阅读说明：本技术 四氧化三铁纳米酶的新用途 (New application of ferroferric oxide nanoenzyme ) 是由 颜丙春 高利增 高满满 许卓斌 王莉 张洁 于 2019-10-30 设计创作，主要内容包括：本发明涉及四氧化三铁纳米酶的新用途,属于西药制药领域,具体涉及聚乙二醇包裹的四氧化三铁纳米酶(PEG-Fe3O4 nanozyme)在促进神经母细胞增殖和分化中的应用。本发明通过日常饮用水中给予小剂量PEG-Fe3O4 nanozyme后能使海马区成神经母细胞分化增加,保护血脑屏障,增强自噬,减少氧自由基水平。由本发明可知,聚乙二醇包裹的四氧化三铁纳米酶(PEG-Fe3O4 nanozyme)从多方面促进神经母细胞分化作用,对神经退行性疾病的治疗有一定价值。(The invention relates to a new application of ferroferric oxide nanoenzyme, belongs to the field of western medicine pharmacy, and particularly relates to an application of polyethylene glycol coated ferroferric oxide nanoenzyme (PEG-Fe3O4 nanozyme) in promoting proliferation and differentiation of neuroblast cells. The invention can increase the differentiation of hippocampal neuroblast cells, protect blood brain barrier, enhance autophagy and reduce the level of oxygen free radicals by giving a small dose of PEG-Fe3O4nanozyme to daily drinking water. According to the invention, the polyethylene glycol-coated ferroferric oxide nanoenzyme (PEG-Fe3O4 nanozyme) has the function of promoting the differentiation of the neuroblast from multiple aspects, and has certain value for treating neurodegenerative diseases.)

1. The new application of the ferroferric oxide nanoenzyme is characterized in that the ferroferric oxide nanoenzyme coated by polyethylene glycol is applied to promoting the proliferation and differentiation of neuroblast cells.

2. The new application of the ferroferric oxide nanoenzyme according to claim 1, wherein the polyethylene glycol coated ferroferric oxide nanoenzyme has a diameter of 200 nm.

3. The new application of the ferroferric oxide nanoenzyme according to claim 1 or 2, wherein after the ferroferric oxide nanoenzyme coated by a small dose of polyethylene glycol is given to daily drinking water, the differentiation of hippocampal neuroblasts is increased, the blood brain barrier is protected, autophagy is enhanced, and the oxygen free radical level is reduced.

Technical Field

The invention belongs to the field of western medicine preparations, relates to a new medicinal application of ferroferric oxide nanoenzyme, and particularly relates to an application of ferroferric oxide nanoenzyme in preparation of a medicine for promoting differentiation of neuroblast cells.

Background

Neural Stem Cells (NSCs) have self-renewal property and multidirectional differentiation potential, and the characteristic of the NSCs is utilized to effectively stimulate and promote the process by improving microenvironment, so that damaged nerve cells are repaired and replaced, and the neural stem cells have important application prospect in prevention and treatment of nervous system diseases. Neuronal differentiation in the subvranular zone (SGZ) of the hippocampal dentate gyrus (Dg) is associated with cerebral cognitive stores and brain plasticity. Reactive oxygen species are considered to be the most important oxidative molecules in cells, including superoxide (O2-), hydroxyl radical (OH-), and hydrogen peroxide (H2O 2). Under physiological conditions, ROS are involved in a variety of biological processes, such as inflammatory responses, apoptosis and autophagy induction, as well as synaptic plasticity, learning and memory. However, when reactive oxygen species are overproduced, these molecules cause oxidative stress, reducing neural differentiation, leading to cognitive and memory disorders.

In the past decades, Fe3O4 nanoparticles have shown wide application prospects in biological technologies such as biosensors, Magnetic Resonance Imaging (MRI) contrast agents, hyperthermia, and radiation delivery. Chinese scientists in 2007 report that Fe3O4 nanoparticles can catalyze 3,3', 5' -Tetramethylbenzidine (TME), O-phenylenediamine (OPD), Diazoaminobenzene (DAB) and other peroxidase substrates to develop color for the first time, and the nanoparticles have catalytic activity similar to natural enzymes. Polyethylene glycol (PEG), a polymer that can improve biosafety, prolong in vivo biological half-life, and increase permeability of nanoparticles in brain by prolonging blood circulation time, we prepared PEG-encapsulated Fe3O4 nanoenzyme by heat absorption method, and determined that the enzyme has peroxidase, catalase, and superoxide dismutase. Through observing the influence of long-term application of Fe3O4 nanoenzyme on D-gal induced mouse hippocampal neuron differentiation and BBB integrity, and observing the levels of antioxidant, autophagy and apoptosis-related proteins after nanoenzyme treatment, the action mechanism of PEG-Fe3O4 nanoenzyme in promoting hippocampal neural differentiation is discussed.

Disclosure of Invention

The invention aims to solve the technical problems, provides a new medicinal application of PEG-Fe3O4nanozyme (ferroferric oxide nanoenzyme wrapped by polyethylene glycol), and particularly relates to a therapeutic application of PEG-Fe3O4nanozyme in a nerve differentiation promoting drug.

The technical scheme of the invention is as follows:

the new application of the ferroferric oxide nanoenzyme is characterized in that the ferroferric oxide nanoenzyme coated by polyethylene glycol is applied to promoting the proliferation and differentiation of neuroblast cells.

Preferably, the diameter of the ferroferric oxide nanoenzyme coated by polyethylene glycol is 200 nm.

Preferably, after a small dose of polyethylene glycol-coated ferroferric oxide nanoenzyme is given to daily drinking water, the differentiation of hippocampal neuroblast cells can be increased, the blood brain barrier is protected, autophagy is enhanced, and the oxygen radical level is reduced.

The invention observes the promoting effect of PEG-Fe3O4nanozyme on mouse hippocampal neural differentiation.

The invention uses immunohistochemistry to observe the influence of PEG-Fe3O4nanozyme on the differentiation of mouse hippocampal neuroblast. The experimental result shows that PEG-Fe3O4nanozyme has the effect of promoting neural differentiation.

The invention uses immunohistochemical staining and Western Blot to observe the influence of PEG-Fe3O4nanozyme on the blood brain barrier of mice. Immunohistochemistry results show that PEG-Fe3O4nanozyme significantly increases the expression of PECAM-1 in the mouse hippocampus; the Western Blot result shows that PEG-Fe3O4nanozyme significantly increases the expression of mouse hippocampal tight junction proteins ZO-1 and claudin-5.

The invention uses a Western Blot method to detect the expression of PEG-Fe3O4nanozyme on mouse hippocampal antioxidant enzyme SOD1, SOD2 and catalase and apoptosis-related proteins Caspase-3and Bcl-2; autophagy-related proteins BECN-1, LC3A/B, ATg7 and AKT/MTOR signaling pathway. The experimental result shows that PEG-Fe3O4nanozyme obviously improves the expression of antioxidant enzyme protein in the brain of a mouse, inhibits an AKT/MTOR signal channel, increases the autophagy level and reduces the apoptosis.

The invention comprehensively clarifies the nerve differentiation promoting effect of PEG-Fe3O4nanozyme by applying multi-system and multi-angle research. The PEG-Fe3O4nanozyme has the promotion effect on neural differentiation, and is related to the fact that the PEG-Fe3O4nanozyme has the activities of resisting oxidases, increasing the level of the resisting oxidases in brain, enhancing autophagy, reducing apoptosis and protecting blood brain barrier.

The invention has the beneficial effects that: the PEG-Fe3O4nanozyme plays a role in promoting neural differentiation through the activity of the antioxidant enzyme, and is expected to provide a basis for the research and development of new neural differentiation medicines.

The invention relates to application of polyethylene glycol coated ferroferric oxide nanoenzyme (PEG-Fe3O4 nanozyme) in promoting proliferation and differentiation of neuroblast cells. According to the invention, the polyethylene glycol-coated ferroferric oxide nanoenzyme (PEG-Fe3O4 nanozyme) has the function of promoting the differentiation of the neuroblast from multiple aspects, and has certain value for treating neurodegenerative diseases.

Drawings

FIG. 1: the effect of PEG-Fe3O4nanozyme on mouse hippocampal neural differentiation; FIGS. 1A-1E are DCX + neuroblasts (X200), FIGS. 1A-1E are the results of the amplification of FIGS. 1A-1E, respectively; n is 7 and P<0.01, compared to a control group;^#P＜0.05，^##P<0.01, compared to a model set; (A) normal mice; (B) d-gal treatment of mice; (C) the method comprises the following steps Adding nano enzyme into normal mice; (D) d-gal treated mice are added with nano enzyme; (E) d-gal treated mice plus Melatonin; d-gal is D-galactose; mel: melatonine; PFe3O4 PEG-Fe3O4 nanozyme; PL is a multi-layer; GCL granular cell layer; SGZ: a lower layer of particles; ML is molecular layer;

FIG. 2: the effect of PEG-Fe3O4nanozyme on mouse hippocampal platelet endothelial cell adhesion molecule-1 (PECAM-1); n is 7 and P<0.01, compared to a control group;^#P＜0.05，^##P<0.01, compared to a model set; (A) normal mice; (B) d-gal treatment of mice; (C) d-gal mice plus melatonin (D): adding nano enzyme into normal mice; (E) d-gal treated mice are added with nano enzyme; d-gal is D-galactose; mel: melatonine; PFe3O4 PEG-Fe3O4 nanozyme;

FIG. 3: the effect of PEG-Fe3O4nanozyme on the expression of blood brain barrier related proteins ZO-1 and claudin-5 in the hippocampal region of a mouse; wherein, the data compares the target protein with the internal reference beta-actin to n 7, P <0.05 and P <0.01, and compares with the control group; # P <0.05, # P <0.01, compared to model group; (A) normal mice; (B) d-gal treatment of mice; (C) d-gal mice plus Melatonin; (D) the method comprises the following steps Adding nano enzyme into normal mice; (E) d-gal treated mice are added with nano enzyme; d-gal is D-galactose; mel: melatonine; PFe3O4 PEG-fe3o4nanobzyme. Physiological saline;

FIG. 4: the effect of PEG-Fe3O4nanozyme on the expression of mouse hippocampal antioxidant enzyme related marker proteins SOD1, SOD2 and catalase; wherein, the data compares the target protein with the internal reference beta-actin to n 7, P <0.05 and P <0.01, and compares with the control group; # P <0.05, # P <0.01, compared to model group; (A) normal mice; (B) d-gal treatment of mice; (C) d-gal mice plus Melatonin; (D) the method comprises the following steps Adding nano enzyme into normal mice; (E) d-gal treated mice are added with nano enzyme; d-gal is D-galactose; mel: melatonine; PFe3O4 PEG-Fe3O4nanozyme. Physiological saline;

FIG. 5: the effect of PEG-Fe3O4nanozyme on the expression of mouse hippocampal apoptosis-related marker proteins Caspase-3and Bcl-2; wherein, the data compares the target protein with the internal reference beta-actin to n 7, P <0.05 and P <0.01, and compares with the control group; # P <0.05, # P <0.01, compared to model group; (A) normal mice; (B) d-gal treatment of mice; (C) d-gal mice plus Melatonin; (D) the method comprises the following steps Adding nano enzyme into normal mice; (E) d-gal treated mice are added with nano enzyme; d-gal is D-galactose; mel: melatonine; PFe3O4 PEG-fe3o4nanobzyme. Physiological saline;

FIG. 6: the effect of PEG-Fe3O4nanozyme on the expression of mouse hippocampal autophagy-associated marker protein Atg7, Beclin-1and LC3 II/I; wherein, the data compares the target protein with the internal reference beta-actin to n 7, P <0.05 and P <0.01, and compares with the control group; # P <0.05, # P <0.01, compared to model group; (A) normal mice; (B) d-gal treatment of mice; (C) d-gal mice plus Melatonin; (D) the method comprises the following steps Adding nano enzyme into normal mice; (E) d-gal treated mice are added with nano enzyme; d-gal is D-galactose; mel: melatonine; PFe3O4 PEG-Fe3O4nanozyme. Physiological saline;

FIG. 7: the effect of PEG-Fe3O4nanozyme on AKT/mTOR signaling pathway in mouse hippocampus; wherein, the data compares the target protein with the internal reference beta-actin to n 7, P <0.05 and P <0.01, and compares with the control group; # P <0.05, # P <0.01, compared to model group; (A) normal mice; (B) d-gal treatment of mice; (C) d-gal mice plus Melatonin; (D) the method comprises the following steps Adding nano enzyme into normal mice; (E) d-gal treated mice are added with nano enzyme; d-gal is D-galactose; mel: melatonine; PFe3O4 PEG-Fe3O4nanozyme. Physiological saline;

FIG. 8 is a schematic diagram of PEG-Fe3O4nanozyme according to the present invention.

Detailed Description

Experimental materials and instruments:

experimental materials:

preparation of PEG-Fe3O4nanozyme

The Fe3O4nano enzyme coated with PEG is prepared by a heat absorption method.

2. Melatonin (Melatonin) was purchased from abcam corporation. The antibody was used as follows: murine anti-PECAM-1 was purchased from Bio-Technology, USA, rabbit anti-Caspase-3, LC3II/I, Atg7, AKT, P-AKT, mTOR, P-mTOR was purchased from CellSignaling Technology, USA; immunohistochemical secondary antibodies Rabbit anti-sheep and sheep anti-mouse, Rabbit anti-ZO-1, Claudin5, SOD1and2 were purchased from abcam, and sheep anti-DCX, BCL-2, BECN-1, Rabbit anti-beta-actin were purchased from Santa Cruz Biotechnology.

An experimental instrument: computer-based microscopes (Nikon Corporation; Tokyo, Japan); super Signal West Pico chemistry subsystem (Thermo Scientific, Rockford, USA)

The source of the mice is: male ICR mice, 8 weeks old, were purchased at the university of yangzhou, comparative medicine center (yangzhou, china).