A caspase-1 responsive fluorescent probe based on FRET, preparation method, application and product

Abstract

The application discloses a caspase-1 responsive fluorescent probe based on FRET, a preparation method, application and products, and belongs to the technical field of FRET fluorescent probes. The technical problem to be solved is to provide a FRET fluorescent probe which can simultaneously detect caspase-1 enzyme in vitro and detect caspase-1 activity in vivo. The specific technical scheme is a FRET fluorescent probe with a chemical structure of formula I. The FRET fluorescent probe provided by the application has a short reaction time, is suitable for detecting caspase-1 activity and imaging cell pyroptosis under a fluorescence confocal microscope, and can realize multi-scale imaging of pyroptosis in cell balls and animals.

Description

Technical Field

[0001] This invention relates to the field of FRET fluorescent probe technology, specifically to a FRET-based caspase-1 responsive fluorescent probe, its preparation method, application, and product. Background Technology

[0002] For understanding the technical content of this invention: Pyroptosis is a programmed, pro-inflammatory form of cell death, distinct from apoptosis and necroptosis. Studies have shown that pyroptosis is closely related to a variety of diseases, including bacterial infections, viral infections, autoimmune diseases, neurological disorders (such as Alzheimer's disease), cardiovascular diseases, and cancer.

[0003] Caspase-1, or caspase-1, is an important member of the caspase family of proteases. Caspase-1 acts as an "initiator and executor" of the inflammatory response, releasing strong inflammatory signals and clearing pathogen habitats by killing infected cells. Caspase-1 typically exists in its inactive proenzyme form in the cytoplasm. Its activation depends on a multi-protein complex called the inflammasome. When cells sense pathogen infection, cell damage, or other danger signals, they assemble the inflammasome, thereby activating caspase-1. The core functions of caspase-1 include: ① Cleavage and activation of inflammatory cytokines: This is caspase-1's most classic function; it cleaves inactive interleukin-1 and interleukin-18 precursors to produce active forms of IL-1β and IL-18. These two are potent pro-inflammatory factors that play a central role in initiating and amplifying inflammatory responses; ②, they perform pyroptosis: caspase-1 can cleave a protein called Gasdermin D. After cleavage, the N-terminal fragment of Gasdermin D forms a hole in the cell membrane, leading to pyroptosis.

[0004] Fluorescence resonance energy transfer (FRET) is a non-radiative energy transfer process. When two fluorescent molecules (chromophores) meet specific conditions, if the emission spectrum of one molecule (donor) overlaps sufficiently with the absorption spectrum of the other molecule (acceptor), and the two molecules are close enough, then upon excitation, the excited state energy of the donor is not released through fluorescence emission, but rather transferred directly to the acceptor through non-radiative dipole-dipole coupling. Because FRET places strict requirements on the spectral overlap, microscopic distance, and spatial orientation of functional groups in the donor and acceptor, it has been used in fields such as protein-protein interactions, nucleic acid conformational changes, biosensing, membrane biology, and enzyme activity detection.

[0005] Relevant patent documents retrieved: The publication is from China, publication number CN119061113A, publication date 2024.12.3, national priority data 202310640693.X 2023.05.31 CN. This document discloses a system and method for detecting toxin activity. The system for detecting toxin activity includes one or more of the following components: HEPES, NaCl, DTT, surfactant, or Zn(OAc)₂; preferably, the surfactant is selected from Triton X. The FRET probe contains one or more of 100, SDS, Span, or Tween, and also includes an enzymatically cleaved toxin and substrate protein that are active by cleavage with a first-specific protease. The toxin's activity is detected by cleaving the peptide chain in the FP1-FP2 region of the FRET probe through substrate cleavage with the detected toxin, thereby altering the molecule's fluorescence properties. This system can also detect caspase-14 activity.

[0006] Relevant non-patent literature retrieved: Journal title: *Journal of Molecular Structural Research*; Article title: "A novel xanthene-based fluorescence turn-on probe for highly selective detection of Hg" 2+ The paper, "Inwater Samples and Living Cells," Volume 1254 (2022) 132312, published on January 1, 2022, discloses a xaton compound. The synthetic route of the compound (RANS) is shown in the following formula: .

[0007] When this molecule reacts with Hg 2+ Upon interaction, RANS removes the HgS group, undergoing an intramolecular cyclization reaction to form a five-membered ring structure of 1,3,4-oxodiazole. This ring forms a large conjugated structure with the xanthine core, producing fluorescence. RANS molecules can be used to effectively detect Hg. 2+ It has high sensitivity, good linearity, and strong specificity.

[0008] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: Existing technologies have not yet provided a FRET fluorescent probe that uses xatonide and rhodamine compounds as acceptors and donors in the fluorescence resonance energy transfer (FRET) system and can detect caspase-1 activity at the cellular, cell cluster, and in vivo levels. Summary of the Invention

[0009] The purpose of this invention is to provide a caspase-1 responsive fluorescent probe and related technologies to solve technical problems such as further improving the detection sensitivity of caspase-1 enzyme, reducing reaction time, improving selectivity, and expanding application scenarios to cell clusters and in vivo detection, or combinations thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specified. It should also be noted that, unless otherwise specified, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “containing” are not limiting.

[0012] Definitions of the relevant standard terms can be found in the following references: Inorganic Chemistry (Volumes 1 & 2), edited by Wuhan University, Jilin University, etc., 3rd edition, January 1994; Basic Organic Chemistry (Volumes 1 & 2), Xing Qiyi, Higher Education Press, 3rd Edition, June 2005; Molecular Biology, Robert F. Weaver, Science Press, 2013-3, 5th Edition; Principles of Biochemistry, Zhang Chufu, Higher Education Press, 2011-2, Second Edition.

[0013] Unless otherwise defined, the chemical reactions described herein are carried out at room temperature.

[0014] The term "room temperature" refers to the ambient temperature in a laboratory under normal conditions, including but not limited to any temperature value or sub-range between 20-30°C.

[0015] Unless otherwise stated, this document employs conventional methods within the scope of the art, such as solid-phase peptide synthesis (SPSS), high-resolution mass spectrometry, liquid chromatography-mass spectrometry, cell resuscitation, cell expansion, cell incubation, fluorescence confocal microscopy, and animal husbandry. Specifically, the described solid-phase peptide synthesis method is a classic synthetic method documented in the prior art, such as the classic synthetic method described in the literature "Peptide Synthesis: Methods and Protocols" (Humana Press, 2nd edition, June 2025).

[0016] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0017] The terms “optional / arbitrary” or “optionally / arbitrarily” refer to events or situations described subsequently that may or may not occur, including both the occurrence and non-occurrence of the events or situations. For example, according to the definition below, “arbitrarily solvated” means “solventized” (including single solvation and mixed solvation) or “non-solventized”.

[0018] The term "pharmaceutical acceptable" means that something is safe and harmless to subjects and suitable for use in drug preparation.

[0019] As used herein, the term "solvent" refers to a molecular complex formed by the non-covalent bonding of a compound and a solvent molecule under solid-state conditions. Unless otherwise specified, "solvent" refers to any solvated compound, particularly hydrates, organic solvates, and mixed solvates of water and organic solvents.

[0020] The term “selected independently” as used in this article means that each element within the range can be selected independently and is not related to the others.

[0021] The term "hydrocarbon group" as used in this article refers to a chemical group composed solely of carbon and hydrogen elements. Common hydrocarbon groups include alkanes, alkenes, alkynes, cycloalkanes, aromatics, etc.

[0022] The term "hydrocarbon group containing xy carbon atoms" as used in this article refers to the range of groups including: hydrocarbon groups containing x carbon atoms, hydrocarbon groups containing x+1 carbon atoms, hydrocarbon groups containing x+2 carbon atoms, ... hydrocarbon groups containing y-1 carbon atoms, and hydrocarbon groups containing y carbon atoms. For example, "hydrocarbon groups containing 1-3 carbon atoms" specifically includes methyl, ethyl, vinyl, ethynyl, n-propyl, isopropyl, 1-propenyl, 2-propenyl, allyl, propynyl, propynyl propargyl, cyclopropyl, etc.

[0023] The term "caspase-1" used in this article refers to caspase-1.

[0024] As used in this article, the term "caspase-1 sensitive linker" refers to a chain-like chemical group that includes the following characteristics: ① It can connect two chemical groups at the molecular structure level. The "two chemical groups" can have the same or different chemical structures; in FRET fluorescent probes, the "two chemical groups" often have different chemical structures.

[0025] ② It contains chemical bonds that can be cleaved by caspase-1, and the cleavage of these chemical bonds leads to the two chemical groups mentioned in ① breaking into two molecules from a molecular structure perspective.

[0026] As used in this article, the term "(fluorescent probe) pair ion" refers to an ion that can partially or completely balance the charge on a fluorescent probe.

[0027] The term "chirality," also known as "enantiomerism," refers to the property that an object or molecule cannot be perfectly superimposed on its mirror image. The most common reason for chirality in molecules is that a carbon atom is bonded to four distinct atoms or groups. This carbon atom is called the chiral center or asymmetric carbon atom.

[0028] The term "L / S configuration" refers to a nomenclature system used to label the absolute configuration of chiral molecules, particularly amino acids and sugars. It is determined based on a comparison with the stereostructure of the standard reference glyceraldehyde.

[0029] The term "oligopolymer" refers to a short-chain polymer formed by a small number of monomers (usually 2-20) linked together by covalent bonds.

[0030] The term "peptide" or "polypeptide" refers to a linear molecule formed by the condensation of multiple amino acids linked end-to-end by peptide bonds. It is the structural basis of proteins.

[0031] Unless otherwise defined, all chiral amino acids mentioned in this article are L-amino acids, which are the basic amino acids that make up human proteins. Unless otherwise defined, the amino acid abbreviations used in this article are as follows: glycine (Gly / G), alanine (Ala / A), valine (Val / V), leucine (Leu / L), isoleucine (Ile / I), proline (Pro / P), phenylalanine (Phe / F), tyrosine (Tyr / Y), tryptophan (Trp / W), serine (Ser / S), threonine (Thr / T), cysteine ​​(Cys / C), methionine (Met / M), asparagine (Asn / N), glutamine (Gln / Q), aspartic acid (Asp / D), glutamic acid (Glu / E), lysine (Lys / K), arginine (Arg / R), and histidine (His / H).

[0032] In a first aspect, the present invention provides a FRET-based caspase-1 responsive fluorescent probe, wherein the caspase-1 responsive fluorescent probe is a compound as shown in Formula I or a pharmaceutically acceptable salt or solvate thereof: Formula I; Linker is a caspase-1 sensitive linker group, and R1-R8 are each independently selected from hydrocarbon groups containing 1-6 carbon atoms.

[0033] The caspase-1 sensitive linker includes, but is not limited to, containing one or more polypeptide fragments that can be cleaved by caspase-1.

[0034] R1-R8 are each independently selected from hydrocarbon groups containing 1-6 carbon atoms. Specifically, they can be independently selected from groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, vinyl, allyl, cyclopentyl, cyclohexyl, ethynyl, and phenyl.

[0035] Furthermore, R1-R8 are each independently selected from hydrocarbon groups containing 1-3 carbon atoms.

[0036] The hydrocarbon groups containing 1-3 carbon atoms can be individually selected from methyl, ethyl, n-propyl, isopropyl, vinyl, ethynyl, allyl, cyclopropyl, etc.

[0037] Furthermore, the structure of the Linker is selected from one of the following structures: , , ; Among them, R a and R bEach is independently selected from chemical bonds or groups formed by connecting 1 to 10 structural units end to end. , , , , , , or ; Among them, R c1 R c2 R c3 R c4 R c5 Each is independently selected from hydrocarbon groups containing 1-3 carbon atoms.

[0038] The hydrocarbon groups containing 1-3 carbon atoms can be individually selected from methyl, ethyl, n-propyl, isopropyl, vinyl, ethynyl, allyl, cyclopropyl, etc.

[0039] Furthermore, the R a R b Each group is independently selected from 3 to 7 structural units connected end to end. , , or .

[0040] Furthermore, the R a R b Each is independently selected from the following structures: , , , or .

[0041] Furthermore, the caspase-1 responsive fluorescent probe also contains a p-ion.

[0042] Furthermore, the ion is an anion.

[0043] Furthermore, the ion is one or more of the following: trifluoroacetate ion, acetate ion, perchlorate ion, hexafluorophosphate ion, tetrafluoroborate ion, nitrate ion, chloride ion, tetraphenylborate ion, methanesulfonate ion, p-toluenesulfonate ion, trifluoromethanesulfonate ion, and tetra(3,5-bis(trifluoromethylphenyl)borate ion.

[0044] The FRET-based caspase-1 responsive fluorescent probe provided by this invention can have 0-2 pharmaceutically acceptable anti-anions. As a specific example, for Formula I: ; If the Linker group itself has 2 units of negative charge, the fluorescent probe can achieve electroneutrality with 0 pairs of anions; if the Linker group itself has 1 unit of negative charge, the fluorescent probe can achieve electroneutrality with 1 pair of anions containing 1 unit of negative charge; if the Linker group itself does not contain negative charge, the fluorescent probe can achieve electroneutrality with 2 pairs of anions, which can be selected to be the same and contain 1 unit of negative charge, or with 1 pair of anions containing 2 units of negative charge.

[0045] It should be noted that if the FRET-based caspase-1 responsive fluorescent probe provided by this invention contains a pair of ions, their function is only to achieve overall electroneutrality of the fluorescent probe; the pair of ions have no significant effect on the FRET-based caspase-1 responsiveness of the fluorescent probe itself (including excitation spectrum, emission spectrum, specificity, detection linearity, and other properties).

[0046] Furthermore, and as a specific embodiment of the present invention, the caspase-1 responsive fluorescent probe is selected from: , or .

[0047] Most preferably, the chemical structure of the caspase-1 responsive fluorescent probe is as follows: .

[0048] Secondly, the present invention provides a method for preparing the above-mentioned caspase-1 responsive fluorescent probe, comprising the following steps: [The method involves...] carboxyl group and The carboxyl group is linked through a compound containing a linker to obtain a FRET fluorescent probe; Among them, X1 - for The ion; X2 - for The linker is a caspase-1 sensitive linker group, and R1-R8 are each independently selected from hydrocarbon groups containing 1-6 carbon atoms.

[0049] Thirdly, the present invention provides the application of the above-mentioned caspase-1 responsive fluorescent probe in non-disease diagnostic caspase-1 detection.

[0050] Fourthly, the present invention provides a product for detecting caspase-1, comprising the above-mentioned caspase-1 responsive fluorescent probe or its pharmaceutically acceptable salt or solvate.

[0051] Furthermore, the product is a pyroptosis detection reagent, a pyroptosis detection kit, a caspase-1 detection reagent, or a caspase-1 detection kit.

[0052] The pyrolysis detection reagent is available in the form of powder, solution, and test strip; the caspase-1 detection reagent is available in the form of powder, solution, and test strip.

[0053] Fifthly, the present invention provides a non-disease diagnostic method for detecting pyroptosis, comprising the following steps: The above-mentioned caspase-1 responsive fluorescent probe was used as a probe for caspase-1 detection to detect caspase-1.

[0054] The present invention has at least the following beneficial effects: 1. This invention provides a novel fluorescent probe for caspase-1 activity detection based on the principle of fluorescence resonance energy transfer. This probe has the advantages of high sensitivity, fast response time, and good selectivity in caspase-1 activity detection.

[0055] 2. This invention provides a method for preparing the novel caspase-1 activity detection fluorescent probe. Utilizing existing SPPS technology, this novel fluorescent probe was successfully synthesized. The method is highly versatile and easy to use and promote.

[0056] 3. Cell experiments and in vivo animal experiments show that the caspase-1 fluorescent probe provided by this invention can achieve multi-scale pyroptosis imaging in cells, cell spheres and in animals. Attached Figure Description

[0057] Figure 1 This is a schematic diagram of the principle of the FRET fluorescent probe for detecting caspase-1 according to the present invention.

[0058] Figure 2 The graph shows the sensitivity test results for the FPy1, YPy1, and WPy1 probes, with λex = 510 nm and λem = 585 nm.

[0059] Figure 3 The graphs show the response time detection results of the FPy1, YPy1, and WPy1 probes, where a represents the response time detection result of FPy1, b represents the response time detection result of YPy1, and c represents the response time detection result of WPy1, with λex = 510nm.

[0060] Figure 4The graphs show the selectivity test results of the FPy1, YPy1, and WPy1 probes. In the graphs, a represents the selectivity test result of FPy1 in solution, b represents the selectivity test result of YPy1 in solution, c represents the selectivity test result of WPy1 in solution, and d represents the selectivity test result of FPy1, YPy1, and WPy1 for caspase-1, caspase-3, caspase-4, and caspase-8, respectively. λex = 510 nm and λem = 585 nm.

[0061] Figure 5 The figure shows the results of detecting the effect of FPy1 on the cell viability of THP-1.

[0062] Figure 6 This image shows the effect of FPy1 on the fluorescence detection of intracellular caspase-1 in THP-1 cells. In the image, a represents the experimental flowchart; b represents the confocal fluorescence imaging of intracellular caspase-1 in THP-1 cells after different durations (0h, 1h, 2h) of stimulation with FPy1 in the control group (without LPS stimulation); c represents the bar chart of normalized fluorescence intensity of FPy1 channels in b; d represents the flow cytometry detection of intracellular caspase-1 in THP-1 cells after different durations (0h, 1h, 2h) of stimulation with FPy1 in the control group (without LPS stimulation); and e represents the Western spectral density of intracellular caspase-1 in THP-1 cells after different durations (0h, 1h, 2h) of stimulation with FPy1 in the control group (without LPS stimulation); The blotting results are shown in f, which represents the statistical graph of the relative content of truncated caspase-1 in each group in e. In the confocal fluorescence detection of cells, the excitation wavelength of the FPy1 channel is 514 nm, and the fluorescence receiving band is 540-600 nm.

[0063] Figure 7This is an evaluation diagram of the effectiveness of FPy1 in detecting pyroptosis in multi-scale biological samples. In the diagram, a represents the experimental flowchart; b represents confocal fluorescence imaging of caspase-1 in adherent nucleus pulposus cells after stimulation with H2O2 (control group) and at different durations (30 min, 60 min, 90 min) using FPy1; the scale bar represents 50 μm; and c represents the effect of FPy1 on caspase-1 in adherent nucleus pulposus cells after stimulation with H2O2 (control group) and at different durations (30 min, 60 min, 90 min). Confocal fluorescence imaging of caspase-1 fluorescence detection in nucleus pulposus cell spheres after 90 min. The scale bar represents 200 μm. d represents the normalized fluorescence intensity statistical bar chart of FPy1 channel in b, e represents the normalized fluorescence intensity statistical bar chart of FPy1 channel in c, f represents the relative fluorescence intensity map of the tail modeling area of ​​the rat model of intervertebral disc degeneration characterized by FPy1, and g represents the normalized fluorescence intensity statistical bar chart of f. In the cell / cell sphere confocal fluorescence detection, the excitation wavelength of FPy1 channel is 514 nm, and the fluorescence receiving band is 540-600 nm.

[0064] In the above figures, the definition of significance markers is as follows: ns: There was no significant difference between the two groups being compared, p > 0.05; *: There is a significant difference between the two groups being compared, and p < 0.05; **: There is a significant difference between the two groups being compared, and p < 0.01; ***: There was a significant difference between the two groups being compared, and p < 0.001; ****: There was a significant difference between the two groups being compared, and p < 0.0001. Detailed Implementation

[0065] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0066] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0067] In the following specific embodiments, the Fmoc protected amino acids and starting chemicals used in solid-phase peptide synthesis (SPPS) were purchased from Sigma-Aldrich (Shanghai, China) and Bidepharm (Shanghai, China). All chemicals were reagent grade. The resin used in SPPS was purchased from GL Biochem (Shanghai, China). Lipopolysaccharide (LPS, L2630), nigrin, and ATP (11140965001) were purchased from Merck (Shanghai, China) and Invitrogen (Shanghai, China), respectively. Cell culture plates were provided by NEST Biotechnology Co., Ltd. (Wuxi). Recombinant human caspase-1, caspase-3, caspase-4, and caspase-8 were purchased from Abcam and MCE (Shanghai). Anti-cleavage caspase-1 antibody, anti-GAPDH antibody, and goat anti-rabbit / anti-mouse IgG / HRP markers were purchased from CST (USA) and Bristol-Myers Squibb (Shanghai). Fetal bovine serum, DMEM medium, penicillin / streptomycin, and RPMI 1640 medium were all purchased from Thermo Fisher Scientific (Shanghai). SD rats were purchased from the Animal Center of Hangzhou Medical College. Experimental animals were housed, managed, and used under specific pathogen-free conditions at the animal facilities of Hangzhou Medical College. All animal experiments were approved by the Ethics Review Committee of Hangzhou Medical College (Ethics Approval No.: ZJCLA-IACUC-20011003). 1 H NMR and 13 CNMR spectra were measured using a 500 MHz Bruker AV 500 NMR spectrometer. UV-Vis spectra were measured using a Tecan Infinite M1000 Pro multi-plate reader, and fluorescence spectra were acquired using a Hitachi F-2700 spectrophotometer. Mass spectrometry data were acquired using an Agilent 6546 mass spectrometer. Fluorescence images of cells, cell spheroids, and tissue sections were captured using an Olympus IX83-FV3000 microscope.

[0068] In the following examples, some of the English abbreviations and their corresponding Chinese meanings are shown in Table 1.

[0069] Table 1

[0070] Example 1 Synthesis and characterization of FRET probe molecules FPy1, YPy1 and WPy1.

[0071] The chemical structure of FPy1 is as follows: .

[0072] The chemical structure of YPy1 is as follows: .

[0073] The chemical structure of WPy1 is as follows: .

[0074] The specific method for synthesizing FPy1 is as follows: Compounds RhoB-FLTDG (232 mg, 0.2 mmol) and HBTU (76 mg, 0.2 mmol) were dissolved in DMF (10 mL), and then DIPEA (99 μL, 0.6 mmol) was added. The mixture was stirred for 0.5 h. Then, compound hNR-NH2 (272 mg, 0.4 mmol) was added, and the mixture was stirred continuously for 24 h until the reaction was complete. The mixture was then poured into deionized water (50 mL) to form a precipitate. The precipitate was filtered, washed with water, dried, and redissolved in anhydrous dichloromethane. Then, TFA (0.5 mL) was added and mixed. The mixture was stirred for 3 h, and DCM was removed under vacuum. The residue was purified by silica gel column chromatography (mobile phase DCM / MeOH, 180 / 1 (v / v)) to give a deep purple solid FPy1 (48 mg, 14% yield).

[0075] The characterization results of FPy1 are as follows: HRMS (ESI) + ): calcd for C 91 H 111 N 13 O 142 2+ 805.4197 m / z; found, 805.4203 m / z.

[0076] The synthetic route for the compound hNR-NH2 in the above synthesis process is as follows: .

[0077] The specific synthesis method is as follows: 1. Synthesis of RhoB1.

[0078] Rhodamine B (479 mg, 1 mmol) and HBTU (379 mg, 1 mmol) were dissolved in DMF (10 mL), and then DIPEA (495 μL, 3 mmol) was added. The resulting mixture was stirred for 0.5 h. Then, sarcosine tert-butyl hydrochloride (272 mg, 1.5 mmol) was added and mixed, and the mixture was stirred continuously for 24 h. After the reaction was complete, the mixture was poured into deionized water (50 mL) to form a precipitate. The precipitate was filtered, washed with water, and a pink solid crude product RhoB1 (373 mg, 62% yield) was obtained.

[0079] Characterization of RhoB1: 1 H NMR (500 MHz, DMSO-) d 6) δ 7.77-7.74 (m, 2H), 7.64 (dd, J =7.1, 1.7 Hz, 1H), 7.54 (dd, J =6.5, 2.3 Hz, 1H), 7.16 (1H), 7.14 (1H), 7.09 (d, J =2.3 Hz, 1H), 7.07 (d, J =2.4 Hz, 1H), 6.93 (d, J =2.3 Hz, 2H), 3.78 (s, 2H), 3.67-3.61 (m, 8H), 2.80 (s, 3H), 1.23 (s, 9H), 1.20 (t, J =7.0 Hz, 12H).

[0080] 13 C NMR (126 MHz, DMSO-) d 6) δ 168.21, 167.21, 157.32, 155.14, 154.89, 135.48, 131.96, 130.19, 130.09, 129.90, 129.49, 127.07, 113.92, 113.36, 95.60, 80.66, 48.88, 45.25, 38.11, 27.44, 12.39.

[0081] HRMS (ESI) + ): calcd for C 35 H 44 N3O4 + 570.3326 m / z, found, 570.3342 m / z.

[0082] 2. Synthesis of RhoB-Sar.

[0083] RhoB1 (303 mg, 0.5 mmol) was dissolved in anhydrous DCM (10 mL), and then TFA (1 mL) was added. The solution was stirred at room temperature for 3 h, and then the solvent was removed under vacuum. The residue was purified by silica gel column chromatography (DCM / MeOH, 150 / 1 (v / v)) to give a pink solid RhoB-Sar (230 mg, 84% yield).

[0084] Characterization of RhoB-Sar: 1 H NMR (500 MHz, DMSO-) d6) δ 7.76-7.62 (m, 4H), 7.51-7.46 (m, 1H), 7.13 (d, J =3.5Hz, 2H), 7.09 (dd, J =9.7, 1.9 Hz, 1H), 6.93-6.90 (m, 2H), 3.75 (s, 2H), 3.66-3.63 (m, 8H), 2.80 (s, 3H), 1.20 (t, J =6.7 Hz, 12H).

[0085] HRMS (ESI) + calcd for C 31 H 36 N3O4 + 514.2700 m / z, found, 514.2715 m / z.

[0086] 3. Synthesize R1.

[0087] 6-Amino-1,2,3,4-Tetrahydro-1-naphthone (1.3 g, 8 mmol) and K₂CO₃ (3.3 g, 24 mmol) were dissolved in DMF (10 mL), and then CH₃I (2.8 g, 20 mmol) was added. The mixture was stirred at 45 °C for 24 h. The reaction solution was then extracted with EA, and the organic phase was dried over Na₂SO₄. EA was then removed under vacuum, and the crude product was purified by silica gel column chromatography (PE / EA, 20 / 1 (v / v)) to give colorless crystals R1 (1.1 g, 73% yield).

[0088] Characterize R1: 1 H NMR (500 MHz, DMSO-) d 6) δ 7.70 (d, J =8.9 Hz, 1H), 6.63 (dd, J =8.9, 2.6 Hz, 1H), 6.48 (d, J =2.6 Hz, 1H), 3.00 (s, 6H), 2.82 (t, J =6.0 Hz, 2H), 2.43 (t, J =6.4 Hz, 2H), 1.98-1.93 (m, 2H).

[0089] 13 C NMR (126 MHz, DMSO-) d6) δ 195.43, 153.40, 146.39, 128.45, 121.00, 110.18, 109.48, 39.68, 38.46, 29.85, 23.27.

[0090] HRMS (ESI) + ): calcd for C 12 H 16 NO [M+H] + : 190.1226 m / z, found, 190.1230 m / z.

[0091] 4. Synthesize hNR.

[0092] 2-[4-Diethylamino-2-hydroxybenzoyl]benzoic acid (627 mg, 2 mmol) and R1 (378 mg, 2 mmol) were dissolved in sulfuric acid (10 mL, 98% by mass) and mixed, then stirred at 90°C for 3 h. After cooling to room temperature, the mixture was poured into ice water, and HClO4 (1.5 mL, 30% by mass) was added. The precipitate was filtered, and the crude product was purified by silica gel column chromatography (DCM / MeOH, 60 / 1 (v / v)) to give a deep purple solid hNR (807 mg, 71% yield).

[0093] Characterizing hNR: 1 H NMR (500 MHz, DMSO-) d 6) δ 13.2 (s, 1H) 8.21-8.17 (m, 2H), 7.86 (ddd, J =7.6, 7.6, 1.3 Hz, 1H), 7.76 (ddd, J =7.7, 7.7, 1.2 Hz, 1H), 7.41 (dd, J =7.6, 1.3 Hz, 1H), 7.23 (d, J =2.5 Hz, 1H), 7.08 (dd, J =9.4, 2.5 Hz, 1H), 6.92 (dd, J =9.2, 2.5 Hz, 1H), 6.87 (d, J =9.4 Hz, 1H), 6.74 (d, J =2.4 Hz, 1H), 3.58 (q, J =7.2 Hz, 4H), 3.18 (s, 6H), 2.95-2.88 (m, 1H), 2.85-2.79 (m, 1H), 2.54-2.40 (m, 2H), 1.20 (t, J=7.0 Hz, 6H).

[0094] 13 C NMR (126 MHz, DMSO-) d 6) δ 166.44, 163.36, 156.12, 155.02, 153.04, 145.04, 134.32, 132.95, 130.85, 129.93, 129.18, 129.03, 128 .58, 118.38, 115.18, 114.27, 112.93, 111.98, 110.68, 95.97, 54.86, 44.77, 39.94, 26.84, 23.54, 12.36.

[0095] HRMS (ESI) + ): calcd for C 30 H 31 N2O3 + 467.2329 m / z, found, 467.2346 m / z.

[0096] 5. Synthesize hNR1.

[0097] hNR (567 mg, 1 mmol) and HBTU (379 mg, 1 mmol) were dissolved in DMF (10 mL), mixed, and then DIPEA (495 μL, 3 mmol) was added. The mixture was stirred for 0.5 h. Then, sarcosine tert-butyl hydrochloride (272 mg, 1.5 mmol) was added, and the mixture was stirred continuously for 24 h. After the reaction was completed, the mixture was poured into deionized water (50 mL), and a precipitate was formed. The precipitate was filtered, washed with water, dried, and purified by silica gel column chromatography (DCM / MeOH, 200 / 1 (v / v)) to give a deep purple solid hNR1 (501 mg, 72% yield).

[0098] Characterization of hNR1: HRMS (ESI) + ): calcd for C 37 H 44 N3O4 + 594.3326 m / z, found, 594.3337 m / z.

[0099] 6. Synthesize hNR2.

[0100] hNR1 (694 mg, 1 mmol) was dissolved in anhydrous DCM (10 mL), and then TFA (1 mL) was added. The solution was stirred at room temperature for 3 h, the solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (DCM / MeOH, 180 / 1) to give a dark purple solid hNR2 (554 mg, 87%).

[0101] Characterization of hNR2: HRMS (ESI) + ): calcd for C 33 H 36 N3O4 + 538.2700 m / z, found, 538.2731 m / z.

[0102] 7. Synthesize hNR3.

[0103] hNR2 (319 mg, 0.5 mmol) and HBTU (190 mg, 0.5 mmol) were dissolved in DMF (10 mL), and then DIPEA (1.5 mmol) was added and mixed. The mixture was stirred for 0.5 h, and then N-tert-butoxycarbonyl-ethylenediamine (136 mg, 0.75 mmol) was added and mixed. The mixture was stirred continuously for 24 h. After the reaction was complete, the mixture was poured into deionized water and H2O (50 mL) to form a precipitate. The precipitate was filtered, washed with water, dried, and purified by silica gel column chromatography (DCM / MeOH, 200 / 1) to obtain a deep purple solid hNR3 (294 mg, 75%).

[0104] Characterization of hNR3: HRMS (ESI) + ): calcd for C 40 H 50 N5O5 + 680.3806 m / z, found, 680.3816 m / z.

[0105] 8. Synthesize hNR-NH2.

[0106] hNR3 (156 mg, 0.2 mmol) was dissolved in anhydrous DCM (5 mL), and then TFA (0.5 mL) was added. The solution was stirred at room temperature for 3 h, the solvent was removed under vacuum, and the residue was purified by silica gel column chromatography (DCM / MeOH, 100 / 1) to give a dark purple solid hNR-NH2 (102 mg, 75%).

[0107] Characterization of hNR-NH2: HRMS (ESI) + ): calcd for C 35 H 42 N5O3+ 580.3282 m / z, found, 580.3309 m / z.

[0108] The synthetic route for the compound RhoB-FLTDG in the above synthesis process is as follows (20% piperidine: 20% piperidine DMF solution, the same below): .

[0109] The specific synthesis method is as follows: RhoB-Phe-Leu-Thr(tBu)-Asp(OtBu)-Gly-COOH (RhoB-FLTDG) was synthesized via a solid-phase synthesis method. First, the first amino acid was loaded onto a 2-chlorotriphenylmethyl resin. Then, a protecting agent (DCM / MeOH / DIPEA = 17 / 2 / 1 (v / v / v), total volume 20 mL) was added to protect the remaining active site on the resin. The Fmoc group was removed using a DMF solution containing 20% ​​piperidine (v / v), exposing the active site. The next Fmoc-protected amino acid was then attached to the exposed active site using HBTU. Using the solid-phase synthesis method described above, 0.4 mmol of Fmoc-Gly-OH, Fmoc-Asp(OtBu)-OH (164 mg), Fmoc-Thr(tBu)-OH (159 mg), Fmoc-Leu-OH (141 mg), Fmoc-Phe-OH (154 mg), and RhoB-Sar (220 mg) were sequentially added to 0.5 g of 2-chlorotriphenylmethyl resin to synthesize RhoB-FLTDG. The RhoB-FLTDG peptide was cleaved from the resin using 0.5% TFA / DCM, and the eluent was collected and concentrated under reduced pressure. Finally, cold diethyl ether was added to form a precipitate. The precipitate was filtered to obtain a pink solid crude product, RhoB-FLTDG (305 mg, 64% yield). The crude RhoB-FLTDG product was used directly in the next reaction without purification.

[0110] Characterization of RhoB-FLTDG: HRMS (ESI) + ): calcd for C 64 H 87 N8O 12 + 1159.6438 m / z, found, 1159.6442 m / z.

[0111] The steps for synthesizing YPy1 are as follows: RhoB-YVADG (226 mg, 0.2 mmol) and HBTU (76 mg, 0.2 mmol) were dissolved in DMF (10 mL), and then DIPEA (99 μL, 0.6 mmol) was added. The mixture was stirred for 0.5 h, and then hNR-NH2 (272 mg, 0.4 mmol) was added. The reaction mixture was stirred continuously for 24 h. After the reaction was complete, the mixture was poured into deionized water (50 mL), resulting in a precipitate. The precipitate was filtered, washed with water, and dried. The dried precipitate was dissolved directly in anhydrous DCM (5 mL) without purification, and then TFA (0.5 mL) was added. The reaction was stirred for 3 h, and DCM was removed under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH, 180 / 1 (v / v)) to give a deep purple solid YPy1 (33 mg, 10% yield).

[0112] Characterization of YPy1: HRMS (ESI) + ): calcd for C 89 H 107 N 13 O 142 2+ 791.4040 m / z; found, 790.9030 m / z.

[0113] In the synthesis of YPy1, the method for synthesizing hNR-NH2 is the same as that for synthesizing hNR-NH2 in FPy1.

[0114] The synthetic route for RhoB-YVADG is as follows: .

[0115] The synthesis method of RhoB-YVADG is as follows: Peptide synthesis was performed using a solid-phase synthesis (SPPS) method. 0.4 mmol of Fmoc-Gly-OH (119 mg), Fmoc-Asp(OtBu)-OH (164 mg), Fmoc-Ala-OH (124 mg), Fmoc-Val-OH (136 mg), Fmoc-Tyr(tBu)-OH (184 mg), and RhoB-Sar (220 mg) were sequentially added to 0.5 g of 2-chlorotriphenylmethyl resin to obtain RhoB-YVADG. The obtained peptide was cleaved from the resin using 0.5% TFA / DCM, the eluent was collected, concentrated under reduced pressure, and then 50 mL of cold diethyl ether was added to form a precipitate. The precipitate was filtered to obtain a pink solid (316 mg, 68% yield), which was the crude RhoB-YVADG product. The crude RhoB-YVADG product was directly used in the next reaction without further purification.

[0116] Characterization of RhoB-YVADG: HRMS (ESI) + ): calcd for C 62 H 83 N8O 12 + 1131.6125 m / z, found, 1131.6122 m / z.

[0117] The steps for synthesizing WPy1 are as follows: RhoB-WEHDG (319 mg, 0.2 mmol) and HBTU (76 mg, 0.2 mmol) were dissolved in DMF (10 mL), and then DIPEA (99 μL, 0.6 mmol) was added. The mixture was stirred for 0.5 h. Then, hNR-NH2 (272 mg, 0.4 mmol) was added, and the mixture was stirred for 24 h. After the reaction was complete, the mixture was poured into deionized water (50 mL), and a precipitate was formed. The precipitate was filtered, washed with water, dried, and directly redissolved in anhydrous DCM (5 mL) without further purification. Then, TFA (0.5 mL) was added. The mixture was stirred for 3 h, and DCM was removed under reduced pressure. The residue was purified by silica gel column chromatography (DCM / MeOH, 180 / 1 (v / v)) to give a dark purple solid WPy1 (21 mg, 6% yield).

[0118] Characterization of WPy1: HRMS (ESI) + ): calcd for C 94 H 108 N 16 O 152 2+ 850.9100 m / z; found, 850.9097 m / z.

[0119] In the synthesis of WPy1, the method for synthesizing hNR-NH2 is the same as that for FPy1.

[0120] The synthetic route of RhoB-WEHDG is as follows:

[0121] The synthesis method of RhoB-WEHDG is as follows: Peptide synthesis was performed using a solid-phase synthesis (SPPS) method. 0.4 mmol of Fmoc-Gly-OH (119 mg), Fmoc-Asp(OtBu)-OH (164 mg), Fmoc-His(Trt)-OH (248 mg), Fmoc-Glu(OtBu)-OH (170 mg), Fmoc-Trp(Boc)-OH (210 mg), and RhoB-Sar (220 mg) were sequentially added to 0.5 g of 2-chlorotriphenylmethyl resin to obtain RhoB-WEHDG. The obtained peptide was cleaved from the resin using 0.5% TFA / DCM, the eluent was collected, concentrated under reduced pressure, and then 50 mL of cold diethyl ether was added to form a precipitate. The precipitate was filtered, and the pink solid (358 mg, 55% yield) was collected as the crude RhoB-WEHDG product. The crude RhoB-WEHDG product was directly used in the next reaction without further purification.

[0122] Characterization of RhoB-WEHDG: HRMS (ESI) + ): calcd for C 91 H 106 N 11 O 15 + 1593.7896 m / z, found, 1593.7901 m / z.

[0123] Based on the molecular structure design approach, the principle of FRET fluorescence in the detection of caspase-1 in this invention is as follows: Figure 1 As shown (using FPy1 as an example). When the probe interacts with caspase-1, the caspase-1-sensitive peptide sequence of the linker in the probe is cleaved by caspase-1, and the FRET process in the system is disrupted, thereby restoring the donor fluorescence.

[0124] Example 2 The effects of the three probes FPy1, YPy1 and WPy1 prepared in Example 1 were characterized and studied, as detailed below.

[0125] 1. Probe sensitivity detection Different concentrations of caspase-1 (final concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.25, 1.5, 1.75, 2, 2.5, and 3 U / mL) were incubated with FPy1 (5 μM), YPy1 (5 μM), and WPy1 (5 μM) in a buffer solution (50 mM HEPES, 50 mM NaCl, 0.1% chaps, 5% glycerol, 10 mM DTT, and 10 mM EDTA) at 37°C for 2 hours. Fluorescence spectra were recorded using an F-2700 fluorometer with an excitation wavelength of 510 nm (photomultiplier tube voltage 700 V; slit width 10 nm × 5.0 nm).

[0126] Experimental results are as follows Figure 2 As shown, the fluorescence intensity of all probes at 585 nm increases with increasing caspase-1 concentration. FPy1 and YPy1 show similar fold increases in response to caspase-1, with fluorescence intensities increasing by approximately 20.2-fold and 19.2-fold, respectively, while WPy1 only shows an approximately 11.5-fold increase in fluorescence intensity.

[0127] Furthermore, the sensitivity of FPy1, YPy1, and WPy1 was tested. The results are shown in Table 2 below.

[0128] Table 2

[0129] 2. Response time test Fluorescence spectra of 5 μM FPy1, YPy1, and WPy1 after incubation with caspase-1 at 37 °C for 0, 5, 10, 20, 30, 40, 50, 60, 80, and 100 minutes were recorded using an F-2700 fluorescence spectrometer. The excitation wavelength was 510 nm (photomultiplier tube voltage 700 V; slit width 10 nm × 5.0 nm). The reaction time of the probes was investigated by monitoring the fluorescence changes of each probe with caspase-1 at different incubation time intervals. Figure 3 As shown, fluorescence change analysis at 585 nm wavelength revealed that FPy1 had the shortest reaction time, reaching saturation fluorescence intensity in approximately 40 minutes, while YPy1 and WPy1 required 60 minutes to reach maximum intensity. These results confirm that caspase-1 has a faster response rate to FPy1.

[0130] 3. Selective testing Probes FPy1, YPy1, and WPy1 were respectively associated with HSA (100 μg / mL), BSA (100 μg / mL), GSH (1 mM), glucosidase (100 μg / mL), lipase (100 μg / mL), collagenase (100 μg / mL), thrombin (100 μg / mL), ATP (1 mM), ADP (1 mM), H2O2 (1 mM), and ClO2. - Caspase-3 (1 mM), caspase-4 (2 U / mL), caspase-8 (2 U / mL), and caspase-1 (2 U / mL) were incubated at 37°C for 2 hours, with a final probe concentration of 5 μM. The fluorescence spectra were recorded using an F-2700 fluorescence spectrometer. The excitation wavelength was 510 nm, and the emission wavelength for recording intensity was 585 nm (photomultiplier tube voltage 700 V; slit width 10 nm × 5.0 nm). Experimental results are as follows: Figure 4 As shown, the three probes FPy1, YPy1, and WPy1 all exhibit good selectivity.

[0131] 4. Evaluation of the effect of FPy1 on THP-1 cell viability The effect of FPy1 on THP-1 cell viability was detected using a CCK-8 assay kit. THP-1 cells were seeded at a density of 5000 cells per well in 96-well plates and cultured (37°C, 24 h in a cell culture incubator). Then, solutions of different concentrations of the FPy1 probe in RPMI-1640 medium were added to the 96-well plates (100 μL / well). The FPy1 concentration gradient was 0 (control concentration), 2.5, 5, 10, 20, 30, 40, and 50 μM (all test concentrations). After co-culturing for 12 h, the culture medium was removed, and the cells were carefully washed with PBS. 100 μL of fresh culture medium and 10 μL of CCK-8 staining solution were added to each well, and staining was performed for 2 h. The absorbance at 450 nm was measured using a Tecan Infinite M1000 multi-plate reader. Cell viability was calculated using the following formula.

[0132] Cell viability (%) = [(OD test - OD blank) ÷ (OD control - OD blank)] × 100%.

[0133] Among them, OD blank is the absorbance of the well without cells, OD test is the absorbance at the test concentration, and OD control is the absorbance when the probe concentration is 0.

[0134] Experimental results are as follows Figure 5 As shown, FPy1 exhibits low toxicity to THP-1 cells within the concentration range of 0-50 μM.

[0135] 5. Test of caspase-1 fluorescence detection effect on THP-1 cells The fluorescence detection performance of the fluorescent probe FPy1 was analyzed using confocal fluorescence imaging and cell fluorescence flow cytometry, respectively. The experimental procedure is as follows.

[0136] THP-1 cells were cultured overnight in confocal culture dishes. After stimulation for 4 hours in RPMI 1640 medium containing LPS (1 μg / mL), cells were further treated with nigericin (Nig, 10 μM) or ATP (2 mM) for 0, 1, and 2 hours, respectively. Finally, the medium was replaced with fresh medium containing FPy1 (5 μM) and incubated at 37°C for 1 hour. After incubation, cells were washed twice with PBS and then subjected to fluorescence imaging. Further, THP-1 cell fluorescence flow cytometry analysis was performed. 5 THP-1 cells were seeded in 6-well plates at a density specified for each well and cultured overnight. The treatment procedure for each group was the same as described for fluorescence imaging. After treatment, the cells were gently rinsed three times with PBS, resuspended in PBS, and then added to flow cytometry tubes placed on ice. Flow cytometry results were analyzed using FlowJo software.

[0137] like Figure 6 As shown. To evaluate the ability of FPy1 to monitor the intrinsic activation of caspase-1 in cells, a classic NLRP3 inflammasome-mediated caspase-1 activation model was established on THP-1 cells. THP-1 cells were stimulated with LPS (1 μg / mL) for 4 hours, followed by stimulation with nigericin (Nig, 10 μM) for different durations to induce intracellular caspase-1 activation. After stimulation, THP-1 cells were stained with FPy1 (5 μM) for 1 hour and subjected to fluorescence imaging. Figure 6 (a) Imaging results showed that intracellular fluorescence intensity was positively correlated with Nig stimulation time (a). Figure 6 (b) Intracellular fluorescence quantitative analysis also showed that fluorescence intensity increased with prolonged Nig stimulation time. Figure 6 c). The results obtained from cell flow cytometry are also consistent with the above imaging results ( Figure 6 (d in the text). Western blot directly confirmed a positive correlation between intracellular activated caspase-1 levels and Nig stimulation time. Figure 6 (e in the text) This also verifies the reliability of FPy1 in fluorescent imaging of activated caspase-1 in cells.

[0138] 6. Pyroptosis imaging of probe FPy1 in multi-scale biological samples Previous reports have shown that excessive reactive oxygen species (ROS) can significantly disrupt the microenvironment homeostasis of the intervertebral disc, thereby inducing pyroptosis of nucleus pulposus cells. Therefore, we selected H2O2 to stimulate primary rat nucleus pulposus cells, followed by incubation with FPy1 and fluorescence imaging. The procedure is as follows: Figure 7 As shown in 'a'. The specific process is as follows: Fresh nucleus pulposus tissue surgically removed from rats was rinsed with sterile saline to remove blood and resuspended in DMEM / F12 medium containing 10 μM ITS-A. For the 2D model of adherent growth, the resuspended cells were placed in a confocal culture dish. For the 3D cell spheroid model, 10 μM ITS-A was directly added to the culture dish. 5 Each cell is placed in a centrifuge tube, and half the volume of culture medium is periodically removed and replaced with fresh culture medium until small cell spheres are visible in the centrifuge tube.

[0139] After culture, the 2D / 3D nucleus pulposus cell models were stimulated with H2O2 for 30 min, 60 min, and 90 min respectively. Cells were then collected and dispersed in a culture medium containing 5 μM FPy1 for 1 h. The stained 2D / 3D nucleus pulposus cells were then analyzed by fluorescence confocal imaging. Results are as follows: Figure 7 As shown in b and c in the figure. The results of quantitative fluorescence are as follows. Figure 7 As shown in d and e.

[0140] Imaging results showed a positive correlation between fluorescence intensity and H2O2 stimulation time. Quantitative fluorescence analysis also clearly demonstrated that intracellular fluorescence intensity increased with increasing H2O2 stimulation time. To further investigate the imaging performance of FPy1, nucleus pulposus cells were cultured into 3D cell spheroids to observe the imaging of FPy1 within the spheroids. The fluorescence imaging and corresponding quantitative analysis results of the cell spheroids were consistent with those of 2D nucleus pulposus cells, but the fluorescence enhancement was more pronounced in the spheroids under the same stimulation conditions.

[0141] Given the excellent imaging results of FPy1 in 2D nucleus pulposus cells and spheroids, we attempted to evaluate its in vivo imaging capabilities.

[0142] A model of intervertebral disc degeneration was established in thirty female SD rats (3 months old, approximately 150 g). Rats were anesthetized by intraperitoneal injection of 0.8% (w / v) sodium pentobarbital (10 μL / g body weight). Intervertebral disc locations were identified and marked between the 5th-6th, 6th-7th, and 7th-8th cervical vertebrae (Co5 / 6, Co6 / 7, Co7 / 8). Disc degeneration was induced by percutaneous puncture at the marked disc sites (using a 29-gauge needle, approximately 2 mm depth). The needle was then rotated 360° and held within the disc for 1 minute before removal. Rats received intradiscal injections of FPy1 targeting the Co5 / 6, Co6 / 7, and Co7 / 8 discs at weeks 0, 1, 2, and 4 post-modeling. Disc tissue was collected after animal sacrifice for analysis.

[0143] After injecting FPy1 into the tail of a rat model of intervertebral disc degeneration, in vivo fluorescence imaging of the rat tail was performed using small animal in vivo imaging. Figure 7 The f-values ​​in the model mouse showed significantly enhanced fluorescence in the tail region. Figure 7 As can be seen from the g-index, quantitative analysis shows that FPy1 can significantly distinguish the degree of intervertebral disc degeneration at different modeling stages based on fluorescence intensity. Combining the above imaging results of biological samples at different scales, FPy1 can achieve multi-scale pyroptosis imaging in cells, cell spheres, and within animals.

[0144] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A FRET-based caspase-1 responsive fluorescent probe, characterized in that, The caspase-1 responsive fluorescent probe is selected from: , or One of them.

2. The application of the caspase-1 responsive fluorescent probe according to claim 1 in non-disease diagnostic caspase-1 detection.

3. A product for detecting caspase-1, characterized in that, Includes the caspase-1 responsive fluorescent probe as described in claim 1.

4. A non-disease diagnostic method for detecting pyroptosis, characterized in that, Includes the following steps: The caspase-1 responsive fluorescent probe of claim 1 is used as a probe for caspase-1 detection to detect caspase-1.

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