Application of COPS5 and inhibitor in preparation of medicine for detecting and treating acute lung injury caused by sepsis

Abstract

The invention discloses application of COPS5 and an inhibitor in preparation of a medicine for detecting and treating sepsis acute lung injury. The invention systematically reveals a molecular mechanism of COPS5 on acute lung injury caused by sepsis by regulating macrophage NLRP3 inflammasome activation for the first time, and provides a new target for research and treatment of acute lung injury caused by sepsis. In addition, COPS5 can be used as a therapeutic target for screening targeted drugs or therapeutic modes for treating sepsis acute lung injury.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the use of COPS5 and an inhibitor in the preparation of drugs for detecting and treating sepsis-induced acute lung injury. Background Art

[0002] Sepsis is a highly heterogeneous disease caused by a dysfunctional host response to infection. Sepsis is currently a major challenge faced by clinicians and researchers in the critical care field due to its high morbidity, high mortality, and difficulty in early diagnosis. Patients with sepsis often suffer from multiple organ damage and even multiple organ failure. The lungs are the most easily affected organ during the development of sepsis, and patients with sepsis often develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) in the early stages.

[0003] The pathogenesis of acute lung injury in sepsis is complex, with high morbidity and mortality. The main reason is the inflammatory factor storm caused by the imbalance between pro-inflammatory and anti-inflammatory factors due to the dysregulated immune response of the body. The pathological manifestations are a syndrome characterized by inflammatory cell infiltration, endothelial cell damage, pulmonary edema and inflammatory cell recruitment, and the clinical manifestations are acute respiratory distress syndrome. The occurrence and development of acute lung injury in sepsis can aggravate the functional damage of other organs and accelerate the progression of sepsis to septic shock, the latter of which has a mortality rate of up to 40%-60%. Therefore, exploring and developing new safe and effective strategies for the treatment of acute lung injury in sepsis remains a key scientific issue that needs to be urgently addressed in this field.

[0004] Macrophages are an important component of innate and adaptive immunity that leads to excessive activation of inflammation, and are also one of the most abundant immune cells in lung tissue. Macrophages participate in immune responses by secreting inflammatory factors, transducing signals, and other pathways. They play a key role in lung development, inflammatory response, and injury repair, and maintain lung pathophysiological homeostasis. The development of new therapies for excessive activation of macrophage inflammation is currently focused on multi-domain immunological data, including genomics, epigenomics, transcriptomics, and proteomics. Protein levels and biological activity are mainly affected by post-translational modifications. There is increasing evidence that deubiquitinating enzymes can target several inflammasomes by changing the stability of inflammasomes and protein-protein interactions, thereby affecting macrophage immune responses. Summary of the invention

[0005] COPS5 is a member of the JAMM deubiquitinase family. Cops5 gene deletion can reduce mortality, reduce weight loss, and improve septic inflammation in mice with cecal ligation and puncture (CLP)-induced sepsis. Mechanistically, COPS5 regulates the activation level of NLRP3 inflammasome by targeting K48 polyubiquitination of NLRP3. In addition, CSN5i-3, a selective inhibitor of COPS5, is expected to provide a new treatment strategy for patients with sepsis with excessive inflammatory activation.

[0006] The present invention analyzed the correlation between COPS5 expression in macrophages and acute lung injury in sepsis based on clinical samples, database analysis and transcriptomic sequencing of animal models, and further used gene-deficient mice to model and verify it. Then, cell and animal experiments were used to detect the promotion of COPS5 in macrophages on NLRP3 inflammasome activation, and finally the COPS5-specific inhibitor CSN5i-3 was used to verify the therapeutic effect of this target in acute lung injury in sepsis. The present invention systematically reveals for the first time the molecular mechanism of COPS5 on acute lung injury in sepsis by regulating the activation of NLRP3 inflammasomes in macrophages, providing a new target for the study and treatment of acute lung injury in sepsis, and COPS5 can be used as a therapeutic target for screening targeted drugs or treatment methods for the treatment of acute lung injury in sepsis.

[0007] When acute lung injury in sepsis occurs, NLRP3 assembles with ASC and caspase-1 into a multimeric protein complex called NLRP3 inflammasome. NLRP3 inflammasome activates caspase-1, which in turn secretes proinflammatory cytokines IL-1β and IL-18, triggering a cascade amplification of the inflammatory response, forming an inflammatory storm, and further aggravating the damage to the lung parenchymal cells in sepsis. At this time, the control of excessive inflammatory response is the key to the treatment of acute lung injury in sepsis. Ubiquitination modification is one of the key steps to prevent excessive activation of NLRP3. Therefore, by inhibiting the expression of the deubiquitinating enzyme COPS5, regulating the activation of macrophage inflammasomes, and alleviating the excessive immunity of acute lung injury in sepsis, the mortality rate of patients with acute lung injury in sepsis can be reduced.

[0008] The present invention is achieved through the following technical solutions:

[0009] Application of COPS5 in the preparation of drugs for detecting acute lung injury in sepsis.

[0010] Preferably, the COPS5 is the expression level of COPS5 in macrophages.

[0011] Application of COPS5 in the preparation of drugs for treating acute sepsis injury.

[0012] Preferably, the COPS5 is used to inhibit K48 ubiquitination-mediated proteasomal degradation of NLRP3.

[0013] Use of COPS5 inhibitors in the preparation of drugs for treating acute sepsis injury.

[0014] Preferably, the COPS5 inhibitor is CSN5i-3.

[0015] The present invention has but is not limited to the following beneficial effects:

[0016] The present invention constructs COPS5 mice to conduct research on acute septic lung injury. The experimental results show that reduced expression level of COPS5 can inhibit the activation level of macrophage inflammasomes, and plays a key role in the immune regulation of septic acute lung injury. The present invention systematically reveals for the first time the molecular mechanism of COPS5 signaling on septic acute lung injury by regulating macrophage inflammasomes, providing a new target for studying and treating septic acute lung injury, and COPS5 can be used as a therapeutic target for screening targeted drugs or treatment methods for treating septic acute lung injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to clearly demonstrate the specific implementation methods of the present invention and certain detection technologies used in the experiments, the implementation methods and the technologies used will be described below, mainly in the form of drawings.

[0018] Figure 1 This is the result of differential expression of transcriptomics in peripheral blood mononuclear cells of patients with sepsis and septic shock;

[0019] Figure 2 Flowchart for obtaining alveolar macrophages in the CLP-induced sepsis mouse model;

[0020] Figure 3 The results of transcriptomic expression differences of alveolar macrophages in mice of the Sham group and CLP group are shown in Figure 2.

[0021] Figure 4 The results show that the inflammatory factors in the bronchoalveolar lavage fluid of Cops5 gene-deficient septic mice were significantly lower than those in WT septic mice;

[0022] Figure 5 The results show that the inflammatory factors secreted by alveolar macrophages in Cops5 gene-deficient septic mice were significantly lower than those in WT septic mice;

[0023] Figure 6 H&E staining shows the results of pathological damage changes in the lung tissues of mice in each group;

[0024] Figure 7This is a confocal image showing the co-localization results of COPS5 and NLRP3 molecules;

[0025] Figure 8 The result of the energy resonance transfer experiment proves that COPS5 and NLRP3 interact with each other;

[0026] Fig. 9 This is the result diagram showing that COPS5 stabilizes NLRP3 protein levels;

[0027] Fig.10 The results show that COPS5 inhibits NLRP3 K48 ubiquitination-mediated proteasome degradation under inflammatory stimulation;

[0028] Fig.11 This is the result diagram showing that the COPS5-specific inhibitor CSN5i-3 reduces the protein level of NLRP3;

[0029] Fig.12 The results show that CSN5i-3 treatment reduced inflammation in septic mice.

[0030] Fig.13 The results show that the inflammation level of AMs in sepsis patients treated with CSN5i-3 was significantly reduced;

[0031] Fig.14 The levels of inflammatory factors in the bronchoalveolar lavage fluid of septic mice treated with CSN5i-3 were also significantly reduced.

[0032] Fig.15 The results showed that the pathological damage of lung tissue, mortality rate and weight loss level of septic mice in the treatment group were reduced. DETAILED DESCRIPTION

[0034] The specific implementation methods of the present invention are explained with the aid of examples. Except that the technology used for detection does not impose any form of limitation on the present invention, some schemes in the described embodiments belong to part of the embodiments of the invention. The embodiments obtained by ordinary technical operators in this field without creative results all belong to the protection scope of the present invention.

[0035] Example 1

[0036] The expression of COPS5 deubiquitinase is significantly increased in PBMCs of patients with sepsis

[0037] like Figure 1As shown, the heat map shows the differential expression of deubiquitinases in transcriptomic sequencing datasets of peripheral blood mononuclear cells from patients with sepsis and septic shock (GSE65682, GSE131761, GSE154918, GSE95233 and GSE57065). Among the 90 deubiquitinase mRNAs analyzed, 29 deubiquitinases were expressed in PBMCs of sepsis patients, including up- and down-regulation, compared with PBMCs of healthy controls. It is worth noting that COPS5 showed significant upregulation in different datasets.

[0038] Example 2

[0039] Obtaining alveolar macrophages from CLP-induced sepsis mouse model

[0040] To further investigate the roles of key deubiquitinase members in animal models of sepsis, e.g. Figure 2 As shown in the figure, we used transcriptomic sequencing RNA-seq analysis to detect the expression of deubiquitinase genes in alveolar macrophages (AMs) in the CLP-induced sepsis mouse model. We extracted bronchoalveolar lavage fluid from septic mice and sham-operated mice (5 mice in each group) 24 hours after CLP surgery, separated the alveolar macrophages of mice by centrifugation, extracted RNA using the TRizol method, and performed transcriptomic sequencing on the alveolar macrophages of mice in the Sham and CLP groups, and compared the differences in gene expression between the two groups to find key deubiquitinases.

[0041] Mouse bronchoalveolar lavage fluid:

[0042] 1. Preparation of alveolar lavage and cell suspension: Use ophthalmic scissors to cut a small hole between the tracheal rings, insert the hose of the intravenous scalp needle, tie it with silk thread and put the head high and feet low. Inject 1ml of 5% penicillin-streptomycin-PBS buffer solution precooled at 4℃, gently massage the chest cavity for 2 minutes, then lie flat and slowly withdraw the lavage fluid. Repeat the lavage 3 times to recover the lavage fluid. (ELISA detects the release of inflammatory factors in alveolar lavage fluid)

[0043] Mouse alveolar macrophage extraction

[0044] 1. Centrifuge the recovered alveolar lavage fluid at 4°C, 500g for 8 minutes. After removing the supernatant, add 1ml of red blood cell lysis buffer to resuspend the liquid, lyse on ice for 2 minutes, and centrifuge at 4°C, 500g for 8 minutes;

[0045] 2. Adherent culture: discard the supernatant, add 10% FBS-1% PS-1640 medium 1ml to resuspend the cells, mix each sample, adjust the cell concentration to 1×106 cells / ml, inoculate in a 12-well plate, aspirate the supernatant after 6 hours, and replace with 1% fetal bovine serum 1640 medium,

[0046] Continue to culture for 24 hours for later use.

[0047] Example 3

[0048] RNA-seq analysis showed that COPS5 expression was significantly upregulated in AMs of septic mice. Figure 3 As shown in the figure, Gene Ontology (GO) analysis showed that differentially expressed genes (DEGs) related to the inflammatory process were significantly enriched, including innate immune response and inflammatory response. Further volcano plot analysis found that 1,309 genes were upregulated in AMs of CLP-induced septic mice, including the key deubiquitinating enzymes Tnfaip3 and Cops5 that were upregulated in the human database.

[0049] Example 4

[0050] The inflammatory factors in bronchoalveolar lavage fluid of Cops5 gene-deficient septic mice were significantly decreased compared with WT septic mice

[0051] like Figure 4 As shown, ELISA was used to measure Cops5 + / + or Cops5 + / - The contents of inflammatory factors in bronchoalveolar lavage fluid (BALF) isolated from mice (7 mice in each group) 24 hours after CLP modeling. + / - 24 hours after the sepsis model was established in mice and WT mice, Cops5 + / - The levels of inflammatory factors IL-1β, IL-6, and TNF-α in bronchoalveolar lavage fluid (BALF) of septic mice were significantly lower than those of WT septic mice.

[0052] Example 3

[0053] The inflammatory factors secreted by alveolar macrophages in Cops5 gene-deficient septic mice were significantly decreased compared with those in WT septic mice. Figure 5 (The right figure is the specific ratio result of the plot) As shown in the figure, WT and Cops5 were extracted 24 hours after CLP modeling. + / - Alveolar macrophages from BALF of septic mice were cultured, and immunoblotting showed that Cops5 + / - AMs culture supernatant mature IL-1β release (the fourth bar, Cops5 + / - +CLP) compared with WT septic mouse AMs (the second bar, Cops5 + / + +CLP) decreased.

[0054] Supernatant protein precipitation experiment:

[0055] 1. Carefully pipette the supernatant of the corresponding stimulated cells into the EP tube, centrifuge at 1000 rpm for 5 minutes at 4 degrees, and then carefully pipette 400 μl (be careful not to suck up the cell debris at the bottom) and transfer it into a new EP tube;

[0056] 2. Add an equal volume (400 μl) of methanol to the EP tube, then add 0.25 times the volume (100 μl)

[0057] Chloroform, shake thoroughly, centrifuge at 15000 rpm for 15 minutes at 4 degrees;

[0058] 3. Use a pipette to carefully aspirate the upper and lower layer solutions, being careful not to touch the middle layer of protein precipitation, and continue to add an equal volume (400 μl) of methanol solution, shake thoroughly to wash the protein precipitation, and centrifuge at 15,000 rpm for 10 minutes at 4 degrees centrifuge;

[0059] 4. After absorbing as much liquid as possible from the surface of the precipitate, place the EP tube in a 65-degree oven with the cover open for 10 minutes to dry the precipitate to a translucent state;

[0060] 5. Dissolve the precipitate with 25 μl 2X loading and heat in a 100°C dry bath for 10 minutes.

[0061] Store in a refrigerator at 4°C for subsequent experiments.

[0062] Example 6

[0063] H&E staining showed the pathological changes of lung tissue in each group of mice

[0064] like Figure 6 As shown, hematoxylin and eosin (H&E) staining of the left lung tissue of Cops5+ / + or Cops5+ / - mice 24 hours after CLP modeling was observed under a light microscope. The scale bar is 200μm. The Mann-Whitney U test was used to analyze the significance of the acute lung injury score data of the lung tissue of 5 Cops5+ / + or Cops5+ / - mice. Histological examination showed that compared with WT sepsis mice, Cops5+ / -septic mice had reduced lung pathological inflammation, reduced pulmonary edema and inflammatory cell infiltration, emphasizing the potential role of COPS5 in CLP-induced sepsis, and proving that Cops5 gene deletion attenuated the inflammatory response in the cell and mouse models of CLP-induced sepsis.

[0065] Example 7

[0066] Confocal microscopy showed colocalization of COPS5 and NLRP3 molecules

[0067] like Figure 7As shown, confocal microscopy images show that HeLa cells were transfected with NLRP3-mcherry for 24 hours and treated or not with LPS (100 ng / mL) for 3 hours (26 cells in each group), and NLRP3 (red) and COPS5 (green) colocalized. Scale bar, 10 μm. Pearson coefficient statistical results show that there is a lot of colocalization (yellow) between mcherry-labeled NLRP3 (red) and endogenous COPS5 (green), and LPS stimulation further enhances this colocalization. Confocal experiment:

[0068] 1. Transfect Mcherry-NLRP3 fluorescent plasmid 24 hours before transfection;

[0069] 2. 24 hours after transfection, the culture medium was removed, and then 1 ml of 4% paraformaldehyde, 1 ml of -20°C pre-cooled methanol, and 6% goat serum (about 200 ul) were used for fixation, cell permeabilization and blocking.

[0070] 3. Wash the cells three times, dilute the anti-COPS5 antibody (1:100) with blocking buffer, and incubate with fluorescently labeled secondary antibodies (Alexa Fluor 488-conjugated antibodies against mouse) at room temperature in the dark for 1 hour;

[0071] 4. Remove the secondary antibody and wash the cells 3 times with 4°C pre-cooled PBS. The samples can be used immediately or stored at 4°C in the dark.

[0072] 5. Confocal images were acquired using a Leica TCS-SP8 STED 3X confocal microscope equipped with a 100×1.40NA oil objective and analyzed using Leica Application Suite Advanced Fluorescence (LAS AF, Version 4.2) software. Colocalization was quantified using ImageJ software using the Pearson correlation coefficient, ranging from -1 to 1.

[0074] Example 8

[0075] Energy resonance transfer experiments have shown that COPS5 and NLRP3 interact with each other. Figure 8As shown, HEK293T cells were transfected with a plasmid expressing CFP-COPS5 as a FRET donor and YFP-NLRP3 as a FRET acceptor for 24 hours, and then treated with LPS (100 ng / mL) for 3 hours to detect the interaction between COPS5 and NLRP3 (18 cells per group). The results showed that the energy transfer from the FRET donor CFP-COPS5 to the FRET acceptor YFP-NLRP3 was within 10 nm, and this energy transfer was significantly enhanced with LPS stimulation. This experimental result further supports the co-localization and interaction between NLRP3-COPS5.

[0076] Fluorescence energy transfer resonance analysis (FRET analysis)

[0077] 1. 12 hours before transfection, 5×104 HEK293T cells were seeded in a 35 mm glass bottom culture dish and transfected as indicated;

[0078] 2. 24 hours after transfection, remove the culture medium and wash the cells 3 times with 4°C pre-cooled PBS on a shaker for 5 minutes each time. Then, fix each dish with 1 ml of 4% paraformaldehyde in a 4°C refrigerator for 5 minutes.

[0079] 3. Repeat washing the cells three times with 4°C pre-cooled PBS;

[0080] 4. Fluorescence resonance energy transfer (FRET) analysis was performed using the Leica software FRET SE (sensitized emission) on a Leica TCS SP8 confocal microscope system (λex = 405 nm, CFP acquisition at 462-510 nm, YFP acquisition at 518-580 nm);

[0081] 5. Under 405nm excitation, FRET is calculated as the ratio of YFP to CFP intensity.

[0082] The efficiency of (EFRET) was calculated by EFRET = TDA / TD, where TDA is the measurement of the donor intensity value in the presence of the acceptor and TD is the donor intensity value alone. Data analysis was performed using GraphPad Prism version 8.0.

[0083] Example 9

[0084] COPS5 stabilizes NLRP3 protein levels

[0085] like Fig. 9As shown in a, HEK293T cells were transfected with Flag-NLRP3 and different doses of Myc-COPS5 (0, 100ng, 300ng, and 600ng) for 24 hours. Cell lysates were collected for immunoblot analysis. We found that increasing doses of exogenous COPS5 resulted in a dose-dependent increase in NLRP3 protein levels.

[0086] like Fig. 9 As shown in b, THP-1-derived human macrophages and mouse macrophage BMDMs were transfected with control siRNA or COPS5 siRNA for 48 hours, and cell lysates were collected for immunoblot analysis. We found that the decrease in COPS5 protein content in both human and mouse macrophages led to a decrease in NLRP3 protein levels, demonstrating that COPS5 has a stabilizing effect on NLRP3.

[0087] Example 10

[0088] COPS5 inhibits NLRP3 K48 ubiquitination-mediated proteasomal degradation under inflammatory stimulation

[0089] like Fig.10 As shown in the figure, since different types of polyubiquitin linkages mediate the various biological functions of NLRP3, we investigated the effects of COPS5 on different polyubiquitin chains of NLRP3. Cells treated with MG132 (to inhibit the degradation pathway of NLRP3) were co-transfected with HA-tagged WT ubiquitin (HA-Ub), HA-tagged K48-linked ubiquitin (K48-Ub), HA-tagged K63-linked ubiquitin (K63-Ub), Myc-COPS5, and Flag-NLRP3 plasmids (a). HEK293T cells (b) were transfected with control siRNA or COPS5 siRNA for 48 hours, then treated with MG132 for 6 hours, followed by transfection with HA-tagged K48 ubiquitin (K48-Ub) and Flag-NLRP3 plasmids. THP-1-derived macrophages (b) were transfected with control siRNA or COPS5 siRNA for 48 hours, then treated with MG132 for 6 hours, with or without LPS treatment (100 ng / mL, 3 hours). Cell lysates in (a) and (b) were immunoblotted and immunoprecipitated with anti-Flagbeads, followed by probing with HA antibody. Cell lysates in (c) were immunoprecipitated with NLRP3 and probed with anti-K48 ubiquitin antibody.

[0090] Co-immunoprecipitation results showed that COPS5 overexpression significantly reduced K48-related NLRP3 polyubiquitination without affecting other types of ubiquitination ( Fig.10 a, 5 is less than 4). In contrast, knockdown of COPS5 significantly increased K48-associated NLRP3 polyubiquitination ( Fig.10 b, 3 is greater than 2). Analysis of endogenous ubiquitination of NLRP3 showed that COPS5 knockdown enhanced endogenous K48-associated NLRP3 ubiquitination ( Fig.10 c, 4 is greater than 3). These results together demonstrate that COPS5 specifically inhibits K48 polyubiquitination of NLRP3.

[0091] Embodiment 11

[0092] The COPS5-specific inhibitor CSN5i-3 effectively enhanced K48 ubiquitination of NLRP3 in macrophages and reduced the protein level of NLRP3

[0093] like Fig.11 As shown, THP-1-derived macrophages were pretreated with CSN5i-3 (1 μM) for 2 hours, and then stimulated with ATP (5 mM) for 3 hours in the presence of MG132 (10 μM, 6 hours) under the initiation of LPS (100 ng / mL, 3 hours). Cell lysates were immunoprecipitated with anti-NLRP3 antibodies and ABE beads, and ubiquitination levels were detected by immunoblotting. We found that after CSN5i-3 intervention in macrophages stimulated by LPS and ATP, the K48 polyubiquitination level of NLRP3 protein was significantly increased (5 greater than 4), which is consistent with the results of the previous COPS5 knockdown experiment.

[0094] CSN5i-3 is a potent, selective and oral CSN5 / Jab1 inhibitor that inhibits CSN-catalyzed Cul1 deneddylation with an IC50 value of 5.8 nM. CSN5i-3 has anticancer activity, inhibits cell proliferation and induces apoptosis in human breast cancer cells. It is commercially available under the product number HY-112134 (MCE).

[0095] CSN5i-3 has been shown to reduce osteosarcoma (OS) cell viability and has specific effects on the ubiquitin-proteasome system of OS. It can also significantly alter the activity of multiple signaling pathways, regulate the expression of specific CRLs in chondrosarcoma cells, and induce apoptosis and G1 cell cycle arrest in breast cancer cells and serous ovarian cancer (SOC) cells.

[0096] CSN5i-3 Reference: CSN5i-3shows a goodpharmacokinetic profile.Treatmentwith CSN5i-3triggers the formation of cleaved PARP and cleaved caspase3indicative of apoptosis induction. (SchlierfA, et al. Targeted inhibition of the COP9 signalosome for treatment of cancer. Nat Commun. 2016 Oct 24; 7: 13166.).

[0097] Example 12

[0098] RNA-seq analysis of AMs in septic mice treated with CSN5i-3 showed a significant decrease in inflammation compared with the placebo group

[0099] Fig.12 As shown in the figure, given the effect of CSN5i-3 on the NLRP3 inflammatory pathway in cells, we verified its effect at the animal experiment level. We injected DMSO (control group) and CSN5i-3 (experimental group) into mice of appropriate age, and 2 hours later, the two groups of mice were subjected to cecal ligation and puncture to induce sepsis. 24 hours later, the alveolar macrophages of the mice were collected by extracting the alveolar lavage fluid of the mice, and the RNA was extracted and sent for transcriptomic sequencing.

[0100] The scatter plot shows the FPKM (Fragments Per Kilobase Million) values ​​and Gene Ontology (GO) annotations of modified genes in AMs of septic mice 24 hours after CLP after pretreatment with placebo or CSN5i-3 (1 mmol / kg). NT represents untreated samples. Enriched pathways such as immune system processes, protein ubiquitination, and inflammatory response are marked in red.

[0101] Compared with the control group, the expression of pro-inflammatory cytokines (such as Cxcl3, Cxcl5, Il-18, etc.) in alveolar macrophages of septic mice treated with CSN5i-3 was significantly downregulated. At the same time, GO analysis showed that inhibitor treatment enriched the immune system process at the cellular transcription level, and the inflammatory response and protein ubiquitination pathway proteins were significantly changed.

[0102] Embodiment 13

[0103] The inflammation level of AMs in sepsis patients treated with CSN5i-3 was significantly reduced

[0104] As shown in 13, alveolar macrophages from healthy controls (HC) and sepsis patients (n = 3 in each group) were collected and plated, pretreated with CSN5i-3 (1 μM) for 2 h, and then the supernatant was changed to RPMI 1640 medium and cultured for 8 h. PBMC IL-1β expression was evaluated by qPCR analysis ( Fig.13 a). Collect cell supernatant and detect IL-1β release level by ELISA ( Fig.13 b). The results of qPCR experiments showed that the level of inflammation in the treated cells was reduced, and the level of mature IL-1β in the cell supernatant measured by ELISA was significantly reduced by CSN5i-3, indicating that CSN5i-3 has an anti-inflammatory effect on human and mouse alveolar macrophages by targeting NLRP3.

[0105] Embodiment 14

[0106] The levels of inflammatory factors in bronchoalveolar lavage fluid of septic mice treated with CSN5i-3 were also significantly reduced.

[0107] like Fig.14 As shown, in order to further test the therapeutic effect of CSN5i-3 in sepsis, we performed CLP modeling on mice pretreated with CSN5i-3 or DMSO. The results of ELISA analysis of the bronchoalveolar lavage fluid of mice extracted 24 hours after CLP modeling showed that the level of inflammatory factor IL-1B in the bronchoalveolar lavage fluid of mice treated with CSN5I-3 was significantly lower than that of the placebo group, proving the therapeutic effect of CSN5I-3 in vivo.

[0108] Embodiment 15

[0109] H&E staining showed that the pathological damage of lung tissues, mortality and weight loss in septic mice in the treatment group were reduced.

[0110] like Fig.15 As shown, H&E staining of mouse lung tissue was performed 24 hours after CLP modeling, and lung damage was observed under light microscopy (a). Scale bar: 200 μm. Weight loss (b) and survival rate data (c) of CLP-induced septic mice (n=9 in each group) treated with DMSO or CSN5i-3 (1 mmol / kg). We found that compared with wild-type (WT) mice, lung tissue damage in septic mice treated with CSN5i-3 was significantly alleviated (a). Continuous observation showed that the survival rate after cecal ligation and puncture was significantly improved (b), and the degree of weight loss was significantly reduced (c), indicating that CSN5i-3 can effectively inhibit the lung and systemic inflammatory response of CLP-induced septic mice, suggesting its therapeutic potential for diseases related to NLRP3 overactivation such as acute lung injury in sepsis.

Claims

1. Application of COPS5 in the preparation of drugs for detecting acute lung injury in sepsis.

2. The use according to claim 1, characterized in that The COPS5 is the expression level of COPS5 in macrophages.

3. Application of COPS5 in the preparation of drugs for treating acute sepsis injury.

4. The use according to claim 3, characterized in that The COPS5 is used to inhibit the K48 ubiquitination-mediated proteasomal degradation of NLRP3.

5. Use of COPS5 inhibitors in the preparation of drugs for treating acute sepsis-induced injuries.

6. The use according to claim 5, characterized in that The COPS5 inhibitor is CSN5i-3.

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