CN112089731A - Application of nano graphene oxide in treatment of beta amyloid protein Abeta related diseases and test method thereof - Google Patents

Abstract

The invention discloses an application of nano graphene oxide in treatment of beta amyloid protein Abeta related diseases, which is characterized by comprising the following steps: in the treatment of beta amyloid protein A beta related diseases, the used medicament or medicament carrier contains nano graphene oxide. The nano graphene oxide can reduce the content of the amyloid beta A beta and improve the cognitive function of a mouse by inhibiting the generation of the amyloid beta A beta and promoting the clearance of the amyloid beta A beta, and provides a new way for developing the prevention and treatment of diseases related to the amyloid beta A beta. The test method powerfully proves the obvious effect of the application of the nano graphene oxide in the treatment of beta amyloid A beta related diseases.

Description

Application of nano graphene oxide in treatment of beta amyloid protein Abeta related diseases and test method thereof

Technical Field

The invention relates to application of nano graphene oxide in treatment of beta amyloid protein A beta related diseases and a test method thereof.

Background

Amyloid beta (a β) plays an important role in the pathological progression of diseases such as Alzheimer's Disease (AD), post-operative cognitive dysfunction (post-operative cognitive dysfunction), Inclusion-body myoinflammation (Inclusion-body myositis), and amyloidosis-associated Hereditary cerebral hemorrhage (heredary cerebral hemorrhage with amyloidosis). A β is produced by hydrolysis of Amyloid Precursor Protein (APP) by β -and γ -secretases. Specifically, APP internalizes into endosomes, beta-secretase (BACE1) cleaves APP on the endosomes to generate beta-carbon terminal fragments (beta-CTF); the latter is cut by gamma-secretase to generate Abeta; and then transported to lysosomes for degradation. When aberrantly produced or impaired clearance of a β, a β accumulates gradually. In the brain, a β accumulation can trigger pathogenic cascades leading to tau hyperphosphorylation, intracellular neurofibrillary tangles, synaptic dysfunction, neuronal death, and ultimately loss of cognitive function. Currently, in studies on a β, intervention is mainly performed using natural products, compounds, short peptides, antibodies, and the like, to reduce a β accumulation. The regulation mechanism is mainly through inhibiting A beta generation or promoting A beta elimination, etc.; however, since studies for simultaneously inhibiting a β production and promoting a β clearance are few, the effect of current intervention means for reducing a β is limited.

Post-operative cognitive dysfunction (POCD) is a common central nervous system complication after anesthesia and surgery, manifested as a decline in cognitive function. POCD is found in elderly patients and often manifests as impaired attention, memory and learning. POCD not only causes serious economic problems and mental stress to patients and their families, but also greatly increases the mortality of patients. The mechanisms of POCD are complex, including accumulation of beta amyloid (a β), hyperphosphorylation of tau protein, inflammation, impaired synaptic function, and inhibition of central cholinergic transmission. Among them, the accumulation of a β is the most widely studied. There is increasing evidence that anesthesia and surgery can increase mouse brain a β levels, leading to a decrease in cognitive function; reduction of a β with drugs can alleviate cognitive impairment in post-operative aged mice. These studies indicate that a β accumulation plays a major role in the development of POCD, and down-regulation of a β may be an effective method for treating POCD.

Graphene is a two-dimensional structure carbon-based material with single atom thickness, has unique chemical, electrical and physical properties, and is a hot spot for research in the biomedical field. Compared with a graphene structure, the graphene oxide introduces hydroxyl, carboxyl and epoxy groups, so that the dispersibility, hydrophilicity and compatibility with polymers and the like of the graphene oxide are improved, and the surface chemical activity of the graphene oxide is enriched. In addition, the graphene oxide has large specific surface area, good hydrophilicity and low toxicity, so that the graphene oxide has wide application prospects in the fields of biological imaging, intracellular probes, promotion of cell growth and differentiation, gene and drug transportation, photothermal therapy and the like. However, whether the nano graphene oxide can reduce A beta and what the mechanism is, and whether the post-operation cognitive dysfunction POCD can be improved is not reported.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an application of nano graphene oxide in treatment of beta amyloid A beta related diseases, wherein the nano graphene oxide can reduce the content of beta amyloid A beta by inhibiting the generation of the beta amyloid A beta and promoting the clearance of the beta amyloid A beta, improve the cognitive function of mice and provide a new way for developing prevention and treatment of the beta amyloid A beta related diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an application of nano graphene oxide in treatment of beta amyloid protein A beta related diseases, which comprises the following steps: in the treatment of beta amyloid protein A beta related diseases, the used medicament or medicament carrier contains nano graphene oxide.

As a further improvement of the invention: in the treatment of beta amyloid a beta related diseases, the route of administration of the drug is injection.

As a further improvement of the invention: in the medicine or the medicine carrier, the concentration of the nano graphene oxide in the formed injection dosage form is less than or equal to 10 mg/mL.

The invention also provides a test method for the application of the nano graphene oxide in the treatment of beta amyloid protein A beta related diseases, which comprises the following steps:

test materials and methods

1) Synthesis of nano graphene oxide GO

2) Cell culture and establishment of APP overexpression cell lines

HKE293T and SHSY5Y cells in Dulbecco's modified Eagle Medium supplemented with 10% fetal bovine serum in the presence of 5% CO₂Culturing at 37 deg.C;

the pLenti-CMV-APP-GFP-Puro plasmid or the empty vector plasmid was transfected into 293T cells with pMD2.G and pPsAX2.0 plasmids using Lipofectamine3000 to produce lentiviruses; after 2 days, SHSY5Y cells were infected with filtered lentivirus-containing medium; then the infected cells are screened for 4 days by 1 mu g/mL puromycin;

3) western Blot

Lysing the cells with sample buffer and boiling for 10 minutes; proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes; incubating the membrane with a primary antibody overnight at 4 ℃, and after washing, incubating the membrane with a secondary antibody for 1 hour at 37 ℃; after washing, the membrane was incubated with the substrate chemiluminescent ECL reagent and observed using a chemiluminescent instrument; preparation of amyloid precursor protein APP 1: 1000 antibodies for the calibration of APP proteins; beta actin beta-actin 1: 1000 antibody, used as internal reference; horseradish peroxidase-conjugated secondary antibody 1: 10000 for calibrating primary antibody;

4) enzyme-linked immunosorbent ELISA assay

Homogenating the hippocampus with guanidine hydrochloride extraction buffer, detecting the total β amyloid a β 42 and β amyloid a β 40 concentration in cultured cells or hippocampal tissue homogenates by ELISA kit according to the manufacturer's instructions;

second, observation of experimental results

Experiment 1: characterization of nano graphene oxide GO

Experiment 2: nano graphene oxide GO (graphene oxide) for reducing beta amyloid protein Abeta in cells

To assess the effect of GO on β amyloid a β levels, HEK293T-APP-GFP and SHSY5Y-APP-GFP cell lines were established; western Blot and ELISA results were observed.

As a further improvement of the invention: the synthesis process of the nano graphene oxide GO is as follows:

firstly, 1g of graphite powder and sodium nitrate NaNO31g are added into cooled concentrated sulfuric acid H2SO423mL, and the mixture is stirred in an ice bath for 30 minutes; then gradually adding potassium permanganate KMnO under stirring and cooling₄3g, carrying out ultrasonic treatment at 40kHz for 5 hours to obtain a uniform liquid suspension, peeling off graphene oxide, gradually adding deionized water, adding 46mL within 15min as a standard, and further adding deionized water; stirring for 10 minutes; by adding 140mL of deionized water and H hydrogen peroxide₂O₂The reaction was stopped with 30% aqueous solution and 10 mL; separating the solid product by centrifugation at 1500rpm for 10 min;

finally, by washing 5 times the suspension suspended in 5% HCl solution in hydrochloric acid, then twice with deionized water, followed by drying; ultrasonic preparation was carried out in an ice bath at 380W for 2 hours.

As a further improvement of the invention: the operation content is specifically as follows:

anesthetizing the mice with 2.5-3.5% sevoflurane; performing skin incision below the knee, exposing the tibia, and inserting a 0.3mm pin into a medullary cavity of the tibia to realize intramedullary fixation; next, fracture at the midpoint using surgical scissors; finally, the wound is closed after the necessary debridement; to relieve pain, a 1.5-2.5% lidocaine solution was applied topically to the wound twice daily with 0.5-1.5% tetracaine hcl mucus prior to incision.

As a further improvement of the invention: the operation content is specifically as follows:

mice were anesthetized with 3.0% sevoflurane; performing skin incision below the knee, exposing the tibia, and inserting a 0.3mm pin into a medullary cavity of the tibia to realize intramedullary fixation; next, fracture at the midpoint using surgical scissors; finally, the wound is closed after the necessary debridement; to alleviate pain, a 2% lidocaine solution was applied topically prior to incision and 1% tetracaine hcl mucus was applied to the wound twice daily.

As a further improvement of the invention: step 8), treating the mice, and injecting the mice by using a microinjector with a fixed needle.

As a further improvement of the invention:

step one, test materials and methods

5) Immunofluorescence

Cells were fixed with 4% paraformaldehyde for 10min, permeabilized with 0.1% triton X-100 for 10min, and blocked with 1% bovine serum albumin BSA for 1 h; cells were incubated with primary antibody overnight at 4 ℃ and labeled with secondary antibody for 1 hour at 37 ℃, images were collected using a confocal fluorescence microscope, and to prepare frozen sections, mice were anesthetized with sodium pentobarbital and perfused with phosphate buffered saline PBS or with 4% paraformaldehyde; after perfusion, the brains were harvested and fixed in fixative overnight, then dehydrated overnight with 30% sucrose solution, then brain tissue was embedded with OCT cryo-section embedding medium and stored frozen at-80 ℃ or cut into 10 μm sections for immunofluorescence manipulation, and the sections were observed under confocal fluorescence microscopy; preparation of beta-secretase 1BACE 11: 200, 3C1C₃(ii) a 1: 200, ab183612 primary antibody, for detecting the localization of BACE1 protein and the binding to GO; early endosomal antigen 1EEA 11: 200, ab2900 primary antibody and Ras-associated protein 7Rab 71: 200, ab50533 primary antibody for labeling early and late endosomes; lysosomal associated membrane protein 1LAMP 11: 100, sc-20011 primary antibody for labeling lysosomes; alexa Fluor 5681: 500 and Alexa Fluor 6471: 500 secondary antibody for fluorescent labeling;

6) determination of protein-nanoparticle interaction

BGO is bovine serum albumin BSA modified GO, 10 μ gGO or BGO was added to 0.6mg/mL purified APP or BACE1 protein, incubated at 4 ℃ for 2 hours, and centrifuged at 14000g for 30 minutes; the supernatant was stored and the pellet was washed 3 times with PBS containing 3% tween 20; boiling the protein solution, the supernatant and the precipitate in SDS loading buffer for 10 minutes, and then carrying out SDS-PAGE and Western blot containing a specified antibody; to prepare BGO, 20mgBSA and 10mgGO were mixed and sonicated for 1 h; the suspension was then centrifuged at 14000g for 2h to remove excess BSA; the precipitate was collected and resuspended in water for 15 minutes by sonication;

7) an animal

10 months old female C57BL/6 mice were purchased; all animals were kept in a temperature, humidity and light controlled chamber and water was provided ad libitum;

8) mouse treatment

Mice were anesthetized with 3.0% sevoflurane; mouse body temperature was maintained at 37 ℃; with reference to bregma, in coordinates: bilateral intracerebral injection is carried out at the position of 2.18mm at the back, 2.30mm at the side and 2.10mm at the abdomen; injecting 2 mu L of graphene oxide or BGO with the concentration of 10mg/mL or performing intracerebral injection by using physiological saline; for chloroquine CQ, mice were injected intraperitoneally with 60 mg/kgCQ;

9) surgical model

Performing intramedullary fixation of tibial fracture under sevoflurane anesthesia on mice after GO, BGO or CQ administration;

10) conditioned fear test

The test comprises a training stage before operation and a scene test stage 1 day after operation; one day before surgery, fear conditioning training was performed on the mice to establish long-term memory; placing each mouse in a test box for 120s, then performing 6 pairs of conditional-unconditional stimulation, and then staying in the test box for 60 s;

1 pair of conditional-unconditional stimuli includes 20s, 70dB sine wave sound as conditional stimulus, up and down interval of 25s and last 2s, 0.70mA electrical stimulus as unconditional stimulus; paired conditional-unconditional stimuli were separated by a random interval of 60 s; contextual fear memory reflects hippocampal-dependent memory; in the context test, the mice were simply placed back in the test chamber for 5min without tone or stimulation; the percentage of stagnation time is defined as the time that the mouse does not move except for breathing, and is recorded and counted by a Panlab conditioned fear and startle reflex combined system and Packwin2.0 software;

step two, experimental observation also comprises:

experiment 3: mechanism for reducing amyloid beta (Abeta) by graphene oxide

Experiment 4: whether graphene oxide can promote lysosomal degradation of beta amyloid protein Abeta

Experiment 5: observation of nano graphene oxide GO for reducing beta amyloid Abeta in mice hippocampus after surgery

Using a mouse model: inducing the up-regulation of the beta amyloid A beta content of the hippocampal tissue after the operation of the intramedullary fixation of the tibial fracture under the anesthesia of the sevoflurane; observing whether GO intervention can lower the content of beta amyloid A beta in the hippocampus of the mouse;

experiment 6: observation of nano graphene oxide GO for improving cognitive function status of mice after operation

The cognitive function of the mice after the fracture surgery is tested by adopting fear conditions, and whether the behavior of the mice after the surgery is influenced by reducing the content of the beta amyloid protein A beta by GO is evaluated.

The application of the nano graphene oxide in the treatment of beta amyloid protein Abeta related diseases provided by the invention has the following beneficial effects of patent law significance:

1. the nano graphene oxide is synthesized, and the nano graphene oxide is found to reduce beta amyloid protein A beta in the brain of a mouse in cells and after an operation for the first time. In particular, the nano graphene oxide can reduce the intracellular Abeta content of SHSY5Y-APP-GFP and HEK-APP-GFP; the hippocampus is injected with the nano graphene oxide, so that the content of beta amyloid A beta in hippocampus tissues of mice receiving tibia fracture intramedullary fixation operation can be reduced.

2. The nano graphene oxide can improve the cognitive function of the mice after operation for the first time; in particular to a method for improving the cognitive function of a mouse subjected to tibia fracture intramedullary fixation by injecting nano graphene oxide into a hippocampus.

3. Pioneering suggestion that inhibition of beta-shear is one of the mechanisms of nano graphene oxide to reduce beta amyloid a beta.

4. Pioneering suggestion that promotion of lysosomal degradation of β amyloid a β is another mechanism by which nano-graphene oxide reduces a β.

Drawings

FIG. 1 is a diagram: characterization diagram of nano graphene oxide

Wherein, in fig. 1: FIG. A is an atomic force microscope image of graphene oxide; FIG. B is a Fourier infrared spectroscopy analysis; FIG. C is a dynamic light scattering diagram; fig. D is an ultraviolet-visible spectrum analysis chart.

FIG. 2 is a diagram of: nano graphene oxide for reducing beta amyloid protein Abeta in cells

Wherein, the graphs A and B are the content of APP and beta-CTF in the cells detected by Western Blot; FIGS. C and D are statistical plots of the relative amounts of APP and β -CTF in cells; FIGS. E and F are graphs in which the levels of beta amyloid A beta 42 and beta amyloid A beta 40 in SHSY5Y-APP cells were measured using ELISA; graphs G and H are for HEK-APP intracellular β amyloid a β 42 and β amyloid a β 40 levels measured using ELISA.

FIG. 3 is a diagram of: inhibition of beta-shear is one of the mechanisms of nano graphene oxide to reduce beta amyloid A beta

Wherein, the graph A is that Western Blot is used for detecting the content of APP and beta-CTF in cells after destroying the functions of lysosomes; panels B and C are statistics of APP and β -CTF content, respectively, in cells after disruption of lysosomal function; panel D is a plot of fluorescence following immunostaining for EEA1, BACE1, and APP in cells; panel E shows the detection of APP and BACE1 content in total protein, in supernatant and in pellet layer using Western Blot; panel F shows the detection of the amount of beta-CTF in cells using Western Blot; FIG. G is a statistical graph of the relative amount of β -CTF in cells; panels H and I are graphs depicting the detection of intracellular β amyloid a β 42 and β amyloid a β 40 levels using ELISA.

FIG. 4 is a diagram of: nano graphene oxide for enhancing degradation of beta amyloid protein Abeta by lysosome

Wherein, the graphs A and B are used for detecting and counting the content of the beta-CTF in the cells without affected beta-shear by using Western Blot; panels C and D are graphs showing the detection and statistics of beta amyloid a β 42 and beta amyloid a β 40 levels in cells unaffected by beta-shear using ELISA; panels E and F are for the detection and statistics of intracellular a β 42 and a β 40 content using ELISA; panel G is a fluorescent image of immunostaining Rab7 and LAMP 1; panel H vs Rab7 and LAMP 1.

FIG. 5 is a diagram: nano graphene oxide for reducing beta amyloid protein Abeta in mouse hippocampus after operation

Wherein panel A is a fluorescence plot of APP and BACE1 immunostaining in mouse hippocampus after surgery; panel B is a fluorescent image of APP and LAMP1 immunostained in the mouse hippocampus post-surgery; the graphs C and D are the detection and statistics of the content of the beta-CTF in the postoperative mouse hippocampus by using Western Blot; and the graphs E and F are used for detecting and counting the content of beta amyloid protein A beta 42 and beta amyloid protein A beta 40 in the postoperative mouse hippocampus by adopting ELISA.

FIG. 6 is a diagram of: nano graphene oxide for improving cognitive function of mice after operation

Wherein, the graph A is a representative graph of the detection of the motion amplitude of the mice in the conditioned fear detection box after the operation; panel B is the percentage of time to fear in mice after surgery as measured by the conditional fear experiment.

Detailed Description

The application of the nano graphene oxide in the treatment of beta amyloid a β related diseases provided by the present invention is further described in more detail by the following specific examples:

the abbreviation A beta amyloid protein A beta.

Experimental materials and methods:

1) synthesis of nano graphene oxide

Graphene oxide was prepared from purified graphite (purchased from Sigma-Aldrich) by a modified Hummers method. First, graphite powder (1g) and NaNO were mixed₃(1g) Addition to cooled concentrated H₂SO₄(23mL), and the mixture was stirred in an ice bath for 30 minutes. KMnO4(3g) was then added gradually with stirring and cooling to obtain a homogeneous liquid suspension after 5h of sonication at 40kHz to exfoliate the graphene oxide, followed by gradual addition of deionized water (46 mL over 15 min) and then further deionized water. Stirred for 10 minutes. By adding deionized water (140mL) and H₂O₂The reaction was stopped with aqueous solution (30%, 10 mL). The solid product was isolated by centrifugation (1500rpm, 10 min). Finally, the suspension suspended in HCl solution (5%) was washed 5 times, then twice with deionized water, and then dehydrated. Preparation by means of tip sonication (Misonix Sonicator3000) in an ice bath at a power of 380W for 2 hours, followed by injection through 0.45 μmFiltration was performed with a filter of the vessel type (Millipore).

2) Cell culture and establishment of APP overexpression cell lines

HKE293T and SHSY5Y cells in Dulbecco's modified Eagle Medium supplemented with 10% Fetal Bovine Serum (FBS) in 5% CO₂The culture was carried out at 37 ℃.

Transfection of pLenti-CMV-APP-GFP-Puro or empty vector with pMD2.G and pPsAX2.0 into 293T cells using Lipofectamine3000 to produce lentivirus; after 2 days, SHSY5Y cells were infected with filtered lentivirus-containing medium; then the infected cells are screened for 4 days by 1 mu g/mL puromycin; the experimental results are shown in fig. 2, 3 and 4.

3) Immunoblotting (Western Blot)

Cells were lysed with sample buffer and boiled for 10 min. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membrane was incubated with the primary antibody overnight at 4 ℃ and after washing, the membrane was incubated with the secondary antibody for 1 hour at 37 ℃. After washing, the membranes were incubated with ECL reagents and observed using a chemiluminescence instrument (ImageQuantLAS4000, GE Healthcare). APP (1: 1000) antibody for the calibration of APP protein; beta-actin (1: 1000) antibody, used as an internal reference; horseradish peroxidase conjugated secondary antibody (1: 10000) was used to calibrate the primary antibody. The results are shown in FIGS. 2A-B, 3A, 3E-F, 4A-B, and 5C.

4) Enzyme-linked immunosorbent assay

Hippocampus was homogenized with guanidine hydrochloride extraction buffer. The total Ab42 and Ab40 concentrations in cultured cells or in hippocampal homogenates were detected by ELISA kits (purchased from cubabio) according to the manufacturer's instructions. The results are shown in FIGS. 2E-H, FIGS. 3H-I, FIGS. 4C-F, and FIGS. 5E-F.

5) Immunofluorescence

Cells were fixed with 4% paraformaldehyde for 10min, permeabilized with 0.1% TritonX-100 for 10min, and blocked with 1% BSA for 1 h. Cells were incubated with primary antibody overnight at 4 ℃ and labeled with secondary antibody for 1 hour at 37 ℃. Images were acquired using a confocal fluorescence microscope (LSM800, zeiss). To prepare frozen sections, mice were anesthetized with sodium pentobarbital and perfused with PBS or with 4% paraformaldehyde. After perfusion, brains were harvested and fixed in fixative overnight, then dehydrated overnight with 30% sucrose solution. Thereafter, brain tissue was embedded using tissue tek OCT and either-80 ℃ cryopreserved or cut into 10 μm sections for immunofluorescence procedures. The sections were observed under a confocal fluorescence microscope (LSM800, zeiss). BACE1 (1: 200, 3C1C 3; 1: 200, ab183612) primary antibody, used for detecting the localization of BACE1 protein and the binding with GO; EEA1 (1: 200, ab2900) primary antibody and Rab7 (1: 200, ab50533) primary antibody for labeling early and late endosomes; LAMP1 (1: 100, sc-20011) primary antibody for labeling lysosome; alexa Fluor 568 (1: 500) and Alexa Fluor 647 (1: 500) secondary antibodies were used for fluorescent labeling. The results of the experiments are shown in FIG. 3D, FIG. 4G, FIG. 4I, FIGS. 5A-B.

6) Determination of protein-nanoparticle interaction

Add 10 μ g Graphene Oxide (GO) or BGO (BSA modified graphene oxide, containing 10 μ gGO) to 0.6mg/mL purified APP or BACE1 protein purchased from Sino Biological, incubate for 2 hours at 4 ℃ and centrifuge for 30 minutes at 14000 g; the supernatant was stored and the pellet was washed 3 times with PBS containing 3% tween 20; the protein solution, supernatant and pellet were boiled in SDS loading buffer for 10 minutes, and then subjected to SDS-PAGE and Western blot containing the designated antibody, and for the preparation of BGO, 20mg BSA and 10mg GO were mixed and sonicated for 1 h. The suspension was then centrifuged at 14000g for 2h to remove excess BSA. The pellet was collected and resuspended in water for 15 minutes by sonication. The results of the experiment are shown in FIG. 3E.

7) An animal

Female C57BL/6 mice, 10 months of age, were purchased from the university of Nanjing, model animal research center. All animals were kept in a temperature, humidity and light controlled chamber and provided water ad libitum. Animal welfare and experimental procedures were performed according to the ethical code for the care and use of laboratory animals at the university of medical, Anhui, and approved by the animal experimentation Committee of school. The results of the experiment are shown in FIGS. 5 and 6.

8) Mouse treatment

Mice were anesthetized with 3.0% sevoflurane. The mouse body temperature was maintained at 37 ℃. With reference to bregma, in coordinates: at the position of the back 2.18mm, the side 2.30mm and the abdomen 2.10mm, 2 mu L of graphene oxide (10mg/mL) or BGO (graphene oxide concentration is 10mg/mL) is injected by a micro-injector with a fixed needle for bilateral intracerebral injection or intracerebral injection is carried out by physiological saline. For Chloroquine (CQ), mice were injected intraperitoneally with 60 mg/kgCQ. The results of the experiment are shown in FIGS. 5 and 6.

9) Surgical model

Following GO, BGO or CQ administration, mice were subjected to intramedullary fixation of tibial fractures under sevoflurane anesthesia. Briefly, mice were anesthetized with 3.0% sevoflurane. A skin incision was made below the knee, the tibia was exposed, and a 0.3mm pin was inserted into its medullary cavity to achieve intramedullary fixation. Next, the fracture is made at the midpoint using surgical scissors. Finally, the wound is closed after the necessary debridement has taken place. To alleviate pain, a 2% lidocaine solution was applied topically prior to incision and 1% tetracaine hcl mucus was applied to the wound twice daily. The results of the experiment are shown in FIGS. 5 and 6.

10) Conditioned fear test

The test included a pre-operative training phase and a post-operative 1 day testing phase. One day prior to surgery, mice were conditioned for fear to establish long-term memory. Each mouse was placed in the conditioning chamber for 120s, then placed for 6 pairs of conditioned-unconditioned stimuli, and then left in the test chamber for 60 s. The 1 pair of conditional-unconditional stimuli included a 20s, 70dB sinusoidal sound (conditional stimulus), an up-down interval of 25s and a final 2s, 0.70mA shock (unconditional stimulus). Paired conditional-unconditional stimuli were separated by a random interval of 60 s. Contextual fear memory reflects hippocampal-dependent memory. In the context test, the mice were simply placed back in the test chamber for 5min without tone or stimulation. Percent time to arrest (defined as the time the mouse has no movement except for breathing) was recorded and counted by the Panlab conditioned fear & startle reflex (startle) combined system and the packwin2.0 software. The results of the experiment are shown in FIG. 6.

Experiment 1: characterization of Nano graphene oxide

Graphene Oxide (GO) was prepared from natural graphite using a modified Hummers method. Atomic Force Microscopy (AFM) results indicated that the GO nanoplates had thicknesses of about 0.8-1.0nm (FIG. 1A). The surface states of GO were characterized by Fourier Transform Infrared (FTIR) spectroscopy. As shown in FIG. 1B, the absorption band near 1743cm-1 is due to carboxyl groups, while the absorption bands near 3431cm-1 and 1604cm-1 are due to hydroxyl stretching and deformation vibrations, indicating that the GO surface contains various chemical functional groups. Consistent with the AFM data, Dynamic Light Scattering (DLS) analysis showed water in deionized water and a radius size of about 200nm (fig. 1C), and ultraviolet-visible (UV-VIS) spectroscopy showed a broad light absorption peak at 230nm (fig. 1D).

Experiment 2: nano graphene oxide for reducing beta amyloid protein Abeta in cells

To assess the effect of GO on A β levels, HEK293T-APP-GFP and SHSY5Y-APP-GFP cells commonly used for A β mechanism studies were established. The Western Blot results showed no significant difference in APP content in GO treated cells, but reduced levels of β -CTF (FIGS. 2A-D). In the cells, the total content of a β in soluble and insoluble form was further examined; the results show that GO significantly reduced the levels of A β 42 and A β 40 in SHSY5Y and HEK293T cells (FIGS. 2E-H).

Experiment 3: inhibition of beta-shear is one of the mechanisms by which graphene oxide reduces beta amyloid A beta

GO induces a decrease in intracellular Α β levels, probably due to decreased Α β production or accelerated Α β degradation. First, this experiment tested the production of a β. It is well known that β amyloid a β is produced by the proteolytic pathway of APP, where in the amyloidogenic pathway APP is internalized into early endosomes and cleaved by BACE1 to form β -CTF. Then, β -CTF is cleaved by γ -secretase to produce a β. Thus, β -cleavage of APP is the rate-limiting step in a β production. Reduction of β -CTF in GO-treated cells was previously observed in this experiment (FIGS. 2A-B). Considering that β -CTF may be degraded by lysosomes, CQ was further used to disrupt the lysosomal degradation function. Under CQ treatment, GO still decreased β -CTF but increased APP levels (FIGS. 3A-C). These results indicate that β -splicing of APP is inhibited in GO-treated cells. BACE1 mediates beta-cleavage of APP; however, BACE1 expression levels did not change significantly after GO treatment. Therefore, this experiment further investigated the location of BACE1 in SHSY5Y-APP-GFP cells. EEA1 is a marker for early endosomes. It was found that BACE1 was co-localized with APP/β -CTF and early endosomes in control cells (FIG. 3D). Further observation revealed that co-localization of BACE1 and APP/β -CTF was reduced in GO-treated cells (FIG. 3D), confirming that BACE1 inhibits β -cleavage of APP. It is generally reported that nanomaterials can induce cells to phagocytose them and form endosomes. A large number of endosomes were consistently observed in GO-treated cells (fig. 3D). Furthermore, since previous studies demonstrated that GO can interact with Α β, it was speculated that GO may also interact with its precursor protein APP, resulting in co-localization of most endosomes with APP, but rarely with BACE1 in GO-treated cells. Indeed, in the interaction assay experiments of proteins and nanoparticles, most APP was detected in GO precipitates, whereas BACE1 was detected only in the supernatant, but not in GO precipitates, indicating that GO interacts with APP (fig. 3E). Bovine Serum Albumin (BSA) may block GO affinity; thus, the assay constructed BSA modified go (bgo). Western blot results showed that APP was barely detectable in BGO precipitates; instead, it was enriched into the supernatant (fig. 3E). These results demonstrate that BSA modification prevents the interaction between GO and APP. In addition, it was further observed that BACE1 and APP/β -CTF were co-localized in BGO-treated cells, similar to control cells (fig. 3D), indicating that disruption of co-localization of APP and BACE1 was caused by the interaction of GO and APP. Thus, co-localization of APP and BACE1 was disrupted, blocking β -cleavage of APP, resulting in decreased β -CTF in GO-treated cells (FIG. 3G) and decreased A β production (FIG. 3H-I). However, BSA modification restored co-localization of APP and BACE1 and significantly increased A β levels (FIGS. 3D and H-I), suggesting that inhibition of β -cleavage is one of the mechanisms by GO for A β reduction.

Experiment 4: nano graphene oxide for promoting lysosome to degrade beta amyloid protein Abeta

Although BSA modification significantly inhibited GO from reducing levels of β -CTF and a β, levels of β -CTF and a β in BGO-treated cells were still significantly lower than in BSA-treated cells (fig. 3F-I). These results indicate that there may be other mechanisms for GO to reduce Α β. As described above, in addition to reducing a β production, acceleration of a β degradation can also reduce the intracellular a β content, and thus it was decided to test it. Early endosomes mature into late endosomes and fuse with lysosomes to degrade phagosomes such as APP, β -CTF and a β. BGO was used to exclude intervention of β -splicing and CQ was used to disrupt lysosomal degradation. The effect of BGO on β -CTF, a β 42 and a β 40 levels with or without CQ intervention was then further compared. Experiments found that BGO decreased β -CTF, a β 42 and a β 40 under CQ treatment (fig. 4A-D), indicating that lysosomal degradation is involved in GO decreasing a β effects. Furthermore, GO was further detected to significantly reduce the levels of β -CTF, a β 42 and a β 40, but CQ treatment attenuated the above effect (4E-F), suggesting that the effect of lysosomal degradation of a β was enhanced by GO. Notably, inhibition of β -scission also contributed to GO reduction of Α β 42 and Α β 40 in CQ treated cells. Next, to further confirm that GO can enhance Α β degradation, the abundance of APP/β -CTF in late endosomes and lysosomes was observed. Rab7 is a marker protein for late endosomes. As shown in FIG. 4G, a large number of APP/β -CTFs were localized in Rab 7-labeled late endosomes. GO significantly increased co-localization of Rab7 and LAMP1 (fig. 4G-H), suggesting that GO treatment can enhance endosomal cargo delivery to lysosomes. Furthermore, fewer APP/β -CTFs were observed in the lysosomes of GO-treated cells than control cells (fig. 4I), suggesting that GO enhances Α β degradation. In addition, additional CQ treatment decreased a β delivery and lysosomal degradation (fig. 4G-I). Collectively, these results indicate that GO promotes lysosomal degradation of a β.

Experiment 5: graphene oxide for reducing beta amyloid Abeta in mouse hippocampus after surgery

Intramedullary fixation of tibial fractures under sevoflurane anesthesia is commonly used to induce post-operative cognitive dysfunction (POCD) in rodents, and this mouse model was used in studies. In this model, a significant increase in hippocampal APP/β -CTF levels was observed in mice post-surgery compared to sham-operated mice (FIGS. 5A-B). In addition, Western Blot and ELISA results showed that surgery and anesthesia significantly increased hippocampal levels of β -CTF, A β 42 and A β 40 (FIGS. 5C-F). These results demonstrate the induction of acute a β increase in post-operative mice. In addition, it was further observed that GO administration reduced co-localization of APP/β -CTF and BACE1 in the hippocampus of mice after surgery and reduced lysosomal APP/β -CTF. However, the above changes can be eliminated by BSA modification of GO, and additional CQ treatment, respectively (fig. 5A-B). These results indicate that GO inhibits β -splicing and enhances lysosomal degradation of APP in vivo. Thus, GO administration inhibited surgical increases in β -CTF, A β 42 and A β 40 levels in the hippocampus of mice, while BSA modification and additional CQ treatment significantly reversed GO effects (FIGS. 5C-F).

Experiment 6: graphene oxide for improving cognitive function of mice after operation

It was evaluated whether GO reduced beta amyloid a β levels would affect the behavior of mice after surgery. Fear Conditioning Tests (FCTs) are commonly used to test cognitive function in mice following fracture surgery. FCT includes a pre-operative training phase and a post-operative 1 day testing phase. During the training phase, mice were stimulated with a 70dB sine wave tone and a 0.70mA foot click to establish long-term memory. During training and testing, the change in gravity of the mice on the deck was recorded as the amplitude of movement. When the fear memory of the mice was evoked, they exhibited a freezing behavior and almost no change in gravity was recorded. If the cognitive function of the mouse is impaired, fear memory is not elicited and the mouse is active and a continuously higher amplitude of movement can be detected. As shown in fig. 6A, the surgically treated mice were more active than the sham operated mice, indicating impaired fear memory. GO treated mice exhibited longer freezing behavior compared to surgery treated mice and other groups of mice (fig. 6A). Furthermore, statistical results show that surgery and anesthesia reduce the lag time in contextual testing, while GO administration restores the lag time. BSA modification and additional CQ treatment significantly reduced the lag time in GO-treated mice (fig. 6B). GO significantly reduced hippocampal β amyloid a β content, while BSA modification or additional CQ treatment restored a β content. Thus, these results demonstrate that GO improves post-operative cognitive dysfunction in mice by reducing beta amyloid a β content.

Example 1

The application of the nano graphene oxide in the treatment of beta amyloid a beta related diseases of the present embodiment: in the treatment of beta amyloid protein A beta related diseases, the used medicament or medicament carrier contains nano graphene oxide.

In the treatment of beta amyloid a beta related diseases, the route of administration of the drug is injection.

The dosage for human body is less than the dosage for animal body by converting the dosage of medicament between animal and human, and the concentration of the nano graphene oxide in the formed injection dosage form in the medicament or the medicament carrier is 6 mg/mL.

A test method for application of nano graphene oxide in treatment of beta amyloid A beta related diseases comprises the following steps:

test materials and methods

1) Synthesis of nano graphene oxide GO

2) Cell culture and establishment of APP overexpression cell lines

HKE293T and SHSY5Y cells in Dulbecco's modified Eagle Medium supplemented with 10% fetal bovine serum in the presence of 5% CO₂Culturing at 37 deg.C;

the pLenti-CMV-APP-GFP-Puro plasmid or the empty vector plasmid was transfected into 293T cells with pMD2.G and pPsAX2.0 plasmids using Lipofectamine3000 to produce lentiviruses; after 2 days, SHSY5Y cells were infected with filtered lentivirus-containing medium; then the infected cells are screened for 4 days by 1 mu g/mL puromycin;

3) western Blot

Lysing the cells with sample buffer and boiling for 10 minutes; proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes; incubating the membrane with a primary antibody overnight at 4 ℃, and after washing, incubating the membrane with a secondary antibody for 1 hour at 37 ℃; after washing, the membrane was incubated with the substrate chemiluminescent ECL reagent and observed using a chemiluminescent instrument; preparation of amyloid precursor protein APP 1: 1000 antibodies for the calibration of APP proteins; beta actin beta-actin 1: 1000 antibody, used as internal reference; horseradish peroxidase-conjugated secondary antibody 1: 10000 for calibrating primary antibody;

4) enzyme-linked immunosorbent ELISA assay

Homogenating the hippocampus with guanidine hydrochloride extraction buffer, detecting the total β amyloid a β 42 and β amyloid a β 40 concentration in cultured cells or hippocampal tissue homogenates by ELISA kit according to the manufacturer's instructions;

second, observation of experimental results

Experiment 1: characterization of nano graphene oxide GO

Experiment 2: nano graphene oxide GO (graphene oxide) for reducing beta amyloid protein Abeta in cells

To assess the effect of GO on β amyloid a β levels, HEK293T-APP-GFP and SHSY5Y-APP-GFP cell lines were established; western Blot and ELISA results were observed.

The synthesis process of the nano graphene oxide GO is as follows:

first, 1g of graphite powder and sodium nitrate NaNO31g were added to cooled concentrated sulfuric acid H₂SO₄23mL, and the mixture was stirred in an ice bath for 30 minutes; then gradually adding potassium permanganate KMnO under stirring and cooling₄3g, carrying out ultrasonic treatment at 40kHz for 5 hours to obtain a uniform liquid suspension, peeling off graphene oxide, gradually adding deionized water, adding 46mL within 15min as a standard, and further adding deionized water; stirring for 10 minutes; by adding 140mL of deionized water and H hydrogen peroxide₂O₂The reaction was stopped with 30% aqueous solution and 10 mL; separating the solid product by centrifugation at 1500rpm for 10 min;

finally, by washing 5 times the suspension suspended in 5% HCl solution in hydrochloric acid, then twice with deionized water, followed by drying; ultrasonic preparation was carried out in an ice bath at 380W for 2 hours.

The operation content is specifically as follows:

mice were anesthetized with 3.20% sevoflurane; performing skin incision below the knee, exposing the tibia, and inserting a 0.3mm pin into a medullary cavity of the tibia to realize intramedullary fixation; next, fracture at the midpoint using surgical scissors; finally, the wound is closed after the necessary debridement; to alleviate pain, a 2.2% lidocaine solution was applied topically to the wound prior to incision, and 1% tetracaine hcl mucus was applied to the wound twice daily.

Step 8), treating the mice, and injecting the mice by using a microinjector with a fixed needle.

Further, the air conditioner is provided with a fan,

step one, test materials and methods

5) Immunofluorescence

Cells were fixed with 4% paraformaldehyde for 10min, permeabilized with 0.1% triton X-100 for 10min, and blocked with 1% bovine serum albumin BSA for 1 h; cells were incubated with primary antibody overnight at 4 ℃ and labeled with secondary antibody for 1 hour at 37 ℃, images were collected using a confocal fluorescence microscope, and to prepare frozen sections, mice were anesthetized with sodium pentobarbital and perfused with phosphate buffered saline PBS or with 4% paraformaldehyde; after perfusion, the brains were harvested and fixed in fixative overnight, then dehydrated overnight with 30% sucrose solution, then brain tissue was embedded with OCT cryo-section embedding medium and stored frozen at-80 ℃ or cut into 10 μm sections for immunofluorescence manipulation, and the sections were observed under confocal fluorescence microscopy; preparation of beta-secretase 1BACE 11: 200, 3C1C 3; 1: 200, ab183612 primary antibody, for detecting the localization of BACE1 protein and the binding to GO; early endosomal antigen 1EEA 11: 200, ab2900 primary antibody and Ras-associated protein 7Rab 71: 200, ab50533 primary antibody for labeling early and late endosomes; lysosomal associated membrane protein 1LAMP 11: 100, sc-20011 primary antibody for labeling lysosomes; alexa Fluor 5681: 500 and Alexa Fluor 6471: 500 secondary antibody for fluorescent labeling;

6) determination of protein-nanoparticle interaction

BGO is bovine serum albumin BSA modified GO, 10 μ gGO or BGO was added to 0.6mg/mL purified APP or BACE1 protein, incubated at 4 ℃ for 2 hours, and centrifuged at 14000g for 30 minutes; the supernatant was stored and the pellet was washed 3 times with PBS containing 3% tween 20; boiling the protein solution, the supernatant and the precipitate in SDS loading buffer for 10 minutes, and then carrying out SDS-PAGE and Western blot containing a specified antibody; to prepare BGO, 20mgBSA and 10mgGO were mixed and sonicated for 1 h; the suspension was then centrifuged at 14000g for 2h to remove excess BSA; the precipitate was collected and resuspended in water for 15 minutes by sonication;

7) an animal

10 months old female C57BL/6 mice were purchased; all animals were kept in a temperature, humidity and light controlled chamber and water was provided ad libitum;

8) mouse treatment

Mice were anesthetized with 3.0% sevoflurane; mouse body temperature was maintained at 37 ℃; with reference to bregma, in coordinates: bilateral intracerebral injection is carried out at the position of 2.18mm at the back, 2.30mm at the side and 2.10mm at the abdomen; injecting 2 mu L of graphene oxide or BGO with the concentration of 10mg/mL or performing intracerebral injection by using physiological saline; for chloroquine CQ, mice were injected intraperitoneally with 60 mg/kgCQ;

9) surgical model

Performing intramedullary fixation of tibial fracture under sevoflurane anesthesia on mice after GO, BGO or CQ administration;

10) conditioned fear test

The test comprises a training stage before operation and a scene test stage 1 day after operation; one day before surgery, fear conditioning training was performed on the mice to establish long-term memory; placing each mouse in a test box for 120s, then performing 6 pairs of conditional-unconditional stimulation, and then staying in the test box for 60 s;

1 pair of conditional-unconditional stimuli includes 20s, 70dB sine wave sound as conditional stimulus, up and down interval of 25s and last 2s, 0.70mA electrical stimulus as unconditional stimulus; paired conditional-unconditional stimuli were separated by a random interval of 60 s; contextual fear memory reflects hippocampal-dependent memory; in the context test, the mice were simply placed back in the test chamber for 5min without tone or stimulation; the percentage of stagnation time is defined as the time that the mouse does not move except for breathing, and is recorded and counted by a Panlab conditioned fear and startle reflex combined system and Packwin2.0 software;

step two, experimental observation also comprises:

experiment 3: mechanism for reducing amyloid beta (Abeta) by graphene oxide

Experiment 4: whether graphene oxide can promote lysosomal degradation of beta amyloid protein Abeta

Experiment 5: observation of nano graphene oxide GO for reducing beta amyloid Abeta in mice hippocampus after surgery

Using a mouse model: inducing the up-regulation of the beta amyloid A beta content of the hippocampal tissue after the operation of the intramedullary fixation of the tibial fracture under the anesthesia of the sevoflurane; observing whether GO intervention can lower the content of beta amyloid A beta in the hippocampus of the mouse;

experiment 6: observation of nano graphene oxide GO for improving cognitive function status of mice after operation

The cognitive function of the mice after the fracture surgery is tested by adopting fear conditions, and whether the behavior of the mice after the surgery is influenced by reducing the content of the beta amyloid protein A beta by GO is evaluated.

Example 2

This embodiment is basically the same as embodiment 1 except that:

the operation content is specifically as follows: the dosage for human body is less than the dosage for animal body by converting the dosage of medicament between animal and human, and the concentration of the nano graphene oxide in the formed injection dosage form in the medicament or the medicament carrier is 5 mg/mL.

The operation content is specifically as follows:

mice were anesthetized with 2.5% sevoflurane; performing skin incision below the knee, exposing the tibia, and inserting a 0.3mm pin into a medullary cavity of the tibia to realize intramedullary fixation; next, fracture at the midpoint using surgical scissors; finally, the wound is closed after the necessary debridement; to reduce pain, a 1.5% lidocaine solution was applied topically to the wound prior to incision, and 0.5% tetracaine hcl mucus was applied to the wound twice daily.

Example 3

This embodiment is basically the same as embodiment 1 except that:

the operation content is specifically as follows: the dosage for human body is smaller than the dosage for animal body by converting the dosage of medicament between animal and human, and the concentration of the nano graphene oxide in the formed injection dosage form in the medicament or the medicament carrier is 8 mg/mL.

Mice were anesthetized with 3.5% sevoflurane; performing skin incision below the knee, exposing the tibia, and inserting a 0.3mm pin into a medullary cavity of the tibia to realize intramedullary fixation; next, fracture at the midpoint using surgical scissors; finally, the wound is closed after the necessary debridement; to reduce pain, a 2.5% lidocaine solution was applied topically to the wound prior to incision, and 1.5% tetracaine hcl mucus was applied to the wound twice daily.

It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should also be understood that various alterations, modifications and/or variations can be made to the present invention by those skilled in the art after reading the technical content of the present invention, and all such equivalents fall within the protective scope defined by the claims of the present application.

It will be appreciated by those skilled in the art that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments disclosed above are therefore to be considered in all respects as illustrative and not restrictive. All changes which come within the scope of or equivalence to the invention are intended to be embraced therein.

Claims (9)

1. The application of the nano graphene oxide in the treatment of beta amyloid protein Abeta related diseases is characterized in that: in the treatment of beta amyloid protein A beta related diseases, the used medicament or medicament carrier contains nano graphene oxide.

2. The use of the nanographene oxide according to claim 1 for the treatment of a β -amyloid a β -related disease, wherein: in the treatment of beta amyloid a beta related diseases, the route of administration of the drug is injection.

3. The use of the nanographene oxide according to claim 1 for the treatment of a β -amyloid a β -related disease, wherein: in the medicine or the medicine carrier, the concentration of the nano graphene oxide in the formed injection dosage form is less than or equal to 10 mg/mL.

4. An assay method for the use of nanographene oxide according to any one of claims 1 to 3 in the treatment of a β -amyloid a β -related disease, comprising the steps of:

test materials and methods

1) Synthesis of nano graphene oxide GO

2) Cell culture and establishment of APP overexpression cell lines

HKE293T and SHSY5Y cells in Dulbecco's modified Eagle Medium supplemented with 10% fetal bovine serum in the presence of 5% CO₂Culturing at 37 deg.C;

the pLenti-CMV-APP-GFP-Puro plasmid or the empty vector plasmid was transfected into 293T cells with pMD2.G and pPsAX2.0 plasmids using Lipofectamine3000 to produce lentiviruses; after 2 days, SHSY5Y cells were infected with filtered lentivirus-containing medium; then the infected cells are screened for 4 days by 1 mu g/mL puromycin;

3) western Blot

Lysing the cells with sample buffer and boiling for 10 minutes; proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes; incubating the membrane with a primary antibody overnight at 4 ℃, and after washing, incubating the membrane with a secondary antibody for 1 hour at 37 ℃; after washing, the membrane was incubated with the substrate chemiluminescent ECL reagent and observed using a chemiluminescent instrument; preparation of amyloid precursor protein APP 1: 1000 antibodies for the calibration of APP proteins; beta actin beta-actin 1: 1000 antibody, used as internal reference; horseradish peroxidase-conjugated secondary antibody 1: 10000 for calibrating primary antibody;

4) enzyme-linked immunosorbent ELISA assay

Homogenating the hippocampus with guanidine hydrochloride extraction buffer, detecting the total Α β 42 and Α β 40 concentration in the cultured cells or in the hippocampal tissue homogenates by means of an ELISA kit according to the manufacturer's instructions;

second, observation of experimental results

Experiment 1: characterization of nano graphene oxide GO

Experiment 2: nano graphene oxide GO (graphene oxide) for reducing beta amyloid protein Abeta in cells

To assess the effect of GO on β amyloid a β levels, HEK293T-APP-GFP and SHSY5Y-APP-GFP cell lines were established; western Blot and ELISA results were observed.

5. The test method for the application of the nano graphene oxide in the treatment of beta amyloid A beta related diseases according to claim 4, characterized in that: the synthesis process of the nano graphene oxide GO is as follows:

first, 1g of graphite powder and NaNO sodium nitrate were mixed₃1g of concentrated sulfuric acid H₂SO₄23mL, and the mixture was stirred in an ice bath for 30 minutes; then gradually adding potassium permanganate KMnO under stirring and cooling₄3g, carrying out ultrasonic treatment at 40kHz for 5 hours to obtain a uniform liquid suspension, peeling off graphene oxide, gradually adding deionized water, adding 46mL within 15min as a standard, and further adding deionized water; stirring for 10 minutes; by adding 140mL of deionized water and H hydrogen peroxide₂O₂The reaction was stopped with 30% aqueous solution and 10 mL; separating the solid product by centrifugation at 1500rpm for 10 min;

finally, by washing 5 times the suspension suspended in 5% HCl solution in hydrochloric acid, then twice with deionized water, followed by drying; ultrasonic preparation was carried out in an ice bath at 380W for 2 hours.

6. The test method for the application of the nano graphene oxide in the treatment of beta amyloid A beta related diseases according to claim 4, characterized in that the surgical content is specifically as follows:

anesthetizing the mice with 2.5-3.5% sevoflurane; performing skin incision below the knee, exposing the tibia, and inserting a 0.3mm pin into a medullary cavity of the tibia to realize intramedullary fixation; next, fracture at the midpoint using surgical scissors; finally, the wound is closed after the necessary debridement; to relieve pain, a 1.5-2.5% lidocaine solution was applied topically to the wound twice daily with 0.5-1.5% tetracaine hcl mucus prior to incision.

7. The test method for the application of the nano graphene oxide in the treatment of beta amyloid A beta related diseases according to claim 6, characterized in that the surgical content is specifically as follows:

mice were anesthetized with 3.0% sevoflurane; performing skin incision below the knee, exposing the tibia, and inserting a 0.3mm pin into a medullary cavity of the tibia to realize intramedullary fixation; next, fracture at the midpoint using surgical scissors; finally, the wound is closed after the necessary debridement; to alleviate pain, a 2% lidocaine solution was applied topically prior to incision and 1% tetracaine hcl mucus was applied to the wound twice daily.

8. The test method for the application of the nano graphene oxide in the treatment of beta amyloid A beta related diseases according to claim 4, characterized in that: step 8), treating the mice, and injecting the mice by using a microinjector with a fixed needle.

9. The test method for the application of the nano graphene oxide in the treatment of beta amyloid A beta related diseases according to claim 4, characterized in that:

step one, test materials and methods

5) Immunofluorescence

Cells were fixed with 4% paraformaldehyde for 10min, permeabilized with 0.1% triton X-100 for 10min, and blocked with 1% bovine serum albumin BSA for 1 h; cells were incubated with primary antibody overnight at 4 ℃ and labeled with secondary antibody for 1 hour at 37 ℃, images were collected using a confocal fluorescence microscope, and to prepare frozen sections, mice were anesthetized with sodium pentobarbital and perfused with phosphate buffered saline PBS or with 4% paraformaldehyde; after perfusion, the brains were harvested and fixed in fixative overnight, then dehydrated overnight with 30% sucrose solution, then brain tissue was embedded with OCT cryo-section embedding medium and stored frozen at-80 ℃ or cut into 10 μm sections for immunofluorescence manipulation, and the sections were observed under confocal fluorescence microscopy; preparation of beta-secretase 1BACE 11: 200,3C1C₃(ii) a 1: 200, ab183612 primary antibody, for detecting the localization of BACE1 protein and the binding to GO; early endosomal antigen 1EEA 11: 200, ab2900 primary antibody and Ras-associated protein 7Rab 71: 200, ab50533 primary antibody for labeling early and late endosomes; lysosomal associated membrane protein 1LAMP 11: 100, sc-20011 primary antibody for labeling lysosomes; alexa Fluor 5681: 500 and Alexa Fluor 6471: 500 secondary antibody for fluorescent labeling;

6) determination of protein-nanoparticle interaction

BGO is bovine serum albumin BSA modified GO, 10 μ gGO or BGO was added to 0.6mg/mL purified APP or BACE1 protein, incubated at 4 ℃ for 2 hours, and centrifuged at 14000g for 30 minutes; the supernatant was stored and the pellet was washed 3 times with PBS containing 3% tween 20; boiling the protein solution, the supernatant and the precipitate in SDS loading buffer for 10 minutes, and then carrying out SDS-PAGE and Western blot containing a specified antibody; to prepare BGO, 20mgBSA and 10mgGO were mixed and sonicated for 1 h; the suspension was then centrifuged at 14000g for 2h to remove excess BSA; the precipitate was collected and resuspended in water for 15 minutes by sonication;

7) an animal

10 months old female C57BL/6 mice were purchased; all animals were kept in a temperature, humidity and light controlled chamber and water was provided ad libitum;

8) mouse treatment

Mice were anesthetized with 3.0% sevoflurane; mouse body temperature was maintained at 37 ℃; with reference to bregma, in coordinates: bilateral intracerebral injection is carried out at the position of 2.18mm at the back, 2.30mm at the side and 2.10mm at the abdomen; injecting 2 mu L of graphene oxide or BGO with the concentration of 10mg/mL or performing intracerebral injection by using physiological saline; for chloroquine CQ, mice were injected intraperitoneally with 60 mg/kgCQ;

9) surgical model

Performing intramedullary fixation of tibial fracture under sevoflurane anesthesia on mice after GO, BGO or CQ administration;

10) conditioned fear test

The test comprises a training stage before operation and a scene test stage 1 day after operation; one day before surgery, fear conditioning training was performed on the mice to establish long-term memory; placing each mouse in a test box for 120s, then performing 6 pairs of conditional-unconditional stimulation, and then staying in the test box for 60 s;

1 pair of conditional-unconditional stimuli includes 20s, 70dB sine wave sound as conditional stimulus, up and down interval of 25s and last 2s, 0.70mA electrical stimulus as unconditional stimulus; paired conditional-unconditional stimuli were separated by a random interval of 60 s; contextual fear memory reflects hippocampal-dependent memory; in the context test, the mice were simply placed back in the test chamber for 5min without tone or stimulation; the percentage of stagnation time is defined as the time that the mouse does not move except for breathing, and is recorded and counted by a Panlab conditioned fear and startle reflex combined system and Packwin2.0 software;

step two, experimental observation also comprises:

experiment 3: mechanism for reducing amyloid beta (Abeta) by graphene oxide

Experiment 4: whether graphene oxide can promote lysosomal degradation of beta amyloid protein Abeta

Experiment 5: observation of nano graphene oxide GO for reducing beta amyloid Abeta in mice hippocampus after surgery

Using a mouse model: inducing the up-regulation of the beta amyloid A beta content of the hippocampal tissue after the operation of the intramedullary fixation of the tibial fracture under the anesthesia of the sevoflurane; observing whether GO intervention can lower the content of beta amyloid A beta in the hippocampus of the mouse;

experiment 6: observation of nano graphene oxide GO for improving cognitive function status of mice after operation

The cognitive function of the mice after the fracture surgery is tested by adopting fear conditions, and whether the behavior of the mice after the surgery is influenced by reducing the content of the beta amyloid protein A beta by GO is evaluated.

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