Novel target P2X1 for demyelination disease treatment and application thereof

Abstract

The invention discloses a novel target P2X1 for demyelination disease treatment and application thereof, particularly relates to the technical field of biomedicine, and finds that in the later period of demyelination, P2X1 is knocked out from astrocytes, and oligodendrocyte precursor cells OPC are promoted to be differentiated into newborn oligodendrocytes OL. According to the novel target point P2X1 for treating the demyelination disease and the application thereof, the differentiation capacity of oligodendrocyte precursor cells can be promoted by knocking out P2X1 in astrocytes, the novel target point P2X1 has an important position on myelination regeneration, the importance of P2X1 on myelination regeneration in the demyelination disease is defined, and the application of the novel target point P2X1 for treating the demyelination disease is provided. Through targeted intervention of primary astrocyte P2X1 receptor expression, it is found that the differentiation capacity of oligodendrocyte precursor cells (OPC) can be affected, then treatment of demyelination diseases is promoted, and myelin sheath repair is promoted.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a novel target P2X1 for treating demyelinating diseases and an application thereof. Background Art

[0002] Myelin is an important structural basis for the vertebrate nervous system to maintain normal physiological activities. Its appearance is a milestone event in the history of biological evolution. It enables nerve impulses to be transmitted quickly in a "jumping" manner, greatly improving the working efficiency of the nervous system while saving energy and physical space. Not only that, the myelin sheath also plays an important nutritional support role for the axons it wraps, including maintaining their structural and functional integrity.

[0003] The myelin sheath of the central nervous system is formed by oligodendrocytes (OL) cell membranes tightly wrapping around axons. OL and myelin are highly susceptible to a variety of damaging factors including ischemia and hypoxia, leading to poor myelination during development or adult demyelination changes, that is, brain white matter / myelin damage diseases, and a series of neurological or mental dysfunctions. In addition, OL is the direct responsible cell for the formation and regeneration of central myelin, which itself is ultimately generated by its precursor cells (Oligodendrocyte precursor cell, OPC) through a series of intermediate differentiation and development stages. Therefore, OPC is the source cell of myelin regeneration.

[0004] OPCs are the source cells for myelin regeneration, and they express various different marker proteins in the process of differentiation, such as PDGFRa, O4, O1 / GalC, CC1, and MBP. Cells at all stages of this differentiation process are collectively referred to as oligodendrocyte lineage cells. Currently, the main role of OPCs that has been fully revealed and recognized is as the source cells for myelin regeneration in the adult central nervous system, and their successful differentiation into mature OLs is the key to successful myelin regeneration. However, how to promote myelin regeneration is currently the main goal of demyelinating disease treatment, and it is also a difficult problem that needs to be solved urgently.

[0005] Previous studies on demyelinating diseases have indeed focused on the development and function of OL and its precursor cells OPC, because these cells play a key role in the formation and regeneration of myelin, ignoring that other types of cells can also regulate the development and differentiation of OL and OPC through interaction. However, during the occurrence and development of the disease, there will be interactions and synergies between multiple types of cells. Therefore, from the perspective of other cells regulating OL and OPC, studying their joint participation in myelin regeneration can provide multiple potential targets for the treatment of demyelinating diseases. Summary of the invention

[0006] The main object of the present invention is to provide a novel target P2X1 for the treatment of demyelinating diseases and its applications, which can effectively solve the problems mentioned in the background art.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] In the first aspect of the present invention, there is provided an application of knocking out the P2X1 gene in astrocytes as a target in the treatment of demyelinating diseases.

[0009] Preferably, after targeting and intervening in the expression of the P2X1 receptor, it affects the differentiation of OPCs in a paracrine manner.

[0010] Preferably, the targeted intervention is to transfect an overexpressed P2X1 virus, activate astrocytes with Bz-ATP, and after 72 hours of virus expression, collect the conditioned medium and add it to the isolated primary OPCs at a ratio of 1:1 with the OPC differentiation medium.

[0011] Preferably, knocking out P2X1 in astrocytes promotes the differentiation of oligodendrocyte progenitor cells (OPCs) into newborn oligodendrocytes (OLs) in the late stage of demyelination.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. By targeting and intervening in the expression of the P2X1 receptor in primary astrocytes, the present invention discovers that it can affect the differentiation ability of oligodendrocyte progenitor cells (OPCs), thereby promoting the treatment of demyelinating diseases and promoting myelin repair.

[0014] 2. The present invention discovers that specific upregulation of P2X1 in astrocytes may affect the differentiation of OPCs through a paracrine manner, and knocking out P2X1 in astrocytes can promote the differentiation ability of oligodendrocyte progenitor cells, which has an important position in myelin regeneration. The aim is to clarify the importance of P2X1 in myelin regeneration in demyelinating diseases, and it is expected to provide a novel therapeutic target for demyelinating diseases.

[0015] 3. The present invention reveals that the P2X1 receptor in the reactive astrocyte pathway affects the differentiation of OPCs in a paracrine manner after central demyelinating injury, revealing its potential for development as a new target for precision treatment of demyelinating diseases. Description of the Drawings

[0016] Figure 1 It is a schematic diagram for analyzing the demyelination degree of the standard cuprizone (CPZ) model of the present invention;

[0017] Figure 2 It is a schematic diagram of mouse hybridization of the present invention;

[0018] Figure 3Schematic diagram of specific knockout of P2X1 gene in astrocytes of the present invention;

[0019] Figure 4 Schematic diagram of the effect of P2X1 knockout on OPC differentiation during demyelination process of the present invention;

[0020] Figure 5 Schematic diagram for detecting the differentiation state of OPC of the present invention. Detailed implementation manners

[0021] To make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the present invention will be further described below in conjunction with specific implementation manners.

[0022] Example 1: Feed mice with 0.2% cuprizone to establish a demyelination disease model.

[0023] Refer to Figure 1 and use C57BL / 6J mice, male, 8 weeks old, purchased from Zhuhai Bestong Biotechnology Co., Ltd. Breeding conditions: temperature 21±3°C, humidity 50%, circadian rhythm, free access to food and water; number the mice before the experiment and randomly divide them into a control group (Control) and an experimental group (CPZ). Among them, feed the mice in the experimental group with a feed powder containing 0.2% cuprizone for 5 consecutive weeks to cause demyelination mainly in the corpus callosum. Then, anesthetize the mice by intraperitoneal injection of 1% sodium pentobarbital, decapitate and sacrifice them, dissect the brain tissue, fix it with buffered formalin, and perform immunofluorescence staining after routine tissue embedding and frozen sectioning. Add rat anti-MBP antibody (BioRad; MCA409S; 1:200), incubate overnight at 4°C, add the corresponding secondary antibody, incubate at room temperature for 2 h, wash 3 times with PBS and then seal the slides. Observe with a fluorescence microscope (Leica) and perform statistical analysis with ImageJ. It is found that after treatment with cuprizone CPZ for 5 weeks, the myelin basic protein MBP level in the corpus callosum is significantly reduced (P<0.01).

[0024] It shows that the standardized demyelination model used in the experiment is successfully constructed.

[0025] Example 2: Preparation and verification of mice with specific knockout of P2X1 gene in astrocytes.

[0026] Prepare P2X1 by CRISPR / Cas9 technology floxConditional gene knockout mice. This transgenic mouse carries the P2X1 receptor gene, and recombinant genes with loxp sites are inserted upstream of exon 2 and downstream of exon 7 respectively. This mouse is crossed with Aldh1l1-CreERT2 mice that specifically express Cre enzyme in astrocytes to obtain homozygous mice with inducible knockout of the P2X1 receptor in astrocytes. Subsequently, tamoxifen is administered intraperitoneally to induce the expression of Cre enzyme and knockout the P2X1 receptor. For the specific details of the mouse cross, refer to Figure 2 ;

[0027] Moreover, using the same method as above, the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, decapitated, the brain tissues were dissected, perfused and fixed with formalin, routinely embedded in paraffin, and frozen sections were prepared for immunofluorescence staining. Mouse anti-GFAP antibody (Sigma; G3893; 1:200) and rabbit anti-P2X1 antibody (Alomonela; APR-001; 1:200) were added, incubated overnight at 4°C, the corresponding secondary antibody was added, incubated at room temperature for 2 h, washed 3 times with PBS and then sealed. Observation was carried out using a fluorescence microscope (Leica), and it was found that the expression of P2X1 in the experimental group was significantly lower than that in the control group. For the specific details, refer to Figure 3 .

[0028] It was shown that the P2X1 gene in astrocytes was successfully knocked out.

[0029] Example 3: Knocking out the P2X1 gene in astrocytes affects OPC differentiation during the demyelination process.

[0030] Homozygous mice with specific knockout of the P2X1 gene in astrocytes, male, about 8 weeks old, were divided into a control group P2X1 - / - and an experimental group Aldh1l1-CreER T2 ; P2X1 - / - . Subsequently, tamoxifen was administered intraperitoneally to induce the expression of Cre enzyme and knockout the P2X1 receptor. According to the method in Example 1 above, the mice were fed with a diet powder containing 0.2% cuprizone for adult mouse demyelination treatment. After 4 weeks of cuprizone (CPZ) treatment, the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, decapitated, the brain tissues were dissected, perfused and fixed with formalin, and frozen sections were prepared after routine tissue embedding.

[0031] The above-mentioned mouse brain slices were taken, rinsed in 1× enzyme-free PBS solution, and then preheated at 65°C in a hybridization oven; secondly, the hybridization solution was taken out from -20°C, preheated at 65°C, and the in situ hybridization probe was taken out from -80°C, incubated in a metal bath at 70°C for 10 min, diluted 1:800 into the hybridization solution, and dropped onto the brain slices and incubated overnight in the hybridization oven.

[0032] Afterwards, the brain slices were placed in the elution buffer washbuffer and rinsed 3 times at 65°C for 30 minutes each time; then the brain slices were transferred to the MABT solution and rinsed 3 times at room temperature on a shaker for 15 minutes each time; after blocking at room temperature for 2 hours, the primary antibody (Anti-DIG Ab Fragment) was added at a ratio of 1:800 and incubated overnight at 4°C; after rinsing with the MABT solution, the secondary antibody was incubated and the slices were sealed. Photographs were taken and observed using a Leica optical microscope. It was found that 4 weeks after demyelination treatment, specifically knocking out P2X1 in astrocytes increased the expression of the neonatal oligodendrocyte marker Enpp6. For specific reference, see Figure 4 , and there was a statistical difference (P < 0.05);

[0033] The above indicates that knocking out P2X1 in astrocytes promotes the differentiation of oligodendrocyte progenitor cells OPCs into neonatal oligodendrocytes OLs in the late stage of demyelination.

[0034] Furthermore, since OLs are the cells that form myelin in the central nervous system, an increase in the differentiation of OPCs into neonatal OLs means that more OLs can participate in the myelin repair process. In the late stage of demyelination, more neonatal OLs can synthesize and secrete myelin-related proteins, such as myelin basic protein (MBP), etc., thereby promoting myelin regeneration, restoring the myelin structure of nerve fibers, and improving the insulation and efficiency of nerve signal conduction;

[0035] At the same time, with the repair of myelin, the conduction speed and accuracy of nerve impulses will be improved. The damaged nerve pathways are restored, and the nervous system can transmit information more effectively, which helps to improve various neurological dysfunction symptoms caused by demyelination, such as limb movement incoordination and sensory abnormalities, etc., may be alleviated.

[0036] Furthermore, neonatal OLs can not only form myelin, but also provide nutritional support and protection for neurons by secreting various neurotrophic factors and other substances. In the late stage of demyelination, the generation of more OLs can enhance the supportive effect on surrounding neurons, reduce neuronal damage and death, and help maintain the structural and functional integrity of the nervous system.

[0037] The repair of myelin and the increase in OLs can regulate the microenvironment of neurons and reduce neuronal hyperexcitation and excitotoxicity caused by demyelination. The normal myelin structure helps to maintain the stability of the neuronal membrane potential, reduce abnormal conditions such as calcium ion influx caused by neuronal hyperexcitation, and thus reduce neuronal damage.

[0038] Example 4, Targeted Intervention on the Effect of Overexpression of Astrocytic P2X1 on OPC Differentiation

[0039] Newborn rats at 1 - 2 days old were used to extract their cortical tissues for primary mixed glial cell culture. After culturing for about 10 - 12 days, differential centrifugation was used to separate various glial cells. OPCs were separated at 190 rpm for standby, and astrocytes were separated at 230 rpm. They were divided into a blank group (Control), a control group (Vector), and an experimental group (P2X1OE). Among them, the blank group received no treatment; the control group was transfected with a control virus and astrocytes were activated with Bz - ATP; the experimental group was transfected with a virus overexpressing P2X1 and astrocytes were activated with Bz - ATP. After 72 hours of virus expression, the conditioned medium was collected and added to the isolated primary OPCs at a ratio of 1:1 with the OPC differentiation medium. After culturing for 5 days, cell immunofluorescence detection was performed.

[0040] The specific operation was as follows: The medium in the cell plate was removed, and the cells were washed 3 times with pre - cooled PBS solution. Then, 500 μl of 4% paraformaldehyde solution was added and fixed for 15 min, followed by blocking at room temperature for 2 h. Rat anti - MBP antibody (BioRad; MCA409S; 1:200) and rabbit anti - NG2 antibody (Millipore; AB5320; 1:200) were added and incubated overnight at 4°C. The corresponding secondary antibody was added and incubated at room temperature for 2 h. After washing 3 times with PBS, the cells were sealed and observed under a fluorescence microscope. It was found that after overexpressing the P2X1 receptor in astrocytes, myelin basic protein MBP + in the entire oligodendrocyte lineage cell SOX10 + showed a significant decrease in proportion, specifically referring to Figure 5 , and there was a statistical difference (P < 0.05).

[0041] It was shown that overexpressing the P2X1 receptor in astrocytes in vitro reduced the differentiation ability of OPCs.

[0042] Therefore, after central demyelinating injury, the P2X1 receptor in the reactive astrocyte pathway affects OPC differentiation in a paracrine manner.

[0043] The above - mentioned paracrine manner is a common inter - cellular communication method in the prior art, in which cells secrete chemical signal substances (such as cytokines, growth factors, etc.) into the extracellular environment and act on neighboring cells through diffusion, thereby affecting the functions and behaviors of neighboring cells.

[0044] In this protocol, when astrocytes specifically upregulate P2X1, they secrete certain bioactive substances (such as specific cytokines, chemokines, growth factors and other signaling molecules) into the microenvironment surrounding the cells. These signaling molecules secreted outside the cells diffuse in the extracellular fluid, reach the adjacent oligodendrocyte precursor cells (OPCs), and bind to the corresponding receptors on the surface of OPCs, thereby activating a series of signaling pathways within OPCs, ultimately affecting the differentiation process of OPCs.

[0045] Therefore, after overexpression of P2X1 receptor in astrocytes in vitro, myelin basic protein MBP + SOX10 in cells across the oligodendrocyte lineage + The proportion in the myelin sheath is significantly reduced, which means that the number or proportion of oligodendrocytes that can produce MBP and participate in myelin formation is reduced, which directly affects the normal production and repair of myelin sheath, and may lead to the occurrence and development of abnormal myelin development or demyelinating diseases.

[0046] At the same time, overexpression of P2X1 receptors in astrocytes in vitro reduces the differentiation ability of OPCs and leads to insufficient myelin formation, affecting the myelination process of nerve fibers, which may slow down the speed of nerve conduction and cause abnormalities in neural connections and signal transmission during the development of the nervous system, thereby affecting the establishment of normal functions of neural organs such as the brain.

[0047] Therefore, this scheme uses the Cre / LoxP system to construct a mouse model of specifically knocking out the P2X1 gene in astrocytes, and uses 0.2% copper hydrazone to feed mice to induce demyelinating diseases. In situ hybridization and other techniques found that copper hydrazone treatment can promote the differentiation of oligodendrocyte precursor cells (OPC) into oligodendrocytes (OL) after 4 weeks. On this basis, by targeted intervention of the expression of P2X1 receptors in primary astrocytes, it was found that the differentiation ability of oligodendrocyte precursor cells (OPC) can be affected, thereby promoting the treatment of demyelinating diseases and promoting myelin repair. The present invention finds that after astrocytes specifically upregulate P2X1, it may affect OPC differentiation through paracrine, and knocking out P2X1 in astrocytes can promote the differentiation ability of oligodendrocyte precursor cells, which plays an important role in myelin regeneration. It aims to clarify the importance of P2X1 to myelin regeneration in demyelinating diseases, and is expected to provide a new therapeutic target for demyelinating diseases.

[0048] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. Use of knocking out the P2X1 gene in astrocytes as a target in the treatment of demyelinating diseases.

2. The application according to claim 1, wherein: By targeting and intervening in the expression of P2X1 receptor, it affects OPC differentiation in a paracrine manner.

3. The application according to claim 2, wherein: The said targeted intervention is to transfect the virus overexpressing P2X1, and give Bz-ATP to activate astrocytes. After 72 hours of virus expression, collect the conditioned culture medium and add it to the isolated primary OPCs at a ratio of 1:1 with the OPC differentiation medium.

4. The application according to claim 1, wherein: Knocking out P2X1 in the astrocytes promotes the differentiation of oligodendrocyte progenitor cells (OPCs) into newborn oligodendrocytes (OLs) in the late stage of demyelination.

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