CN116008558B - Application of extracellular matrix elastin degradation product in preparation of products for diagnosing or delaying neurodegenerative diseases - Google Patents
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- CN116008558B CN116008558B CN202211354536.4A CN202211354536A CN116008558B CN 116008558 B CN116008558 B CN 116008558B CN 202211354536 A CN202211354536 A CN 202211354536A CN 116008558 B CN116008558 B CN 116008558B
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Abstract
The invention provides application of extracellular matrix elastin degradation products in preparation of products for diagnosing or delaying neurodegenerative diseases, and relates to a biomedical technical scheme. The invention adopts the technologies of three-dimensional positioning injection, high-throughput screening and the like, explores the role of EDPs in nervous system aging in a mouse and microglial cell-neuron co-culture system, and clarifies the transduction mechanism of extracellular EDPs for regulating microglial cell activation and downstream channels; agents blocking the activation of microglia by EDPs are proposed to be beneficial in restoring neuronal function and cognitive behavioral abnormalities, slowing down the progression of AD disease. The invention is beneficial to deeply understanding the relationship between aging and neuroinflammation and neurodegenerative diseases, and provides potential therapeutic targets and biomarkers for diseases related to cognitive dysfunction.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of extracellular matrix elastin degradation products in preparation of products for diagnosing or delaying neurodegenerative diseases.
Background
Extracellular matrix (Extracellular matrix, ECM) is a dynamic three-dimensional structure that constitutes the cellular microenvironment, involved in the regulation of cellular functions. Elastin (Elastin) is an important component in maintaining the dynamic structure of the ECM, in the form of elastic fibers. As the body ages, the elastic fibers are digested to produce free elastin polypeptides (Elastin derived polypeptides, EDPs) 1-4 . Clinical studies have found that patients with Alzheimer's Disease (AD) have elevated levels of EDPs in cerebrospinal fluid, the extent of which has a correlation with age, AD progression (Braak stage) 5-8 . However, the functional studies of EDPs in the nervous system are not yet comprehensive. Previous studies reported that injection of high concentrations of EDPs analogs into the ventricles of AD mice aggravated mouse brain Amyloid beta (Abeta) deposition and cognitive dysfunction, while detecting brain microglial activation 9-10 。
AD is a central nervous system degenerative disease that is dominated by cognitive disorders and memory impairment, and synaptic loss and dysfunction in the brain of a patient are direct causes of cognitive disorders. It is thought that microglial-mediated neuroinflammation occurs earlier than cognitive behavioral abnormalities and promotes neuronal synaptic loss and progression of neurodegenerative diseases 11-12 . Early detection, incubation of high concentrations of EDPs promoted BV2 microglial cell polarization to M1 type and induced the EDPs receptors Neu1, gal-3 to coat. Notably, neu1, gal-3 are important signaling molecules in microglial-mediated neuronal synaptic plasticity pathways, involved in "eat-me" signaling to activate microglial phagocytic neuronal synapses 13-14 。
With the increasing global aging level, the position of neurodegenerative diseases associated with aging in the disease spectrum is continuously advancing. The biological mechanism of EDPs in microglial activation and cognitive dysfunction is explained, the effect of ECM proteins in the nervous system is well understood, and theoretical basis and experimental basis are provided for clinical early diagnosis and treatment and targeted treatment of neurodegenerative diseases.
Dynamic regulation of ECM components of the nervous system is closely related to various physiological and pathological processes, and many studies have found that changes in ECM protein expression profile can be detected early in neurodegenerative diseases 15-16 . Elastin (Elastin) is an important component of the brain ECM, expressed in high levels in the ventriculus plexus, and present in small amounts in the interneuronal matrix (Neural interstitial matrix) 17 . The choroid plexus is the main part of cerebrospinal fluid, EDPs generated by elastin degradation enter the cerebrospinal fluid and can be clinically detected, and the abnormally-increased EDPs level can reach the range of mu g/mL from ng/mL 7-8 。
With age, ECM elastin degradation in the brain increases, there is a significant correlation between the extent of degradation and AD occurrence, and it is independent of atherosclerosis and cerebral infarction caused by vascular wall elastin degradation 4-5 . Compared with healthy subjects, the AD patients have reduced elastin levels in frontal lobe and hippocampal brain regions and increased elastase expression. This phenomenon is detected in the early stages of AD (Braak II stage) and is associated with the pathological stages of Braak 6 。
Microglia are immune effector cells of the central nervous system, have a variety of morphology and function, and can be divided into resting state, M1 pro-inflammatory type and M2 anti-inflammatory type for convenience of study (FIG. 1) 18 . Activation status of microglia during development and aging of the nervous system directly affects neuronal survival, synaptic maturation, transmission and plasticity regulation 19 。
Transcriptome sequencing found that the transition of microglial cells from resting to disease-related states was parallel to AD disease progression; aging causes the proportion of microglia related to diseases to be increased, and is considered as an inflammation background for the occurrence and development of neurodegenerative diseases 11,20 . Neurons (strong-but-living neurons) that are chronically under chronic inflammatory stress recruit microglia and regulate their activity by secreting signals such as cytokines, nucleotides and chemokines, wherein a signal called "eat me" assists microglia in clearing these neurons and synapses (FIG. 1) 13,21 。
It is thought that microglia in the aging brain are more activated (Primed) and are difficult to recover to a resting state 22 . Therefore, continuous activation of aging-related microglia is detrimental to neuronal synaptic pruning, leading to loss of neuronal synapses and functional impairment, which in turn exacerbates the progression of cognitive impairment diseases such as AD.
Elastin receptor complex (Elastin receptor complex, ERC) is the main receptor of EDPs, consisting of elastin binding protein (Elastin binding protein, EBP), cathepsin A (Protective protein/cathepsin A, PPCA) and Neuraminidase/sialidase 1 (Neu 1) (FIG. 2) 2-3 . Neu1 is the core of ERC action, and EDPs bind ERC to activate Neu1 sialidase function and hydrolyze sialic acid at the end of substrate glycoprotein 23-25 。
Sialic acid modification of cell membrane proteins is critical in regulating cell-cell, cell-matrix interactions, and high levels of sialylation are positive regulatory factors that promote brain development and nerve regeneration, and desialylation can lead to reduced synaptic plasticity in the hippocampus, and reduced learning and memory capacity 26 . Activated microglia release sialidases increased, desialylating self and neuronal surface glycoproteins. The phagocytic capacity of the microglial cell membrane protein after desialylation is improved; after desialylation, the neuronal membrane proteins are recognized by Complement proteins (C) C1q, C3 and Galectin-3 (Gal-3), releasing an "eat-me" signal to microglial cells, triggering microglial cells to phagocytose neuronal synapses (FIG. 1). Notably, gal-3 is also one of the receptors for EDPs and can bind EDPs-VGVAPG, VAPG 27 . However, in the nervous system, the occurrence and development of diseases in which EDPs bind to the receptor Neu1/Gal-3 are not known.
Reference to the literature
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Disclosure of Invention
In view of the above, the present invention aims to provide an application of an extracellular matrix elastin degradation product in preparing a product for diagnosing or delaying neurodegenerative diseases.
The invention provides application of extracellular matrix elastin degradation products as markers in preparation of a kit for diagnosing brain aging, neuroinflammation or neurodegenerative diseases.
The invention provides application of extracellular matrix elastin degradation products serving as targets in preparation of medicaments for delaying brain aging, neuroinflammation or neurodegenerative diseases.
The invention provides an application of a reagent for blocking extracellular matrix elastin degradation products to activate microglia in preparation of drugs for delaying brain aging, neuroinflammation or neurodegenerative diseases.
Preferably, the agent comprises at least one of: agents that silence elastase genes, EDPs-EBP binding antagonists, sialidase inhibitors, and Gal-3 inhibitors.
Preferably, the agent for silencing elastase gene comprises shRNA, siRNA or sgRNA;
the nucleotide sequence of shRNA for silencing elastase gene is shown as SEQ ID NO. 6.
Preferably, the EDPs-EBP binding antagonists include V14 peptide or lactose;
the nucleotide sequence of the V14 peptide is shown as SEQ ID NO. 7;
the sialidase inhibitor comprises ddNeu5Ac or oseltamivir;
the Gal-3 inhibitor comprises TD139 or citrus fructose.
Preferably, the amino acid sequence of the extracellular matrix elastin degradation product is glycine-X-proline-glycine, wherein X represents any amino acid.
Preferably, the nucleotide sequence of the extracellular matrix elastin degradation product is shown as one or more of SEQ ID NO. 2-SEQ ID NO. 5.
Preferably, the neurodegenerative disease comprises alzheimer's disease.
Preferably, the extracellular matrix elastin degradation product has the ability to reduce microglial clearance of beta amyloid in the brain;
the extracellular matrix elastin degradation product has the ability to promote activation of microglial cells to an M1 pro-inflammatory state;
the extracellular matrix elastin degradation products activate microglia through receptors Neu1 and Gal-3, and are involved in neuronal synaptic plasticity regulation.
The invention provides application of extracellular matrix Elastin Degradation Products (EDPs) serving as markers in preparation of a kit for diagnosing brain aging, neuroinflammation or neurodegenerative diseases. The present invention demonstrates that high concentrations of EDPs have significant relevance to neuroinflammation and AD for synaptic plasticity regulation in the context of aging and alzheimer's disease. The invention firstly utilizes brain stereotactic injection technology to improve the level of the old mouse cerebrospinal fluid EDPs, observes whether the activation state of microglial cells, the morphology of neuron synapses and electrophysiological functions in the brain of the mouse are changed, and determines the key roles of the EDPs in microglial cell activation and neuron synapse plasticity regulation. Meanwhile, the invention also verifies that the change of the activation state of microglial cells influences the clearance capacity of the microglial cells to extracellular Abeta, so the invention makes clear that EDPs influence the development process of Alzheimer's disease by influencing Abeta level and neuron synaptic plasticity regulation, and therefore, the EDPs can be used as detection markers for diagnosing early neurodegenerative diseases, neuroinflammation or brain aging.
The invention provides application of extracellular matrix elastin degradation products serving as targets in preparation of medicaments for delaying brain aging, neuroinflammation or neurodegenerative diseases. Experiments prove that the method is beneficial to restoring neuron function and cognitive behavioral abnormality and slowing down AD disease process by blocking EDPs from excessively activating microglial cells, and simultaneously defines the relationship between brain aging and neuroinflammation and neurodegenerative diseases, namely, the high-concentration EDPs in the aging brain are induction factors causing neuroinflammation and synaptic loss, and the microglial cells are blocked to activate the microglial cells, so that the clearance of the microglial cells to Abeta deposition is improved, and simultaneously, the microglial cells are lightened to phagocytize neuron synapses, so that a new idea is provided for delaying the neurodegenerative disease process.
Drawings
FIG. 1 is a schematic representation of signals associated with microglial activation and phagocytic neurons;
FIG. 2 is a schematic diagram of signal transduction paths of EDPs in the prior art;
FIG. 3 is a schematic diagram showing the mechanism of promotion of AD generation and development by EDPs mediated microglial cell activation;
FIG. 4 is a graph showing that high levels of EDPs affect microglial activation in the brain;
FIG. 5 is that high levels of EDPs reduce neuronal synaptic density in the brain;
FIG. 6 is a graph showing that high levels of EDPs affect neuronal electrophysiological transfer function in the brain;
FIG. 7 is that high levels of EDPs reduce spatial learning and memory in mice;
FIG. 8 is a schematic diagram of a primary neuron-microglial cell co-culture system;
FIG. 9 is that high levels of EDPs reduced spatial learning and memory in mice;
FIG. 10 is a graph showing that incubation of high concentrations of EDPs promotes microglial activation;
FIG. 11 is a graph showing the effect of high concentrations of EDPs on in vitro neuronal dendrites and dendritic spine morphology;
FIG. 12 is a graph showing that high concentrations of EDPs raise microglial EDPs receptor EBP and Gal-3 membrane protein levels;
FIG. 13 is a graph showing that blocking the interaction of EDPs with receptors can inhibit the activation of microglia by EDPs;
fig. 14 shows that blocking the interaction of EDPs with receptors can protect mouse neuronal morphology and cognitive function.
Detailed Description
The invention provides application of extracellular matrix elastin degradation products as markers in preparation of a kit for diagnosing brain aging, neuroinflammation or neurodegenerative diseases.
In the present invention, elastin is an important component of the extracellular matrix in the brain, degrading to EDPs with age, a process that is irreversible. The EDPs produced after elastolysis contain a characteristic sequence, in particular glycine-x-x-proline-glycine, gxxPG, x representing any amino acid, e.g. VGVAPG(SEQ ID NO:2)，GVYPG(SEQ ID NO:3)，GFGPG(SEQ ID NO 4) andGVLPG(SEQ ID NO: 5), wherein hexapeptide VGVAPGThe EDPs are contained in the greatest amount.
In the invention, the high-concentration EDPs generated by brain aging are firstly proposed to promote microglial activation through receptors Neu1 and Gal-3, excessively phagocytose neuron synapses and accelerate the pathological process of neurodegenerative diseases (AD), and the relationship among the EDPs, the prominent plasticity and the occurrence of the neurodegenerative diseases (AD) is clarified. The results of the embodiment of the invention show that by utilizing the brain stereotactic injection technology, the level of the EDPs in the cerebrospinal fluid of the aged mice is improved, microglial cells in the brains of the mice are in an activated state, the original form of neuron synapses is changed, the electrophysiological functions of the neurons are influenced, and the excessive EDPs are determined to serve as key roles in microglial cell activation and neuron synapse plasticity regulation. Therefore, the result of early diagnosis of brain aging or neurodegenerative diseases can be achieved by detecting EDPs.
In the present invention, microglial-mediated neuroinflammation occurs earlier than cognitive behavioral abnormalities and promotes neuronal synaptic loss and development of neurodegenerative diseases. The results of the embodiment of the invention show that the EDPs promote the activation of microglial cells to an M1 pro-inflammatory state, in particular the EDPs promote the increase of secretion of microglial pro-inflammatory factors TNF-alpha and IL-1 beta, so the invention reveals the logical relationship between the EDPs and the pathological development of AD and proves the bridge effect of neuroinflammation in the process. Meanwhile, the EDPs are increased in level, so that neurons are under chronic pressure for a long time, the activation state of microglia is regulated, and the excessive clearance of microglia to neuron synapses is promoted. Thus, increased levels of pro-inflammatory factors and altered morphology and physiological function of neuronal synapses further promote the progression of neurodegenerative diseases.
The invention provides application of extracellular matrix elastin degradation products serving as targets in preparation of medicaments for delaying brain aging, neuroinflammation or neurodegenerative diseases.
In an embodiment of the present invention, the neurodegenerative disease preferably includes alzheimer's disease. The three-dimensional positioning injection technology is utilized to simulate the environment of aging or chronic high-concentration EDPs of an AD model mouse, the phenotypes such as activation of microglial cells in brain, morphology and functions of neuron synapses, learning and memory behaviours of the AD mouse and the like are observed, and the results show that Iba1 immunohistochemical staining shows that the injection of the EDPs enables the microglial cells to be activated, the morphology and original biological functions of the neuron synapses are changed, and the learning capacity and memory behavior of the AD mouse are reduced.
The invention provides an application of a reagent for blocking extracellular matrix elastin degradation products to activate microglia in preparation of drugs for delaying brain aging, neuroinflammation or neurodegenerative diseases.
In the invention, it is clear that the extracellular matrix elastin degradation product accelerates the neurodegenerative disease by activating microglial cells, so the invention provides a reagent for blocking the extracellular matrix elastin degradation product from activating microglial cells to achieve the aim of delaying the neurodegenerative disease. The reagent preferably comprises at least one of the following: agents that silence elastase genes, EDPs-EBP binding antagonists, sialidase inhibitors, and Gal-3 inhibitors.
In the present invention, since the level of extracellular matrix elastin degradation in the brain is an important factor for activating microglia, the radical reduction of the level of extracellular matrix elastin degradation in the brain is one of the blocking strategies. The agent that silences the elastase gene preferably comprises shRNA, siRNA or sgRNA. The nucleotide sequences of the shRNA silencing the elastase gene are preferably set forth in SEQ ID NO. 6 (5'-CACCGCTGAACGACATTGTGATTATCGAAATAATCACAATGTCGTTCAGC-3', NCBI accession No. NM-015779.2 for murine genes) and SEQ ID NO. 8 (5'-CACCGCTCAACGACATCGTGATTCTCGAAAGAATCACGATGTCGTTGAGC-3', NCBI accession No. NM-001972.4 for human genes).
In the present invention, the prior art has reported that the Elastin Receptor Complexes (ERC) and Gal-3 are the main receptors for EDPs, the ERC consisting of elastin binding protein (Elastin binding protein, EBP), cathepsin a (Protective protein/cathepsin a, PPCA) and Neuraminidase/sialidase 1 (Neu 1). EBP is a non-enzymatic sheared form of beta-galactosidase (beta-galactosidase), and binding to EDPs can be competitively inhibited by beta-lactose (lactose), V14 peptide. Thus, the EDPs-EBP binding antagonists preferably comprise V14 peptide or lactose. The nucleotide sequence of the V14 peptide is preferably shown in SEQ ID NO. 9. Furthermore, inhibition of sialidases corresponds to inhibition of EDPs receptor activity and downstream pathways, and activation of the receptor Neu1 may be one of the causes of the microglial activation functional phenotype. Thus, neu1 is the core of ERC action, and EDPs bind to ERC to activate the sialidase function of Neu1 and hydrolyze sialic acid at the end of the substrate glycoprotein. The sialidase inhibitor preferably comprises ddNeu5Ac or oseltamivir. The Gal-3 inhibitor preferably comprises TD139 or citrus fructose.
In the present invention, the extracellular matrix elastin degradation product preferably has the ability to promote the excessive pruning of neuronal dendritic spines/synapses by microglial cells. The extracellular matrix elastin degradation product has the ability to promote activation of microglial cells to an M1 pro-inflammatory state; the extracellular matrix elastin degradation products activate microglia through receptors Neu1 and Gal-3, and are involved in neuronal synaptic plasticity regulation.
In the invention, blocking the excessive activation of microglia by extracellular matrix elastin degradation products can effectively delay the development of neurodegenerative diseases.
The use of the extracellular matrix elastin degradation products provided by the present invention in the preparation of products for diagnosing or delaying neurodegenerative diseases is described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
High concentrations of EDPs accelerate AD progression by activating microglia
The high concentration EDPs environment of AD patients reported in literature was simulated by injecting EDPs-VGVAPG (0.5. Mu.g/. Mu.L, 2. Mu.L/site) into the ventricles of the lateral side of mice (C57 BL/6, 12 months, male) by using a stereotactic injection technique, so as to inject the control polypeptide (VVGPGA, SEQ ID NO: 1) with the same volume as a negative control. The brain stereotactic injection method comprises the following steps: the mice were anesthetized with 1% pentobarbital sodium by intraperitoneal injection, and fixed on a brain stereotactic apparatus, ensuring head stability. Erythromycin ointment is applied to the eyeballs of mice to protect the cornea from damage caused by long-time exposure and light irradiation. The head of the mice was disinfected with 75% alcohol by hair and skin, a long incision of about 1cm was made along the sagittal suture, the skin was carefully peeled off, and the surface fascia was washed with 2% hydrogen peroxide to expose the skull. The mouse Bregma (Bregma) position was marked and recorded, the microinjector position was moved using brain stereotactic coordinates, and the injection position was marked. Lateral ventricle stereotactic: the anterior chimney is-0.34 mm, the side opening is 1.0mm, the needle depth is 2.5mm, the dental drill (diameter is 0.5 mm) is used for carefully drilling, and the surrounding bone fragments are cleaned by a cotton swab. A microinjector (10. Mu.L, hamilton) was fixed to an electronic microinjection pump, and the injection was stopped for 2 minutes after the needle was lowered to the prescribed position, the injection speed was selected to be 0.2. Mu.L/min, and the needle was slowly withdrawn after the injection was completed for 2 minutes. After the injection on both sides is finished, stitching.
After 1 month of injection, phenotypes such as microglial activation, neuronal synaptic morphology and function, AD mice learning and memory behaviours were examined as follows.
(1) Microglial activation: immunohistochemical staining is carried out on the brain sections of the mice, after images are acquired under a laser confocal microscope, the density and the morphology of the micro glial cells positive to the Iba1 of the hippocampus and cortex brain areas of the mice are analyzed, wherein the density and the morphology comprise the cell number and the cell area under unit area, the average branch length and the branch richness of single cells (Sholl analysis) are compared, and the influence of EDPs on the activation of the micro glial cells in the brain is compared.
The results are shown in FIG. 4. 1 month after injection of EDPs into the lateral ventricle, iba1 immunohistochemical staining showed morphological changes in the cortex and hippocampal microglial cells, and an increase in the microglial cell area and a decrease in the protrusion of the EDPs group were clearly observed, and the cells were in an activated state (A in FIG. 4). Statistical histograms of cortical and hippocampal Iba1 positive microglial density (B in fig. 4). Statistical histograms of cortex and hippocampal Iba1 positive microglial branch length. The increased density of Iba1 positive cells and decreased single cell branch length suggests that EDPs promote microglial activation (C in fig. 4).
(2) Neuronal synaptic density and morphology changes: random labeling of neuronal morphology using Golgi-Cox silver staining was accomplished with FD rapid Golgi staining kit. Mouse brain concussion sections, 200 μm thick, were attached to gelatin (2%) coated slides. The dyeing process is to take care of light shading, the color development time needs to be optimized, gradient alcohol dehydration and xylene permeabilization are carried out after color development, and the gel is sealed. The cortex and hippocampal projection neurons were examined by light microscopy, CCD imaging and statistical analysis of dendritic spine density.
The results are shown in FIG. 5. Statistics of Golgi-Cox silver plating staining results show that the density of the mouse hippocampal neurons dendritic spines is reduced after lateral ventricle injection of EDPs.
(3) Neuronal function change: long-term potentiation (LTP) and Long-term depression (LTD) of activity dependence are the main manifestations of synaptic plasticity in hippocampal neurons, embodying the function of the hippocampal brain region. The impaired hippocampal LTP in the brain of AD patients is considered to be the electrophysiological basis for the development of cognitive impairment. LTP was detected in the hippocampal region using patch clamp technique, and the effect of EDPs on brain function was evaluated at the nerve loop level.
Current patch clamp mode, the stimulating electrode was placed at the hippocampal schaffer side branch fibers, the recording electrode was placed at the hippocampal CA1 radiation layer, about 300 μm apart, and the amplitude and frequency of excitatory postsynaptic current (peefsc) induced at different amplitude conditions were recorded. A DC test stimulus current with a wave width of 100 μs and a strength of 150 μA was applied every 10s until an optimal fEPSP was developed, at which the stimulus strength of the base fEPSP was recorded at a stimulus strength that caused 40% to 50% of the maximum response. The high-frequency stimulation for inducing LTP is 200Hz, the wave width is 100 mu s, one period of stimulation comprises 20 pulses, the total period is 3, the period interval is 30s, and the total recording time is 60min.
The results are shown in FIG. 6. Statistical histograms of current amplitudes after excitatory synapses, high-level EDPs suppressed the amplitude of the sfscs (B in fig. 6). Changes in the levels of EDPs did not cause significant changes (E in fig. 6).
(4) AD mice learn and memory behavioural analysis: new object identification (short term memory) and Morris water maze (long term spatial memory) tests were performed according to standard procedures. In the new object recognition experiment, the exploratory time of the mice was defined as the position where the tip of the nose contacted the object or entered into the 2cm area around the object. In Morris water maze experiments, the activity time of the mice in the platform quadrants, the latency time for searching the platform positions and the shuttle times embody the spatial learning and memory capacity of the mice.
The results are shown in FIG. 7. The levels of EDPs did not have a significant effect on the time of exploration of mice in new object recognition (B in fig. 7). After the platform was removed on day 7 of the water maze experiment, the mice were shuttled to the original platform location, and it was seen that high levels of EDPs reduced the spatial memory capacity of the mice (D in FIG. 7).
Example 2
EDPs modulate synaptic plasticity by activating microglia
The primary microglial cell-neuron co-culture system is established, and is shown in fig. 8, and the specific method is as follows:
mice born for 3 days (C57 BL/6) were stunned, sterilized with 75% alcohol, and the brains were quickly removed, taking care of the low temperature aseptic technique. Hippocampal and cortical tissue was dissected in HBSS buffer, cut into small pieces, and digested with pancreatin (0.125%) at 37 ℃ for 15min. Proper amount of fetal bovine serum is stopped from digestion, the fetal bovine serum is gently blown to no obvious tissue mass by a polished pipettor, centrifuged at 1500rpm for 5min, and the pre-heated DMEM/F12 high sugar medium at 37 ℃ is resuspended and precipitated, and the adherent cells are mixed type glial cells in a polylysine coated culture dish. After 24 hours of culture, the whole amount of liquid is changed, and after 3-4 days, the cells grow to about 70% of fusion, and half amount of liquid is changed. After 7-9 days, the glial cells are gradually mature and layered, and observed under a microscope, the bottom layer is tightly connected astrocytes, the upper layer is round or elliptic microglial cells, and the volume is small and the refractive index is strong. The flask was fixed on a 37℃thermostat shaker at 200rpm for 2h, and the exfoliated microglial cells were collected and seeded into polylysine coated dishes, and these cells continued to proliferate.
Primary neuron culture: hippocampus tissue of the mice on the birth day was obtained by the above method, and pancreatin was digested at 37℃for 15min. Digestion was stopped and centrifuged at 800rpm for 1min. Resuspension with neuron growth medium (Neurobasal A+1% glutamine+2% B-27) containing 1% fetal bovine serum, gently beating with a polished pipette, standing for 5min, and obtaining the supernatant as neuron suspension. Cell count, plating. Cytarabine (3 μm) was added 48h later, and the neuronal growth medium was replaced every 3 days half-dose.
When establishing the co-culture system, care was taken to maintain an initial density ratio of neurons to microglia of about 1:10. after microglial cells are independently cultured for two days, the neurons are cultured in vitro until the 14 th day, the glass slide paved with the neurons is taken out, and the glass slide is reversely buckled in a microglial cell culture dish with paraffin support points. The duration of co-cultivation can be optimized according to experimental results, and is generally 24 hours.
Aβ42 is added into the primary microglial cell-neuron co-culture system constructed above for incubation, microglial cell and neuron morphology, microglial cell inflammatory factor secretion and protein expression difference are observed, and the influence of incubation concentration and time on results is noted.
(1) Microglial inflammatory factor expression: microglial morphology and the type of secreted cytokines reflect their activation status and function. The effect of different concentrations of EDPs-VGVAPG (50 nM, 1. Mu.M and 50. Mu.M) on transcription, expression and secretion of microglial pro-inflammatory factors (TNF-. Alpha., IL-1. Beta.) and anti-inflammatory factors (TGF-. Beta., IL-4) was examined using real-time fluorescent quantitative PCR (after 6 h).
BV2 cell transcription inflammatory factor mRNA is different under the condition of different concentrations of EDP-VGVAPG, and when the EDPs with high concentrations are incubated, the transcription level of pro-inflammatory factors TNF-alpha and IL-1 beta is obviously increased. It is speculated that high concentrations of EDPs promote activation of microglia to the M1 pro-inflammatory state.
Results
FIG. 9 is a graph showing the effect of different concentrations on mRNA levels of in vitro cultured microglial pro-and anti-inflammatory factors. The results show that high concentrations of EDPs promote transcription of the inflammatory factor TNF-alpha, IL-1 beta gene.
(2) Microglial morphology: from AlexaFluor TM 488 ghost pen ringPeptides stained cytoskeleton (F-actin), and the percentage of non-protruding and 2 or more protruding cells in random fields was counted. The microglial cell in a resting state is in the form of a short rod or a slender shuttle and is provided with 2 or more protrusions; in the activated and phagocytic state, microglial cell bodies become enlarged, lengthened or rounded.
The results are shown in FIG. 10.EDP-VGVAPG (50. Mu.M) was incubated in vitro to incubate the stained image of microglial cytoskeleton (A in FIG. 10), and it was seen that the cell processes were shortened, the area was increased, and amoeba-like, indicating that microglial cells were activated. Amoeba-like microglial cell occupancy statistics, incubation of EDPs promoted conversion of microglial cells to amoeba, but the changes were not significant (B in fig. 10). Incubation with high concentrations of EDPs increased microglial area, indicating microglial activation, transformation to amoeba-like (C in fig. 10).
(3) Neuron morphology: to distinguish whether the subject of activated microglial phagocytosis is apoptotic neuronal debris or healthy neuronal synapses, the neuronal morphology in the co-culture system should be analyzed. The antibodies anti-MAP2 and anti-PSD95 respectively mark neuron dendrites and synapses, pictures are randomly collected under a laser confocal microscope, and Image J software analysis comprises dendrite length, richness (Sholl analysis), synapse density and PSD95 fluorescence intensity distribution.
Results
The results are shown in FIG. 11. After DIV14 immunofluorescence staining, the enrichment degree of MAP2 positive dendrites is obviously reduced, the area of PSD95 positive protrusion is reduced, and the protrusion is clung to the dendrite surface.
(4) EDPs activate microglial cells through the receptors Neu1 and Gal-3
The effect of high concentrations of EDPs-VGVAPG (1. Mu.M) on microglial EDPs receptor EBP and Gal-3 levels was examined using immunoblotting (Westernblot) (after 24 h).
The results are shown in FIG. 12. The cytoplasmic and cell Membrane protein western blot results showed that EBP and Gal-3 levels were elevated in microglial cell Membrane (M) proteins after incubation of EDPs (FIG. 12A). Mouse cortex and hippocampal EDPs receptor EBP immunohistochemical staining pictures (B in fig. 12). Mouse cortex and hippocampal EDPs receptor Gal-3 immunohistochemical staining pictures (C in FIG. 12).
Example 3
Blocking EDPs from overactivating microglia and delaying AD development
(1) A primary microglial-neuronal co-culture system was established as in example 2, and EDPs were incubated while applying the same concentration of EDPs-EBP binding antagonist V14 or β -lactose (20 mM) to the in vitro co-culture system, and whether or not to block the excessive activation of microglial cells by EDPs was observed.
The results are shown in FIG. 13. Immunofluorescence staining images of microglia cultured in vitro after 24h of different stimuli were applied, control being the same volume of complete medium (a in fig. 13); it can be seen that both V14 and beta-lactose restore microglial morphology to a multi-bulge shape. Statistics of the proportion of amoeba-like microglia activated under different incubation conditions, V14 and β -lactose reduced amoeba-like morphology, indicating a decrease in the proportion of microglia activated (B in FIG. 13). Microglial area statistics under different incubation conditions, V14 and β -lactose reduced microglial area, further demonstrating the transformation of cells from amoeba-like in activated state to polysemous-like in resting state (fig. 13C).
(2) Animal experiment: the experimental screening result needs to be further verified by utilizing an AD model mouse, and the administration mode capable of being orally and intraperitoneally injected is preferably selected, and then in-situ injection is performed. The observation index and experimental scheme are consistent with the animal experiment, and mainly comprise microglial cell activation, neuronal synapse morphology and function and behavioural remediation effect. Oral drug remedies were preferentially selected by injecting EDPs into the ventricles of AD model mice (APP/PS 1,5 months, male) according to the method of example 1: the blocking group was fed with drinking water containing β -lactose (20 mM) and the control group (control) was fed normally. After one month, microglial activation status, neuronal dendritic spine density and learning memory behavioural performance of mice in the water maze were observed.
The results are shown in FIG. 14. The mouse lateral ventricles were injected with EDPs and hippocampal CA1 microglial activation Iba1 immunohistochemical staining results 1 month after drug intervention (fig. 14 a). Oral β -lactose reduced the rate of Iba1 positive activated microglia (B in fig. 14). Oral β -lactose improved activated microglial morphology and increased protrusion length (C in fig. 14). The mouse lateral ventricles were injected with EDPs and simultaneously given the results of staining of hippocampal CA1 neurons by dendritic spines Golgi-Cox 1 months after drug intervention (D in fig. 14). Oral β -lactose increased the mouse brain hippocampal neuron dendritic spine density (E in fig. 14). Oral β -lactose improved cognitive performance in the water maze in mice with increased number of platform crossing on day 7 (F in fig. 14).
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. Use of an agent that blocks extracellular matrix elastin degradation products from activating microglia in the manufacture of a medicament for delaying brain aging, neuroinflammation or neurodegenerative disease, said neurodegenerative disease being alzheimer's disease.
2. The use according to claim 1, wherein the agent comprises at least one of the following: agents that silence elastase genes, EDPs-EBP binding antagonists, sialidase inhibitors, and Gal-3 inhibitors.
3. The use according to claim 2, wherein the agent for silencing an elastase gene comprises shRNA, siRNA or sgRNA;
the nucleotide sequence of shRNA for silencing elastase gene is shown as SEQ ID NO. 6.
4. The use according to claim 3, wherein the EDPs-EBP binding antagonist comprises V14 peptide or lactose;
the nucleotide sequence of the V14 peptide is shown as SEQ ID NO. 7;
the sialidase inhibitor comprises ddNeu5Ac or oseltamivir;
the Gal-3 inhibitor comprises TD139 or citrus fructose.
5. The use according to any one of claims 1 to 4, wherein the amino acid sequence of the extracellular matrix elastin degradation product is glycine-X-proline-glycine, wherein X represents any amino acid.
6. The use according to claim 5, wherein the nucleotide sequence of the extracellular matrix elastin degradation product is one or more of SEQ ID NO. 2-SEQ ID NO. 5.
7. The use according to any one of claims 1 to 4, wherein the extracellular matrix elastin degradation product has the ability to reduce the clearance of beta amyloid in brain by microglia;
the extracellular matrix elastin degradation product has the ability to promote activation of microglial cells to an M1 pro-inflammatory state;
the extracellular matrix elastin degradation products activate microglia through receptors Neu1 and Gal-3, and are involved in neuronal synaptic plasticity regulation.
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| CN110536682A (en) * | 2017-04-18 | 2019-12-03 | 基恩菲特公司 | The combination of Yi Lafeinuo or derivatives thereof and anti-NASH, anti-fibrosis or anti-cholestasis medicament |
| CN111704653A (en) * | 2020-06-08 | 2020-09-25 | 中山大学 | Inhibitor polypeptide compounds targeted to fibronectin derived peptides and uses thereof |
| CN112345770A (en) * | 2020-11-06 | 2021-02-09 | 苏州大学附属第一医院 | Application of elastin degradation peptide as vascular calcification marker of peritoneal dialysis patient |
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| CN110536682A (en) * | 2017-04-18 | 2019-12-03 | 基恩菲特公司 | The combination of Yi Lafeinuo or derivatives thereof and anti-NASH, anti-fibrosis or anti-cholestasis medicament |
| CN111704653A (en) * | 2020-06-08 | 2020-09-25 | 中山大学 | Inhibitor polypeptide compounds targeted to fibronectin derived peptides and uses thereof |
| CN112345770A (en) * | 2020-11-06 | 2021-02-09 | 苏州大学附属第一医院 | Application of elastin degradation peptide as vascular calcification marker of peritoneal dialysis patient |
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