阅读说明：本技术 运用荧光寿命成像揭示内质网修复机制的荧光探针及其制备方法和用途 (Fluorescent probe for revealing endoplasmic reticulum repair mechanism by using fluorescence lifetime imaging, and preparation method and application thereof ) 是由 张瑞龙 陈娟 韩光梅 刘正杰 张忠平 于 2021-09-17 设计创作，主要内容包括：本发明公开了一种运用荧光寿命成像揭示内质网修复机制的荧光探针及其制备方法和用途,涉及荧光探针技术领域,结构式如下：本发明提供一种荧光探针,该探针能够同时定位溶酶体和内质网,联合共聚焦成像和FLIM,不仅可以实时定量监测内质网的自噬、损伤程度及修复过程,从而揭示内质网的修复机制；而且发现了甘油三酯在内质网自噬过程中的修复作用,以及甘油三酯可能有部分抑制内质网自噬的作用。(The invention discloses a fluorescent probe for revealing an endoplasmic reticulum repair mechanism by using fluorescence lifetime imaging, a preparation method and application thereof, relating to the technical field of fluorescent probes, wherein the structural formula is as follows: the invention provides a fluorescent probe, which can simultaneously position lysosome and endoplasmic reticulum, combines confocal imaging and FLIM (fluorescence imaging microscopy), and can quantitatively monitor autophagy, damage degree and repair process of the endoplasmic reticulum in real time, thereby disclosing a repair mechanism of the endoplasmic reticulum; it was also found that the triglyceride has a repairing effect in the course of endoplasmic reticulum autophagy, and that the triglyceride may partially inhibit the endoplasmic reticulum autophagy.)

1. A compound, abbreviated RHC, of the formula:

2. a process for the preparation of a compound according to claim 1, characterized in that: the method comprises the following steps of (1) taking rhodamine B as an initial raw material, reacting the rhodamine B with ethylenediamine to obtain a compound 1, and reacting the compound 1 with 7-hydroxy-3-acetic acid coumarin to obtain a compound RHC;

the synthetic route is as follows:

3. use of the compound of claim 1 for the preparation of a fluorescent probe.

4. Use according to claim 3, characterized in that: the fluorescent probe simultaneously positions lysosome and endoplasmic reticulum and has different fluorescent life responses to different environments.

5. Use according to claim 3, characterized in that: the fluorescent probe combines confocal imaging and fluorescence lifetime imaging, quantitatively monitors autophagy, damage degree and repair process of the endoplasmic reticulum, and discloses a repair mechanism of the endoplasmic reticulum.

The technical field is as follows:

the invention relates to the technical field of fluorescent probes, in particular to a fluorescent probe for revealing an endoplasmic reticulum repair mechanism by using fluorescence lifetime imaging, and a preparation method and application thereof.

Background art:

the Endoplasmic Reticulum (ER) is a well-organized cytoplasmic membrane organelle rich in ribosomes and other functional proteins, and is encapsulated in a phospholipid bilayer, and the inversion of the phospholipid membrane creates a large surface area to perform various important physiological functions, such as protein synthesis, folding and modification, and intracellular phospholipid and Ca²⁺And storing the ions. In addition, due to the capacity of the endoplasmic reticulum to process and supply phospholipids, most membranes in cells (e.g., plasma membrane, mitochondria, golgi apparatus, endosomes, lysosomes) are closely associated with endoplasmic reticulum activity. Thus, the ER is a key organelle in the entire life cycle of a cell from proliferation, differentiation, senescence to eventual death. Even under normal physiological conditions, the integrity of the intracellular endoplasmic reticulum is often destroyed by biochemical reactions and constantly repaired. Dysfunctional endoplasmic reticulum spontaneously removes stress by autophagy (phagocytosis by lysosomes), such as the addition of Reactive Oxygen Species (ROS) and drugs, enhancing endoplasmic reticulum autophagy and damage, and may even trigger cell death. In response to injury, cells have an intrinsic mechanism to repair the endoplasmic reticulum.

Transmission Electron Microscopy (TEM) can determine the ultrastructure of endoplasmic reticulum and the interaction between the ultrastructure and lysosome, and integrated technologies such as western blotting can quantify the expression level of autophagy-related protein. Despite these advances, these techniques have not been able to monitor dynamic ER behavior in living cells. In this regard, fluorescence imaging can track the behavior of many organelles (e.g., ER, lysosomes, lipid droplets) in living cells using molecular probes in the study of endoplasmic reticulum autophagy, fluorescent probes with unique emissions can label both the endoplasmic reticulum and lysosomes, but these traditional fluorophores have the following problems in the detailed processes and mechanisms of combing endoplasmic reticulum autophagy and recovery. First, current imaging probes in combination with confocal microscopy cannot quantify the extent of endoplasmic reticulum damage because the probe released into the cytoplasm (after lysosomal processing of the damaged endoplasmic reticulum) emits the same fluorescent signal as in the intact endoplasmic reticulum. Second, the use of multiple probes increases endoplasmic reticulum autophagy, and because of the combined cytotoxicity of multiple drugs, addressing these limitations is crucial not only for monitoring the dynamics of endoplasmic reticulum autophagy and destruction in living cells, but also for understanding the mechanisms of endoplasmic reticulum recovery.

Aiming at the problems in various aspects, a multifunctional probe is designed, and has the characteristics that the fluorescence color and the service life change along with the change of the environment. When combined with confocal microscopy, this single probe can label both ER and lysosomes due to their unique emissions at different pH values, and in combination with Fluorescence Lifetime Imaging Microscopy (FLIM) can distinguish between dyes on the ER membrane and released into the cytoplasm after digestion of lysosomes. This "one probe two imaging modality (confocal and FLIM)" approach is an ideal approach to study disruption and recovery of endoplasmic reticulum autophagy in living cells.

The invention content is as follows:

the invention aims to solve the technical problem of providing a fluorescent probe, which can simultaneously position a lysosome and an endoplasmic reticulum, combines confocal imaging and FLIM (confocal imaging and fluorescence imaging), and can quantitatively monitor autophagy, damage degree and repair process of the endoplasmic reticulum in real time, thereby disclosing a repair mechanism of the endoplasmic reticulum; it was also found that the triglyceride has a repairing effect in the course of endoplasmic reticulum autophagy, and that the triglyceride may partially inhibit the endoplasmic reticulum autophagy.

The technical problem to be solved by the invention is realized by adopting the following technical scheme:

it is a first object of the present invention to provide a compound, abbreviated to RHC, of the formula:

the second purpose of the invention is to provide a preparation method of a compound RHC, which takes rhodamine B as a starting material, the rhodamine B reacts with ethylenediamine to obtain a compound 1, and the compound 1 reacts with 7-hydroxy-3-acetic acid coumarin to obtain the compound RHC.

The synthetic route is as follows:

the third purpose of the invention is to provide the application of the compound RHC in the preparation of a fluorescent probe.

The fluorescent probe simultaneously positions lysosome and endoplasmic reticulum and has different fluorescent life responses to different environments.

The fluorescent probe combines confocal imaging and fluorescence lifetime imaging, quantitatively monitors autophagy, damage degree and repair process of the endoplasmic reticulum, and discloses a repair mechanism of the endoplasmic reticulum.

The invention has the beneficial effects that:

1. the ability of the RHC to enter acidic lysosomes is enhanced by the ethylenediamine bridge and the multiple basic amino groups in rhodamine B, the RHC molecule is overall amphiphilic with a log P value of 1.68, facilitating its binding to phospholipid membranes.

2. The RHC dye only emits blue fluorescence, but the rhodamine B group does not emit red fluorescence at the time, because the spiro ring destroys the conjugated structure of the rhodamine B, and the probe also emits blue fluorescence when being connected with the ER membrane. In acidic environments, such as the luminal side of lysosomes (pH-5.0), the RHC fluoresces red, as the helical ring is cleaved by acid, thereby restoring the conjugation to rhodamine B, while the coumarin group barely fluoresces.

3. When the endoplasmic reticulum is damaged, the probe is released from the lysosome into the cytoplasm, and the probe released from the lysosome changes from red to blue due to the reversibility of the spiro ring. Due to hydrogen bond formation between coumarin group and water environment (such as cytoplasm), a non-radiative process is generated, compared with phospholipid environment, the service life is greatly shortened, the service life value (tau) of RHC blue emission on ER membrane is obviously longer than that of RHC in cytoplasm, and the method provides another method for quantitatively measuring the degree of ER damage.

Description of the drawings:

FIG. 1 is a schematic representation of two imaging modes of an RHC probe: (a) the molecular structure of the RHC probe and the fluorescence emission thereof change along with the change of the pH value, the RHC emits blue fluorescence centered at 455nm at a neutral pH value, and the fluorescence emission thereof is transferred to orange-red at 580nm at an acidic pH value, such as the inside of a lysosome; (b) a schematic of disruption of the endoplasmic reticulum by autophagy (phagocytosis by lysosomes), when lysosomes break, the RHC digested from the initially labeled endoplasmic reticulum is released into the cytoplasm; (c) RHC lifetime changes, which allow discrimination between RHC in the cytoplasm and RHC molecules re-linked to the ER, based on the fluorescence lifetime (τ) of RHC under FLIM;

FIG. 2 shows RHC fluorescence in solution: (a) fluorescence spectra at 365nm and 540nm excited by RHC (10 μ M) in PBS buffer (pH 7.0) and liposome (1mg/mL lecithin) solutions with the images in the inset taken under a 365nm UV lamp; (b) fluorescence decay of RHC (10 μ M) at 455nm after addition of liposome solution; (c) concentration-dependent fluorescence lifetime of RHC at 455nm, error bar represents the average error of 5 test results; (d) integral FLIM images and fitted images of different life values of 10 μ M RHC liposome vesicles; (e) FLIM images of 10 μ M RHC droplets; (f) the ratio of the two fitted components at 4.08 + -0.22 and 1.48 + -0.15 ns for FLIM excitation at 405nm and emission at 460nm was collected;

FIG. 3 is fluorescence imaging of HeLa cells using RHC and commercial dyes, the cells incubated with RHC, then treated with endoplasmic reticulum green commercial stain (top panel) and lysosome deep red commercial stain (bottom panel), the excitation wavelengths of endoplasmic reticulum commercial stain and lysosome commercial stain were 504 nm and 630nm, respectively, and the emission wavelengths were 510-530nm and 640-660nm, respectively;

FIG. 4 shows FLIM for HeLa cells: (a) incubate with 4 μ M RHC alone for 15 min; (b) treatment with 20 μ M TB for 5h, followed by incubation with 4 μ M RHC for 15 min;

figure 5 is the dynamics of endoplasmic reticulum autophagy: (a, b) time-dependent confocal and FLIM imaging of TB-treated cells, HeLa cells incubated with RHC for 15min and then treated with 20 μ M TB; (c) fluorescence intensity of red channel and fluorescence lifetime of blue channel in a and b; (d) pearson correlation coefficient between the fitted FLIM image and the red channel image; (e) western blot experiments of autophagy marker proteins in TB-treated cells, error bars represent standard deviation (± SD);

figure 6 FLIM imaging of Triglyceride (TG) promoting ER regeneration: (a) fluorescence and FLIM images of TG and Tat-Beclin 1(TB) -treated cells incubated with TG-loaded micelles (F127) for 12h and sequentially treated with 20 μ M TB for 5h and 4 μ M RHC for 15 min; (b) the fluorescence intensity of the red channel and the fluorescence lifetime of the blue channel of the TB-treated group, TG and TB co-treated group; (c) immunoblot experiments of autophagy proteins in cells of control, TB-treated, TG and TB-combination treated groups; (d) fluorescence imaging of intracellular Lipid Droplets (LDs); (e) evolution of intracellular LDs region, cells were incubated with TG loaded micelles (F127) for 12h, followed by lipid droplet-mediated incubation for 10min and 20 μ M TB, error bars representing standard deviation (± SD);

figure 7 reveals the endoplasmic reticulum regeneration pathway for FLIM imaging: (a) TG and TB treated cell negative control experiments, group 1 cells were incubated with TG-loaded micelles (F127) for 12h, then cells were treated with 20. mu.M TB for 5h, and 4. mu.M RHC for 15min in sequence; group 2 is hormone sensitive lipase inhibitor (30 μ M HSL-IN-3) pretreatment for 12h, TG-containing liposome (F127) retreatment for 12h, 20 μ M TB treatment for 5h, 4 μ M RHC treatment for 15 min; group 3 is lipase inhibitor (20. mu.M Atglistin) pretreatment for 12h, TG-containing liposome (F127) retreatment for 12h, 20. mu.M TB treatment for 5h, 4. mu.M RHC treatment for 15 min; group 4 was 20. mu.M Atglistidin, 30. mu.M HSL-IN-3 co-pretreatment group; (b) fluorescence intensity of red channel and fluorescence lifetime of blue channel in a; (c) western blot experiments of autophagy marker proteins in TG and TB treated cells in the presence and absence of lipase inhibitors; (d) the triglyceride is a precursor material of endoplasmic reticulum membrane through partial inhibition of autophagy, and a schematic diagram of promotion of endoplasmic reticulum regeneration; (e) schematic representation of ER disruption without triglyceride and error bars represent standard deviation (+ -SD).

The specific implementation mode is as follows:

in order to make the technical means, the original characteristics, the achieved purposes and the effects of the invention easy to understand, the invention is further explained by combining the specific embodiments and the drawings.

Example 1: synthesis of Probe RHC

Firstly dissolving rhodamine B (0.44g, 1mmol) into 50mL ethanol, heating and refluxing until the rhodamine B is completely dissolved, and then500 μ L of ethylenediamine was slowly added dropwise to the above solution. Heating and refluxing for 12h, cooling the reaction liquid to room temperature, then spin-drying a large amount of solution, adding a large amount of distilled water into the remaining small amount of liquid to generate a large amount of pink precipitates, filtering and drying the separated pink precipitates to obtain a product, namely a compound 1, which can be directly put into the next step without subsequent treatment, wherein the yield is 84%.¹H NMR(400MHz,Chloroform-d)δ7.95-7.86(m,1H),7.52-7.41(m,2H),7.15-7.06(m,1H),6.45(d,J＝8.9Hz,2H),6.39(d,J＝2.6Hz,2H),6.29(dd,J＝8.9,2.6Hz,2H),3.35(q,J＝7.2Hz,8H),1.18(t,J＝7.0Hz,12H).HR-MS(m/z,ESI)Calculated for C₃₀H₃₆N₄O₂ m/z＝484.2838[M+H].Foundm/z＝485.2775.

Compound 1(0.48g,1mmol) was dissolved in CH₂Cl₂After adding N, N-diisopropylethylamine (DIEA, 0.135g, 2mmol) to a DMF (V: V ═ 5: 1) mixed solvent to accelerate dissolution, and then adding O-benzotriazole-N, N' -tetramethyl-urea-hexafluorophosphate (HBTU, 0.48g, 2mmol) to activate carboxyl groups for 1h, 7-hydroxy-3-acetic acid coumarin (0.22g, 1mmol) was added to the above solution, followed by stirring at room temperature for 24 h. The reaction is completed until the reaction of the raw materials is completed, and a large amount of solvent is dried by spinning (CH)₂Cl₂) And after a large amount of distilled water is poured into the remaining small amount of liquid (DMF), a large amount of white precipitate is generated, the separated solid is filtered, dried and then further purified by column chromatography, and the eluent is ethyl acetate: methanol 20: 1, obtaining white solid which is the target molecule RHC, and the yield is 50%.¹H-NMR(d₆-DMSO,400MHz,δ),¹H NMR(400MHz,)δ＝7.99(t,J＝5.5,1H),7.79–7.69(m,1H),7.49–7.33(m,3H),6.97–6.87(m,1H),6.70–6.56(m,2H),6.31(d,J＝9.6,7H),5.93(s,1H),3.41(s,2H),3.25(q,J＝7.1,8H),3.04(t,J＝7.3,2H),2.80(dd,J＝13.1,7.0,2H),1.18(s,1H),1.02(t,J＝7.0,13H).¹³C-NMR(CDCl₃100 MHz,δ):170.23,168.60,161.92,155.50,153.65,153.34,149.73,149.09,133.16,130.03,128.47,128.35,125.74,123.92,123.14,114.05,112.20,111.32,108.44,104.41,103.53,97.84,65.94,44.41,40.96,40.00,39.39,12.63.HR-MS(m/z,ESI)Calculated for C₃₉H₂₆N₇O₆ m/z＝686.3401[M+H].Foundm/z＝687.3183.

Example 2: biological investigation of Probe RHC

1. The probe RHC and the endoplasmic reticulum and the lysosome are used for co-localization respectively, so that the probe RHC can localize the lysosome and the endoplasmic reticulum simultaneously, and a foundation is laid for observing endoplasmic reticulum autophagy.

2. Inducing endoplasmic reticulum autophagy by using a classical autophagy drug TB, and observing the fluorescence lifetime change of a blue channel (a channel for positioning the endoplasmic reticulum) of a cell in a normal state and an abnormal state; the RHC probe can quantitatively monitor the damage degree of endoplasmic reticulum in real time through the fluorescence lifetime.

3. When the cells are cultured by using TG and TB together, the change of the fluorescence lifetime of a blue channel is observed, the change is small, and the fact that the TG has a repairing effect on autophagy of an endoplasmic reticulum and the TG inhibits the autophagy to a certain extent is proved.

4. Using the triglyceride lipase decomposing TG and the hormone sensitive lipase inhibitor atglistitin and HSL-IN-3, it was confirmed that TG provides a synthetic precursor for endoplasmic reticulum structure by diglycerides and fatty acids generated by the decomposition of triglyceride lipase and hormone sensitive lipase and promotes the mechanism of endoplasmic reticulum regeneration by partially inhibiting autophagy.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.