A medicament for treating type i hypersensitivity

Abstract

The application provides a medicine for treating type I hypersensitivity, and belongs to the field of pharmacy. The tetrahedral framework nucleic acid can effectively inhibit mast cell degranulation, can stabilize the level of mitochondrial energy metabolism, can restore the decrease of mitochondrial membrane potential and metabolic change caused after the allergy is triggered, and can effectively inhibit type I hypersensitivity in vivo. The application further modifies siRNA targeting Gab2 and Syk on the basis of the tetrahedral framework nucleic acid, and obtains a complex TSS. The complex TSS can stabilize mitochondrial energy metabolism, inhibit the outflow of calcium ions in mitochondria into cells, inhibit the occurrence of mast cell degranulation, and plays a role in inhibiting type I hypersensitivity. It is particularly worth noting that the complex TSS plays a synergistic role in inhibiting type I hypersensitivity. The tetrahedral framework nucleic acid and the complex TSS of the application are not only simple to synthesize and stable in structure, but also have a wide application prospect in the preparation of medicines for preventing and treating type I hypersensitivity.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a drug for treating type I hypersensitivity reactions. Background Technology

[0002] Hypersensitivity reactions can be classified into four types according to the Gale-Coombs classification, with Type I hypersensitivity being the most common. The term "allergy" usually refers to this type. It is estimated that 20%-30% of the population suffers from allergic diseases such as asthma, food allergies, allergic rhinitis, allergic conjunctivitis, urticaria, angioedema, and anaphylactic shock. Their incidence is on the rise worldwide, placing a significant burden on the economy. It was reported that in 2013, the total cost of asthma alone in the United States reached $81.9 billion. Allergic diseases have a serious negative impact on human quality of life.

[0003] Type I hypersensitivity is a complex immune response. Upon exposure to an external antigen, B cells produce immunoglobulin E (IgE), which then binds to the high-affinity IgE receptor (FcεRI) on mast cells. At this point, mast cells are sensitized. Upon re-exposure to the same antigen, mast cells rapidly degranulate, releasing histamine, trypsin-like enzymes, leukotrienes, tumor necrosis factor-α (TNF-α), and other factors, leading to increased vascular permeability, muscle spasms, and increased glandular secretion. Mast cells are widely distributed in the skin, mucous membranes, gastrointestinal tract, glands, and around nerves and blood vessels; therefore, degranulation can cause various allergic symptoms. Mast cells are a key component in the development of type I hypersensitivity, and their degranulation process involves two stages. First, particles far from the cell membrane are transported towards the cell membrane via microtubules. Then, the particles fuse with the cell membrane and release their contents extracellularly. These two stages are controlled by the Fyn / Gab2 and Lyn / Syk pathways, respectively. Therefore, developing drugs that can effectively inhibit mast cell degranulation is an important direction in the research and development of drugs to treat type I hypersensitivity reactions.

[0004] Currently, most patients can achieve some degree of control over type I hypersensitivity reactions by avoiding allergen exposure, receiving traditional allergen-specific immunotherapy (AIT), and symptomatic drug treatment. However, a small number of patients still experience persistent symptoms and are at risk of life-threatening allergic reactions. Developing new drugs that can effectively and safely treat type I hypersensitivity reactions is of great significance. Summary of the Invention

[0005] In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a drug for treating type I hypersensitivity reactions.

[0006] This invention provides a tetrahedral framework nucleic acid, which is self-assembled from four DNA single strands, the sequences of which are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0007] The present invention also provides a method for preparing the above-mentioned tetrahedral framework nucleic acid, the method comprising the following steps: mixing four DNA single strands, heating the mixture, and then cooling the mixture to obtain the tetrahedral framework nucleic acid.

[0008] Furthermore, the heating reaction is carried out at a temperature of 80-100°C for a time of 5-15 minutes;

[0009] The cooling reaction is carried out at a temperature of 0-10℃ for a time of 10-30 minutes.

[0010] Preferably, the heating reaction is carried out at a temperature of 95°C for 10 minutes.

[0011] The cooling reaction was carried out at a temperature of 4°C for 20 minutes.

[0012] The present invention also provides a tetrahedral framework nucleic acid complex, which is a product prepared using a reagent that inhibits mast cell degranulation and the above-mentioned tetrahedral framework nucleic acid as raw materials.

[0013] Furthermore, the reagent that inhibits mast cell degranulation is a reagent that targets and inhibits the expression of Gab2 and / or Syk.

[0014] Furthermore, the reagent for targeting and inhibiting Gab2 expression is siRNA that targets and inhibits Gab2 expression, and the siRNA that targets and inhibits Gab2 expression is composed of two single strands with sequences as shown in SEQ ID NO.5 and SEQ ID NO.6, respectively, through complementary base pairing;

[0015] The reagent for targeting and inhibiting Syk expression is an siRNA that targets and inhibits Syk expression. The siRNA that targets and inhibits Syk expression is composed of two single strands with complementary base pairing, as shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0016] Furthermore, the reagent for inhibiting mast cell degranulation is a reagent that targets and inhibits Syk expression and a reagent that targets and inhibits Gab2 expression, wherein the molar ratio of the reagent that targets and inhibits Syk expression, the reagent that targets and inhibits Gab2 expression, and the tetrahedral framework nucleic acid is 1:1:(0.1-1), preferably 1:1:0.5.

[0017] The present invention also provides a method for preparing the above-mentioned tetrahedral framework nucleic acid complex, the method comprising the following steps: mixing a reagent for inhibiting mast cell degranulation with tetrahedral framework nucleic acid, heating the mixture to react, and then cooling the mixture to react, thereby obtaining the tetrahedral framework nucleic acid complex.

[0018] Furthermore, the heating reaction is carried out at a temperature of 80-100°C for a time of 5-15 minutes;

[0019] The cooling reaction is carried out at a temperature of 0-10℃ for a time of 10-30 minutes.

[0020] Preferably, the heating reaction is carried out at a temperature of 95°C for 10 minutes.

[0021] The cooling reaction was carried out at a temperature of 4°C for 20 minutes.

[0022] The present invention also provides the use of the above-mentioned tetrahedral framework nucleic acid and the above-mentioned tetrahedral framework nucleic acid complex in the preparation of medicaments for the prevention and / or treatment of type I hypersensitivity reactions.

[0023] Furthermore, the drug is a pharmaceutical preparation made with the tetrahedral framework nucleic acid or tetrahedral framework nucleic acid complex as the active ingredient, plus pharmaceutically acceptable excipients.

[0024] Furthermore, the drug is a drug that inhibits mast cell degranulation.

[0025] The present invention has achieved the following beneficial effects:

[0026] 1. The tetrahedral framework nucleic acid provided by this invention can effectively inhibit RBL-2H3 cell degranulation when administered alone, stabilize mitochondrial energy metabolism levels, restore the mitochondrial membrane potential reduction and metabolic changes caused by allergic reactions, and effectively inhibit type I hypersensitivity reactions in vivo. It has broad application prospects in the preparation of drugs for the prevention and / or treatment of type I hypersensitivity reactions.

[0027] 2. This invention further modifies tetrahedral framework nucleic acids to target Gab2 and Syk siRNAs, resulting in the complex TSS. The TSS complex can carry siRNA into cells to exert RNA interference (RNAi) for the treatment of type I hypersensitivity reactions. In anti-DNP-IgE and DNP-BSA-induced RBL-2H3 cell models, after the Gab2 and Syk-targeting siRNAs enter the cells, they inhibit the Fyn / Gab2 and Lyn / Syk pathways and their downstream targets, thus inhibiting mast cell degranulation. The TSS complex of this invention can stabilize mitochondrial energy metabolism, inhibit the efflux of calcium ions from mitochondria into cells, and further inhibit mast cell degranulation. In a mouse passive cutaneous anaphylaxis model, the TSS complex of this invention can enter mice via intraperitoneal injection and transdermal administration, inhibiting mast cell degranulation through multiple routes of administration, reducing swelling and inflammation caused by increased local vascular permeability, and thus inhibiting type I hypersensitivity reactions. It is particularly noteworthy that the TSS complex exhibits a synergistic effect in inhibiting type I hypersensitivity reactions. The TSS complex of this invention is not only simple to synthesize and structurally stable, but also has excellent potential for multi-route drug delivery and has broad application prospects.

[0028] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0029] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0030] Figure 1 Experimental results for the synthesis, validation, and characterization of siRNA-modified tetrahedral framework nucleic acids (TSS) targeting Gab2 and Syk (A: TSS synthesis process; B: High-performance capillary electrophoresis of tFNAs; C: Polyacrylamide gel electrophoresis of tFNAs; D: Dynamic light scattering of TSS; E: Transmission electron microscopy of TSS; F: Atomic force microscopy of TSS; G: Stability of TSS in cell lysis buffer; H: Stability of TSS in serum; I: Stability of TSS at -4°C).

[0031] Figure 2Experimental results for TSS entry into cells and its RNA interference effect (A: The process of TSS entering cells and exerting RNAi effect; B: Laser confocal microscopy test of siRNA being carried into cells by tFNAs; C: Flow cytometry detection of TSS; D: Cell viability test of TSS; E: Western blot detection of TSS on Gab2 expression; F: Western blot detection of TSS on Syk expression; G: qPCR detection of TSS on Gab2 and Syk expression; H: Immunofluorescence detection of TSS on Gab2 expression; I: Immunofluorescence detection of TSS on Syk expression).

[0032] Figure 3 The experimental results of TSS inhibiting RBL-2H3 cell degranulation (A: Cell morphology after toluidine blue staining; B: Cell morphology under scanning electron microscopy; C: Apoptosis under fluorescence detection; D: Calcium ion concentration under fluorescence detection; E: Effects of different treatments on apoptosis and calcium ion concentration; F: β-hexosinosinase assay results; G: Histamine, TNF-α and IL-6 assay results).

[0033] Figure 4 Experimental results on the role of TSS in maintaining mitochondrial energy metabolism stability (A: Schematic diagram of TSS in mitochondrial energy metabolism; B: Mitochondrial membrane potential results under fluorescence assay; C: Effects of different treatments on OCR; D: Effects of TSS on basal respiratory capacity; E: Effects of TSS on maximal respiratory capacity; F: Results of ATP production rate; G: Effects of TSS on reserve respiratory capacity; H: Effects of TSS on ATP production capacity).

[0034] Figure 5 The experimental results of TSS inhibiting passive cutaneous anaphylaxis by intraperitoneal injection are as follows: (A: Schematic diagram of intraperitoneal injection of TSS in mice; B: Schematic diagram of drug distribution in mice; C: Evans blue exudation map and quantitative detection results in the ear; D: HE section image of mouse ear and ear thickness results; E: Mast cell infiltration map of mouse ear; F: Expression of Syk in tissue under fluorescence detection; G: Expression of Gab2 in tissue under fluorescence detection; H: Effect of different treatments on the expression of Syk and Gab2 in tissue; I: Effect of different treatments on leukocytes and neutrophils in mouse blood).

[0035] Figure 6The experimental results of TSS inhibiting passive cutaneous anaphylaxis via transdermal administration are as follows: (A: TSS transdermal administration process in mice; B: Evans blue exudation in mouse ears; C: Quantitative detection results of Evans blue exudation; D: Fluorescence test results of Cy5 and DAPI-Cy5; E: Quantitative statistical results of tissue thickness; F: HE section of mouse ears; G: Mast cell infiltration in mouse ears; H: Effect of different treatments on Syk expression in tissues; I: Effect of different treatments on Gab2 expression in tissues). Detailed Implementation

[0036] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0037] Example 1: Synthesis of Tetrahedral Framework Nucleic Acids

[0038] Four DNA single strands (S1, S2, S3, S4; sequences shown in Table 1) were dissolved in TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0) to control the final concentration of the four DNA single strands to 1000 nM. After thorough mixing, the mixture was rapidly heated to 95 °C and held for 10 minutes, then rapidly cooled to 4 °C and held for 20 minutes to self-assemble into a tetrahedral framework nucleic acid (tFNA) solution.

[0039] Table 1. Specific sequences of the four single-stranded DNA strands

[0040]

[0041] Example 2: Synthesis of the complex TSS

[0042] Two types of siRNA double strands (Gab2-siRNA and Syk-siRNA; sequences are shown in Table 2) were added to the tFNAs solution prepared in Example 1 and mixed well. The final concentration of each siRNA was controlled at 2000 nM, and the mixture was incubated at 37°C for 1 hour to obtain a tetrahedral framework nucleic acid complex (abbreviated as TSS) modified with siRNA targeting Gab2 and Syk.

[0043] Table 2. Specific sequences of the two siRNA double strands

[0044]

[0045]

[0046] The following experimental examples demonstrate the beneficial effects of the present invention.

[0047] Example 1: Structural characterization of tetrahedral framework nucleic acid and its complex TSS

[0048] 1. Experimental Methods

[0049] The successful synthesis of tFNAs and the shape, particle size, and zeta potential of TSS were verified by high-performance capillary electrophoresis (HPCE), polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM).

[0050] 2. Experimental Results

[0051] Figure 1 A demonstrates the synthesis process of TSS. Figure 1 The successful synthesis of tFNAs was verified by high-performance capillary electrophoresis (HPCE) and polyacrylamide gel electrophoresis (PAGE) in B and 1C. Figure 1 D detected that the TSS particle size was approximately 21.9 nm and the potential was approximately -9.59 mV using dynamic light scattering (DLS). Figure 1 E and 1F used transmission electron microscopy (TEM) and atomic force microscopy (AFM) to visually observe the tetrahedral morphology of the TSS with side chains connected to its vertices. Figure 1 G, 1H, and 1I were used to verify the stability of TSS in cell lysate, serum, and at -4°C storage conditions using PAGE. The results showed that TSS maintained stability at 6 × 10⁻⁶ cells / day. 4 It can be stably stored for 6 hours in cell lysate, 12 hours at 37°C in 4% FBS, and one week at -4°C.

[0052] Experimental results demonstrate the successful synthesis of TSS, and the complex exhibits good stability.

[0053] Experiment Example 2: In vitro cell experiments of tetrahedral framework nucleic acids and their complex TSS

[0054] I. Entering cells and exerting RNAi effects

[0055] 1. Experimental Methods

[0056] (1) Cell culture and in vitro model preparation

[0057] RBL-2H3 cells were cultured in MEM medium containing 15% fetal bovine serum, 1% sodium pyruvate, 1% NEAA, and 1% penicillin-streptomycin, and incubated at 37°C with 5% CO2. Subculture was performed every 2-3 days. The in vitro experimental model preparation consisted of two steps: Sensitization: 0.1 μg / mL anti-DNP-IgE was added to the cell culture medium overnight. Challenge: The original culture medium was removed, the cells were washed three times with PBS, and 10 μg / mL DNP-BSA was added before incubation for 1 hour. Other drug treatments should be performed after sensitization and before challenge.

[0058] (2) CCK-8 test

[0059] To determine the appropriate dosage of TSS, RBL-2H3 cells were cultured to the logarithmic growth phase and then injected with 1×10⁻⁶ TSS. 5 Cells were seeded at a concentration of [specific concentration] per well into 96-well plates. Cells were cultured in an incubator for 24 hours to allow cell adhesion. Cells were then treated with different concentrations of TSS for 24 hours. After removing the original culture medium, 10% CCK-8 solution (MCE, USA) was added, and the cells were incubated in the dark for 1-2 hours. The absorbance at 450 nm was measured.

[0060] (3) Cellular uptake of TSS

[0061] The uptake of TSS by RBL-2H3 cells was assessed by flow cytometry and confocal microscopy. After culturing RBL-2H3 cells to the logarithmic growth phase, they were cultured at 2.5 × 10⁻⁶ cells / cells. 6 RBL-2H3 cells were seeded into 6-well plates at a concentration of 2.5 × 10⁶ cells / mL. The next day, Cy5-labeled tFNA and TSS (250 nM) were added to serum-free medium, replacing the original medium. Cells were incubated for different times (6, 12, 24 h), washed three times with PBS, and resuspended in PBS. Fluorescence signals within RBL-2H3 cells were detected using flow cytometry. After reaching the logarithmic growth phase, RBL-2H3 cells were seeded at 2.5 × 10⁶ cells / mL. 6 Cells were seeded into confocal dishes at a concentration of cells per well. The next day, cells were cultured for 24 hours in serum-free medium containing 250 nM Cy5-labeled tFNA, Cy5-labeled Syk-siRNA, Cy3-labeled Gab2-siRNA, and TSS containing both of these siRNAs. After washing the cells three times with PBS, they were stained with Hoechst solution (Solarbio, China), and then fluorescent images of the cells were captured using a confocal microscope (Olympus, Japan).

[0062] (4) Real-time quantitative PCR (qPCR)

[0063] qPCR was used to detect the expression levels of relevant pathway mRNAs to determine the success of siRNA in exerting its RNAi effect after entering cells. Cells were divided into six groups: blank group (sensitized, unchallenged), model group (sensitized, no drug treatment, challenged), ketotifen fumarate group (sensitized, 25 μg / mL ketotifen fumarate for 12 h, challenged), tFNA group (sensitized, 250 nM tFNA for 12 h, challenged), siRNA group (sensitized, 250 nM siRNA for 12 h, challenged), and TSS group (sensitized, 250 nM TSS for 12 h, challenged). After cell treatment, total RNA was extracted using a total RNA extraction kit (Vazyme, China). Then, the RNA samples were reverse transcribed into cDNA using a reverse transcription kit (Vazyme, China). Samples were mixed. After Green IPCR master mix, the relative expression of the target mRNA was measured using a qPCR analysis system (ABIQuantStudio 7, Thermo Fisher, USA).

[0064] (5) Western blot (WB)

[0065] Western blotting was used to detect the expression levels of proteins in the relevant pathways. RBL-2H3 cells were treated as described in (4) and then cellular proteins were extracted using a total protein extraction kit (KeyGen, China). The samples were mixed with 5× protein loading buffer at a ratio of 4:1 and boiled at 100°C for 10 minutes. Primary antibodies were used according to previously reported procedures: anti-β-Actin (3700S, CST, USA), anti-Syk (A2123, ABclonal, China), anti-PLCγ1 (A8899, ABclonal, China), anti-phospho-PLCγ1 (AP0827, ABclonal, China), anti-Gab2 (HA500292, HUABIO, China), anti-pan-Akt (A18675, ABclonal, China), and anti-phospho-Akt (AP1208, ABclonal, China). The PVDF membrane was incubated with the primary antibody overnight at 4°C. On the second day, after washing three times with TBST, the PVDF membrane was incubated with the corresponding secondary antibody (L3012, SAB, USA) at room temperature for 1 hour. After TBST washing, developing solution (RM00021, ABclonal, China) was added. Each membrane was analyzed using a chemiluminescence imaging system (Bio-Rad, USA).

[0066] (6) Immunofluorescence staining

[0067] Immunofluorescence was used to detect the localization and expression of related proteins. RBL-2H3 cells were cultured at a concentration of 1×10⁻⁶. 6 Samples were seeded in confocal dishes at a concentration of cells / mL and grouped and processed according to the method described in (4). Samples were fixed with 4% paraformaldehyde, cleared with Triton X-100, and blocked with 5% goat serum. Samples were incubated overnight at 4°C with the following primary antibodies: anti-Syk (A2123, ABclonal, China) and anti-Gab2 (HA500292, HUABIO, China). After washing three times with PBS, samples were incubated with 594 fluorescently labeled secondary antibody for 1 h. The nuclei and cytoplasm of RBL-2H3 cells were stained with DAPI and FITC-labeled phalloidin. The localization and fluorescence intensity of intracellular proteins were observed using a confocal laser microscope.

[0068] 2. Experimental Results

[0069] Figure 2 A demonstrates the process by which TSS enters the cell and exerts its RNAi effect.

[0070] Figure 2 B used laser confocal microscopy to capture images of two siRNAs labeled with fluorescent groups being carried into cells by tFNAs.

[0071] Figure 2 C used flow cytometry to detect the cell entry efficiency of TSS, which showed no significant difference compared with ordinary tFNAs, achieving 95.4% cell entry in 12 hours.

[0072] Figure 2 D showed that TSS had no biotoxicity and promoted cell activity.

[0073] Figure 2 E and Figure 2 Western blot analysis showed that TSS inhibited the expression of Gab2, Syk, and their corresponding downstream proteins.

[0074] Figure 2 G showed by qPCR that TSS inhibited the expression of Gab2 and Syk and their corresponding downstream genes.

[0075] Figure 2 H and Figure 2 I verified by immunofluorescence that TSS inhibited the expression of intracellular Gab2 and Syk proteins.

[0076] The above experimental results demonstrate that TSS can carry siRNA into cells and silence the expression of Gab2 and Syk.

[0077] II. Inhibition of RBL-2H3 cell degranulation

[0078] 1. Experimental Methods

[0079] (1) Detection of RBL-2H3 cell morphological changes

[0080] RBL-2H3 cells were loaded at 1×10 6 Cells were seeded at a concentration of [cells / mL] into 6-well plates, and the cells were divided into six groups: blank group (sensitized, no challenge), model group (sensitized, no drug treatment, challenge), ketotifen fumarate group (sensitized, 25 μg / mL ketotifen fumarate for 12 h, challenge), tFNA group (sensitized, 250 nM tFNA for 12 h, challenge), siRNA group (sensitized, 250 nM siRNA for 12 h, challenge), and TSS group (sensitized, 250 nM TSS for 12 h, challenge). Sensitization: 0.1 μg / mL anti-DNP-IgE was added to the cell culture medium overnight. Challenge: The original culture medium was removed, the cells were washed three times with PBS, and 10 μg / mL DNP-BSA was added before incubation for 1 h. After challenge, the cells were washed three times with PBS. Toluidine blue staining solution (Solarbio, China) was added to the first batch of samples according to the instructions. Cell color and morphology were observed using an optical microscope. Meanwhile, another batch of samples was fixed overnight at 4°C after adding 4% paraformaldehyde. A series of alcohol concentration gradients (30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%) were added sequentially to the samples for 15 minutes each time to dehydrate them. Changes in cell surface morphology were observed using scanning electron microscopy.

[0081] (2) Cell liveness / death staining

[0082] Cells were stained using the Calcein / PI Cell Viability and Cytotoxicity Assay Kit (Beyotime, China) according to the manufacturer's instructions. Cell fluorescence signals were observed using a fluorescence microscope. Live cells stained green fluorescence, and apoptotic cells stained red fluorescence. Apoptosis rate = live cells / (live cells + apoptotic cells).

[0083] (3) Calcium ion concentration detection

[0084] Cells were stained using the Fluo-3,AM calcium ion concentration assay kit (Solarbio, China) according to the manufacturer's instructions. Intracellular fluorescence signals were observed using a fluorescence microscope. Quantitative analysis of the fluorescence patterns was performed using ImageJ software.

[0085] (4) β-hexosamine glycoside enzyme assay

[0086] RBL-2H3 cells were loaded at 5 × 10 5Cells were seeded at a density of 100% in 24-well plates. After stimulation with DNP-BSA for 1 hour, 50 μL of cell supernatant was mixed with 50 μL of chromogenic solution (1 mM 4-Nitrophenyl N-acetyl-β-D-galactosaminide, 0.1 M Sodium Citrate Buffer, pH 4.5) and incubated at 37°C for 1 hour. After 1 hour, 200 μL of stop solution (0.1 M Na₂CO₃ / NaHCO₃, pH 10.0) was added, and the absorbance at 405 nm was measured using a microplate reader. β-hexosinosinase release rate = (OD of drug-treated group - OD of blank group) / (OD of model group - OD of blank group)

[0087] (5) Enzyme-linked immunosorbent assay (ELISA)

[0088] To detect cytokine expression, the concentrations of histamine, TNF-α, and IL-6 in the cell supernatant were measured using an ELISA kit (mlbio, China).

[0089] 2. Experimental Results

[0090] Figure 3 A demonstrates that TSS can inhibit the lightening of toluidine blue staining after RBL-2H3 cells are induced to become hypersensitive.

[0091] Figure 3 B used scanning electron microscopy to show that TSS could inhibit the irregular morphological changes of RBL-2H3 cells after allergic stimulation.

[0092] Figure 3 C and Figure 3 E showed that tFNAs and TSS can reduce apoptosis in RBL-2H3 cells after stimulating allergy.

[0093] Figure 3 D and Figure 3 E showed that tFNAs and TSS could reduce the increase in calcium ion concentration after RBL-2H3 cells were induced to become hypersensitive.

[0094] Figure 3 F and Figure 3 G showed that tFNAs and TSS could reduce the release of β-hexosinolates, histamine, TNF-α and IL-6 after RBL-2H3 cells were stimulated to become hypersensitive.

[0095] RBL-2H3 cells exhibit biological characteristics similar to mast cells, and experimental results have demonstrated that tFNAs and TSS can inhibit RBL-2H3 cell degranulation.

[0096] III. Stabilizing mitochondrial energy metabolism levels

[0097] 1. Experimental Methods

[0098] (1) Detection of mitochondrial membrane potential

[0099] RBL-2H3 cells were loaded at 1×10 6 Cells were seeded at a density of [number] cells / mL in confocal dishes. The cells were divided into six groups: blank group (sensitized, no challenge), model group (sensitized, no drug treatment, challenge), ketotifen fumarate group (sensitized, 25 μg / mL ketotifen fumarate for 12 h, challenge), tFNA group (sensitized, 250 nM tFNA for 12 h, challenge), siRNA group (sensitized, 250 nM siRNA for 12 h, challenge), and TSS group (sensitized, 250 nM TSS for 12 h, challenge). Sensitization: 0.1 μg / mL anti-DNP-IgE was added to the cell culture medium overnight. Challenge: The original culture medium was removed, the cells were washed three times with PBS, and 10 μg / mL DNP-BSA was added before incubation for 1 h. After challenge, the original culture medium was removed. Mito-Tracker Green and TMRE (Beyotime, China) were added to the cells according to the manufacturer's instructions and incubated at 37°C for 30 min each. Fluorescence signals were observed under a laser confocal microscope. The fluorescence images were quantitatively analyzed using ImageJ.

[0100] (2) Detection of ATP production rate and oxygen consumption rate

[0101] RBL-2H3 cells were seeded into XF24 cell culture microplates and treated as described in (1). After stimulation, the cells were washed with Seahorse buffer and cultured in Agilent Seahorse XF DMEM medium containing 1 mM sodium pyruvate, 2 mM glutamine, and 1 mM D-glucose for 1 h in a non-CO2 incubator at 37°C. ATP rate and OCR were measured using a Seahorse XFe24 analyzer. The procedure for ATP rate measurement was to inject 1.5 μM oligomycin and 0.5 μM rotenone / antimycin A (Rot / AA). The procedure for OCR measurement was to inject 1.5 μM oligomycin, 1.0 μM FCCP, and 0.5 μM Rot / AA in sequence. The procedures were strictly performed according to the manufacturer's (Agilent, USA) recommended procedure.

[0102] 2. Experimental Results

[0103] Figure 4 A demonstrates the process by which the TSS stabilizes mitochondrial energy metabolism levels.

[0104] Figure 4 B showed that tFNAs and TSS can stabilize the mitochondrial membrane potential after RBL-2H3 cells are induced to become hypersensitive.

[0105] Figure 4 F showed that tFNAs and TSS could restore the reduced ATP production rate after RBL-2H3 cells were induced to hypersensitivity, especially by inhibiting oxidative phosphorylation.

[0106] Figure 4 C Figure 4 D、 Figure 4 E, Figure 4 G and Figure 4 H showed that tFNAs and TSS had a good effect on maintaining the basal respiration, maximal respiration, reserve respiration capacity and ATP production capacity of RBL-2H3 cells.

[0107] Experimental results demonstrate that tFNAs and TSS can stabilize mitochondrial energy metabolism levels and restore the decreased mitochondrial membrane potential and metabolic changes caused by allergic reactions.

[0108] Experiment Example 3: In vivo animal experiments

[0109] 1. Experimental Methods

[0110] (1) In vivo distribution detection

[0111] Male BALB / c mice, 6-8 weeks old and weighing 20-25g, were purchased from Dossy Science (Chengdu, China). To investigate the distribution of TSS throughout the mice, Cy5-labeled TSS was injected intraperitoneally. Fluorescence distribution was captured using a small animal in vivo optical three-dimensional imaging system (IVIS Spectrum, Perkin Elmer, USA) at time points of 24h, 12h, 6h, 3h, 1h, and 0h. To investigate the local transdermal effect of TSS, Cy5-labeled TSS was mixed with an equal volume of cream. The cream was applied to the mouse ears, and samples were collected at 24h, 12h, 6h, and 0h and fixed in 4% paraformaldehyde for 24h. The ears were then frozen in liquid nitrogen, fixed with embedding gel, and sectioned. Cell nuclei in the sections were stained with DAPI, and the sections were scanned using a whole-slide scanner (VS200, Olympus, Japan).

[0112] (2) Passive skin allergic reaction

[0113] Anti-DNP-IgE sensitization is a known method for constructing a type I hypersensitivity reaction model.

[0114] Male BALB / c mice were housed in an SPF-grade environment for one week and then divided into eight groups: blank control group, model group, tFNAs group, siRNA group, ketotifen fumarate injection group, TSS injection group, ketotifen fumarate topical application group, and TSS topical application group (n=3). Mice were anesthetized with ether, and sensitization was induced by intradermal injection of 0.5 μg anti-DNP-IgE into the ear. 24 hours later, sensitization was induced by intravenous injection of 0.5 mg DNP-BSA (containing 1% Evans blue) into the tail vein. Six hours prior to challenge, mice in the blank control group (200 μL saline), model group (200 μL saline), tFNAs group (200 μL 1000 nM tFNAs), siRNA group (200 μL 1000 nM siRNA), ketotifen fumarate injection group (200 μL 20 μg / g ketotifen fumarate), and TSS injection group (200 μL 1000 nM TSS) were administered intraperitoneal injections. Twenty-four hours prior to challenge, mice in the ketotifen fumarate topical group and the TSS topical group had ketotifen fumarate (10 μg / g mixed in cream) and TSS (mixed with an equal volume of cream) applied to their ears, respectively. One hour after allergic reaction was triggered, the mice were euthanized and samples were collected.

[0115] (3) Evans Blue Exudation Detection

[0116] The Evans blue exudate in mouse ears was photographed and recorded. Ears from each group of mice were collected, and ears of the same size were dried at 60°C for 24 hours. The ears were cut into small pieces and placed in 1 mL of formamide, incubated in a water bath at 85°C for 2 hours, centrifuged at 3000 rpm for 15 minutes, and the absorbance of the supernatant at 620 nm was measured.

[0117] (4) Histological examination

[0118] Mouse ears from each group were collected and immersed in 4% paraformaldehyde. Tissue samples were dehydrated, embedded in paraffin, and sectioned. HE staining and toluidine blue staining were performed on the samples to observe tissue edema and mast cell infiltration. In addition, immunofluorescence staining was performed on the sections to assess the expression of Syk and Gab2 proteins in the tissues. The thickness and fluorescence intensity of each sample were the average of measurements taken at three random sites within that sample.

[0119] (5) Complete blood count

[0120] Blood was collected from the eyeballs of mice and thoroughly mixed in EDTA anticoagulant tubes. The condition of various cell types in the blood was then analyzed using a hematology analyzer.

[0121] 2. Experimental Results

[0122] Figure 5 A demonstrates the process by which intraperitoneal injection of TSS inhibits passive cutaneous anaphylactic reactions.

[0123] Figure 5 B shows that 6 hours after intraperitoneal injection of TSS, the drug can be distributed throughout the mouse system.

[0124] Figure 5 C shows that tFNAs and TSS effectively inhibit Evans blue exudation in the ear.

[0125] Figure 5 D showed that tFNAs and TSS effectively inhibited edema in the mouse ear.

[0126] Figure 5 E showed that tFNAs and TSS effectively reduced mast cell infiltration in the mouse ear.

[0127] Figure 5 F, Figure 5 G and Figure 5 H showed that tFNAs and TSS suppressed the expression of Syk and Gab2 in the tissue.

[0128] Table 3. Syk expression after different experimental group treatments

[0129]

[0130] Table 3 shows that Syk expression decreased after treatment with Ketotifen, siRNA, tFNAs, and TSS. Further comparison of the percentage decrease in Syk expression relative to the Model group after treatment with siRNA, tFNAs, and TSS in Table 3 reveals that the reduction in Syk expression after TSS treatment was greater than the sum of the reductions after siRNA and tFNAs treatment, indicating that TSS exerted a synergistic effect in reducing Syk expression.

[0131] Table 4. Gab2 expression after different experimental group treatments

[0132]

[0133]

[0134] As shown in Table 4, Gab2 expression decreased after treatment with Ketotifen, siRNA, tFNAs, and TSS. Further comparison of the percentage decrease in Gab2 expression relative to the Model group after treatment with siRNA, tFNAs, and TSS in Table 4 revealed that the reduction in Gab2 expression after TSS treatment was greater than the combined reduction after siRNA and tFNAs treatment, indicating that TSS exerted a synergistic effect in reducing Gab2 expression.

[0135] Figure 5 I showed that tFNAs and TSS reduced the increase of white blood cells, especially neutrophils, in the blood of mice.

[0136] Figure 6 A demonstrates the process by which TSS is administered transdermally to inhibit passive cutaneous anaphylactic reactions.

[0137] Figure 6 B shows that TSS effectively inhibits the exudation of Evans blue in the ear.

[0138] Figure 6 C is a quantitative detection of Evans blue exudate, and the results show that TSS effectively inhibits the exudation of Evans blue in the ear.

[0139] Figure 6 D shows that 24 hours after topical application of TSS, drug fluorescence can be observed extensively in the tissue.

[0140] Figure 6 E is... Figure 6 Quantitative analysis of tissue thickness in F showed that TSS effectively inhibited edema in the mouse ear.

[0141] Figure 6 F is a HE section of the mouse ear, which shows that TSS effectively inhibits edema in the mouse ear.

[0142] Figure 6 G showed that TSS effectively reduced mast cell infiltration in the mouse ear.

[0143] Figure 6 H and Figure 6 I showed that TSS suppressed the expression of Syk and Gab2 in the tissue.

[0144] Experimental results demonstrate that the tFNAs of this invention, administered alone, can effectively inhibit type I hypersensitivity reactions. The complex TSS, obtained by further modifying siRNAs targeting Gab2 and Syk based on tFNAs, can also effectively inhibit type I hypersensitivity reactions. Furthermore, the complex TSS plays a synergistic role in inhibiting type I hypersensitivity reactions.

[0145] In summary, this invention provides a drug for treating type I hypersensitivity reactions. The tetrahedral framework nucleic acid of this invention can effectively inhibit mast cell degranulation, stabilize mitochondrial energy metabolism levels, restore the decreased mitochondrial membrane potential and metabolic changes caused by induced allergy, and effectively inhibit type I hypersensitivity reactions in vivo. This invention further modifies the tetrahedral framework nucleic acid with siRNA targeting Gab2 and Syk, obtaining the complex TSS. The complex TSS can stabilize mitochondrial energy metabolism, inhibit the influx of intramitochondrial calcium ions into cells, and inhibit mast cell degranulation, thus inhibiting type I hypersensitivity reactions. Particularly noteworthy is that the complex TSS exhibits a synergistic effect in inhibiting type I hypersensitivity reactions. The tetrahedral framework nucleic acid and the complex TSS of this invention are not only simple to synthesize and structurally stable, but also have broad application prospects in the preparation of drugs for the prevention and / or treatment of type I hypersensitivity reactions.

Claims

1. The use of a tetrahedral framework nucleic acid in the preparation of a drug for treating type I hypersensitivity reactions, said drug being a drug for inhibiting mast cell degranulation, wherein the tetrahedral framework nucleic acid is self-assembled from four DNA single strands, the sequences of which are shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

2. The use according to claim 1, characterized in that, The preparation method of the tetrahedral framework nucleic acid includes the following steps: mixing four DNA single strands, heating the mixture, and then cooling it to obtain the tetrahedral framework nucleic acid.

3. The use according to claim 2, characterized in that, The heating reaction is carried out at a temperature of 80-100°C for 5-15 minutes. The cooling reaction is carried out at a temperature of 0-10°C for a time of 10-30 min.

4. The use according to claim 3, characterized in that, The heating reaction was carried out at a temperature of 95°C for 10 minutes. The cooling reaction was carried out at a temperature of 4°C for 20 minutes.

5. Use of a tetrahedral framework nucleic acid complex in the preparation of a drug for treating type I hypersensitivity reactions, wherein the drug is a drug for inhibiting mast cell degranulation, and the tetrahedral framework nucleic acid complex is a product prepared from a reagent for inhibiting mast cell degranulation and the tetrahedral framework nucleic acid as described in any one of claims 1-4; the reagent for inhibiting mast cell degranulation is a reagent for targeting and inhibiting Syk expression and a reagent for targeting and inhibiting Gab2 expression, wherein the reagent for targeting and inhibiting Gab2 expression is a siRNA for targeting and inhibiting Gab2 expression, and the siRNA for targeting and inhibiting Gab2 expression is composed of two single strands with sequences as shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively, through complementary base pairing; the reagent for targeting and inhibiting Syk expression is a siRNA for targeting and inhibiting Syk expression, and the siRNA for targeting and inhibiting Syk expression is composed of sequences as shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively. The two single strands shown in .8 are formed by complementary base pairing; the molar ratio of the reagent that targets and inhibits Syk expression, the reagent that targets and inhibits Gab2 expression, and the tetrahedral framework nucleic acid is 1:1:0.

5.

6. The use according to claim 5, characterized in that, The preparation method of the tetrahedral framework nucleic acid complex includes the following steps: mixing a reagent that inhibits mast cell degranulation with tetrahedral framework nucleic acid and reacting the mixture to obtain the tetrahedral framework nucleic acid complex.

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