Use of lcn2 in the preparation of a drug for alleviating the toxicity of microplastic exposure

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to application of Lcn2 in preparation of a medicine for relieving microplastic exposure toxicity. The present application discloses a mouse model of macrophage transplantation with Lcn2 knockout, and discloses a key regulation role of Lcn2 in microplastic exposure toxicity. When microplastics are exposed, the mouse model of macrophage transplantation with Lcn2 knockout can block the NF-kappa B-Lcn2 signal axis due to the lack or reduction of Lcn2 expression, reduce the release of proinflammatory factors, significantly reduce the vascular endothelial injury, systemic inflammation and multiple organ injury, and thus relieve the toxicity caused by microplastic exposure. Meanwhile, the present application proves that the targeted inhibition of the expression or function of Lcn2 in macrophages is an effective way to relieve the toxicity of microplastic exposure based on the above model. The present application provides direct experimental evidence for the development of an intervention means with Lcn2 as a target, promotes the research and development of related medicines, provides theoretical support for the preparation or screening of medicines for relieving microplastic toxicity, and has important application value for the prevention and treatment of health risks caused by microplastic pollution.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to the application of Lcn2 in the preparation of a drug for relieving the toxicity of microplastic exposure. BACKGROUND

[0002] Microplastics (MPs) generally refer to plastic particles with a particle size of less than 5 mm, as a new environmental pollutant, it is widely distributed in the ecological environment, and can enter the human body through diet, respiration and other ways, and poses a potential threat to health. Studies have shown that microplastic exposure can cause multi-system damage, especially in the digestive, immune and cardiovascular systems, but the intervention means for its toxicity is still very limited, resulting in a lack of targeted and mechanism-specific prevention and treatment drugs.

[0003] Microplastics show organ-specific accumulation in the body, and their concentration in tissues rich in macrophages such as the spleen and liver is significantly higher than that in other organs, which suggests that macrophages may mediate the toxic effects of microplastics. Lipocalin 2 (Lcn2) is an acute phase protein secreted by macrophages in large quantities, and plays an important role in inflammatory response and tissue damage. Although abnormal expression of Lcn2 has been reported to be associated with various inflammatory diseases, its role in the toxicity caused by microplastic exposure has not been clearly defined. SUMMARY

[0004] An object of the present application is to solve at least the above problems and / or defects, and to provide the application of Lcn2 in the preparation of a drug for relieving the toxicity of microplastic exposure.

[0005] The inventors found through mouse experiments that significant red fluorescence signals can be observed in the spleen, liver and aorta tissues, indicating that microplastics have undergone trans-barrier transfer and accumulation. Among them, the fluorescence signal of the spleen is the strongest, suggesting that it is the main target organ of microplastics. In contrast, almost no fluorescence signal is detected in the heart and kidney tissues, indicating that the deposition of microplastics in these organs is very low. The area rich in microplastics is where macrophages are distributed in large quantities. This distribution characteristic suggests that macrophages may play a key role in the in vivo transport, clearance and subsequent biological effects of microplastics. Subsequently, the inventors unexpectedly found in their research that by knocking out Lcn2 in macrophages, systemic inflammation and multi-organ damage caused by microplastic exposure can be reduced. Based on this, the inventors have completed the following invention.

[0006] The first aspect of the present application provides the application of Lcn2 in the preparation of a drug for relieving the toxicity of microplastic exposure. In this application, the toxicity includes but is not limited to vascular endothelial damage, systemic inflammation and multi-organ damage caused by microplastic exposure.

[0007] According to a preferred scheme of the present application, the application of Lcn2 in the preparation of a drug for alleviating the toxicity of microplastic exposure includes the following two aspects: (1) applying Lcn2 as a target to the preparation of a drug for alleviating the toxicity of microplastic exposure; and (2) applying Lcn2 as a target to the screening of a drug for alleviating the toxicity of microplastic exposure.

[0008] According to a preferred scheme of the present application, the application of Lcn2 as a target to the preparation of a drug for alleviating the toxicity of microplastic exposure includes applying Lcn2 as a target of a drug or preparation, and developing a drug or preparation for the toxicity of microplastic exposure, which can inhibit the expression of Lcn2 in macrophages.

[0009] According to a preferred scheme of the present application, the application of Lcn2 as a target to the screening of a drug for alleviating the toxicity of microplastic exposure includes applying Lcn2 as a target of a drug or preparation, and screening the drug or preparation to find a drug or preparation that can inhibit the expression of Lcn2 in macrophages as a drug or preparation for alleviating the toxicity of microplastic exposure.

[0010] According to a preferred scheme of the present application, the drug includes an agent that inhibits the expression of Lcn2 in macrophages. Further, the present inventors constructed a Lcn2 gene knockout macrophage stable strain by CRISPR-Cas9 technology, and found that knocking out Lcn2 can significantly reduce the damage effect of macrophages on vascular endothelial cells; at the same time, through a lentivirus-mediated Lcn2 interference experiment (the virus contains an interference sequence targeting the Lcn2 gene shown in SEQ ID No. 1), it was confirmed that inhibiting the expression of Lcn2 can reduce the secretion of Lcn2 in macrophages, thereby alleviating the toxicity caused by microplastic exposure.

[0011] According to a preferred scheme of the present application, other agents (such as nucleic acid molecules, carbohydrates, lipids, small molecule chemicals, antibody drugs, polypeptides, and proteins) that interfere with, inhibit, or silence Lcn2 can also obtain the same or similar effects through similar mechanisms. Therefore, the use of other agents that can interfere with, inhibit, or silence Lcn2 is within the scope of protection of the present application.

[0012] According to a preferred scheme of the present application, when the agent that interferes with, inhibits, or silences the expression of Lcn2 in macrophages is a nucleic acid molecule, the nucleic acid molecule includes but is not limited to: an antisense oligonucleotide, a double-stranded RNA, a small interfering RNA, or a short hairpin RNA. Such nucleic acid molecules can specifically target the Lcn2 gene (SEQ ID No. 1), interfere with, inhibit, or silence the expression of Lcn2, reduce the secretion of Lcn2 in macrophages, and thereby alleviate the toxicity caused by microplastic exposure.

[0013] According to a preferred scheme of the present application, the drug blocks the NF-κB-Lcn2 signaling axis by inhibiting the expression or secretion of Lcn2 in macrophages, specifically including inhibiting endoplasmic reticulum stress and activation of the NF-κB pathway in endothelial cells, restoring the tube formation ability of vascular endothelial cells; reducing the levels of pro-inflammatory factors (IL-6, IL-1β) in peripheral blood and tissues, reducing systemic inflammation; reducing inflammation infiltration and tissue structure destruction of target organs such as spleen and liver, and relieving multiple organ damage.

[0014] According to a preferred scheme of the present application, the drug also contains a pharmaceutically acceptable carrier, which includes but is not limited to stabilizers for maintaining the structural integrity and stability of the agent for inhibiting the expression of Lcn2 in macrophages, such as glucose, glycerol, etc.; transfection enhancers for enhancing the efficiency of the agent for inhibiting the expression of Lcn2 in macrophages into cells, such as polyethyleneimine; protective agents for protecting the stability and activity of the agent for inhibiting the expression of Lcn2 in macrophages during preparation and storage, such as cryogenic stabilizers, buffers; solvents and regulators for regulating the solubility of the agent for inhibiting the expression of Lcn2 in macrophages and the preparation process of the drug preparation, such as water, hydrochloric acid

[0015] The second aspect of the present application provides a Lcn2 knockdown or knockout macrophage transplanted mouse model (for resisting microplastic exposure toxicity) in screening drugs for alleviating microplastic exposure toxicity. The present application first proposes to construct a macrophage transplanted mouse model for resisting microplastic toxicity by knocking down or knocking out Lcn2, which will help to promote more research on drugs for alleviating microplastic toxicity, and provide a model basis for the research and development of drugs for treating and alleviating microplastic exposure toxicity.

[0016] According to a preferred scheme of the present application, the construction of the macrophage transplanted mouse model comprises:

[0017] S1, construct a knockout or knockdown plasmid targeting the Lcn2 gene shown in SEQ ID No. 1, and perform lentivirus packaging on the plasmid to obtain a lentivirus particle carrying a Lcn2 knockout or knockdown element;

[0018] S2, extract primary peritoneal macrophages from a donor mouse, introduce the lentivirus obtained in S1 into the peritoneal macrophages, and obtain Lcn2 gene knockdown or knockout macrophages through screening;

[0019] S3, continuously orally gavage microplastics to a recipient mouse for 30 days to construct a microplastic exposure mouse model;

[0020] S4, inject the Lcn2 gene knockdown or knockout macrophages in S2 into the microplastic exposure mouse model through the abdominal cavity to obtain a macrophage transplanted mouse model.

[0021] The present application at least includes the following beneficial effects compared with the prior art:

[0022] Firstly, the present application firstly reveals the key regulatory role of Lcn2 in the toxicity of microplastic exposure by constructing a macrophage transplantation mouse model with Lcn2 knockdown or knockout. Compared with the control mice transplanted with normal macrophages, in the mice transplanted with Lcn2 knockdown or knockout macrophages, due to the lack or reduction of Lcn2 expression, the release of pro-inflammatory factors is reduced through the NF-κB-Lcn2 signaling axis, which significantly reduces the vascular endothelial injury, systemic inflammation and multiple organ injury, thereby alleviating the toxicity caused by microplastic exposure.

[0023] Secondly, based on the research of the above-mentioned macrophage transplantation model, it is confirmed that targeted inhibition of the expression or function of Lcn2 in macrophages is an effective way to alleviate the toxicity of microplastic exposure. This finding provides direct experimental basis for the development of intervention means targeting Lcn2, promotes the research and development of drugs for alleviating the toxicity of microplastic exposure, provides theoretical basis for the preparation or screening of drugs and reagents for alleviating the toxicity of microplastics, and has important application value for the prevention and treatment of health risks caused by microplastic pollution.

[0024] Other advantages, objects and features of the present application will be partly embodied by the following description, and partly understood by those skilled in the art through research and practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a schematic diagram for the experimental design of Example 1 of the present application; Figure 2 It is a representative fluorescence microscope image of the spleen and liver frozen sections provided in Example 1 of the present application, wherein A is the spleen and B is the liver; Figure 3 It is a representative fluorescence microscope image of the aorta, heart and kidney frozen sections provided in Example 1 of the present application, wherein A is the aortic arch, B is the kidney and C is the heart.

[0026] Figure 4 It is a system analysis diagram of the transcriptome difference and functional enrichment induced by microplastic exposure provided in Example 2 of the present application, wherein A is a principal component analysis (PCA) diagram provided in Example 2 of the present application, B is a volcano plot of differentially expressed genes (DEGs) provided in Example 2 of the present application, C is a hierarchical clustering heat map of DEGs provided in Example 2 of the present application, and D is a GO functional enrichment analysis diagram provided in Example 2 of the present application; Figure 5 It is a KEGG pathway enrichment and gene set enrichment analysis diagram provided in Example 2 of the present application, wherein A is a KEGG pathway enrichment analysis provided in Example 2 of the present application, and B is a gene set enrichment analysis diagram provided in Example 2 of the present application; Figure 6A protein-protein interaction (PPI) network diagram provided in Example 2 of the present application.

[0027] Figure 7 A multi-dimensional analysis diagram of the response of macrophages to microplastics provided in Example 3 of the present application, wherein A is a phagocytosis diagram of macrophages to microplastics provided in Example 3 of the present application, B is a principal component analysis (PCA) diagram provided in Example 3 of the present application, and C is a volcano plot of differentially expressed genes (DEGs) provided in Example 3 of the present application. Figure 8 A transcriptome difference and functional enrichment analysis diagram of macrophages after phagocytosis of microplastics provided in Example 3 of the present application, wherein A is a hierarchical clustering heat map of DEGs provided in Example 3 of the present application, B is a GO functional enrichment analysis diagram provided in Example 3 of the present application, and C is a KEGG pathway enrichment analysis diagram provided in Example 3 of the present application. Figure 9 A gene set enrichment and protein diagram of macrophages responding to microplastics provided in Example 3 of the present application, wherein A is a gene set enrichment analysis diagram provided in Example 3 of the present application, and B is a protein-protein interaction (PPI) network diagram provided in Example 3 of the present application.

[0028] Figure 10 A transcription factor enrichment analysis diagram of up-regulated genes in macrophages treated with MPs provided in Example 4 of the present application. Figure 11 A bioinformatics prediction and experimental verification diagram of NF-kB regulating the Lcn2 gene in MPs-treated macrophages provided in Example 4 of the present application, wherein A is a bioinformatics prediction diagram of the NF-κB binding site in the promoter region of the mouse Lcn2 gene provided in Example 4 of the present application, B is a NF-κB binding motif predicted by the JASPAR database provided in Example 4 of the present application, and C is a diagram of the regulation of Lcn2 promoter by NF-κB verified by dual luciferase reporter gene experiment provided in Example 4 of the present application.

[0029] Figure 12 A schematic diagram of the flow of the macrophage adoptive transfer experiment provided in Example 5 of the present application. Figure 13 A diagram of the inter-group difference in the tube formation experiment for detecting the tube formation ability of HUVECs provided in Example 5 of the present application. Figure 14 A diagram of ELISA detection of the levels of inflammation and blood vessel regulation-related factors secreted by HUVECs provided in Example 5 of the present application. Figure 15 A diagram of Western Blot detection of the levels of IκBα and GRP78 proteins in HUVECs provided in Example 5 of the present application, wherein A is a diagram of Western Blot detection of the levels of IκBα proteins in HUVECs provided in Example 5 of the present application, and B is a diagram of Western Blot detection of the levels of GRP78 proteins in HUVECs provided in Example 5 of the present application.

[0030] Figure 16 Schematic diagram of the procedure of the macrophage adoptive transfer experiment provided for Example 6 of the present application; Figure 17 Representative HE staining images of the main organs of the mice in each group provided for Example 6 of the present application; Figure 18 ELISA detection of the protein levels of inflammatory factors IL-6 and IL-1β in the homogenates of the liver, spleen and kidney tissues of the mice in each group provided for Example 6 of the present application. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present application.

[0032] In the embodiments, the drugs and reagents are all ordinary commercially available products unless otherwise specified. In the embodiments, the experimental operations are all performed according to the conventional operations in the art unless otherwise specified.

[0033] Example 1, Distribution and localization of microplastics in the main organs of mice

[0034] 1.1 Experimental materials

[0035] Experimental animals: C57BL / 6 mice (6-8 weeks old, humidity 50%~60%, free water and food intake); microplastics: 1μm red fluorescent polystyrene microspheres (purity ≥99%), and the rest of the reagents are all ordinary commercially available products.

[0036] 1.2 Experimental methods

[0037] The mice were randomly divided into a control group (CON) and a microplastic exposure group (MP-TG), with 6 mice in each group.

[0038] See Figure 1 , the mice in the microplastic exposure group (MP-TG) were orally gavaged with 20mg / kg of 1μm red fluorescent polystyrene microspheres (MPs) per day, and the mice in the control group (CON) were gavaged with an equal amount of normal saline, for 30 consecutive days; 24h after the last treatment, the mice were sacrificed, and the spleen, liver, aorta, heart and kidney tissues were taken for preparation of frozen sections; the distribution of microplastics (red fluorescence) in each tissue was observed by fluorescence microscopy, the cell nuclei were counterstained with DAPI (blue), and the colocalization of microplastics and macrophage-rich areas was analyzed.

[0039] 1.3 Experimental results

[0040] Figure 2 and Figure 3Representative fluorescence microscopy images of frozen sections of various organs; Figure 2 In the diagram, A represents the spleen and B represents the liver. Figure 3 In the diagram, A represents the aortic arch, B represents the kidneys, and C represents the heart.

[0041] Observations showed that the spleen of mice in the microplastic exposure group (MP-TG) was... Figure 2 A) Liver ( Figure 2 B) Aorta ( Figure 3 A) shows a significant red fluorescent signal, indicating that microplastics have undergone cross-barrier migration and accumulation. Among them, the spleen ( Figure 2 A) showed the strongest fluorescence signal, suggesting it is a major target organ for microplastics. In contrast, the heart ( Figure 3 C) Kidneys ( Figure 3 The near absence of fluorescence signal in B) indicates extremely low levels of microplastic deposition in these organs. Further observation in the spleen ( Figure 2 Microplastics accumulated in A) are mainly concentrated in the red marrow region. Similarly, in the liver ( Figure 2 A significant distribution of microplastic particles was also observed in sample B). Notably, the areas of microplastic accumulation (spleen red pulp and liver) are precisely where macrophages are abundant. This distribution characteristic suggests that macrophages may play a crucial role in the in vivo transport, clearance, and subsequent biological effects of microplastics.

[0042] Example 2: Effects of microplastic exposure on the splenic transcriptome and immune inflammatory pathways in mice

[0043] 2.1 Experimental Materials

[0044] Experimental animals: Same as mice in Example 1; all other reagents were conventional commercial products.

[0045] 2.2 Experimental Methods

[0046] The control group (CON) and the microplastic exposure group (MPs) were set up, with 3 biological replicates in each group. Mice in the microplastic exposure group (MPS) were given microplastics (1μm red fluorescent polystyrene microspheres, 20mg / kg / day) by gavage for 30 days.

[0047] Spleen tissues from mice were collected, total RNA was extracted and quality controlled; RNA-seq was performed using the Illumina platform to screen differentially expressed genes (DEGs, |log2FC|>1, P<0.05); GO functional enrichment analysis, KEGG pathway analysis, GSEA enrichment analysis and PPI network construction were performed on DEGs.

[0048] 2.3 Experimental Results

[0049] Figure 4In Fig. 1, A is a principal component analysis (PCA) plot; B is a volcano plot of differentially expressed genes (DEGs); C is a hierarchical clustering heatmap of DEGs; D is a GO functional enrichment analysis plot; Figure 5 In Fig. 2, A is a KEGG pathway enrichment analysis; B is a gene set enrichment analysis plot; Figure 6 Fig. 3 is a protein-protein interaction (PPI) network plot.

[0050] Figure 4 The principal component analysis result showed that the microplastic exposure group (MPs) and the control group (CON) showed clear separation in the transcriptional profile, and the two groups of samples were clustered on the two sides of the PC1 axis without overlapping area. This indicates that the oral intake of microplastics can cause significant and consistent changes in the overall gene expression pattern of the spleen;

[0051] Figure 4 B The differential expression analysis showed that there were 289 differentially expressed genes in the microplastic exposure group (MPs) compared with the control group (CON), of which 175 genes were up-regulated and 114 were down-regulated, which further confirmed the findings in A; Figure 4

[0052] Figure 4 C The hierarchical clustering heatmap of DEGs showed the expression pattern of 289 DEGs between the two groups (red: high expression, blue: low expression), which confirmed the significant difference between the groups and the good consistency within the groups. It intuitively showed the different expression patterns of DEGs between the two groups and verified the good repeatability of the samples within the groups.

[0053] Figure 4 D The GO functional enrichment analysis showed that DEGs were significantly enriched in “inflammatory response”, “immune response”, “extracellular region” and “antigen binding” and other items. This result strongly proves that the spleen has an active immune response to microplastic exposure.

[0054] Figure 5 A The KEGG pathway enrichment analysis showed that the differentially expressed genes were significantly enriched in “Phagosome” and “Fc gamma R-mediated phagocytosis” and other pathways, indicating the activation of macrophage function.

[0055] Figure 5 ​B Gene Set Enrichment Analysis shows that "cytokine-cytokine receptor interaction (CYTOKINE-CYTOKINE RECEPTOR INTERACTION, Figure 5 B corresponds to orange) and "TGF-beta signaling pathway (TGF-BETA SIGNALING PATHWAY, Figure 5 B corresponds to blue) and other key immunomodulatory pathways are significantly enriched in the microplastic exposure group (MPs). These findings collectively point to the possibility that microplastics may exert their biological effects by activating the phagocytic function of macrophages in the spleen and the associated immune signaling network.

[0056] Figure 6 In the protein-protein interaction (PPI) network, the key node gene Lcn2 (lipocalin 2, indicated by an arrow in the figure) is highlighted. Lcn2 is not only significantly up-regulated, but also interacts with multiple genes in this immune network. Given that Lcn2 is a recognized acute phase protein secreted by immune cells such as macrophages in an inflammatory state, its prominent position in the network suggests that it may be a key effector molecule mediating the inflammatory damage of the spleen caused by microplastics.

[0057] Example 3: Direct effect of microplastics on macrophages and the core regulatory position of Lcn2

[0058] 3.1 Experimental materials

[0059] Mouse primary peritoneal macrophages: extracted from C57BL / 6 mice peritoneal cavity according to standard operating procedures; microplastics: same as in Example 1; and the rest of the reagents, etc. are all conventional commercially available products.

[0060] 3.2 Experimental methods

[0061] S1, Construct an in vitro model of macrophages co-cultured with microplastics (MPs):

[0062] Set up a control group (CON, containing only macrophages and culture medium, with a microplastic concentration of 0) and a microplastic exposure group (MPs, macrophages co-cultured with microplastic particles). In the microplastic exposure group, add fluorescently labeled microplastic particles with a final concentration of 100 μg / mL, 300 μg / mL, and 500 μg / mL, and co-incubate with peritoneal macrophages for 24 hours. Evaluate the phagocytic capacity of peritoneal macrophages for different concentrations of microplastics (MPs, 100 μg / mL, 300 μg / mL, 500 μg / mL) to determine the optimal exposure dose.

[0063] S2, Transcriptomics and functional network analysis:

[0064] Control group (CON, untreated macrophages) and microplastics exposure group (MPs, 300 μg / mL, macrophages treated with microplastics for 24 h), each group with 3 biological replicates; followed by transcriptome sequencing, differential expression gene (DEGs) analysis, functional enrichment analysis, PPI network construction.

[0065] 3.3 Experimental results

[0066] Figure 7 Figure 2. Macrophage phagocytosis of microplastics, principal component analysis (PCA), and differential expression genes (DEGs) analysis. A, phagocytosis of microplastics by macrophages; B, principal component analysis (PCA); C, volcano plot of differential expression genes (DEGs); Figure 8 Figure 3. Hierarchical clustering heatmap of DEGs, GO functional enrichment analysis, and KEGG pathway enrichment analysis. A, hierarchical clustering heatmap of DEGs; B, GO functional enrichment analysis; C, KEGG pathway enrichment analysis; Figure 9 Figure 4. Gene set enrichment analysis and protein-protein interaction (PPI) network. A, gene set enrichment analysis; B, protein-protein interaction (PPI) network;

[0067] Figure 7 Figure 2. Macrophage phagocytosis of microplastics, principal component analysis (PCA), and differential expression genes (DEGs) analysis. A, phagocytosis of microplastics by macrophages; B, principal component analysis (PCA); C, volcano plot of differential expression genes (DEGs);

[0068] Figure 7 Figure 2. Macrophage phagocytosis of microplastics, principal component analysis (PCA), and differential expression genes (DEGs) analysis. A, phagocytosis of microplastics by macrophages; B, principal component analysis (PCA); C, volcano plot of differential expression genes (DEGs);

[0069] Figure 7 Figure 2. Macrophage phagocytosis of microplastics, principal component analysis (PCA), and differential expression genes (DEGs) analysis. A, phagocytosis of microplastics by macrophages; B, principal component analysis (PCA); C, volcano plot of differential expression genes (DEGs);

[0070] Figure 8 Figure 3. Hierarchical clustering heatmap of DEGs, GO functional enrichment analysis, and KEGG pathway enrichment analysis. A, hierarchical clustering heatmap of DEGs; B, GO functional enrichment analysis; C, KEGG pathway enrichment analysis;

[0071] Figure 8B GO functional enrichment analysis showed that the up-regulated DEGs were mainly enriched in "inflammatory response", "response to lipopolysaccharide" and "response to cytokine", and other biological processes closely related to macrophage activation and inflammatory functions;

[0072] Figure 8 C KEGG pathway analysis further revealed that these DEGs were significantly enriched in classical inflammatory signaling pathways, including "Cytokine-cytokine receptor interaction", "TNF signaling pathway" and "MAPK signaling pathway";

[0073] Figure 9 A Gene Set Enrichment Analysis (GSEA) further confirmed the activation of immune-related pathways, such as "LEUKOCYTE_TRANSENDOTHELIAL_MIGRATION, Figure 9 corresponding to green in A)", and suggested the possibility of metabolic reprogramming, such as the enrichment of "OXIDATIVE_PHOSPHORYLATION, Figure 9 corresponding to blue in A)" pathway.

[0074] Figure 9 B In the protein-protein interaction (PPI) network, the results of the analysis were highly consistent with the findings in the spleen, again positioning Lcn2 (position indicated by the arrow in the figure) as a key central node. In the network, Lcn2 interacted closely with multiple important inflammation-related genes such as Tnf and Sqstm1. Lcn2 showed significant up-regulation and was in the central position of the regulatory network both in vivo (spleen) and in vitro (macrophages), indicating that Lcn2 was a key effector molecule mediating the activation of macrophages and subsequent inflammatory cascade reactions induced by microplastics.

[0075] Example 4, NF-κB regulates the expression of Lcn2 by binding to its promoter

[0076] 1) Transcription factor enrichment analysis predicts core regulatory factors

[0077] Based on the transcriptome sequencing results of Example 3, the differentially expressed genes (DEGs) up-regulated in macrophages after microplastics (MPs) treatment were analyzed, and the enriched analysis of known transcription factor binding motifs for the differentially expressed genes (DEGs) up-regulated after microplastics (MPs) treatment was performed using Homer software;

[0078] The results are shown in Figure 10 , Figure 10 The transcription factor enrichment analysis of the up-regulated genes in the MPs-treated macrophages showed that the binding motif of NF-κB / RelA (p65) was most significantly enriched in the promoter regions of these genes, which strongly suggested that NF-κB was the core transcription factor mediating the microplastic-induced inflammatory response of macrophages;

[0079] 2) Functional experiment to verify the direct regulation of NF-κB on Lcn2

[0080] Since lipocalin 2 (Lcn2) is a key gene that is significantly up-regulated in both in vivo and in vitro models, it is hypothesized that its expression is directly regulated by NF-κB, and the direct regulation of NF-κB on Lcn2 is verified by the following experiments;

[0081] Bioinformatics prediction: please refer to Figure 11 A, the potential NF-κB binding site in the promoter region of murine Lcn2 was determined by JASPAR database; please refer to Figure 11 B, which shows the sequence motif of the predicted NF-κB binding site in the Lcn2 promoter region;

[0082] Dual luciferase reporter experiment verification: based on the predicted NF-κB binding site, pcDNA3.1 was used as a blank, and wild-type (WT) and mutant (MUT) Lcn2 promoter reporter plasmids were constructed according to the standard procedure; and wild-type (WT) and mutant (MUT) Lcn2 promoter reporter plasmids were co-transfected with p65 overexpression plasmid (purchased from Addgene #23255 (p65-EGFP-N1)) into cells;

[0083] The results are shown in Figure 11 C, pcDNA3.1-NC in the figure is the transfection of blank plasmid pcDNA3.1 group; pcDNA3.1-Lcn2 is the transfection of wild-type (WT) Lcn2 promoter reporter plasmid; pcDNA3.1-Lcn2+NFκB-WT is the transfection of p65 overexpression plasmid and wild-type (WT) Lcn2 promoter reporter plasmid group; pcDNA3.1-Lcn2+NFκB-MUT is the transfection of p65 overexpression plasmid and mutant (MUT) Lcn2 promoter reporter plasmid group.

[0084] Results showed that p65 overexpression plasmid significantly enhanced the activity of wild type (WT) Lcn2 promoter reporter plasmid, while the effect was significantly inhibited after the binding site was mutated. Data are expressed as mean ± SD (P <0.05). This result provides direct experimental evidence to confirm that NF-κB positively regulates the transcription of Lcn2 gene by binding to specific sites on the promoter of Lcn2 gene.

[0085] Example 5, Effect of Macrophage NF-κB-Lcn2 Axis on Endothelial Cell Function

[0086] 5.1 Experimental materials

[0087] Macrophages (Raw264.7); HUVECs (CRL-1730); the rest of the reagents are conventional commercial products.

[0088] 5.2 Experimental methods, please refer to Figure 12 , including:

[0089] S1, construction of gene knockout macrophage strain

[0090] Knock out the NF-κB (RelA) gene and Lcn2 gene in wild type macrophages (Raw264.7) respectively, obtain NF-κB gene knockout macrophages (NF-κB-KO) and Lcn2 gene knockout macrophages (Lcn2-KO); The specific steps are as follows:

[0091] 1) Design of gRNA

[0092] Using CRISPR-Cas9 technology, according to the specificity of gene sequence and off-target risk screening target sites, gRNA is designed for Lcn2 gene (SEQ ID No. 1) and NF-κB (RelA), denoted as gRNA1 and gRNA2; Among them, the sequences of gRNA1 and gRNA2 are as follows:

[0093] gRNA1 (SEQ ID NO. 2): 5'-GGCGAACTGGTTGTAGTCCG-3';

[0094] gRNA2 (SEQ ID NO. 3): 5'-TCATCGAACAGCCGAAGCAA-3';

[0095] 2) Synthesis and verification of gRNA

[0096] Specifically entrusted to Shengye (Suzhou) Biotechnology Co., Ltd.;

[0097] Lentiviral expression vectors containing gRNA1-Cas9 and gRNA2-Cas9 were constructed respectively; at the same time, specific PCR primers were designed for subsequent verification of gene knockout efficiency, and the primer information is as follows:

[0098] Table 1 Lcn2 gene knockout verification PCR primers

[0099]

[0100] Table 2 NF-κB (RelA) gene knockout verification PCR primers

[0101]

[0102] 3) Cell transfection and screening

[0103] Macrophages (Raw264.7) were divided into two groups and infected with lentivirus containing gRNA1-Cas9 and lentivirus containing gRNA2-Cas9, respectively. After single clone screening, PCR sequencing verification (using primers in Table 1 and Table 2) and Western blot protein level verification, NF-κB gene knockout macrophages and Lcn2 gene knockout macrophages with stable knockout were obtained.

[0104] S2, macrophages treated with microplastics (MPs) and collection of supernatant

[0105] Wild-type macrophages, NF-κB (RelA) gene knockout macrophages (NF-κB-KO), and Lcn2 gene knockout macrophages (Lcn2-KO) were inoculated in culture plates and treated with culture medium containing 300 μg / mL microplastics (1 μm red fluorescent polystyrene microspheres) for 24 h to simulate microplastic exposure. After treatment, the conditioned supernatant of each group of macrophages was collected, centrifuged to remove cell debris, and aliquoted for storage.

[0106] S3, co-culture of vascular endothelial cells (HUVECs) and macrophage supernatant

[0107] HUVECs were inoculated in culture plates, and after the cells adhered, the original culture medium was discarded. The following supernatants were added to treat HUVECs: WT group: supernatant of wild-type macrophages (Raw264.7) treated with microplastics; NF-κB-KO group: supernatant of NF-κB-KO macrophages treated with microplastics; Lcn2-KO group: supernatant of Lcn2-KO macrophages treated with microplastics; and a microscope was used to observe and record the formation of capillary-like structures at 2h, 6h, 12h and 24h after culture.

[0108] 5.3 Endothelial cell function and molecular mechanism detection

[0109] 1) Collect the culture medium of HUVECs treated by WT group, NF-κB-KO group, Lcn2-KO group, and mark them as WT+MP group, NF-κB-KO+MP group and Lcn2-KO+MP group, and use ELISA to detect the levels of pro-inflammatory factors (IL-6, IL-1β, PGE2), anti-angiogenic factor (Ang-2), pro-fibrotic factor (TGF-β), pro-angiogenic factor (VEGF), oxidative stress marker (8-OHdG) and part of chemotactic factor (IL-8).

[0110] 2) Collect the culture medium of HUVECs treated by wild type macrophages and mark them as WT+MP, and use Western Blot to detect the following pathway markers: endoplasmic reticulum stress pathway (marker GRP78), endothelial cell's own NF-κB pathway (marker IκBα).

[0111] 5.4 Experimental results

[0112] Figure 13 To detect the differences in the tube formation ability of HUVECs between groups for the vascularization experiment; Figure 14 To detect the levels of inflammation and blood vessel regulation related factors secreted by HUVECs by ELISA;

[0113] Figure 13 The results of the vascularization experiment showed that the supernatant of wild type (WT) macrophages treated with microplastics could significantly inhibit the tube formation ability of HUVECs. However, when HUVECs were cultured with the supernatant from NF-κB-KO or Lcn2-KO macrophages (also treated with microplastics), their tube formation ability at 6, 12 and 24h was significantly restored, far superior to the WT supernatant treatment group. This result strongly proves that the activation of NF-κB and the secretion of Lcn2 in macrophages are necessary conditions for MPs to indirectly damage the vascularization function of endothelial cells.

[0114] Figure 14 The ELISA results showed that compared with the wild type (WT) macrophage supernatant group, the supernatant from NF-κB-KO and Lcn2-KO macrophages could significantly reduce the secretion levels of pro-inflammatory factors (IL-6, IL-1β, PGE2), anti-angiogenic factor (Ang-2) and pro-fibrotic factor (TGF-β) in HUVECs, while restoring the expression of the key pro-angiogenic factor VEGF. Notably, the oxidative stress marker 8-OHdG and part of the chemotactic factor (IL-8) did not show significant changes, suggesting that Lcn2 from macrophages may damage endothelial cells through specific inflammation and blood vessel regulation pathways rather than directly inducing oxidative stress.

[0115] Figure 15 Western Blot detection results of A and B show that the supernatant of microplastic-treated WT macrophages can significantly induce endoplasmic reticulum stress of HUVECs, which is manifested as the increase of protein level of stress marker GRP78 Figure 15 A). At the same time, the supernatant also activates the NF-κB pathway of HUVECs itself, which is manifested as the significant degradation of NF-κB inhibitor IκBα Figure 15 B).

[0116] In summary, this series of gene knockout and functional rescue experiments, from cell function to molecular mechanism level, completely verify the previous hypothesis that the "NF-κB-Lcn2" signal axis in macrophages is the core mechanism of mediating the paracrine effect of microplastics and causing vascular endothelial injury.

[0117] Example 6, Lcn2 alleviates the damage of microplastic-exposed mouse tissues

[0118] 6.1 Experimental method, please refer to Figure 16 , including:

[0119] S1, extract primary macrophages from donor mice: according to the standard operation procedure;

[0120] S2, construct Lcn2 gene knockout macrophage cell line: same as Example 5, so it is not repeated here;

[0121] S3, establish microplastic-exposed model mice: continuously gavage MP (20 mg / kg / day, 1 μm red fluorescent polystyrene microspheres) for 30 days

[0122] S4, macrophage reinfusion: the microplastic-exposed model mice are further divided into 3 groups (8 in each group):

[0123] NC group: microplastic-exposed model mice without treatment;

[0124] WT group: intraperitoneal injection of WT primary macrophages;

[0125] Lcn2-KO group: intraperitoneal injection of Lcn2 gene knockout macrophages;

[0126] After reinfusion, continue to feed normally for 7 days, then sacrifice and sample to evaluate the damage of the recipient mice tissues.

[0127] 6.2, tissue damage and molecular level detection

[0128] 1) Histopathological analysis (HE staining): sacrifice the mice, collect the main target organs (liver, spleen, kidney, aorta); after tissue fixation, paraffin embedding and sectioning, perform HE staining;

[0129] 2) Measurement of pro-inflammatory cytokines (ELISA): The liver, spleen, and kidney tissues were homogenized and the supernatant was collected. The concentrations of pro-inflammatory cytokines IL-6 and IL-1 β in the tissues were measured by ELISA kits to reflect the degree of inflammatory response.

[0130] 6.3 Experimental results

[0131] Figure 17 Representative HE staining images of major organs (liver, spleen, kidney, and aorta) of mice in each group; Figure 18 Protein levels of inflammatory cytokines IL-6 and IL-1 β in the homogenate of liver, spleen, and kidney tissues of mice in each group were detected by ELISA;

[0132] Figure 17 Histopathology (HE staining) analysis showed that microplastic exposure could cause significant pathological damage to the main target organs. In the microplastic model group (NC) mice, the liver lobule structure was severely damaged, accompanied by necrosis and congestion in the central vein area. The normal structure of the spleen was also severely affected, showing extensive hemorrhage and cell necrosis. The kidney showed obvious glomerular lesions, renal tubular damage, and interstitial inflammatory cell infiltration. In addition, the adventitia of the aorta also showed significant inflammation. It is worth noting that the reinfusion of WT macrophages showed a slight alleviating effect on tissue damage, but the pathological improvement was limited. In contrast, the reinfusion of Lcn2 knockout macrophages could more significantly alleviate the tissue damage caused by MPs, and the tissue structure of the liver, spleen, kidney, and aorta in this group of mice was significantly improved, closer to the normal state. These morphological observation results showed that Lcn2 secreted by macrophages is an important factor in mediating MP-induced multi-organ damage.

[0133] Figure 18 ELISA detection of key pro-inflammatory cytokines showed that compared with the MP model group (NC) without cell reinfusion and the WT macrophage reinfusion group, the reinfusion of Lcn2-KO macrophages could significantly reduce the levels of IL-6 and IL-1 β in the liver and spleen tissues. In the kidney, the level of IL-6 was also significantly reduced, while the decline in IL-1 β did not reach statistical significance, which may suggest that in the kidney tissue, MP-induced IL-1 β production may also involve other non-Lcn2-dependent regulatory pathways. Even so, the overall data clearly supports that blocking the Lcn2 secretion of macrophages can effectively inhibit the inflammatory response induced by MPs in target organs.

[0134] In summary, this series of in vivo functional rescue experiments provide strong in vivo support for the core role of the "macrophage-Lcn2" axis in mediating systemic inflammation and multi-organ pathological damage caused by microplastic exposure; provide direct experimental basis for the development of intervention means targeting Lcn2, which can promote the research and development of drugs to alleviate the toxicity of microplastic exposure, provide a theoretical basis for the preparation or screening of drugs and reagents to alleviate the toxicity of microplastics, and have important application value for the prevention and treatment of health risks caused by microplastic pollution.

[0135] Although embodiments of the present application have been disclosed as above, they are not limited only to the uses listed in the specification and embodiments, and can be fully applied to various fields suitable for the present application, and additional modifications can be easily made by those skilled in the art, and therefore the present application is not limited to specific details and the figures shown and described herein, without departing from the general concept defined by the claims and the equivalent scope.

Claims

1. Use of a viral vector carrying a coding sequence of an Lcn2 gene interference element in the preparation of a drug for alleviating the toxicity of microplastic exposure, wherein the viral vector carries a coding sequence of an interference element which is an sgRNA for an Lcn2 gene, the sequence of the Lcn2 gene is SEQ ID No. 1, and the sequence of the sgRNA is SEQ ID No.

2.

2. Use according to claim 1, characterized in that, The toxicity of microplastic exposure includes vascular endothelial injury, systemic inflammation, and multiple organ injury.

3. Use according to claim 1, characterized in that, The drug alleviates the vascular endothelial injury, systemic inflammation, and multiple organ injury caused by microplastic exposure by inhibiting the expression or secretion of Lcn2 in macrophages and blocking the NF-κB-Lcn2 signaling axis.

4. Use according to claim 1, characterized in that, The drug also contains a pharmaceutically acceptable carrier, which includes a diluent, an excipient, or a solvent.

5. Use of Lcn2 as a target for screening drugs for alleviating the toxicity of microplastic exposure, characterized in that, The sequence of Lcn2 is SEQ ID No.

1.

6. Use according to claim 5, characterized in that, The drug is screened by taking Lcn2 as the action target of the drug to find a drug that can inhibit the expression or secretion of Lcn2 in macrophages as a drug for alleviating the toxicity of microplastic exposure.

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