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Application of ferroptosis-based key marker in high-throughput screening of anti-epileptic regulators

A marker, ferroptosis technology, applied in the field of medicine to achieve the effect of inhibiting neuronal ferroptosis

Pending Publication Date: 2021-12-21
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report that will target Fe 2+ Probe for Fe in the epileptic brain 2+ Perform distribution imaging

Method used

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  • Application of ferroptosis-based key marker in high-throughput screening of anti-epileptic regulators
  • Application of ferroptosis-based key marker in high-throughput screening of anti-epileptic regulators
  • Application of ferroptosis-based key marker in high-throughput screening of anti-epileptic regulators

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Imaging Fe in living neurons using FeP 2+ dynamic fluctuations.

[0065] To further determine whether FeP can track endogenous Fe in living cells 2+ Variations, respectively, with CuSO 4 (100μM, 30min), MnSO 4 (100μM, 30min), FeCl 3 (100μM, 30min), ZnSO 4 (100μM, 30min), lipopolysaccharide (LPS) (1ug / mL, 12h), KA (500μM, 12h), glutamic acid (2mM, 30min), H 2 o 2 (100μM, 1h) or 2,2'-bipyridine (BPY) (1mM, 1h) to incubate the SHSY-5Y cells with a growth confluence of 80% in the well plate, wash with PBS (pH7.4), and then wash with containing The cells were incubated in DMEM medium with 10 μM FeP for 30 minutes before imaging (the medium should completely cover the cells), and the cells were kept at 37° C. during all incubations.

[0066] The result is as figure 1 , with Cu 2+ , Mn 2+ , Fe 3+ and Zn 2+ May lead to more Fe 2+ Dissociated, cells treated with these ions showed higher fluorescence signals than control cells treated with DMSO. Significantly highe...

Embodiment 2

[0068] neuronal Fe 2+ Steady-state regulation.

[0069] Establishment of a high-throughput cellular screening platform to screen for potential compounds modulating iron homeostasis. A high-throughput screening platform was constructed by combining FeP with a high-content assay (HCA). The SHSY-5Y cells with a growth confluence of 80% were mixed with 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 Oxazole, celecoxib, naproxen, quercetin, genistein) in DMEM medium (natural product final concentration 20μM) pre-incubated for 12 hours, washed with PBS (pH7.4), the cells were mixed with FeP (10 μM) in fresh DMEM medium (it is enough that the medium completely covers the cells) and incubate at 37°C for another 30 minutes. Quantification of fluorescenc...

Embodiment 3

[0073] Two-photon imaging of living hippocampal neurons with FeP.

[0074] To further investigate whether DHA can protect neurons from glutamate / KA-induced oxidative stress, FeP two-photon imaging studies were used to directly observe primary neurons extracted from the hippocampal tissue of 18-day-gestational BABL / C fetal mice. Intracellular Fe in metacellular cells under oxidative stress 2+ Steady state change. Live primary hippocampal neuron cells with or without BPY (1mM, 1h), Glu (2mM, 12h), KA (500μM, 12h), DHA (20μM, 12h), first DHA (20μM, 12h) After treatment with KA (500 μM, 12h) or KA (500 μM, 12h) first and then DHA (20 μM, 12h) after stimulation, the cells were washed with PBS, and then the cells were incubated with DMEM medium containing FeP (10 μM) at 37°C for 30 minutes before imaging. min, commercially available nuclear dye Hoechst 33342 and neuronal dye Neuron-Dio for 30 min localized staining of cells. Then image the three dyes separately by two-photon conf...

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Abstract

The invention discloses application of a ferroptosis-based key marker in high-throughput screening of an anti-epileptic regulating agent. The ferrous ions are used as the marker and can be used for preparing drugs, reagents or tools for screening anti-epileptic regulators, such as a near-infrared fluorescent probe FeP for detecting the ferrous ions. The near-infrared excitation two-photon fluorescent probe FeP is utilized to directly image the change of Fe < 2 + > in the brain of an epilepsy mouse living body, high spatial and temporal resolution is achieved, the dynamic change of Fe < 2 + > flux in the epilepsy generation and treatment process can be directly observed by utilizing the in-vivo three-dimensional dynamic two-photon imaging of FeP, a high-throughput screening platform combining FeP and high content analysis is constructed, it is found that a series of anti-epileptic compounds and the like can effectively regulate and control cell iron balance, it is proved that the anti-epileptic compounds have the remarkable anti-epileptic effect through behavior research and electroencephalogram monitoring analysis of an epileptic mouse model, and help is provided for screening drugs for treating epilepsy.

Description

technical field [0001] The invention belongs to the field of medicine, and specifically relates to the application of key markers of ferroptosis in high-throughput screening of anti-epileptic regulators. Background technique [0002] Iron is an essential element of life. It is widely involved in the synthesis of protein cofactors, heme, and iron-sulfur clusters, and then participates in a series of biochemical processes such as nucleotide biosynthesis, cell cycle regulation, oxygen transport, and mitochondrial oxidative respiratory chain. Intracellular iron content is closely monitored by iron regulatory proteins (IRPs), which control the expression of a series of iron-related genes, such as transferrin receptor 1 (TfR1), Lipocalin-2, divalent metal transporter-1 (DMT1 ) and FPN, etc., to maintain the balance of iron input, storage and output. In fact, iron deficiency anemia, one of the most common nutritional deficiencies, is caused by iron deficiency; abnormally high iron...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64A61B5/00
CPCG01N21/6458A61B5/0071A61B5/0042A61B5/4094A61B2503/40A61B2503/42
Inventor 钱勇邵晨雯
Owner NANJING NORMAL UNIVERSITY
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