Application of CD177 protein glycosylation modification in acute brain injury neuroinflammation treatment

Abstract

The invention provides application of CD177 protein glycosylation modification in treatment of acute brain injury neuroinflammation. In particular, the invention provides an application of the St3gal5 protein or the St3gal5 protein up-regulating agent in preparation of the CD177 protein up-regulating agent.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and relates to the application of glycosylation modification of CD177 protein in the treatment of acute brain injury neuroinflammation. Background Art

[0002] Acute brain injuries, including traumatic brain injury (TBI), acute ischemic stroke (AIS), and intracranial hemorrhage (ICH), affect millions of people worldwide every year. Although pre-hospital emergency care and neurocritical care can significantly improve the survival rate of such diseases, secondary neuroinflammation caused by primary brain injury can seriously affect neural repair and cause neurological complications. Neuroprotective therapy is a very promising method for treating and preventing secondary injuries of acute brain injury. Hundreds of neuroprotective drugs have been evaluated in preclinical studies and have been found to have good effects, but have all failed in clinical trials. Therefore, based on the complex pathophysiological responses after acute brain injury, there is an urgent need to have in-depth and innovative insights into the pathological mechanisms of neuroinflammation and to propose promising neuroprotective treatments.

[0003] After acute brain injury occurs, surrounding viable tissues are activated and immune cells are recruited from the peripheral circulation system, initiating sterile inflammation. The first to respond are microglia, which migrate to the site of injury and release proinflammatory cytokines and chemokines, promoting the infiltration of leukocytes in the peripheral circulation. Neutrophils, as the first peripheral immune cells activated after brain injury, play an important role in the development of neuroinflammation and the process of injury repair after injury. More and more studies have found that neutrophils have significant heterogeneity, among which CD177-positive neutrophils are a newly discovered neutrophil subtype that increases in inflammatory conditions in recent years. Studies have reported that compared with CD177-negative subpopulations, CD177-positive neutrophils produce lower levels of proinflammatory cytokines (interferon-γ, interleukin (IL)-6, IL-17A). The loss of CD177 gene and protein can aggravate the symptoms of mice with colitis and damage the intestinal barrier. Moreover, as the main immunosuppressive cell type in tumors, regulatory T cells (Treg) also express CD177 protein. The presence of CD177 protein has been confirmed to be closely related to the immunosuppressive ability of Treg cells. Blocking CD177 reduces the inhibitory activity of Treg cells, while the absence of CD177 leads to reduced tumor growth in mice. Therefore, CD177 protein has immunosuppressive function and plays a certain role in suppressing inflammation.

[0004] Therefore, there is a need in the art to provide mechanisms and methods for regulating CD177 protein or CD177-positive neutrophils, thereby providing drugs or methods for treating CD177-mediated related diseases. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preventing / treating neuroinflammation by drug intervention based on glycosylation modification of CD177 protein.

[0006] In a first aspect, the present invention provides use of St3gal5 protein or a St3gal5 protein up-regulator in the preparation of a CD177 protein up-regulator.

[0007] In another preferred embodiment, the CD177 protein upregulator is used to upregulate the level of CD177-positive neutrophils or upregulate the expression or surface quantity of CD177 in neutrophils.

[0008] In another preferred embodiment, the CD177 protein upregulator is used to downregulate the levels of interferon-γ, interleukin (IL)-6, IL-17A, TNF-α, IL-17A and / or sema4d.

[0009] In another preferred embodiment, the CD177 protein upregulator is used to prevent or treat colitis or intestinal barrier damage.

[0010] In another preferred embodiment, the St3gal5 protein upregulator is selected from the following group: substances that promote the expression of St3gal5 protein gene, substances that increase the activity of St3gal5 protein and / or substances that inhibit the degradation or inactivation of St3gal5 protein.

[0011] In another preferred embodiment, the St3gal5 protein upregulator is selected from the following group: small molecule compounds, proteins, nucleic acids, or a combination thereof.

[0012] In another preferred embodiment, the St3gal5 protein upregulator is selected from the following group of substances or their pharmaceutically acceptable salts: butyrate, phenyl butyrate, valproic acid, romidepsin (FK-228), vorinostat (Vorinostat), trichostatin, apicidin, epoxy oxime, entinostat (MS-275), mosetinol (MGCD0103) or a combination thereof.

[0013] The second aspect of the present invention provides the use of St3gal5 protein or St3gal5 protein upregulator in the preparation of a pharmaceutical composition for preventing or treating neuroinflammation with low CD177 expression.

[0014] In another preferred embodiment, low expression of CD177 means having a low level of CD177-positive neutrophils.

[0015] In another preferred embodiment, the St3gal5 protein up-regulator includes a substance that promotes the expression of the St3gal5 protein gene, a substance that increases the activity of the St3gal5 protein, and / or a substance that inhibits the degradation or inactivation of the St3gal5 protein.

[0016] In another preferred embodiment, the St3gal5 protein upregulator is selected from the following group of substances or pharmaceutically acceptable salts thereof: butyrate, phenyl butyrate, valproic acid, romidepsin (FK-228), vorinostat, trichostatin, apicidin, or a combination thereof.

[0017] In another preferred embodiment, the St3gal5 protein upregulator is valproic acid or a pharmaceutically acceptable salt thereof.

[0018] In another preferred embodiment, the neuroinflammation is neuroinflammation caused by acute brain injury.

[0019] In another preferred embodiment, the acute brain injury is traumatic brain injury (TBI), acute ischemic stroke (AIS), intracranial hemorrhage (ICH), or a combination thereof.

[0020] In another preferred embodiment, the prevention or treatment of the neuroinflammation includes reducing the infarct volume, reducing blood-brain barrier damage, reducing intracranial hemorrhage, reducing the number of pro-inflammatory CCL2-positive microglia, IL-1α microglia, or a combination thereof.

[0021] In another preferred embodiment, preventing or treating the neuroinflammation comprises inhibiting or reducing microglial apoptosis.

[0022] In another preferred embodiment, the prevention or treatment of the neuroinflammation comprises downregulating the levels of interferon-γ, interleukin (IL)-6, IL-17A, TNF-α, IL-17A and / or sema4d.

[0023] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0024] The third aspect of the present invention provides the use of St3gal5 protein inhibitors and / or glycosylation inhibitors in the preparation of CD177 protein down-regulators.

[0025] In another preferred embodiment, the St3gal5 protein (enzyme) inhibitor is a St3gal5 antibody, such as a monoclonal antibody or a polyclonal antibody, or P-3FAX-Neu5AC.

[0026] In another preferred embodiment, the glycosylation inhibitor is an inhibitor that inhibits the formation of N-glycosidic bonds in protein glycosylation modification.

[0027] In another preferred embodiment, the glycosylation inhibitor is tunicamycin (such as tunicamycin A, tunicamycin B, tunicamycin C, tunicamycin D, or a mixture thereof), triamcinolone, or a combination thereof.

[0028] In a fourth aspect, the present invention provides the use of St3gal5 protein as a catalyst for glycosylation at position 115 of CD177 protein.

[0029] In another preferred embodiment, St3gal5 protein serves as a catalyst for sialylation of the 115th glycosyl group of CD177 protein.

[0030] In a fifth aspect, the present invention provides a method for prolonging the half-life of CD177 protein, wherein the CD177 protein is contacted with St3gal5 protein and a sialylated substrate to increase the sialylation level of the 115th glycosyl group of the CD177 protein, thereby prolonging the half-life of the CD177 protein.

[0031] In another preferred embodiment, the CD177 protein is human wild-type CD177 protein.

[0032] In another preferred embodiment, the sialylation substrate is cytidine-5'-monophospho-N-acylneuraminic acid (CMP-Neu5Ac).

[0033] In a sixth aspect, the present invention provides the use of a glycosylation or sialylation detection reagent in the preparation of a kit for detecting the glycosylation or sialylation level of asparagine 115 of CD177 protein.

[0034] In another preferred embodiment, the kit is used to detect the expression or quantity level of CD177 in neutrophils.

[0035] In another preferred embodiment, the detection reagent is a mass spectrometry detection reagent.

[0036] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This shows that N-glycosylation is essential for maintaining the stable expression of CD177 and anti-inflammatory maintenance. A. Immunofluorescence staining shows CD177 in brain slices 1 day after MCAO + Increased neutrophils and CD177 in brain slices 3 days after MCAO + Neutropenia; BD. Obtained CD45.1 by flow cytometry + CD177 + Neutrophils were infused into CD45.2 syngeneic mice via tail vein. Multiple tissues including brain tissue, peripheral blood, bone marrow, spleen, lung, and liver were collected from CD45.2 mice 3 days after MCAO and analyzed by flow cytometry for CD45.1 +Changes in neutrophils. CD45.1 appeared in the brain tissue of CD45.2 mice + CD177 - Neutrophils, and CD45.1 in brain tissue + CD177 - The proportion of neutrophils is much higher than that of other tissues; E. Primary neutrophils were treated with different concentrations of triamcinolone for 24 hours in vitro, and the CD177 protein on the neutrophil membrane was detected by flow cytometry. The results showed that after triamcinolone inhibited the glycosylation reaction of neutrophils, the expression of CD177 protein decreased significantly; F. LC-MS / MS protein spectrum technology was used to analyze the CD177 protein at the N115 position of wild-type neutrophils. G. Graphical fragmentation diagram of CD177 protein. Blue = N-acetylglucosamine, green = mannose, yellow = galactose, purple = N-acetylneuraminic acid; H. Schematic diagram of mouse CD177-NQ mutant plasmid construction; I. Flow cytometry diagram and flow cytometry statistics of CD177 expression in CD177KO neutrophils transfected with different plasmids. CD177KO neutrophils transfected with CD177 wild-type gene plasmid successfully expressed CD177 protein, but CD177KO neutrophils transfected with CD177 mutant gene plasmid could not express CD177 protein. JL. CD177KO neutrophils transfected with CD177 wild-type gene plasmid significantly reduced the production of proinflammatory cytokines TNF-α, IL-17A and sema4d.

[0038] Figure 2It shows that St3gal5 is a key enzyme that mediates the sialylation and stable expression of CD177 on neutrophils. AB. After primary neutrophils were treated with the sialidase inhibitor 3Fax-Neu5AC, flow cytometry results showed that CD177 protein expression was significantly reduced; CD CD177+ and CD177- neutrophils were isolated from mouse bone marrow and stained with biotinylated lectins SNA, MAAII and streptavidin FITC. Flow cytometry detected that compared with CD177- neutrophils, both SNA and MAAII lectins on CD177+ neutrophils had significantly high affinity binding, indicating that both 2,3-glycosidic bonds and 2,6-glycosidic bonds on CD177 protein were significantly highly expressed; E. Single-cell sequencing results show the UMAP diagram of st3gal5 expression in neutrophils of wild-type mouse bone marrow. F. Gene expression of sialidase on neutrophils. Single-cell sequencing results showed that st3gal5 is the main sialidase expressed on neutrophils. G. Schematic diagram of the expression of St3gal5 in various cell types in the bone marrow of wild-type mice. St3gal5 is specifically highly expressed in the CD177-positive neutrophil population. H. Flow cytometry detection showed that CD177+ neutrophils expressed st3gal5 protein more than CD177- neutrophils in both sham-operated and MCAO groups. I. Flow cytometry detection of S100a8 cre St3gal5 fl / fl and St3gal5 fl / fl Differences in the number of CD177+ neutrophils in mice, results show S100a8 cre St3gal5 fl / fl CD177+ neutrophil population disappears in mice; JK. Flow cytometry detection of S100a8 cre St3gal5 fl / fl and St3gal5 fl / fl Fluorescence intensity of SNA and MAAII in mice. The results showed that after St3gal5 knockout, SNA was significantly downregulated and MA was compensatory upregulated; L. Flow cytometry detection of S100a8 cre St3gal5 fl / fl and St3gal5 fl / fl Fluorescence intensity of proinflammatory cytokines sema4d, IFN-y, TNF-α, and IL-17A on mouse neutrophils. The results showed that after St3gal5 knockout, the production of sema4d, IFN-y, TNF-α, and IL-17A on neutrophils increased. M. Flow cytometry was used to detect the difference in the expression of st3gal5 between CD177+ / + and CD177- / - mice. The results showed that after CD177 knockout, there was no statistical difference in the expression of st3gal5 protein.

[0039] Figure 3 It shows that St3gal5 upregulator valproic acid promotes sialylation of neutrophils and increases CD177+ neutrophils. AB. Mouse primary neutrophils were treated with valproic acid in vitro for 24 hours, and the expression of CD177 protein on neutrophils was detected by flow cytometry. The results showed that valproic acid can effectively increase the expression of CD177 protein; C. Flow cytometry results showed that valproic acid can effectively increase the 2,6-glycosidic bond represented by SNA on mouse neutrophils; D. Flow cytometry results showed that valproic acid can effectively increase the 2,3-glycosidic bond represented by MAAII on mouse neutrophils; EF. Flow cytometry results showed that valproic acid can effectively increase the expression of CD177 protein in human neutrophils (unstimulated state); GH. Flow cytometry results showed that valproic acid can effectively increase the expression of CD177 protein in human neutrophils (stimulated state).

[0040] Figure 4 The effects of VPA on CD177KO or S100a8 cre st3gal5 fl / fl There was no effect on CD177 of neutrophils. AB.VPA treated wild-type, CD177KO and S100a8 in vitro cre st3gal5 fl / fl Primary neutrophils, flow cytometry detection VPA can effectively increase the expression of st3gal5 and S100a8 on wild-type and CD177KO neutrophils cre st3gal5 fl / fl There was no significant change in neutrophils; C. VPA treated wild-type, CD177KO and S100a8 in vitro cre st3gal5 fl / fl Primary neutrophils, flow cytometry detection VPA can effectively increase the expression of CD177 on wild-type neutrophils, S100a8 cre st3gal5 fl / fl There was no significant change in neutrophils; D. VPA treated wild type, CD177KO and S100a8 in vitro cre st3gal5 fl / fl VPA can effectively detect wild-type, CD177KO and S100a8 in primary neutrophils by flow cytometry cre st3gal5 fl / fl Expression of Sema4D on primary neutrophils; E.F. vs. S100a8 cre St3gal5 fl / flAfter the MCAO model was established in mice, VPA was given and the protein expression of CD177 and st3gal5 on neutrophils in peripheral blood was detected by flow cytometry. The results showed that VPA treatment did not increase the protein expression of CD177 and st3gal5 on neutrophils; there was no difference in the expression of sema4dG.

[0041] Figure 5 It shows that VPA upregulation of st3gal5 can promote the stable expression of CD177 in neutrophils and alleviate acute brain injury. AB. After the MCAO model was established in mice, VPA was given. Flow cytometry detection of the mouse bone marrow and peripheral blood showed that CD177-positive neutrophils increased significantly; C. After the MCAO model was established in mice, VPA was given. Immunofluorescence staining showed that VPA could reduce the infarction volume of MCAO mice; D. After the MCAO model was established in mice, VPA was given. Immunofluorescence staining showed that VPA could reduce the number of pro-inflammatory CCL2-positive microglia and IL-1α microglia. E. After the TBI model was established in mice, VPA was given. Flow cytometry detection of the mouse bone marrow and peripheral blood showed that CD177-positive neutrophils increased significantly; F. After the TBI model was established in mice, VPA was given. The results of immunofluorescence staining showed that VPA could reduce the blood-brain barrier damage in TBI mice; G. After the TBI model was established in mice, VPA was given. Immunofluorescence staining showed that VPA could reduce the number of pro-inflammatory CCL2-positive microglia and IL-1α microglia. H. After the ICH model was established in mice, VPA was given. Flow cytometry showed that the number of CD177-positive neutrophils in the mouse bone marrow and peripheral blood increased significantly. I. After the ICH model was established in mice, VPA was given. Immunofluorescence staining showed that VPA could reduce the blood-brain barrier damage in ICH mice. J. After the ICH model was established in mice, VPA was given. Immunofluorescence staining showed that VPA could reduce the number of pro-inflammatory CCL2-positive microglia and IL-1α microglia. K. After the ICH model was established in mice, VPA was given to mice, which could effectively improve the survival rate of mice with intracranial hemorrhage. DETAILED DESCRIPTION

[0042] After extensive and in-depth research, the inventors have provided the application of glycosylation modification of CD177 protein in the treatment of acute brain injury neuroinflammation through a large number of screening and testing. The present invention unexpectedly discovered for the first time that the 115th asparagine of CD177 protein is glycosylated, and further discovered that the glycosylation can be used as a target for regulating CD177 protein, especially the sialylation of the glycosyl mediated by St3gal5 protein plays an important role in maintaining the stability of CD177 protein, and proved that the regulation of CD177 protein can be achieved by regulating St3gal5 protein, thereby affecting the physiological processes related to the level of CD177 protein. The present invention was completed on this basis.

[0043] the term

[0044] 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 invention belongs.

[0045] As used herein, when used in reference to a specific recited numerical value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0046] As used herein, the term "comprising" or "including (comprising)" may be open, semi-closed and closed. In other words, the term also includes "consisting essentially of" or "consisting of".

[0047] As used herein, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 25±5°C.

[0048] As used in the present invention, the term "St3gal5" refers to ST3β-galactosideα-2,3-sialyltransferase 5 (ST3GAL5). The St3gal5 protein that can be used in the present invention includes wild-type St3gal5 protein, recombinant St3gal5 protein, active fragments of St3gal5 protein, or other forms that have the activity of catalyzing sialic acid to be attached to the glycosyl terminal of protein with α2,3-glycosidic bonds. Methods for modifying or transforming proteins to make them more suitable for pharmaceutical use are readily available to those skilled in the art. Preferably, the St3gal5 protein can be derived from humans or non-human mammals, such as rats or mice. Preferably, the St3gal5 protein has the human St3gal5 amino acid sequence shown in SEQ ID No: 1, or the mouse St3gal5 amino acid sequence shown in SEQ ID No: 2, or an amino acid sequence that is more than 80%, more than 90%, or more than 95% identical to SEQ ID No: 1 and SEQ ID No: 2.

[0049] The amino acid sequence of human St3gal5 is shown in SEQ ID No: 1:

[0050] MRTKAAGCAERRPLQPRTEAAAAPAGRAMPSEYTYVKLRSDCSRPSLQWYTRAQSKMRRPSLLLKDILKCTLLVFGVWILYILKLNYTTEECDMKKMHYVDPDHVKRAQKYAQQVLQKECRPKFAKTSMALLFEHRYSVDLLPFVQKAPKDSEAESKYDPPFGFRKFSSKVQTLLELLPEHDLPEHLKAKTCRRCVVIGSGGILHGLELGHTLNQFDVVIRLNSAPVEGYSEHVGNKTTIRMTYPEGAPLSDLEYYSNDLFVAVLFKSVDFNWLQAMVKKETLPFWVRLFFWKQVAEKIPLQPKHFRILNPVIIKETAFDILQYSEPQSRFWGRDKNVPTIGVIAVVLATHLCDEVSLAGFGYDLNQPRTPLHYFDSQCMAAMNFQTMHNVTTETKFLLKLVKEGVVKDLSGGIDREF(SEQ ID No:1)

[0051] The amino acid sequence of mouse St3gal5 is shown in SEQ ID No:2:

[0052] MHTEAVGGAARRPQKLRSQAAAPACRAMPSEFTSAKLRSDCSRTSLQWYTRTQHKMRRPSLLIKDICKCTLVAFGVWLLYILILNYTAEECDMKRMHYVDPDRIKRAQSYAQEVLQKECRPRYAKTAMALLFEDRYSINLEPFVQKVPTASEAELKYDPPFGFRKFSSKVQSLLDMLPEHDFPEHLRAKACKRCVVVGNGGILHGLELGHALNQFDVVIRLNSAPVEGYSEHVGNKTTIRMTYPEGAPLSDVEYYANDLFVTVLFKSVDFKWLQAMVKNESLPFWVRLFFWKQVAEKVPLQPKHFRILNPVIIKETAFDILQYSEPQSRFWGHDKNIPTIGVIAVVLATHLCDEVSLAGFGYDLSQPRTPLHYFDSQCMGAMHWQVMHNVTTETKFLLKLLKEGVVEDLSGGIH(SEQ ID No:2)

[0053] As used herein, the term "CD177" or "CD177 protein" refers to cluster of differentiation 177 antigen (CD177, also known as human neutrophil alloantigen 2a or NB1), which is a glycosylated phosphatidylinositol (GPI-coupled N-glycosylated transmembrane protein) that is primarily expressed on the surface of neutrophils. CD177 is a polymorphic gene that is associated with a variety of important clinical diseases and plays an important role in both positive and negative regulation of CD177 levels.

[0054] The amino acid sequence of human wild-type CD177 protein is shown in SEQ ID No: 3 ( N represents asparagine at position 115):

[0055] MNSIPVLTLLGVTALLPCVPALTCQKSSAQAVRNVAELPLRWWGAGEKTCEVSEGCQDLIMLLYNGPKVNLVIIKGCTEVEDQEPKVIWLRTPGGLSVVSYTRVCRHGDLCNDV N(SEQ ID No:3)

[0056] uses

[0057] The present invention unexpectedly discovered for the first time that the 115th asparagine of the CD177 protein peptide chain is glycosylated, and further discovered that the sialylation of the glycosyl group mediated by the St3gal5 protein plays an important role in maintaining the stability of the CD177 protein, thereby affecting the physiological processes related to changes in the CD177 protein level. In particular, St3gal5 can be used as a regulatory "switch" for the CD177 protein. When the St3gal5 level is increased, the CD177 protein level is also increased, and when the St3gal5 level is decreased, the CD177 protein level is also decreased; therefore, St3gal5 can be used as a target molecule for regulating the CD177 protein, and specifically regulates the CD177 protein and the physiological processes mediated by it.

[0058] The present invention provides use of St3gal5 protein or St3gal5 protein upregulator in preparing CD177 protein upregulator.

[0059] Preferably, the St3gal5 protein upregulator is selected from the following group: substances that promote St3gal5 protein gene expression, substances that increase St3gal5 protein activity and / or substances that inhibit St3gal5 protein degradation or inactivation. The St3gal5 protein upregulator can be a small molecule compound, protein, nucleic acid, or a combination thereof.

[0060] "Upregulation" of CD177 protein refers to an increase in the level (concentration or quantity) of CD177 protein compared to not using St3gal5 protein or St3gal5 protein upregulator. St3gal5 protein acts as a sialidase to mediate sialylation of CD177. Experiments have shown that the higher the level of CD177 sialylation, the higher its stability, thereby increasing the level of CD177 or maintaining CD177 at a higher level.

[0061] Since CD177 protein is mainly expressed on the surface of neutrophils, preferably, the upregulation of CD177 protein includes upregulation of the level of CD177-positive neutrophils and / or upregulation of the number of CD177 on neutrophils (such as the number of CD177 per unit cell membrane area).

[0062] As used in the present invention, "upregulation of CD177-positive neutrophil levels" refers to an increase in the level of CD177 protein-positive neutrophils compared to not using St3gal5 protein or St3gal5 protein upregulators, including an increase in the unit number of CD177 protein-positive neutrophils in blood and tissues, or an increase in the ratio of CD177 protein-positive neutrophils / CD177 protein-negative neutrophils; the term "downregulation of CD177 protein-positive neutrophil levels" can be understood similarly.

[0063] In another preferred embodiment, the CD177 protein upregulator can be used to downregulate the levels of interferon-γ, interleukin (IL)-6, IL-17A, TNF-α, IL-17A and / or sema4d.

[0064] In another preferred embodiment, the CD177 protein upregulator is used to prevent or treat colitis or intestinal barrier damage.

[0065] Preferably, the St3gal5 protein upregulator is selected from the following group of substances or their pharmaceutically acceptable salts: butyrate, phenyl butyrate, valproic acid, romidepsin (FK-228), vorinostat, trichostatin, apicidin, or a combination thereof; preferably, the St3gal5 protein upregulator is valproic acid or its pharmaceutically acceptable salt. The acid that forms a salt with the St3gal5 protein upregulator of the present invention can be selected from the following group: hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid, etc.; or the sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt formed by the St3gal5 protein upregulator and an inorganic base; or the methylamine salt, ethylamine salt or ethanolamine salt formed by the St3gal5 protein upregulator and an organic base.

[0066] Furthermore, the present invention provides the use of St3gal5 protein or St3gal5 protein upregulator in the preparation of a pharmaceutical composition for preventing or treating neuroinflammation with low expression of CD177.

[0067] As used in the present invention, the term "neuroinflammation with low CD177 expression" refers to neuroinflammation accompanied by the symptoms of "low CD177 expression", wherein the "low level" means that the CD177 level of the subject is lower than that of a subject not suffering from neuroinflammation or lower than a medically defined threshold.

[0068] In another preferred embodiment, low expression of CD177 means having a low level of CD177-positive neutrophils.

[0069] As used in the present invention, the term "neuroinflammation with low levels of CD177-positive neutrophils" refers to neuroinflammation accompanied by symptoms of "low levels of CD177-positive neutrophils". Wherein, the "low level" means that the level (such as individuals / unit volume) of the subject's CD177-positive neutrophils in body fluids or tissues (such as blood, brain tissue, bone marrow, spleen, lung, liver, etc.) is lower than that of subjects who do not suffer from neuroinflammation or lower than a medically defined threshold; or the ratio of CD177-positive neutrophils / CD177-negative neutrophils in the subject's body fluids or tissues (such as blood, brain tissue) is lower than that of subjects who do not suffer from neuroinflammation or lower than a medically defined threshold. Methods for determining the level of CD177-positive neutrophils are known to those skilled in the art, for example, they can be determined by flow cytometry.

[0070] As used in the present invention, the "medically defined threshold value" can be obtained by measuring the corresponding indicators of subjects who do not have and / or do not have neuroinflammation through statistical methods commonly used in the art, which is easy to obtain for those skilled in the art. Preferably, the medically defined threshold value is CD177 or CD177-positive neutrophil levels that effectively reduce the level of proinflammatory cytokines (such as interferon-γ, interleukin (IL)-6, IL-17A, TNF-α, IL-17A and / or sema4d). In the present invention, the neuroinflammation can be treated in a targeted manner through the concomitant diagnosis of CD177 protein, CD177-positive neutrophils and / or CD177-negative neutrophils.

[0071] In another preferred embodiment, the St3gal5 protein up-regulator includes a substance that promotes the expression of the St3gal5 protein gene, a substance that increases the activity of the St3gal5 protein, and / or a substance that inhibits the degradation or inactivation of the St3gal5 protein.

[0072] In another preferred embodiment, the St3gal5 protein upregulator is selected from the following group of substances or their pharmaceutically acceptable salts: butyrate, phenyl butyrate, valproic acid, romidepsin (FK-228), vorinostat (Vorinostat), trichostatin, apicidin, epoxy oxime, entinostat (MS-275), mosetinol (MGCD0103) or a combination thereof.

[0073] Preferably, the St3gal5 protein upregulator is valproic acid or a pharmaceutically acceptable salt thereof.

[0074] In another preferred embodiment, the neuroinflammation is neuroinflammation caused by acute brain injury.

[0075] The present invention also found that the loss of CD177-positive neutrophils leads to apoptosis of microglia, and therefore, the upregulation of CD177-positive neutrophil levels has a protective effect on nerve cells.

[0076] In addition, the present invention has proved through experiments that the St3gal5 protein upregulator valproic acid can effectively increase the protein expression level of CD177, significantly reduce brain damage, prevent the excessive activation of microglia, improve neuroinflammation, and provide a new drug therapy for the neuroprotective treatment of acute brain injury neuroinflammation.

[0077] In another preferred embodiment, the acute brain injury is traumatic brain injury (TBI), acute ischemic stroke (AIS), intracranial hemorrhage (ICH), or a combination thereof.

[0078] In another preferred embodiment, the prevention or treatment of the neuroinflammation includes reducing the infarct volume, reducing blood-brain barrier damage, reducing intracranial hemorrhage, reducing the number of pro-inflammatory CCL2-positive microglia, IL-1α microglia, or a combination thereof.

[0079] In another preferred embodiment, preventing or treating the neuroinflammation comprises inhibiting or reducing microglial apoptosis.

[0080] The present invention also provides the use of St3gal5 protein inhibitor and / or glycosylation inhibitor in preparing CD177 protein down-regulator.

[0081] In another preferred embodiment, the glycosylation inhibitor is an inhibitor that inhibits the formation of N-glycosidic bonds in protein glycosylation modification.

[0082] In another preferred embodiment, the glycosylation inhibitor is tunicamycin, such as tunicamycin A, tunicamycin B, tunicamycin C, tunicamycin D, or a mixture thereof.

[0083] Experiments have shown that when st3gal5 on neutrophils is knocked out, the CD177-positive neutrophil population disappears. This shows that st3gal5-mediated sialylation plays a vital role in maintaining or increasing CD177 levels, and also indicates that inhibiting St3gal5 can downregulate CD177 protein levels.

[0084] In another preferred embodiment, the St3gal5 protein (enzyme) inhibitor is a St3gal5 antibody, such as a monoclonal antibody or a polyclonal antibody; or the St3gal5 protein (enzyme) inhibitor is P-3FAX-Neu5AC.

[0085] Preferably, the down-regulation of CD177 protein includes down-regulation of the level of CD177-positive neutrophils and / or down-regulation of the number of CD177 on neutrophils (such as the number of CD177 per unit cell membrane area).

[0086] The present invention also provides the use of St3gal5 protein as a catalyst for glycosylation at position 115 of CD177 protein.

[0087] More specifically, the St3gal5 protein acts as a catalyst for the sialylation of the 115th glycosyl group of the CD177 protein.

[0088] The experiments of the present invention prove that St3gal5 mediates the stable expression of CD177 protein by catalyzing sialic acid modification of CD177 protein in the form of α2,3-glycosidic bond connection.

[0089] The present invention also provides a method for prolonging the half-life of CD177 protein, wherein the CD177 protein is contacted with St3gal5 protein and sialic acid substrate to increase the sialylation level of the 115th glycosyl group of the CD177 protein, thereby prolonging the half-life of the CD177 protein.

[0090] The present invention also provides a use of a glycosylation or sialylation detection reagent in preparing a kit for detecting the glycosylation or sialylation level of asparagine at position 115 of CD177 protein.

[0091] Composition

[0092] The protein, up-regulator, down-regulator, inhibitor of the present invention is preferably provided in the form of a composition. In addition to the active ingredient, the composition generally includes a pharmaceutically acceptable carrier.

[0093] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, a substance with a reasonable benefit / risk ratio. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0094] There is no particular limitation on the administration of the hair-raising composition, and representative administrations include (but are not limited to): oral, intralesional, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration. The composition is in liquid or solid dosage form, and the dosage form of the composition can be (but is not limited to) tablets, capsules, granules, pills, powders, oral liquids, buccal tablets, or aerosols, injections, powder injections, etc.

[0095] As used herein, the term "effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. It should be understood by those skilled in the art that the "effective amount" may vary depending on the form of the pharmaceutical composition, the severity of the disease, and the combination of other drugs.

[0096] The "prevention", "treatment", "relief" or "improvement" described in the present invention includes delaying and stopping the progression of the disease, or eliminating the disease, and does not require 100% inhibition, elimination and reversal. In some embodiments, the composition or pharmaceutical composition of the present invention can prevent the occurrence of polar brain damage and / or neuroinflammation, or reduce, inhibit and / or reverse the existing polar brain damage and / or neuroinflammation by, for example, at least about 10%, at least about 30%, at least about 50%, or at least about 80%, compared to the level observed in the absence of the composition or pharmaceutical composition of the present invention.

[0097] The main advantages of the present invention include:

[0098] The present invention discovered for the first time that St3gal5 sialidase mediates the important role of glycosylation modification of CD177 protein in the expression of CD177 protein. St3gal5 sialidase catalyzes sialic acid modification of CD177 protein in the form of α2,3-glycosidic bond, maintains the stable expression of CD177 protein, makes neutrophils inhibit inflammation, and reduces the release of proinflammatory cytokines. By using the currently marketed drug St3gal5 upregulator valproic acid, the protein expression level of CD177 can be effectively increased, brain damage can be significantly reduced, excessive activation of microglia can be prevented, neuroinflammation can be improved, and a new safe drug can be provided for the neuroprotective treatment of acute brain injury neuroinflammation.

[0099] The present invention will be further described below in conjunction with specific implementation. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0100] The present invention determines the sample size by power calculation of mean square error and standard deviation for the main parameters (power 80%, α=0.05). Continuous data are expressed as mean ± standard error (SEM). The data of two groups are analyzed using t test. The data of three or more groups are analyzed using one-way analysis of variance, and the Bonferroni test is performed for multiple comparisons. The differences in the mean values ​​of multiple groups measured multiple times over time are analyzed using two-way ANOVA. In all analyses, p≤0.05 is considered statistically significant. Statistical analysis is performed using GraphPad Prism software (version 7.0).

[0101] Example 1. Mouse cerebral ischemia model:

[0102] All animal experiments were approved by the Animal Care and Use Committee of Renji Hospital and were performed in accordance with the Institutional Guide for the Care and Use of Laboratory Animals. Male 8- to 10-week-old C57 / BL6 mice were selected to establish the middle cerebral artery occlusion model (MCAO). The mice were anesthetized with 2% isoflurane. A skin incision was made in the neck to expose the left internal carotid artery and ligated. The left external carotid artery was isolated and ligated at the proximal and distal ends, and the vessel was cut open between the two ligatures and a suture plug was inserted. The suture plug was inserted into the internal carotid artery by pulling the external carotid artery parallel to the internal carotid artery, further reaching the occluded middle cerebral artery and fixed. Sham-operated animals received anesthesia and internal carotid artery ligation surgery, but no middle cerebral artery occlusion surgery. Body temperature was maintained at 37±0.5℃ with a heating pad during the surgical procedure.

[0103] Example 2. Mouse acute craniocerebral trauma model:

[0104] Male 8- to 10-week-old C57 / BL6 mice were selected to establish a craniocerebral trauma model (CCI). The mice were anesthetized with 2% isoflurane and fixed on a stereotaxic frame. Then a sagittal incision was made on the scalp, and a circular craniotomy (4 mm in diameter) was made in the middle of the left anterior and posterior bregma, with the medial edge 2 mm lateral to the midline. A 3.0 mm circular impact head was connected to an electromagnetically controlled impact device and adjusted to an angle of 15°-20° with the vertical plane so that the impact head was perpendicular to the dura mater. Then the mice were subjected to moderate cortical contusion by a single impact (piston speed: 3.0 m / s, deformation depth: 1.0 mm, dwell time: 180 ms). The sham group received the same procedure except for the CCI injury.

[0105] Example 3. Mouse intracranial hemorrhage model

[0106] Male 8- to 10-week-old C57 / BL6 mice were selected to establish a craniocerebral trauma model (CCI). The mice were anesthetized with 2% isoflurane and fixed on a stereotaxic frame. A burr hole was drilled 2.3 mm lateral to the midline on the right side of the skull and 0.5 mm anterior to the bregma. 0.0375 U collagenase was dissolved in 0.5 μl of saline and injected into the striatum at a rate of 0.5 μl / min (0.5 mm anterior, 2.3 mm right, and 3.5 mm deep relative to the bregma). The control group was injected with an equal amount of saline. The animal body temperature was maintained at 37°C with a warming blanket throughout the operation.

[0107] Example 4. Single-cell sequencing

[0108] The mice were killed on the third day after the establishment of the cerebral ischemia model, and the bone marrow and brain tissue were processed into single cell suspensions to detect cell viability:

[0109] Wild-type mouse bone marrow - control group 90.8967%

[0110] Wild-type mice bone marrow-stroke group 85.9009%

[0111] The BD Rhapsody system is used to obtain transcriptome information of single cells. Single cell capture can be achieved by randomly distributing single cell suspensions in >200,000 microwells using a limited dilution method. Oligonucleotide barcodes are added to beads to saturation to allow beads to pair with cells in microwells. Cell lysis buffer is added to allow polyadenylated RNA molecules to hybridize with microbeads. The beads are collected into a single tube for reverse transcription. In cDNA synthesis, each cDNA molecule is labeled with a unique molecular identifier (UMI) and a cell marker at the 5' end (i.e., the 3' end of the mRNA transcript) to indicate its origin cell. The whole transcriptome library single cell whole transcriptome amplification workflow was prepared using BDRhapsody. In short, the second-strand cDNA was synthesized and then connected to the WTA adapter for universal amplification. 18 PCR cycles were used to amplify the cDNA product connected to the adapter. Random primer PCR was used to prepare a sequencing library for the whole transcriptome amplification product to enrich the 3' end of the transcript connected to the cell marker and UMI. High sensitivity quantitative sequencing library was used. DNA chips (Agilent) and Qubit High Sensitivity DNA Assay (Thermo Fisher Scientific) on Bioanalyzer 2200. The libraries of each sample were sequenced by HiSeq Xten (Illumina, San Diego, CA) with 150 bp paired-end sequencing.

[0112] In this sequencing process, a total of 10,610 cells were captured from the wild-type mouse bone marrow-control group and the wild-type mouse bone marrow-stroke group to establish a sequencing library. Clustering and projection were performed based on the transcriptome profile differences of each cell, resulting in a total of 18 cell populations and 10 cell types. Groups 0, 1, 2, 3, and 6 are all neutrophils, of which groups 0 and 1 mainly express genes related to pro-inflammatory granulocytes, and groups 2, 3, and 6 mainly express genes related to anti-inflammatory granulocytes. Therefore, groups 0 and 1 are pro-inflammatory neutrophils, and groups 2, 3, and 6 are anti-inflammatory neutrophils. CD177 is mainly expressed in groups 2, 3, and 6, and groups 0 and 1 express low levels of CD177 ( Figure 1 ). By comparing the transcriptome data of CD177-positive and CD177-negative neutrophils, it was found that compared with CD177-negative neutrophils, CD177-positive neutrophils expressed higher levels of genes related to inflammation inhibition (Chil3, Ltf, Ngp, Camp); the GO enriched by differential genes focused on downregulation of inflammatory response, including the production of IFN-γ and IL-6; the enriched pathways focused on downregulation of inflammation-related pathways such as NF-KB, TNF, and JAK-STAT. This proves that CD177-positive neutrophils are neutrophils with anti-inflammatory properties (see Figure 1 ).

[0113] Subsequently, the present invention captured 12176 cells in the brain-stroke group of wild-type mice and the brain-stroke group of CD177KO mice to establish a sequencing library, and clustered and projected the transcriptome profile differences of each cell, generating a total of 19 cell populations and 12 cell types. By analyzing the microglia of the two groups, it was found that the microglia in the brain of CD177KO mice and wild-type mice had large differences. In particular, the 4 groups and 5 groups of cells completely from CD177KO mice specifically highly expressed genes related to inflammation (IL-1a, TNF, Nlrp3), and the pathways enriched by the differential genes were concentrated on TNF, IL-6-JAK-STAT3, P53 and other signaling pathways, proving that the absence of CD177-positive neutrophils led to a significant enhancement of the pro-inflammatory ability of microglia.

[0114] Example 5. Protein profiling and glycoproteomic analysis

[0115] Place the sample in the lysis buffer, sonicate in an ice bath, then centrifuge and collect the supernatant protein lysis buffer. Add pre-cooled acetone to the sample, precipitate overnight, collect the precipitate by centrifugation and redissolve in urea, dilute with ABC solution. Add TCEP to the protein solution for reduction, then add IAA for reaction. Add trypsin to the protein solution and cleave overnight on a shaker. Use C18 (Phenomenex, 15μm, ) The enzymatically hydrolyzed peptides were desalted and gradient eluted. The intact N-glycopeptides were enriched and gradient eluted using ZIC-HILIC (Merck Millipore, 5μm, 200) and concentrated to dryness under vacuum. (1) HPLC separation parameters - analytical column: 360μod×75μid, 75cm long; filler: Phenomenex Jupiter C18, 5μm, ; Trapped column: 360μod×200μid, 5cm long; Filler: Phenomenex Jupiter C18, 5μm, ; Mobile phase: Buffer A is a mixture of 99.9% H2O and 0.1% FA, Buffer B is a mixture of 99.9% ACN and 0.1% FA. Flow rate: Loading pump mobile phase flow rate (loading): 5μL / min; Nano pump mobile phase flow rate (separation): 300nL / min

[0116] Gradient: Buffer B ratio is 9% for sample loading within 12 min; then Buffer B ratio increases linearly from 9% to 40% within 188 min for elution; then Buffer B ratio is increased to 95% within 10 min, and Buffer B ratio is kept at 95% for 5 min for impurity removal; Buffer B ratio is reduced to 9% within 5 min, and Buffer B ratio is kept at 9% until the end of the gradient. (2) Nano source electrospray ionization parameters, the temperature of the ion transfer tube is set to 300 ° C, and the spray voltage is 1.9 kV. (3) Mass spectrometry and tandem mass spectrometry setting parameters, primary mass spectrometry MS: m / z range is 700-2000, mass resolution is 60k (m / z 200), automatic gain control target value (Automatic Gain Control Target, AGC Target) is 3×106, and the maximum ion injection time (Maximum Injection Time) is 20 ms. Tandem mass spectrometry MS / MS: mass resolution 30k, data-dependent acquisition (DDA) Top 20, automatic gain control target value 2×105, maximum ion injection time 250ms, parent ion isolation window 1.4m / z, dynamic exclusion 30.0s, HCD step collision normalized energy (stepped NCEs) set to 20%, 30%, 31%. Establishment and search of gPeptide database: Based on the mouse whole protein library and N-glycosylation modification database, trypsin was selected as the protease, the allowed missed cleavage sites were set to 4, the static modification was alkylation, and the MS m / z range was 700-2000. The N-glycosylation analysis database was established using gPeptide, and the mass spectrometry data was matched and identified for complete N-glycopeptides. According to the search results, the number of matching peptides (p-MPs) was screened ≥ 5, and the FDR was controlled ≤ 1%. The peptide sequence (p-Seq.), post-translational modification (p-PTMs), and monosaccharide linkage mode (g-Linkage) were removed, and the glycopeptides with site and structure scores were screened. Finally, the IDs identification list was obtained, including the number of complete N-glycopeptide identifications (Identification, IDs), peptide sequence (p-Seq.), N-glycosylation site (N-glycosite), N-sugar chain composition (Composition) and linkage mode (g-Linkage), and sugar chain structure diagnostic fragment ions (Structure-diagnostic ions) and other qualitative results. The results showed that there was a sugar chain modification on the asparagine at the N115 site on the wild-type mouse CD177 protein (see Figure 1).

[0117] Example 6. Flow cytometry

[0118] For bone marrow, peripheral blood, and brain tissue, single cell suspensions were prepared with red blood cell lysis buffer (BD) and filtered through a 70 μm nylon membrane. According to the manufacturer's instructions (eBioscience), surface molecules were directly stained with anti-mouse CD45, CD11b, Ly6g, CD177 and appropriate isotype controls for single cell suspensions, and intracellular molecules IFN-γ, TNF-α, IL-17, and St3gal5 were stained after cell membrane fixation and cell permeabilization buffer (BD). Counting was performed on a FACS Verse cell analyzer (BD Biosciences) and using FlowJo software (TreeStar).

[0119] In order to evaluate the changes of CD177 protein in the brain, CD45.1+CD11b+Ly6g+CD177+ cells were obtained by flow cytometry sorting technology. After the MCAO model of ischemic brain injury was established in CD45.2 mice, the cells were infused into the body through the tail vein. The peripheral blood and brain tissue were detected and analyzed. It was found that CD177- cells appeared in the CD45.1+ cells infused into CD45.2 mice, and the proportion in brain tissue was higher than that in peripheral blood (see Figure 1 ), indicating that CD177-positive neutrophils differentiated into CD177-negative neutrophils and CD177 protein was degraded.

[0120] To evaluate the effects of glycosylation and sialylation on CD177 protein expression, different doses of glycosylation inhibitor Tunicamycin (Selleck) and sialidase inhibitor P-3FAX-Neu5AC (R&Dsystem) were administered, and CD177 protein expression on primary neutrophils decreased in a dose-dependent manner. The proportion of CD177-positive neutrophils was significantly reduced (see Figure 1 and Figure 2 ). Different types of plasmids were transfected into primary neutrophils with knockout CD177 gene. After 24 hours of culture, it was found that CD177 wild-type gene plasmid could make neutrophils re-express CD177 protein, the proportion and number of CD177 positive neutrophils increased significantly, and the average fluorescence intensity of CD177 protein also increased. The present invention detected the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-17 in neutrophils, and found that the expression of pro-inflammatory cytokines in neutrophils overexpressing CD177 protein was significantly reduced. However, the gene plasmid transfected with CD177 glycosylation site mutant could not express CD177 protein (see Figure 1), directly proving that glycosylation modification is very important for maintaining the stable expression of CD177 protein.

[0121] To evaluate the sialic acid modification level of CD177 protein, the samples were incubated with biotinylated lectins (SNA specifically binds to α2,6 glycosidic bonds, and MAII specifically binds to α2,3 glycosidic bonds) in PBS for 20 minutes, followed by staining with FITC-labeled streptavidin, counting on a FACS Verse cell analyzer (BD Biosciences), and using FlowJo software (TreeStar). The results showed that compared with CD177-negative neutrophils, CD177-positive neutrophils had significantly higher sialylation levels (SNA, MAII), and the mean fluorescence intensity of st3gal5 protein was higher. After knocking out st3gal5 on neutrophils, the CD177-positive neutrophil population disappeared, SNA on neutrophils decreased, MA was upregulated, and the production of proinflammatory cytokines IFN-γ, TNF-α, and IL-17 increased. However, there was no statistical difference in the expression of st3gal5 on neutrophils after knocking out CD177 (see Figure 2 ), demonstrating that St3gal5-mediated sialylation is critical for the stable expression of CD177 protein.

[0122] In order to evaluate the effect of valproic acid on the level of neutrophil CD177 protein by up-regulating the expression of st3gal enzyme, the present invention treated primary wild-type neutrophils in vitro with valproic acid (100 μg / ml), and the results showed that CD177 protein increased in a dose-dependent manner. The proportion and number of CD177-positive neutrophils increased significantly, and the mean fluorescence intensity of CD177 protein increased. Sialic acid levels SNA and MAII were also significantly upregulated (see Figure 3 ). However, in CD177 knockout neutrophils, st3gal5 expression was upregulated after valproic acid treatment, while CD177 did not change. Treatment with valproic acid in st3gal5 knockout neutrophils did not change the protein level of st3gal5, and the level of CD177 did not change significantly (see Figure 4 ). After establishing an acute brain injury model in wild-type mice, valproic acid was intraperitoneally injected. The results showed that the proportion and number of CD177-positive neutrophils in peripheral blood and the mean fluorescence intensity of CD177 protein increased significantly, proving that valproic acid can effectively promote the expression of CD177 protein on neutrophils by upregulating the expression of st3gal enzyme.

[0123] Example 7. Immunofluorescence staining

[0124] In order to evaluate the changes of CD177 positive neutrophils in the brain after acute brain injury, the present invention uses immunofluorescence staining to detect the change trend of CD177 positive neutrophils over time after injury. Take the brain tissue 3 days after the model is established, fix it with 10% paraformaldehyde, dehydrate it with 30% sucrose, and place it on a frozen slicer for sectioning. Immunostaining is performed on 20 μm slices. The slices are incubated overnight at 4 ° C in the following antibodies: rabbit anti-CD177 (Invitrogen), rat anti-Ly6g (Abcam), rabbit anti-IL-1α (Abcam), rabbit anti-CCL2 (Abcam), goat anti-Iba-1 (Abcam). Use a fluorescent microscope (Olympuc) or a confocal microscope (Olympus Fluoview FV3000, Olympus, Central Valley, Pennsylvania) to capture images of 3 consecutive slices of each brain. Quantitative statistics are performed by Image J software.

[0125] Immunofluorescence results showed that after ischemic brain injury, the number of CD177-positive neutrophils infiltrating into the brain increased on the first day after injury and gradually decreased on the third day.

[0126] In order to evaluate the effect of valproic acid on neuroinflammation in acute brain injury, the present invention took brain tissue sections to detect the number of pro-inflammatory microglial cells Iba-1+IL-1α+ and Iba-1+CCL2+.

[0127] The results showed that in the MCAO, TBI and ICH models, valproic acid (135 mg / kg) was administered for three consecutive days after surgery. The number of Iba-1+IL-1α+ and Iba-1+CCL2+ pro-inflammatory microglia in the valproic acid group was significantly reduced, proving that the use of valproic acid can effectively reduce neuroinflammation after acute brain injury (see Figure 5 ).

[0128] Example 8. Cresyl violet staining

[0129] In order to evaluate the changes in the volume of cerebral hemorrhage, the present invention uses cresyl violet (CV) staining. The brain tissue 3 days after the establishment of the model was fixed with 10% paraformaldehyde, dehydrated with 30% sucrose, and then placed on a freezing microtome for slices and dried at 4°C for 24 hours. The slices were placed in CV stain (0.1%, Solarbio) for 1.5 to 2 minutes. Subsequently, they were placed in a 100% alcohol concentration solution and hydrated for 5 minutes; the slices were placed in xylene for 5 minutes. Finally, the slices were covered with Permount (Sinopharm Chemical Reagent Co., Ltd.) and the lid was closed. Images were captured on a laser scanning microscope (Olympus DP80, Olympus), and the hemorrhage area in the ipsilateral hemisphere was quantified using Image J (1.52a). The results showed that valproic acid can significantly reduce the volume of cerebral hemorrhage (see Figure 5).

[0130] discuss

[0131] In the past, the development of neuroprotective therapeutic drugs for acute brain injury has failed. The present invention first provides a new protein modification intervention target and an available marketed drug for the treatment of acute brain injury including ischemic stroke, intracranial hemorrhage, and neuroinflammation caused by traumatic brain injury.

[0132] CD177 protein is a cell surface glycoprotein linked to glycosylphosphatidylinositol, which is mainly expressed on neutrophils. CD177 protein has a certain immunosuppressive effect, but the mechanism of how to regulate the expression of CD177 protein is still unclear. Glycosylation is the most common form of post-translational modification of proteins, and plays an important regulatory role in the folding, stability, transport and function of proteins. The purpose of the present invention is to discover the mechanism of sialylation modification in glycosylation in regulating the stable expression of CD177 protein, and to provide an intervention method based on regulating the expression of CD177 protein on neutrophils to reduce the neuroprotective treatment method of neuroinflammation in acute brain injury.

[0133] In order to achieve the above-mentioned purpose, the present invention first clarifies the effect of glycosylation modification on the expression of CD177 protein. The present invention inhibits the glycosylation reaction of neutrophils, and uses flow cytometry to detect the expression level of CD177 protein. The results show that after glycosylation inhibition, the level of CD177 protein on the surface of neutrophils decreases significantly. It was subsequently detected by protein spectrum that CD177 protein has sugar chain modification at the 115th site of its protein sequence. The present invention constructs a wild-type plasmid of the CD177 protein gene and a mutant plasmid at the 115th site, and then transfects neutrophils with CD177 gene knockout. It is found that the plasmid with site mutation cannot form a neutrophil to express CD177 protein, and neutrophils produce more pro-inflammatory cytokines, which indicates that glycosylation modification is crucial for the expression and inflammation inhibition function of CD177.

[0134] In order to reveal the regulatory mechanism of glycosylation modification mediating CD177 protein expression, the present invention finds that CD177 positive neutrophils specifically highly express sialyltransferase gene St3gal5 through single cell sequencing analysis. Sialylation is a modification of a sialic acid terminal residue occurring on glycoproteins, mediated by sialyltransferase, transferring sialic acid from donor cytidine-5'-monophosphate-N-acylneuraminic acid (CMP-Neu5Ac) to the end of the acceptor sugar chain. In order to verify the effect of sialyltransferase St3gal5 on CD177 protein expression, the present invention constructs mice (S100a8creSt3gal5fl / fl) with neutrophil-specific knockout of St3gal5 gene, and finds by flow cytometry that compared with the same litter control St3gal5fl / fl mice, CD177 positive neutrophil group is missing in the neutrophil knockout St3gal5, and neutrophil produces more pro-inflammatory cytokines. The above evidence indicates that St3gal5-mediated sialylation modification is critical for the expression of CD177 protein.

[0135] In order to further reveal the expression regulation mechanism of CD177 protein mediated by St3gal5, the present invention detects the difference in sialic acid-linked glycosidic bonds formed by mice (S100a8creSt3gal5fl / fl) with neutrophil-specific knockout of St3gal5 gene and the littermate control St3gal5fl / fl mice by flow cytometry. The present invention finds that after knocking out St3gal5, the α2,3-glycosidic bonds on neutrophils are significantly reduced, and the α2,6-glycosidic bonds are compensatory increased, which suggests that St3gal5 modifies CD177 protein by affecting the connection mode of sialic acid with 2,3-glycosidic bonds.

[0136] In order to explore the effect of St3gal5-mediated sialylation modification on the expression of CD177 protein as a drug treatment target in the treatment of acute brain injury, the present invention uses St3gal5 up-regulator valproic acid to perform intraperitoneal injection on mice with acute brain injury model after surgery, and finds that it can significantly increase the number of CD177 positive neutrophils in the peripheral circulation of mice with acute brain injury, alleviate the symptoms of brain injury, reduce the number of pro-inflammatory microglia in the brain, and alleviate neuroinflammation. This suggests that drugs such as valproic acid that upregulate St3gal5 can be used as a candidate drug for increasing CD177 expression to treat acute brain injury and alleviate neuroinflammation. Valproic acid, as a drug currently on the market for preventing epileptic seizures, has long-term clinical medication experience and is very suitable as a neuroprotective drug that promotes CD177 expression and improves neuroinflammation.

[0137] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of St3gal5 protein or St3gal5 protein upregulator in the preparation of CD177 protein upregulator.

2. The use according to claim 1, characterized in that The CD177 protein upregulator is used to upregulate the level of CD177-positive neutrophils or upregulate the expression or surface quantity of CD177 in neutrophils.

3. The use according to claim 1, characterized in that The St3gal5 protein upregulator is selected from the following group of substances or their pharmaceutically acceptable salts: butyrate, phenyl butyrate, valproic acid, romidepsin (FK-228), vorinostat (Vorinostat), trichostatin, apicidin, epoxy oxime, entinostat (MS-275), mosetinol (MGCD0103) or a combination thereof.

4. Use of St3gal5 protein or St3gal5 protein upregulator in the preparation of a pharmaceutical composition for preventing or treating neuroinflammation with low CD177 expression.

5. The use according to claim 4, characterized in that The St3gal5 protein upregulator is selected from the following group of substances or their pharmaceutically acceptable salts: butyrate, phenyl butyrate, valproic acid, romidepsin (FK-228), vorinostat, trichostatin, apicidin, or a combination thereof; preferably, the St3gal5 protein upregulator is valproic acid or a pharmaceutically acceptable salt thereof.

6. The use according to claim 4, characterized in that The neuroinflammation is neuroinflammation caused by acute brain injury; preferably, the acute brain injury is traumatic brain injury (TBI), acute ischemic stroke (AIS), intracranial hemorrhage (ICH), or a combination thereof.

7. Use of St3gal5 protein inhibitors and / or glycosylation inhibitors in the preparation of CD177 protein downregulators.

8. Use of St3gal5 protein as a catalyst for glycosylation at position 115 of CD177 protein.

9. A method for extending the half-life of CD177 protein, comprising contacting CD177 protein with St3gal5 protein and sialylated substrate to increase the sialylation level of the 115th glycosyl group of CD177 protein, thereby extending the half-life of CD177 protein.

10. Use of a glycosylation or sialylation detection reagent in the preparation of a kit for detecting the glycosylation or sialylation level of asparagine 115 of CD177 protein.

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