CN110680831A - New application of ferroferric oxide nanoenzyme - Google Patents

Abstract

The invention relates to a new use of ferric tetroxide nanozyme, belongs to the field of western medicine pharmacy, and particularly relates to the application of polyethylene glycol-wrapped ferric tetroxide nanozyme (PEG-Fe3O4 nanozyme) in promoting the proliferation and differentiation of neuroblasts. The invention can increase the differentiation of neuroblasts in the hippocampus, protect the blood-brain barrier, enhance autophagy, and reduce the level of oxygen free radicals by administering a small dose of PEG-Fe3O4 nanozyme in daily drinking water. It can be seen from the present invention that the polyethylene glycol-coated iron tetroxide nanozyme (PEG-Fe3O4 nanozyme) promotes the differentiation of neuroblasts from various aspects, and has certain value for the treatment of neurodegenerative diseases.

Description

New uses of ferric oxide nanozymes

technical field

The invention belongs to the field of western medicine preparations, relates to a new medicinal use of ferric tetroxide nanometer enzyme, and particularly relates to the application of ferric tetroxide nanometer enzyme in preparing a drug for promoting neuroblast differentiation.

Background technique

Neural stem cells (NSCs) have self-renewal characteristics and multi-directional differentiation potential. Using the characteristics of NSCs, by improving the microenvironment, effectively stimulate and promote this process, repair and replace damaged nerve cells in the prevention and treatment of nervous system diseases. has important application prospects. Neuronal differentiation in the subgranular zone (SGZ) of the dentate gyrus (Dg) of the hippocampus is associated with brain cognitive reserve and brain plasticity. Reactive oxygen species are considered to be the most important oxidative molecules in cells, including superoxide (O2-), hydroxyl radicals (OH-), and hydrogen peroxide (H2O2). 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 can cause oxidative stress, reduce neural differentiation, and lead to cognitive and memory impairments.

Over the past few decades, Fe3O4 nanoparticles have shown broad application prospects in biotechnologies such as biosensors, magnetic resonance imaging (MRI) contrast agents, hyperthermia, and radiation drug delivery. In 2007, Chinese scientists reported for the first time that Fe3O4 nanoparticles can catalyze various peroxides such as 3,3'5,5'-tetramethylbenzidine (TME), o-phenylenediamine (OPD), and diazoaminobenzene (DAB). The enzyme substrate is colored and has catalytic activity similar to that of natural enzymes. Polyethylene glycol (PEG), a polymer that improves biosafety, prolongs biological half-life in vivo, and increases the permeability of nanoparticles in the brain by prolonging blood circulation time, we prepared PEG by thermal absorption The encapsulated Fe3O4 nanozyme is determined to have peroxidase, catalase and superoxide dismutase. By observing the effect of long-term application of Fe3O4 nanozyme on the differentiation and BBB integrity of mouse hippocampal neurons induced by D-gal, and observing the levels of antioxidant, autophagy and apoptosis-related proteins after nanozyme treatment, we explored the Mechanism of PEG-Fe3O4 nanozyme in promoting neural differentiation of hippocampus.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the above-mentioned technical problems, and provides a new medicinal use of PEG-Fe3O4 nanozyme (polyethylene glycol-wrapped iron tetroxide nanozyme), and specifically relates to the use of PEG-Fe3O4 nanozyme in neural differentiation-promoting drugs. therapeutic application.

The technical scheme of the present invention is as follows:

The new application of the ferric oxide nanozyme is characterized by the application of the ferric oxide nanozyme wrapped by polyethylene glycol in promoting the proliferation and differentiation of neuroblasts.

Preferably, the polyethylene glycol-wrapped iron tetroxide nanozyme has a diameter of 200 nm.

Preferably, after administering a small dose of polyethylene glycol-coated ferric oxide nanozyme in daily drinking water, it can increase the differentiation of neuroblasts in the hippocampus, protect the blood-brain barrier, enhance autophagy, and reduce the level of oxygen free radicals .

The invention observes the promoting effect of PEG-Fe3O4 nanozyme on the neural differentiation of mouse hippocampus.

The present invention applies immunohistochemistry to observe the effect of PEG-Fe3O4 nanozyme on the differentiation of mouse hippocampal neuroblasts. The experimental results show that PEG-Fe3O4 nanozyme can promote neural differentiation.

In the present invention, immunohistochemical staining and Western Blot are used to observe the effect of PEG-Fe3O4 nanozyme on the blood-brain barrier of mice. Immunohistochemical results showed that PEG-Fe3O4 nanozyme significantly increased the expression of PECAM-1 in mouse hippocampus; Western Blot results showed that PEG-Fe3O4 nanozyme significantly increased the expression of tight junction proteins ZO-1 and claudin-5 in mouse hippocampus.

In the present invention, Western Blot method is used to detect the expression of antioxidant enzymes SOD1, SOD2 and catalase in mouse hippocampus by PEG-Fe3O4 nanozyme and apoptosis-related proteins Caspase-3 and Bcl-2; autophagy-related proteins BECN-1, LC3A/B , ATg7 and AKT/MTOR signaling pathways. The experimental results showed that PEG-Fe3O4 nanozyme significantly increased the protein expression of antioxidant enzymes in the mouse brain, inhibited the AKT/MTOR signaling pathway, increased the level of autophagy, and reduced cell apoptosis.

The invention uses multi-system and multi-angle research to comprehensively elucidate the neural differentiation-promoting effect of PEG-Fe3O4 nanozyme. It can be seen from the present invention that PEG-Fe3O4 nanozyme has a promoting effect on neural differentiation, which is related to its antioxidase-like activity, increasing the level of antioxidant enzymes in the brain, enhancing autophagy, reducing cell apoptosis, and protecting the blood-brain barrier.

Beneficial effects of the present invention: The PEG-Fe3O4 nanozyme in the present invention promotes neural differentiation through its antioxidant enzyme-like activity, and is expected to provide a basis for the research and development of new drugs for neural differentiation.

The invention relates to the application of polyethylene glycol-wrapped iron tetroxide nanozyme (PEG-Fe3O4 nanozyme) in promoting the proliferation and differentiation of neuroblasts. It can be seen from the present invention that the polyethylene glycol-coated ferric tetroxide nanozyme (PEG-Fe3O4 nanozyme) promotes the differentiation of neuroblasts from various aspects, and has certain value for the treatment of neurodegenerative diseases.

Description of drawings

Figure 1: Effects of PEG-Fe3O4 nanozyme on neural differentiation in mouse hippocampus; Figure 1A-1E are DCX+ neuroblasts (X200), Figures 1a-1e are the enlarged results of Figures 1A-1E; n=7, ** P<0.01, compared with the control group; ^# P<0.05, ^## P<0.01, compared with the model group; (A) normal mice; (B): D-gal treated mice; (C): normal mice Add nanozyme; (D): D-gal treated mice add nanozyme; (E): D-gal treated mice add Melatonin; D-gal: D-galactose; Mel: Melatonin; PFe3O4:PEG -Fe3O4nanozyme; PL: polymorphic layer; GCL: granular cell layer; SGZ: subgranular layer; ML: molecular layer;

Figure 2: The effect of PEG-Fe3O4 nanozyme on platelet endothelial cell adhesion molecule-1 (PECAM-1) in mouse hippocampus; n=7, **P<0.01, compared with the control group; ^# P<0.05, ^## P <0.01, compared with the model group; (A) normal mice; (B): D-gal mice; (C): D-gal mice plus Melatonin (D): normal mice plus nanozyme; (E ): D-gal treated mice plus nanozyme; D-gal: D-galactose; Mel: melatonine; PFe3O4: PEG-Fe3O4 nanozyme;

Figure 3: The effect of PEG-Fe3O4 nanozyme on the expression of blood-brain barrier-related proteins ZO-1 and claudin-5 in the mouse hippocampus; the data is compared with the target protein and the internal reference β-actin, n=7, *P<0.05, **P<0.01, compared with the control group; #P<0.05, ##P<0.01, compared with the model group; (A) normal mice; (B): D-gal treated mice; (C): D -gal mice plus Melatonin; (D): normal mice plus nanozyme; (E): D-gal treated mice plus nanozyme; D-gal: D-galactose; Mel: melatonin; PFe3O4: PEG-Fe3O4nanozyme.Saline: normal saline;

Figure 4: The effect of PEG-Fe3O4 nanozyme on the expression of antioxidant enzyme-related marker proteins SOD1, SOD2 and catalase in the mouse hippocampus; among which, the data is compared with the target protein and the internal reference β-actin, n=7, *P<0.05, **P<0.01, compared with the control group; #P<0.05, ##P<0.01, compared with the model group; (A) normal mice; (B): D-gal treated mice; (C): D -gal mice plus Melatonin; (D): normal mice plus nanozyme; (E): D-gal treated mice plus nanozyme; D-gal: D-galactose; Mel: melatonin; PFe3O4: PEG-Fe3O4 nanozyme.Saline: normal saline;

Figure 5: The effect of PEG-Fe3O4 nanozyme on the expression of apoptosis-related marker proteins Caspase-3 and Bcl-2 in the hippocampus of mice; in which, the data were compared with the target protein and the internal reference β-actin, n=7, *P<0.05,* *P<0.01, compared with the control group; #P<0.05, ##P<0.01, compared with the model group; (A) normal mice; (B): D-gal treated mice; (C): D- gal mice plus Melatonin; (D): normal mice plus nanozyme; (E): D-gal treated mice plus nanozyme; D-gal: D-galactose; Mel: melatonin; PFe3O4:PEG -Fe3O4nanozyme.Saline: normal saline;

Figure 6: The effect of PEG-Fe3O4 nanozyme on the expression of autophagy-related marker proteins Atg7, Beclin-1 and LC3II/I in the mouse hippocampus; the data were compared with the target protein and the internal reference β-actin, n=7, *P<0.05, **P<0.01, compared with the control group; #P<0.05, ##P<0.01, compared with the model group; (A) normal mice; (B): D-gal treated mice; (C): D -gal mice plus Melatonin; (D): normal mice plus nanozyme; (E): D-gal treated mice plus nanozyme; D-gal: D-galactose; Mel: melatonin; PFe3O4: PEG-Fe3O4 nanozyme.Saline: normal saline;

Figure 7: The effect of PEG-Fe3O4 nanozyme on the AKT/mTOR signaling pathway in the hippocampus of mice; among which, the data were compared with the target protein and the internal reference β-actin, n=7, *P<0.05, **P<0.01, compared with the control Group comparison; #P<0.05, ##P<0.01, compared with model group; (A) normal mice; (B): D-gal mice; (C): D-gal mice plus Melatonin; ( D): normal mice plus nanozyme; (E): D-gal treated mice plus nanozyme; D-gal: D-galactose; Mel: melatonine; PFe3O4: PEG-Fe3O4 nanozyme.Saline: physiological brine;

Figure 8 is a schematic diagram of the PEG-Fe3O4 nanozyme in the present invention.

Detailed ways

Experimental materials and instruments:

Experimental Materials:

1. Preparation of PEG-Fe3O4 nanozyme

PEG-encapsulated Fe3O4 nanozymes were prepared by thermal absorption method.

2. Melatonin was purchased from abcam company. Antibodies were used as follows: mouse anti-PECAM-1 was purchased from Bio-techne, USA, rabbit anti-Caspase-3, LC3II/I, Atg7, AKT, P-AKT, mTOR, P-mTOR were purchased from CellSignaling Technology, USA; The secondary antibodies rabbit anti-goat, goat anti-mouse, rabbit anti-ZO-1, Claudin5, SOD1and2 were purchased from abcam company, goat anti-DCX, BCL-2, BECN-1, rabbit anti-β-actin were purchased from Santa Cruz Biotechnology company.

Experimental apparatus: computer-based microscope (Nikon Corporation; Tokyo, Japan); Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, USA)

Mice source: 8-week-old male ICR mice were purchased at the Center for Comparative Medicine of Yangzhou University (Yangzhou, China).

Example 1: Preparation of animal model

The present invention adopts male 8-week-old male ICR mice recognized by the international academic community, weighing 18-22 g, 2-3 months old, purchased from the Comparative Medicine Center of Yangzhou University, and maintaining an appropriate temperature (23° C.) and humidity (60%), 12 hours light/12 hours dark environment, after 1 week of adaptive rearing. The mice in the normal control group ate and drank normally, and the animals were randomly divided into 5 groups (14 mice in each group):

1) control group (0.9% saline), 2) 100 mg/kg D-galactose treatment group (D-gal-group), 3) 50 mg/kg melatonin d-gal group (Mel-gal group), 4 ) 10μg/ml PEG-Fe3O4 nanozyme treatment group (pfe3O4-group); 5) 10μg/ml PEG-Fe3O4 nanozyme+100mg/kg D-gal treatment group (pFe3O4+D-gal group).

D-galactose was injected intraperitoneally at 100 mg/kg, once a day, for a total of 12 weeks to prepare a mouse model. It has been confirmed that melatonine is one of the positive endogenous regulators of neurogenesis, and has an important role in improving memory and promoting neural differentiation in the hippocampus. In our study, we chose melatonine as a positive control, daily Gavage once, 50mg/kg body weight each time, for 4 weeks. The PEG-Fe3O4 nanozyme was administered by drinking Fe3o4 nanozyme (10 μg/ml, diluted with ddh2O) daily for 4 weeks.

Example 2: Protective effect of PEG-Fe3O4 nanozyme on neurogenesis in the hippocampal DG area of mice treated with D-gal

This part adopts the animal model described in Example 1, drinking Fe3o4 nanozyme every day for 4 weeks, deeply anesthetizing the mice 1 hour after the last administration, rapidly stripping the brain tissue, perfusion with 4% paraformaldehyde, dehydrating the brain tissue with gradient sucrose, Frozen sections were performed, and hippocampal brain slices were selected according to the brain atlas for immunohistochemical staining, and the morphology of neuroblasts was observed by electron microscope.

The results are shown in Figure 1: Doublecortin (DCX), a microtubule-associated protein present in migrating neuroblasts, is one of the specific markers of early neurons. The number of DCX+ cells is correlated with neurogenesis. DCX-positive cells were mainly located in the subgranular layer of the dentate gyrus of the hippocampus (Figure 1). In the control group, many DCX+ cell bodies were observed in the subgranular layer (Fig. 1A). DCX immunoreactive positive cells in the D-gal group were much less than those in the control group (Fig. 1B). However, we found that the number of DCX-positive cells was significantly increased in Mel+D-gal and PFe3O4+D-gal groups compared with D-gal (Fig. 1D, 1E). In contrast, DCX immunoreactive positive cells were significantly reduced in the PFe3O4 group compared with the control group (Fig. 1C).

In addition, we classified the dendrites of DCX-immunopositive neurons into three categories according to previously described methods. In the control group, the majority of DCX immunopositive cells had "α"-type dendrites, which were very long and protruded into the molecular layer (ML) of the DG (Fig. 1a). In the D-gal and PFe3O4 groups, the dendrites of many DCX immunoreactive cells were of the "beta" and "gamma" type (Fig. 1b, 1c). However, the dendrites of most DCX-immunopositive cells in Mel-gal and PFe3O4+D-gal were of the "α" and "β" types. (Fig. 1d, 1e)

Example 3: The effect of PEG-Fe3O4 nanozyme on platelet endothelial cell adhesion molecule-1 (PECAM-1) in the DG region of the hippocampus of mice treated with D-gal.

This part adopts the animal model described in Example 1, drinking Fe3O4 nanozyme every day for 4 weeks, deeply anesthetizing the mice 1 hour after the last administration, rapidly exfoliating the brain tissue, perfusion with 4% paraformaldehyde, dehydration with gradient sucrose, and brain tissue. Frozen sections were performed, and hippocampal brain slices were selected according to the brain map for immunohistochemical staining, and the expression of vascular endothelial platelet endothelial cell adhesion molecule-1 (PECAM-1) was observed by electron microscope.

The results are shown in Figure 2: PECAM-1 expression was easily detected in the hippocampal DG region of the control group (Figure 2A). In the D-gal group, we noticed a significant decrease in PECAM-1 immunoreactivity in the DG region of the hippocampus compared with the control group (Fig. 2B). However, PECAM-1 immunoreactivity was significantly higher in the Mel+D-gal and PFe3O4+D-gal groups than in the D-gal group (Fig. 2C, 2E). In the PFe3O4 group, PECAM-1 immunoreactivity was significantly lower than that in the control group.

Example 4: The effect of PEG-Fe3O4 nanozyme on the expression of tight junction proteins ZO-1 and claudin-5 in the hippocampal brain tissue of mice treated with D-gal

In this part, the animal model described in Example 1 was used, and Fe3O4 nanozyme was drank daily for 4 weeks. The mice were deeply anesthetized 1 hour after the last administration, and the hippocampal tissue was rapidly separated. Connexin ZO-1 and claudin-5 were expressed. The tissue was pretreated with a whole protein extraction kit, lysed on ice for 30min, 2500rpm×4℃×15min, and the supernatant was aspirated. 1 μL was taken for BCA protein quantification (Biyuntian, Shanghai), and the remaining supernatant protein was added to 5X loading buffer according to the volume ratio and denatured at 95°C for 5min, and stored at -20°C after aliquoting. According to the quantitative results of BCA, 30 μg of sample/lane was loaded, and the concentration was selected according to the molecular weight of the protein to prepare SDS-PAGE gel for separation by constant pressure (80-120V) gel electrophoresis, and electrotransfer to PVDF membrane (Millipore, USA). 5% (mass percent skim milk powder in TBST (pH 7.4, 10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) was blocked by shaking for 1 h at room temperature, and 5% (mass percent) BSA-TBST was added to the primary antibody: rabbit Anti-ZO-1, Claudin5 (1:1000, abcam), rabbit anti-β-actin (1:1000, Santa Cruz Biotechnology). Incubate overnight at 4°C. Rinse 3 times with TBST for 10 min, add horseradish peroxidase-labeled Antibody: goat anti-rabbit-HRP (1:3000, Santa Cruz, USA), incubated at room temperature for 60 min, added chemiluminescent substrate for color development (Thermo Scientific, Rockford, USA), and analyzed using Quantity One analysis software (Bio-Rad) Quantitative densitometric analysis, the software is used to calculate the ratio of relative optical density (ROD): calibrated ROD in %, the control group is designated as 100%. Performed with the ratio of the gray value of the target protein to the gray value of the respective internal reference β-actin Semi-quantitative analysis (ImageJ.) The above 1:1000 all refer to a 1000-fold dilution.

Figure 3 shows that compared with the control group, the levels of ZO-1 and Claudin-5 proteins in the D-gal group were significantly decreased. Compared with the D-gal group, the protein levels of ZO-1 and Claudin-5 in the Mel+D-gal and PFe3O4+D-gal groups were significantly increased, while the protein levels of ZO-1 and Claudin-5 in the PFe3O4 group were decreased compared with the control group .

Example 5: Effect of PEG-Fe3O4 nanozyme on the content of antioxidant enzyme-labeled protein in D-gal-treated hippocampal brain tissue

This part adopts the animal model described in Example 1, drinking Fe3O4 nanozyme every day for 4 weeks, deeply anesthetizing the mice 1h after the last administration, rapidly separating the hippocampal tissue, adding protein lysis solution, tissue protein extraction, and applying Western blot method (Same as Example 4) The content of antioxidant enzyme-labeled protein in the hippocampus of mice was detected.

The results are shown in Figure 4. Compared with the control group, the levels of SOD1, SOD2 and catalase proteins in the D-gal group were significantly decreased. However, compared with the D-gal group, Mel+D-gal and PFe3O4+D-gal groups were significantly increased; compared with the control group, SOD1, SOD2 and catalase protein levels in PFe3O4 were significantly decreased.

Example 6: Effect of PEG-Fe3O4 nanozyme on apoptosis-related proteins in mouse hippocampal brain tissue.

This part adopts the animal model described in Example 1, drinking Fe3O4 nanozyme every day for 4 weeks, deeply anesthetizing the mice 1h after the last administration, rapidly separating the hippocampal tissue, adding protein lysis solution, tissue protein extraction, and applying Western blot method (Same as Example 4) The content of apoptosis-related proteins in the hippocampus of mice was detected.

The results are shown in Figure 5, in the PFe3O4 group, the Caspase-3 protein level was significantly increased, while the Bcl-2 protein level was greatly decreased.

Example 7: Effects of PEG-Fe3O4 nanozyme on autophagy-related proteins in mouse hippocampal brain tissue.

This part adopts the animal model described in Example 1, drinking Fe3O4 nanozyme every day for 4 weeks, deeply anesthetizing the mice 1h after the last administration, rapidly separating the hippocampal tissue, adding protein lysis solution, tissue protein extraction, and applying Western blot method (Same as Example 4) The content of autophagy-related proteins in the hippocampus of mice was detected.

The results are shown in Figure 6, in the PFe3O4 group, we noticed that the levels of these proteins were significantly reduced compared to the control group.

Example 8: Effect of PEG-Fe3O4 nanozyme on Akt/mTOR signaling pathway in mouse hippocampus.

The Akt/mTOR pathway plays a key role in neural differentiation and is an important pathway that affects the level of autophagy. The results are shown in Figure 7, and there are differences in the ratios of p-Akt/Akt and p-mTOR/mTOR between the control and experimental groups . Compared with the control group, p-Akt and p-mTOR were significantly enhanced in the pFe3O4 group, and autophagy was inhibited.

Claims (3)

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.

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