阅读说明：本技术 骨桥蛋白在缺氧缺血性脑损伤中的应用 (Application of osteopontin in hypoxic and ischemic brain injury ) 是由 王贞 李婷婷 刘德祥 柯鸿飞 于 2019-12-02 设计创作，主要内容包括：本发明提供骨桥蛋白在缺氧缺血性脑损伤中的应用,属于生物医药和分子生物学技术领域。本发明利用蛋白质组学发现在HI新生小鼠损伤皮层中骨桥蛋白变化最为明显,进一步通过试验证明骨桥蛋白可能是抑制缺氧缺血性脑损伤中外周巨噬细胞入侵与神经炎症之间的恶性循环的潜在靶点,由此证明骨桥蛋白可作为缺氧缺血性脑损伤中诊断、预后的标志物和潜在治疗靶点,因此具有良好的实际应用之价值。(The invention provides an application of osteopontin in hypoxic and ischemic brain injury, belonging to the technical field of biological medicine and molecular biology. The invention discovers that the osteopontin in the damaged cortex of the HI newborn mouse changes most obviously by utilizing proteomics, and further proves that the osteopontin can be a potential target point for inhibiting malignant circulation between peripheral macrophage invasion and neuroinflammation in the hypoxic-ischemic brain injury through tests, so that the osteopontin can be used as a marker and a potential treatment target point for diagnosis and prognosis in the hypoxic-ischemic brain injury, and has good value of practical application.)

1. The application of the substance for detecting the expression level of osteopontin in preparing a reagent for detecting, diagnosing or predicting the progress of hypoxic-ischemic brain injury.

2. The use of claim 1, wherein the progression of hypoxic-ischemic brain injury comprises an adverse prognosis of hypoxic-ischemic brain injury.

3. A composition for detecting, diagnosing or predicting the progression of hypoxic-ischemic brain injury, comprising a substance that detects the expression of the OPN gene and its expression products based on a high-throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method; or a substance for detecting the expression of OPN protein based on an immunoassay.

4. The composition of claim 3, wherein the OPN gene expression is detected by any one of Northern hybridization, miRNA expression profiling, ribozyme protection analysis, RAKE, and in situ hybridization.

5. The composition of claim 3, wherein the immunodetection methods comprise immunoblotting, immunofluorescence, immunoenzyme labeling, affinity immunohistochemistry, and protein chips.

6. Use of a substance inhibiting the reduction of the OPN gene and its expression products and/or activity in at least one or more of the following a) -h):

a) inhibiting HI-induced brain damage;

b) inhibiting the malignant cycle between peripheral macrophage invasion and neuroinflammation in hypoxic-ischemic brain injury;

c) reducing the cerebral infarction area caused by hypoxic-ischemic brain injury;

d) reducing cerebral edema caused by hypoxic-ischemic brain injury;

e) inhibiting the expression of inflammatory factors caused by hypoxic-ischemic brain injury;

f) reducing the number of peripheral invading macrophages;

g) inhibiting OPN produced by peripheral invading macrophages;

h) inhibition of hypoxic-ischemic brain injury-induced CD11b ⁺/CD45 ^highRecruitment of immune cells.

7. The use of claim 6, wherein said inflammatory factors include CD16, CD11b, CD86, IL-1 β, and TNF α.

8. A pharmaceutical composition for treating hypoxic-ischemic brain injury, comprising a substance that inhibits reduction in OPN gene and its expression product and/or activity;

preferably, the treatment of hypoxic-ischemic brain injury comprises at least one or more of the following a) -h):

a) inhibiting HI-induced brain damage;

b) inhibiting the malignant cycle between peripheral macrophage invasion and neuroinflammation in hypoxic-ischemic brain injury;

c) reducing the cerebral infarction area caused by hypoxic-ischemic brain injury;

d) reducing cerebral edema caused by hypoxic-ischemic brain injury;

e) inhibiting the expression of inflammatory factors caused by hypoxic-ischemic brain injury;

f) reducing the number of peripheral invading macrophages;

g) inhibiting OPN produced by peripheral invading macrophages;

h) inhibition of hypoxic-ischemic brain injury-induced CD11b ⁺/CD45 ^highRecruitment of immune cells;

preferably, the inflammatory factors include CD16, CD11b, CD86, IL-1 β and TNF α.

9. The pharmaceutical composition of claim 8, wherein the substance that inhibits the reduction of the OPN gene and its expression products and/or activity comprises an antibody specific for the OPN protein, an RNA interfering molecule or antisense oligonucleotide directed against the OPN mRNA, a small molecule inhibitor, an siRNA, and a substance that effects a lentiviral infection or gene knock-out; further, the antibody is a human antibody or a murine antibody.

10. The pharmaceutical composition of claim 8, further comprising a carrier, an excipient, and a diluent; or the like, or, alternatively,

the composition is in the form of one or more of oral preparation such as powder, granule, tablet, capsule, suspension, emulsion, syrup, spray, etc., external preparation, suppository and sterile injection solution.

Technical Field

The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of osteopontin in hypoxic and ischemic brain injury.

Background

The information disclosed in this background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Perinatal hypoxic-ischemic (HI) brain injury is fetal, neonatal brain injury due to perinatal hypoxic ischemia, which can lead to disruption of brain metabolism and function, abnormal brain blood flow, vascular leakage, tissue damage and necrosis and corresponding inflammatory changes, energy failure caused by ischemia, abnormal maintenance of ion gradients, lactic acid accumulation, increased production of oxygen radicals, altered lipid metabolism, triggering of arachidonic acid linkage, release of excitatory neuro mediators, imbalance in calcium ion homeostasis, etc., and thus remains one of the important causes of neonatal death and long-term mental and motor disorders, however, the underlying mechanisms thereof are still unclear.

Osteopontin (OPN) is a secreted phosphorylated glycoprotein, an important cell adhesion and chemotactic factor, which can be synthesized and secreted by various cells in the body (e.g., osteoblasts, epithelial cells, activated T lymphocytes, monocyte/macrophage lineage, nerve cells, etc.). As a cytokine, OPN is involved in various life phenomena, such as immune regulation and inflammatory reactions, infiltration and metastasis of tumors, and the like. It acts as a cell signaling molecule primarily through two mechanisms: firstly, an intramolecular glutamic acid-glycine-aspartic acid (Arg-Gly-Asp, RGD) motif is combined with an integrin family molecule; the second is the RGD-independent binding with the cell surface adhesive glycoprotein CD 44. Both modes of action mediate cell adhesion, migration and proliferation by activating specific signaling systems within the cell. At present, the role and underlying mechanism of OPN in hypoxic-ischemic brain injury is unclear.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the application of osteopontin in hypoxic and ischemic brain injury. The invention discovers that Osteopontin (OPN) in the damaged cortex of the HI newborn mouse changes most obviously by utilizing proteomics, and further tests prove that osteopontin can be a potential target point for inhibiting malignant circulation between peripheral macrophage invasion and neuroinflammation in hypoxic-ischemic brain injury, so that osteopontin can be used as a marker and a potential treatment target point for diagnosis and prognosis in the hypoxic-ischemic brain injury.

In a first aspect of the present invention, there is provided use of a substance for detecting an expression level of osteopontin in the preparation of a reagent for detecting, diagnosing or predicting progression of hypoxic-ischemic brain injury.

According to the tests of proteomics, immunohistochemistry and the like, the expression of OPN in a HI focal region is obviously increased; meanwhile, immunofluorescence shows that the OPN and the positive microglia are co-located in the HI focal region, and the astrocytes and the neurons are not overlapped with the OPN; microglial immunofluorescent staining shows that hypoxic-ischemic brain injury can cause activation of microglia and release of inflammatory factors, so that OPN can be used for detecting, diagnosing or predicting the progression of hypoxic-ischemic brain injury.

Wherein the progression of hypoxic-ischemic brain injury comprises an adverse prognosis of hypoxic-ischemic brain injury.

In a second aspect of the present invention, there is provided a composition for detecting, diagnosing or predicting the progression of hypoxic-ischemic brain injury, comprising a substance for detecting the expression of the OPN gene and its expression products based on a high-throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method; or a substance for detecting the expression of OPN protein based on an immunoassay.

In a third aspect of the present invention, there is provided a use of a substance inhibiting reduction in the OPN gene and expression products (including OPN mRNA and OPN protein) and/or activity thereof for at least one of the following a) to h):

a) inhibiting HI-induced brain damage;

b) inhibiting the malignant cycle between peripheral macrophage invasion and neuroinflammation in hypoxic-ischemic brain injury;

c) reducing the cerebral infarction area caused by hypoxic-ischemic brain injury;

d) reducing cerebral edema caused by hypoxic-ischemic brain injury;

e) inhibiting the expression of inflammatory factors caused by hypoxic-ischemic brain injury;

f) reducing the number of peripheral invading macrophages;

g) inhibiting OPN produced by peripheral invading macrophages;

h) inhibition of hypoxic-ischemic brain injury-induced CD11b ⁺/CD45 ^highRecruitment of immune cells.

Wherein, in the application of e),

such inflammatory factors include, but are not limited to, CD16, CD11b, CD86, IL-1 β, and TNF α.

In a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising a substance that inhibits reduction of the OPN gene and its expression product and/or activity.

The substance for inhibiting reduction of OPN gene and expression product and/or activity thereof comprises an antibody specific to OPN protein, RNA interference molecule or antisense oligonucleotide aiming at OPN mRNA, small molecule inhibitor, siRNA, and a substance for implementing lentivirus infection or gene knockout; further, the antibody is a human antibody or a murine antibody.

The pharmaceutical composition has the function of treating hypoxic-ischemic brain injury, and specifically, the treatment of hypoxic-ischemic brain injury at least comprises any one or more of the following a) to h):

a) inhibiting HI-induced brain damage;

b) inhibiting the malignant cycle between peripheral macrophage invasion and neuroinflammation in hypoxic-ischemic brain injury;

c) reducing the cerebral infarction area caused by hypoxic-ischemic brain injury;

d) reducing cerebral edema caused by hypoxic-ischemic brain injury;

e) inhibiting the expression of inflammatory factors caused by hypoxic-ischemic brain injury;

f) reducing the number of peripheral invading macrophages;

g) inhibiting OPN produced by peripheral invading macrophages;

h) inhibition of hypoxic-ischemic brain injury-induced CD11b ⁺/CD45 ^highImmune cellRecruitment of (1).

Wherein, in the application of e),

such inflammatory factors include, but are not limited to, CD16, CD11b, CD86, IL-1 β, and TNF α.

Further, the pharmaceutical composition may further comprise a proper amount of carriers, excipients and diluents, which are generally used.

The invention has the beneficial technical effects that:

the invention discovers and proves the application of osteopontin in hypoxic and ischemic brain injury for the first time. Specifically, experiments show that hypoxic-ischemic brain injury can cause microglial cell activation and release of inflammatory factors, immunohistochemistry shows that the expression of OPN in a HI focal region is obviously increased, and the result is consistent with the proteomics result; immunofluorescence shows that OPN and positive microglia are co-localized at a HI focal region, and astrocytes and neurons do not coincide with OPN; flow analysis showed that OPN was predominantly expressed by peripherally invading macrophages with less resident microglia.

Meanwhile, after the expression of the OPN is blocked by using the OPN antibody, the cerebral infarction area can be obviously reduced, the expression of inflammatory factors is inhibited, the number of peripheral invading macrophages is reduced, and the OPN generated by the peripheral invading macrophages is reduced. It can thus be demonstrated that OPN may be a potential target for inhibiting the vicious circle between peripheral macrophage invasion and neuroinflammation in hypoxic-ischemic brain injury. Therefore, the osteopontin can be used as a marker and a potential treatment target for diagnosis and prognosis in hypoxic-ischemic brain injury, and has good value in practical application.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a graph relating to differential protein expression in an embodiment of the present invention. Wherein (a) the heat map shows that the HI group differentially expressed proteins from the Sham group. (B) Volcano plots, each point in the plot representing a protein.

Fig. 2 is a graph relating to the activation of microglia and the release of inflammatory factors by HI in an example of the present invention, wherein (a) is immunofluorescent staining graph, scale 50 μm, N4/group, (B) injured lateral cortex was taken 72 hours after HI, and inflammatory factors CD16, CD11B, IL-1 β expression graph was measured by qRT-PCR, N4/group, statistical values mean ± standard deviation, p <0.05, p <0.01, and data were analyzed using independent sample T-test.

FIG. 3 is a diagram showing the correlation between the cell localization of OPN in the example of the present invention. Wherein, (A) is an immunohistochemical result chart, and the scale is 1 mm. (A1) To (a) show an enlarged view of the area within the frame, scale 50 μm. (B) Shows the co-staining pattern of immunofluorescence Iba-1 and OPN, (B1) shows the in-frame region Iba-1 ⁺/OPN ⁺And (5) dyeing and enlarging. (C) The immunofluorescence GFAP and NeuN were respectively stained with OPN, and (C1) shows the in-frame region GFAP ⁺/OPN ⁺Or NeuN ⁺/OPN ⁺And (5) dyeing and enlarging. Scale 50 μm and N4/group.

FIG. 4 shows an example of the present invention in which OPN is mainly impregnated with CD11b ⁺/CD45 ^highImmune cells produce the relevant figures. Wherein (a) the lateral cortex cells were injured 72 hours after HI was taken and the sample was gradually analyzed using a flow analyzer according to cell size (FSC) and particle size (SSC). (B) Represents CD11b ⁺/OPN ⁺A cell. (C) By CD11b ⁺/OPN ⁺Drawing a door to enclose infiltrated mononuclear/macrophage (CD11 b) ⁺/OPN ⁺/CD45 ^highCells) and resident microglia (CD11 b) ⁺/OPN ⁺/CD45 ^lowA cell). (D) Represents CD11b ⁺/OPN ⁺Quantitative analysis of cell number. (E) Represents CD11b ⁺/OPN ⁺/CD45 ^highQuantitative analysis of cell number. (F) Represents CD11b ⁺/OPN ⁺/CD45 ^lowQuantitative analysis of cell number. N is 4/group. The statistical value is the mean value plus or minus standard deviation; p<0.001, data were analyzed using independent sample T-test.

FIG. 5 is a graph relating to HI-induced brain injury by blocking OPN expression in an example of the present invention. (A) OPN mRNA levels were measured for each group 72 hours after HI by RT-PCR, with N being 4/group. (B) At 72 hours post-HI, the OPN protein levels were measured in each group using Western blot, N3/group. (C) Flow cytometerAnalysis of HI post-lesional lateral cortex CD11b ⁺/OPN ⁺Cell population, N-4/group. (D) Brain water content was measured 72 hours after HI, N-4/group. (E) TTC stained white areas indicate cerebral infarct size, N-4/group. The statistical value is the mean value plus or minus standard deviation; p<0.05,**p<0.01,***p<0.001. Data were analyzed using one-way anova and corrected with Bonferroni.

FIG. 6 is a graph showing that blocking OPN expression inhibits HI-induced CD11b in example of the present invention ⁺/CD45 ^highImmune cell correlation plot. Wherein (A) is through CD11b ⁺/CD45 ^lowAnd CD11b ⁺/CD45 ^highGated recognition of resident microglia and monocytes/macrophages with activated monocytes/macrophages in the upper right quadrant (CD11 b) ⁺/CD45 ^high) The lower left quadrant is primordial microglial cells (CD11 b) ⁺/CD45 ^low) And N is 4/group. (B) 72 hours after HI, the mRNA expression profiles of the inflammatory factors in each group were examined by RT-PCR. N is 4/group. The statistical value is the mean value plus or minus standard deviation; p<0.01,***p<0.001, data were analyzed using one-way anova and corrected with Bonferroni.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

The present invention will now be further described with reference to specific examples, which are provided for the purpose of illustration only and are not intended to be limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.

As previously mentioned, at present, the role and underlying mechanism of osteopontin in hypoxic-ischemic brain injury is not clear.

In view of the above, in one embodiment of the present invention, there is provided a use of a substance for detecting an expression level of osteopontin in the preparation of a reagent for detecting, diagnosing or predicting progression of hypoxic-ischemic brain injury.

According to the tests of proteomics, immunohistochemistry and the like, the expression of OPN in a HI focal region is obviously increased; meanwhile, immunofluorescence shows that the OPN and the positive microglia are co-located in the HI focal region, and the astrocytes and the neurons are not overlapped with the OPN; microglial immunofluorescent staining shows that hypoxic-ischemic brain injury can cause activation of microglia and release of inflammatory factors, so that OPN can be used for detecting, diagnosing or predicting the progression of hypoxic-ischemic brain injury.

Wherein the progression of hypoxic-ischemic brain injury comprises an adverse prognosis of hypoxic-ischemic brain injury.

In still another embodiment of the present invention, there is provided a composition for detecting, diagnosing or predicting the progression of hypoxic-ischemic brain injury, comprising a substance for detecting the expression of OPN gene and its expression product based on a high-throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method; or a substance for detecting the expression of OPN protein based on an immunoassay.

Preferably, a Northern hybridization method, a miRNA expression profile chip, a ribozyme protection analysis technology, a RAKE method and in-situ hybridization are adopted to detect the OPN gene expression (transcription) condition;

the immunodetection method preferably includes, but is not limited to, immunoblotting (Western Blot), immunofluorescence, immunoenzyme assay (ELISA), affinity immunohistochemistry, protein chips, and the like;

in still another embodiment of the present invention, there is provided a use of a substance inhibiting reduction in the OPN gene and expression products thereof (including OPN mRNA and OPN protein) and/or activity, in at least one of the following a) to h):

a) inhibiting HI-induced brain damage;

b) inhibiting the malignant cycle between peripheral macrophage invasion and neuroinflammation in hypoxic-ischemic brain injury;

c) reducing the cerebral infarction area caused by hypoxic-ischemic brain injury;

d) reducing cerebral edema caused by hypoxic-ischemic brain injury;

e) inhibiting the expression of inflammatory factors caused by hypoxic-ischemic brain injury;

f) reducing the number of peripheral invading macrophages;

g) inhibiting OPN produced by peripheral invading macrophages;

h) inhibition of hypoxic-ischemic brain injury-induced CD11b ⁺/CD45 ^highRecruitment of immune cells.

In still another embodiment of the present invention, in said e) application,

such inflammatory factors include, but are not limited to, CD16, CD11b, CD86, IL-1 β, and TNF α.

In still another embodiment of the present invention, there is provided a pharmaceutical composition comprising a substance inhibiting reduction in OPN gene and expression products and/or activity thereof.

In still another embodiment of the present invention, the substance inhibiting reduction of OPN gene and expression products and/or activity thereof comprises an antibody specific for OPN protein, an RNA interfering molecule or antisense oligonucleotide against OPN mRNA, a small molecule inhibitor, siRNA, and a substance effecting lentiviral infection or gene knock-out; further, the antibody is a human antibody or a murine antibody.

In another embodiment of the present invention, the pharmaceutical composition has an effect of treating hypoxic-ischemic brain injury, and specifically, the treatment of hypoxic-ischemic brain injury at least includes any one of the following a) to h):

a) inhibiting HI-induced brain damage;

b) inhibiting the malignant cycle between peripheral macrophage invasion and neuroinflammation in hypoxic-ischemic brain injury;

c) reducing the cerebral infarction area caused by hypoxic-ischemic brain injury;

d) reducing cerebral edema caused by hypoxic-ischemic brain injury;

e) inhibiting the expression of inflammatory factors caused by hypoxic-ischemic brain injury;

f) reducing the number of peripheral invading macrophages;

g) inhibiting OPN produced by peripheral invading macrophages;

h) inhibition of hypoxic-ischemic brain injury-induced CD11b ⁺/CD45 ^highRecruitment of immune cells.

In still another embodiment of the present invention, in said e) application,

such inflammatory factors include, but are not limited to, CD16, CD11b, CD86, IL-1 β, and TNF α.

In another embodiment of the present invention, the pharmaceutical composition may further comprise a proper amount of carriers, excipients and diluents, which are generally used. Further, the composition can be prepared into oral preparations such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and spray, external preparations, suppositories, and sterile injectable solutions according to a conventional method.

In yet another embodiment of the present invention, the non-pharmaceutically active ingredients such as carriers, excipients and diluents which may be included are well known in the art and can be determined by one of ordinary skill in the art to meet clinical criteria.

In still another embodiment of the present invention, the carrier, excipient and diluent include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In yet another embodiment of the present invention, the pharmaceutical composition of the present invention can be administered into the body by known means. For example, by intravenous systemic delivery or local injection into the tissue of interest. Optionally via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are indicated, and are generally carried out under conventional conditions.