Application of LAP3 and agonist thereof in macrophage immune inflammation mediated diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an application of LAP3 and an agonist thereof in macrophage immune inflammation mediated diseases. The invention provides application of leucine aminopeptidase 3 or leucine aminopeptidase 3 agonist substances in preparation of drugs for prevention, treatment and / or adjuvant treatment of macrophage immune inflammation mediated diseases. According to the invention, it is found for the first time that the expression level of LAP3 in macrophages is increased under an acute stress condition, and the limiting effect of LAP3 on generation and development of sepsis and acute hepatitis is disclosed; the invention discloses that the knockout of the LAP3 can remodel a macrophage metabolic network, reduce oxidative phosphorylation, promote ROS (reactive oxygen species) production and activate TAK1, thereby promoting the activation of a downstream inflammation signal channel and finally promoting macrophage inflammation. A new target spot and a theoretical basis can be provided for clinical diagnosis and treatment of macrophage-mediated inflammation by illuminating the effect of LAP3 on regulating and controlling macrophage inflammation and a molecular mechanism of LAP3.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of LAP3 and its agonists in macrophage immune inflammation-mediated diseases. Background Art

[0002] Macrophages (Mφ) play a crucial role in the human immune system. They are not only an important line of innate immunity but also core cells for antigen presentation, with high plasticity. In a complex and variable microenvironment, macrophages can sensitively recognize various danger signals and then initiate innate immune responses. Among them, polarization is a key mechanism for macrophages to exert their immune regulatory functions. Under different stimuli, macrophages can polarize into multiple phenotypes with different functions. Currently, the relatively well-studied ones are the M1 type (inflammatory or classically activated type) and the M2 type (healing or alternatively activated type).

[0003] The inflammatory response is a self-protection mechanism of the host in the face of infection and tissue damage. In the initial or acute stage of inflammation, macrophages or mast cells in tissues will quickly recognize pathogen-associated molecular patterns (PAMPs), and then activate the secretion of a series of pro-inflammatory substances, such as pro-inflammatory cytokines, chemokines, vasoactive amines, and prostaglandins. The release of these substances can enhance the immune response, increase vascular permeability, and cause a large amount of plasma rich in soluble components such as antibodies to pour into the damaged area. At the same time, a variety of signal molecules released at the injury site will recruit neutrophils and monocytes. As the inflammation develops, monocytes and lymphocytes continue to accumulate at the inflammation site to assist in neutralizing harmful substances. Subsequently, apoptotic inflammatory cells are cleared by macrophages. However, the inflammatory response needs to be maintained at a reasonable level. Once it is excessive, too low, or lasts for too long, serious consequences may occur. Diseases such as sepsis, acute hepatitis, and even chronic inflammation-related liver cancer are all related to it.

[0004] Sepsis, as a systemic inflammatory response syndrome (SIRS) caused by infection, has a very complex pathogenesis. The imbalance of the inflammatory response runs through the entire course of sepsis. Under pathogen stimulation, the body usually goes through three main states: first, the overactivation of inflammatory cells leads to the imbalance between pro-inflammatory and anti-inflammatory responses, triggering an out-of-control inflammatory cascade reaction and the release of a large number of pro-inflammatory mediators, which is a key factor for sepsis to cause multiple organ dysfunction; if the condition is not controlled in time, it will enter the stage of immune disorder, that is, a mixed immune state; finally, as the condition worsens, the body's immunity turns into an inhibitory state, greatly increasing the risk of repeated infections. Therefore, regulating the inflammatory response is crucial for alleviating organ damage in sepsis. In-depth study of the macrophage polarization regulation mechanism at different stages of sepsis helps to maintain immune homeostasis and treat complications.

[0005] Liver diseases are also a group of diseases affected by multiple factors. Drugs, chemical agents, viral infections, excessive alcohol consumption, etc. can all trigger them. Liver injury, especially acute liver injury (ALI), has a relatively high mortality rate. The pathogenesis of ALI involves processes such as hepatocyte apoptosis and necrosis, immune homeostasis disorder, oxidative stress, and autophagy. Long-term liver injury may also develop into liver fibrosis, cirrhosis, and even hepatocellular carcinoma. In acute liver injury, immune cells infiltrate the liver. As a key component of innate immunity, macrophages play an important role in coordinating the inflammatory response. Studies have found that regulating macrophage polarization is expected to become a potential strategy for treating liver diseases.

[0006] The polarization process of macrophages is regulated by multiple factors. On the one hand, pathogen-sensing families in the innate immune system, such as Toll-like receptors (TLRs), NOD-like receptors (NLRs), and retinoic acid-inducible gene-like receptors (RLRs), will activate intracellular signaling pathways after binding to pathogens or endogenous risk factors, prompting the release of a large number of pro-inflammatory mediators. On the other hand, macrophage polarization is closely linked to metabolic pathway reprogramming. M1 macrophages mainly rely on glycolysis and the pentose phosphate pathway to obtain ATP, while oxidative phosphorylation and fatty acid oxidation are inhibited; M2 macrophages maintain metabolic activity by enhancing fatty acid oxidation and oxidative phosphorylation. Metabolic differences significantly affect the ability of M1 and M2 macrophages to generate reactive oxygen species (ROS), and ROS are involved in cell signal regulation and the regulation of various macrophage functions.

[0007] Leucine Aminopeptidase 3 (LAP3) belongs to the aminopeptidase family, participates in protein degradation and immune function regulation, can catalyze the removal of N-terminal amino acids, and also plays a role in glutathione metabolism. It is speculated that it has an impact on the cellular redox state. Currently, the research on LAP3 mainly focuses on tumor proliferation and invasion. Its expression has also been found to be upregulated in chronic inflammatory diseases and immune-related diseases, but its role and molecular mechanism in macrophage-mediated immune inflammation are still unclear and urgently need to be explored in depth, which will provide important clues for revealing the pathogenesis of immune inflammation and developing new treatment methods. Summary of the Invention

[0008] In view of the above deficiencies, the present invention provides the application of LAP3 and its agonists in macrophage immune inflammation-mediated diseases. The present invention provides the application of leucine aminopeptidase 3 or a leucine aminopeptidase 3 agonist in the preparation of a medicament for preventing, treating and / or adjuvantly treating macrophage immune inflammation-mediated diseases. The present invention discovers for the first time that the expression level of LAP3 in macrophages increases under acute stress conditions, revealing the restrictive effect of LAP3 on the occurrence and development of sepsis and acute hepatitis; it reveals that knocking out LAP3 can reshape the macrophage metabolic network, reduce oxidative phosphorylation, promote the production of ROS, activate TAK1, and further promote the activation of downstream inflammatory signaling pathways, ultimately promoting macrophage inflammation. By clarifying the role and molecular mechanism of LAP3 in regulating macrophage inflammation, new targets and theoretical bases can be provided for the clinical diagnosis and treatment of macrophage-mediated inflammation.

[0009] The technical solution of the present invention is as follows: On the one hand, the present invention provides the application of leucine aminopeptidase 3 (LAP3) or a leucine aminopeptidase 3 agonist in the preparation of a medicament for preventing, treating and / or adjuvantly treating macrophage immune inflammation-mediated diseases.

[0010] Specifically, the macrophage immune inflammation-mediated diseases include: one or more of systemic inflammatory response syndrome, acute hepatitis, and chronic inflammation-related liver diseases.

[0011] Preferably, the systemic inflammatory response syndrome includes, but is not limited to: one or more of sepsis, septic shock, multiple organ dysfunction syndrome, and disseminated intravascular coagulation.

[0012] More preferably, the systemic inflammatory response syndrome is sepsis.

[0013] Preferably, the acute hepatitis includes, but is not limited to: one or more of viral hepatitis, drug-induced liver injury, autoimmune hepatitis, and toxic hepatitis.

[0014] Preferably, the chronic inflammation-related liver diseases include, but are not limited to: one or more of chronic viral hepatitis, autoimmune liver diseases, alcoholic liver diseases, and non-alcoholic fatty liver diseases.

[0015] Preferably, the leucine aminopeptidase 3 agonist includes, but is not limited to: one or more of small molecule agonists, small molecule compounds, macromolecular compounds, bioactive peptides, antibody fragments, and nucleic acid aptamers.

[0016] Preferably, the medicament exerts its effect through any of the following aspects: (1) By inhibiting the TLR4-NF-κB signaling pathway, thereby inhibiting the expression level of macrophage inflammatory factors; Or (2) by inhibiting the activation of TAK1 / TAB1, inhibiting macrophage-mediated inflammatory responses; Or (3) by enhancing its own enzyme activity, inhibiting macrophage-mediated inflammatory responses; Or (4) by inhibiting glycolysis, promoting oxidative phosphorylation, inhibiting ECAR, and promoting OCR; Or (5) by inhibiting the remodeling of the macrophage metabolic network; Or (6) maintaining mitochondrial function to inhibit ROS activation.

[0017] Specifically, the dosage form of the drug includes administration via the gastrointestinal tract or parenteral administration.

[0018] Preferably, the dosage form of the drug includes, but is not limited to, one or more of tablets, pills, powders, suspensions, gels, emulsions, creams, granules, capsules, suppositories, injections, sprays.

[0019] Specifically, the drug further includes pharmaceutically acceptable excipients.

[0020] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, one or a combination of two or more of wetting agents, emulsifiers, preservatives, antioxidants, buffers, excipients, diluents, lubricants, bacteriostatic agents, solutes that make the preparation isotonic with the recipient's blood, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners, and fragrances.

[0021] On the other hand, the present invention provides the use of leucine aminopeptidase 3 in the preparation of a diagnostic product for macrophage immune inflammation-mediated diseases.

[0022] Specifically, the diagnostic product for macrophage immune inflammation-mediated diseases includes reagents, reagent kits, chips, or detection systems.

[0023] Specifically, the diagnostic product for macrophage immune inflammation-mediated diseases determines the progression of macrophage immune inflammation-mediated diseases by detecting the expression level of leucine aminopeptidase 3 in a sample.

[0024] Preferably, the expression level of leucine aminopeptidase 3 is negatively correlated with macrophage immune inflammation-mediated diseases.

[0025] The beneficial effects of the present invention are as follows: (1) The present invention has for the first time demonstrated the regulatory role of leucine aminopeptidase LAP3 in macrophage-mediated inflammatory responses. It has determined that the expression level of LAP3 in macrophages increases under acute stress conditions, revealing the restrictive effect of LAP3 on the occurrence and development of sepsis and acute hepatitis.

[0026] (2) The present invention discloses the "metabolism-natural immunity" molecular mechanism by which LAP3 regulates macrophage inflammation. Knocking out LAP3 remodels the metabolic network of macrophages, reduces oxidative phosphorylation, promotes the production of ROS, activates TAK1, and further promotes the activation of downstream inflammatory signaling pathways, and ultimately promotes macrophage inflammation.

[0027] (3) The present invention explores the regulatory effect of LAP3 on macrophage inflammation from multiple aspects such as cells, animals, and molecules. By clarifying the role and molecular mechanism of LAP3 in regulating macrophage inflammation, it can provide new targets and theoretical basis for the clinical diagnosis and treatment of macrophage-mediated inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 LAP3 expression is upregulated when macrophages are treated with inflammatory stimuli in vitro; in the figure, A shows the mRNA expression levels of Lap3 and inflammatory factors Il-6, Il-1β in wild-type PMs after stimulation with LPS (200 ng / ml) for 0 - 24 h detected by RT-PCR; B shows the protein levels of LAP3 and inflammatory factor IL-1β in wild-type PMs after stimulation with LPS (200 ng / ml) for 0 - 24 h detected by WB; C shows the mRNA expression levels of Lap3 in wild-type PMs after stimulation with Poly(I:C) (20 μg / ml) for 12 h detected by RT-PCR; D shows the mRNA expression levels of Lap3 in wild-type PMs after stimulation with CpG (6 μg / ml) for 12 h; the values are expressed as mean ± SEM, **p < 0.01, ***p < 0.001, two-tailed Student's t-test.

[0029] Figure 2 LAP3 expression is upregulated under inflammatory stimulation in vivo; in the figure, A shows wild-type mice intraperitoneally injected with PBS or E.coli , and after 6 h, bone marrow cells of the mice were extracted, and the mRNA expression levels of Lap3 were detected by RT-PCR (n = 7 - 9); B shows the collection of blood from healthy volunteers, SIRS patients, and sepsis patients, extraction of PBMC, and detection of the mRNA expression levels of LAP3 by RT-PCR (n = 21 - 28); the values are expressed as mean ± SEM, **p < 0.01, ***p < 0.001, two-tailed Student's t-test.

[0030] Figure 3LPS promotes the expression of LAP3 by activating the TLR4-NF-κB pathway; in the figure, A shows the prediction of several potential NF-κB protein binding sites in the promoter region of the LAP3 gene by sequence analysis using the TSEARCH program; B-D show that in PMs, after transfection with siControl (siCtrl) or siMyd88 (B), siTrif (C), and siP65 (D), and stimulation with 200 ng / ml LPS for 6 h, the corresponding knockdown efficiency in cells and Lap3 and inflammatory factors Il-6 mRNA expression levels were detected by RT-PCR (n = 3); the values are expressed as mean ± SEM, **p<0.01, ***p<0.001, two-tailed Student's t-test.

[0031] Figure 4 LAP3 inhibits the expression levels of LPS-induced macrophage inflammatory factors; in the figure, A and B show that in RAW264.7, after transfection with siControl (siCtrl) or siLAP3, and stimulation with 200 ng / ml LPS for 6 h, the Lap3 knockdown efficiency in cells and Il-6, Il-1β and Ifn-β mRNA expression levels were detected by RT-PCR; C and D show that in RAW264.7, after transfection with GFP-Flag or LAP3-Flag plasmids, and stimulation with 200 ng / ml LPS for 6 h, the overexpression efficiency of LAP3 in cells was detected by WB and the Il-6, Il-1β and Tnf-α mRNA expression levels were detected by RT-PCR; the values are expressed as mean ± SEM, ***p<0.001, two-tailed Student's t-test.

[0032] Figure 5 Construction and verification of LAP3 myeloid-specific knockout mice; in the figure, A shows the breeding strategy of the Lap3 gene with Lyz2 -cre. Two loxP sites are located upstream of exon 2 and downstream of exon 4 of the Lap3 gene respectively. By crossing with Lyz2 -cre mice, the sequences of exons 2-4 were knocked out to generate myeloid-specific knockout mice; B shows the breeding scheme of LAP3-KO-Mφ and LAP3-WT-Mφ mice; C shows the genotype identification by PCR using the DNA of mouse toes or tails as templates; D shows the knockout efficiency of LAP3 in cells detected by RT-PCR and WB; the values are expressed as mean ± SEM, ***p<0.001, two-tailed Student's t-test.

[0033] Figure 6Myeloid-specific knockout of LAP3 does not affect the proliferation and differentiation of macrophages; in the figure, A shows the number of cells singly positive for F4 / 80 + (left panel of A) and CD11b + (right panel of A) in peritoneal cells of LAP3-WT-Mφ and LAP3-KO-Mφ mice detected by flow cytometry; B shows the proportion of macrophages double positive for F4 / 80 + and CD11b + in peritoneal cells of LAP3-WT-Mφ and LAP3-KO-Mφ mice detected by flow cytometry; C shows the cell count of macrophages double positive for F4 / 80 + and CD11b + in peritoneal cells of LAP3-WT-Mφ and LAP3-KO-Mφ mice detected by flow cytometry; n = 3; values are presented as mean ± SEM, NS indicates no statistical difference, two-tailed Student's t-test.

[0034] Figure 7 Myeloid-specific knockout of LAP3 affects multiple signal transduction pathways such as immunity, inflammation, and cell metabolism; specifically, RNA-seq analysis was performed to screen for differentially expressed genes between LAP3-WT-Mφ and LAP3-KO-Mφ and KEGG pathway enrichment analysis was carried out.

[0035] Figure 8 Myeloid-specific knockout of LAP3 promotes the inflammatory response mediated by the TLR4 pathway; in the figure, A and B show the knockout efficiency and the mRNA expression levels of Lap3 and Il-6, Il-1β, Nos2 and Tnf-α detected by RT-PCR in LAP3-WT-Mφ and LAP3-KO-Mφ PMs after stimulation with 200 ng / ml LPS for 6 h; C and D show the knockout efficiency and the mRNA expression levels of Lap3 and Il-6, Il-1β, Nos2 and Tnf-α detected by RT-PCR in LAP3-WT-Mφ and LAP3-KO-Mφ BMDMs after stimulation with 200 ng / ml LPS for 6 h; n = 3; values are presented as mean ± SEM, **p<0.01, ***p<0.001, two-tailed Student's t-test.

[0036] Figure 9 Myeloid-specific knockout of LAP3 promotes the inflammatory response mediated by the TLR3 / TLR9 pathway; in the figure, A shows the mRNA expression levels ofIl-6, Il-1β and Ifn-β mRNA expression levels; B shows the intracellular Il-1β, Nos2 and Ifn-β mRNA expression levels detected by RT-PCR after 12 h of stimulation with 20 μg / ml Poly(I:C) in LAP3-WT-Mφ and LAP3-KO-Mφ BMDMs; C shows the intracellular Il-6, Il-1β and Tnf-α mRNA expression levels detected by RT-PCR after 12 h of stimulation with 6 μg / ml CpG in LAP3-WT-Mφ and LAP3-KO-Mφ PMs; n = 3; values are expressed as mean ± SEM, *p<0.05, **p<0.01, two-tailed Student's t-test.

[0037] Figure 10 Systemic knockout of LAP3 exacerbated the systemic response in septic mice; A in the figure shows the survival rates of wild-type and LAP3 systemic knockout mice after intraperitoneal injection of LPS (25 mg / kg) (n = 11-12), Mantel-Cox test; B shows the collection of ocular blood from wild-type and LAP3 systemic knockout mice 6 h after intraperitoneal injection of LPS (25 mg / kg), and the secretion of inflammatory factors IL-6, IL-1β, and TNF-α in the serum was detected by ELISA, n = 6, two-tailed Student's t-test; values are expressed as mean ± SEM, *p<0.05, ***p<0.001.

[0038] Figure 11 Systemic knockout of LAP3 exacerbated organ damage in septic mice; A-C in the figure show the isolation of lung tissues from wild-type and LAP3 systemic knockout mice 6 h after intraperitoneal injection of LPS (25 mg / kg). A shows the detection of the lung wet / dry weight ratio of the mice, B shows the evaluation of the degree of pulmonary inflammatory infiltration by H&E staining (scale bar: 100 μm), and C shows the detection of the mRNA expression levels of cytokines and chemokines in the lung tissues by RT-PCR; D-E show the collection of ocular blood from wild-type and LAP3 systemic knockout mice 6 h after intraperitoneal injection of LPS (25 mg / kg). D shows the detection of the expression levels of AST and ALT in the blood by an enzyme-linked immunosorbent assay, and E shows the detection of the expression levels of CRE and Urea in the blood by an enzyme-linked immunosorbent assay; values are expressed as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed Student's t-test.

[0039] Figure 12 Myeloid-specific knockout of LAP3 exacerbated E.coliSepsis in mice induced by CLP or LPS; A-B in the figure show the survival rates of LAP3-WT-Mφ and LAP3-KO-Mφ mice after intraperitoneal injection E.coli of saline or LPS (25 mg / kg) (n = 11-15); C shows the collection of orbital blood from mice 6 h after intraperitoneal injection of LPS (25 mg / kg) in LAP3-WT-Mφ and LAP3-KO-Mφ mice, and the secretion of inflammatory factors IL-6, IL-1β, and TNF-α in serum was detected by ELISA (n = 3); D-E show the isolation of lung tissues from LAP3-WT-Mφ and LAP3-KO-Mφ mice 6 h after intraperitoneal injection of LPS (25 mg / kg). D shows the evaluation of the degree of pulmonary inflammatory infiltration by H&E staining (scale bar: 100 μm), and E shows the detection of the mRNA expression levels of cytokines and chemokines in lung tissues by RT-PCR (n = 3); Mantel-Cox test (A and B); values are expressed as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed Student's t test (C and E).

[0040] Figure 13 Myeloid-specific knockout of LAP3 exacerbated sepsis in mice induced by CLP; A in the figure shows the survival rates of LAP3-WT-Mφ and LAP3-KO-Mφ mice after CLP surgery (n = 11); B shows the collection of orbital blood from mice 20 h after CLP surgery in LAP3-WT-Mφ and LAP3-KO-Mφ mice, and the secretion of inflammatory factors IL-6, IL-1β, and TNF-α in serum was detected by ELISA (n = 3); C shows the isolation of lung tissues from LAP3-WT-Mφ and LAP3-KO-Mφ mice 20 h after CLP surgery, and the degree of pulmonary inflammatory infiltration was evaluated by H&E staining (scale bar: 100 μm); D shows the isolation of lungs, livers, kidneys, and spleens from LAP3-WT-Mφ and LAP3-KO-Mφ mice 20 h after CLP surgery, and the mRNA expression levels of cytokines Il-6 in each tissue were detected by RT-PCR (n = 3); Mantel-Cox test (A); values are expressed as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed Student's t test (B and D).

[0041] Figure 14Myeloid-specific knockout of LAP3 exacerbated CCl4-induced acute hepatitis; in the figure, A shows the collection of ocular blood from LAP3-WT-Mφ and LAP3-KO-Mφ mice 48 h after intraperitoneal injection of CCl4, and the expression levels of AST and ALT in the blood were detected by an enzyme-linked immunosorbent assay (ELISA); B-C show that 48 h after intraperitoneal injection of CCl4 in LAP3-WT-Mφ and LAP3-KO-Mφ mice, part of the liver tissue was isolated. In B, HE staining was used to evaluate the degree of liver inflammatory infiltration (scale bar: 100 μm), and in C, RT-PCR was used to detect the mRNA expression levels of cytokines and chemokines in the liver tissue; the values are expressed as mean ± SEM, *p<0.05, **p<0.01, two-tailed Student's t-test.

[0042] Figure 15 Myeloid-specific knockout of LAP3 increased macrophage inflammatory infiltration in the liver tissue in CCl4-induced acute hepatitis; in the figure, A and B show that 48 h after intraperitoneal injection of CCl4 in LAP3-WT-Mφ and LAP3-KO-Mφ mice, part of the liver tissue was isolated, and immunofluorescence staining was used to evaluate the proportion of inflammatory macrophages in the liver. In A, it is IL-6 + Macrophage infiltration ratio, and in B, it is IL-1β + Macrophage infiltration ratio. The values are expressed as mean ± SEM, **p<0.01, ***p<0.001, two-tailed Student's t-test.

[0043] Figure 16 Myeloid-specific knockout of LAP3 led to an increase in the number and volume of tumors in chronic inflammation-related liver cancer; in the figure, A shows the protocol for the DEN and CCl4-induced chronic inflammation-related liver cancer model. LAP3-WT-Mφ and LAP3-KO-Mφ mice were used, and DEN (25 mg / kg) was intraperitoneally injected once every two weeks for a total of two times. Six weeks later, CCl4 (0.5 ml / kg, dissolved in corn oil) was intraperitoneally injected once a week for 12 weeks. At the 30th week, the mice were euthanized, and serum and liver tissue were collected for biochemical and histological analysis; B shows the tumor situation in the livers of the mice 30 weeks after induction; C shows the liver weight / body weight ratio of LAP3-WT-Mφ and LAP3-KO-Mφ mice (n = 6); D shows the measurement of the number, maximum size, and average size of tumors in the livers of LAP3-WT-Mφ and LAP3-KO-Mφ mice (n = 6); the values are expressed as mean ± SEM, **p<0.01, ***p<0.001, two-tailed Student's t-test.

[0044] Figure 17Myeloid-specific knockout of LAP3 led to increased inflammatory infiltration, aggravated liver injury and fibrosis in chronic inflammation-related liver cancer; in the figure, A shows the secretion of inflammatory factors IL-6, IL-1β, and TNF-α in the sera of LAP3-WT-Mφ and LAP3-KO-Mφ mice detected by ELISA; B shows the expression levels of AST and ALT in the blood of LAP3-WT-Mφ and LAP3-KO-Mφ mice detected by microplate reader; C-D show the mRNA expression levels of related cytokines and chemokines in liver tissues detected by RT-PCR; E shows the degree of inflammatory infiltration in liver tissues evaluated by HE staining (scale bar: 100 μm); F shows the degree of liver tissue fibrosis evaluated by Sirius red staining (scale bar: 50 μm); G shows immunohistochemical staining of liver tissue sections of LAP3-WT-Mφ and LAP3-KO-Mφ mice with the indicated antibodies (scale bar: 50 μm); values are expressed as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed Student's t-test.

[0045] Figure 18 LAP3 inhibition of macrophage inflammation depends on the TLR4-NF-κB signaling pathway; in the figure, A shows the protein expression of related signaling pathways detected by WB in PMs of LAP3-WT-Mφ and LAP3-KO-Mφ mice treated with LPS (200 ng / ml) for 0, 15, and 30 min; B shows WB analysis of cytoplasmic and nuclear proteins extracted from PMs of LAP3-WT-Mφ and LAP3-KO-Mφ mice to detect the nuclear entry of p65 after LPS treatment; C shows extraction of PMs from LAP3-WT-Mφ and LAP3-KO-Mφ mice and immunofluorescence analysis of the nuclear entry of p65, P65 (red), DAPI (blue), scale bar: 20 μm; values are expressed as mean ± SEM, **p<0.01, ***p<0.001, two-tailed Student's t-test.

[0046] Figure 19 Myeloid-specific knockout of LAP3 promoted the expression of NF-κB downstream target genes; the mRNA expression levels were detected by RT-PCR in PMs of LAP3-WT-Mφ and LAP3-KO-Mφ mice treated with LPS (200 ng / ml) for 6 h Cxcl15, Ccl5, Ier3, Lta, Bcl2 , n = 3; values are expressed as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed Student's t-test.

[0047] Figure 20LAP3 inhibits the activation of NF-κB dual luciferase reporter gene and the production of inflammatory factors by regulating the activation of TAK1. In Figure A, HEK293T cells were transfected with plasmids expressing NF-κB luciferase reporter gene, Renilla, TRAF6 (left) / TRIF (right), and simultaneously transfected with increasing doses of LAP3 siRNA to detect the luciferase activity of the cells. In Figure B, plasmids expressing NF-κB luciferase reporter gene, Renilla, GFP or LAP3 plasmid, and plasmids co-expressing MyD88, TRAF6, TRIF, TAK1, TAB1, IKKα, IKKβ or p65 were transfected into HEK293T cells respectively. After 24 h, the cells were collected and lysed to detect the luciferase activity. In Figure C, PMs of LAP3-WT-Mφ and LAP3-KO-Mφ mice were extracted, TAK1 in the cells was knocked down by siRNA, and RT-PCR was performed to detect the expression changes of inflammatory cytokines after LPS treatment, n = 3; the values are expressed as mean ± SEM, NS: no statistical difference, *p<0.05, **p<0.01, ***p<0.001, two-tailed Student's t-test. Il-6, Il-1β The expression changes, n = 3; the values are expressed as mean ± SEM, NS: no statistical difference, *p<0.05, **p<0.01, ***p<0.001, two-tailed Student's t-test.

[0048] Figure 21 LAP3 has no interaction with downstream signal transduction molecules of TLR4. In Figure A, HEK293T cells were transfected with overexpressed LAP3-Flag plasmid and GFP-Flag control plasmid respectively. After 24 h of transfection, the cells were stimulated with LPS for 20 min, collected and lysed, and Co-IP was used to verify the interaction between LAP3 and downstream molecular proteins of TLR4. In Figure B, HEK293T cells were transfected with overexpressed GFP-Flag control plasmid, LAP3-Flag plasmid, TAK1-Flag plasmid and TAB1-HA plasmid respectively. After 24 h of transfection, the cells were stimulated with LPS for 20 min, collected and lysed, and Co-IP was used to verify the interaction between LAP3 and TAK1 and TAB1.

[0049] Figure 22LAP3 inhibits macrophage inflammatory response through its enzymatic activity; in the figure, A - B show that GFP-Flag control plasmid, LAP3-WT-Flag plasmid and LAP3-R368K-Flag plasmid were transfected into RAW264.7 cells respectively, the enzymatic activity of LAP3 was detected by microplate reader, and the expression of LAP3 in each group was detected by WB; C - D show that PMs of LAP3-WT-Mφ and LAP3-KO-Mφ mice were transfected with GFP-Flag control plasmid, LAP3-WT-Flag plasmid and LAP3-R368K-Flag plasmid respectively. After transfection for 24 h, the cells were stimulated with LPS for 6 h, and then the cells were collected. Among them, C shows the protein level of IL-1β detected by WB, and D shows the Il-6, Il-1β and Tnf-α mRNA expression levels. n = 3 - 4 (A and D). Values are expressed as mean ± SEM, **p < 0.01, ***p < 0.001, two-tailed Student's t-test.

[0050] Figure 23 Macrophage LAP3 knockout inhibits oxidative phosphorylation and promotes glycolysis; in the figure, A - D show that LAP3-WT-PMs and LAP3-KO-PMs were stimulated with LPS for 4 h, and OCR or ECAR was measured by Seahorse XFe-24 software; among them, A - B show that the cells were treated with oligomycin, mitochondrial uncoupler (FCCP), antimycin A plus rotenone and rotenone to measure OCR; C - D show that glucose, oligomycin and 2-deoxy-D-glucose were added to the drug-added plate to measure ECAR, and the measurement was carried out after adding each drug and before adding the next drug; two-tailed Student's t-test.

[0051] Figure 24 Myeloid-specific knockout of LAP3 remodels the macrophage metabolic network; in the figure, A shows non-targeted metabolomics analysis of PMs of LAP3-WT-Mφ and LAP3-KO-Mφ mice; B shows that after extracting PMs of LAP3-WT-Mφ and LAP3-KO-Mφ mice, RNA-seq sequencing was carried out after LPS treatment to find differentially expressed genes after LAP3 deletion and perform enrichment analysis.

[0052] Figure 25Myeloid-specific knockout of LAP3 led to a significant decrease in the expression of mitochondrial complex-related markers and the activity of mitochondrial complexes; in the figure, A-B: After extracting PMs from LAP3-WT-Mφ and LAP3-KO-Mφ mice and treating them with LPS, RNA-seq sequencing was performed. Among them, A was the GO enrichment analysis of differentially expressed genes, and B was the comparison of the expression levels of differentially expressed genes related to the electron respiratory chain in the RNA-seq data; C: After extracting PMs from LAP3-WT-Mφ and LAP3-KO-Mφ mice and treating them with LPS, the activity of mitochondrial complexes was detected using related kits; the values are expressed as mean ± SEM, *p<0.05, **p<0.01, two-tailed Student's t-test.

[0053] Figure 26 Myeloid-specific knockout of LAP3 led to a decrease in mitochondrial membrane potential and a concomitant decrease in the expression levels of multiple mitochondrial-related markers; in the figure, A: After extracting PMs from LAP3-WT-Mφ and LAP3-KO-Mφ mice and treating them with LPS, JC-1 staining was performed to detect changes in mitochondrial membrane potential; B: After extracting PMs from LAP3-WT-Mφ and LAP3-KO-Mφ mice and treating them with LPS, the mRNA levels of mitochondrial-related markers were detected by RT-PCR; the values are expressed as mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, two-tailed Student's t-test.

[0054] Figure 27 Myeloid-specific knockout of LAP3 led to an increase in ROS levels; in the figure, A and B: After extracting PMs from LAP3-WT-Mφ and LAP3-KO-Mφ mice and treating them with LPS, the production levels of intracellular ROS were detected by FACS; in the figure, A is the flow cytometry histogram; B is the bar chart; the values are expressed as mean ± SEM, **p<0.01, two-tailed Student's t-test.

[0055] Figure 28 Deletion of LAP3 further activated TAK1 by promoting the production of ROS and promoted the occurrence and development of inflammation; PMs from LAP3-WT-Mφ and LAP3-KO-Mφ mice were extracted, pretreated with GSH (5 mM) for 2 h, and after 6 h of LPS stimulation, the expression changes of inflammatory cytokines were detected by RT-PCR Il-6, Il-1β n = 3; the values are expressed as mean ± SEM, NS indicates no statistical difference, **p<0.01, two-tailed Student's t-test. Detailed implementation methods

[0056] The present invention will be further clearly and completely described below through examples. The following examples are only a part of the embodiments of the present invention, which are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following examples are all conventional experiments unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0057] Example 1 Role of LAP3 in Inflammatory Response 1. Experimental Methods 1.1 Construction of Myeloid-Specific LAP3 Knockout Mice Myeloid-specific LAP3 gene knockout mice Lap3 fl / fl (C57BL / 6J) mice were purchased from Cyagen Biosciences Inc., Guangzhou, China, and were bred using CRISPR-Cas9-mediated genome editing technology. The myeloid knockout tool mice Lyz2 -Cre (C57BL / 6J) was provided by the team of Professor Tan Xiaoyue at Nankai University. It was crossed with Lap3 fl / fl mice to obtain myeloid cell-specific LAP3 gene knockout mice Lap3 fl / fl Lyz2 -Cre + / - , and littermate-bred Lap3 fl / fl Lyz2 -Cre - / - mice were used as experimental control mice. All mice were housed under specific pathogen-free (SPF) conditions in the Department of Laboratory Animal Science, Tianjin Medical University. The environmental temperature was maintained at 22 - 24 °C, and free access to food and water was provided, with a 12 h light-dark cycle. Male and female mice were randomly divided into an experimental group and a littermate control group. During the experiment, the requirements of the "Guidelines for the Ethical Review of Laboratory Animal Welfare (GB / T 35892-2018)" in China were strictly followed, and the welfare ethics of laboratory animals were standardized. All experiments have been approved by the Institutional Animal Care and Use Committee (IACUC) of Tianjin Medical University.

[0058] 1.2 LPS Injection, E.coli Infection and CLP-Induced Sepsis Model (1) Intraperitoneal injection of Escherichia coli E.coli (1×10 6 CFU) was used to induce septic shock in mice. E.coli It was introduced into mice by intraperitoneal injection to simulate sepsis caused by bacteria.

[0059] (2) Intraperitoneal injection of LPS (25 mg / kg) was used to induce septic shock in mice. LPS can activate the immune response in mice, leading to the rapid progression of inflammatory response and the occurrence of sepsis.

[0060] (3)The surgical protocol of cecal ligation and puncture (CLP) refers to the existing literature. The operation is as follows: Select male WT and KO mice at 8-10 weeks of age with matched body weights, and anesthetize the mice with tribromoethanol (200 mg / kg, intraperitoneal injection); shave the abdominal hair and disinfect the skin with 75% alcohol. Incise the skin in the middle of the abdomen, expose and remove the cecum, ligate the cecum at about 1 / 3 of the distal end with 4-0 sterile surgical silk thread, and at the same time use a sterile 21-gauge needle to make a cross-shaped through-puncture at the central part of the ligated distal cecum, and gently squeeze the cecum to release a small amount of feces to ensure complete perforation. Then replace the cecum and suture the incision in two layers. Immediately after the operation, inject warm saline (0.5 mL) subcutaneously and wait for the mice to wake up. The control group of mice only received anesthesia, laparotomy, cecum exposure and wound closure, but no cecal ligation and puncture.

[0061] The above method can establish a sepsis model in mice. The septic shock model mice after induction usually show symptoms such as decreased body temperature, rapid breathing, and circulatory failure. To evaluate the severity of sepsis, further biochemical analysis, pathological examination, immunohistochemical staining and other tests can be performed by collecting blood, organ or tissue samples. The mice in the group for detecting survival rate need to be observed and recorded regularly for their survival status.

[0062] 1.3 CCl4-induced acute hepatitis model The acute hepatitis model is induced by intraperitoneal injection of carbon tetrachloride (CCl4, 2.0 ml / kg). CCl4 is first diluted with corn oil at a ratio of 1:3 to ensure its solubility and reduce its toxicity to mice. After 48 h of injection, the mice are euthanized, and the serum and liver tissues of the mice are collected for biochemical and histochemical analysis. Take paraffin-embedded liver sections for HE staining, and evaluate liver injury by observing the degree of inflammatory infiltration of hepatocytes. Take frozen liver sections for immunofluorescence staining to observe the expression of pro-inflammatory cytokines IL-6 and IL-1β. Perform the determination of liver AST (aspartate aminotransferase) and ALT (alanine aminotransferase). Extract RNA from the liver and use RT-PCR to detect the expression of related genes.

[0063] 1.4 Hematoxylin-eosin staining (H&E staining, taking lung tissue as an example) After euthanizing the mice, take an appropriate amount of lung tissue and immerse it in 4% paraformaldehyde solution for fixation for no less than 48 h. The lung tissue is dehydrated and cleared by gradient, infiltrated with wax, embedded, sectioned, dewaxed, stained with hematoxylin, de-differentiated with hydrochloric acid alcohol, stained with eosin, dehydrated and cleared to complete the hematoxylin-eosin staining. Observe the sections under the microscope and photograph and record the results.

[0064] 1.5 Tissue immunofluorescence To detect the co - localization of F4 / 80 with IL - 1β or EGFP, immunofluorescence staining of 5 - μm - thick frozen liver sections was performed using specific antibodies against F4 / 80 (1:200; #ab16911, Abcam), IL - 6 (1:200; R&D Systems), or IL - 1β (1:200; #9722, Abcam). Subsequently, secondary antibodies labeled with Alexa Fluor 594 or Alexa Fluor 488 were used to detect the primary antibodies. Nuclei in all images were stained with DAPI (S36939; Invitrogen; Carlsbad, California, USA). Images were acquired by fluorescence microscopy.

[0065] 1.6 Detection of biochemical indexes The expression levels of AST, ALT, creatinine (CRE), and urea (Urea) in the serum samples to be measured were determined using kits, all of which were purchased from Nanjing Jiancheng Bioengineering Institute.

[0066] 2. Experimental results 2.1 Up - regulation of LAP3 expression in primary macrophages under inflammatory stimulation Macrophages play a crucial role in the inflammatory response. They not only participate in immune defense but also maintain the balance of the immune response by regulating the production of inflammatory factors. This invention explores the expression of LAP3 in macrophage - mediated inflammation.

[0067] Peritoneal macrophages of wild - type mice were extracted and treated with LPS to simulate the inflammatory response caused by bacterial stimulation. The changes in the expression levels of LAP3 and pro - inflammatory cytokines were detected by RT - PCR and WB. It was found that under the stimulation of LPS (activating the TLR4 signaling pathway), Lap3 showed a time - dependent increase in expression, while the expression of pro - inflammatory cytokines such as Il-6 and Il-1β showed a trend of first increasing and then decreasing (A and B in Figure 1 ). This invention detected the changes in LAP3 in PMs by exogenously adding Poly(I:C) (activating the TLR3 signaling pathway) and CpG (activating the TLR9 signaling pathway) to extract RNA, and found that, consistent with the LPS stimulation, Lap3 the expression level was significantly up - regulated (C and D in Figure 1 ). The above results indicate that the expression level of LAP3 increases after in vitro inflammatory stimulation.

[0068] 2.2 Up - regulation of LAP3 expression induced by in - vivo inflammatory stimulation Mice were intraperitoneally injected with E.coliA sepsis model was constructed, and mouse bone marrow-derived cells (BMs) were collected for RT-PCR detection. It was found that the expression level of LAP3 was significantly upregulated (A in Figure 2 ). In addition, by collecting the blood of healthy volunteers, patients with systemic inflammatory response syndrome (SIRS), and sepsis patients, PBMCs were extracted for RT-PCR detection. It was found that the mRNA expression levels of LAP3 in the blood of SIRS and sepsis patients were both increased (B in Figure 2 ). The above results indicate that the expression level of LAP3 is increased after inflammatory stimulation in vivo and in vitro, suggesting that LAP3 may be involved in macrophage-mediated inflammatory responses.

[0069] 2.3 LPS promotes the expression of LAP3 by activating the TLR4-NF-κB pathway After macrophages recognize LPS or Gram-negative bacteria, TLR4 on the cell membrane specifically recognizes and transmits the signal into the cell. This signal transduction process may activate the downstream IKKα / β-NF-κB signaling pathway in a MyD88-dependent or MyD88-independent manner. To study the reason for the increased expression of LAP3 after LPS stimulation, the following experiments were designed.

[0070] Sequence analysis using the TSEARCH program predicted that there were several potential NF-κB protein binding sites in the promoter region of the LAP3 gene (A in Figure 3 ). There are two main activation pathways of the NF-κB signaling pathway: one is the classical pathway dependent on Myd88, and the other is the non-classical pathway independent of Myd88. To study which NF-κB signaling pathway LPS uses to promote the expression of LAP3, the present invention constructed five siRNAs, including siMyd88#1, siMyd88#2, siTrif, siP65#1, and siP65#2, and transfected them into PMs cells respectively to knock out Myd88, Trif, and P65. LPS was added to each transfection well at a final concentration of 200 ng / mL. The experimental results showed that knocking out Myd88, Trif, and P65 could significantly inhibit the Lap3 increased expression induced by LPS, and also inhibit the expression levels of inflammatory factors Il-6 induced by LPS (B-D in Figure 3 ). In summary, it can be concluded that LPS promotes the expression of LAP3 in mouse macrophages through the TLR4-NF-κB signaling pathway.

[0071] 2.4 LAP3 inhibits the expression levels of LPS-induced macrophage inflammatory factors In RAW264.7 macrophages, siRNA was transfected to knockdown LAP3, and some cell samples were collected. RT-PCR was used to detect the knockdown efficiencyFigure 4 in A) of [reference]. The remaining transfected cells were divided into four groups: siCtrl group, siLAP3 group, siCtrl + LPS group, and siLAP3 + LPS group. The final concentration of the LPS treatment group was 200 ng / mL. After 6 h of LPS treatment, all cell samples were collected, and the expression levels of inflammatory cytokines were detected by RT-PCR. The results showed that knockdown of LAP3 significantly promoted the expression of inflammatory cytokines Il-6, Il-1β and Ifn-β in the LPS-stimulated group ( Figure 4 in B) of [reference].

[0072] To determine the mechanism of action of LAP3 therein, an LAP3 overexpression plasmid was transfected into RAW264.7 macrophages, and some cell samples were collected. The overexpression efficiency was detected by WB ( Figure 4 in C) of [reference]. The remaining transfected cells were also divided into four groups: fluorescent protein-Flag (GFP-Flag) group, LAP3-Flag group, GFP-Flag + LPS group, and LAP3-Flag + LPS group. The final concentration of the LPS treatment group was 200 ng / mL. After 6 h of LPS treatment, all cell samples were collected, and the expression levels of inflammatory factors were detected by RT-PCR. The results showed that the expression levels of inflammatory factors Il-6, Il-1β and Tnf-α in the LPS treatment group with overexpressed LAP3 were significantly lower than those in the control group ( Figure 4 in D) of [reference].

[0073] In summary, in the mouse macrophage cell line RAW264.7, LAP3 inhibited the expression of inflammatory cytokines induced by LPS stimulation.

[0074] 2.5 Construction and verification of LAP3 myeloid-specific knockout mice 2.5.1 Construction and verification of LAP3 myeloid-specific knockout mice To study the role of LAP3 in macrophage-mediated inflammatory responses, Cyagen Biosciences Inc. in Guangzhou was commissioned to construct mice with myeloid-specific LAP3 knockout. Exons 2-4 in the LAP3 gene were selected as the region for conditional knockout ( Figure 5 in A) of [reference]. By crossing the tool mouse Lysozyme2 (Lyz2) -Cre mouse with Lap3 fl / fl mice, mice with myeloid-specific LAP3 knockout Lap3 fl / fl Lyz2 -Cre + (LAP3-KO-Mφ) were generated, and the littermate Lap3 fl / fl Lyz2 -Cre- The (LAP3-WT-Mφ) mice were used as controls. After breeding the mice, the genotypes were identified by PCR using the DNA from the toes or tails as templates ( Figure 5 B and C in). Subsequently, peritoneal macrophages (PMs) were isolated from LAP3-WT-Mφ and LAP3-KO-Mφ mice, and the knockout efficiency was detected by RT-PCR and WB. The results showed that the mRNA and protein levels of LAP3 were significantly decreased ( Figure 5 D in).

[0075] 2.5.2 Myeloid-specific knockout of LAP3 does not affect the proliferation and differentiation of macrophages To investigate whether myeloid-specific knockout of LAP3 affects the proliferation and differentiation of macrophages, peritoneal macrophages (PMs) were isolated and incubated with F4 / 80 and CD11b fluorescent antibodies. F4 / 80 + and CD11b + double-positive macrophages were analyzed by flow cytometry.

[0076] The results showed that there were no significant changes in the number and proportion of double-positive macrophages in the LAP3-KO-Mφ group compared with the LAP3-WT-Mφ group ( Figure 6 A-C in). These results indicate that myeloid-specific knockout of LAP3 in mice does not affect the proliferation and differentiation of macrophages, which also lays a foundation for subsequent experiments.

[0077] 2.6 Myeloid-specific knockout of LAP3 promotes inflammatory responses To investigate the role of LAP3 in macrophage-mediated immune inflammation, PMs from LAP3-WT-Mφ and LAP3-KO-Mφ mice were isolated, and RNA-seq was performed after LPS treatment to search for differentially expressed genes after LAP3 deletion and perform KEGG signaling pathway enrichment analysis.

[0078] The results showed that myeloid-specific knockout of LAP3 significantly affected the NF-κB signaling pathway, positive regulation of cytokine production, inflammatory response, and mitogen-activated protein kinase (MAPK) signaling pathway ( Figure 7 ).

[0079] 2.6.2 Myeloid-specific knockout of LAP3 promotes TLR4 pathway-mediated inflammatory responses LPS can induce inflammatory responses through the TLR4 signaling pathway. This invention studied the effects of myeloid-specific knockout of LAP3 on macrophage-mediated immune inflammation under the stimulation of LPS.

[0080] Results showed that in PMs, after LPS stimulation, myeloid-specific knockout of LAP3 in mice showed upregulation of the mRNA levels of inflammatory genes Il- 6, Il-1β, Nos2 and Tnf-α (A and B in Figure 8 ). The same results were obtained in BMDMs (C and D in Figure 8 ). The above results further verified at the cellular level that myeloid-specific knockout of LAP3 could promote the inflammatory response of macrophages.

[0081] 2.6.3 Myeloid-specific knockout of LAP3 promotes inflammatory responses mediated by the TLR3 / TLR9 pathway Toll-like receptors (TLRs) are a type of pattern recognition receptor (PRRs) in the innate immune system that can recognize different PAMPs, such as LPS, Poly(I:C), and CpG, and activate downstream factors through their respective specific signaling pathways to initiate different types of immune responses. By studying different TLRs and their ligands, the immune response can be better understood and regulated. TLR3 mainly recognizes the mimic of viral double-stranded RNA (dsRNA), Poly(I:C), TLR4 mainly recognizes the outer membrane components of Gram-negative bacteria, LPS, while TLR9 mainly recognizes unmethylated CpG islands in bacterial and viral DNA, as well as synthetic CpG oligodeoxynucleotides. The present invention investigated the role of LAP3 in the inflammatory responses induced by Poly(I:C) and CpG.

[0082] Eight- to twelve-week-old LAP3-WT-Mφ and LAP3-KO-Mφ mice (3 mice per group) were selected, and their primary peritoneal macrophages (PMs) or bone marrow-derived macrophages (BMDMs) differentiated from bone marrow cells were used to repeat the previous cell experiments. The results showed that in PMs or BMDMs, after Poly(I:C) stimulation, myeloid-specific knockout of LAP3 in mice showed upregulation of the mRNA levels of inflammatory genes Il-1β、 Ifn-β, Il-6 or Nos2 (A and B in Figure 9 ). And in PMs, after CpG stimulation, myeloid-specific knockout of LAP3 in mice showed upregulation of the mRNA levels of inflammatory genes Il-6, Il-1β and Tnf-α (C in Figure 9 ). The above results indicate that by activating different TLRs and their ligands, myeloid-specific knockout of LAP3 can promote the inflammatory response of macrophages.

[0083] 2.7 LAP3 knockout promotes the occurrence and development of sepsis 2.7.1 Systemic knockout of LAP3 exacerbates the systemic response of septic mice In a mouse model, the manifestations of sepsis are similar to those in humans, including decreased activity; lethargy and fatigue; reduced food and water intake; decreased body temperature; rough hair; increased respiratory rate or tachypnea; diarrhea; elevated pro-inflammatory cytokines; elevated levels of ALT and AST, indicating hepatocyte damage; elevated levels of blood urea nitrogen and creatinine, indicating impaired renal function; pulmonary edema or ARDS (acute respiratory distress syndrome), indicating impaired lung function; severe sepsis may lead to ischemic shock and multiple organ failure, ultimately resulting in death. The present invention used wild-type and LAP3 knockout male mice aged 8-12 weeks and weighing approximately 20-25 g, and intraperitoneally injected LPS (25 mg / kg) to construct a mouse sepsis model.

[0084] First, the survival of a group of mice was observed and recorded, and it was found that compared with wild-type mice, the survival rate of LAP3 knockout mice was lower ( Figure 10 A in). In another group of mice, LPS (25 mg / kg) was intraperitoneally injected, and an equal dose of PBS was injected in the control group. After 6 h, blood was collected from the orbital cavity of the mice, the mice were sacrificed, and lung tissues were isolated for subsequent serum detection and pathological analysis. The secretion of inflammatory factors in the serum was detected, and the results showed that after LAP3 knockout, the secretion levels of IL-6, IL-1β, and TNF-α in the serum were higher ( Figure 10 B in).

[0085] 2.7.2 Global knockout of LAP3 exacerbates organ damage in septic mice Part of the mouse lung tissue was isolated for wet weight and dry weight measurement, and the wet / dry weight ratio of the mouse lung tissue was compared. The study found that compared with wild-type mice, the wet / dry weight ratio of LAP3 knockout mice was higher, indicating more severe pulmonary edema in LAP3 knockout mice ( Figure 11 A in).

[0086] The lung tissue was stained with H&E to observe the inflammatory infiltration in the lungs, and it was found that the degree of inflammatory infiltration in LAP3 knockout mice was more severe than that in wild-type mice ( Figure 11 B in).

[0087] Lung RNA was extracted from each group, and the mRNA levels of pro-inflammatory cytokines ( Il-6, Il-1β and Tnf- α ) and chemokines ( Ccl2, Ccl5, Cxcl1 and Cxcl9 ) in the lungs of mice were detected by RT-PCR. The mRNA levels of these factors were increased in LAP3 knockout mice, while the mRNA level of the anti-inflammatory cytokine ( Il-10 ) was decreased in LAP3 knockout mice ( Figure 11 C in).

[0088] By detecting the secretion of ALT and AST in the serum, it was shown that the secretion levels of ALT and AST in LAP3 knockout mice were higher than those in wild-type mice, indicating more severe liver injury ( Figure 11 as shown in D of Figure 11 ). By detecting the levels of creatinine (CRE) and urea (Urea) in the blood, it was shown that the levels of CRE and Urea in LAP3 knockout mice were higher than those in wild-type mice, indicating more severe kidney injury (

[0089] as shown in E of E.coli ). In summary, global knockout of LAP3 can exacerbate LPS-induced sepsis in mice. E.coli To investigate the effect of myeloid-specific knockout of LAP3 on sepsis in mice, male mice of LAP3-WT-Mφ and LAP3-KO-Mφ, 8-12 weeks old and weighing about 20-25 g, were intraperitoneally injected with Figure 12 or LPS (25 mg / kg) to establish a mouse sepsis model. By observing the survival of the mice, it was found that the survival rate of LAP3-KO-Mφ mice was lower than that of LAP3-WT-Mφ mice ( as shown in A and B of

[0090] ). In another group of mice, LPS (25 mg / kg) was intraperitoneally injected, and an equal dose of PBS was injected into the control group. After 6 h, blood was collected from the eyes of the mice, and the mice were sacrificed. The lung tissues were isolated for subsequent serum detection and pathological analysis. ELISA was used to detect the secretion of inflammatory factors in the serum, and it was found that the secretion levels of IL-6, IL-1β, and TNF-α in the serum of LAP3-KO-Mφ mice were higher ( Figure 12 as shown in C of

[0091] ). The lung tissues were stained with H&E to observe the inflammatory infiltration in the lungs. It was found that the degree of inflammatory infiltration in LAP3-KO-Mφ mice was more severe than that in LAP3-WT-Mφ mice ( Figure 12 as shown in D of

[0092] ). Lung RNA was extracted from each group, and RT-PCR was used to detect the mRNA levels of pro-inflammatory cytokines ( Il-6, Il-1β and Tnf- α ) and chemokines ( Ccl2, Ccl5, Cxcl1 and Cxcl9 ) in the lungs of mice. The mRNA levels were increased in LAP3-KO-Mφ mice, while the mRNA level of the anti-inflammatory cytokine ( Il-10 ) was decreased in LAP3-KO-Mφ mice ( Figure 12 as shown in E of

[0093] The results showed that myeloid-specific knockout of LAP3 exacerbated E.coli or LPS-induced sepsis in mice.

[0094] 2.7.4 Myeloid-specific knockout of LAP3 exacerbated sepsis in CLP-induced mice In this invention, the cecal ligation and puncture model was used to simulate sepsis. LAP3-WT-Mφ and LAP3-KO-Mφ male mice aged 8 - 12 weeks and weighing about 20 - 25 g were used in the experiment. After anesthesia recovery after the operation, their survival was observed. Compared with LAP3-WT-Mφ mice, the survival rate of LAP3-KO-Mφ mice was lower ( Figure 13 A in

[0095] In another group of experiments, orbital blood of mice was collected 20 h after the operation, the mice were sacrificed, and the lungs, livers, kidneys and spleens were isolated for subsequent serum detection and pathological analysis. First, the levels of IL-6, IL-1β and TNF-α in the serum were detected, and the results showed that the secretion levels of these inflammatory factors in the serum of LAP3-KO-Mφ mice were significantly increased ( Figure 13 B in

[0096] The lung tissues were stained with H&E to observe the inflammatory infiltration. The results showed that the degree of pulmonary inflammatory infiltration in LAP3-KO-Mφ mice was significantly higher than that in LAP3-WT-Mφ mice ( Figure 13 C in

[0097] The mRNA levels of pro-inflammatory factors in the lungs, livers, kidneys and spleens of mice in each group were detected by RT-PCR Il-6 and it was found that the Il-6 mRNA levels in LAP3-KO-Mφ mice were significantly increased ( Figure 13 D in

[0098] 2.8 LAP3 knockout promoted the occurrence and development of acute hepatitis 2.8.1 Myeloid-specific knockout of LAP3 exacerbated CCl4-induced acute hepatitis To verify the regulatory role of LAP3 in the inflammatory response induced by other chemicals, this invention explored the role of LAP3 in the CCl4-induced acute hepatitis model. Male mice of LAP3-WT-Mφ and LAP3-KO-Mφ aged 8 - 12 weeks and weighing about 20 - 25 g were used to construct a mouse acute hepatitis model by intraperitoneal injection of CCl4 for 48 h.

[0099] By using a related kit to detect the secretion of ALT and AST in serum, it was shown that mice with LAP3-KO-Mφ had higher secretion levels of ALT and AST than mice with LAP3-WT-Mφ, suggesting more severe liver injury ( Figure 14 A) in

[0100] Part of the liver tissue was stained with H&E to observe the inflammatory infiltration in hepatitis. It was found that the degree of inflammatory infiltration in mice with LAP3-KO-Mφ was more severe than that in mice with LAP3-WT-Mφ ( Figure 14 B) in

[0101] Liver tissue RNA was extracted from each group, and the mRNA levels of pro-inflammatory cytokines ( Il-6, Il-1β and Tnf-α ) and chemokines ( Ccl2, Cxcl1 and Cxcl9 ) in the liver of mice were detected by RT-PCR. The mRNA levels were increased in LAP3-KO-Mφ mice, while the mRNA level of the anti-inflammatory cytokine ( Il-10 ) was decreased in LAP3-KO-Mφ mice (C) in Figure 14

[0102] 2.8.2 Myeloid-specific knockout of LAP3 increased macrophage inflammatory infiltration in liver tissue in CCl4-induced acute hepatitis Subsequently, part of the liver was made into frozen sections and immunofluorescently stained with IL-6, IL-1β, and F4 / 80 antibodies to observe the proportion of inflammatory macrophages in the liver. The results showed that the proportion of inflammatory macrophages in mice with LAP3-KO-Mφ was higher than that in mice with LAP3-WT-Mφ (A and B) in Figure 15

[0103] 2.9 LAP3 knockout promotes the occurrence and development of chronic inflammation-related liver cancer 2.9.1 Myeloid-specific knockout of LAP3 led to an increase in the number and volume of tumors in chronic inflammation-related liver cancer Sustained inflammatory responses can not only lead to cell carcinogenesis but also promote tumor development. Hepatocellular carcinoma is one type of cancer caused by chronic inflammation. The present invention aims to further investigate the role of LAP3 in the occurrence and development of chronic inflammation-related liver cancer. A chronic inflammation-related liver cancer model was established by inducing mice with diethylnitrosamine (DEN) and CCl4. Eight-week-old mice with LAP3-WT-Mφ and LAP3-KO-Mφ were intraperitoneally injected with DEN (25 mg / kg) once every two weeks for a total of two times. Six weeks later, CCl4 (0.5 ml / kg, dissolved in corn oil) was intraperitoneally injected once a week for 12 weeks. At the 30th week, the mice were euthanized, and serum and liver tissues were collected for biochemical and histological analysis ( Figure 16 as shown in A).

[0104] The results showed that compared with LAP3-WT-Mφ mice, the liver weight / body weight ratio of LAP3-KO-Mφ mice was significantly increased, the number of tumors was more, and the maximum and average sizes of the tumors were larger ( Figure 16 as shown in B-D). These results indicate that the knockout of LAP3 can promote the occurrence of chronic inflammation-related liver cancer.

[0105] 2.9.2 Myeloid-specific knockout of LAP3 leads to increased inflammatory infiltration, more severe liver injury and fibrosis in chronic inflammation-related liver cancer In the present invention, ELISA was used to detect the levels of inflammatory factors IL-6, IL-1β and TNF-α in serum. The results showed that after the knockout of LAP3 in myeloid cells, the secretion of these inflammatory factors increased significantly ( Figure 17 as shown in A).

[0106] The corresponding kits were used to detect ALT and AST in serum. It was found that the levels of ALT and AST in LAP3-KO-Mφ mice were higher than those in LAP3-WT-Mφ mice, indicating more severe liver injury ( Figure 17 as shown in B).

[0107] Liver tissue RNA of each group was extracted, and RT-PCR was used to analyze the mRNA levels of inflammatory factors ( Il-6, Il-1β, Tnf-α and Nos2 ) and chemokines ( Ccl5 and Cxcl2 ) in liver tissue. The results showed that the expression of these factors increased in LAP3-KO-Mφ mice ( Figure 17 as shown in C), while the mRNA levels of M2-related markers ( Arg1, Retnla, Mgl1, Mgl2 ) and chemokines ( Ccl17 and Ccl22 ) decreased ( Figure 17 as shown in D).

[0108] Partial liver tissues were stained with H&E and Sirius red to observe inflammatory infiltration and fibrosis. The results showed that the degrees of inflammatory infiltration and fibrosis in LAP3-KO-Mφ mice were higher than those in LAP3-WT-Mφ mice ( Figure 17 E and F in

[0109] Immunohistochemical staining was used to detect the infiltration of immune cells in liver tissues. It was found that the numbers of F4 / 80-positive cells (macrophages), CD11b-positive cells (myeloid cells), CD4-positive cells (CD4 + T cells), and CD8a-positive cells (CD8 + T cells) increased in LAP3-KO-Mφ mice; while the staining results of the specific marker FoxP3 of regulatory T cells (Treg cells) showed that the number of regulatory T cells decreased ( Figure 17 G in

[0110] In summary, the present invention found that LPS treatment and various stimuli promoted the expression of LAP3 in macrophages, and this inhibitory effect was dependent on the TLR4-NF-κB signaling pathway. Through in vitro cell experiments and E.coli infection, LPS stimulation, CLP-induced sepsis model, and CCl4-induced acute hepatitis model in mice, it was found that LAP3 could alleviate macrophage-mediated immune inflammation both in vivo and in vitro and participate in the immune regulation of the body.

[0111] Example 2 Mechanism study of LAP3 in inflammatory response 1. Experimental methods 1.1 Untargeted metabolomics detection In the present invention, untargeted metabolomics detection was performed on mouse PMs. Mouse PMs (5 × 10 6 ) were inoculated into a 10 cm cell plate and cultured in complete DMEM medium. After 24 h, the cells were washed with ice-cold PBS, 500 μL of cold methanol:H2O (4:1, v / v) was added to completely cover the cells, and the cells were scraped off on dry ice after standing at -80°C for 20 min and collected into a pre-cooled 1.5 mL EP tube. Another 500 μL of methanol aqueous solution was added to rinse the remaining cells and collect them. LC-MS mass spectrometry analysis of the samples was performed by LipidALL Technologies (Changzhou, China). According to the internal standard metabolite standard protocol, the relative abundances of metabolites in the samples were compared, and the significant changes were determined by the t-test method after data quality control and normalization.

[0112] 1.2 Mitochondrial stress detection (1) PMs were inoculated into a 24-well Seahorse XFe-24 detection plate and stimulated with LPS for 6 h.

[0113] (2) Wash the cells and culture them in XF RPMI medium (pH 7.4) for 1 h.

[0114] (3) Perform mitochondrial stress tests using Seahorse XFe-24 software, automatically calculate and record the oxygen consumption rate (OCR). Determine ATP production using 2.0 μM oligomycin, determine maximal respiration using 2.0 μM FCCP, and determine non-mitochondrial respiration by treating cells with 1.0 μM antimycin A / rotenone. Measurements must be taken after each addition of a mitochondrial inhibitor and before adding the next inhibitor.

[0115] 1.3 Glycolysis stress detection (1) Seed PM in a 24-well Seahorse XFe-24 assay plate and stimulate with LPS for 6 h.

[0116] (2) Wash the cells and culture them in XF RPMI medium (pH 7.4) for 1 h.

[0117] (3) Perform glycolysis stress tests using Seahorse XFe-24 software, automatically calculate and record the extracellular acidification rate (ECAR). Determine basal glycolytic capacity using 10 μM glucose, determine maximal glycolytic capacity using 1.5 μM oligomycin. Stimulate cells with 50 μM 2-DG (2-deoxyglucose) to verify that the ECAR generation in the experiment is derived from the glycolytic pathway.

[0118] 1.4 Luciferase reporter gene assay Transfect HEK293T cells with NF-κB luciferase reporter gene plasmid and Renilla plasmid, and measure luciferase readings using a dual-luciferase reporter gene assay system according to the instructions of the manufacturer Promege. Refer to the instructions for the specific experimental procedures.

[0119] 1.5 Mitochondrial membrane potential detection (JC-1) Aspirate the culture medium in the 6-well plate, wash the cells twice with PBS. Then, add 1 ml of cell culture medium. Add 1 ml of JC-1 staining working solution, mix well, and incubate the cells in a 37 ºC cell culture incubator for 20 min. After incubation, aspirate the supernatant and wash the cells twice with JC-1 staining buffer. Add 2 ml of cell culture medium to the wells. Observe using a fluorescence microscope or a laser confocal microscope.

[0120] 2. Experimental results 2.1 LAP3 inhibits macrophage inflammation depending on the TLR4-NF-κB signaling pathway The production of inflammatory cytokines is an important part of the body's immune response, and its activation mechanism involves multiple signaling pathways. One of the classical pathways is through the activation of nuclear transcription factor kappa B (NF-κB), which further induces the expression of various inflammation-related genes. When LPS binds to TLR4, it further activates the MyD88-dependent pathway within the cell. MyD88 further recruits IRAKs and TNF receptor-associated factors (TRAFs), ultimately activating the IκB kinase (IKK) complex. The activation of the IKK complex leads to the phosphorylation and degradation of IκB protein, enabling NF-κB to be released and translocated into the nucleus, further regulating the expression of a series of genes related to immune and inflammatory responses. Therefore, it is verified whether the deletion of LAP3 promotes the production of inflammatory cytokines by promoting the TLR4-NF-κB signaling pathway.

[0121] Extract PMs from LAP3-WT-Mφ and LAP3-KO-Mφ mice, and perform WB analysis after LPS treatment for 0, 15, and 30 min to detect the activation of the TLR4 downstream signaling pathway. It was found that in the PMs of LAP3-KO-Mφ mice, the phosphorylation levels of TAK1, IKKα / β, ERK, JNK, and P38 were all significantly increased ( Figure 18 A in). Extract the cytoplasmic and nuclear proteins of PMs from LAP3-WT-Mφ and LAP3-KO-Mφ mice for WB analysis, and it was found that under the stimulation of LPS, the nuclear localization of p65 in LAP3-KO-Mφ mice was significantly increased ( Figure 18 B in). In addition, LPS was used to treat LAP3-WT-Mφ and LAP3-KO-Mφ mouse PMs for 0, 15, and 30 min respectively, and the activation and nuclear entry of p65 were detected by cellular immunofluorescence experiments. The results showed that after LPS treatment, p65 in KO mice was significantly translocated into the nucleus and showed a time-dependent effect in the short term ( Figure 18 C in).

[0122] In addition, in the present invention, by extracting PMs from LAP3-WT-Mφ and LAP3-KO-Mφ mice and performing RT-PCR detection after LPS treatment, it was found that after myeloid-specific knockout of LAP3, the mRNA levels of NF-κB downstream target genes Cxcl15, Ccl5, Ier3, Lta, Bcl 2 were all increased ( Figure 19 ). To sum up, LAP3 plays a crucial role in the activation of NF-κB induced by TLR4.

[0123] 2.2 LAP3 inhibits the activation of the NF-κB dual luciferase reporter gene and the production of inflammatory factors by regulating the activation of TAK1 In this invention, a luciferase reporter plasmid of NF-κB-promotor-Luc was constructed, and the luciferase reporter gene assay was used to deeply explore the mechanism of action of LAP3 on TLR4-NF-κB activation in MyD88-dependent and -independent signaling pathways. The NF-κB-promotor-Luc plasmid and LAP3 siRNA were co-transfected into HEK293T cells together with downstream protein molecules of TLR4, and then the activity level of luciferase in the cells was detected. The results showed that compared with the transfection of TRAF6 or TRIF alone, the co-transfection of siLAP3 significantly increased the luciferase activity, and this increase showed a concentration-dependence of siLAP3 ( Figure 20 as shown in A).

[0124] In further experiments, when LAP3 was co-expressed with TGF-β-activated kinase 1 (TAK1) inhibitor, TGF-β binding protein 1 of activated kinase 1 (TAB1), NF-κB kinase α / β (IKKα / β), and p65, the luciferase activity did not increase further. This result suggests that the role of LAP3 in regulating the TLR4-NF-κB signaling pathway may be upstream of TAK1 / TAB1 and downstream of TRIF ( Figure 20 as shown in B).

[0125] To further verify this hypothesis, peritoneal macrophages (PMs) of LAP3-WT-Mφ and LAP3-KO-Mφ mice were extracted, and the expression of TAK1 in the cells was down-regulated by siRNA. After LPS treatment, the expression levels of inflammatory cytokines (such as Il-6 and Il-1β ) were detected by RT-PCR. The results showed that the siTAK1 group significantly inhibited the increase in the levels of inflammatory cytokines caused by LAP3 knockout, making the expression of inflammatory cytokines in the WT group and the KO group show no significant difference ( Figure 20 as shown in C).

[0126] The above results indicate that LAP3 inhibits the activation of the NF-κB signaling pathway by regulating the activation of TAK1, thereby reducing the expression of downstream inflammatory factors. This finding provides a new perspective for understanding the specific mechanism of action of LAP3 in immune signal transduction.

[0127] 2.3 LAP3 has no interaction with downstream signal transduction molecules of TLR4 The study found that the phosphorylation levels of TAK1, IKKα / β, ERK, JNK, and P38 were significantly increased in the PMs of LAP3-KO-Mφ mice. To verify whether LAP3 further regulates the immune response by interacting with downstream molecular proteins in the TLR4 signaling pathway, the present invention conducted an immunoprecipitation experiment (Co-IP). By separately transfecting the overexpressed LAP3-Flag plasmid and the GFP-Flag control plasmid into HEK293T cells, stimulating with LPS for 20 min 24 h after transfection, collecting and lysing the cells, incubating with the tagged magnetic beads FLAG-Beads for 12 h, performing WB after competitive elution, and incubating with different antibodies to detect the binding proteins.

[0128] The results showed that LAP3 did not interact with the protein molecules related to the downstream signaling pathway of TLR4 ( Figure 21 A in). By further separately transfecting the overexpressed GFP-Flag control plasmid, LAP3-Flag plasmid, TAK1-Flag plasmid, and TAB1-HA plasmid into HEK293T cells, it was found that LAP3 did not interact with TAB1, while the interaction between TAK1 and TAB1 confirmed the credibility of the Co-IP experiment results ( Figure 21 B in). Combining the experimental data of the luciferase reporter gene, it is considered that LAP3 may regulate macrophage-mediated inflammatory responses through the upstream of TAK1 / TAB1.

[0129] 2.4 LAP3 inhibits macrophage inflammatory responses through its enzymatic activity LAP3 is an enzyme involved in intracellular protein metabolism and usually regulates protein functions by hydrolyzing and removing amino acid residues. By mutating the arginine at the 368th site of LAP3 to lysine, the enzymatic activity of LAP3 can be changed, resulting in the loss of the enzymatic function of LAP3. Therefore, the present invention evaluated whether the role of LAP3 in regulating macrophage immune inflammation also depends on its enzymatic activity. The present invention successfully constructed the enzymatic mutant plasmid LAP3-R368K of LAP3 and conducted subsequent experiments.

[0130] First, by separately transfecting the GFP-Flag control plasmid, LAP3-WT-Flag plasmid, and LAP3-R368K-Flag plasmid into RAW264.7 cells and using a related kit to detect the activity of LAP3, the results showed that the enzymatic activity of LAP3 increased with the overexpression of the LAP3-WT-Flag plasmid, while the enzymatic activity of LAP3 decreased with the overexpression of the LAP3-R368K-Flag plasmid. WB was used to detect the expression of LAP3 in each group, indicating that the mutation at the LAP3-R368K site indeed affected the enzymatic activity of LAP3 ( Figure 22 A in).

[0131] Next, in the present invention, GFP-Flag control plasmid, LAP3-WT-Flag plasmid and LAP3-R368K-Flag plasmid were respectively transfected into PMs of LAP3-WT-Mφ and LAP3-KO-Mφ mice. After 24 h of transfection, LPS was used to stimulate for 6 h, and then the cells were collected for WB and RT-PCR detection. First, the overexpression efficiency of LAP3 was verified, and it was found that both LAP3-WT-Flag plasmid and LAP3-R368K-Flag plasmid could be overexpressed ( Figure 22 B in Figure 22 ). The results showed that after overexpressing the LAP3-WT-Flag plasmid, the mRNA and protein levels of inflammatory cytokines decreased, but the same phenomenon did not occur after overexpressing the LAP3-R368K-Flag enzyme activity mutant plasmid (

[0132] C and D in ). In summary, LAP3 inhibits the production of inflammatory cytokines through its enzyme activity.

[0133] The OCR results showed that after knocking out LAP3, the level of oxidative phosphorylation in macrophages decreased ( Figure 23 A and B in Figure 23 ); on the contrary, the ECAR results showed that the glycolytic ability of PMs with LAP3 knockout increased regardless of LPS stimulation (

[0134] C and D in To study the potential mechanism of LAP3 in suppressing inflammatory responses, the present invention first performed untargeted metabolomics analysis on the PMs of LAP3-WT-Mφ and LAP3-KO-Mφ mice and conducted enrichment analysis on the data. The results showed that after LAP3 knockout, the tricarboxylic acid cycle (TCA cycle) was significantly enriched in the cell metabolic pathway ( Figure 24 A in

[0135] In addition, the PMs of LAP3-WT-Mφ and LAP3-KO-Mφ mice were further extracted and RNA sequencing was performed after LPS stimulation to identify genes with differential expression after LAP3 deletion and conduct relevant enrichment analysis. The results showed that after myeloid-specific knockout of LAP3, the gene expression of the oxidative phosphorylation pathway and the thermogenesis pathway was significantly enriched ( Figure 24 B in

[0136] These results indicate that the deletion of LAP3 leads to the remodeling of the macrophage metabolic network and may play an important role in the immune response by regulating cellular oxidative phosphorylation. 2.7 Myeloid-specific knockout of LAP3 leads to mitochondrial dysfunction 2.7.1 Myeloid-specific knockout of LAP3 leads to a significant decrease in the expression of mitochondrial complex-related markers and the activity of mitochondrial complexes In the present invention, by extracting the PMs of LAP3-WT-Mφ and LAP3-KO-Mφ mice and performing RNA-seq sequencing after LPS treatment, genes with differential expression after LAP3 deletion were searched for GO enrichment analysis ( Figure 25 A in Figure 25 It was found that many pathways related to mitochondrial structure, electron respiratory chain transfer, and oxidative phosphorylation were enriched. By comparing the expression of genes related to the electron respiratory chain in the RNA-seq data, it was found that the expression of electron respiratory chain-related markers was downregulated in the PMs of LAP3-KO-Mφ mice ( Figure 25 B in

[0137] Next, by extracting the PMs of LAP3-WT-Mφ and LAP3-KO-Mφ mice and detecting the activity of mitochondrial complexes with related kits after LPS treatment, the activities of mitochondrial complexes I, II, III, IV, and V all showed varying degrees of decline in LAP3 knockout mice (

[0137] C in Figure 25 ), indicating that LAP3 plays a crucial role in maintaining mitochondrial function and metabolic stability. 2.7.2 Myeloid-specific knockout of LAP3 leads to a decrease in mitochondrial membrane potential and is accompanied by a decrease in the expression levels of various mitochondrial-related markers Mitochondrial dysfunction is usually caused by multiple factors, such as damage to the mitochondrial membrane, inhibition of the respiratory chain, reduction of enzyme activity, damage to mitochondrial DNA (mtDNA), etc. These factors trigger disorders in energy metabolism and further initiate a series of cascading cellular damage responses. Specifically, mitochondrial dysfunction leads to a decline in the activity of respiratory chain enzymes, a decrease in mitochondrial membrane potential, a reduction in ATP synthesis, and disruption of intracellular calcium homeostasis. In addition, the opening of the mitochondrial permeability transition pore (mPTP) changes the permeability of the mitochondrial membrane, hinders β-oxidation of fatty acids, and causes the accumulation of fatty acids in cells. During this process, the excessive generation of reactive oxygen species (ROS) triggers oxidative stress and further leads to oxidative damage of mtDNA, thereby inhibiting the mitochondrial biogenesis ability, exacerbating mitochondrial dysfunction, and ultimately inducing apoptosis or death of cells.

[0138] The present invention first detected the change in mitochondrial membrane potential after LPS treatment using the JC-1 staining method. The results showed that after LPS treatment, the mitochondrial membrane potential of PMs in LAP3-KO-Mφ mice decreased significantly ( Figure 26 A in). The study also found that after LPS stimulation, the mRNA expression levels of key genes involved in mitophagy, ATP synthesis, and mitochondrial biogenesis in PMs of LAP3-KO-Mφ mice decreased significantly ( Figure 26 B in). These results indicate that myeloid-specific knockout of LAP3 significantly affects mitochondrial function and leads to mitochondrial dysfunction.

[0139] 2.8 Myeloid-specific knockout of LAP3 leads to an increase in ROS levels Normal mitochondrial membrane potential is the basis for maintaining the normal operation of the electron transport chain. When the electron transport chain of mitochondria is dysfunctional, electrons are prone to leakage during the transfer process, resulting in more electrons reacting with oxygen molecules to produce ROS, significantly increasing the production of ROS in mitochondria. With the accumulation of ROS, intracellular oxidative stress increases, further damaging mitochondria and forming a vicious cycle. Flow cytometry was used to detect the intracellular ROS level. Both the flow cytometry histogram and column chart showed that myeloid-specific knockout of LAP3 led to an increase in ROS levels after LPS stimulation ( Figure 27 A and B in).

[0140] 2.9 Deletion of LAP3 further activates TAK1 by promoting the production of ROS, promoting the occurrence and development of inflammation Mitochondria-derived ROS can affect the intracellular redox state, and the tilting of the redox balance towards the oxidative direction can promote the expression of inflammatory cytokines. To investigate whether mitochondria-derived ROS regulate the inhibition of inflammatory cytokines by LAP3 in macrophages, the present invention selected GSH, which can, to a certain extent, scavenge the ROS generated by cells due to stimulating factors, thereby reducing the ROS level. After ROS is generated in macrophages, it can activate the TLR4 signaling pathway, and further promote the activation of TAK1. After TAK1 is activated, it can further activate the downstream MAPK (such as JNK, p38) and NF-κB (nuclear factor kappa B) signaling pathways, which play key roles in cell stress response, inflammatory factor secretion, and cell proliferation and apoptosis. Therefore, by extracting PMs from LAP3-WT-Mφ and LAP3-KO-Mφ mice, knocking down TAK1 in cells by siRNA, pretreating with 5 mM GSH for 2 h, and then stimulating with LPS for 6 h, the expression changes of Il-6 and Il-1β were detected by RT-PCR. The results showed that GSH pretreatment partially antagonized the increase in the level of inflammatory cytokines caused by LAP3 gene knockout, and through siTAK1 intervention, the increase in the level of inflammatory factors caused by LAP3 gene deletion was significantly controlled, ( Figure 28 ). In summary, LAP3 inhibits the secretion of inflammatory cytokines by reducing the activation of TAK1 by inhibiting the production of ROS.

[0141] The above results indicate that LAP3 knockout promotes NF-κB transcriptional activity and thus regulates the inflammatory response of macrophages; LAP3 knockout promotes the activation of the classical signaling pathway in the inflammatory response of macrophages; LAP3 knockout regulates the inflammatory response of macrophages depending on its enzyme activity; LAP3 knockout alters macrophage energy metabolism (LAP3 knockout promotes ECAR and inhibits OCR) and remodels the macrophage metabolic network; LAP3 knockout promotes the production of ROS by causing mitochondrial dysfunction, further activates TAK1, and thus promotes the inflammatory response of macrophages.

[0142] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. Use of leucine aminopeptidase 3 or a leucine aminopeptidase 3 agonist in the preparation of a drug for preventing, treating and / or adjuvantly treating macrophage immune inflammation-mediated diseases.

2. The application according to claim 1, wherein The macrophage immune inflammation-mediated diseases include one or more of: sepsis, acute hepatitis, and chronic inflammation-related liver diseases.

3. The application according to claim 1, characterized in that The leucine aminopeptidase 3 agonist includes one or more of: small molecule agonists, small molecule compounds, macromolecular compounds, bioactive peptides, antibody fragments, and nucleic acid aptamers.

4. The application according to claim 1, wherein The drug exerts its effect through any of the following aspects: (1) By inhibiting the TLR4-NF-κB signaling pathway, thereby inhibiting the expression level of macrophage inflammatory factors; or (2) By inhibiting the activation of TAK1 / TAB1, inhibiting the macrophage-mediated inflammatory response; or (3) By enhancing its own enzyme activity, inhibiting the macrophage-mediated inflammatory response; or (4) By inhibiting glycolysis, promoting oxidative phosphorylation, inhibiting ECAR, and promoting OCR; or (5) By inhibiting the remodeling of the macrophage metabolic network; or (6) By maintaining mitochondrial function to inhibit ROS activation.

5. The application according to claim 1, characterized in that, The dosage form of the drug includes one or more of: tablets, pills, powders, suspensions, gels, emulsions, creams, granules, capsules, suppositories, injections, sprays.

6. The application according to claim 1, characterized in that The drug also includes pharmaceutically acceptable excipients.

7. Use of leucine aminopeptidase 3 in the preparation of a diagnostic product for macrophage immune inflammation-mediated diseases.

8. The application according to claim 7, characterized in that The macrophage immune inflammation-mediated disease diagnostic product includes reagents, kits, chips or detection systems.

9. The application according to claim 7, characterized in that, The macrophage immune inflammation-mediated disease diagnostic product determines the disease progression of macrophage immune inflammation-mediated diseases by detecting the expression level of leucine aminopeptidase 3 in a sample.

10. The application according to claim 9, wherein, The expression level of leucine aminopeptidase 3 is negatively correlated with macrophage immune inflammation-mediated diseases.

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