Fluorescent probe for non-alcoholic fatty liver disease diagnosis and drug screening and preparation method thereof

Abstract

According to the fluorescent probe disclosed by the invention, the response to the pH is realized through intramolecular cyclization and ring opening mechanisms, and the response to the pH is reversible. Under the condition of pH triggering, the fluorescent probe LD-Nu-APP can dye the lipid droplets and the nucleolus and emit green fluorescence and red fluorescence respectively. The advantages of two-parameter detection (lipid content and nucleolus size) of the fluorescent probe LD-Nu-APP are utilized, the non-alcoholic fatty liver tissue and the normal tissue can be successfully distinguished, and the method is more accurate than Nile red which only depends on lipid change. Besides, based on the Bayesian theorem, the fluorescent probe LD-Nu-APP is used for screening and evaluating the two drugs for treating the non-alcoholic fatty liver disease, and it is found that pioglitazone is more effective than metformin, and the application range is wider. In conclusion, it is believed that the fluorescent probe LD-Nu-APP has unique lipid content-nucleolus size two-parameter detection characteristics and has huge value in non-alcoholic fatty liver disease diagnosis and drug screening.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a fluorescent probe for diagnosing non-alcoholic fatty liver disease and screening drugs, and a preparation method thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] The liver is an important and complex organ involved in protein and fat metabolism, bile production, detoxification, immune regulation, and other functions. Non-alcoholic fatty liver disease (NAFLD) is now considered one of the most common liver diseases worldwide.

[0004] Lipid accumulation is a major factor in the pathogenesis of NAFLD and has become a popular diagnostic parameter. Lipid droplets (LDs) are composed of a monolayer of phospholipids and a core of neutral lipids. Nucleoli are rich in proteins and serve as the primary storage site for neutral lipids within cells, playing important roles in lipid metabolism and storage, membrane transport, protein degradation, and signal transduction. The nucleolus, the site of ribosome biogenesis, contains a large amount of acidic phosphates. These nucleoli enlarge and increase during cellular inflammation or carcinogenesis; therefore, the nucleolus can be considered a subcellular organelle closely associated with NAFLD. Therefore, the detection of subcellular organelles and their microenvironment in cells is also of great significance for NAFLD and other biological processes related to subcellular organelles.

[0005] In recent years, fluorescent probes have been widely used in biological detection due to their advantages such as high sensitivity and real-time in situ detection. Fluorescent probes designed for lipid droplet detection have been disclosed for the study of NAFLD. For example, the probe DCIQ was used to study changes in lipid droplet levels in NAFLD tissues; the probe fluorescent probe G1 was used to track lipid droplets in cells and liver tissues of high-fat mice. However, to date, the known probes used to study NAFLD only target a single parameter, mainly lipid droplets. Compared with such single analyte detection, dual detection targeting two independent analytes associated with the disease has advantages because it improves the sensitivity, specificity and reliability of diagnosis and reduces the possibility of diagnostic errors (e.g., false positives or false negatives). Summary of the Invention

[0006] In order to overcome the above problems, the present invention provides a fluorescent probe for diagnosis of non-alcoholic fatty liver disease and drug screening and a preparation method thereof.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a fluorescent probe, the structural formula of which is shown in formula (I):

[0009]

[0010] The second aspect of the present invention provides a method for preparing the fluorescent probe described in the first aspect, comprising the following steps:

[0011] Compound 1 and compound 2 react in an organic solvent to obtain the fluorescent probe;

[0012] The structural formula of compound 1 is shown below:

[0013]

[0014] The structural formula of compound 2 is shown below:

[0015]

[0016] In one or more embodiments, the molar ratio of Compound 1 to Compound 2 is 1:1.1 to 1.5, preferably 1:1.28.

[0017] In one or more embodiments, the organic solvent is selected from alcohol solvents, preferably methanol.

[0018] In one or more embodiments, the concentration of the compound 2 in the organic solvent is 0.02 to 0.026 mol / L, preferably 0.022 mol / L.

[0019] In one or more embodiments, the reaction temperature is 70-80° C., and the reaction time is 40-60 h.

[0020] In one or more embodiments, the preparation method of compound 1 comprises the following steps:

[0021] 2,3,3-Trimethylbenzindole reacts with 2-iodoethanol in chloroform to obtain compound 1.

[0022] Preferably, the molar ratio of 2,3,3-trimethylbenzindole to 2-iodoethanol is 1:(0.9-1.2), preferably 1:1.

[0023] Preferably, the concentration of 2-iodoethanol in chloroform is 0.75 to 1 mol / L, preferably 0.84 mol / L.

[0024] Preferably, the reaction temperature is 70-80° C., and the reaction time is 40-60 h.

[0025] The third aspect of the present invention provides the fluorescent probe described in the first aspect or the fluorescent probe prepared by the preparation method described in the second aspect for use in imaging intracellular lipid droplets and nucleoli.

[0026] A fourth aspect of the present invention provides use of the fluorescent probe described in the first aspect or the fluorescent probe prepared by the preparation method described in the second aspect in preparing a diagnostic product for non-alcoholic fatty liver disease.

[0027] In one or more embodiments, the diagnostic product includes a detection reagent or a detection kit.

[0028] A fifth aspect of the present invention provides a diagnostic product for non-alcoholic fatty liver disease, comprising the fluorescent probe described in the first aspect or the fluorescent probe prepared by the preparation method described in the second aspect.

[0029] In one or more embodiments, the diagnostic product includes a detection reagent or a detection kit.

[0030] A sixth aspect of the present invention provides use of the fluorescent probe described in the first aspect or the fluorescent probe prepared by the preparation method described in the second aspect in preparing a product for screening drugs for non-alcoholic fatty liver disease.

[0031] In one or more embodiments, products for screening non-alcoholic fatty liver disease drugs include screening reagents and screening kits.

[0032] In a seventh aspect, the present invention provides a product for screening drugs for non-alcoholic fatty liver disease, comprising the fluorescent probe described in the first aspect or the fluorescent probe prepared by the preparation method described in the second aspect.

[0033] In one or more embodiments, products for screening non-alcoholic fatty liver disease drugs include screening reagents and screening kits.

[0034] The beneficial effects of the present invention are:

[0035] The fluorescent probe of the present invention achieves a response to pH through an intramolecular cyclization and ring-opening mechanism, and its response to pH is reversible. When triggered by pH, the fluorescent probe LD-Nu-APP can stain lipid droplets and nucleoli, and emit green and red fluorescence, respectively. By utilizing the dual-parameter detection (lipid content and nucleolus size) advantage of the fluorescent probe LD-Nu-APP, non-alcoholic fatty liver tissue can be successfully distinguished from normal tissue, and it is more accurate than Nile red, which relies solely on lipid changes. In addition, based on Bayes' theorem, the fluorescent probe LD-Nu-APP was used to screen and evaluate two drugs for non-alcoholic fatty liver disease, and it was found that pioglitazone was more effective than metformin and had a wider range of applications. In summary, it is believed that the fluorescent probe LD-Nu-APP has a unique lipid content-nucleolus size dual-parameter detection characteristic, which is of great value in the diagnosis of non-alcoholic fatty liver disease and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0037] Figure 1 Characterization of the fluorescence characteristics of LD-Nu-APP, where (a) is the variation of the absorption spectrum of LD-Nu-APP (10 μM) with pH value (3.0-9.0), (c) is the variation of the emission spectrum of LD-Nu-APP (10 μM) under 305 nm excitation with pH value (3.0-9.0), (d) is the variation of the emission spectrum of LD-Nu-APP (10 μM) under 550 nm excitation with pH value (3.0-9.0), and (e) is the fluorescence intensity ratio I under pH change. 600nm / I 418nm (ratio of peak height at 600 nm to peak height at 418 nm); (e) is pH / log curve [(I max -I) / (II min )], (f) pH reversibility of LD-Nu-APP (10 μM) in the pH range of 3.0–9.0;

[0038] Figure 2 The results of fluorescence stability study, where (a) is the photostability of LD-Nu-app (10 μM) at pH 3.0 and 9.0; (b) is the photostability of LD-Nu-app (10 μM) in ClO - (50 μM), H2O2 (50 μM) and ONOO - Stability in (50 μM), Ex: = 550 nm, Em = 600;

[0039] Figure 3 The pH response mechanism of LD-Nu-APP, where (a) (b) (c) represent the pH response mechanism of LD-Nu-APP in DMSO-d6, DMSO-d6 and NaOH (20 μL), and DMSO-d6 and trifluoroacetic acid-d1 (20 μL), respectively. 1 H NMR spectrum; (d) is the optimal geometry calculated by Gaussian 09W, C is gray, H is white, N is blue, and O is red;

[0040] Figure 4 HOMO-LUMO energy levels (a) and electrostatic potential (b) of LD-Nu-APP based on B3LYP / 6-31G;

[0041] Figure 5 MTT results of HeLa, HepG2 and Hacat cell viability after incubation with different concentrations of LD-Nu-APP for 24 h;

[0042] Figure 6 Fluorescence images of HepG2 cells stained with LD-Nu-APP (2 μM, 10 min), (a) is the green channel image; (b) is the differential interference contrast (DIC) image; (c) is the merged image of panels a and b; (d) is the red channel image; (e) is the DIC image; (f) is the merged image of panels d and e; (g~i) are the magnified images of panels a, b, and c, marked with solid line frames; (j~l) are the magnified images of panels d, e, and f marked with solid line frames; wherein the green channel: Ex = 405 nm, Em = 420~480 nm; the red channel: Ex = 532 nm, Em = 570-640 nm; scale bars (a~f) = 20 μm, scale bar (g~i) = 1 μm, scale bar (j~l) = 5 μm;

[0043] Figure 7 Fluorescence images of HeLa cells stained with LD-Nu-APP (2 μM, 10 min), where (a) is the green channel image, (b) is the differential interference contrast (DIC) image, (c) is the merged image of panels a and b, (d) is the red channel image, (e) is the differential interference contrast (DIC) image, (f) is the merged image of panels d and e, (g-i) are magnified images of panels a, b, and c, marked with solid line frames, and (j-l) are magnified images of panels d, e, and f marked with solid line frames, green channel: Ex = 405 nm, Em = 420-480 nm; red channel: Ex = 532 nm, Em = 570-640 nm; scale bars (a-f) = 20 μm, scale bar (g-i) = 1 μm, scale bar (j-l) = 5 μm;

[0044] Figure 8The RNA response characteristics of LD-Nu-APP; (a) is the fluorescence titration spectrum of LD-Nu-APP (10 μM) in response to RNA (0-4500 μg / mL), Ex = 550 nm; (b) is the fitting curve of the graph according to the Scatchard equation, and the RNA binding constant is 2.89×10 6 M -1 , goodness of fit R 2 =0.924; (c) is the fluorescence spectrum of LD-Nu-APP (10 μM) to RNA and DNA at the same concentration of 1400 μg / mL and RNA concentration of 4500 μg / mL; (d) is the molecular docking of LD-Nu-APP and RNA;

[0045] Figure 9 Figure 2 shows the DNA response characteristics of LD-Nu-APP. (a) Fluorescence titration spectrum of LD-Nu-APP (10 μM) against DNA (0-1400 μg / mL), Ex = 550 nm. (b) The curve fitted by the Scatchard equation shows that the DNA binding constant is 2.18 × 10 5 M -1 ;

[0046] Figure 10 Fluorescence (red channel), DIC, and merged images of HepG2 cells stained with 2 μM LD-Nu-APP after 4 hours of DNase and RNase digestion. Red channel: Ex = 532 nm, Em = 570-640 nm, scale bar = 20 μm.

[0047] Figure 11 Fluorescence (red channel), DIC, and merged images of HeLa cells stained with 2 μM LD-Nu-APP after 4 hours of DNase and RNase digestion. Red channel: Ex = 532 nm, Em = 570-640 nm, scale bar = 20 μm.

[0048] Figure 12Fluorescence imaging data of normal mice, NAFLD mice, and NAFLD mice treated with pioglitazone or metformin, where (a) is the in vivo fluorescence image of the mouse, (b) is the average fluorescence intensity data in a; (c) is the fluorescence / color image of the mouse liver tissue in a: upper lower, tissue stained with LD-Nu-APP; middle lower, tissue stained with Nile red; LD-Nu-APP (green channel: Ex = 405 nm, Em = 420–480 nm) (red channel: Ex = 532 nm, Em = 570–640 nm), Nile red (Ex = 532 nm, Em = 560–600 nm) m); scale bar (LD-Nu-APP and Nile Red) = 20 μm, scale bar (Oil Red O) = 1.5 cm; (d) and (e) are the mean fluorescence intensities of LD-Nu-APP and Nile Red in c; (f) is the comparison of the detection accuracy of LD-Nu-APP and Nile Red with that of Nile Red, and n is the number of detection parameters; where Ⅰ is the control group, II is the NAFLD mice treated with FFA, III is the NAFLD mice treated with pioglitazone for two weeks, IV is the NAFLD mice treated with pioglitazone for three weeks, V is the NAFLD mice treated with metformin for two weeks, and VI is the NAFLD mice treated with metformin for three weeks;

[0049] Figure 13 Fluorescence images and fluorescence intensity distribution diagrams of different mice stained with LD-Nu-APP (10 μM, 10 minutes) and RNA Select Green fluorescent probe (10 μM, 10 minutes), where (a) is a fluorescence image, and (b) to (g) are the fluorescence intensity distributions in groups I to VI; I is the control group, II is NAFLD mice treated with FFA, III is NAFLD mice treated with pioglitazone for two weeks, IV is NAFLD mice treated with pioglitazone for three weeks, V is NAFLD mice treated with metformin for two weeks, and VI is NAFLD mice treated with metformin for three weeks; LD-Nu-APP: Ex (red channel): = 532 nm, Em (red channel) = 570–640 nm; RNA Select Green: Ex = 488 nm, Em = 500–530 nm; Scale bar = 20 μm;

[0050] Figure 14 These are comparative examples of the docking of DNVI and DNVB with RNA molecules, where (a) is DNVI and (b) is DNVB. DETAILED DESCRIPTION

[0051] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0052] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0053] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0054] Cell culture and imaging

[0055] All cells were cultured at 37°C in a 5% CO2 incubator. HepG2 and HeLa cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin. Hacat cells were cultured in RPMI1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin. The toxicity of the probe to mammalian cells was determined using the methylthiazolyldiphenyltetrazolium bromide (MTT) assay. HepG2 and HeLa cells were incubated with 2 μM LD-Nu-APP in culture medium for 20 minutes and observed using confocal microscopy.

[0056] Example 1

[0057] Preparation of fluorescent probe LD-Nu-APP:

[0058] (1) 2,3,3-Trimethylbenzindole (4 mL, 25.2 mmol) and chloroform (30 mL) were added to a 100 mL round-bottom flask and stirred thoroughly. 2-iodoethanol (2 mL, 25.2 mmol) was then added and refluxed at 75°C for more than 48 hours. The reaction was monitored by TLC. After the reaction was completed, the system was cooled to room temperature and filtered to obtain compound 1. 1 H NMR(DMSO–d6,400MHz)δ(ppm):8.39(d,J=8.4Hz,1H),8.29(d,J=8.9Hz,1H),8.22(d,J=8.2Hz,1H),8.14(d,J=8.9Hz,1 H), 7.76 (dt, J = 24.2, 7.4Hz, 2H), 5.32 (s, 1H), 4.72 (t, J = 5.1Hz, 2H), 3.94 (t, J = 5.1Hz, 2H), 2.92 (s, 3H), 1.78 (s, 6H).

[0059] (2) Compound 1 (0.2 g, 0.525 mmol) and methanol (30 mL) were added to a three-necked flask and heated at 75°C until compound 1 dissolved. Compound 2 (0.1 g, 0.671 mmol) was then added until completely dissolved and refluxed at 75°C for at least 48 h. The reaction was monitored by TLC. After the reaction was complete, the system was cooled to room temperature and filtered to obtain the fluorescent probe LD-Nu-APP, i.e., the compound represented by formula (I). 1 H NMR(400MHz, DMSO–d6)δ7.98(d,J=8.5Hz,1H),7.85(d,J=8.0Hz,1H),7.77(d,J=8.7Hz,1H),7.48–7.34(m,3H),7.28(t,J=8.0Hz,2 H),6.76–6.66(m,3H),6.13(d,J=15.9Hz,1H),3.90(s,1H),3.72(s,1H),3.51–3.36(m,2H),2.91(s,6H),1.68(s,3H),1.25(s,3H).

[0060] Example 3

[0061] In order to verify whether the fluorescent probe LD-Nu-APP can sense pH, a pH titration experiment was performed. Figure 1 As shown in (a) to (c), two maximum absorption peaks were observed at 305 nm and 550 nm in alkaline and acidic environments, respectively. Under 305 nm excitation light, the fluorescence intensity of LD-Nu-APP increased with increasing pH, with the maximum emission peak at 418 nm. When the probe was excited at 550 nm, its fluorescence intensity increased with increasing pH, with a maximum peak at 600 nm. These emission characteristics can be explained by the pH-dependent ring closing and opening behavior of the probe. 600nm / I 418nm ) versus pH, the pKa of LD-Nu-APP was calculated to be 6.13 ( Figure 1 (e)). Figure 1 It can be seen from (f) that in the continuous acid-base cycle, under acid-base conditions (I 600nm / I 418nm ) ratio changes are negligible, which indicates the photochemical stability of the probe at different pH values. In addition, the photostability of LD-Nu-APP was also studied, such as Figure 2 As shown in (a), at pH 3.0 and 9.0, even if the xenon lamp is continuously irradiated for 30 min, the fluorescence intensity ratio (I 600nm / I 418nm) remained stable. In addition, the stability of the probe was tested under oxidative conditions ( Figure 2 (b)). Under oxidative conditions, the fluorescence intensity of LD-Nu-APP is relatively stable, facilitating long-term imaging of cells and mice using LD-Nu-APP. These experimental results preliminarily confirm that the fluorescence of the probe LD-Nu-APP undergoes a blue shift in acidic environments relative to alkaline environments, enabling sensitive detection of pH changes.

[0062] To further confirm that the red shift of LD-Nu-APP in acidic environment is caused by pH-dependent ring closing and opening equilibrium, the red shift of LD-Nu-APP in DMSO-d6 was compared in the presence of trifluoroacetic acid and NaOH, respectively. 1 H NMR spectrum. Figure 3 As shown in (a) and (b), the LD-Nu-APP before and after adding NaOH to DMSO-d6 1 The H NMR spectra were identical, and the two methyl protons (1 and 2) on the benzindole ring appeared as two singlet peaks (1 and 2) because the two groups are diastereoisomers in the closed ring form. However, when the probe solution was treated with deuterated trifluoroacetic acid, the two methyl peaks appeared as a singlet ( Figure 3 (c)) (3 and 4), because in the ring-opened form of the probe under acidic conditions, they are now enantiomeric groups.

[0063] Density functional theory (DFT) was used to calculate the energy and electrostatic potential of LD-Nu-APP. Figure 4 As shown, the HOMO-LUMO energy gaps of LD-Nu-APP in the closed and open ring states are 9.9778 eV and 3.2692 eV, respectively. This energy gap difference is consistent with the pH-dependent absorption and emission behavior of the probe. The much smaller energy gap in the open ring form supports its highly pi-expanded nature.

[0064] Lipid droplets are weakly alkaline or neutral, so the fluorescent probe LD-Nu-APP in its closed ring form is localized in lipid droplets. Nucleoli are acidic, so the fluorescent probe LD-Nu-APP in its open ring form is localized in the nucleolus.

[0065] Example 4

[0066] Experiments on the cytotoxicity and living cell staining ability of the fluorescent probe LD-Nu-APP:

[0067] Cytotoxicity is a key parameter for evaluating fluorescent probes, so the MTT assay of the probe LD-Nu-APP was performed. Figure 5As shown, HeLa, HepG2, and Hacat cells were stained with LD-Nu-APP (2, 4, 6, 8, and 10 μM) for 24 hours, and the results showed that the viability of all cells remained above 80%. Therefore, the LD-Nu-APP probe shows negligible cytotoxicity for live cell imaging within a short period of time (such as 30 minutes) and can be used to analyze biological behavior inside cells.

[0068] According to the designed mechanism, LD-Nu-APP can stain lipid droplets and nucleoli simultaneously. The staining performance of LD-Nu-APP was first evaluated using HepG2 cells. Figure 6 As shown in Figure 2, fluorescence was observed from the green (420-480 nm) and red (570-640 nm) channels when excited at 405 nm and 532 nm, respectively. The fluorescence images have different distributions, shapes, and sizes. Green fluorescence is randomly distributed in a dot-like pattern, while red fluorescence is concentrated in the cell nucleus and covers a larger area. The average diameter of the green fluorescence dots is 1-2 μm (see the enlarged image). Figure 6 (g) to (i)), which is comparable to the size of lipid droplets. Figure 6 The black image in the DIC image in h is the lipid droplet. The green fluorescence is well integrated with the DIC lipid droplet image ( Figure 6 (g) to (l)). The red fluorescence is well integrated with the larger subcellular organelles in the cell nucleus ( Figure 6 (j) to (1). Similar results were observed using HeLa cells ( Figure 7 ). In summary, LD-Nu-APP can stain lipid droplets and nucleoli with green and red fluorescence, respectively.

[0069] Example 5

[0070] Experiments for LD-Nu-APP specific RNA responses:

[0071] In order to study the RNA targeting mechanism of the probe LD-Nu-APP in detail, a series of experiments were conducted in this example. First, in vitro fluorescence titration assays of LD-Nu-APP responses to RNA and DNA were performed. Figure 8 As shown in (a) to (b), the fluorescence intensity of LD-Nu-APP gradually increases with the addition of RNA, and the RNA binding constant of LD-Nu-APP is 2.89×10 6 M -1 , indicating that it has a high affinity for RNA. At the same time, at the same concentration, the fluorescence response of LD-Nu-APP to RNA is much higher than that to DNA ( Figure 8 (c)), and the binding constant of LD-Nu-APP to DNA is small ( Figure 9 ), which is 2.18×10 5 M-1 Therefore, in the presence of both RNA and DNA, LD-Nu-APP can preferentially bind to RNA, which provides a prerequisite for the probe to specifically stain nucleoli in the cell nucleus.

[0072] In order to understand the binding mechanism between RNA and LD-Nu-APP in the open ring state, molecular docking analysis was performed in this example. Figure 8 As shown in (d), RNA and the probe LD-Nu-APP interact primarily through three interactions in the ring-open state. First, π-π stacking interactions play an important role in probe-RNA binding. These interactions can be found between the benzindole ring and the G5 base ring, and between the naphthalene and G4 base rings. Second, the hydroxyl group of LD-Nu-APP forms hydrogen bonds with the oxygen of the phosphate on A15 and the oxygen anion of the phosphate on G5, respectively. Furthermore, the hydroxyl group and dimethylamino group on G4 are also connected by hydrogen bonds. Finally, a weak polar contact exists between the P=O group on A15 and the methylene group on the indole N atom. Similar interactions can be found between the benzene and P=O groups on A14, and between the indole N and -OH groups on the G4 sugar. Therefore, the ring-opened form of LD-Nu-APP can strongly interact with RNA through the three molecular interactions described above.

[0073] In addition, to further support that LD-Nu-APP can target RNA to stain nucleoli, RNA and DNA enzyme digestion experiments were performed in HepG2 and HeLa cells. Figures 10-11 As shown in the figure, after 4 hours of deoxyribonuclease (DNase) digestion of HeLa cells, bright red fluorescence was still observed in the nucleolus, while after the same period of ribonuclease (RNase) digestion, no red fluorescence of LD-Nu-APP was observed in the nucleolus. From the above experiments, it can be seen that the LD-Nu-APP probe can specifically stain the nucleolus because it targets RNA and there are strong hydrogen bonds and electrostatic interactions between them.

[0074] Example 6

[0075] LD-Nu-APP NAFLD detection and drug screening experiments

[0076] NAFLD was detected by in vivo fluorescence imaging and the effectiveness of the drugs pioglitazone and metformin in normal mice and NAFLD mice was evaluated. First, compared with mice eating normal feed (I), mice fed with a high-fat diet (II) showed bright red fluorescence. Combined with the previous description, it can be seen that the number of nucleoli in this group of mice increased significantly and their size also became larger, indicating the presence of NAFLD. We dissected the mice and stained them with LD-Nu-APP, Nile Red (a commercially available LD probe) and Oil Red O (a commonly used staining dye to visualize lipid accumulation in liver tissue, especially in the case of NAFLD), and then performed fluorescence imaging on the corresponding tissues. All tissue imaging results showed that the lipid content in NAFLD mice increased significantly and the number of nucleoli also increased. Based on this, the probe LD-Nu-APP detected NAFLD through dual parameters such as lipid content and nucleolar number and size ( Figure 12 ).

[0077] Compared with normal mice, NAFLD mice showed a significant increase in red fluorescence from the cationic form of the probe in the nucleolus (due to enlarged nucleoli under disease conditions) ( Figure 12 (a), (b)). In addition, after feeding mice with pioglitazone and metformin for 2 to 3 weeks, red fluorescence was significantly reduced. The results show that these drugs improved the severity of NAFLD and reduced the size of the nucleoli. In addition, the fluorescence intensity of mice treated with pioglitazone (Groups V and VI) was weaker than that of mice treated with metformin (Groups III and IV). Similarly, the red fluorescence signal observed using LD-Nu-APP completely overlapped with the red fluorescence signal of RNASelectGreen ( Figure 13 ). Therefore, LD-Nu-APP emits stronger red fluorescence in NAFLD liver tissue, likely due to the increased size and number of nucleoli caused by the disease. Furthermore, tissue imaging results showed that lipid content in NAFLD was significantly reduced after treatment. In summary, the probe LD-Nu-APP evaluated the therapeutic effects of pioglitazone and metformin using dual parameters—lipid content and nucleolar number and size—and the results suggest that both can be used in vivo to treat NAFLD, with the former appearing to have a greater therapeutic effect.

[0078] Example 7

[0079] This example performs docking analysis of similar probe molecules DNVI and DNVB. Figure 14Both probes (a) and (b) bind to RNA only through weak hydrogen bonding, preventing DNVI and DNVB from entering the nucleus and binding to the nucleolus. However, the LD-Nu-APP probe strongly interacts with RNA through three molecular interactions: π-π stacking, hydrogen bonding, and weak polar contacts. Therefore, the LD-Nu-APP probe is a more effective tool for RNA binding than DNVI and DNVB.

[0080] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A fluorescent probe, characterized in that Its structural formula is shown in formula (I), 2. The method for preparing the fluorescent probe according to claim 1, wherein The steps include: Compound 1 and compound 2 react in an organic solvent to obtain the fluorescent probe; The structural formula of compound 1 is shown below: The structural formula of compound 2 is shown below:

3. The preparation method according to claim 2, wherein The molar ratio of compound 1 to compound 2 is 1:1.1-1.5, preferably 1:1.28; Alternatively, the organic solvent is selected from alcohol solvents, preferably methanol; Alternatively, the concentration of compound 2 in the organic solvent is 0.02 to 0.026 mol / L, preferably 0.022 mol / L; Alternatively, the reaction temperature is 70-80° C.; the reaction time is 40-60 h.

4. The preparation method according to claim 2, wherein The preparation method of compound 1 comprises the following steps: 2,3,3-Trimethylbenzindole reacts with 2-iodoethanol in chloroform to obtain compound 1; Preferably, the molar ratio of 2,3,3-trimethylbenzindole to 2-iodoethanol is 1:(0.9-1.2), preferably 1:1; Preferably, the concentration of 2-iodoethanol in chloroform is 0.75 to 1 mol / L, preferably 0.84 mol / L; Preferably, the reaction temperature is 70-80° C., and the reaction time is 40-60 h.

5. The fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 4 is used for imaging intracellular lipid droplets and nucleoli.

6. Use of the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 4 in the preparation of a diagnostic product for non-alcoholic fatty liver disease.

7. A diagnostic product for non-alcoholic fatty liver disease, characterized in that: The invention comprises the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 4.

8. Use of the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 4 in the preparation of a product for screening drugs for non-alcoholic fatty liver disease.

9. A product for screening drugs for non-alcoholic fatty liver disease, characterized in that: The invention comprises the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 4.

10. The product according to claim 9, characterized in that Includes screening reagents and screening kits.

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