF membrane, filter paper, sponge, positive transfer plate; each layer needs to be centrifuged Roll out the bubbles in the interlayer to avoid movement after placement; after placement, install the transfer plate, place it in the transfer tank, add an appropriate amount of transfer solution, install the tank cover, and connect the power supply; place the transfer tank in a foam box, and fill it with crushed ice around the transfer tank and the cover; transfer the membrane at a steady voltage of 100V for 2h; 8) Wash the membrane: after the transfer is completed, cut off a small corner of the upper right corner of the front of the PVDF membrane as a front mark, put it in a small box, add an appropriate amount of 1×TBST buffer (prepared by diluting 10×TBST buffer with deionized water), wash the membrane on a decolorization shaker for 5min, and discard it.Repeat the washing twice, each time on a decolorizing shaker for 5 minutes; 9) Blocking and washing: Use fast blocking solution (P30500, Suzhou Xinsaimei Biotechnology) to block the PVDF membrane for 30 minutes; after blocking, wash the membrane again 3 times with TBST buffer, each time for 5 minutes; 10) Primary antibody incubation: Each primary antibody is diluted to the optimal concentration recommended by the instructions with primary antibody diluent; add the corresponding primary antibody and incubate at 4°C overnight; 11) Secondary antibody incubation: After the primary antibody incubation, recover the primary antibody and wash the membrane 3 times with TBST buffer, each time for 5 minutes; the corresponding species of secondary antibody is diluted with secondary antibody diluent (P0258, Beyotime Biotechnology) according to the concentration recommended in the antibody manual; add secondary antibody, place on a shaker, and incubate at room temperature for 1 hour; after the incubation, recover the secondary antibody and wash the membrane 3 times with TBST buffer, 5 minutes each time; 12) Development: Use SuperECLPlus ultra-sensitive luminescent solution (strong) (P1050, Applygen) to prepare the colorimetric solution, use tweezers to clamp the target PVDF membrane, absorb the residual liquid on the surface with filter paper, incubate the colorimetric solution in the dark, develop the image, take pictures, and store the results in the Tianneng Tanon-5200 imaging system; 13) Result analysis: Use ImageJ software to analyze the optical density value of the target band, and GraphPadPrism8.0 software to analyze the data and draw the picture; 14) Membrane regeneration: After the development, use Stripping Buffer was incubated at room temperature for 30 minutes to regenerate the membrane; after regeneration, the membrane was washed three times with TBST buffer for 5 minutes each time, and the above steps of blocking, washing, incubating other primary antibodies overnight, incubating secondary antibodies, and developing were continued to display other target bands; each PVDF membrane can be repeatedly regenerated and incubated with antibodies for about 4-5 times to obtain other target protein bands.

[0057] 8. Immunofluorescence detection:

[0058] Caspase-8 antibody (Cell Signaling Technology, #8592) and NLK antibody (Cell Signaling Technology, #94350) were used in combination with DAPI dye to indicate the generation of NLK and Caspase-8 in BMDMs, respectively, and their fluorescence co-localization was observed using a confocal microscope. 1) Cell plating and treatment: The cells were resuspended and mixed with complete culture medium and then inoculated on a 20mm cell slide in a 12-well plate. They were incubated in a 37°C, 5% CO2 incubator for seven days until the BMDMs were stimulated to differentiate and mature and given corresponding treatments; 2) Fixation and permeabilization: After the stimulation treatment, the culture medium supernatant was discarded, and the cell slides were washed with PBS buffer, which was washed twice and then discarded; 4% paraformaldehyde was used to fix the cell slides for 15 minutes; 0.5% Triton X-100 permeabilization for 15 minutes; wash the slides with PBS buffer 3 times and discard; 3) Blocking: incubate the slides with 1% BSA (prepared with PBS buffer) and block at room temperature for 30 minutes; discard the blocking solution after blocking; 4) Primary antibody incubation: add 500μL rabbit anti-Caspase-8 primary antibody to the slides, incubate them in a light-proof humid box at 4°C overnight; after incubation, add PBS buffer and wash the cell slides on a decolorizing shaker 3 times, 5 minutes each time; 5) add mouse anti-NLK antibody and incubate them in a light-proof humid box at 4°C overnight; after incubation, add PBS buffer and wash the cell slides on a decolorizing shaker 3 times, 5 minutes each time; 6) Secondary antibody incubation: add 500μL Cy3-goat anti-rabbit and 500μL FITC-goat anti-mouse fluorescent secondary antibody, incubate at room temperature in the dark for 60 minutes; 7) DAPI nuclear staining: After completion, use antibody elution solution to wash the slides, wash the slides again for 3 times with PBS, then add DAPI and incubate at room temperature in the dark for 10 minutes to counterstain the cell nuclei; wash the slides again for 3 times with PBS, each time for 5 minutes; 8) Sealing and image acquisition: Use anti-fluorescence quenching sealing agent to seal the slides; place the slides under a confocal microscope for observation and image acquisition.

[0059] 9. Co-Immunoprecipitation (CO-IP):

[0060] Bioinkedin Classic IP / Co-IP Kit (Biolinkedin, IK-1018) was used according to the instructions of the kit: 1) Lysis solution preparation: 250 μL lysis solution (IP Lysis / Wash Buffer+Phosphatase Inhibitor Cocktail+PMSF were added in proportion) was used for each 6 cm cell culture dish, mixed well, and placed in an ice box for standstill; 2) Cell lysis: After the experimental stimulation was completed, the cell culture medium supernatant was discarded, and the cells were washed twice with 1 mL PBS buffer, discarded, and cell lysis solution was added. The cells were scraped into a 1.5 mL EP tube using a cell scraper, shaken, and placed in an ice box for standstill for 30 min. During this period, the cells were repeatedly shaken several times to fully digest and lyse the cells; 3) Lysis solution supernatant collection: The EP tube was placed in a high-speed low-temperature centrifuge, centrifuged at 4°C, 12000 rpm for 15 min to obtain the lysate supernatant; 4) Preparation of the positive control Input group: 100 μL lysate supernatant was taken as the Input tube, and 1× SDS-PAGE Sample Loading was added. Buffer was used to make protein samples. After protein denaturation in a metal bath at 100°C for 10 minutes, the EP tube was placed in an ice box and allowed to stand for 5 minutes to obtain the Input group protein sample (positive control). The remaining lysate was used as the IP tube. 5) Negative control preparation: The HA / Flag empty tube (negative control) was obtained by lysing 293T cells that were only transfected with the HA / Flag empty plasmid. 6) Magnetic bead washing: Anti-HA and / or Anti-Flag magnetic beads were prepared according to the number of IP tubes and HA / Flag tubes. 40-50 μL of magnetic beads were pipetted into a new 1.5 mL EP tube for each tube. IP Lysis / Wash was used. Wash the magnetic beads 3 times with buffer. After each wash, fix the EP tube on the magnetic rack and discard the supernatant after the magnetic beads are completely adsorbed on the magnetic rack; 7) Magnetic bead binding protein: After the magnetic beads are washed, add the cell lysis supernatant to the EP tube containing 40-50μL magnetic beads, seal it with sealing film, place it in a silent mixer, and incubate it at 4℃ overnight; 8) Magnetic bead washing: On the second day, after the magnetic beads are fully bound to the cell lysate, use the magnetic rack to adsorb the magnetic beads and discard the unbound samples.Add 500μL IP Lysis / Wash Buffer and repeat the above washing steps to wash the magnetic beads 5 times, and discard the supernatant; 9) Protein sample preparation: Add 1×50μL SDS-PAGE Sample Loading Buffer to each EP tube; Place the EP tube in a 100℃ metal bath for protein denaturation for 10 minutes, insert the EP tube into an ice box and let it stand for 5 minutes, then use a magnetic stand to adsorb the magnetic beads, transfer the protein sample to another corresponding new EP tube, and obtain HA / Flag histone samples and IP histone samples; 10) Western Blot verification: The primary antibody used in Western Blot is Flag and / or HA, and the secondary antibody is used corresponding to the species of the primary antibody.

[0061] The components of the kit are described in Table 6 below:

[0062] Table 6

[0063]

[0064] 10. Statistical methods

[0065] All data in this experiment are presented 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 groups, and all data were normalized relative to the control group. P < 0.05 was considered significant and statistically significant.

[0066] 11. Experimental Results

[0067] (1) LPS stimulation induces increased co-localization of NLK and Caspase-8 in BMDMs

[0068] Without LPS stimulation, BMDMs showed uniform fluorescence staining, and no obvious intracellular fluorescent spots were found after co-localization. However, after LPS stimulation, the production of NLK and Caspase-8 fluorescent proteins in BMDMs increased, showing as dot-shaped fluorescent particles. After the fluorescence channels were merged, the red NLK fluorescent spots and the green Caspase-8 fluorescent spots were co-localized in the BMDMs, forming a large number of yellow fluorescent spots (such as the attached Figure 1 ). The results showed that NLK and Caspase-8 had protein-protein interaction under LPS stimulation and co-localized in BMDMs.

[0069] (2) Protein interaction between NLK and Caspase-8 under LPS stimulation

[0070] The inventors of the present application transfected HA-NLK and Flag-Caspase-8 plasmids separately and simultaneously into different 293T cells. The immunoprecipitation complex obtained by detecting the Flag magnetic beads using HA antibody was found to have the presence of HA-NLK protein in both the Input group and IP group of 293T transfected with HA-NLK alone and co-transfected with HA-NLK and Flag-Caspase-8. Similarly, the co-precipitation complex obtained by detecting the HA magnetic beads using the Flag antibody was found to have the presence of Flag-Caspase-8 protein in both the Input group and IP group of 293T transfected with Flag-Caspase-8 alone and co-transfected with HA-NLK and Flag-Caspase-8. The experimental results show that protein interactions also exist between NLK and Caspase-8 in 293T exogenous CO-IP.

[0071] (3) Effect of NLK gene knockout on the expression of pan-apoptotic proteins in BMDMs stimulated by LPS

[0072] Western Blot results showed that LPS significantly induced the production of NLK (2.14±0.15 vs. 1.00±0.05, P<0.01) and Caspase-8 (1.50±0.04 vs. 1.00±0.03, P<0.01) (Appendix Figure 3 A, Figure 4 AB); when NLK was knocked out, the production of Caspase-8p18 (1.27±0.03 vs. 1.50±0.04, P<0.01) in the NKO-LPS group was significantly reduced compared with that in the WT-LPS group (Appendix Figure 3 A, Figure 4 B).

[0073] Compared with the Control group, the expressions of pyroptosis-related proteins NLRP3 (4.43±0.63 vs. 1.00±0.13, P<0.01), Caspase-1p10 (2.93±0.17 vs. 1.00±0.05, P<0.01), and GSDMD-N (2.63±0.08 vs. 1.00±0.04, P<0.01) in the WT group were significantly increased under LPS stimulation (Appendix Figure 3 A, attached Figure 4CE); the expression of apoptosis-related proteins Caspase-3 and Caspase-7, Caspase-3p19, P17 (1.67±0.08 vs. 1.00±0.03, P<0.01) and Caspase-7p20 (1.64±0.04 vs. 1.00±0.04, P<0.01) were significantly increased compared with the WT-Control group, and the phosphorylation of necroptosis executioner protein MLKL (1.64±0.03 vs. 1.00±0.05, P<0.01) was significantly upregulated (Appendix Figure 3 B, Attachment Figure 4 FH). After NLK gene knockout, compared with the WT-LPS group, the expression levels of pyroptosis-related proteins NLRP3 (2.68±0.37 vs. 4.43±0.63, P<0.05), Caspase-1p10 (1.87±0.10 vs. 2.93±0.17, P<0.01) and GSDMD-N (1.68±0.06 vs. 2.63±0.08, P<0.01) in BMDMs of the NKO-LPS group were significantly reduced (Appendix Figure 3 A, attached Figure 4 CE); the expression of apoptosis-related proteins Caspase-3p19, p17 (1.32±0.05 vs. 1.67±0.08, P<0.05) and Caspase-7p20 (1.23±0.02 vs. 1.64±0.04, P<0.01) was also significantly reduced; on the contrary, the phosphorylated form of necroptosis MLKL (1.40±0.03 vs. 1.64±0.03, P<0.05) was significantly increased (Appendix Figure 3 B, Attachment Figure 4 FH). The results showed that BMDMs under LPS stimulation underwent pan-apoptosis, which was manifested by increased production of pyroptosis, apoptosis, and necroptosis; while pan-apoptosis of BMDMs was significantly reduced after NLK gene deficiency.

[0074] (4) Effect of NLK gene knockout on the pan-apoptotic cell morphology of BMDMs stimulated by LPS

[0075] Electron microscopic observation revealed that the morphology of macrophages in the normal group was normal, with highly irregular cell shapes, more wrinkles on the cell surface, and various pseudopodia extending outward. Lysosomes at all levels were abundant in the cytoplasm, and the Golgi apparatus and mitochondria were well developed; the nucleus was irregular in shape. After LPS treatment, the cells in the WT group showed obvious morphological changes in cell pyroptosis, characterized by cell membrane perforation (indicated by red circles), extravasation of cytoplasmic contents, increased intracellular vesicle generation (indicated by red arrows), and obvious swelling of organelles; there was obvious apoptosis characterized by cell atrophy, increased nuclear / cell ratio, nuclear membrane invagination, and chromatin condensation (indicated by blue arrows); and there was cell necrosis characterized by cell membrane rupture (indicated by yellow arrows), translucent cytoplasm, expanded organelles, and retained nuclear membrane. However, under LPS stimulation, the cell damage of BMDMs in the NLK knockout group was mainly morphological changes of necroptosis and apoptosis, which were manifested by obvious cell membrane rupture, translucent cytoplasm, obvious damage to intracellular organelles, and the presence of nuclear membrane. The morphological changes of cell pyroptosis and apoptosis were significantly reduced, and the morphology of most cells was relatively normal (see Appendix). Figure 5 ). The cell morphological changes in this result were consistent with the trend detected by Western Blot.

[0076] (5) Effect of NLK gene knockout on BMDM cell death rate under LPS stimulation

[0077] Calcein-PI staining and detection showed that without LPS stimulation, there was no obvious cell death in the WT-Control group and the NKO-Control group, and there was no statistical difference between the groups (0.77±0.39% vs. 0.98±0.57%, P>0.05) (Appendix Figure 6 Compared with the WT-Control group, LPS stimulation significantly increased the BMDMs mortality rate in the WT-LPS group (27.42±1.45% vs. 0.77±0.39%, P<0.01) and the cell mortality rate in the NKO-LPS group (18.84±0.81% vs. 0.98±0.57%, P<0.01); and after NLK knockout, the cell mortality rate in the NKO-LPS group was significantly decreased compared with the WT-LPS group (18.84±0.81% vs. 27.42±1.45%, P<0.01) (Appendix Figure 6 The results showed that LPS-stimulated pan-apoptosis could induce increased macrophage cell death (increased proportion of red blood cells), and that NLK knockout effectively reduced the macrophage cell death rate (significantly reduced proportion of red blood cells).

[0078] The present invention uses a variety of experimental methods to prove that under LPS-induced sepsis, upregulated NLK can regulate the occurrence of Caspase-8-dependent macrophage pan-apoptosis through protein interaction; after silencing the NLK gene, Caspase-8-mediated cell pan-apoptosis is significantly inhibited and cell death is significantly reduced. The above results suggest that targeting Caspase-8 can effectively inhibit the occurrence of macrophage pan-apoptosis, thereby reducing sepsis-induced cell death.

[0079] 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 inhibiting Caspase-8 expression in the preparation of drugs for improving macrophage pan-apoptosis.

2. Application of inhibiting Caspase-8 expression in the preparation of drugs for improving pan-apoptosis of septic macrophages.

3. The use according to claim 1 or 2, wherein the expression of Caspase-8 protein is inhibited by NLK gene interference, thereby alleviating Caspase-8-dependent macrophage pan-apoptosis.

4. The use according to claim 3, wherein the expression of Caspase-8 protein is inhibited by NLK gene interference, thereby alleviating Caspase-8-dependent pan-apoptosis of septic macrophages.

5. The use according to claim 4, wherein the expression of Caspase-8 protein is inhibited by NLK gene interference, thereby alleviating Caspase-8-dependent pan-apoptosis of mammalian septic macrophages.

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

7. The drug according to claim 6, characterized in that The drug is a reagent for interfering with NLK, a drug intermediate and / or an NLK antibody.

8. The drug according to claim 6, characterized in that The drug comprises a pharmaceutically acceptable carrier, which comprises a buffer or a culture solution or a preservative.

9. A method for improving Caspase-8-dependent macrophage pan-apoptosis in mammalian sepsis, characterized in that: The method comprises the following steps: interfering with NLK gene expression to alleviate Caspase-8-dependent macrophage pan-apoptosis during sepsis, significantly reducing macrophage cell death rate, and relieving sepsis symptoms.

10. The method according to claim 9, characterized in that The method of interfering with the NLK gene is any 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.

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