Application of Mgst2 as target spot in preparation of rheumatoid arthritis related interstitial lung disease treatment product

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of an Mgst2 gene serving as a target to preparation of a product for treating rheumatoid arthritis related interstitial lung diseases. According to the invention, it is found for the first time that Mgst2 can be used as a biomarker for treatment of rheumatoid arthritis related interstitial lung diseases, targeted knock-down is carried out on Mgst2 in neutrophils through an AAV9-shRNA vector (AAV9-shRNA-Mgst2) driven by a Cd11b promoter, and lung lesion progress can be effectively relieved by inhibiting NET formation. The Mgst2 inhibitor provided by the invention can directly intervene in the occurrence and development process of diseases from a molecular level, and has the advantages of accurate effect, remarkable curative effect, low side effect and the like. Compared with a traditional non-specific treatment mode, the product takes effect only aiming at specific molecular targets, damage to normal cells and tissues is remarkably reduced, and therefore the risk of toxic and side effects is reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to application of Mgst2 as a target point in preparation of a treatment product for rheumatoid arthritis-related interstitial lung disease. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a chronic, systemic inflammatory autoimmune disease, and its main pathological manifestations are inflammation and destruction of the synovial membrane of the joint. However, RA is not limited to the joint, but can also affect multiple organ systems, among which the lung is one of the most commonly affected organs. RA-associated interstitial lung disease (RA-ILD) is one of the most severe pulmonary complications of RA, and is characterized by extensive fibrosis, inflammatory infiltration of the lung interstitium, and gradual decline in lung function. Patients can have symptoms such as progressive dyspnea and dry cough, and severe cases can lead to respiratory failure and even death.

[0003] Currently, the treatment of RA-ILD mainly relies on immunosuppressive agents (such as glucocorticoids, methotrexate, cyclophosphamide, etc.) and anti-fibrotic drugs (such as pirfenidone, nintedanib, etc.). Although these methods can slow down the progression of inflammation and fibrosis to some extent, there are still significant shortcomings: on the one hand, long-term use of immunosuppressive agents can easily cause infection and other systemic side effects; on the other hand, the existing anti-fibrotic drugs have limited efficacy and are difficult to significantly improve lung function or reverse the fibrosis process. In addition, due to the complexity and heterogeneity of the pathological mechanism of RA-ILD, the existing treatment regimens lack specificity and precision, and the prognosis of patients is still not ideal.

[0004] Therefore, it is of great clinical significance to develop new, safe and effective treatment products for RA-ILD. SUMMARY

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide the use of Mgst2 as a target point in the preparation of a treatment product for rheumatoid arthritis-related interstitial lung disease.

[0006] In order to achieve the above-mentioned purposes and other related purposes, the present application adopts the following technical solutions:

[0007] The present application provides, in a first aspect, the use of Mgst2 gene as a target for the preparation of a treatment product for rheumatoid arthritis-related interstitial lung disease.

[0008] The Mgst2 gene as a target for preparing a rheumatoid arthritis associated interstitial lung disease treatment product specifically refers to: designing biological agents that can inhibit the function / expression of Mgst2, as a rheumatoid arthritis associated interstitial lung disease treatment product.

[0009] Microsomal glutathione S-transferase 2 (MGST2) is encoded by Mgst2 gene, belonging to MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) family. This family contains several targets that are crucial for the development of anti-inflammatory and anti-tumor drugs, especially involving the regulation of prostaglandin and leukotriene biosynthesis. In a mouse model of kidney injury, MGST2 elevation can exacerbate oxidative stress-related cell damage by promoting endoplasmic reticulum stress, DNA damage, and apoptosis. The present application identifies a MGST2-high-expressing neutrophil subpopulation (Mgst2hi Neu) through single-cell RNA sequencing (scRNA-seq), which releases NETs through the NOX2 signaling pathway; further found that these NETs promote pericyte to myofibroblast transformation (PMT) through TGF-β signal. The formation of neutrophil extracellular traps (NETs) is involved in the occurrence and development of rheumatoid arthritis associated interstitial lung disease (RAILD).

[0010] The optional Mgst2 gene is a mammalian gene. For example, human, mouse. The Genbank number of human Mgst2 gene is: KR711145.1, and the Genbank number of mouse Mgst2 gene is NCBI Reference Sequence: NM_001310482.1.

[0011] Optionally, the Mgst2 gene is derived from neutrophils.

[0012] Preferably, the neutrophils are a Mgst2-high-expressing neutrophil subpopulation.

[0013] The Mgst2-high-expressing neutrophil subpopulation refers to a type of neutrophil in which the expression level of the Mgst2 gene and / or its encoded product is significantly higher than the normal reference range or background level in a neutrophil population derived from a test individual. And in the application, it should mean at least 5%, 10% or 20% higher than the normal reference range or background level, preferably at least 30% or 50% higher, more preferably at least 80% or 100% or more significantly higher.

[0014] Optionally, the rheumatoid arthritis associated interstitial lung disease treatment product comprises a Mgst2 inhibitor.

[0015] The Mgst2 inhibitor generally includes a substance capable of inhibiting the transcription or translation of the Mgst2 gene, or capable of inhibiting the expression or activity of the Mgst2 protein. The administration amount of the therapeutic product is a dose sufficient to inhibit the transcription or translation of Mgst2, or sufficient to inhibit the expression or activity of the Mgst2 protein. The expression of the Mgst2 gene is reduced by at least 50%, 80%, 90%, 95%, or 99%.

[0016] Optionally, the Mgst2 inhibitor is selected from a nucleic acid molecule, a small molecule chemical drug, an antibody drug, a polypeptide, a protein, a nucleic acid construct, a lentivirus, an adenovirus, or a CRISPR / Cas9 genome editing system.

[0017] The form of the product is not particularly limited, and can be various forms of substances such as solids, liquids, gels, semi-liquids, and aerosols.

[0018] The second aspect of the present application provides the use of the Mgst2 inhibitor in the preparation of a therapeutic product for rheumatoid arthritis-related interstitial lung disease.

[0019] The target Mgst2 gene of the Mgst2 inhibitor is derived from neutrophils.

[0020] Preferably, the neutrophils are a subpopulation of neutrophils with high expression of Mgst2.

[0021] The Mgst2 inhibitor generally includes a substance capable of inhibiting the transcription or translation of the Mgst2 gene, or capable of inhibiting the expression or activity of the Mgst2 protein. The administration amount of the therapeutic product is a dose sufficient to inhibit the transcription or translation of Mgst2, or sufficient to inhibit the expression or activity of the Mgst2 protein. The expression of the Mgst2 gene is reduced by at least 50%, 80%, 90%, 95%, or 99%.

[0022] The Mgst2 inhibitor refers to a molecule having an inhibitory effect on the Mgst2 gene.

[0023] The Mgst2 inhibitor is the only effective component or one of the effective components of the product.

[0024] Further, the Mgst2 inhibitor has at least one of the following effects:

[0025] (1) treating rheumatoid arthritis-related interstitial lung disease;

[0026] (2) improving inflammatory cell infiltration and extracellular matrix deposition in lung tissue;

[0027] (3) reducing the expression of any one or more of NOX2 (GenBank: KJ596435.1), PADI4 (GenBank: KJ898514.1), or Cit-H3 (GenBank: HM485559.1);

[0028] (4) reducing the expression of MPO-DNA in the lung alveoli. MPO-DNA refers to the complex of myeloperoxidase (MPO) and DNA. Myeloperoxidase is an enzyme produced by neutrophils, monocytes, and macrophages, which mainly participates in immune response, helping to resist the invasion of pathogens. In the study, the MPO-DNA complex is used as an indicator to detect the level of neutrophil extracellular traps (NETs), which are network structures of DNA and antibacterial proteins released by neutrophils, involved in inflammation and immune response.

[0029] (5) improving the nuclear membrane rupture, chromatin decondensation, and extracellular fiber structure formation in lung tissue;

[0030] (6) reducing the formation of NETs;

[0031] (7) reducing the conversion of pericytes to myofibroblasts;

[0032] (8) reducing the expression of ACTA2 and / or COL1A1.

[0033] Preferably, in the present application, "improving the inflammatory cell infiltration and extracellular matrix deposition in lung tissue" means that the abnormal aggregation and infiltration of inflammation-related cells (including but not limited to neutrophils, macrophages, lymphocytes, etc.) in lung tissue are significantly reduced by the inhibitor of the present application, thereby reducing the tissue damage caused by inflammatory reaction. At the same time, the improvement also includes the inhibition or reversal of the abnormal deposition of extracellular matrix (ECM) in lung tissue, which is specifically manifested as the reduction of the content or the normal distribution of matrix components such as collagen, fibronectin, and laminin. Through this effect, the inflammatory reaction and fibrosis process of lung tissue can be effectively alleviated, and the alveolar structure integrity and lung function stability can be maintained. For example, based on the lung tissue of the subject before treatment, the product can make the number of inflammation-related cells in the lung tissue of the subject decrease by at least 30%, 50%, 70%, 80%, 90%, or 100%.

[0034] The number of inflammation-related cells in the lung tissue of the subject decreases by at least 30%, 50%, 70%, 80%, 90%, or 100%.

[0035] Reducing the expression of any one or more of NOX2, PADI4, or Cit-H3 means inhibiting the expression or activity of any one or more of NOX2, PADI4, or Cit-H3. The amount of the therapeutic product administered is a dose sufficient to inhibit the transcription or translation of any one or more of NOX2, PADI4, or Cit-H3, or a dose sufficient to inhibit the expression or activity of any one or more of NOX2, PADI4, or Cit-H3. The expression of any one or more of NOX2, PADI4, or Cit-H3 is reduced by at least 50%, 80%, 90%, 95%, or 99%.

[0036] Reducing the expression of MPO-DNA in alveoli means a substance that inhibits the transcription or translation of extracellular DNA in alveoli and inhibits the expression or activity of MPO protein. The amount of the therapeutic product administered is a dose sufficient to inhibit the transcription or translation of extracellular DNA in alveoli, a dose sufficient to inhibit the expression or activity of MPO protein. The expression of MPO-DNA is reduced by at least 30%, 50%, 80%, 90%, 95%, or 99%.

[0037] The "improving the nuclear membrane rupture, chromatin decondensation, and extracellular fiber structure formation in lung tissue" of the present application means inhibiting the key pathological changes in the process of neutrophil extracellular traps (NETs) formation by means of drug or gene intervention. The process generally includes: nuclear membrane rupture: the nuclear membrane structure of neutrophils is damaged under abnormal activation, and the nuclear content is released into the cytoplasm; chromatin decondensation: chromatin loses its normal compressed state and diffuses and expands; extracellular fiber structure formation: decondensed DNA and granular proteins such as myeloperoxidase (MPO) and neutrophil elastase (NE) are released to form a fibrous network structure.

[0038] "Improving" means reducing the frequency or degree of the above-mentioned process, thereby reducing the over-formation of NETs and reducing the inflammatory response and fibrotic damage of lung tissue. For example, based on healthy lung tissue, the product can restore the nuclear membrane, chromatin, and extracellular fibers of the target lung tissue to at least 30%, 50%, 70%, 80%, 90%, or 100% of the healthy lung tissue.

[0039] Reducing the formation of NETs means inhibiting the process of neutrophil extracellular traps released by neutrophils in lung tissue under abnormal activation by the inhibitor of the present application. For example, based on the neutrophil NETs of the target lung tissue before treatment, the product can reduce the neutrophil NETs of the target lung tissue by at least 30%, 50%, 70%, 80%, 90%, or 100%.

[0040] Reducing the pericyte-to-myofibroblast transition refers to reducing or blocking the process of pericytes located in the wall of microvessels being abnormally activated, migrating and differentiating into myofibroblasts under tissue injury or pathological stimulation by the inhibitor of the present application.

[0041] Reducing the expression of ACTA2 and / or COL1A1 refers to inhibiting the expression or activity of ACTA2 and / or COL1A1 protein. The amount of the therapeutic product to be administered is a dose sufficient to inhibit the transcription or translation of ACTA2 and / or COL1A1 protein, or a dose sufficient to inhibit the expression or activity of ACTA2 and / or COL1A1 protein. The expression of the ACTA2 and / or COL1A1 gene is reduced by at least 50%, 80%, 90%, 95%, or 99%.

[0042] In the present application, the meaning of NET and NETs is the same.

[0043] The product includes, but is not limited to, drugs, health products, food, etc.

[0044] Optionally, the Mgst2 inhibitor is selected from a nucleic acid molecule, a small molecule chemical drug, an antibody drug, a polypeptide, a protein, a nucleic acid construct, a lentivirus, an adenovirus, or a CRISPR / Cas9 genome editing system.

[0045] The nucleic acid molecule is selected from any one or more of the following: an antisense oligonucleotide, double-stranded RNA, or shRNA.

[0046] Preferably, the double-stranded RNA contains a nucleotide sequence capable of hybridizing to the Mgst2 gene.

[0047] The shRNA contains a nucleotide sequence capable of hybridizing to the Mgst2 gene.

[0048] The shRNA includes a sense strand segment and an antisense strand segment, and a stem loop structure connecting the sense strand segment and the antisense strand segment, the sequences of the sense strand segment and the antisense strand segment are complementary, and the sequence of the sense strand segment is substantially identical to the target sequence in the Mgst2 gene.

[0049] Further, the sequence of the stem loop structure of the shRNA can be selected from any one of the following: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU, and CCACACC.

[0050] Preferably, the nucleotide sequence for preparing the shRNA is shown in SEQ ID NO: 1 and SEQ ID NO: 2:

[0051] hMGST2-564-s: AGGUGCCCUGGGAAUUGCA / dT / / dT / (SEQ ID NO: 1)

[0052] hMGST2-564-a: UGCAAUUCCCAGGGCACCU / dT / / dT / (SEQ ID NO: 2).

[0053] / dT / / dT / : indicates that the sequence has two deoxythymidine nucleotides at the 3' end.

[0054] Optionally, the double-stranded RNA comprises a first strand and a second strand, the first strand and the second strand are complementary to each other to form an RNA duplex together, and the sequence of the first strand is substantially the same as the target sequence in the Mgst2 gene.

[0055] The target sequence in the Mgst2 gene is a fragment in the Mgst2 gene corresponding to the mRNA fragment recognized and silenced by the nucleic acid molecule when the nucleic acid molecule is used to specifically silence the expression of the Mgst2 gene.

[0056] Further, the double-stranded RNA is small interfering RNA (siRNA).

[0057] Optionally, the first strand and the second strand of the siRNA sequence are as shown in SEQ ID NO: 3 and SEQ ID NO: 4. Specifically:

[0058] hMGST2-220-s:

[0059] CUGCUGGCUGCUGUCUCUA / TT (SEQ ID NO: 3)

[0060] hMGST2-220-a:

[0061] UAGAGACAGCAGCCAGCAG / TT (SEQ ID NO: 4)

[0062] Optionally, the first strand and the second strand of the siRNA sequence are as shown in SEQ ID NO: 5 and SEQ ID NO: 6.

[0063] hMGST2-441-s:

[0064] UUGUCUGGGUCUGGUGUAC / TT (SEQ ID NO: 5)

[0065] hMGST2-441-a:

[0066] GUACACCAGACCCAGACAA / TT (SEQ ID NO: 6)

[0067] The 3' end of each strand is capped with a deoxythymidine dinucleotide (TT) overhang to enhance its stability within the cell. In the above sequence, all the nucleotides are ribonucleotides except for the 3' end overhang which is a deoxythymidine. Those skilled in the art know that the siRNA can comprise one or more modified nucleotides (such as 2'-O-methyl modification, phosphorothioate linkage, etc.) to further modulate its stability, specificity or immunogenicity, and these modified equivalents fall within the scope of the present application.

[0068] The Mgst2 inhibitor of the present application can achieve specific silencing of the Mgst2 gene by shRNA, and the inhibitory effect is attributed to the RNA interference activity of the nucleic acid molecule itself. In order to achieve efficient delivery and long-term expression in vivo or in specific tissues, the shRNA can be loaded into a delivery vector. Therefore, the gene inhibitor is essentially a nucleic acid molecule inhibitor, and the delivery vector does not change the molecular class of the inhibitor.

[0069] The delivery vector can be an adeno-associated virus. For example, AAV9.

[0070] The form of the product is not particularly limited, and can be various forms of substances such as solids, liquids, gels, semi-liquids, aerosols, etc.

[0071] The third aspect of the present application provides the use of Mgst2 inhibitors and NOX2 inhibitors in the preparation of NETs inhibiting products.

[0072] The NOX2 inhibitor generally includes a substance that can inhibit the transcription or translation of the NOX2 gene, or can inhibit the expression or activity of the NOX2 protein. The amount of the therapeutic product administered is a dose sufficient to inhibit the transcription or translation of NOX2, or a dose sufficient to inhibit the expression or activity of the NOX2 protein. So that the expression of the NOX2 gene is at least reduced by 50%, 80%, 90%, 95% or 99%.

[0073] Alternatively, the NOX2 inhibitor is selected from nucleic acid molecules, small molecule chemicals, antibody drugs, polypeptides, proteins, nucleic acid constructs, lentiviruses, adenoviruses or CRISPR / Cas9 genome editing systems.

[0074] Alternatively, the NOX2 inhibitor is DPI (diphenyl iodonium chloride salt).

[0075] The NETs inhibiting product refers to a product that can inhibit the release of extracellular trap webs by neutrophils in the lung tissue in an abnormally activated state. For example, based on the neutrophil NETs in the lung tissue of the subject before treatment, the product can reduce the neutrophil NETs in the lung tissue of the subject by at least 30%, 50%, 70%, 80%, 90% or 100%.

[0076] The NETs-inhibiting product has one or more of the following effects:

[0077] (1) reducing pericyte-to-myofibroblast conversion;

[0078] (2) reducing expression of ACTA2 and / or COL1A1.

[0079] The fourth aspect of the present application provides use of a NETs inhibitor in the preparation of a product having at least one of the following effects:

[0080] (1) reducing pericyte-to-myofibroblast conversion;

[0081] (2) reducing expression of ACTA2 and / or COL1A1.

[0082] The fifth aspect of the present application provides use of a Mgst2 inhibitor in the preparation of a product having at least one of the following effects:

[0083] (1) improving lung tissue inflammatory cell infiltration and extracellular matrix deposition;

[0084] (2) reducing expression of any one or more of NOX2, PADI4 or Cit-H3;

[0085] (3) reducing expression of MPO-DNA in alveoli;

[0086] (4) improving nuclear membrane rupture, chromatin decondensation and extracellular fibrous structure formation in lung tissue;

[0087] (5) reducing formation of NETs;

[0088] (6) reducing pericyte-to-myofibroblast conversion;

[0089] (7) reducing expression of ACTA2 and / or COL1A1.

[0090] In the present application, the technical features, definitions and the like of the same professional terms can be used in the invention content of different aspects, and to avoid repetition, they will not be described one by one.

[0091] Compared with the prior art, the present application has the following beneficial effects:

[0092] The first discovery of the present application Mgst2 can be used as a biomarker for the treatment of rheumatoid arthritis associated interstitial lung disease, by targeting Mgst2 knockdown in neutrophils with Cd11b promoter driven AAV9-shRNA vector (AAV9-shRNA-Mgst2), which can effectively alleviate the progression of lung lesions by inhibiting NET formation. The Mgst2 inhibitor described in the present application can directly intervene in the occurrence and development process of the disease at the molecular level, which has the advantages of precise action, significant effect and low side effect. Compared with traditional non-specific treatment methods, this product only acts on specific molecular targets, significantly reduces damage to normal cells and tissues, and thus reduces the risk of toxic side effects. BRIEF DESCRIPTION OF DRAWINGS

[0093] Figure 1 . Constructed a model of interstitial pneumonia with joint swelling in SKG mice. A. Joint swelling and interstitial pneumonia were induced in SKG mice by intraperitoneal injection of 7.5 mg / kg ZYM or PBS (phosphate buffered saline) for 16 weeks. The results of HYP detection, immunofluorescence, pulmonary function test (PFT), and RT-PCR in SKG mice are shown in the figure. Some graphs were generated by FigDraw.com. B. The concentration of HYP in the lung tissue of ZYM-treated or PBS control SKG mice is shown (n=5). C, D. Immunofluorescence and statistical analysis of the expression of ACTA2, COL1A1, and CTHRC1 in the lung tissue of ZYM-treated or PBS control SKG mice (n=5). Nuclei were stained with DAPI (blue). The scale bar is 100 μm. E. RT-PCR analysis of the expression of Acta2, Col1a1, and Cthrc1 genes in the lung tissue of ZYM-treated or PBS control SKG mice (n=5). F-L. Mouse lung function results, including inspiratory capacity (IC) (F), vital capacity (VC) (G), forced vital capacity (FVC) (H), forced expiratory flow at 50% of vital capacity (FEV50) (I), expiratory reserve volume (ERV) (J), peak expiratory flow (PEF) (K), and FEV50 / FVC ratio (L). Statistical significance was assessed by paired t-test. *P<0.05; **P<0.01; ***P<0.001; ****P<0.001 compared with the PBS-treated control group.

[0094] Figure 2. Transcriptional heterogeneity of mouse lung neutrophils was revealed by single-cell RNA sequencing (scRNA-seq) analysis. A. Uniform Manifold Approximation and Projection (UMAP) plot showing 7 neutrophil subsets in mouse lung at 16 weeks of PBS (207 cells) or ZYM (3,729 cells) modeling. Each dot represents a single cell, and colors are distinguished according to subsets. B-H. Gene expression patterns projected onto the UMAP plot with corresponding violin plots showing Nrlp12 (anti-inflammatory Neu, N1), Osginl (proliferative Neu, N2), Cxcl3 (chemotactic Neu, N3), Mgst2 (Mgst2hi Neu, N4), Olfm4 (Olfm4 + Neu, N5), Prok2 (Prok2hi Neu, N6), and Stfa3 (mature Neu, N7) gene expression (scale: gene expression was log-transformed). n values for N1-N7 were 1,499, 702, 678, 371, 304, 193, and 189, respectively. I. Bar plot showing the changes in the proportion of cell subsets in PBS control and ZYM-treated groups. J. Up- and down-regulated genes in the 7 subsets according to differential gene expression analysis. K. Trajectory analysis showing the evolutionary direction of all neutrophil subsets; the evolutionary direction is from the center to the edge. L. Neutrophil UMAP plot with RNA flow direction labeled. M. Latent period heat map showing specific genes during neutrophil differentiation.

[0095] Figure 3Mgst2hi Neu promotes NET formation. A. KEGG pathway analysis of the top 200 marker genes of Mgst2hi Neu. B. The profile of NET genes in the seven neutrophil subpopulations. C-E. Representative immunofluorescence images and statistical analysis of cells co-expressing Ly6G (green), Mgst2 (red), and Padi4 (white) proteins (C, D) and Ly6G (green), MGST2 (red), Cit-H3 (white) in the alveoli and septa, airways, and lung tissue of mice in the ZYM-treated group (lower panel) and PBS control group (upper panel) (n = 6). F-I. Representative immunofluorescence images and statistical analysis of cells co-expressing Ly6G (green), Mgst2 (red), and Padi4 (white) proteins in BALF (F, G) (n = 3) and PB (H, I) (n = 3) of mice in the ZYM-treated group (right) and PBS control group (left). The inset indicates the highlighted area with a white full box. DAPI nuclei (blue). J. This schematic represents the experimental workflow of AAV intervention in the lungs of mice. K. Changes in the body weight of mice during the experiment (n = 5). L. Representative histopathology images of H&E staining (upper panel) and Masson’s trichrome staining (lower panel) of the lungs of mice infected with AAV vectors. M. Representative images of immunohistochemical staining of PADI4 and Cit-H3 in the lungs of mice infected with AAV vectors. N. The content of MPO-DNA in lung tissue, BALF (n = 5), and PB (n = 5) of mice infected with AAV vectors (n = 5). O. Representative electron microscopy images of NET structures in the lungs of mice infected with AAV vectors. Scale bars in C, F, H, L, M are equal to 50 pm. Statistical significance was assessed by unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.001 as indicated.

[0096] Figure 4 Mgst2hi Neu promotes NET formation through activation of NOX2. A. The distribution of Nox2 genes in the seven neutrophil subpopulations. B. ROS / oxidative stress gene markers in the seven neutrophil subpopulations. C, D. Representative immunofluorescence images and statistical analysis of cells showing co-expression of Ly6G (green), Mgst2 (red), and Nox2 (white) proteins in the alveolar space and septa, airways, and blood vessels of mice in the ZYM-treated group (lower panel) and PBS control group (upper panel) (n = 6). E-H. Representative immunofluorescence images and statistical analysis of cells showing co-expression of Ly6G (green) and Mgst2.

[0097] Figure 5 NETs from Mgst2hi Neus promote pericyte-myofibroblast transition. A. Cell-cell interaction network illustrating ligand-receptor interactions between Mgst2hi Neus and various cell types, including proliferative Neus, B cells, chemotactic Neus, dendritic cells, endothelial cells, epithelial cells, fibroblasts, macrophages, mature Neus, monocytes, NK cells, Olfm4+ Neus, pericytes, plasma cells, anti-inflammatory Neus, Prok2hi Neus, smooth muscle cells, and T cells. b. Quantification of the number of pericytes and the combined fraction of inflammatory fibroblasts and fibroblasts in lung tissue of ZYM-treated and PBS control mice. C. Trajectory analysis illustrating the differentiation trajectory of pericytes to myofibroblasts and eventually to inflammatory myofibroblasts. D-F. Immunofluorescence and statistical analysis of CD146 (yellow) and Acta2 (white) protein, and Ng2 (yellow) and acta2 (white) co-expression in lung tissue of PBS control (upper panels) and ZYM-treated (lower panels) mice (n=6). G. Pseudotrajectories and enrichment of extracellular region-associated genes and TGF-b signaling pathway. H. Heatmap showing differential expression of fibrosis-associated genes in pericytes of ZYM-treated and PBS control mice. I. Workflow showing human lung microvascular pericytes under different experimental conditions. J. Immunofluorescence images showing ACTA2 (green) and COL1A1 (red) protein expression in human lung microvascular pericytes after 24 hours with or without NETs generated using PMA and DNase I treatment (n=3). K. Representative bands and quantification of ACTA2, COL1A1, TGFBR1, and TGFBR2 protein in human lung microvascular pericytes after 24 hours with or without NETs generated using PMA and DNase I treatment (n=3). L. RT-PCR analysis of Col1a1, Acta2, Tgfbr1, and Tgfbr2 in human lung microvascular pericytes under different conditions (n=3). Scale bars in D equal 50 pm and in J equal 10 pm. DETAILED DESCRIPTION

[0098] In order to make the invention purposes, technical solutions and beneficial technical effects of the present application clearer, the following embodiments are further described in detail. Those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in the present specification.

[0099] Before particular embodiments of the present application are further described, it is to be understood that the application is not limited to the particular specific embodiments described below; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the application. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of the present application, unless expressly identified otherwise.

[0100] When numerical ranges are given, it should be understood that every numerical range encompasses any number falling within the range, unless otherwise indicated. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Except in the Examples, or where otherwise explicitly indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, exemplary methods and materials are described below. All publications mentioned herein are incorporated herein by reference to disclose and allow for the use of techniques described therein.

[0101] Unless otherwise indicated, the experimental methods, detection methods, preparation methods disclosed in the present application all use conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields.

[0102] Example 1

[0103] 1. Materials and Methods

[0104] 1.1 Animals, Cells and Vectors

[0105] SKG mice were purchased from Jiangsu Jicui Yaoke Company. HL-60 differentiated neutrophil model, primary human lung microvascular pericyte were purchased from iCell. AAV9-shRNA vector driven by Cd11b promoter (AAV9-shRNA-Mgst2) was commissioned from Jikai Gene,

[0106]

[0107] Construction method: linearized vector is obtained by restriction enzyme digestion. The target fragment is prepared by primer annealing. The designed primer adds enzyme cutting sites at both ends. After annealing, the primer contains the same enzyme cutting sites at both ends of the linearized cloning vector. The reaction system is prepared with linearized vector and annealing product, and the product is directly transformed. Single colonies on the plate are picked for PCR identification, and positive clones are sequenced and analyzed. The correct clone broth is cultured, extracted, and high-purity plasmid is obtained for downstream virus packaging.

[0108] wherein the oligonucleotide sequence for preparing the shRNA is shown as SEQ ID NO: 1 and SEQ ID NO: 2,

[0109] hMGST2-564-s: AGGUGCCCUGGGAAUUGCA / dT / / dT /

[0110] hMGST2-564-a: UGCAAUUCCCAGGGCACCU / dT / / dT / .

[0111] The siRNA sequence numbers are shown as SEQ ID NO: 3 and SEQ ID NO: 4 and SEQ ID NO: 5 and SEQ ID NO: 6.

[0112] 1.2 Main reagents

[0113] Zymosan A (ZYM) was purchased from Sigma-Aldrich.

[0114] MPO-DNA complex and ROS, detection reagent / antibody were purchased from Wuhan Sanying.

[0115] DPI (diphenyl iodine chloride salt) was purchased from MCE.

[0116] The primer sequences used in the application are shown in Table 1, and the primary antibodies are shown in Table 2.

[0117] Table 1. List of primer sequences used in the application.

[0118]

[0119] Table 2. List of primary antibodies used in the application.

[0120]

[0121] 1.3 Experimental method:

[0122] 1.3.1 Establishment of model mice and model cells

[0123] 1.3.1.1 Establishment of arthritis mice with concurrent interstitial lung disease

[0124] Information of experimental animals: gender and age (8-week-old male and female mice)

[0125] Housing environment (SPF barrier environment of Ningxia Medical University Experimental Animal Center)

[0126] Induction specific steps:

[0127] Induction drug: Zymosan A (ZYM)

[0128] Dosing method: intraperitoneal injection

[0129] Dose and volume: 0.05g / ml, the injection volume corresponding to the mouse body weight is 7.5mg / kg.

[0130] Dosing interval: SKG mice were injected once at the eighth week, lasting for 16 weeks.

[0131] 1.3.1.2 Establishment of mice with Mgst2 gene knockout in neutrophils

[0132] AAV9-shRNA-Mgst2 was administered by tracheal instillation at the tenth week of ZYM administration, and evaluated after 16 weeks.

[0133] Knockout effect was verified by flow cytometry, H&E (Hematoxylin and Eosin Staining, immunofluorescence staining.

[0134] Flow cytometry: After obtaining the lung tissue, cut it into 2-3mm, add 2ml of collagenase type 1 / DNase I digestion solution (collagenase: 5mg / ml DNase I 1 mg / ml), 37℃, 125rpm digestion for 30min. Use an 18-gauge needle and a 5ml syringe to extract and discharge the digested tissue twice, and finally pass through a 75um sieve. If there are still large pieces of tissue, use a 2ml syringe piston to gently grind on the 75um sieve, and resuspend in a pre-cooled PBS containing 1ml of 0.5% BSA. Then add fluorescently labeled primary antibodies and incubate for 1h before analysis.

[0135] HE staining:

[0136] 1. Xylene dewaxing 5-10 minutes per step 2. Absolute ethanol I 5min II 5min 3. 95% ethanol I 5min II 5min 4. 80% ethanol 5min 5. 70% ethanol 5min 6. Pure water 3-5min 7. Harris hematoxylin solution 5min (in winter, it can be appropriately extended, such as 20min) 8. Water washing 1-3min 9. 0.5% hydrochloric acid alcohol differentiation 3-10 seconds, observed under a microscope 10. Saturated sodium hydrogen phosphate solution counterstain 10min 11. 70% ethanol 5min 12. 80% ethanol 5min 13. Eosin solution (95% ethanol solution) 3-30 seconds 14. Baking, neutral resin mounting. Knockout results are shown in Figure 3 (L, M) as shown.

[0137] 1.3.1.3 Construction of a neutrophil model in which the Mgst2 gene is knocked out

[0138] A neutrophil model in which the Mgst2 gene is knocked out was constructed using HL-60 cells of a certain number of generations, stimulating with retinoic acid for three days after resuscitation, adding siRNA small interference in a measured proportion, and stimulating for 60-72h. The construction effect was detected by immunoblotting analysis.

[0139] 1.3.1.4 Construction of a neutrophil model using an Mgst2 gene inhibitor

[0140] In the neutrophil model in which the Mgst2 gene was knocked out, DPI was added and stimulated for 24h, and then the cells were collected for immunoblotting analysis verification.

[0141] 1.3.1.5 Construction of a model of the effect of neutrophil extracellular traps (HL-60 NETs) on lung pericytes

[0142] Primary human lung pericytes purchased from iCell were resuscitated and then added with PMA-induced HL-60 NETs or with finished DNase I enzyme and stimulated for 24h, and then whole protein was extracted for immunoblotting analysis verification.

[0143] The HL-60 NET construction method is as follows: HL-60 cells were differentiated into neutrophils by retinoic acid stimulation, and then PMA (phorbol-12-myristate-13-acetate) finished solution was added and stimulated for 2h.

[0144] 1.3.2 Immunofluorescence

[0145] Dewaxing and rehydration: The dewaxing and rehydration of the tissue is completed in the order of xylene I (10 min), xylene II (10 min), xylene III (10 min), anhydrous ethanol I (5 min), anhydrous ethanol II (5 min), 95% alcohol (3 min), 90% alcohol (3 min), 80% alcohol (2 min), 70% alcohol (2 min), distilled water I (3 min), and distilled water II (3 min) (Note: The operation process is not fixed, and appropriate adjustment can be made according to the tissue itself).

[0146] Antigen retrieval: Prepare citrate antigen retrieval solution (PH 6.0), and use a microwave oven for antigen retrieval. First, high heat (80% heat) for 8 min, then stop heating for 8 min, and finally, low heat (50% heat) for 7 min. After natural cooling, wash with PBS buffer for 3 times, each for 5 min.

[0147] Blocking: After washing, gently absorb the water, draw a circle around the tissue with a histological pen, cover the entire tissue, then add goat serum and block at room temperature for 1 h. After blocking, wash with PBS buffer for 3 times, each for 5 min.

[0148] Primary antibody incubation: Add primary antibody to cover the entire tissue, and incubate at 4°C overnight. After incubation, wash with PBS buffer for 3 times, each for 5 min.

[0149] Secondary antibody incubation: Add fluorescent secondary antibody to cover the entire tissue, and incubate in a dark box at room temperature for 1 h (Note: All subsequent steps need to be operated in the dark). After incubation, wash with PBS buffer for 3 times, each for 5 min.

[0150] DAPI staining: Use DAPI staining solution to stain at room temperature in the dark for 10 min, and wash with PBS for 3 times, each for 5 min. Gently dab the water, add anti-fluorescence quenching mounting medium, and cover with a cover glass.

[0151] Observation: Observe the distribution of the target protein under a fluorescence microscope or a confocal microscope.

[0152] 1.3.3 Hydroxyproline determination (Nanjing Jiancheng):

[0153] 1. Sample hydrolysis:

[0154] ① Serum (plasma): Take 0.5 mL of serum (plasma) and accurately add 1 mL of hydrolysis solution, mix well. Place the test tube in a 95°C or boiling water bath for hydrolysis.

[0155] 20 minutes.

[0156] ②, urine (culture solution): take 1.0 mL of urine (culture solution) and accurately add 1 mL of hydrolysis solution, mix well. After placing the test tube in a 95°C water bath or boiling water bath, hydrolyze for 20 minutes.

[0157] ③, tissue: accurately take 30-100 mg of wet tissue and place it in a test tube, accurately add 1 mL of hydrolysis solution, mix well. After placing the test tube in a 95°C water bath or boiling water bath, hydrolyze for 20 minutes (mix once after 10 minutes of hydrolysis, the purpose is to make the hydrolysis more complete).

[0158] 2. Adjust the pH value to about 6.0-6.8.

[0159] ①, after cooling the test tubes with running water, add 10 μL of indicator to each tube, shake well;

[0160] ②, accurately add 1.0 mL of pH adjusting solution A to each tube, mix well (at this time the solution should be red);

[0161] ③, use a 200 μL pipette to draw pH adjusting solution B and add it drop by drop to each tube, mix well after each drop is added, until the color of the indicator in the liquid changes to yellow-green (i.e. when the red color disappears). At this time

[0162] The pH value is about 6.0-6.8 (about 100-500 μL of pH adjusting solution B is added); When adding pH adjusting solution B, mix well after each drop is added, to prevent liquid from spilling, if you do not have a glass ground test tube with a cover, you can use a regular glass test tube instead, and you can use plastic film or refrigerator preservative film to press the test tube opening when mixing each time, and mix well by vortexing. If your sample is cell culture solution, it contains phenol red, so the mixed indicator in the liquid is orange-red when the pH is about 6.0-6.8, not yellow-green.

[0163] ④, then add double distilled water to 10 mL, mix well;

[0164] ⑤, take 3-4 mL of diluted hydrolysis solution, add an appropriate amount of activated carbon (about 20-30 mg, the above clear supernatant is clear and colorless), mix well, centrifuge at 3500 rpm for 10 minutes, carefully take 1 mL of supernatant for detection.

[0165] 1.3.4 Lung function:

[0166] AniRes 2005 animal lung function respiratory system is used, the hardware includes body plethysmograph, signal conditioner, animal respirator, negative pressure generator, vacuum pump, etc., and the software is AniRes 2005.

[0167] (1) Turn on the instrument, usually set the ratio of inspiration to expiration at 20:10, and the respiratory rate at 90 breaths / min for mice. Open the software and create a new experiment: select the animal type and the size of the body plethysmograph according to the actual situation (use a small body plethysmograph for mice). Confirm the new experiment and turn it on. The data area of the software displays the airway pressure (P), lung volume (V), and flow rate (Flow), and the parameter area displays the airway resistance (Ri, Re) and lung compliance (Cdyn).

[0168] (2) Prepare 8 mg / ml pentobarbital sodium solution and 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, and 0.8 mg / ml acetylcholine solutions on the day of the experiment. The concentrations of the above solutions can be adjusted according to the results of the preliminary experiment, the breed of the mice, and the brand of the reagent.

[0169] (3) Anesthesia and tracheal intubation: anesthetize the mice by intraperitoneal injection of 0.1 ml / 10 g pentobarbital sodium solution. Fix the mice and, when the mice do not respond to pinching of the toes with forceps, disinfect the neck of the mice, cut open the skin and muscles of the neck in sequence, and expose the trachea and jugular vein of the mice. Isolate the trachea, cut a small opening in the upper part of the trachea for tracheal intubation, and connect it to the respirator. Start a new experiment in the software, and after the mice are connected to the respirator, the P column of the software starts to show a peak shape. Adjust the flow valve to make the peak top about 10.

[0170] (4) Jugular vein needle insertion: carefully insert the tip of a 1-ml syringe into the jugular vein of the mice, and connect the other end of the needle tip to a thin tube and a 1-ml syringe to form a vein administration channel. Fill the vein administration channel with physiological saline. After the needle tip is inserted into the jugular vein, draw blood, immediately inject the drawn blood into the vein, and then close the body plethysmograph and completely seal it. At this time, the V column starts to show a peak shape, and the parameter column starts to show a slightly fluctuating baseline.

[0171] (5) Drug administration: inject acetylcholine at concentrations of 0, 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, and 0.8 mg / kg through the jugular vein in sequence, 50 μl each time, and then inject 50 μl of physiological saline to push the remaining drug in the pipeline into the jugular vein. Use a computer with AniRes 2005 software to continuously calculate and store Ri, Re, and Cdyn from the digitized pressure and flow signals. After each administration, wait until the data in the parameter column of the software are stable before the next administration.

[0172] 1.3.5 RT-PCR:

[0173] Specific steps:

[0174] 1) Take 50-100 mg of lung tissue from each group, and cut the tissue into small pieces with scissors (avoid RNA contamination).

[0175] 2) Add the tissue pieces into 1 ml Tripure (the volume of the tissue pieces should not exceed 10% of the volume of the Tripure), and grind the mixture in a glass homogenizer until there are no obvious large tissue pieces. The grinding process should be performed in ice water at 0°C to prevent RNA degradation.

[0176] 3) Then transfer the homogenate into a 1.5 ml Eppendorf tube, and incubate the homogenate at 15°C-25°C for 5 min to completely separate the nucleoprotein complex.

[0177] 4) Add 0.2 ml of chloroform, tightly cap the tube, and shake it vigorously on a shaker for about 15 seconds, and then incubate it at 15°C-25°C for 2 min-15 min.

[0178] 5) Centrifuge the mixture in a 2°C-8°C ice-freezer centrifuge (12000 g / min, 15 min). At this time, the liquid is layered: the bottom layer is the red phenol-chloroform phase; the middle layer is the interface phase; and the upper layer is the colorless liquid phase.

[0179] 6) The RNA is only in the supernatant. Transfer the supernatant to a new tube (do not suck the middle layer into the tube),

[0180] 7) Add 0.5 ml of isopropanol, mix well, and incubate at room temperature for 5 min-15 min.

[0181] 8) Centrifuge the mixture in a 2°C-8°C ice-freezer centrifuge (12000 g / min, 10 min).

[0182] 9) Discard the supernatant (move slowly and gently), and a small white precipitate can be seen at the bottom of the tube, which is the RNA.

[0183] 10) Add 1 ml of 75% ethanol, mix and shake, (it can be stored at 2°C-8°C for one week or at -15°C-25°C for one year)

[0184] 11) Centrifuge the mixture in a 2°C-8°C ice-freezer centrifuge (7500 g / min) for 5 min, and discard the supernatant,

[0185] 12) Ventilate, and dry the tube at room temperature for 5 min-10 min to allow the ethanol to evaporate (according to the visual observation of the moisture in the tube, it should not be too dry, as the RNA will be difficult to dissolve after drying).

[0186] 13) Add 30ul-50ul of 0.1% DEPC, blow several times,

[0187] 14) Incubate at 55-60°C for 10 min, cool on ice,

[0188] 15) Use for next experiment or store at -70°C.

[0189] Detection of RNA integrity:

[0190] 1) Prepare agarose gel, weigh 0.34 g of agarose gel, add 0.5x TBE buffer 20 ml, heat to melt in microwave oven.

[0191] 2) Finally make the final concentration of 1.7% agarose gel liquid.

[0192] 3) Add bromoethane (0.5 mg / ml) 2ul, mix well.

[0193] 4) Cool to room temperature.

[0194] 5) Prepare gel on a horizontal electrophoresis tank, after solidification, immerse in 0.5x TBE buffer, remove sample comb. Take 1ug RNA and mix with 1.5ul formaldehyde, 4.5ul formamide, 60°C water bath for 10 min, add 1ul loading buffer (0.25% bromophenol blue; 40% sucrose), 75mv electrophoresis to bromophenol blue at 2 / 3 of the agarose gel.

[0195] Detection of RNA purity and concentration:

[0196] 1) Preheat GeneQuant and adjust parameters,

[0197] 2) Zero with 1000ul of inactivated DEPC water.

[0198] 3) Take 1ul of RNA sample, add 1000ul of inactivated DEPC water, mix well.

[0199] 4) Directly read the RNA concentration and Ratio, and according to the dilution, get the actual RNA concentration, repeat the operation three times and take the average value.

[0200] 5) The Ratio of RNA is between 1.7-2.0. The concentration of RNA is between 0.5ug / ul-1ug / ul, aliquot 10ul per tube, store at -70°C in the freezer.

[0201] RT-PCR:

[0202] (I) Preparation of primers:

[0203] 1. Dissolve the synthesized target gene and GAPDH upstream and downstream primers (1 OD) in 0.1% DEPC water.

[0204] 2. According to the different molecular weights of each primer, calculate the amount of 0.1% DEPC water.

[0205] 3. The amount of 0.1% DEPC water added to the target gene and GAPDH upstream and downstream primers is respectively: 488ul, 431ul, ul, ul, so that the final concentration is 20pmol / ul.

[0206] 4. Store in -20°C refrigerator for standby.

[0207] RT-PCR reaction system (25ul):

[0208] Enzyme-free water: 14.4ul

[0209] AMV Buff target gene: 5ul

[0210] dNTP: 0.5ul

[0211] Target gene upstream primer: 0.75ul

[0212] Target gene downstream primer: 0.75ul

[0213] GAPDH upstream primer: 0.75ul

[0214] GAPDH downstream primer: 0.75ul

[0215] MgSO4: 0.8ul

[0216] Tfl DNA Poly: 0.5ul

[0217] RNA: 0.8ul

[0218] Mix each component above and cover with paraffin oil 20ul, label number and put into gene amplifier.

[0219] RT-PCR amplification conditions and parameters: 48°C, 45min, 94°C, 2min; 94°C 30s, 58°C 60s, 68°C 2min, a total of 40 cycles; After the cycle is completed, 68°C extension for 7min. The amplification product is electrophoresed or stored at -20°C.

[0220] RT-PCR product analysis:

[0221] (1) RT-PCR product electrophoresis analysis

[0222] 1. Take 6 ul RT-PCR product and mix with 1 ul loading buffer, then load on 2% agarose gel, with 0.8ul Ladd target gene sample, and 0.5xTBE as buffer for electrophoresis.

[0223] 2. 75mv electrophoresis to bromophenol blue at 2 / 3 of agarose gel, observe under UV light, scan, take pictures and analyze images.

[0224] 3. Image analysis processing system for luminance scanning, and GAPDH correction for relative quantity analysis, the value is expressed as the ratio of integral absorbance of the two.

[0225] PCR product sequencing:

[0226] Sequencing of the amplified target gene PCR product (50ul).

[0227] 1.3.6 Immunoblotting

[0228] Protein extraction (RIPA lysis buffer)

[0229] 1) Dissolve RIPA lysis buffer and mix well. Take appropriate amount of lysis buffer and add PMSF within a few minutes before use to make the final concentration of PMSF 1 mM.

[0230] 2) Tissue samples: add lysis buffer according to the ratio of 150-250 μL lysis buffer per 20 mg tissue. Homogenize with a glass homogenizer until fully lysed.

[0231] 3) After complete lysis, centrifuge at 10000-14000 g for 3-5 min, and take the supernatant.

[0232] Electrophoresis: 1) Cut the package and take out the precast gel. Tear off the deep green tape at the bottom of the gel plate, slowly pull out the comb, and fix the precast gel in the electrophoresis tank. Fill the inner tank with electrophoresis buffer, and add the outer tank liquid to a height of 1 / 3 higher than the electrophoresis tank. Use a pipette or other tools to aspirate the electrophoresis buffer and gently blow it into the sample well to remove the residual storage buffer and impurities in the sample well

[0233] 2) Dissolve 5x SDS-PAGE loading buffer in a water bath at room temperature or not more than 37°C. Store at room temperature immediately after water bath dissolution.

[0234] 3) Mix the protein sample and 5x SDS-PAGE protein loading buffer according to the ratio of 1 μL 5x SDS-PAGE protein loading buffer per 4 μL protein sample.

[0235] 4)100℃ or boiling water bath heating 3-5 min, to fully denature the protein, then cool to room temperature for standby.

[0236] 5)In each sample well, loading 10-30 μg protein, and in one well, loading protein marker, cover the electrophoresis tank cover, turn on the power, recommended 150 V, running 50-70 min, usually electrophoresis to the blue dye near the bottom end of the gel can stop electrophoresis.

[0237] 6)After electrophoresis, remove the gel, use the gel opener or other appropriate tools to insert into the gap between the two sides of the gel plate, slowly pry up, down, and the middle three different positions, and then repeat the operation on the other side, until the two sides of the gel plate are completely opened.

[0238] 7)After the gel plate is opened, the gel may be stuck on either side of the gel plate, tilt the gel plate with the gel to the water, gently move the gel, so that the gel falls freely into the container filled with water, shake the gel, and then remove it.

[0239] Transfer membrane:

[0240] 1)The gel is immersed in pre-cooled transfer buffer for 5 min.

[0241] 2)According to the size of the gel, cut the membrane and filter paper 6 pieces, and place them in pre-cooled transfer buffer for 10 min, such as PVDF membrane, refer to the instructions, first soak in pure methanol for 1-2 min, then incubate in pre-cooled transfer buffer.

[0242] 3)Assemble the transfer sandwich: sponge / 3 layers of filter paper / gel / membrane / 3 layers of filter paper / sponge, after each layer is placed, use a roller or test tube to remove air bubbles.

[0243] 4)Place the transfer tank in an ice bath, place the sandwich, the membrane close to the anode, and the gel close to the cathode, add transfer buffer 300 mA, 60 min transfer treatment, or use fast transfer liquid 400 mA, only 15-35 min to complete the transfer.

[0244] 5)After transfer, turn off the power and remove the membrane.

[0245] 6)Submerge the membrane in 5 mL of ponceau staining solution, place it on an orbital shaker at room temperature for 5-10 min or longer until the membrane shows protein bands.

[0246] 7)Wash: remove the membrane, rinse with distilled water, PBS or other appropriate solution until the background is clear, and take a photo, about 2-3 times, 5 min each time.

[0247] 8) Decolorization: Rinse the membrane in 0.1M NaOH solution for 5 min; discard the eluent and repeat once.

[0248] 9) Cleaning: Clean the membrane with TBST 2-3 times, 5 minutes each time.

[0249] 10) Place the membrane in 25 mL of blocking buffer and incubate at room temperature for 1 hour or use rapid blocking solution for 10 min to complete the blocking.

[0250] 11) Wash three times with 5 mL TBST, 15 min each time.

[0251] Primary antibody incubation:

[0252] 1) Place the membrane and primary antibody (at the recommended dilution for product application) in 10 mL of primary antibody dilution buffer and incubate overnight at 4ºC with gentle shaking from time to time.

[0253] 2) Wash three times with 5 mL TBST for 15 min each time to remove residual primary antibody.

[0254] 3) Place the membrane and secondary antibody (at the recommended dilution for product application) in 10 mL of blocking buffer and incubate for 1-2 hours, gently shaking occasionally.

[0255] 4) Wash three times with 5 mL TBST, 15 min each time.

[0256] Protein detection:

[0257] 1) During the final membrane wash, prepare the luminescent working solution fresh according to the ECL hypersensitive reagent kit instructions.

[0258] 2) Use flat-tipped tweezers to remove the membrane and place it on filter paper to drain the washing solution, being careful not to let the membrane dry completely. Immerse the membrane completely in the luminescent working solution, ensuring full contact. Incubate at room temperature for 3 minutes, then prepare for immediate pelleting and exposure.

[0259] 3) Use tweezers to pick up the membrane and place it on filter paper to drain the luminescent working solution.

[0260] 4) Lay a piece of cling film larger than the film on the inner surface of the X-ray film cassette. Place the imprint film on the cling film, fold the cling film to completely cover the imprint film, removing air bubbles and wrinkles. Trim any excess cling film from the edges. Blot away excess luminescent working solution with filter paper. Secure the cling film covering the imprint film to the cassette with tape, protein strip side up.

[0261] 5) Press the X-ray film in the darkroom and expose it for different times, such as from a few seconds to a few minutes. Develop and wash.

[0262] The remaining experimental procedures are the same as in 1.3.2.

[0263] The expression of ACTA2 and COL1A1, TGFβR1 and TGFβR2, and the detection of p-SMAD2 / 3 levels were all performed using immunoblotting analysis.

[0264] 1.3.7 Clustering and Subpopulation Marker Identification of Mouse Lung Neutrophils

[0265] Dimensionality reduction and cluster analysis: The highly variable genes (HVGs) in the Scanpy package were screened using the highly variable genes function. PCA (principal component analysis) was performed using the expression profiles of the highly variable genes. The harmony_integrate analysis in the harmonyPy package (version 0.0.10) was used to correct for batch effects. The results were visualized in two-dimensional space using UMAP (non-linear dimensionality reduction).

[0266] Marker gene identification: Identify the specific representative markers for each cell type from the literature, and use the rank_genes_groups function (method=wilcoxon) in the Scanpy package to identify marker genes. That is, find the genes that are upregulated in each cell type relative to other cell populations. These genes are the potential marker genes for each cell type. The identified marker genes are visualized using the VInPlot and FeaturePlot functions in the Seurat (version 4.0.0) package.

[0267] 1.3.8 Regarding Mgst2 hi KEGG enrichment of the first 200 marker genes of Neu

[0268] After marker gene screening, functional enrichment analysis was performed using the top 200 marker genes with genediff values, and the significance of functional enrichment was analyzed using the hypergeometric distribution test.

[0269] 2. Results

[0270] 2.1 Establishment of Arthritis Mice with Interstitial Lung Disease

[0271] Under SPF conditions, SKG mice were induced to develop arthritis complicated by interstitial lung disease using Zymosan A (ZYM). The success rate of the model was assessed using immunofluorescence (IF), hydroxyproline (HYP) assay, pulmonary function testing (PFT), and real-time quantitative PCR (RT-PCR) (Figure 1A). Histological results showed ( Figure 1C) Immunofluorescence showed the expression of ACTA2, COL1A1, and CTHRC1 in the lung tissues of mice in the ZYM-treated group and the PBS control group. The HYP content in the lung tissue of ZYM-treated mice was significantly increased (Figure 1B). Simultaneously, IF and RT-PCR showed that the expression of fibrosis-related genes ACTA2, COL1A1, and CTHRC1 was increased in the ZYM group (Figures 1D, 1E, 1F). PFT results further indicated that compared with the PBS control, the inspiratory volume (IC), vital capacity (VC), forced vital capacity (FVC), and forced expiratory flow (FEV50) at 50 s were significantly reduced in the ZYM group (Figures 1F-1I). While expiratory reserve (ERV), peak expiratory flow (PEF), and the FEV50 / FVC ratio did not differ significantly, they showed a decreasing trend (Figures 1J-1L). These data suggest that, under SPF conditions, continuous ZYM treatment for 16 weeks can induce RA-ILD-like changes in SKG mice, exhibiting both significant joint swelling and interstitial pneumonia phenotypes.

[0272] 2.2 Transcriptional and functional heterogeneity of neutrophils in lung tissue of arthritic mice with interstitial lung disease

[0273] ZYM exposure significantly promoted neutrophil infiltration; however, the increased number did not reflect phenotypic or functional heterogeneity. Neutrophils are highly heterogeneous and may play different pro-fibrotic roles in autoimmune-related lung diseases. Clustering and subset marker identification of mouse lung neutrophils revealed seven different functional subsets: anti-inflammatory Neutrophils, proliferating Neutrophils, chemotactic Neutrophils, and Mgst2-positive Neutrophils. hi Neu、Olfm4 + Neu、Prok2 hi Neutrophil maturity score indicates a maturation lineage roughly from Mgst2. hi Gradually transition to chemotactic, anti-inflammatory, and Prok2-based therapies. hi Olfm4 + Proliferate, eventually reaching a mature subpopulation. Figure 2A). Among them: the anti-inflammatory type is enriched with Nlrp12, Pik3cd, Stat5b, etc., and its GO function is mainly related to "inhibition of NIK / NF-κB signaling and IL-1 production"; the proliferative type upregulates Osgin1 (accompanied by Olr1, Nr4a1, etc.), which is involved in the regulation of type B pancreatic cell proliferation; the chemotactic type is enriched with Cxcl3, Icam1, Ly6i, etc., which are associated with chemotaxis and CXCR receptor binding activity; Mgst2 hi High expression of Mgst2, Camp, and Chil3 is associated with leukotriene biosynthesis and CYP exogenous substance metabolism; Olfm4 + Enrichment of Olfm4 and Zcchc2, associated with chitin binding and hexosinosinase activity; Prok2 hi Enrichment of Prok2 and Gm2511 is associated with positive regulation of smooth muscle contraction and differentiation of vascular-associated smooth muscle cells; mature cells upregulate Stfa3 and Csta3, characterizing the late stage of neutrophil maturation. Figure 2 BH). After ZYM treatment, the proportion of anti-inflammatory phenotypes decreased, while the proportions of proliferation, chemotaxis, and Mgst2 increased. hi Olfm4 + Prok2 hi The proportion of mature individuals increased (Figure 2I). Common differentially expressed genes showed high heterogeneity among different subpopulations. Figure 2 J). Pseudo-temporal analysis shows that differentiation progresses from the center to the periphery, Mgst2 hi Early marker, chemotactic marker terminal ( Figure 2 K), consistent with RNA velocity (Figure 2L). Potential time heatmaps suggest: early activation primarily activates Xkr4, Resp18, and CD177; late activation activates Col5a2, Klf4, and Gbp5 (Figure 2M). These results suggest that Mgst2... hi Neutrophils may play an initiating role in neutrophil activation during the pathogenesis of RA-ILD.

[0274] 2.3Mgst2 hi Neutrophil subsets promote NET formation

[0275] For Mgst2 hi KEGG enrichment of the top 200 marker genes of Neu showed that they were significantly associated with peroxisome metabolism, pentose phosphate pathway, platelet activation, and complement / coagulation pathway. Figure 3 A). These functional characteristics suggest that Mgst2 hiNeu possesses the "precursor potential" to induce NET formation, consistent with previous findings on the link between peroxisome metabolism, platelet activation, ROS-related pathways, and NETosis. Figure 3 B). Based on the scoring of NETosis-related genes in each subgroup, the results showed that this gene set was mainly enriched in Mgst2. hi Neu. Immunofluorescence confirmed a significant increase in cells co-expressing Ly6G and Mgst2, and simultaneously expressing Padi4 or the NET marker Cit-H3, in the alveolar cavities, septa, bronchi, and vascular regions of the ZYM group; Mgst2 was also observed in BALF and PB. + Ly6G + Padi4 + With Mgst2 + Ly6G + Cit-H3 + Increase in cells ( Figure 3 To examine whether targeting Mgst2 affects NET formation, mice were administered AAV9-shRNA-Mgst2 at week 10 of ZYM treatment, and the results were evaluated at week 16. Figure 3 JM). Compared to AAV9-EGFP, knockdown of Mgst2 shRNA increased mouse body weight, reduced inflammatory cell infiltration and extracellular matrix deposition in lung tissue, and decreased PADI4 and Cit-H3. ELISA showed that AAV9-shRNA-Mgst2 reduced MPO-DNA (quantitative NET index) in BALF, but had no significant effect on MPO-DNA in lung tissue and PB. Figure 3 N). Electron microscopy also showed that Mgst2 knockdown reduced nuclear membrane rupture, chromatin decondensation, and extracellular fiber formation. Figure 3 O). The above results all indicate that: Mgst2 hi Neu promotes NET formation in RA-ILD.

[0276] 2,4Mgst2 hi Neutrophils promote NET formation by activating NOX2 signaling.

[0277] caspase-11 / gasdermin D and NOX2 / ROS are the two main pathways of NETosis. Figure 4 A). scRNA analysis also showed that the key NOX2 and ROS-related genes mainly expressed Mgst2. hi Neu ( Figure 4B). Immunofluorescence further confirmed the presence of Mgst2 in the lung tissue, BALF, and PB of the ZYM group. + Ly6G + NOX2 + A significant increase in triple-positive cells suggests Mgst2 hi Neu may promote NET formation via the NOX2 pathway. Figure 4 CH). In vitro, using an HL-60 differentiated neutrophil model, MGST2 was knocked down using siRNA: Under PMA stimulation, MGST2 knockdown significantly reduced the expression of MPO-DNA complex and ROS, and downregulated the expression of NOX2, PADI4, and Cit-H3. The NOX2 inhibitor DPI also significantly inhibited the above indicators, and showed a synergistic inhibitory effect when combined with MGST2 knockdown. Figure 4 IR). SYTOX Green staining further showed that both MGST2 knockdown and DPI treatment reduced cfDNA release; the combined use of the two resulted in more significant inhibition. Figure 4 These results suggest that Mgst2 hi Neu facilitates NET formation through NOX2 dependency.

[0278] 2.5 is derived from Mgst2 hi Neu's NET promotes pericyte-myofibroblast transdifferentiation

[0279] NETs can promote the massive aggregation of myofibroblasts. This invention compared cell-cell interactions in lung tissues from the ZYM and PBS groups. Results showed that Mgst2 in the ZYM group... hi The number and intensity of ligand-receptor interactions between neutrophils and pericytes were significantly increased, suggesting the existence of a dominant interaction. Figure 5 A). Meanwhile, the total number of inflammatory myofibroblasts and myofibroblasts in the lung tissue of the ZYM group was significantly increased, while the number of pericytes was significantly decreased. Figure 5 B). Monocle trajectory shows differentiation starting from pericytes, transitioning to myofibroblasts, and ultimately to inflammatory myofibroblasts (B). Figure 5 C). Immunofluorescence confirmed that the number of CD146 / Acta2 and Ng2 / Acta2 co-positive cells was significantly increased in the ZYM group. Figure 5 DF). The pseudo-temporal enrichment suggests that during the transition of pericellular cells to a fibrotic phenotype, extracellular genes and TGF-β signaling are gradually activated. Figure 5G). Furthermore, pericytes in the ZYM group showed higher expression of TGF-β signaling and fibrosis-related genes (Tgfb1, Tgfbi, Col1a1, Col4a1, Col4a2, Fn1, Timp3, Acta2, Tagln, Pdgfb) compared to the PBS group. Figure 5 In vitro: Primary human lung microvascular perivascular cells were exposed to PMA-induced HL-60 NETs, ​​or NETs were degraded with DNase I. Immunofluorescence, Western blotting, and RT-qPCR consistently showed that NET treatment significantly upregulated ACTA2 and COL1A1, and this upregulation was significantly attenuated after DNase I degradation of NETs. Simultaneously, the expression of TGFβR1 and TGFβR2, as well as the level of p-SMAD2 / 3, increased after NET treatment, suggesting that NETs promote PMT (perivascular microvascular perivascular microvessels) by activating TGF-β signaling. Figure 5 IL).

[0280] This invention identified a neutrophil subset with high MGST2 expression (Mgst2). hi Neutrophils (Mgst2) have a stronger tendency to form NETs and may initiate neutrophil activation during the pathogenesis of RA-ILD. In the lung tissue and BALF of mice and patients, Mgst2... hi The proportion of NETs formed by the Neu subpopulation increased significantly, suggesting their involvement in local lung pathology. Notably, Mgst2... hi The Neu subgroup is phenotypically similar to previously reported "aging" CXCR4. hi Neutrophils share similarities. ALDH2 deficiency can enhance NET formation and exacerbate myocardial injury through endoplasmic reticulum stress-Mgst2 / LTC4 pathway and NOX2 activation. Mgst2 hi The NET generated by Neu tends to rely heavily on the NOX2 signal.

[0281] Cellular interactions within the lungs are crucial for maintaining homeostasis, and their disruption is involved in various interstitial lung diseases, including RA-ILD. Among these, the interaction between neutrophils and pericytes is particularly critical for the regulation of inflammatory responses and vascular function. This invention demonstrates that Mgst2... hi Neu interacts directly with pericytes, promoting their transdifferentiation into myofibroblasts. PMT promotes fibrous matrix deposition and vascular remodeling, consistent with the view that "pericytes are important myofibroblast precursors in pulmonary fibrosis." Furthermore, Mgst2 was confirmed. hi Neu-derived NETs can promote PMT by activating TGF-β signaling.

[0282] This invention is the first to identify Mgst2 hi Neutrophil subsets release NETs through the NOX2 pathway and participate in the occurrence and progression of RA-ILD; at the same time, the released NETs can promote the transdifferentiation of perivascular cells into myofibroblasts via TGF-β signaling.

[0283] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. The use of the Mgst2 gene as a target in the preparation of products for the treatment of rheumatoid arthritis-related interstitial lung disease.

2. The use according to claim 1, characterized in that, It also includes one or more of the following features: (1) The Mgst2 gene is derived from neutrophils; preferably, the neutrophils are a subset of neutrophils that highly express Mgst2. (2) The rheumatoid arthritis-related interstitial lung disease treatment products include Mgst2 inhibitors.

3. Use of Mgst2 inhibitors in the preparation of treatment products for rheumatoid arthritis-related interstitial lung disease.

4. The use according to claim 3, characterized in that, It also includes one or more of the following features: (1) The target gene of the Mgst2 inhibitor is derived from neutrophils; preferably, the neutrophils are a subset of neutrophils that highly express Mgst2. (2) The Mgst2 inhibitor refers to a molecule that has an inhibitory effect on the Mgst2 gene; (3) The Mgst2 inhibitor is the only active ingredient or one of the active ingredients of the product; (4) The Mgst2 inhibitor is selected from nucleic acid molecules, small molecule chemical drugs, antibody drugs, peptides, proteins, nucleic acid constructs, lentiviruses, adenoviruses, or CRISPR / Cas9 genome editing systems.

5. The use as described in claim 3, wherein the Mgst2 inhibitor has at least one of the following effects: (1) Treatment of rheumatoid arthritis-related interstitial lung disease; (2) Improves inflammatory cell infiltration and extracellular matrix deposition in lung tissue; (3) Reduce the expression of one or more of the following proteins: NOX2, PADI4, or Cit-H3; (4) Reduces the expression of MPO-DNA in the alveoli; (5) Improves the rupture of the nuclear membrane in lung tissue, chromatin decondensation, and the formation of extracellular fiber structures; (6) Reduce the formation of NETs; (7) Reduces the transformation of pericytes into myofibroblasts; (8) Reduce the expression of ACTA2 and / or COL1A1.

6. The use according to claim 4, characterized in that, In feature (4), the nucleic acid molecule is selected from any one or more of the following: antisense oligonucleotides, double-stranded RNA or shRNA.

7. The use according to claim 6, characterized in that, It also includes one or more of the following features: (1) The nucleotide sequence used to prepare the shRNA is shown in SEQ ID NO: 1 and SEQ ID NO: 2; (2) The double-stranded RNA is siRNA; preferably, the first and second strands of the siRNA sequence are as shown in SEQ ID NO: 3 and SEQ ID NO: 4, and / or the first and second strands of the siRNA sequence are as shown in SEQ ID NO: 5 and SEQ ID NO:

6.

8. Use of Mgst2 inhibitors and / or NOX2 inhibitors in the preparation of NETs inhibitory products.

9. Use of NETs inhibitors in the preparation of products having at least one of the following effects: (1) Reduce the transformation of pericytes into myofibroblasts; (2) Reduce the expression of ACTA2 and / or COL1A1.

10. Use of Mgst2 inhibitors in the preparation of products having at least one of the following effects: (1) Improves inflammatory cell infiltration and extracellular matrix deposition in lung tissue; (2) Reduce the expression of one or more of the following proteins: NOX2, PADI4, or Cit-H3; (3) Reduces the expression of MPO-DNA in the alveoli; (4) Improves the rupture of the nuclear membrane in lung tissue, chromatin decondensation, and the formation of extracellular fiber structures; (5) Reduce the formation of NETs; (6) Reduces the transformation of pericytes into myofibroblasts; (7) Reduce the expression of ACTA2 and / or COL1A1.

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