CN119818678A - Application of TFEB in preparation of medicines for allergic rhinitis - Google Patents
Application of TFEB in preparation of medicines for allergic rhinitis Download PDFInfo
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Abstract
本发明属于生物医药技术领域，公开了TFEB在制备变应性鼻炎的药物中的应用，具体公开了TFEB抑制剂在制备治疗和/或预防鼻炎的药物中的应用。本发明首次公开了TFEB抑制剂在防治鼻炎中的应用，通过抑制剂靶向下调TFEB，可以抑制ILC2细胞的增殖和功能效应，从而达到治疗鼻炎的目的，即通过靶向TFEB抑制ILC2细胞的增殖和功能效应治疗变应性鼻炎。The present invention belongs to the field of biomedicine technology, and discloses the use of TFEB in the preparation of a drug for allergic rhinitis, and specifically discloses the use of a TFEB inhibitor in the preparation of a drug for treating and/or preventing rhinitis. The present invention discloses for the first time the use of a TFEB inhibitor in the prevention and treatment of rhinitis, and by targeting and downregulating TFEB with an inhibitor, the proliferation and functional effects of ILC2 cells can be inhibited, thereby achieving the purpose of treating rhinitis, that is, allergic rhinitis is treated by inhibiting the proliferation and functional effects of ILC2 cells by targeting TFEB.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an application of TFEB in preparing a medicine for allergic rhinitis.
Background
Allergic Rhinitis (AR) is characterized by the induction of an immune response promoted by immunoglobulin E (IgE) by allergens in the air. Epidemiological data indicate that AR affects more than 10% of the population worldwide, and recent research reports that the prevalence of this disease is increasing.
Type II innate lymphocytes (ILC 2) belong to innate immune cells, which are predominantly distributed in mucosal tissues and whose differentiation and function are predominantly dependent on specific transcription factors GATA3 and rorα. ILC2 is poorly distributed in peripheral blood, but they are critical for the development of type 2 inflammatory responses, an indispensable prerequisite for the development of allergic diseases. Activation is mainly dependent on cytokines such as IL-25, IL-33 and Thymic Stromal Lymphopoietin (TSLP) from the nasal epithelium. Type 2 cytokines (IL-4, IL-5 and IL-13) are the primary mediators of inflammation that regulate allergic reactions.
Transcription Factor EB (TFEB) is a transcription factor that binds to the E mu heavy chain immunoglobulin enhancer. More recently, TFEB was identified during autophagy as a key transcriptional activator and lysosomal gene. Phosphorylation of TFEB by mTORC1, ERK2 and GSK3 signaling pathways is closely related to its function. TFEB plays a role in mediating innate immunity by directly modulating pro-inflammatory/cytoprotective genes, as well as up-regulating the autophagy/lysosomal system after intracellular infection and phagocytosis. In AR, there is no report on how TFEB-mediated autophagy exerts metabolic and functional regulation of ILC2 to participate in the pathogenesis of AR.
Disclosure of Invention
It is an object of a first aspect of the present invention to provide the use of a TFEB inhibitor.
The object of the second aspect of the invention is to provide an sgRNA.
The object of the third aspect of the invention is to provide a biomaterial which is identical to the sgRNA of the second aspect of the invention.
The object of the fourth aspect of the invention is to provide a product.
The object of the fifth aspect of the invention is to provide the use of the sgrnas of the second aspect of the invention, the biomaterials of the third aspect of the invention or the products of the fourth aspect of the invention.
The sixth aspect of the invention aims to provide the application of TFEB as a target point in developing medicines for preventing and treating rhinitis.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided the use of a TFEB inhibitor in any of (1) to (7):
(1) Preparing a medicament for treating and/or preventing rhinitis;
(2) Inhibiting the expression of IL-5 and/or IL-13;
(3) Preparing an agent that inhibits the expression of IL-5 and/or IL-13;
(4) Inhibit proliferation of ILC2 cells;
(5) Preparing an agent that inhibits proliferation of ILC2 cells;
(6) Inhibiting the expression of ILC 2-specific transcription factors GATA3 and/or rorα;
(7) Agents are prepared that inhibit the expression of the ILC 2-specific transcription factors GATA3 and/or ROR alpha.
In some embodiments of the invention, the TFEB inhibitor is at least one of a substance that inhibits TFEB activity, a substance that degrades TFEB, a substance that reduces/knocks down the expression level of TFEB, a substance that knocks out TFEB.
In some embodiments of the invention, the TFEB inhibitor is a substance that reduces/knocks down the expression level of TFEB.
In some embodiments of the invention, the TFEB inhibitor is at least one of a 1) to a 5):
a1 sgRNA, siRNA, dsRNA, miRNA, ribozyme, or shRNA targeting TFEB;
a2 A nucleic acid molecule encoding sgRNA, siRNA, dsRNA, miRNA, a ribozyme or shRNA of a 1) that targets TFEB;
a3 An expression cassette, vector or transgenic cell line comprising the nucleic acid molecule of a 2);
a4 Small molecule drugs targeted to inhibit TFEB;
a5 A TFEB inhibitory antibody that blocks TFEB proteins and/or TFEB protein intermediates.
In some embodiments of the invention, the TFEB inhibitor is at least one of a 1) to a 5):
a1 Sgrnas targeting TFEB;
a2 A nucleic acid molecule encoding a TFEB-targeted sgRNA as described in a 1);
a3 An expression cassette, vector or transgenic cell line comprising the nucleic acid molecule of a 2);
a4 Small molecule drugs targeted to inhibit TFEB;
a5 A TFEB inhibitory antibody that blocks TFEB proteins and/or TFEB protein intermediates.
In some embodiments of the invention, the TFEB inhibitory antibody is selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a resurfaced antibody, a reconstituted antibody, a fully human antibody, or an antigen binding portion thereof, specific for TFEB.
In some embodiments of the invention, the TFEB inhibitory antibody is a monoclonal antibody specific to TFEB.
In some embodiments of the invention, the small molecule drug targeted to inhibit TFEB comprises eltrombopag (Eltrombopag, EO).
In some embodiments of the invention, the nucleotide sequence of the sgRNA is shown in SEQ ID NO. 3.
In some embodiments of the invention, (1) the rhinitis comprises allergic rhinitis.
In some embodiments of the invention, the inhibiting the expression of IL-5 and/or IL-13 in (2) - (3) is inhibiting the expression of IL-5 and/or IL-13 in ILC2 cells.
In some embodiments of the invention, the pharmaceutical formulation described in (1) is an injection, a spray, a nasal drop, an inhalant or an oral formulation.
In a second aspect of the invention, there is provided an sgRNA having a nucleotide sequence as shown in SEQ ID NO. 3.
In a third aspect of the invention, there is provided a biomaterial associated with the sgRNA of the second aspect of the invention, the biomaterial comprising any one of b 1) to b 12):
b1 A nucleic acid molecule encoding an sgRNA of the second aspect of the invention;
b2 An expression cassette comprising b 1) the nucleic acid molecule;
b3 A vector comprising b 1) the nucleic acid molecule;
b4 A vector comprising b 2) said expression cassette;
b5 A transgenic cell line comprising b 1) said nucleic acid molecule;
b6 A transgenic cell line comprising b 2) said expression cassette;
b7 A transgenic cell line comprising b 3) the vector;
b8 A transgenic cell line comprising b 4) the vector;
b9 A recombinant microorganism comprising the nucleic acid molecule of b 1);
b10 A recombinant microorganism comprising the expression cassette of b 2);
b11 A recombinant microorganism comprising b 3) said vector;
b12 A recombinant microorganism comprising the vector of b 4).
In some embodiments of the invention, the transgenic animal cell line does not comprise propagation material.
In some embodiments of the invention, the expression cassette comprises a 5' transcriptional control region, an open reading frame encoding a fusion protein of the first aspect of the invention, a translational control signal, a 3' untranslated region (3 ' utr), and a transcriptional termination signal.
In some embodiments of the invention, the 5' transcriptional control region comprises a promoter (a general purpose promoter such as a viral promoter (SV 40 promoter) or mammalian "housekeeping" promoter may be used), a transcription initiation site, an enhancer, and/or a silencer element.
In some embodiments of the invention, the 3'utr may encode AU-rich elements that ARE common regulators of mRNA stability via the 3' -5 'exosome pathway, and ARE typically located in the 3' utr. The AU-rich element may comprise one or more repetitions of the sequence AUUUA. It may also comprise one or more so-called US2B elements having the sequence AUAUAU.
In some embodiments of the invention, the vector comprises a promoter operably linked to the nucleic acid molecule.
In some embodiments of the invention, the vector is independently selected from the group consisting of a non-pathogenic viral vector and a viral vector.
In some embodiments of the invention, the viral vector comprises at least one of a lentiviral vector, an adenoviral vector, a baculovirus vector, a retroviral vector, a poxviral vector, a sendai viral vector, a herpes simplex viral vector.
In some embodiments of the invention, the non-viral vector comprises at least one of a plasmid vector, a cationic polymer vector, chitosan, polyethylenimine, a nanoparticle vector, a liposome.
In some embodiments of the invention, the vector is a plasmid vector, phagemid, viral vector, cellular vector, phage, cosmid, F cosmid, artificial chromosome.
In some embodiments of the invention, the plasmid vector may be an optional plasmid and the viral vector may be an optional virus.
In some embodiments of the invention, the cells include prokaryotic cells, eukaryotic cells, and the cells are not new plant or animal varieties.
In some embodiments of the invention, the prokaryotic cells include E.coli, streptomyces, bacillus subtilis, and the like, bacteria known in the art that can be used to express the protein of interest.
In some embodiments of the invention, the eukaryotic cell comprises at least one of a yeast cell, a mammalian cell, a plant cell, and an insect cell.
In a fourth aspect of the invention there is provided a product comprising an sgRNA of the second aspect of the invention, a biological material of the third aspect of the invention or a TFEB inhibitory antibody targeting a TFEB protein and/or a TFEB protein intermediate.
In some embodiments of the invention, the TFEB inhibitory antibody is selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a resurfaced antibody, a reconstituted antibody, a fully human antibody, or an antigen binding portion thereof, specific for TFEB.
In some embodiments of the invention, the TFEB inhibitory antibody is a monoclonal antibody specific to TFEB.
In some embodiments of the invention, the product comprises a medicament, a reagent and/or a kit.
In some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable excipient.
In some embodiments of the invention, the pharmaceutically acceptable excipients include at least one of diluents, wetting agents, binders, disintegrants, lubricants, color and flavor modifiers, solvents, solubilizing agents, co-solvents, emulsifiers, absorption enhancers, surfactants, antioxidants, excipients, stabilizers, flavoring agents, slow release agents, metal complexing agents, inert gases, preservatives, fillers, topical analgesics, pH modifiers, and isotonic or isotonic agents.
In some embodiments of the present invention, the diluent includes, but is not limited to, at least one of starches, sugars, celluloses, inorganic salts. The wetting agent includes, but is not limited to, at least one of water, ethanol, propanol. The binder includes, but is not limited to, at least one of starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol. The disintegrating agent comprises at least one of microcrystalline cellulose, crosslinked polyvinylpyrrolidone, crosslinked sodium carboxymethyl cellulose starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, crosslinked sodium carboxymethyl cellulose, crosslinked povidone, surfactant (such as sodium dodecyl benzene sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan or polysorbate, etc.), and effervescent disintegrating agent. The lubricant comprises at least one of talcum powder, calcium stearate, magnesium dodecyl sulfate, micro-powder silica gel, calcium carbonate, sodium bicarbonate and polyethylene glycol. The color, smell and taste regulator includes at least one of pigment, perfume, sweetener, mucilage and corrigent. The solvent includes, but is not limited to, at least one of water, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil, ethyl acetate, vegetable oil (such as soybean oil, castor oil, peanut oil, blend oil, etc.). The solubilizer includes, but is not limited to, at least one of tweens, sellers, polyoxyethylene fatty alcohol ethers, soaps, sulfates, sulfonates. The cosolvent comprises at least one of organic acid and salts thereof, amide and amine compounds, inorganic salts, polyethylene glycol, povidone and glycerin. The emulsifier includes, but is not limited to, at least one of span, tween, herba Euphorbiae Helioscopiae, benzyl Euphorbiae, glycerin fatty acid ester, higher fatty acid salt, sulfate, sulfonate, acacia, tragacanth, gelatin, pectin, phospholipid, agar, sodium alginate, hydroxide, silica, bentonite. The suspending agent comprises at least one of glycerol, syrup, acacia, tragacanth, agar, sodium alginate, cellulose derivative, povidone, carbopol, polyvinyl alcohol and thixotrope. The antioxidant includes, but is not limited to, at least one of sulfite, metabisulfite, bisulfite, ascorbic acid, gallic acid and esters thereof. The metal complexing agent comprises at least one of disodium ethylenediamine tetraacetate and a polycarboxylic acid compound, and the inert gas comprises at least one of nitrogen and carbon dioxide. The preservative comprises at least one of nipagin, organic acid and salts thereof, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols and volatile oil. The filler comprises at least one of starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, or glucose. The flavoring agent comprises at least one of aspartame, sucralose, essence, steviosin, acesulfame potassium, citric acid or saccharin sodium. The local analgesic includes, but is not limited to, at least one of benzyl alcohol, chlorobutanol, lidocaine, procaine. The pH regulator includes, but is not limited to, at least one of hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate, and citrate. The isotonic or isotonic regulator includes, but is not limited to, at least one of glucose, sodium chloride, sodium citrate, sorbitol, xylitol.
In some embodiments of the invention, the medicament may be administered orally, parenterally (e.g., intravenously, intramuscularly, intraperitoneally), intracranially, intrathecally, intraarterially (e.g., via carotid artery), rectally, dermally, nasally, vaginally, inhalally, dermally (patch), or ocularly, etc., the effective dose administered being a pharmaceutically acceptable dose.
In some embodiments of the invention, the pharmaceutical composition is in a dosage form selected from the group consisting of tablets (e.g., normal tablets, bilayer tablets, multilayer tablets, sustained release tablets, single-compartment controlled release tablets, dual-compartment controlled release tablets, microporous controlled release tablets, sublingual tablets, orally rapidly disintegrating tablets, dispersible tablets, enteric-coated tablets), pills, patches, powders, suspensions, gels, emulsions, creams, granules, solutions, nanoparticles, drops, capsules (e.g., normal capsules, sustained release capsules, controlled release capsules, capsules containing pellets or minipills, pH-dependent capsules containing pellets or minipills, gastrointestinal complex capsules), enemas, suppositories, granules, injections, sprays and injections.
In a fifth aspect, the invention provides the use of an sgRNA according to the second aspect of the invention, a biomaterial according to the third aspect of the invention or a product according to the fourth aspect of the invention in any one of (1) to (7):
(1) Preparing a medicament for treating and/or preventing rhinitis;
(2) Inhibiting the expression of IL-5 and/or IL-13;
(3) Preparing an agent that inhibits the expression of IL-5 and/or IL-13;
(4) Inhibit proliferation of ILC2 cells;
(5) Preparing an agent that inhibits proliferation of ILC2 cells;
(6) Inhibiting the expression of ILC 2-specific transcription factors GATA3 and/or rorα;
(7) Agents are prepared that inhibit the expression of the ILC 2-specific transcription factors GATA3 and/or ROR alpha.
In some embodiments of the invention, (1) the rhinitis comprises allergic rhinitis.
In some embodiments of the invention, the inhibiting the expression of IL-5 and/or IL-13 in (2) - (3) is inhibiting the expression of IL-5 and/or IL-13 in ILC2 cells.
In a sixth aspect of the invention, there is provided the use of TFEB as a target in the development of a medicament for the prevention and treatment of rhinitis.
In some embodiments of the invention, the rhinitis comprises allergic rhinitis.
The beneficial effects of the invention are as follows:
The invention discloses an application of a TFEB inhibitor in preventing and treating rhinitis for the first time, and can inhibit proliferation and functional effects of ILC2 cells by targeting TFEB, thereby achieving the purpose of treating rhinitis, namely, treating allergic rhinitis by inhibiting proliferation and functional effects of ILC2 cells by targeting TFEB.
Drawings
FIG. 1 shows differences in ILC2 ratios and functional phenotypes in the nasal mucosa of AR and control groups, wherein A is a statistical chart of flow cytometry detection results of A comparing the ratio of ILC2 cells in the nasal mucosa of AR patients (10 cases) and normal control (10 cases), B is a statistical chart of flow cytometry detection results of A comparing the ratio of IL-5+ILC2 and IL-13+ILC2 cells in the nasal mucosa of C flow cytometry detection AR patients and normal control groups, and D-E is a statistical chart of flow cytometry detection results of IL-5+ILC2 (D) and IL-13+ILC2 cells (E) in the nasal mucosa of AR patients and normal control groups.
FIG. 2 shows the correlation of autophagosomes and autophagy-related proteins expressed in the nasal mucosa ILC2 of AR and control patients with the ratio of ILC2 cells and the functional phenotype, wherein A is the difference in the number of autophagosomes in the nasal mucosa of transmission electron microscopy comparison AR patients and normal control patients, B is the comparison of the expression of autophagy-related proteins (LC 3II, P62, ATG5 and TFEB) in the nasal mucosa ILC2 of Western blot detection AR patients and normal control patients, C is the correlation of the expression of autophagy-related proteins (LC 3II, P62, ATG 5) in the nasal mucosa ILC2 of AR and the ratio of ILC2 cells, D is immunofluorescence to show the difference in the expression of TFEB in the nasal mucosa ILC2 of AR and control groups with the ratio of 1:800, HC in the figure, normal control group, AR, allergic rhinitis patients, and x represents P <0.05.
FIG. 3 shows the difference in TFEB expression in AR nasal mucosa ILC2 between AR patients and normal controls and their correlation with the ratio of IL-5+ILC2, IL-13+ILC2 cells in the nasal mucosa.
FIG. 4 is an illustration of the expression level of TFEB gene in overexpressed or knocked-down ILC2 cells.
Fig. 5 shows changes in proliferation and function of ILC2 following intervention of lentiviral vectors in ILC2 by TFEB expression, where TFEB-OV is the TFEB over-expression group, TFEB-KD is the TFEB knockout group, representing P <0.05.
FIG. 6 shows therapeutic effect and immune modulation of TFEB antagonists on AR mice models, wherein A is a comparison of the levels of Der p 1-specific IgE in each group, B is a comparison of the number of sneezes recorded during 15 minutes in each group, C is a comparison of the number of scratchings recorded during 15 minutes in each group, D is a comparison of the levels of ILC2 in each group, E is a comparison of the levels of IL-5+ILC2 in each group, and F is a comparison of the levels of IL-13+ILC2 in each group.
Detailed Description
The following describes the present invention in further detail by way of specific examples.
It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
1) Patient incorporation
According to the AR guidelines of the China medical society 2022, the inclusion criteria for AR patients are as follows, typical perennial nasal symptoms (nasal congestion, nasal itching, sneezing and nasal discharge, at least 1 year), age 18 years old, skin prick and serum specific IgE detection suggesting mite allergy (at least one of house dust mites or dust mites), exclusion criteria are as follows, recent (within 2 weeks) systemic or nasal glucocorticoid, with upper respiratory tract infections, with respiratory tract diseases such as acute and chronic sinusitis, cystic fibrosis, asthma, and with severe systemic diseases. The normal control group was patients over 18 years of age, negative for skin prick and serum-specific IgE detection allergen, no respiratory disease and systemic disease, and a partial subturbinate excision. The patient was from a affiliated women child medical center at Guangzhou medical university. Peripheral blood (20 mL) spares and nasal mucosa spares were collected from both groups of patients, approved by the patient's informed consent and ethical committee.
2) Sample preparation, preparation of human Peripheral Blood Mononuclear Cells (PBMC) and ILC2 sorting
① Human peripheral blood fresh heparin anti-venous blood (20 mL) from AR patient was mixed well with RPMI-1640 medium 1:1, slowly added to a tube containing appropriate amount of lymphocyte separation solution (Ficoll) and centrifuged at 2200rpm for 25 minutes at room temperature. PBMC were collected and resuspended in RPMI-1640 after washing. Peripheral blood lineage negative (Lin -) cells (CD 2, CD3, CD14, CD16, CD19, CD56, CD235a and fceri) were sorted from PBMCs by LINEAGE CELL depletion kit (Miltenyi Biotec), then stained with PE-labeled CRTH2 and PE-Cy7 labeled CD127 and flow cytometry sorted to obtain Lin -CD127+CRTH2+, i.e., ILC2 cells.
② Human nasal mucosa, nasal cavity curettage was performed 1 to 3 times on the patient's inferior turbinates with a Rhino probe (Arlington Scientific, SPRINGVILLE, utah) and each sample was placed in RPMI medium for use. Li n- cells (CD 2, CD3, CD14, CD16, CD19, CD56, CD235a and fceri) were sorted from PBMCs by lineage cell removal kit (Miltenyi Biotec), and flow cytometry was further sorted for Lin -CD127+CRTH2+, i.e., ILC2 cells were obtained.
The flow cell sorting procedure described above was followed by purification of PBMC using Ficoll-Paque PLUS (China Feng Hao). To mitigate non-specific binding, cells were pre-incubated with 5% fetal bovine serum (FBS, fenghao, china) for 10 minutes on ice, then stained with a fixable vital dye (Fenghao, china) to exclude non-viable cells. ILC2 cells are defined as human ILC2 cells Lin -CD127+CRTH2+, wherein the Lin - markers include CD2, CD3, CD14, CD16, CD19, CD56, CD235a, and Fc epsilon RI. Murine ILC2 cells Lin -ST2+CD45+CD90.2+CD25+Sca1+.
Comparison of AR patient and normal control nasal mucosa ILC2 cells by flow cytometry showed differences in IL-5+ilc2 and IL-13+ilc2 cell ratios, with AR patient nasal mucosa derived ILC2, IL-5+ilc2 and IL-13+ilc2 ratios significantly higher than normal control, suggesting importance of ILC2 cell proliferation and function in AR development.
3) Transmission electron microscope
Tissues (nasal mucosa) or cells were fixed overnight with 4% glutaraldehyde solution, then treated with 1% oso 4 (osmium tetroxide) in 0.1mol/L diformylidene buffer containing 0.1% cacl 2 for 2 hours (4 ℃) before staining the samples with uranyl acetate and dehydrating the samples with increasing concentrations of ethanol. Epoxy resin embedding at 60 ℃ for 48 hours and cutting into ultrathin sections. Staining was performed with 0.1% lead citrate and 4% uranyl acetate, and the number of autophagosomes of ILC2 was counted using a transmission electron microscope (model H-7100FA; hitachi, tokyo, japan).
The difference in the number of autophagosomes in AR patients and normal control ILC2 was compared by transmission electron microscopy, and as shown in the figure, autophagy of ILC2 derived from nasal mucosa of AR patients was significantly stronger than that of normal control group, and autophagosomes in ILC2 were increased (fig. 2 a).
4)Western Blot
The expression of autophagy-related proteins (LC 3II, P62, ATG5 and TFEB) in AR patients and normal control nasal mucosa ILC2 was examined using Western Blot and the correlation of autophagy-related protein expression (LC 3II, P62, ATG 5) in AR nasal mucosa ILC2 with ILC2 cell ratios was further analyzed. The Western Blot experiment was performed by treating the sample with a cell lysis buffer, adding protease inhibitors, and centrifuging to obtain the supernatant. The protein sample is added into polyacrylamide gel, the protein is separated in the gel by the action of an electric field, and the protein after electrophoresis separation is transferred from the gel to a membrane by using a wet transfer membrane method. The membrane is treated with blocking buffer to prevent non-specific binding. The membrane was incubated with specific antibodies (primary antibodies, LC3II, P62, ATG5 and TFEB) to bind the primary antibodies to the target protein, and antibodies (goat anti-human IgG (1:10000), thermo Fisher, AH 1109925) were added to bind the primary antibodies to form an antibody complex. Color development was performed using a chemiluminescent method, then imaging was performed using a fluorescence imager, and quantitative analysis was performed.
The results showed that autophagy-related proteins LC3II, P62, ATG5 and TFEB expression in ILC2 were significantly upregulated compared to the normal control group (B and C in fig. 2).
5) Immunofluorescence
Detecting expression difference of Transfer Factor EB (TFEB) in the nasal mucosa ILC2 of the AR group and the control group by adopting immunofluorescence technology, and further analyzing correlation of TFEB expression in the nasal mucosa ILC2 of the AR and the proportion of the nasal mucosa IL-5+ILC2 cells and IL-13+ILC2 cells. The experiments were performed using immunofluorescent staining kit (P0188, biyun biotechnology) and were performed by centrifugation of a sample tissue cell suspension followed by smear, fixation of cells or tissue with fixative and treatment of cells with permeabilizer. Cells or tissues were blocked using blocking solution, proportioned primary antibodies (TFEB (3. Mu.g/mL), GATA3 (5. Mu.g/mL), DAPI (0.1. Mu.g/mL), all purchased from R & D systems), incubated at room temperature or overnight at 4℃and then proportioned secondary antibodies (NL 001 (1:200), NL002 (1:200), NL003 (1:200), all purchased from R & D systems) labeled with the corresponding species of primary antibody were added and incubated at room temperature protected from light. After each antibody incubation, multiple washes with PBS buffer were performed, autofluorescence quenchers were added, and the tablets were capped with anti-fluorescence quench. Observed under a fluorescence microscope and photographed, and the experimental results were recorded.
The results showed that TFEB expression was positively correlated with the proportion and functional phenotype of ILC2 cells, IL-5+ILC2 cells and IL-13+ILC2 cells in the nasal mucosa (FIG. 2D and FIG. 3)
Example 2
(1) TFEB expression in peripheral blood sorted ILC2 cells of AR patients is regulated (over-expressed/knockdown) by lentiviral vectors. The specific process is as follows:
ILC2 cells (sorted in example 1) were resuscitated and cultured in advance, ensuring that the cells were in good growth. The TFEB regulatory sequence is inserted into proper site of slow virus vector to construct expression vector. The primers were designed based on the human TFEB gene sequences in NCBI database, and EcoRI and BamHI cleavage sites and protecting bases were added at both ends by alignment, and the primer sequence was 5'-CGGAATTCTGATGGCGTCACGCATAGGGTTGC-3' (SEQ ID NO: 1) upstream and 5'-CGGGATCCCGCAGCACATCGCCCTCCTCCATG-3' (SEQ ID NO: 2) downstream. Wherein the knockdown expression uses a sgRNA having a nucleotide sequence 5'-CATTGACAACATTATGCGTC-3' (SEQ ID NO: 3). Transfecting the constructed expression vector into specific packaging cells, inoculating HEK 293T cells with good growth state into a 6-hole plate 1 day before transfection, changing fresh complete culture medium containing 10% FBS after the cell confluence reaches about 70%, adding 105 mug of TFEB-DNA3.1-Flag-PCDH, 3.75 mug of packaging plasmid pSPAX and 1.5 mug of pMD2G into 500 mug L Optimum, uniformly mixing to prepare DNA tissue, standing at room temperature for 5 minutes, simultaneously uniformly mixing 500 mug L Optimum and 20 mug L Lipofactamine2000, standing at room temperature for 20 minutes, uniformly mixing the DNA tissue with the DNA tissue, uniformly dripping the mixture into the HEK 293T cells, placing the cells into fresh complete culture medium containing 10% FBS after the cell confluence reaches about 70%, continuously culturing for 48 hours, collecting cell supernatant after the cell culture is continuously used for 0.45 mu m filter, filtering at 4 ℃ for 12000 r/min, and obtaining TFEB over-expressed slow virus. TFEB knockdown lentiviruses were obtained in the same manner. Viral particles are produced on a large scale by cell culture and virus amplification processes.
Inoculating 2X 10 6 THP-1 cells to a 6-hole plate, adding a corresponding volume of virus stock solution according to the virus infection number and the cell number when the cell grows to 50% -60%, continuously culturing for 24 hours in a cell culture box by using an adaptive culture medium containing ampicillin (2 mug/ml), continuously screening the cells infected by the virus, continuously culturing for 3-4 days until the residual few THP-1 cells survive, and stably passaging the THP-1 containing TFEB-DNA3.1-Flag-PCDH10 lentivirus, thereby obtaining a THP-1 cell strain with high expression of TFEB genes, namely an over-expression group (OV), transferring the THP-1 cell strain with low-knocking of the TFEB genes, and taking the THP-1 cell strain without the TFEB genes as a control group.
And detecting the expression level of the TFEB gene in the ILC2 cells by RT-PCR and Western blot to determine whether the ILC2 cells are knocked down or over-expressed.
As shown in fig. 4, ILC2 cells were successfully knocked down or overexpressed TFEB.
(2) CFSE detection of proliferation of ILC2 cells after the above-described TFEB knockdown
CFSE was used to detect proliferation of the above TFEB knocked-down ILC2 cells, and the method comprises taking out CFSE reagent (Abmole, cat# M5117) from the refrigerator, cooling to room temperature, opening, dissolving CFSE in DMSO to obtain a stock solution, and diluting with serum-free DMEM culture solution to obtain working solution. The cell concentration was adjusted to 1X 10 5/mL, an appropriate amount of CFSE working solution (1 mL out of 10mM CFSE in DMSO) was added, gently stirred and mixed, and incubated in a 37℃incubator for 20 minutes. After the incubation is finished, the supernatant is removed by centrifugation, the cells are washed by adding PBS solution, the operation is repeated for 2-3 times, and redundant CFSE is removed. The washed cells are added into RPMI 1640 medium to prepare cell suspension, and then are placed into a culture dish for culture. CFSE fluorescence intensity of cells was analyzed to track cell division.
As shown in FIG. 5, the proliferation and functional effects of ILC2 were enhanced by overexpressing TFEB expression in ILC2 sorted in peripheral blood of AR patients by lentiviral vectors, whereas the proliferation and functional effects of ILC2 were inhibited by knocking down TFEB expression.
(3) RT-PCR detection of expression differences of ILC 2-specific transcription factors GATA3 and ROR alpha
The expression difference of ILC2 specific transcription factors GATA3 and ROR alpha is detected by RT-PCR, and the detection process is specifically as follows:
30mg of tissue is ground by liquid nitrogen, total RNA is extracted by an RNA extraction kit (Guangzhou fly biological engineering Co., ltd.) according to the specification, and when A 260/A280 is 1.8-2.1, the subsequent experiment can be carried out. cDNA was synthesized by reverse transcription from 1. Mu.g of total RNA, amplified under the conditions described in the specification (reagents were purchased from Toyobo Biotechnology Co., ltd.) and tested in Aoolied Biosustems system. The relative expression levels of the ILC 2-specific transcription factors GATA3 and ROR alpha were calculated using the 2 -△△Ct method with GADPH as an initial reference correction sample.
RT-PCR detection results are shown in FIG. 5, after TFEB expression in ILC2 sorted in peripheral blood of AR patients is knocked down by a lentiviral vector, ILC 2-specific transcription factors GATA3 and ROR alpha are down-regulated, and overexpression of TFEB enhances ILC 2-specific transcription factors GATA3 and ROR alpha.
(4) ELISA detection of IL-5 expression differences in ILC2 cells
ELISA was used to detect differences in IL-5 expression in ILC2 cells, and the specific detection procedure was as follows:
30mg of tissue was taken and added with an appropriate amount of RIPA (tissue/cell lysate) on ice for complete grinding according to instructions. The lysed sample was centrifuged at 12,000 r/min for 5min and the supernatant was taken. The protein concentration was then determined by the BCA method (bi yun tian BCA protein concentration assay kit). The protein expression levels of IL-5 and IL-13 in all samples were detected using an ELISA kit (Shanghai Jiang Lai Biotechnology Co., ltd.) and the procedure was performed as described in the kit.
ELISA detection results are shown in FIG. 5, and IL-5 expression is enhanced after TFEB expression in peripheral blood sorted ILC2 of patients with excessive AR by using a lentiviral vector, and IL-5 expression can be inhibited after TFEB expression is knocked down.
The experimental conclusion suggests that the proliferation of ILC2 cells and the expression of the main inflammatory cell factor IL-5 thereof are obviously inhibited by targeted inhibition of TFEB expression in ILC2, so that the occurrence and development of allergic inflammation are obviously inhibited.
Example 3
1) Experimental mice
Female BALB/c mice at 8 weeks of age were randomly divided into normal, AR and anti-TFEB groups of 6. The AR and anti-TFEB groups construct sensitized AR models by induction of Ovalbumin (OVA). The sensitized AR model was constructed by intraperitoneally injecting 0.5mL of physiological saline suspension containing 100. Mu.g OVA (0.2 mg/mL, invivogen, U.S.) and 2mg aluminum hydroxide (4 mg/mL, sigma-Aldrich, U.S.) on days 0, 2, 4, 6, 8, 10 and 12, and by nasal instilling 10% OVA, 10. Mu.L each on days 14-27. Normal groups were injected with physiological saline at the corresponding time points. The success of constructing the sensitized AR model was determined by recording the number of times the mice were scratched and sneezed to determine whether modeling was successful. The AR group and the anti-TFEB group were instilled with 10% OVA, 10. Mu.L/mouse in the nasal cavity on days 14-27. Normal groups were injected with physiological saline at the corresponding time points. anti-TFEB groups were given by intraperitoneal injections of 0.4mg/mL of anti-TFEB (anti-TFEB antibodies were purchased from Abcam, inc., USA, cat No. ab 245349) at 10mL/kg 30 minutes prior to nasal instillation of OVA on days 14, 18, 22 and 26. All groups were sampled the next day by recording the number of nasal scratchings and sneezes within 10 minutes of the end of the last dose. After all mice are anesthetized, the eyesockets are sampled for blood, heparin sodium in blood samples is anticoagulated, and alveolar lavage liquid and nasal lavage liquid are collected after euthanasia, and complete nasal mucosa and lungs are taken.
The confirmation of the injection concentration of the anti-TFEB antibody mainly depends on the early pre-experiment, the inventor injects the concentrations of 0.1mg/mL, 0.2mg/mL, 0.4mg/mL, 0.8mg/mL, 1.0mg/mL, 2.0mg/mL and the like, and the comparison finds that 0.4mg/mL is optimal for improving the symptoms of AR mice, and the mice have no adverse events under all concentrations.
2) Sorting of ILC2 cells in mouse nasal mucosa the sensitized AR model mice were euthanized, and then the entire mice were nasal stripped and peeled and cut into small pieces with scissors. The sections were digested with 1mg/mL collagenase IV and 10U/mL DNase I with continuous stirring at 37℃for 40 minutes. After filtration through a 70 μm cell filter, the cells were resuspended in 40% percoll medium and gently added to 80% percoll medium. Centrifuge at 400 Xg for 15 min at room temperature. Monocytes (MNCs) were collected from the interface between 40% and 80% percoll medium. MNCs were then washed twice with ice-cold PBS. Lin - cells (CD 2, CD3, CD14, CD16, CD19, CD56, CD235a and Fc εRI) were sorted from PBMC by lineage cell removal kit (Miltenyi Biotec), and further sorted by flow cytometry (experimental procedure same as example 1) to obtain murine ILC2 cells.
3) Sorting of ILC2 cells in mouse peripheral blood fresh heparin anti-venous blood from source was added to a tube containing lymphocyte isolate (Ficoll), centrifuged for 25 minutes (2200 rpm), and PBMC were collected and washed and suspended in RPMI-1640. The sorting strategy is the same as that of the nasal mucosa of the mice. Murine ILC2 cells were obtained.
The results are shown in fig. 6, where TFEB antagonists (anti-TFEB antibodies) have therapeutic effects on AR (remission of AR symptoms after TFEB administration) and immune modulation. Specifically, the anti-TFEB group was significantly reduced in Der p1 specific IgE content (fig. 4 a), nasal mucosa ILC2 cells (fig. 4D), nasal mucosa IL-5+ilc2 cells (fig. 4E) and IL-13+ilc2 cells (fig. 4F) compared to the AR group, and the number of sneezes (fig. 4B) and nasal deflection (fig. 4C) in the mice was also significantly reduced.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
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| CN103458970A (en) * | 2011-03-07 | 2013-12-18 | 泰莱托恩基金会 | Tfeb phosphorylation inhibitors and uses thereof |
| CN113316453A (en) * | 2019-01-03 | 2021-08-27 | 匹兹堡大学联邦系统高等教育 | Methods and materials for increasing transcription factor EB polypeptide levels |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103458970A (en) * | 2011-03-07 | 2013-12-18 | 泰莱托恩基金会 | Tfeb phosphorylation inhibitors and uses thereof |
| CN113316453A (en) * | 2019-01-03 | 2021-08-27 | 匹兹堡大学联邦系统高等教育 | Methods and materials for increasing transcription factor EB polypeptide levels |
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| CHEN WANG ET AL: "Role of autophagy and mitophagy of group 2 innate lymphoid cells in allergic and local allergic rhinitis", WORLD ALLERGY ORGAN J., vol. 17, 17 January 2024 (2024-01-17), pages 1 - 13 * |
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