CN113820297A

CN113820297A - Application of iron death key marker in high-throughput screening of anti-epileptic regulator

Info

Publication number: CN113820297A
Application number: CN202110410177.9A
Authority: CN
Inventors: 钱勇; 邵晨雯
Original assignee: Nanjing Normal University
Current assignee: Nanjing University; Nanjing Normal University
Priority date: 2021-04-16
Filing date: 2021-04-16
Publication date: 2021-12-21

Abstract

The invention discloses an application of an iron death key marker in high-throughput screening of an anti-epileptic regulator. The invention can be used for preparing medicines, reagents or tools for screening the anti-epileptic regulating agent by using ferrous ions as markers, such as a near-infrared fluorescent probe FeP for detecting the ferrous ions. The invention utilizes near infrared to excite a two-photon fluorescent probe FeP, and can treat Fe in living brains of epileptic mice²⁺The change is used for direct imaging, the spatial and temporal resolution is higher, and the Fe in the epileptogenesis and treatment process can be directly observed by utilizing the in-vivo three-dimensional dynamic two-photon imaging of FeP²⁺Dynamic change of flux, a high-flux screening platform combining FeP and high content analysis is constructed, a series of antiepileptic compounds and the like can be found to effectively regulate and control the iron balance of cells, and the remarkable antiepileptic effect is proved through behavioral research and electroencephalogram monitoring analysis of an epileptic mouse model, so that the screening and treatment are realized for screening and treatingThe epilepsy treating medicine provides help.

Description

Application of iron death key marker in high-throughput screening of anti-epileptic regulator

Technical Field

The invention belongs to the field of medicines, and particularly relates to an application of an iron death key marker in high-throughput screening of an anti-epileptic regulator.

Background

Iron is a basic element of life, and is widely involved in the synthesis of protein cofactors, heme and iron-sulfur clusters, and then in a series of biochemical processes such as nucleotide biosynthesis, cell cycle regulation, oxygen transport and mitochondrial oxidation respiratory chain. Iron content in cells is closely monitored by Iron Regulatory Proteins (IRPs), and cells maintain a balance of iron import, storage and export by controlling the expression of a range of iron-related genes, such as transferrin receptor 1(TfR1), Lipocalin-2, divalent metal transporter-1 (DMT1), FPN, and the like. In fact, one of the most common nutritional deficiencies, iron deficiency anemia, is caused by iron deficiency; abnormally high iron overload is closely related to various diseases, such as cancer, cardiovascular diseases, neurodegenerative diseases and the like. Since the brain is the major accumulating organ for iron, there is increasing evidence that excessive accumulation and abnormal metabolism of iron is present in many neurological diseases, including epilepsy. Therefore, understanding the status of iron metabolism and iron stability in the brain of a living body is crucial to understanding its relevance to the onset of epilepsy. The discovery of new chemical modulators related to iron metabolism may provide new avenues for exploring the regulation of iron balance and the prevention and treatment of epilepsy.

Within neurons, cellular iron is stored predominantly in an oxidized state, while minute amounts of free iron with redox activity loosely bind to cellular ligands and are present at intersections of cellular iron metabolism, known as labile iron pools. Maintenance of intracellular iron (Fe)³⁺) And ferrous iron (Fe)²⁺) The dynamic balance of redox cycling between them is critical to the normal structure and function of neurons. However, active Fe²⁺The excessive accumulation of (b) can promote the disproportionation of hydrogen peroxide (Fenton reaction) to generate hydroxyl radicals, leading to excessive oxidation of lipids and other biomolecules, and ultimately leading to apoptosis or iron death of neuronal cells. Therefore, development of monitoring Fe in neurons²⁺State of the art, particularly for Fe in the brain of a living subject²⁺Imaging spatial and temporal distribution in the body is crucial to understanding the iron balance in epileptic brain, which will facilitate understanding the pathological mechanism of epilepsy. Fluorescence imaging technology, especially two-photon fluorescence imaging technology, plays a central role in vivo imaging research of biomolecules due to its advantages of deep penetration depth, extremely high sensitivity, low fluorescence background, three-dimensional (3D) space-time high resolution imaging, etc. As a non-invasive imaging method, the development of a chemical probe suitable for two-photon fluorescence imaging in the brain is crucial to measure the subtle changes in the biomolecule concentration in the brain of neurological diseases, and its application in neurobiological research is becoming more and more widespread. At present, there is no report that would be directed to Fe²⁺Application of probe in epilepsia brain to Fe²⁺And carrying out distribution imaging.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides the application of the high-throughput screening of the novel anti-epileptic regulation and control agent based on the iron death key marker, in particular the application of ferrous ions as the marker in the screening of the anti-epileptic regulation and control agent, and can provide a brand-new and effective method and strategy for screening the novel anti-epileptic regulation and control agent.

The invention also provides application of the near-infrared fluorescent probe FeP for detecting ferrous ions in screening the anti-epileptic regulation and control agent.

The technical scheme is as follows: in order to achieve the purpose, the ferrous ions are used as markers for screening anti-epileptic modulators.

The application of the ferrous ions as a marker in preparing a medicament, a reagent or a tool for screening the anti-epileptic regulating agent is provided.

The invention discloses an application of a near-infrared fluorescent probe FeP for detecting ferrous ions in preparing a medicine, a reagent or a tool for screening an anti-epileptic regulator, wherein the structure of the near-infrared fluorescent probe FeP is shown as a structural formula I:

the near-infrared fluorescent probe FeP is used for preparing Fe in epileptic brain²⁺Use in agents for distributed imaging.

The near-infrared fluorescent probe FeP aiming at ferrous ions is screened aiming at Fe²⁺The inducer and the inhibitor of (1). Finds that natural products such as DHA, curcumin, apigenin, quercetin, celecoxib, ibuprofen and the like can effectively inhibit Fe in cells²⁺The concentration can inhibit iron death and can be used as a potential epilepsia inhibitor, and rhein, luteolin and other natural products can induce Fe in cells²⁺Thereby inducing cell iron death as a potential anticancer compound.

Wherein, the near-infrared fluorescent probe FeP of ferrous ions visually traces Fe in nerve cells²⁺Is dynamically changed.

Wherein the infrared fluorescent probe FeP can be used for endogenous Fe of in-vivo epileptic patient²⁺The horizontal change is imaged. In particular, the invention can form a simple and efficient endogenous Fe in tracing KA-induced epileptic disease²⁺Method of signalling, Fe²⁺Abnormal aggregation in epileptic brain, e.g. DHA was found to be effective in modulating Fe in epileptic mice²⁺Abnormal up-regulation of (c).

Wherein, the infrared fluorescent probe FeP can carry out in-vivo three-dimensional dynamic two-photon imaging on the mouse, and can directly observe the generation of epilepsy and Fe in the treatment process²⁺Dynamic variation of flux.

Wherein the infrared fluorescent probe FeP is used for 3D imaging in the living brain to find that the screened anti-epileptic regulator is obtained by down-regulating Fe in the brain of an epileptic patient²⁺The levels achieve an effective therapeutic effect on epilepsy. The invention relates to a method for screening an anti-epileptic inhibitor and an anti-cancer drug in high throughput.

Specifically, experiments show that, for example, dihydroartemisinin DHA can effectively improve the survival rate of neuron cells under KA-induced stress. DHA significantly inhibits the abnormal internal flow of calcium ions of hippocampal neurons under KA-induced stress, effectively protects the synaptic structure of the hippocampal neurons, relieves the damage of synaptic length, increases the number of synapses of the neurons, and can reduce the grade, frequency and energy of epileptic seizures, thereby achieving the anti-epileptic effect.

The near-infrared fluorescent probe FeP is applied to the preparation of a reagent or a tool for screening DHA, curcumin, apigenin, quercetin, celecoxib or ibuprofen as a regulation and control drug of ferrous ions abnormally expressed in epileptic diseases.

The near-infrared fluorescent probe FeP is used for preparing and screening medicines, reagents or tools for screening the influence of the epilepsy inhibitors DHA, curcumin, apigenin, quercetin, celecoxib or ibuprofen on the expression level of LCN2/FPN/GPX 4. The invention realizes the endogenous Fe in the brain of the living body²⁺Non-invasive real-time monitoring of fluctuations reveals Fe²⁺A positive correlation between abnormal clustering and severe neuronal damage during seizures. On the molecular level, DHA inhibits iron death by regulating the LCN2/FPN/GPX4 pathway to play the role of antiepileptic.

The design principle is as follows: by using inductively coupled plasma mass spectrometry (ICP-MS) studies, the present invention determines that the total iron content in the brain of an epileptic is significantly higher than that of the normal control group; however, cytotoxic Fe²⁺The distribution of (biomarkers characteristic of iron death) in the brain remains unclear. Furthermore, there is increasing evidence that iron-containing protein degradation within lysosomes mediates lysosomal membrane permeation and leakage of contents into the cytoplasm, which in turn can lead to iron death. Thus, exploring iron homeostasis in neuronal lysosomes may provide a more direct insight into the mechanisms of neuronal iron death and pathological mechanisms associated with epilepsy.

The invention utilizes a two-photon fluorescent probe FeP to firstly treat Fe in the brain of the live epilepsy animal model²⁺Performing three-dimensional imaging of spatial and temporal distribution to obtain brain Fe in pathological process of epilepsy²⁺Provides direct evidence support. In addition, the invention also discovers that Dihydroartemisinin (DHA) can effectively regulate unstable iron pools in neurons under KA stress.

According to the invention, a high-throughput cell fluorescence imaging screening platform is constructed, and Dihydroartemisinin (DHA) and the like can effectively regulate and control intracellular iron balance. The Fe in the living brain of the epileptic mouse can be detected by using a near infrared excited two-photon fluorescent probe (FeP)²⁺The change is directly imaged, and has higher spatial and temporal resolution. By utilizing the in-vivo three-dimensional dynamic two-photon imaging of FeP, the Fe in the epileptogenesis and treatment process can be directly observed²⁺Dynamic variation of flux. The high-throughput screening platform combining FeP and high content analysis is constructed for the first time, and the Dihydroartemisinin (DHA) can effectively regulate and control the iron balance of cells. And the remarkable anti-epileptic effect of DHA is verified through behavioral research and electroencephalogram monitoring analysis of an epileptic mouse model, and a potential chemical regulator is provided for treating epilepsy. Through the research of molecular mechanism, the DHA is further revealed to be capable of reducing the expression of LCN2,regulating LCN2/FPN/GPX4 related pathway and relieving Fe in brain²⁺And finally inhibiting iron death to exert its anti-epileptic effect. The work is epileptic intracerebral Fe2⁺The three-dimensional dynamic two-photon imaging provides a reliable chemical tool, clarifies the role of iron balance in epilepsia pathogenesis, and reveals the potential function of DHA in regulating and controlling the iron balance in brain, thereby providing a new and promising candidate drug for the treatment of epilepsy. This will be to study Fe in brain²⁺And screening the antiepileptic drug, and provides a simple and effective product and method, and shows that the DHA has good application prospect in the field of antiepileptic drugs.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention utilizes a near-infrared fluorescent probe FeP aiming at ferrous ions to prove that the probe can effectively and selectively image Fe in vivo²⁺Can cross the Blood Brain Barrier (BBB), has the characteristics of targeting the brain, and is the direct observation of Fe during KA-induced epilepsy for the first time in vivo and in vitro²⁺Up-regulation of (a) for the first time effective to dynamically track endogenous Fe in KA-induced epileptic disease in the living brain²⁺The signal can be used as a convenient minimally invasive intuitive long-term detection platform. In addition, the combination of high content analysis and FeP can be used for researching Fe in biological system²⁺And screening the antiepileptic drug, a high-throughput screening method is provided, and the inhibitor of the antiepileptic drug can be simply and effectively screened out. In addition, the imaging agent, electroencephalogram monitoring and animal behavioral research are combined for the first time, a high-flux cell fluorescence imaging screening platform is constructed, and the fact that compounds with the fluorescence intensity being reduced, such as DHA and the like, have an effective behavior treatment effect on a mouse epilepsy model induced by KA is proved, and the DHA and the like can effectively regulate and control the iron balance in cells is found. Further molecular mechanism researches show that, for example, DHA significantly down-regulates LCN2 expression and up-regulates FPN expression, thereby regulating brain iron homeostasis, further inhibiting neuronal iron death, and disclosing a molecular mechanism for regulating epileptogenesis development by DHA. Monitoring Fe related to iron metabolism in nervous system disease brain for 3D brain imaging for the first time²⁺Provide a study ofThe platform also provides a new method platform for screening the iron homeostasis regulator, provides help for discovering new chemical entities for epilepsy treatment, and simply and effectively screens out the regulator of the antiepileptic drug.

Drawings

FIG. 1A shows that FeP of the present invention reacts with endogenous stimuli in living SH-SY5Y nerve cells with Fe²⁺An imaging study schematic diagram of content dynamic changes, wherein 1B is the quantitative analysis data of FIG. 1A;

FIG. 2A shows that FeP of the present invention stimulates Fe in living SH-SY5Y nerve cells with various natural products²⁺A schematic of an imaging study of the change in content, fig. 2B is the quantitative analysis data of fig. 2A;

FIG. 3 shows FeP and Fe stimulated by the reported antiepileptic natural product in living SH-SY5Y nerve cells²⁺Fluorescence quantification of the change in content;

FIG. 4 shows FeP and Fe stimulated by non-reported anti-epileptic natural products in living SH-SY5Y nerve cells²⁺Fluorescence quantification of the change in content;

FIG. 5 is a graph of the determination of the effective permeability (Pe) of FeP and candidate natural products in the PAMPA-BBB method;

FIG. 6 is a graph of the relative fluorescence intensity of KA-treated hippocampal primary neurons of FeP of the present invention under the modulation of different artemisinin analogs in living SH-SY5Y neural cells;

FIG. 7 shows that DHA is effective in increasing the survival rate of neuronal cells under KA-induced stress in a cell survival experiment;

FIG. 8A is a two-photon image of FeP of the invention in hippocampal primary neurons under glutamate/KA-induced stress, and FIG. 8B is the quantitative analysis data of FIG. 8A;

FIG. 9 shows that DHA can significantly inhibit Fe in neurons during KA-induced oxidative stress²⁺Flow charts and quantitative data charts of abnormal upregulation;

FIG. 10A is a graph of the quantitative analysis of the fluorescence signal of BODIPY 581/591 in hippocampal neurons incubated with KA and DHA detected by flow cytometry, and FIG. 10B is a graph of the quantitative analysis of the fluorescence of JC-1 analyzing the effect of KA and DHA on mitochondrial membrane potential;

FIG. 11 is a graph of changes in intracellular activity of KA and DHA induced by the microplate reader in hippocampal, cortical, hippocampal and cortical neurons, TrxR (A), GSH (B) and ROS (C);

FIG. 12 is a graph of the kinetics of ECAR (extracellular acidification rate) and OCR (mitochondrial pressure) of hippocampal neurons in the presence of KA, Glu, various natural antioxidants, or artemisinin analogs;

FIG. 13 is a graph of the kinetics of hippocampal neuron response to oligomycin, FCCP (mitochondrial oxidative phosphorylation uncoupler), rotenone, and antimycin A, ECAR, after stimulation with KA and DHA;

FIG. 14 is a graph of the kinetics of hippocampal neuron response to glucose, oligomycin and 2-DG (2-deoxy-D-glucose), i.e., OCR, after stimulation with KA and DHA;

FIG. 15 is quantitative data showing that DHA reduces the production of lactate (A) and pyruvate kinase (B) by KA;

FIG. 16 is a quantitative plot of DHA significantly inhibiting abnormal influx of calcium ions into hippocampal neurons under KA-induced stress;

FIG. 17 is a graph of the quantitative analysis of DHA's ability to effectively protect the synaptic structure of hippocampal neurons, reduce synaptic length damage, and increase the number of neuronal synapses;

FIG. 18 is a diagram showing Western blot method for detecting the levels of GPx4, Ac-p53, TfR1 and ferritin;

FIG. 19 is a schematic representation of western blot method for detecting expression levels of TFR1 and ferritin in hippocampal neurons and hippocampal tissues for different treatments;

FIG. 20 shows the total iron content in hippocampal tissues of the head of mice in the KA stimulating group and the normal group before and after DHA treatment by ICP-MS;

FIG. 21 shows the determination of Fe in mouse brain homogenate by FeP²⁺A fluorescence quantification scheme of (a);

FIG. 22 is a fluorescence image and quantification schematic of in vivo FeP imaging of mice;

FIG. 23 is a fluorescent image and quantitative schematic of in vitro brain imaging of mice with FeP;

FIG. 24 is a representative TEM micrograph of synapses and neurons in hippocampal regions of mice induced by KA and DHA;

FIG. 25 shows representative immunofluorescence quantitation analyses of dcx (immature neurons), neun (neurons), gfap (mature astrocytes) and iba-1 (microglia) positive cells in mouse hippocampal slice DG (dentate gyrus);

FIG. 26 is a fluorescence image of hippocampal brain slices incubated with FeP in mice;

fig. 27 is a three-dimensional two-photon fluorography of different depths of brain sections of epileptic mice incubated with FeP;

figure 28 is in vivo two-photon (2PM) imaging of brain in live epileptic mice. (A) Schematic experimental design is described. Mice were imaged in vivo in real time with KA, DHA and FeP. (B) Carrying out real-time three-dimensional two-photon fluorescence imaging on the living brain by using a Zeiss LSM 880NLO two-photon confocal microscope;

FIG. 29 is a graph of the quantitative fluorescence analysis of FIG. 28B;

fig. 30 is a quantitative data analysis of Local Field Potential (LFP) recordings showing that DHA significantly reduced the event rate, mean duration, energy and peak-to-peak amplitude in hippocampal slices;

fig. 31A is a schematic diagram of experimental design describing the procedure of video photographing and eeg-emg recording of mice, fig. 31B is a statistical graph of seizure grade of mice evaluated with Racine scale, fig. 31C is a statistical graph of electroencephalogram representative of mice of different treatment groups, which is a statistical graph of data of amplitude (31D), seizure duration (31E), line length (31F) and energy (31G) of seizures of mice;

FIG. 32 shows the detection of ferritin expression-related gene expression levels in hippocampal homogenate tissue (A) and cortical tissue (B) of each group using real-time fluorescent quantitative PCR;

FIG. 33 is a primer sequence for real-time quantitative PCR;

FIG. 34 is a graph of (A) western blot measurements of the expression levels of FPN1, LCN2 and GPX4 in hippocampal and cortical tissues versus the gray scale values of FPN1, LCN2 and GPX4 in hippocampal (B) and cortical (C) tissues of differently treated mice.

Detailed Description

The invention will be further described with reference to specific embodiments and the accompanying drawings.

The experimental methods described in the examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

BABL/C mice were purchased from the national laboratory animal seed center, Shanghai province center, or Jiangsu Jiejiaokang Biotech limited.

SHSY-5Y cells were purchased from Gexin Biotech, Inc. in Shanghai.

The synthesis method of FeP refers to Chinese patent CN 109912540A.

Example 1

Imaging Fe in living nerve cells using FeP²⁺Dynamic fluctuation of (2).

To further determine whether FeP could track endogenous Fe in living cells²⁺Respectively using CuSO₄ (100μM，30min)、MnSO₄(100μM，30min)、FeCl₃(100μM，30min)、ZnSO₄(100. mu.M, 30min), Lipopolysaccharide (LPS) (1ug/mL, 12H), KA (500. mu.M, 12H), glutamic acid (2mM, 30min), H₂O₂SHSY-5Y cells with a growth fusion of 80% in the well plate were incubated (100. mu.M, 1h) or 2, 2' -Bipyridine (BPY) (1mM, 1h), washed with PBS (pH7.4), and imaged after incubating the cells for 30 minutes in DMEM medium containing 10. mu.M FeP (medium covering cells completely), all incubation being at 37 ℃.

The results are shown in FIG. 1, using Cu²⁺、Mn²⁺、Fe³⁺And Zn²⁺Possibly resulting in more Fe²⁺Dissociation, cells treated with these ions showed higher fluorescence signals than control cells treated with DMSO. In the presence of LPS, a known inflammatory activator, a significantly higher fluorescence is also observed, which stimulates Fe in the cells²⁺Is accumulated. Importantly, a significant increase in cellular fluorescence was observed with Glu and KA treatment, suggesting that neuronal cells may release more Fe in the presence of Glu or KA induced stress²⁺(FIG. 1). In contrast, in BPY (Fe)²⁺Chelating agent of (b) was significantly inhibited in fluorescence intensity, which highlights that FeP is responsible for Fe in living cells²⁺With selectivity (figure 1). Overall, these observations suggest that FeP can be used to probe and image endogenous Fe under oxidative stress²⁺And reveals that the method can rapidly detect endogenous Fe in the cellular microenvironment² ⁺Potential for dynamic changes.

Example 2

DHA on neuron Fe²⁺Regulation of the steady state.

A high-throughput cell screening platform was established to screen potential compounds that modulate iron homeostasis. A high throughput screening platform was constructed by combining FeP with High Content Assay (HCA). SHSY-5Y cells with growth fusion degree of 80% are preincubated with DMEM medium (20 μ M final concentration of natural product) of natural products (phenethyl caffeate, artesunate, dihydroartemisinin, apigenin, luteolin, formononetin, daidzein, 3, 4-dihydroxybenzaldehyde, protocatechuic acid, naringenin, curcumin, rhein, 10-hydroxycamptothecin, chrysin, perillyl alcohol, ibuprofen, guaiacol, sulfamethoxazole, celecoxib, naproxen, quercetin, genistein) for 12 hours, washed with PBS (pH7.4), and incubated with FeP (10 μ M) in fresh DMEM medium (complete coverage of medium on cells) at 37 deg.C for 30 minutes. The fluorescence intensity of the cells was quantified after high content fluorescence imaging. Rhein and other natural products can induce intracellular Fe²⁺The up-regulation of (1); while some natural products such as DHA, curcumin and the like can effectively reduce Fe in cells²⁺Concentration (fig. 2).

To further investigate whether potential anti-epileptic compounds can be screened based on a similar strategy, a model of KA (renoic acid, a classical inducer in animal models of epilepsy) induced neuronal cell stress was constructed. First, neuronal cells under KA stress were treated with a series of reported natural products with anti-epileptic potential (isochrysene acid, asarone, borneol, saikoside, decursin, 7-hydroxycoumarin, cannabidiol) to assess whether these compounds have the ability to modulate iron homeostasis. The specific method comprises the following steps: cells with 80% confluency were induced by 20. mu. mol of each compound at 500. mu. mol of KA, incubated, and probed with FePAfter staining the cells with a needle (10. mu.M), the fluorescence intensity was analyzed by flow cytometry. As shown in FIG. 3, it was found that these compounds all inhibit KA-induced intracellular Fe²⁺Up-regulation of (1) indicates Fe²⁺Homeostasis is associated with the presence of epilepsy. To find new, more promising chemical modulators of epilepsy, the high-throughput screening strategy was extended to more natural products (apigenin, quercetin, celecoxib, ibuprofen, 10-hydroxycamptothecin, curcumin, 3, 4-dihydroxybenzaldehyde, dihydroartemisinin, 3,4, 5-trimethoxycinnamic acid). It is found that apigenin, Dihydroartemisinin (DHA), quercetin, celecoxib and ibuprofen inhibit Fe²⁺A great potential for accumulation. Of these potential modulators, DHA (the major active metabolite of artemisinin derivatives) effectively down-regulates intracellular Fe under KA-induced stress²⁺The level was most significant (fig. 4), other compounds were also more effective, but incubation with NaSH did not enhance the inhibitory effect of the natural product.

Since BBB permeability is a prerequisite for the development of antiepileptic drugs, the permeability of BBB candidates was evaluated using the classical BBB in vitro parallel artificial membrane permeability assay (PAMPA-BBB) (specific experimental methods refer to Yang J, Zhang X, Yuan P, Yang J, Xu Y, Grutzendler J, Shao Y, Moore A, Ran C. Oxalate-currmin-based probe for micro-and macrologic of reactive oxidative genes species in Alzheimer' S disease. Proc Natl Acad Sci U A.2017Nov 21; 114(47):12384-12389.doi: 10.1073/pnas.1706248114.). Respectively weighing 5mg of different compounds (apigenin, dihydroartemisinin, quercetin, celecoxib, ibuprofen, atenolol and carbamazepine), dissolving with 1mL of n-propanol, and respectively adding 9mL of PBS solution containing 20% acetonitrile to prepare 500 mug/mL of drug solution to be tested. After 5. mu.L of dodecane solution was added to the filters of the PAMPA model donor plates, 150. mu.L of the solution to be tested was added immediately, and 300. mu.L of PBS buffer solution was added to each receiver plate, and each set of experiments was repeated 3 times. Placing the supply plate on a receiving plate, covering with an upper layer cover, taking out the receiving plate at room temperature in dark place for 16h, measuring absorption intensity in the receiving solution with microplate reader, respectively detecting peak area of the receiving solution with high performance liquid chromatography, and determining the substance according to the substanceAnd calculating the mass concentration of the compounds according to the quantitative ratio, and calculating the permeability coefficient Pe and the permeability of the blood-brain barrier of each compound. The results show that the BBB permeability of quercetin and ibuprofen is poor; while apigenin, DHA, and celecoxib had better BBB permeability, with DHA being the most effective (fig. 5). Artemisinin and its derivatives have been widely used clinically as effective antimalarial drugs, so other representative artemisinin analogs have been evaluated for modulating neuronal cell Fe²⁺Effects on steady state; DHA was also more potent than the other derivatives (fig. 6). In addition, cell survival experiments showed that DHA was effective in increasing neuronal cell survival under KA-induced stress (fig. 7). DHA was therefore chosen as Fe²⁺The potential regulator of the steady state, researches the anti-epileptic effect of the regulator and deeply discusses the potential molecular mechanism of the regulator. This example screens inhibitors of ferrous ion, a key marker of iron death, at high throughput to alleviate the oxidative stress level of cells under simulated status epilepticus.

Example 3

Two-photon imaging of live hippocampal neurons was performed with FeP.

To further investigate whether DHA can protect neurons from glutamate/KA-induced oxidative stress, FeP two-photon imaging studies were used to directly observe intracellular Fe in primary neuronal cells under oxidative stress extracted from hippocampal tissue of 18-day-pregnant BABL/C fetal mice²⁺A steady state change. Viable primary hippocampal neurons were stimulated with or without BPY (1mM, 1h), Glu (2mM,12h), KA (500. mu.M, 12h), DHA (20. mu.M, 12h) first followed by KA (500. mu.M, 12h) or DHA (20. mu.M, 12h) first followed by PBS, washed with PBS, and cells were incubated with DMEM medium containing FeP (10. mu.M) for 30min at 37 ℃ before imaging, and cells were subjected to localized staining with the commercial nuclear dye Hoechst 33342 and the neuronal dye Neuron-Dio for 30 min. Three dyes were then imaged separately with two-photon confocal (FeP: two-photon 880nm excitation, Hoechst 33342: blue channel, Neuron-Dio: green channel). As shown in FIG. 8, primary hippocampal neurons treated with glutamate or KA exhibited a comparable profile to the control group (hippocampal neurons incubated with medium containing DMSO only at a final concentration of 1%) (Gilles de la Tokya)Stronger Fe²⁺Red fluorescence signals indicate that FeP can sensitively detect Fe in neuron cells under stress state²⁺Is increased. In contrast, neurons treated with BPY showed lower Fe than the control group²⁺A fluorescent signal; likewise, DHA-treated neurons also showed weaker Fe²⁺A fluorescent signal. In addition, to determine whether DHA is effective in controlling Fe under KA stimulation²⁺Was pre-treated/post-treated with DHA under KA-stimulated conditions (fig. 9). The result proves that DHA can obviously inhibit Fe in neurons in KA-induced oxidative stress process²⁺Abnormal up-regulation of (c). These results indicate that the Fe in neurons under external stress can be directly observed by FeP two-photon fluorescence imaging²⁺Fluctuating changes, DHA as a potential chemical modulator can maintain neuronal Fe²⁺And (4) steady state.

Example 4

DHA inhibits iron death and protects hippocampal neurons.

To investigate how DHA reduced KA-induced neuronal stress, the status of neuronal cell membranes under various stimulation conditions (1% DMSO-treated control, KA (500 μ M,12h), DHA (20 μ M,12h) first followed by KA (500 μ M,12h) or DHA (500 μ M,12h) first followed by DHA (20 μ M,12 h)) was first examined. After drug stimulation, the cells were washed with PBS, incubated with 5. mu. M C11-BODIPY (oxidation sensitive lipid peroxidation probe) for 30min and then confocal imaged with a laser confocal microscope. The results show that KA stimulation leads to abnormal accumulation of lipid peroxidation in hippocampal neuronal cell membranes, whereas treatment with DHA effectively attenuated lipid peroxidation and exhibited lower fluorescence signals (fig. 10A). Cells treated with the same drug (1% DMSO-treated control, KA (500. mu.M, 12h), DHA (20. mu.M, 12h) first followed by KA (500. mu.M, 12h) or drug-induced group treated with KA (500. mu.M, 12h) first followed by DHA (20. mu.M, 12 h)) after stimulation were incubated for 30min with 5. mu.M of commercial dye JC-1 (5,5 ', 6, 6' -Tetrachloro-1,1 ', 3, 3' -tetraethylene-iodocynine) and then subjected to fluorescence quantification using a flow cytometer. A significant decrease in membrane potential was observed for hippocampal neuronal mitochondria under KA stimulation, whereas DHA-combination treated groups were similar to normal controls (cells treated with 1% DMSO alone) (fig. 10B). In addition, it is observed that DHA can effectively improve the antioxidant capacity of hippocampal neurons, possibly by inducing thioredoxin reductase (TrxR) and Glutathione (GSH) levels and inhibiting Reactive Oxygen Species (ROS) levels (fig. 11), and the specific experimental method refers to total glutathione detection kit (S0052) of picnic and thioredoxin reductase (TrxR) detection kit (BC1155-100 tubes/96 samples) of ACMEC.

To further determine whether DHA contributed to the maintenance of hippocampal mitochondrial function, cellular respiration of hippocampal neurons was evaluated using a hippocampal XFe96 analyzer. The 4 groups of cells (1% DMSO-treated control, KA (500. mu.M, 12h), DHA (20. mu.M, 12h) first followed by KA (500. mu.M, 12h) or drug-induced group treated KA (500. mu.M, 12h) first followed by DHA (20. mu.M, 12 h)) were analyzed using Agilent's kit (103020-. The results in fig. 12 show that under KA-induced stress, mitochondrial respiration in hippocampal neurons is significantly inhibited, reducing basal cell respiration, ATP production, maximal respiration, and backup respiration (fig. 13). At the same time, glycolytic capacity and glycolytic reserve of neurons were also partially inhibited (fig. 14). DHA combination therapy was effective in maintaining or improving mitochondrial function, which was also consistent with corresponding changes in pyruvate kinase and lactate accumulation (fig. 15). In addition, DHA was observed to significantly inhibit abnormal influx of calcium ions into hippocampal neurons under KA-induced stress (fig. 16). DHA was also effective in protecting the synaptic structure of hippocampal neurons, reducing synaptic length damage, and increasing neuronal synaptic number (FIG. 17).

Because DHA can obviously improve Fe²⁺And accumulation of lipid peroxidation (two key biomarkers of iron death), suggesting that DHA may play a role in the regulation of neuronal cell iron death. Western blot studies were performed on GPX4, a key protein regulatory site for iron death, Ac-p53 and TfR 1. After incubation of primary neuronal cells with growth confluency of 80% with KA (500. mu.M, 12h) alone or in combination with DHA (20. mu.M, 12h) before and after, the medium was aspirated from the culture, the cells were washed with PBS and aspirated, lysis was added to lyse the cells, the cells were immediately scraped from the plate and the plates were removedPlaced on ice. Sonication for 10 seconds to complete cell lysis and shear the DNA (to reduce sample viscosity). Taking 20 mu l of sample, adding a loading buffer, and boiling for 6 minutes at 96 ℃; cooling on ice. Centrifuging in a microcentrifuge, and performing electrophoresis on the SDS-PAGE gel. After electrophoresis, the membrane is transferred and sealed, and then primary antibody and secondary antibody are incubated and then imaged by developing solution. The results show that GPX4 expression was reduced under KA stimulation, while DHA combination treatment increased GPX4 levels and was slightly superior to the regulatory effect of the iron chelator Desferrioxamine (DFO) (fig. 18). In addition, DHA has been shown to down-regulate Ac-p53 expression levels under KA stress, thus inhibiting Ac-p 53-mediated iron death. Treatment with DHA also down-regulated transferrin receptor 1(TfR1) expression on neuronal cell membranes, thereby reducing iron absorption; it promoted iron storage in neurons and up-regulated ferritin levels (figure 19). Taken together, these observations suggest that KA-induced stress leads to Fe in hippocampal neurons²⁺Leading to impairment of mitochondrial function and neuronal synaptic structure, ultimately triggering the onset of iron death in hippocampal neurons. Importantly, the research shows that the DHA can effectively relieve the damage of the hippocampal neurons and maintain the function of the hippocampal neurons by regulating the protein level of GPX4/Ac-p53/TfR 1. This example demonstrates that selected inhibitors modulate key proteins in the iron death pathway and the marker lipid peroxide.

Example 5

Epilepsy Fe in brain²⁺Fluctuating in vivo two-photon imaging.

Since FeP has an obvious effect on imaging hippocampal neurons, the applicability of FeP and DHA in vivo studies is studied. Induction of changes in endogenous ferrous ion content by intraperitoneal (i.p.) injection of KA and DHA; the first group was injected with 100. mu.L of physiological saline containing 5% DMSO, the second group was injected with 100. mu.L of 20mg/kg KA, the third group was injected with DHA (60mg/kg) three times every 12h in advance and then with 100. mu.L of 20mg/kg KA, and the fourth group was injected with 100. mu.L of 20mg/kg KA and then with DHA (60mg/kg) three times every 12h, and 5-week-old male BALL/C nude mice were subjected to drug pretreatment. First, two mice in each group were anesthetized, then the neck was cut off, the brain was sacrificed and the hippocampal tissue was treated with MirabilitumAcid and hydrogen peroxide are digested to colorless transparent liquid at 115 ℃, ICP-MS (inductively coupled plasma mass spectrometry) is used for directly and quantitatively displaying that iron overload exists in epileptic brain (figure 20), then a wild mouse and 20mg/kgKA are taken to induce three mice with 12 hours of disease respectively, PBS is added, the brain tissue is ground by a tissue grinder and taken out, centrifugal constant volume is carried out, and F-7000 fluorescence photometer is used for carrying out fluorescence monitoring to obtain the content of ferrous ions in the brain homogenate of the normal and epileptic disease mice. The results show that FeP is used for Fe in brain homogenate²⁺The trend was consistent with ICP-MS (FIG. 21). The potential of FeP was then directly detected using a small animal in vivo imaging system (IVIS spectroscopy) to track Fe in the brain²⁺A change in level. After injection of FeP (0.5mg/kg) separately into the tail vein of 4 groups of mice, fluorescence signals of the mice were monitored with a small animal live imager at five different time points (5, 15, 30, 45 and 60min) (fig. 22), and a significant increase in fluorescence signals was detected in the brains of KA-induced epileptic mice and a significant decrease by DHA combination treatment (fig. 22). In addition, after 60min in vivo imaging, the brain of the mouse is rapidly stripped, the PBS is used for cleaning and drying the roar, and the in vivo imaging instrument is used for carrying out in vitro imaging on the brain, and the result clearly shows that FeP easily penetrates through BBB and carries out Fe in vivo and in vitro epileptic brain²⁺The change in level was imaged (fig. 23).

To assess the effect of DHA treatment on hippocampal architecture, hippocampal tissue was rapidly isolated from the brain dissected out in fig. 23, soaked in 2.5% glutaraldehyde fixation solution, sent to beijing, china, assistant technology, ltd, for routine electron microscopy sectioning and imaging. Brain tissue sections from different treatment groups were examined using Transmission Electron Microscope (TEM) imaging (fig. 24). In the KA treatment group, significant damage to neuronal structures, including rupture of mitochondrial membrane structures and disappearance of ridge structures, can be clearly observed. In contrast, the DHA combination group reduced the damage and maintained mitochondrial and neuronal synaptic structures. In addition, KA induced the number of astrocytes and microglia in hippocampal region of epileptic mice to be increased significantly, and the number of neurons to be decreased (fig. 25); DHA treatment can effectively inhibit abnormal proliferation of glial cells and protect neurons from being damaged. In addition, to clarify Fe in this process²⁺The steady state isIf not, a two-photon confocal microscope is used for finding that FeP has a bright fluorescence signal in a two-photon wavelength excitation mode, a confocal microscope is used for detecting the fluorescence signal of a corresponding brain tissue section, the isolated brain in the figure 23 is subjected to liquid nitrogen quick freezing, a freezing microtome is used for carrying out 15-micrometer tissue slice section and 80-micrometer tissue depth section, and a Leica two-photon fluorescence confocal microscope is used for imaging the tissue section under the excitation of two photons with the wavelength of 880 nm. Bright, granular red fluorescence signals were observed around nuclei of brain tissue sections from epileptic mice, again confirming BBB permeability and NIR properties of FeP (fig. 26). Furthermore Z-stack depth imaging of epileptic mouse brain tissue sections showed very significant fluorescence signals (fig. 27). These observations directly reveal that FeP has two-photon excitation properties for deep tissue penetration, Fe²⁺Abnormal accumulation in the epileptic brain; DHA can effectively regulate and control Fe in epileptic mice²⁺Abnormal up-regulation of (c).

In order to visually observe the Fe in the living brain of the epileptic mouse²⁺Spatial and temporal distribution of flux and its dynamic changes, three-dimensional dynamic imaging studies of the classical KA model under two-photon near-infrared excitation and groups of mice treated with the same drugs in fig. 22 were monitored for fluorescence signals in the 800X 500 μm (X Y Z) region in live brains with confocal laser monitoring under 880nm excitation after simple and careful skull abrasion. In a 3D dynamic imaging mode, the Fe in the brain of the mouse is continuously monitored for 60min²⁺The flux changes dynamically. In the brains of mice in KA-induced epilepsy model, stronger red fluorescence signals were observed at different depths after intravenous injection of (iv) FeP (0.5mg/kg) compared to normal control (wild-type mice injected with FeP only) mice, and the brain fluorescence signals gradually decreased with the passage of time (fig. 28). In contrast, the fluorescence signal was much weaker in the brain of mice treated before/after with DHA (three DHA injections every 12h (60mg/kg) followed by 100. mu.L of 20mg/kg KA, or 100. mu.L of 20mg/kg KA followed by three DHA injections every 12h (60mg/kg)), further supporting DHA as intracerebral Fe²⁺Effect of steady state regulator (fig. 29). Taken together, these in vivo studies demonstrate that FeP two-photon brain imaging is a means of monitoring endogenous Fe in the brain of epileptic mice²⁺Provides a promising approach. Importantly, these findings also underscore the potential of DHA in the prevention and treatment of epilepsy.

Example 6

The anti-epileptic activity of DHA was confirmed by in vitro and in vivo electrophysiological recording.

To confirm the therapeutic effect of DHA on epilepsy, the anti-epileptic activity of DHA was studied in vitro and in vivo models of epilepsy. After 4 to 5 weeks old BABL/C mice were anesthetized with isoflurane gas, the brains were rapidly stripped after neck amputation. Acute coronary hippocampal slices (300 μm) were prepared using a vibrating microtome. The slices were placed in oxygen (95% O)₂/5％CO₂) In ACS of (1), incubated at 35. + -. 0.5 ℃ for at least 1 hour and then stored at room temperature. Then, high potassium, magnesium-free ACSF (artificial cerebrospinal fluid: 124mM NaCl, 26mM NaHCO) is applied₃，6mM KCl，1.6mM CaCl₂and 10mM D-glucose) is continuously perfused for about 15min at the room temperature of 35 ℃ to induce the brain slices of the mice to generate acute epileptic discharge, epileptic discharge of each region of the hippocampus can be recorded on the multi-electrode array, and then the isolated hippocampus slices are perfused for 3min or 30min by magnesium-free ACSF containing 20 mu M DHA to observe epileptic discharge of each region of the hippocampus. Local Field Potential (LFP) recordings showed that DHA significantly reduced the event rate, mean duration, Ifp event energy and peak-to-peak amplitude in hippocampal slices (fig. 30). Then, it was tested whether DHA treatment could inhibit epileptic spikes in an in vivo model of epilepsy. After the brain surgery of the brain electrode monitor implanted mice was resumed for 7 days, the mice were randomly divided into 4 groups, the KA group (20mg/kg, 0 or 36 hours), the DHA post-treatment group (first injection of KA:20mg/kg, 12 hours later injection of DHA: 60mg/kg, 12 hours x 3 times) and the DHA pre-treatment group (first injection of DHA: 60mg/kg, 12 hours x 3 times, later injection of KA:20mg/kg, 12 hours). Each mouse was placed individually in a transparent cage and EEG (electroencephalogram) -EMG (electromyogram) signals and synchronized video were recorded using the Athena system (fig. 31A). Meanwhile, the Racine scale is used for evaluating the epilepsy symptoms of the mice (grade 1: facial clonus including blinking, hair movement, rhythmic chewing and the like; grade 2: grade 1 plus rhythmic nodding; grade 3: grade 2 plus forelimb clonus; grade 4: grade 3 plus hind limb standing; grade 5: grade 4 plus falling) (fig. 31B), and the mice with the KA-induced epilepsy are found to have obvious convulsion behaviors with accompanying convulsionThere was significant forelimb contracture. The epilepsy of the mice treated by the DHA combination is obviously inhibited, and the convulsion symptom is effectively relieved. In addition, EEG-EMG signal monitoring showed that KA-induced epileptic mice produced significant epileptic spikes (31C). DHA pretreatment significantly attenuated epileptic spikes compared to the KA group alone; importantly, DHA post-treatment further suppressed abnormal discharges (fig. 31D-G), consistent with seizure rating tests in mice. To better understand DHA against epilepsy and Fe²⁺Molecular mechanism of regulatory capacity, proteins and RNA were extracted from hippocampal and cortical regions of the brain of each group of mice treated with the same drug as in fig. 31A. Changes in gene expression of a panel of ferritin proteins in brain homogenates were analyzed by RT-PCR (real-time fluorescent quantitative PCR) (fig. 32, fig. 33). A significant increase in LCN2 expression was found in the KA model. LCN2 is a ferroportin and a neurotoxic factor secreted by reactive astrocytes, selectively promoting neuronal death. In KA-induced epileptic brain, transcription of LCN2 was not inhibited by DHA pretreatment, but was significantly inhibited by DHA post-treatment (fig. 34). LCN2 secreted by reactive astrocytes may play an important role in the regulation of iron homeostasis and neuronal damage in epileptic brains. Furthermore, in epileptic mice treated with DHA before and after, the expression level of iron transporter (FPN) was slightly increased, which contributed to active Fe²⁺To output of (c). In conclusion, data of both an isolated hippocampal slice and an in vivo animal model prove the potential antiepileptic activity of DHA, and the application of ferrous ions based on a key iron death marker in screening an antiepileptic regulator is further demonstrated by proving that a new chemical regulator for epilepsy can be screened at high throughput by detecting the content change of ferrous ions by using a FeP probe. Meanwhile, the embodiment shows that the screened inhibitor regulates epileptic diseases and in-vivo proteins and genes related to ferrous expression.

Claims

1. Based on the application of ferrous ions as key markers of iron death in screening anti-epileptic modulators.

2. The use according to claim 1, wherein the ferrous ion is used as a marker in the preparation of a medicament, an agent or a tool for screening an anti-epileptic modulator.

3. The application of the near-infrared fluorescent probe FeP for detecting ferrous ions based on the key iron death marker in preparing the medicines, reagents or tools for screening the anti-epileptic regulating and controlling agent is disclosed, wherein the structure of the near-infrared fluorescent probe FeP is shown as a structural formula I:

4. fe in preparation of epileptic brain by using near-infrared fluorescent probe FeP as defined in claim 3²⁺Use in agents for distributed imaging.

5. The use according to claim 3 or 4, characterized in that the near infrared fluorescent probe FeP for ferrous ions visually traces Fe in nerve cells²⁺Is dynamically changed.

6. The use according to claim 3 or 4, wherein the infrared fluorescent probe FeP can be endogenous to Fe in live epileptic patients²⁺The horizontal change is imaged.

7. The use according to claim 3 or 4, wherein the infrared fluorescent probe FeP can be used for in vivo three-dimensional dynamic two-photon imaging of mice, and can be used for directly observing the occurrence of epilepsy and the Fe in the treatment process²⁺Dynamic variation of flux.

8. The use as claimed in claim 3 or 4, wherein the infrared fluorescent probe FeP is used for 3D imaging in vivo brain to find that the screened antiepileptic regulator is obtained by down-regulating Fe in brain of epileptic patient²⁺The levels achieve an effective therapeutic effect on epilepsy.

9. The use of the near-infrared fluorescent probe FeP as claimed in claim 3 in the preparation of a reagent or a tool for screening out epileptic inhibitors DHA, curcumin, apigenin, quercetin, celecoxib or ibuprofen as a drug for regulating and controlling ferrous ions abnormally expressed in diseases.

10. The use according to claim 9, wherein the near infrared fluorescent probe FeP is preferably used in the preparation of a medicament, agent or tool for screening the influence of epilepsy inhibitor DHA, curcumin, apigenin, quercetin, celecoxib or ibuprofen on the expression level of LCN2/FPN/GPX 4.